Triazole compound having novel ring structure, method for preparing same, and use thereof

Abstract

The present invention relates to a triazole compound having a novel ring structure, a preparation method therefor, and a use thereof. The triazole compound having a novel ring structure according to the present invention exhibits excellent ASK1 inhibiting ability and inhibits apoptosis of neurons, and thus can be used for the prevention or treatment of ASK1-related diseases, including neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, and liver diseases.

Description

Technical Field

[0001] This invention relates to a triazole compound with a novel ring structure, its preparation method, and its uses. Background Technology

[0002] ASK1 (apoptosis signal-regulated kinase 1) is a c-Jun N-terminal protein kinase (JNK) and a member of the stress-responsive mitogen-activated protein kinase (MAP3K) family, also known as MAP3K5. ASK1 is a core regulator of apoptosis and is known to participate in multiple stress-induced and receptor-mediated apoptosis pathways triggered by various forms of stress, including oxidative stress, reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, unfolded protein responses (UPRs), and mitochondrial stress.

[0003] Furthermore, ASK1 plays a crucial role not only in the apoptosis pathway but also in inflammatory and innate immune responses, including cytokine responses and cell differentiation. Depending on cell type, phosphorylation of the ASK1 protein can lead to apoptosis or other cellular responses. In particular, ASK1 activation and signaling have been reported to play important roles in a wide range of diseases, including neurodegenerative diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, and metabolic diseases. In addition, ASK1 is known to be involved in mediating organ damage caused by ischemia and reperfusion in the heart, brain, and kidneys (Zhang et al. J Clin Invest. 2003; 111(12): 1933-1943.).

[0004] On the other hand, Parkinson's disease is a neurodegenerative disease that primarily affects the elderly. Parkinson's disease leads to the death of dopaminergic neurons in the substantia nigra of the midbrain, causing behavioral disorders. Furthermore, Lewy bodies, formed by the deposition of α-synuclein in various parts of the brain and peripheral nerves, are a major neuropathological feature of Parkinson's disease. Typical symptoms of Parkinson's disease include bradykinesia (abnormally slowed body movement), tremors in the hands and feet, rigidity leading to muscle and joint stiffness, and non-motor symptoms including mental disorders and autonomic dysfunction.

[0005] Current reports indicate that excessive ASK1 is closely associated with heart disease, inflammatory diseases, liver disease, infectious diseases, and degenerative brain diseases, and have proposed the possibility of ASK1-based therapy for Parkinson's disease from multiple perspectives. When Parkinson's disease is induced by α-synuclein pre-fibers (PFFs), the accumulation of phosphorylated α-synuclein (p-alpha-synuclein) in the striatum and cerebral cortex of ASK1-deficient mice is reduced. Furthermore, neuroinflammation is alleviated, leading to behavioral improvement. In addition, p-ASK1 activation levels in neurons of the substantia nigra region of Parkinson's disease patients are observed to be more than four times higher than normal.

[0006] Therefore, in the process of studying ASK1 derivatives, the inventors completed this invention by confirming that the novel triazole compound has excellent ASK1 inhibitory activity, protects nerve cells, easily penetrates the cell membrane, and is not an efflux transport protein.

[0007] Detailed description of the invention

[0008] Technical issues

[0009] The purpose of this invention is to provide a triazole compound with a novel ring structure.

[0010] The purpose of this invention is to provide a method for preparing triazole compounds with novel cyclic structures.

[0011] The object of this invention is to provide a pharmaceutical composition comprising a triazole compound having a novel ring structure as an active ingredient.

[0012] The purpose of this invention is to provide a pharmaceutical composition for the prevention or treatment of ASK1-related diseases, comprising a novel cyclic triazole compound as an active ingredient.

[0013] The object of this invention is to provide a method for preventing or treating ASK1-related diseases, comprising the step of administering a novel cyclic triazole compound to a subject in need.

[0014] The purpose of this invention is to provide the use of a novel cyclic triazole compound in the prevention or treatment of ASK1-related diseases.

[0015] The purpose of this invention is to provide the use of a novel cyclic triazole compound in the preparation of a medicament for the prevention or treatment of ASK1-related diseases.

[0016] Solution to the problem

[0017] In one aspect of the invention, a compound represented by the following chemical formula 1 is provided, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof: [Chemical Formula 1]

[0018] In the above chemical formula 1, For substituted or unsubstituted phenyl or substituted or unsubstituted C 3-8 Cycloalkenyl, Wherein, the substituted phenyl and the substituted C 3-8 Each cycloalkenyl group is independently converted by C 1-6 Alkyl, C 1-6 Alkoxy or halogen substitution; and R1, R2, and R3 are each independently H and C. 1-6 Alkyl, halogen, C 3-6 cycloalkyl or halogenated C 1-6 alkyl.

