Novel compound as UCHL-5 inhibitor and uses thereof

Abstract

The present invention relates to a novel compound as an ubiquitin C-terminal hydrolase L5 (UCHL -5) inhibitor and uses thereof.

Description

Novel compounds as UCHL-5 inhibitors and their uses

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0096697, filed July 22, 2024, and Korean Patent Application No. 10-2025-0098299, filed July 21, 2025, the entire contents of which are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a novel compound as a UCHL-5 inhibitor and its use.

[0005]

[0006] The ubiquitin proteasome system is an important regulatory mechanism in cell growth and division, cell cycle, intracellular signaling, and apoptosis. Through this regulatory mechanism, proteins that act as substrates are degraded by the proteasome. That is, multi-ubiquitin protein chains are covalently linked to substrates, and these are recognized and degraded by the 26S proteasome, which is composed of the 20S CP (catalytic core particle) and the 19S RP (regulatory particle). The 19S has deubiquitinating activity and is involved in distinguishing between long and short multi-ubiquitin chains. It is known to be particularly involved in several processes, including cell cycle progression, differentiation, DNA replication and repair, transcription, protein quality control, immune response, and apoptosis. Examples include UCHL-5 and USP14.

[0007] The ubiquitin-proteasome system has been reported to influence the induction of various cancers, neurodegenerative diseases, metabolic disorders, viral diseases, heart diseases, and age-related diseases. Inhibition of proteasome activity is known to induce cancer cell apoptosis and suppress cancer cell proliferation, leading to a significant increase in interest in and development of proteasome inhibitors (PIs) as anticancer agents. In particular, the treatment of patients with multiple myeloma, a blood cancer arising from circulating plasma cells, relies on the development of novel therapeutics such as proteasome inhibitors (PIs).

[0008] Bortezomib, a 20S proteasome inhibitor recently approved by the US FDA, serves as a prime example of the efficacy of proteasome inhibitor (PI) anticancer agents. However, numerous cancers have developed resistance to this drug. While many chemicals target enzymes in the UB pathway, ubistatin has been identified as an inhibitor that directly binds to K47-linked polyubiquitin, thereby blocking the interaction between ubiquitin and proteins. Furthermore, ubistatin has been demonstrated to inhibit proteasome-dependent degradation of cell cycle components, thereby halting cell cycle progression. However, ubistatin's membrane impermeability significantly limits its development in applications including therapeutics and intracellular probes. Therefore, the development of ubiquitin-pathway inhibitors that overcome these limitations remains a pressing need.

[0009]

[0010] One object of the present invention is to provide a composition useful for preventing or treating UCHL-5 mediated diseases.

[0011]

[0012] In order to achieve the above purpose, one aspect of the present invention provides a pharmaceutical composition for preventing or treating a UCH-5 (Ubiquitin C-Terminal Hydrolase L5)-mediated disease, comprising a compound having a chemical structure represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof.

[0013] [Chemical Formula 1]

[0014]

[0015]

[0016] The compound of the present invention has the effect of inhibiting the activity of UCHL-5 (Ubiquitin C-Terminal Hydrolase L5), a proteasome deubiquitinating enzyme, thereby causing ubiquitinated degradation target proteins to accumulate in cancer cells and causing cancer cells to die (apoptosis) due to endoplasmic reticulum stress.

[0017] Furthermore, the compound of the present invention exhibits anticancer activity even in cancers that are resistant to existing drugs such as bortezomib that target the conventional proteasome, and thus can be usefully used as an alternative or combination anticancer agent to overcome such bortezomib-resistant cancers.

[0018] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0019]

[0020] Figure 1 is a diagram showing the chemical structures of 15 compounds of the present invention.

[0021] Figure 2 is a diagram showing the analysis of the survival rate of cancer cells after treating bortezomib with bortezomib in a bortezomib-resistant cancer cell line constructed in an embodiment of the present invention.

[0022] Figure 3 is a diagram analyzing whether 15 compounds according to the present invention exhibit cell viability inhibition activity in multiple myeloma wild-type and bortezomib-resistant cell lines.

[0023] Figure 4 is a diagram analyzing the cell viability inhibition activity of compounds according to the present invention in multiple myeloma wild-type and bortezomib-resistant cell lines. In Figure 4, DK-3664 refers to a compound having the chemical structure of Chemical Formula 5, and DK-3870 refers to a compound having the chemical structure of Chemical Formula 14.

[0024] Figure 5 shows the compounds according to the present invention treated multiple myeloma wild-type and bortezomib-resistant cell lines, and the induction of caspase 3 / 7, an intracellular cell death signal, and EC of the compounds. 50 This is a diagram showing the analysis of the values. In the above Figure 5, DK-3724 refers to a compound having a chemical structure of chemical formula 13, and DK-3870 refers to a compound having a chemical structure of chemical formula 14.

[0025] Figure 6 is a diagram analyzing the UCHL-5 proteasome inhibitory activity of the compounds of the present invention. In Figure 6, DK-3664 refers to a compound having a chemical structure of Chemical Formula 5, DK-3724 refers to a compound having a chemical structure of Chemical Formula 13, and DK-3870 refers to a compound having a chemical structure of Chemical Formula 14.

[0026] Figure 7 is a diagram confirming the tumor growth inhibitory effect of the compounds of the present invention in a multiple myeloma cell line mouse model. In Figure 7, DK-3664 refers to a compound having the chemical structure of Chemical Formula 5.

[0027] Figure 8 is a diagram confirming the tumor growth inhibitory efficacy of the compounds of the present invention in a colon cancer cell line mouse model. In Figure 8, DK-3724 refers to a compound having the chemical structure of Chemical Formula 13.

[0028] Figure 9 is a diagram showing the accumulation of intracellular ubiquitin proteins and the induction of endoplasmic reticulum stress in various solid tumor types after treatment with the compounds of the present invention, as confirmed by Western blotting. In Figure 9, DK-3724 refers to a compound having the chemical structure of Chemical Formula 13, and DK-3870 refers to a compound having the chemical structure of Chemical Formula 14.

[0029] Figure 10 is a diagram showing the results of measuring the residual amounts of compounds according to the present invention after subjecting them to an enzymatic reaction. In Figure 9, DK-3611 to DK-3613, DK-3664 to DK-3666, DK-3712, DK-3715, DK-3717, DK-3719, DK-3722, DK-3724, DK-3870, DK-3926, and DK-4083 represent compounds having chemical structures represented by Chemical Formulas 2 to 16, respectively.

[0030]

[0031] First, the terms used in the present invention are defined.

[0032]

[0033] The following terms used in the present invention have the following meanings unless otherwise specified. Any undefined term has the meaning understood in the art.

[0034] Throughout the specification, reference to an element as "including" means that it may include other elements, but does not exclude other elements, unless otherwise specifically stated.

[0035] In the structural formula of this specification, the symbol "-" that binds atoms and / or groups may indicate a single bond, and the symbol "=" may indicate a double bond. The above symbols may be omitted, and may also be indicated when necessary, such as when specifying a bonding atom or bonding position.

[0036] The term 'halogen' referred to in the present invention means fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0037] The 'alkyl' referred to in the present invention is an aliphatic hydrocarbon group that does not contain a double bond or a triple bond, and unless otherwise indicated, may have 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10 carbon atoms, for example, 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, and may be straight or branched. Specific examples of the above alkyl group include a methyl group, an ethyl group, a propyl group, an n-propyl group, an isopropyl group, a butyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, a 1-methylbutyl group, a 1-ethylbutyl group, a pentyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, an n-hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 4-methyl-2-pentyl group, a 3,3-dimethyl butyl group, a 2-ethylbutyl group, a heptyl group, an n-heptyl group, a 1-methylhexyl group, an octyl group, an n-octyl group, a tert-octyl group, a 1-methylheptyl group, a 2-ethylhexyl group, a 2-propylpentyl group, a n-nonyl group, a 2,2-dimethylheptyl group, Examples include, but are not limited to, 1-ethylpropyl group, 1,1-dimethylpropyl group, isohexyl group, 4-methylhexyl group, 5-methylhexyl group, and benzyl group.

[0038] The 'alkenyl' referred to in the present invention is an aliphatic hydrocarbon group containing at least one double bond, and unless otherwise indicated, may have 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10 carbon atoms, for example, 2 to 6 carbon atoms, especially 2 to 4 carbon atoms, and may be straight or branched. Specific examples of the above alkenyl group include, but are not limited to, a vinyl group, a 1-propenyl group, an isopropenyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, a 1-pentenyl group, a 2-pentenyl group, a 3-pentenyl group, a 3-methyl-1-butenyl group, a 1,3-butadienyl group, an allyl group, a 1-phenylvinyl-1-yl group, a 2-phenylvinyl-1-yl group, a 2,2-diphenylvinyl-1-yl group, a 2-phenyl-2-(naphthyl-1-yl)vinyl-1-yl group, a 2,2-bis(diphenyl-1-yl)vinyl-1-yl group, a stilbenyl group, and a styrenyl group.

[0039] The 'alkynyl' referred to in the present invention is an aliphatic hydrocarbon group containing at least one triple bond, and unless otherwise indicated, may have 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10 carbon atoms, for example 1 to 6 carbon atoms, particularly 1 to 3 carbon atoms, and may be linear or branched. Specific examples of the alkynyl group include, but are not limited to, an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, etc.

[0040] The 'cycloalkyl' referred to in the present invention is a cyclic aliphatic hydrocarbon group that does not contain a double bond or a triple bond, and unless otherwise indicated, may have 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 22, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10 carbon atoms, for example, 3 to 8 carbon atoms, and particularly 3 to 6 carbon atoms, and may be monocyclic or polycyclic. The polycyclic group refers to a group in which a cycloalkyl group is directly connected to or condensed with another ring group, and wherein the other ring group may be a cycloalkyl group, but may also be another type of ring group, for example, a heterocycloalkyl group, an aryl group, a heteroaryl group, etc. Specific examples of the cycloalkyl group include, but are not limited to, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a 3-methylcyclopentyl group, a 2,3-dimethylcyclopentyl group, a cyclohexyl group, a 3-methylcyclohexyl group, a 4-methylcyclohexyl group, a 2,3-dimethylcyclohexyl group, a 3,4,5-trimethylcyclohexyl group, a 4-tert-butylcyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, a cyclodecyl group, a cycloundecyl group, a cyclododecyl group, a bicyclo[2.2.1]heptyl group, a bicyclo[2.2.2]octyl group, a bicyclo[3.2.2]nonyl group; a bicyclo[4.4.0]decyl group; a bicyclo[4.1.0]heptyl group, etc.

[0041] The term 'cycloalkenyl' used in the present invention refers to a cyclic aliphatic hydrocarbon group containing at least one double bond, and unless otherwise indicated, may have 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 22, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10 carbon atoms, for example, 3 to 8 carbon atoms, and particularly 3 to 6 carbon atoms, and may be monocyclic or polycyclic. The polycyclic refers to a group in which a cycloalkenyl group is directly connected to or condensed with another ring group, wherein the other ring group may be a cycloalkyl group, but may also be another type of ring group, for example, a heterocycloalkyl group, an aryl group, a heteroaryl group, etc. Specific examples of the above cycloalkenyl group include, but are not limited to, a cyclopentenyl group, a cyclohexenyl group, a cyclopenta-1,3-dienyl group, a cycloheptenyl group, a cyclooctenyl group, and a cycloocta-1,4-dienyl group.

[0042] The 'aryl' referred to in the present invention is an aromatic hydrocarbon group, and unless otherwise indicated, may have 6 to 30, 6 to 28, 6 to 26, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 14, for example, 6 to 12 carbon atoms, and may be monocyclic or polycyclic. The polycyclic group refers to a group in which an aryl group is directly connected to or condensed with another ring group, and the other ring group here may be an aryl group, but may also be another type of ring group, for example, a cycloalkyl group, a heterocycloalkyl group, a heteroaryl group, etc. In addition, the aryl group includes a spiro group. Specific examples of the above aryl group include, but are not limited to, a phenyl group, a biphenyl group, a triphenyl group, a naphthyl group, an anthryl group, a chrysenyl group, a phenanthrenyl group, a perylenyl group, a fluoranthenyl group, a triphenylenyl group, a phenalenyl group, a pyrenyl group, a tetracenyl group, a pentacenyl group, a fluorenyl group, an indenyl group, an acenaphthylenyl group, a benzofluorenyl group, a spirobifluorenyl group, a 2,3-dihydro-1H-indenyl group, and condensed ring groups thereof.

[0043] The terms 'heterocycloalkyl', 'heterocycloalkenyl' and 'heteroaryl' referred to in the present invention mean that at least one atom constituting the ring of the cycloalkyl, cycloalkenyl and aryl described above is substituted with a heteroatom such as O, S, Se, N, Si and the like.

[0044] The term 'substitution' as used in the present invention means that a hydrogen atom bonded to a carbon atom in a structure is replaced with another substituent, and the position of substitution is not limited as long as it is a position where a hydrogen atom is replaced, i.e. a position where a substituent can be replaced, and when substitution is made at two or more positions, the two or more substituents may be the same or different from each other.

[0045] As used herein, the term “pharmaceutically acceptable” means that it is capable of being approved or preferably approved by a regulatory agency of the federal or state government for use in animals, and more particularly in humans, by avoiding significant toxic effects when used in conventional medicinal dosages, or is listed in the United States Pharmacopeia, or is otherwise recognized by the general pharmacopeia.