[0019] In one aspect of the invention, a method for preparing a compound represented by chemical formula 1 is provided, comprising: reacting a compound represented by chemical formula 2 with a compound represented by chemical formula 3, as shown in reaction scheme 1 below, to prepare the compound represented by chemical formula 1: [Reaction Scheme 1]

[0020] In the above reaction scheme 1, The definitions of R1, R2, and R3 are the same as those in Chemical Formula 1 above.

[0021] In one aspect of the invention, a pharmaceutical composition for the prevention or treatment of ASK1-related diseases is provided, comprising a compound represented by Formula 1 or a stereoisomer thereof, a solvate, a hydrate or a pharmaceutically acceptable salt as an active ingredient.

[0022] In one aspect of the invention, a method for preventing or treating ASK1-related diseases is provided, comprising administering to a subject a pharmaceutical composition comprising a compound represented by chemical formula 1 or a stereoisomer thereof, a solvate, a hydrate or a pharmaceutically acceptable salt as an active ingredient.

[0023] In one aspect of the invention, there is provided the use of a pharmaceutical composition in the prevention or treatment of ASK1-related diseases, wherein the pharmaceutical composition comprises a compound represented by Formula 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof.

[0024] In one aspect of the invention, there is provided the use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of ASK1-related diseases, wherein the pharmaceutical composition comprises a compound represented by chemical formula 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof.

[0025] Advantages of the present invention

[0026] The novel cyclic triazole compounds of this invention exhibit high ASK1 inhibitory activity and inhibit neuronal cell death, thus they can be used to prevent or treat ASK1-related diseases, including neurodegenerative diseases, cardiovascular diseases, and autoimmune diseases. For example, the compounds of this invention can effectively prevent or treat Parkinson's disease by improving behavioral disorders or errors associated with Parkinson's disease and symptoms of neurological disorders, significantly improving dopaminergic neurodegeneration, and reducing microglia. Furthermore, the compounds of this invention can effectively prevent or treat amyotrophic lateral sclerosis (ALS) by improving neurological disorders or disease symptoms, as well as grip weakness and improving survival rates. Additionally, the compounds of this invention can effectively prevent or treat fibromyalgia by improving pain caused by fibromyalgia. Furthermore, the compounds of this invention can improve liver fibrosis caused by non-alcoholic steatohepatitis (NAH), thereby effectively preventing or treating NHA. Attached Figure Description

[0027] Figure 1 The neuroprotective assay was shown, demonstrating the cellular protective effect as a percentage of the control group (%).

[0028] Figure 2 The cell viability (%) and TNF-α ELISA results of LPS-treated cells are shown.

[0029] Figure 3 This is a schematic diagram of an experiment to confirm the efficacy of a drug in an MPTP-induced PD mouse model.

[0030] Figure 4 This is a graph showing the results of behavioral impairment assessment (pole climbing test) in each group after drug administration in an MPTP-induced PD mouse model, where a, b, and c represent the time to the top turn, the time to descend the pole, and the total time, respectively. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0031] Figure 5a This is a graph showing the degree of TH staining in the striatum of different groups after drug treatment in an MPTP-induced PD mouse model. The significance level relative to G1 is shown. p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0032] Figure 5b The degree of TH staining in the striatum of each group after drug treatment in an MPTP-induced PD mouse model is shown.

[0033] Figure 6a This is a graph showing the degree of TH staining in the substantia nigra of each group after drug treatment in an MPTP-induced PD mouse model. The significance level relative to G1 is shown. p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0034] Figure 6b The degree of TH staining in the substantia nigra of each group after drug treatment in an MPTP-induced PD mouse model is shown.

[0035] Figure 7 This is a graph confirming dopamine concentrations in the striatum and substantia nigra of different groups after drug treatment in an MPTP-induced PD mouse model. Significance level relative to G1 - p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0036] Figure 8 This is a schematic diagram of an experiment confirming the efficacy of a drug in an α-Syn transgenic Parkinson's disease mouse model.

[0037] Figure 9 This is a graph showing the changes in body weight in different groups after drug treatment in an α-Syn transgenic Parkinson's disease mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0038] Figure 10This is a graph showing the neurobehavioral scores of each group after drug treatment in an α-Syn transgenic Parkinson's disease mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0039] Figure 11 This is a graph showing the gait error evaluation results of each group after drug treatment in an α-Syn transgenic Parkinson's disease mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0040] Figure 12a This is a graph showing the efficacy evaluation results of drug treatment in improving dopaminergic neurodegeneration in different groups of α-Syn transgenic Parkinson's disease mouse models. Significance level relative to G1 - p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0041] Figure 12b These are immunofluorescence images showing the efficacy evaluation results of drug treatment in improving dopaminergic neurodegeneration in different groups of α-Syn transgenic Parkinson's disease mouse models.