[0046] The term “pharmaceutically acceptable salt” as used herein means a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired biological or pharmacological activity of the parent compound. Examples of such salts include, but are not limited to, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and acid salts formed with acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, trifluoroacetic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonic acid, and Salts formed with organic acids such as poly-galacturonic acid are included. The compounds may also be administered as pharmaceutically acceptable quaternary salts known to those skilled in the art, particularly chlorides, bromides, iodides, -O-alkyls, toluenesulfonates, methylsulfonates, sulfonates, phosphates, or carboxylates (e.g., benzoates, succinates, acetates, glycolates, maleates, malates, fumarates, citrates, tartrates, ascorbates, cinnamoates, mandeloates, and diphenylacetates).The compound of the chemical formula of the present invention may include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates that can be prepared by conventional methods.

[0047] The term 'hydrate' as used herein refers to a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular force.

[0048] The term "solvate" as used herein refers to a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and / or suitable for human administration.

[0049] The term "prodrug" as used herein refers to a substance that is transformed into a parent drug in vivo. This refers to a compound of the present invention that can be hydrolyzed, oxidized, and undergo other reactions under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the present invention. Examples of prodrugs include, but are not limited to, compounds that contain a biohydrolyzable moiety, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs, which undergo biohydrolysis to produce the compound of the present invention. Such prodrugs include compounds that are easily prepared based on various known documents.

[0050] The term "isomer" as used herein refers to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is structurally or sterically different. Such isomers include structural isomers such as tautomers, stereoisomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and optical isomers (enantiomers). In addition, all of these isomers and mixtures thereof are also included in the scope of the present invention.

[0051] The 'pharmaceutically acceptable carrier' referred to in the present invention refers to a diluent, adjuvant, additive or carrier administered with the compound of the present invention.

[0052] The term “prevention” as used herein refers to a reduction in the risk of acquiring a disease or disorder (i.e., preventing one or more clinical symptoms of a disease from progressing in an individual who is exposed to or susceptible to the disease but has not yet developed the disease or is not showing symptoms of the disease).

[0053] As used herein, the term "treatment" means improving a disease or disorder (i.e., arresting or reducing the progression of the disease or one or more clinical symptoms of the disease), or improving one or more physical parameters that are not perceptible to the subject, or controlling the disease or disorder physically (e.g., stabilizing unrecognized symptoms), mentally (e.g., stabilizing physical parameters), or both.

[0054]

[0055] Hereinafter, the present invention will be described in detail.

[0056]

[0057] 1. Novel pyrazine or quinoxaline derivative compounds

[0058] One aspect of the present invention provides a compound having the chemical structure represented by the following chemical formula 1, or a pharmaceutically acceptable salt, isomer, solvate, hydrate or prodrug thereof.

[0059] [Chemical Formula 1]

[0060]

[0061]

[0062] In the above chemical formula 1, L may be alkynylene. Specifically, L may be alkynylene having 1 to 6 carbon atoms. In particular, L may be any one selected from the group consisting of alkynylene having 1 to 3 carbon atoms.

[0063] In the above chemical formula 1, A may be aryl. For example, A may be aryl having 4 to 7 carbon atoms. Specifically, A may be aryl having 5 to 6 carbon atoms. In particular, A may be phenyl.

[0064] In the above chemical formula 1, R1, R2, and R3 may each independently be any one selected from the group consisting of hydrogen; halogen; nitrile; nitro; substituted or unsubstituted alkyl; hydroxy; and substituted or unsubstituted alkoxy. For example, R1, R2, and R3 may each independently be any one selected from the group consisting of hydrogen; halogen; alkyl substituted or unsubstituted with at least one halogen; and alkoxy substituted or unsubstituted with at least one halogen. Specifically, R1, R2, and R3 may each independently be any one selected from the group consisting of hydrogen; halogen; alkoxy substituted or unsubstituted with at least one halogen. In particular, R1, R2, and R3 may each independently be hydrogen; halogen; methyl; ethyl; propyl; trifluoromethyl; methoxy; ethoxy; or propoxy.

[0065] In the above chemical formula 1, n may be an integer of 1 or 2. Specifically, n may be an integer of 2.

[0066] In the above chemical formula 1, R4 and R5 may each independently be a substituted or unsubstituted alkyl group or may form an aliphatic heterocycle together with N to which R4 and R5 are bonded. For example, R4 and R5 may each independently be a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms or may be an aliphatic heterocycle formed together with N to which R4 and R5 are bonded. In this case, the aliphatic heterocycle may additionally include 1 or 2 heteroatoms. Specifically, R4 and R5 may each independently be a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms or may be morpholine formed together with N to which R4 and R5 are bonded.

[0067] In the above chemical formula 1, R6 may be hydrogen or halogen. For example, R6 may be F or Cl.

[0068] In particular, the chemical structures of specific examples of compounds having the chemical structure of the above chemical formula 1 and the IUPAC names of these chemical structures may be as presented in Table 1 below, but are not limited thereto.

[0069] Chemical formula structure IUPDK name 2 2-(2-((6-Fluoro-3-((3-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine3 2-(2-((6-Fluoro-3-((3-methoxyphenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine4 2-(2-((3-((3-Bromophenyl)ethynyl)-6-fluoroquinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine5 2-(2-((6-Fluoro-3-((4-methoxyphenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine6 2-(2-((6-Fluoro-3-((4-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine7 2-(2-((3-((4-Bromophenyl)ethynyl)-6-fluoroquinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine8 2-(2-((3-((3-Chlorophenyl)ethynyl)-6-fluoroquinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine9 2-(2-((6-Fluoro-3-(phenylethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine10 2-(2-((3-((4-Chlorophenyl)ethynyl)-6-fluoroquinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine11 2-(2-((6-Fluoro-3-((2-methoxyphenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine12 2-(2-((3-((2-Chlorophenyl)ethynyl)-6-fluoroquinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine13 2-(2-((6-Fluoro-3-((2-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine14 2-(2-((6-Chloro-3-((2-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-dimethylethan-1-amine15 2-(2-((6-Chloro-3-((2-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)-N,N-diethylethan-1-amine16 4-(2-(2-((6-chloro-3-((2-fluorophenyl)ethynyl)quinoxalin-2-yl)oxy)ethoxy)ethyl)morpholine

[0070] The compounds of the present invention having the chemical structure of the above chemical formula 1 inhibit the enzymatic activity of UCHL-5 (Ubiquitin C-Terminal Hydrolase L5). Specifically, the compounds of the present invention inhibit the activity of the deubiquitinase DUB (deubiquitinase), which is an activity of UCHL-5, thereby causing ubiquitinated degradation target proteins to accumulate in cancer cells and induce endoplasmic reticulum stress, thereby killing cancer cells. The UCHL-5 is a proteasome enzyme that is a component of the ubiquitin proteasome system, which is an important regulatory mechanism in cell growth and division, cell cycle, intracellular signaling, and apoptosis. This regulatory mechanism is a process in which multiple ubiquitin protein chains covalently bind to substrate proteins, and the ubiquitinated substrate proteins are recognized and degraded by the 26S proteasome, which is composed of a 20S CP (catalytic core particle) and a 19S RP (regulatory particle). UCHL-5 possesses deubiquitin enzyme activity and is involved in the discrimination between long and short multi-ubiquitin chains, and is known to be involved in several processes, including cell cycle progression, differentiation, DNA replication and repair, transcription, protein quality control, immune response, and apoptosis. The ubiquitin-proteasome system has been reported to affect the induction of various types of cancer, neurodegenerative diseases, metabolic disorders, viral diseases, cardiac diseases, and age-related diseases, and inhibition of proteasome activity is known to induce cancer cell apoptosis and suppress cancer cell proliferation. In particular, the treatment of patients with multiple myeloma, a blood cancer arising from circulating plasma cells, relies on treatment with proteasome inhibitors (PIs).In a specific embodiment of the present invention, in an experimental example, it was confirmed that compounds having the chemical structure of chemical formula 1 of the present invention are effective as 'UCHL-5 inhibitors' that inhibit the activity of UCHL-5 proteasome enzyme.

[0071] Therefore, the compound having the chemical structure of the above chemical formula 1 of the present invention or a pharmaceutically acceptable salt, isomer, solvate, hydrate or prodrug thereof can be used as an active ingredient of a pharmaceutical composition for preventing or treating a disease associated with the activity of UCHL-5.

[0072]

[0073] 2. Uses of the new compound

[0074] Another aspect of the present invention provides a pharmaceutical composition comprising a compound having the chemical structure of the above chemical formula 1, or a pharmaceutically acceptable salt, isomer, solvate, hydrate or prodrug thereof, as an active ingredient.

[0075] As described above, the compound having the chemical structure of the above chemical formula 1 has activity as a UCHL-5 inhibitor that inhibits the activity of UCHL-5, and thus the pharmaceutical composition of the present invention can be used for the prevention or treatment of diseases mediated by UCHL-5, so-called UCHL-5-mediated diseases.

[0076] In a specific embodiment of the present invention, it was confirmed that the compounds of the present invention cause the accumulation of ubiquitinated proteins in cancer cells in a concentration-dependent manner, thereby inducing endoplasmic reticulum stress (see Figure 4). In addition, it was confirmed that the compounds of the present invention inhibit the activity of UCHL-5 (see Figure 6).

[0077] In particular, the UCHL-5-mediated disease may be a disease caused by overactivation of UCHL-5, and may be, for example, cancer.

[0078] Accordingly, the compounds of the present invention can be used as an effective ingredient of a composition for preventing, improving, or treating cancer, and another aspect of the present invention provides a composition for preventing or treating cancer comprising the compounds as an effective ingredient.

[0079] The above cancer generally refers to a physiological condition of mammals characterized by abnormal cell growth, and refers to a condition in which a problem occurs in the control function of normal cell division, differentiation, and death, resulting in abnormal excessive proliferation, infiltration into surrounding tissues and organs, formation of a mass, and destruction or deformation of existing structures.

[0080] The cancer may be colon cancer, lung cancer, myeloma, blood cancer, breast cancer, head or neck cancer, uterine cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, esophageal cancer, mesothelioma, neuroblastoma, testicular cancer, lymphoma, leukemia, stomach cancer, liver cancer, skin cancer, brain cancer, neuroma, laryngeal cancer, prostate cancer, thyroid cancer, kidney cancer, pancreatic cancer, and rectal cancer, and specifically, may be multiple myeloma, which is a tumor of malignant plasma cells in the bone marrow, colon cancer, which is a malignant tumor that occurs in the appendix, colon, and rectum, and leukemia, which is a tumor of malignant cells that originate from blood cells, but is not limited thereto, and in general, any cancer that is a physiological state characterized by abnormal cell growth may be included without limitation.

[0081] In a specific embodiment of the present invention, it was confirmed that the compound of the present invention has an excellent effect in inhibiting cancer cell proliferation for multiple myeloma, leukemia, and colon cancer (see FIGS. 5, 7, and 8).

[0082] In the present invention, the cancer may be a cancer that is resistant to bortezomib.

[0083] The cancer resistant to the above bortezomib refers to a cancer that shows extremely low sensitivity to cancer treatment such as chemotherapy using the bortezomib or cancer treatment drugs, especially anticancer treatment, and does not show improvement, relief, alleviation or treatment symptoms by the treatment. The resistant cancer may be resistant to a specific treatment from the beginning, or may not have initially shown resistance but may develop when it no longer shows sensitivity to the same treatment due to genetic mutations in cancer cells after long-term treatment. For example, the cancer resistant to the above bortezomib may be a cancer in which the survival rate of cancer cells is not significantly affected regardless of the concentration of bortezomib, and specifically, it may be a hematological cancer, colon cancer or multiple myeloma that is resistant to bortezomib.

[0084] The compound of the present invention inhibits UCHL-5 activity while simultaneously inhibiting the ubiquitin-proteasome system in cancers that are resistant to bortezomib, thereby exhibiting excellent anticancer effects even against cancers that are difficult to treat with bortezomib.

[0085]

[0086] In a specific embodiment of the present invention, it was confirmed that ubiquitin proteins accumulated in bortezomib-resistant cancer cells treated with the compounds of the present invention, thereby inducing endoplasmic reticulum stress (see FIG. 4). In particular, it was confirmed that the compounds of the present invention could kill cancer cells even in bortezomib-resistant myeloma cells (see FIG. 5).

[0087] In addition, the prevention of the above cancer means all acts that suppress or delay the occurrence or progression of cancer, and the treatment of the above cancer means all effects that do not cause the cancer cells to get worse, such as not only the death of cancer cells but also the reduction of growth of cancer cells, reduction of proliferation, reduction of recurrence, alleviation of one or more symptoms related to cancer to some extent, anticancer activity of the immune system against cancer cells and formation of immune memory or reduction of invasion of cancer cells, but is not limited thereto. The above pharmaceutical composition may further include an active ingredient exhibiting anticancer activity in addition to the above compounds. For example, it may be lenalidomide, dexamethasone, cyclophosphamide, daratumumab, etc., but is not limited thereto, and as long as it is an active ingredient exhibiting an anticancer effect and does not inhibit the anticancer effect of the compound of the present invention, it may be included without limitation.

[0088] In addition to the active ingredient, the pharmaceutical composition may further include a pharmaceutically acceptable carrier or additive.

[0089] The above term "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exhibit toxicity beyond what the subject of application (prescription) can tolerate. The carrier is defined as a compound that facilitates the addition of the compound into cells or tissues.

[0090] The pharmaceutical composition may be administered by mixing with any convenient carrier, etc., and such dosage form may be a single-dose or repeated-dose dosage form. The composition may be a solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid preparations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid preparations include, but are not limited to, solutions such as water or propylene glycol solutions, suspensions, emulsions, etc., and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, etc. For example, a powder may be prepared by simply mixing genipin, which is an active ingredient of the present invention, with an appropriate pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose. Granules can be prepared by mixing the above-described effective ingredient of the present invention with a suitable pharmaceutically acceptable carrier and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropyl cellulose, and then using a wet granulation method using a solvent such as water, ethanol, or isopropanol, or a dry granulation method using compression force. In addition, tablets can be prepared by mixing the above-described granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, and then compressing the granules using a tableting machine. When formulating the above-described pharmaceutical composition into an injection, it can be prepared according to a conventional injection manufacturing method known in the art. When formulating the injection, it can be in a form dispersed in a sterile medium so that it can be used as is when administered to a patient, or it can be in a form that is dispersed in an appropriate concentration by adding distilled water for injection and then administered.