[0042] Figure 13a This is a graph showing the efficacy evaluation results of drug treatment in reducing microglia in different groups in an α-Syn transgenic Parkinson's disease mouse model. Significance level relative to G1 - p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0043] Figure 13bThese are immunofluorescence images showing the efficacy of drug treatment in reducing microglia in different groups of an α-Syn transgenic Parkinson's disease mouse model.

[0044] Figure 14 This is a schematic diagram of an experiment confirming the efficacy of a drug in an SOD1 G93A transgenic ALS mouse model.

[0045] Figure 15 This is a graph showing the changes in body weight in different groups after drug treatment in an SOD1 G93A transgenic ALS mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, p < 0.001.

[0046] Figure 16 This is a graph showing the neurological scores of each group after drug treatment in an SOD1 G93A transgenic ALS mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0047] Figure 17 This is a graph showing the changes in disease scores in different groups after drug treatment in an SOD1 G93A transgenic ALS mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0048] Figure 18 This is a graph showing the changes in grip strength in different groups after drug treatment in an SOD1 G93A transgenic ALS mouse model. The significance level relative to G1 is... p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0049] Figure 19This is a chart showing the survival rates of each group after drug treatment in the SOD1 G93A transgenic ALS mouse model.

[0050] Figure 20 This is a schematic diagram of an experiment confirming the efficacy of a drug in a reserpine-induced fibromyalgia rat model.

[0051] Figure 21 This chart shows the efficacy evaluation results of drug treatment in relieving pain in a reserpine-induced fibromyalgia rat model. Significance level relative to G1 - p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0052] Figure 22 This is a schematic diagram of an experiment confirming the efficacy of the drug in a CDAHFD-induced non-alcoholic steatohepatitis mouse model.

[0053] Figure 23a This is a graph showing the proportion of Picrosirius red staining area in the liver after drug treatment in a CDAHFD-induced non-alcoholic steatohepatitis mouse model. Significance level relative to G1 - p < 0.05, p < 0.01, : p < 0.001; significance level relative to G2 - #: p < 0.05, ##: p < 0.01, ###: p < 0.001.

[0054] Figure 23b This is an image showing the proportion of Picrosirius red staining area in the liver after drug treatment in a CDAHFD-induced non-alcoholic steatohepatitis mouse model.

[0055] Methods of implementing the invention

[0056] One aspect of the present invention provides a compound represented by chemical formula 1, or a stereoisomer, solvate, hydrate or pharmaceutically acceptable salt thereof.

[0057] [Chemical Formula 1]

[0058] In the above chemical formula 1, For substituted or unsubstituted phenyl or substituted or unsubstituted C 3-8 Cycloalkenyl, Wherein, the substituted phenyl and the substituted C 3-8 Each cycloalkenyl group is independently converted by C 1-6 Alkyl, C 1-6 Alkoxy or halogen substitution; and R1, R2, and R3 are each independently H and C. 1-6 Alkyl, halogen, C 3-6 cycloalkyl or halogenated C 1-6 alkyl.

[0059] Furthermore, in the aforementioned chemical formula 1, For substituted or unsubstituted phenyl or substituted or unsubstituted C 5-8 Cycloalkenyl, Wherein, the substituted phenyl and the substituted C 3-8 Each cycloalkenyl group is independently converted by C 1-3 Alkyl, C 1-3 Alkoxy or halogen substitution; and R1, R2, and R3 are each independently H and C. 1-3 Alkyl, halogen, C 3-6 cycloalkyl or halogenated C 1-3 alkyl.

[0060] Furthermore, in the aforementioned chemical formula 1, It is a phenyl group or a C group containing a double bond. 5-8 Cycloalkenyl; R1 is C 3-6 cycloalkyl; R2 is C 1-3 Alkyl; and R3 is a halogen.

[0061] Furthermore, in the aforementioned chemical formula 1, It is a phenyl group or a C group containing a double bond. 5-8 Cycloalkenyl; R1 is cyclopropyl; R2 is a methyl group; and R3 stands for fluorine.

[0062] As used herein, the term "cycloalkenyl" includes any stable cyclic hydrocarbon group containing one or more unsaturated carbon-carbon double bonds at any position on the ring, which may be monosubstituted or polysubstituted, and may be monovalent, divalent, or polyvalent. For the purposes of this specification, it is a divalent group fused to a triazole. Examples of cycloalkenyl groups containing a single double bond include cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.