[0091] The pharmaceutical composition may be administered by injection (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, implant), inhalation, oral administration, nasal administration, vaginal administration, rectal administration, sublingual administration, transdermal administration, topical administration, etc., depending on the disease to be treated and the condition of the subject, but is not limited thereto. Depending on the route of administration, the composition may be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, additive, or vehicle that is commonly used and non-toxic.

[0092] The pharmaceutical composition may be administered at a daily dose of about 0.0001 mg / kg to about 10 g / kg, and may be administered at a daily dosage of about 0.001 mg / kg to about 1 g / kg. The therapeutically effective amount or effective dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time, and / or administration route of the pharmaceutical composition, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. In addition, the total daily dosage may be administered several times a day for convenience, if necessary. The term "therapeutically effective amount" means an amount sufficient to produce a desired effect in a patient with cancer, including reduction of side effects or chemotherapy resistance, improvement of the condition (e.g., one or more symptoms), or delay in disease progression.

[0093] In another aspect, the present invention provides a method of treating a UCHL-5 mediated disorder, e.g., cancer, comprising administering to a subject a therapeutically effective amount of a compound having the chemical structure of Formula 1, or a pharmaceutically acceptable salt, isomer, solvate, hydrate or prodrug thereof.

[0094] In another aspect, the present invention provides a method for inhibiting the activity of UCHL-5, comprising administering to a subject a therapeutically effective amount of a compound having the chemical structure of Formula 1, or a pharmaceutically acceptable salt, isomer, solvate, hydrate or prodrug thereof.

[0095]

[0096] Hereinafter, the present invention will be described in detail by examples.

[0097]

[0098] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0099]

[0100] [Example 1]

[0101] Synthesis of novel compounds of the present invention

[0102] At the request of the Dongguk University Innovative New Drug Library Research Center, 15 compounds having chemical structures of Chemical Formulas 2 to 16 (DK-3611 to DK3613, DK-3664 to DK-3666, DK-3712, DK-3715, DK-3717, DK-3719, DK-3722, DK-3724, DK-3871, DK-3926, and DK-4083 in FIG. 1) were synthesized and prepared, as shown in Table 2 and FIG. 1 below.

[0103] Specifically, 15 compounds of the present invention were synthesized through the processes of manufacturing formulas 1 to 3 below.

[0104]

[0105] [Manufacturing Formula 1]

[0106]

[0107] Reagents and Conditions: (a) i) oxalic acid, silica gel, toluene, 110 ℃, 5 h; ii) POCl3, DMF, toluene, 110°C, 1 h; (b) PdCl2(PPh3)2, CuI, TEA, 80°C, overnight; (c) 2-[2-(dimethylamino)ethoxy]ethanol, t-BuOK, 1,4-dioxane, rt, 2 h.

[0108] Intermediate 5a-ab of Formula 6 was obtained through Sonogashira coupling between intermediate 3 of Formula 1 and various kinds of substituted phenyl acetylenes using PdCl2(PPh3)2 and CuI catalysts in TEA solvent. The Sonogashira coupling reaction was performed in a sealed tube. Subsequently, the final compound 6a-ab of Formula 6 was obtained through substitution reaction between intermediate 5a-ab and 2-[2-(dimethylamino)ethoxy]ethanol using t-BuOK as a base in 1,4-dioxane solvent.

[0109]

[0110] [Manufacturing Formula 2]

[0111]

[0112] Reagents and Conditions: (a) i) oxalic acid, silica gel, toluene, 110 ℃, 5 h; ii) POCl3, DMF, toluene, 110°C, 1 h; (b) 1-ethynyl-2-fluorobenzene, PdCl2(PPh3)2, CuI, TEA, 80°C, overnight; (c) t-BuOK, 1,4-dioxane, rt, 2 h; (d) TFA, DCM, rt, 2 h.

[0113] Intermediate 2 of the above manufacturing method 8 was synthesized by reacting 4-chlorobenzene-1,2-diamine (1) with oxalic acid using silica gel as a catalyst in toluene solvent, and then phosphorous oxychloride was added in situ to obtain intermediate 3. Subsequently, intermediate 4 was obtained through Sonogashira coupling between intermediate 3 and 1-ethynyl-2-fluorobenzene using PdCl2(PPh3)2 and CuI catalysts in TEA solvent. The Sonogashira coupling reaction was performed in a sealed tube. Finally, final compounds 6a-i were obtained through substitution reactions between intermediate 4 and various alcohols using t-BuOK as a base in 1,4-dioxane solvent. In the case of compounds 6g and 6h, the Boc protecting group was deprotected using TFA in DCM solvent to obtain compounds 7g and 7h of the above manufacturing method 8. The 47 compounds of the present invention synthesized through the above process are summarized and shown in Table 2 below.

[0114] Chemical Formula 2 Chemical Formula 3 Chemical Formula 4 Chemical Formula 5 Chemical Formula 6 Chemical Formula 7 Chemical Formula 8 Chemical Formula 9 Chemical Formula 10 Chemical Formula 11 Chemical Formula 12Chemical Formula 13 Chemical Formula 14 Chemical Formula 15 Chemical Formula 16

[0115]

[0116] [Example 2]

[0117] Confirmation of the efficacy of the compounds of the present invention in overcoming bortezomib resistance.

[0118] For the 15 compounds prepared in Example 1 above, the effect of overcoming bortezomib resistance was confirmed.

[0119] 2-1. Establishment of bortezomib-resistant cell lines

[0120] Multiple myeloma cell lines IM-9 and RPMI8226 were treated with bortezomib at gradually increasing concentrations twice a week, and during subculture, the cells were divided into six cell culture dishes; one was used to maintain the concentration and maintain cell survival, and the other five were treated with gradually increasing concentrations for a long period of time. After bortezomib treatment, dead cells were removed by washing with PBS and centrifugation. The IM-9 cell line was cultured at a concentration four times higher than the original bortezomib treatment concentration, and the RPMI8226 cell line was cultured at an concentration eight times higher than the original bortezomib treatment concentration, thereby establishing cell lines with bortezomib resistance. Thereafter, the wild-type IM-9 and RPMI8226 cell lines and the bortezomib-resistant IM-9 and RPMI8226 cell lines established as described above were treated with bortezomib at various concentrations, and the viability of the IM-9 and RPMI8226 cell lines was measured.

[0121] As a result, as shown in Fig. 2, it was confirmed that the wild-type IM-9 and RPMI8226 cell lines were inhibited in proliferation by bortezomib, whereas the bortezomib-resistant IM-9 and RPMI8226 cell lines constructed as described above were no longer inhibited in proliferation by bortezomib even when treated with bortezomib at a high concentration of 66.7 nM.

[0122] 2-2. Inhibitory effect on cancer cell proliferation in bortezomib-resistant cancer cell lines

[0123] In order to confirm whether the 15 compounds prepared in Example 1 above exhibit a therapeutic effect on bortezomib-resistant cancer, the 15 compounds were treated on a bortezomib-resistant cell line and the cancer cell proliferation inhibitory effect was confirmed.

[0124] Specifically, bortezomib was serially diluted 3-fold from a final concentration of 600 nM to 7.4 nM in RPMI1640 medium containing 10% fetal bovine serum to wild-type IM-9 and RPMI8226 cells and the bortezomib-resistant cells constructed in Example 2-1. In addition, compounds of chemical formulas 2 to 16 were serially diluted 2-fold from a final concentration of 10 μM to 0.03125 μM in RPMI1640 medium containing 10% fetal bovine serum, and then treated for 72 hours. At this time, the DK-3503 compound, which is known as a conventional anticancer agent and has the following chemical structure, was used as a control, and the DK-3503 compound was serially diluted 2-fold from a final concentration of 10 μM to 0.03125 μM, and then treated for 72 hours.

[0125]

[0126] Afterwards, the survival inhibition effect of the compounds of the present invention on wild-type multiple myeloma cells and bortezomib-resistant cells was evaluated using IC 50 The values ​​were compared and analyzed, and the IC of bortezomib-resistant cell lines 50 / Wild-type cell line IC 50 The results of calculating the ratio are shown in the table below and Figure 3.

[0127] As a result, it was confirmed that the compounds of chemical formulas 2 to 14 maintained cell death activity in bortezomib-resistant cell lines, thereby exhibiting bortezomib resistance overcoming activity.

[0128] 화합물화학식RPMI8226_WTCell viabilityIC50 (μM)RPMI8226_BRCell viabilityIC50 (μM)BR / WT RatioIM-9_WTCell viabilityIC50 (μM)IM-9_BRCell viabilityIC50 (μM)BR / WT RatioDK-361120.32 ± 0.010.41 ± 0.011.280.21 ± 0.010.21 ± 0.011.00DK-361230.41 ± 0.010.53 ± 0.021.290.41 ± 0.010.39 ± 0.010.95DK-361340.66 ± 0.020.77 ± 0.021.160.42 ± 0.010.40 ± 0.020.95DK-366450.55 ± 0.040.56 ± 0.061.020.49 ± 0.010.42 ± 0.030.86DK-366560.41 ± 0.010.42 ± 0.011.020.36 ± 0.010.34 ± 0.010.94DK-366670.37 ± 0.010.36 ± 0.010.970.22 ± 0.010.21 ± 0.010.95DK-371280.53 ± 0.080.75 ± 0.011.420.78 ± 0.020.44 ± 0.020.56DK-371590.23 ± 0.010.31 ± 0.011.350.42 ± 0.010.31 ± 0.010.74DK-3717100.23 ± 0.010.39 ± 0.011.70.24 ± 0.010.21 ± 0.010.88DK-3719111.65 ± 0.021.91 ± 0.011.163.32 ± 0.101.96 ± 0.080.59DK-3722120.56 ± 0.020.82 ± 0.011.460.90 ± 0.010.65 ± 0.040.72DK-3724130.2 ± 0.010.23 ± 0.011.150.23 ± 0.010.2 ± 0.020.87DK-3870140.11 ± 0.060.17 ± 0.011.540.078 ± 0.010.086 ± 0.0011.1DK-3926150.255 ± 0.0120.29 ± 0.0081.130.187 ± 0.0150.198 ± 0.0041.05DK-4083160.064 ± 0.0030.072 ± 0.0011.130.049 ± 0.0040.060 ± 0.0031.22Bortezomib-9.23 ± 0.25(nM)116.65 ± 1.75(nM)12.609.89 ± 0.70(nM)35.63 ± 2.74(nM)3.6.

[0129] 2-3. Effect of ubiquitin protein accumulation in bortezomib-resistant cancer cell lines

[0130] Among the 15 compounds prepared in Example 1, the compound of Chemical Formula 5 (DK-3664 in Figure 1) and the compound of Chemical Formula 14 (DK-3870 in Figure 1) were dissolved in RPMI1640 medium containing 10% fetal bovine serum to culture wild-type IM-9 and RPMI8226 cells and bortezomib-resistant cells constructed in Example 2-1, at concentrations of 0.5, 1, and 2 μM, respectively, and then cultured for 6 hours. At this time, the DK-3503 compound was used as a control.

[0131] Afterwards, cells were lysed using RIPA lysis buffer to extract proteins, and equal amounts of proteins were separated by polyacrylamide gel electrophoresis. The separated proteins were transferred to a fluorinated polyvinylidene membrane and nonspecific binding was blocked with 5% skim milk. The membrane was incubated overnight at 4°C in a refrigerator with ubiquitin antibody, and the next day, the membrane was incubated with mouse or rabbit secondary antibodies, the same species from which the primary antibodies were extracted, for 1 hour at room temperature, and Western blotting was performed using Luminata Immobilon Crescendo Western HRP substrate reagent from Merck.

[0132] As a result, as shown in FIG. 4, ubiquitinated proteins were accumulated in a concentration-dependent manner not only in wild-type cells but also in bortezomib-resistant cancer cells by the compound having the chemical structure of the above chemical formula 14, confirming that the compounds induce endoplasmic reticulum stress and exhibit activity in overcoming bortezomib resistance.

[0133] From this, it was found that the compounds of the present invention can be used to treat cancers that are resistant to bortezomib.

[0134] 2-4. Apoptotic activity of compounds of the present invention in bortezomib-resistant cell lines

[0135] The multiple myeloma wild-type cell line and bortezomib-resistant cell line of Example 2-1 were treated with various concentrations of the compound having the chemical structure of chemical formula 13 of the present invention (DK-3724 compound of Figure 1) or the compound having the chemical structure of chemical formula 14 (DK-3870 compound of Figure 1), and then it was investigated whether the intracellular cell death signal increased. At this time, the DK-3503 compound was used as a control. The change in the intracellular cell death signal was measured by performing a caspase 3 / 7 glo assay, and caspase 3 / 7 activity and EC 50 The value was measured.

[0136] As a result, as shown in Figure 5, the cell death signal in the cell line increased depending on the treatment concentration of the compound of the present invention, and EC 50 As a result of comparing the values, the compounds of the present invention have an EC of 5 to 10 times higher than that of the DK-3503 compound used as a conventional anticancer agent. 50 It was confirmed that the value was low and the cell death activity was significantly superior.

[0137]

[0138] [Example 3]

[0139] UCHL-5 proteasome activity inhibitory effect of compounds of the present invention

[0140] The compounds prepared in Example 1 were examined to determine what effect they had on the protein activity of the UCHL-5 proteasome. Among the compounds of Example 1, the inhibitory effect on the activity of the target protein, UCHL-5 proteasome, was investigated for three compounds of chemical formulas 5, 13, and 14 (DK-3664, DK-3724, and DK-3870 in Figure 1). As a result, as shown in Figure 6 below, it was confirmed that the three compounds of the present invention had UCHL-5 inhibitory activity that was three times stronger than that of DK-3503, a compound known to have inhibitory activity against UCHL-5.

[0141] Specifically, 1 ng / 10 μL of recombinant UCHL-5 proteasome protein was treated with the compound of formula 5 (DK-3664) at concentrations of 0.25, 0.5, 1, and 2 μM, or the compounds of formulae 13 and 14 (DK-3724 and DK-3870) at concentrations of 0.03, 0.1, 0.3, and 1 μM, and reacted at room temperature for 60 minutes. Then, recombinant human Ubiquitin-AMC (Aminocoumarin) from RnD was added at a final concentration of 1 μM, and reacted at room temperature for 60 minutes, and the generated fluorescence was measured.

[0142] As a result, as shown in Fig. 6, it was confirmed that the three compounds of the present invention had UCHL-5 inhibitory activity that was three times stronger than that of DK-3503, a compound known to have UCHL-5 inhibitory activity.

[0143]

[0144] [Example 4]

[0145] Multiple myeloma growth inhibitory effect of compounds of the present invention

[0146] The compounds prepared in Example 1 were examined to determine their effects on the growth of multiple myeloma. First, to construct a tumor model, a luciferase-GFP stable-expressing multiple myeloma cell line, RPMI8226, and a bortezomib-resistant multiple myeloma cell line (RPMI8226), were constructed using the Firefly Luciferase eGFP lentivirus (puromycin) from BPS bioscience. Subsequently, the compound of Chemical Formula 13 (DK-3724 in Figure 1) was administered at a dose of 10 mg / kg to a tumor model in which the multiple myeloma cell line (RPMI8226) was transplanted into mice. DK-3503 or bortezomib served as the control group. As a result, as shown in Fig. 7, it was confirmed that the tumor weight was lower in mice administered DK-3724 than in mice administered DK-3503 40 mg / kg or bortezomib 0.5 mg / kg.

[0147] In particular, when the tumor mouse model transplanted with a multiple myeloma cell line (RPMI8226) resistant to bortezomib was treated with the same amount of the same compounds, the tumor weight significantly increased compared to the mouse model without bortezomib resistance when treated with bortezomib, whereas the tumor growth inhibition effect was still observed when treated with DK-3724 or DK-3503.

[0148] In addition, the compound of chemical formula 5 (DK-3664 in Figure 1) was administered at 2, 5, and 10 mg / kg, and a concentration-dependent tumor growth inhibitory effect was confirmed in wild-type and bortezomib-resistant cell line transplantation models.

[0149] Through this, it was confirmed that the compounds of the present invention have anticancer activity even against tumors or cancers that exhibit bortezomib resistance.

[0150]

[0151] [Example 5]

[0152] Confirmation of the colon cancer growth inhibitory effect of the compounds of the present invention

[0153] The compounds prepared in Example 1 were examined to determine their effects on the growth of colon cancer. A tumor model in which colon cancer cell lines were transplanted into mice was administered 10 mg / kg of the compound of Chemical Formula 13 (DK-3724 in Figure 1). DK-3503 served as the control group. As a result, as shown in Figure 8, the tumor size was significantly smaller in mice administered DK-3724 than in mice administered 40 mg / kg of DK-3503.

[0154]

[0155] [Example 6]

[0156] Mechanistic studies on three promising compounds in various hematological and solid tumors

[0157] Anticancer effects and mechanism studies of compounds of chemical formula 13 (DK-3724 in Figure 1) and 14 (DK-3870 in Figure 1) were performed on various hematological malignancies and solid tumors. Specifically, five blood cancer cell lines (HL-60, MOLT-4, THP-1, CCRF-CEM, and K562 {cultured in RPMI1640 with 10% fetal bovine serum concentration}), six lung cancer cell lines (H23, H1299, HCC827, H441, A549, and SW1573 {cultured in DMEM with 5% fetal bovine serum concentration}), two colon cancer cell lines (HCT116 and SW460 {cultured in DMEM with 5% fetal bovine serum concentration}), two esophageal cancer cell lines (TE-5 and TE-14 {cultured in RPMI1640 with 10% fetal bovine serum concentration}), and two brain tumor cell lines (T98G {cultured in DMEM with 10% fetal bovine serum concentration} and U87MG {cultured in MEM with 10% fetal bovine serum concentration}). After treating two breast cancer cell lines (MCF-7 and MDA-MB-231 {cultured in RPMI1640 with 10% fetal bovine serum concentration}), two liver cancer cell lines (HepG2 {cultured in DMEM with 5% fetal bovine serum concentration} and Hep3B {cultured in DMEM with 10% fetal bovine serum concentration}), one pancreatic cancer cell line (Mia-PDKa-2 {cultured in DMEM with 10% fetal bovine serum concentration}), one renal cancer cell line (RCC4 {cultured in DMEM with 10% fetal bovine serum concentration}), and one prostate cancer cell line (PC3 {cultured in RPMI1640 with 10% fetal bovine serum concentration}) with the compound of the present invention, the cell viability (IC) of each cancer cell was measured in the same manner as in Example 2. 50 ) was measured. At this time, the above DK-3503 and bortezomib were used as controls. Afterwards, the IC of the compounds of the present invention was used to measure the survival rate inhibition efficacy of various cancer cells. 50 The values ​​were compared and analyzed, and the calculated results are shown in Table 4 below.

[0158] In addition, after treating various cancer types with 1 μM of the compound of the present invention, accumulation of intracellular ubiquitin proteins and induction of endoplasmic reticulum stress were confirmed 6 hours later through Western blot in the same manner as in Example 2 (Fig. 9).

[0159] As a result, as shown in Table 4 below, the compounds of the present invention were found to have an inhibitory effect on cell proliferation in various types of cancer cells, and as shown in Figure 9, they were confirmed to increase the accumulation of intracellular ubiquitinated proteins and endoplasmic reticulum stress. Accordingly, it was found that the compounds of the present invention can be utilized as anticancer agents for various cancers.

[0160] Carcinoma cell line DK-3724 (nM) DK-3870 (nM) DK-3503 (μM) Bortezomib (nM) Lung cancer A549278 ± 793 ± 31.65 ± 0.01 14.35 ± 0.3 H441191 ± 884 ± 10.96 ± 0.02 6.56 ± 0.46 HCC827141 ± 449 ± 30.94 ± 0.05 5.75 ± 0.08 H1299158 ± 445 ± 21.06 ± 0.06 9.26 ± 0.41 H23130 ± 339 ± 20.81 ± 0.03 12.08 ± 0.2 SW1573188 ± 376 ± 81.26 ± 0.06 11.79 ± 0.2 Colon cancer (Colon) HCT116 186 ± 667 ± 31.39 ± 0.05 4.16 ± 0.08 SW470 456 ± 10 147 ± 73.13 ± 0.05 6.21 ± 0.35 Esophageal cancer (Esophagus) TE-5602 ± 19 170 ± 53.16 ± 0.01 7.17 ± 0.18 TE-14370 ± 17 169 ± 102.28 ± 0.05 11.65 ± 0.53 Brain cancer (Brain) T98G164 ± 576 ± 31.5 ± 0.03 12.91 ± 0.21 U87MG497 ± 46 165 ± 52.15 ± 0.06 26.4 ± 3.05 Breast cancer (Breast) MCF-73 24 ± 486 ± 11.8 ± 0.07 449.36 ± 89.36 MDA-MB-23 1350 ± 196 ± 22 ± 0.05 21.36 ± 0.25 Liver cancer (Liver) HepG 2268 ± 993 ± 31.22 ± 0.11 10.57 ± 0.95 Hep3B 156 ± 1450 ± 32.1 ± 0.13 10.95 ± 0.92 Pancreas cancer (Pancreas) Mia-PDKa-2243 ± 26 85 ± 11.8 ± 0.04 22.36 ± 0.34 Kidney cancer (Kidney) RCC 4205 ± 373 ± 51.29 ± 0.0513.51 ± 0.5Prostate cancer (PC-3343 ± 1108 ± 101.92 ± 0.0427.72 ± 0.53Multiple myeloma (Blood, Multiple Myeloma)IM-9183 ± 1543 ± 21.72 ± 0.0521.56 ± 0.841IM-9_BR179 ± 243 ± 11.39 ± 0.0299.36 ± 4.9RPMI8226222 ± 1063 ± 21.4 ± 0.0612.35 ± 0.3RPMI8226_BR314 ± 1079 ± 51.61 ± 0.04130.62 ± 17.61Leukemia(Blood,Leukemia)K562359 ± 298 ± 33.62 ± 0.0254.01 ± 3.7CCRF-CEM150 ± 337 ± 11.84 ± 0.0213.6 ± 0.6THP-1358 ± 1100 ± 23.21 ± 0.0414.18 ± 0.41MOLT-4361 ± 5108 ± 43.51 ± 0.0814.95 ± 0.64HL-60196 ± 251 ± 21.87 ± 0.0129.39 ± 1.9.

[0161]

[0162] [Example 7]

[0163] Confirmation of metabolic stability of the compounds of the present invention

[0164] Conventional proteasome inhibitors have low metabolic stability due to their peptide structure, which makes them susceptible to enzymatic degradation, making their application to solid tumors difficult. Therefore, the metabolic stability of the compounds of the present invention in Example 1 was analyzed. Specifically, mouse and human cells were treated with the compounds of the present invention and enzymes and allowed to react for 30 minutes. The residual amount (%) of the compounds was then measured.

[0165] As a result, as shown in Fig. 10, in mouse and human cells, the compounds of the present invention were found to exist in large numbers even after reacting with an enzyme, confirming that the compounds of the present invention have excellent metabolic stability.

[0166] Through the above experiments, it was found that the compounds of the present invention have excellent UCHL-5 proteasome inhibition activity, exhibit anticancer therapeutic effects even against cancers resistant to bortezomib, and have excellent metabolic stability, so that they can be applied to the treatment of hematological cancers and solid cancers.

[0167]

[0168] Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims

1. A pharmaceutical composition for preventing or treating a UCHL-5 (Ubiquitin C-Terminal Hydrolase L5)-mediated disease, comprising a compound having the chemical structure of the following chemical formula 1, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above L is alkynylene, The above A is aryl, The above R1, R2 and R3 are each independently one selected from the group consisting of hydrogen; halogen; nitrile; nitro; substituted or unsubstituted alkyl; hydroxy; and substituted or unsubstituted alkoxy; The above n is an integer of 1 or 2, The above R4 and R5 are each independently a substituted or unsubstituted alkyl group or an aliphatic heterocycle formed together with N to which the above R4 and R5 are bonded, The above R6 is hydrogen or halogen.

2. In claim 1, The above L is alkynylene, The above A is aryl having 4 to 7 carbon atoms, The above R1, R2 and R3 are each independently one selected from the group consisting of hydrogen; halogen; alkyl substituted or unsubstituted with at least one halogen; and alkoxy substituted or unsubstituted with at least one halogen; The above n is an integer of 1 or 2, The above R4 and R5 are each independently a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms or an aliphatic heterocycle formed together with N to which the above R4 and R5 are bonded, The above R6 is hydrogen or halogen.

3. In claim 1, The above L is alkynylene having 1 to 6 carbon atoms, The above A is aryl having 5 to 6 carbon atoms, The above R1, R2 and R3 are each independently one selected from the group consisting of hydrogen; halogen; alkoxy substituted or unsubstituted with at least one halogen; The above n is an integer of 2, The above R4 and R5 are each independently a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, or an aliphatic heterocycle formed by additionally including 1 or 2 heteroatoms together with N to which the above R4 and R5 are bonded, The above R6 is hydrogen or halogen.

4. In claim 1, The above L is alkynylene having 1 to 3 carbon atoms, The above A is phenyl, The above R1, R2 and R3 are each independently hydrogen; halogen; methyl; ethyl; propyl; trifluoromethyl; methoxy; ethoxy; or propoxy; The above n is an integer of 2, The above R4 and R5 are each independently a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, or an aliphatic heterocycle formed by additionally including 1 or 2 heteroatoms together with N to which the above R4 and R5 are bonded, The above R6 is hydrogen or halogen.

5. In claim 1, The above L is alkynylene having 1 to 3 carbon atoms, The above A is phenyl, The above R1, R2 and R3 are each independently hydrogen; halogen; methoxy; ethoxy; or propoxy; The above n is an integer of 2, The above R4 and R5 are each independently a substituted or unsubstituted alkyl group having 1 to 3 carbon atoms, or a morpholine formed together with N to which the above R4 and R5 are bonded, The above R6 is hydrogen or halogen.

6. In claim 1, A pharmaceutical composition wherein the compound having the chemical structure of the above chemical formula 1 is a compound having any one of the chemical structures of the following chemical formulas 2 to 16: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] [Chemical Formula 16] .

7. In claim 1, A pharmaceutical composition, wherein the above UCHL-5 mediated disease is cancer.

8. In claim 7, A pharmaceutical composition wherein the cancer is a cancer that is resistant to Bortezomib.

9. In claim 7, A pharmaceutical composition further comprising at least one agent selected from the group consisting of lenalidomide, dexamethasone, and daratumumab.

10. In claim 7, A pharmaceutical composition, wherein the cancer is at least one selected from the group consisting of blood cancer, lung cancer, myeloma, cervical cancer, breast cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, colon cancer, and neuroma.

11. In claim 10, A pharmaceutical composition wherein the cancer is multiple myeloma, leukemia or colon cancer.

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