[0063] As used in this specification, the term "alkyl" refers to a straight-chain or branched fully saturated hydrocarbon group, and examples of alkyl groups include methyl (Me), ethyl (Et), propyl (such as n-propyl or isopropyl), butyl (such as n-butyl, isobutyl, sec-butyl or tert-butyl), and pentyl (such as n-pentyl, isopentyl or neopentyl).

[0064] As used in this specification, the term "halogenated alkyl" refers to an alkyl group substituted with one or more halogens, and includes both monosubstituted groups (such as CH2F) and polysubstituted groups (such as -CF3).

[0065] According to the present invention, examples of compounds represented by chemical formula 1 include the following compounds: <1> N-(6-(1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzamide; <2> 5-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide; <3> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)methylbenzamide; <4> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(5,6,7,8-tetrahydrocycloheptan[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)benzamide; and <5> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-4-methylbenzamide.

[0066] One aspect of the present invention provides a method for preparing a compound represented by chemical formula 1, comprising: reacting a compound represented by chemical formula 2 with a compound represented by chemical formula 3, as shown in reaction scheme 1 below, to prepare a compound represented by chemical formula 1: [Reaction Scheme 1]

[0067] In the above reaction scheme 1, The definitions of R1, R2, and R3 are the same as those in Chemical Formula 1 above.

[0068] The above reaction can be carried out in organic solvents such as DMF (dimethylformamide) and NMP (N-methyl-2-pyrrolidone), and can be catalysts such as DMAP (4-dimethylaminopyridine).

[0069] The above reaction can use condensing agents such as HATU (azobenzotriazole tetramethylurea hexafluorophosphate) and T3P (propylphosphonic anhydride).

[0070] The above reaction can be carried out at temperatures ranging from 0°C to 50°C. For example, the reaction can be carried out at temperatures ranging from 0°C to 40°C, 0°C to 30°C, 10°C to 50°C, 10°C to 40°C, 10°C to 30°C, 20°C to 50°C, 20°C to 40°C, or 20°C to 30°C.

[0071] The reaction can proceed from 0.1 hours to 48 hours. For example, the reaction can proceed from 0.1 hours to 36 hours, 0.1 hours to 24 hours, 0.5 hours to 48 hours, 0.5 hours to 36 hours, 0.5 hours to 24 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 2 hours to 48 hours, 2 hours to 36 hours, 2 hours to 24 hours, 3 hours to 48 hours, 3 hours to 36 hours, 3 hours to 24 hours, 4 hours to 48 hours, 4 hours to 36 hours, 4 hours to 24 hours, 6 hours to 48 hours, 6 hours to 36 hours, or 6 hours to 24 hours.

[0072] The compounds represented by Formula 1 of this invention can be used in the form of pharmaceutically acceptable salts, and acid addition salts formed from pharmaceutically acceptable free acids can be used as salts. Acid addition salts can be obtained from the following acids: inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids and alkyl diacids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; and organic acids, such as trifluoroacetic acid, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. These pharmaceutically non-toxic salts include: sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptaates, propynates, oxalates, malonates, succinates, octanoates, sebates, fumarates, maleates, and butyn-1,4-dicarboxylate salts. Hexane-1,6-dicarboxylate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.

[0073] The acid addition salt provided by this invention can be prepared using conventional methods. For example, the derivative of chemical formula 1 is dissolved in organic solvents such as methanol, ethanol, acetone, dichloromethane, and acetonitrile, and an organic or inorganic acid is added to form a precipitate. The precipitate is then filtered and dried to obtain the acid addition salt. Alternatively, the solvent and excess acid can be removed by vacuum distillation to dryness, followed by crystallization in an organic acid to obtain the acid addition salt.

[0074] In addition, alkalis can be used to prepare pharmaceutically acceptable metal salts. For example, a compound is dissolved in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, the undissolved compound salt is filtered out, and the filtrate is evaporated and dried to obtain the alkali metal salt or alkaline earth metal salt. In this case, sodium, potassium, or calcium salts are suitable for preparation as metal salts for pharmaceutical purposes. Furthermore, the corresponding salts can be obtained by reacting the alkali metal or alkaline earth metal salt with a suitable anionic salt (e.g., silver nitrate).

[0075] Furthermore, the present invention includes not only compounds represented by chemical formula 1 and their pharmaceutically acceptable salts, but also solvates, stereoisomers or hydrates that can be prepared therefrom.

[0076] As used herein, the term "hydrate" refers to a compound or its salt that contains stoichiometric or non-stoichiometric amounts of water bound together by non-covalent intermolecular forces. The hydrates of the compounds of Formula 1 of this invention may contain stoichiometric or non-stoichiometric amounts of water bound together by non-covalent intermolecular forces. The hydrates may contain more than one equivalent of water, preferably one to five equivalents. Such hydrates can be prepared by crystallizing the compounds of Formula 1 of this invention or their stereoisomers, solvates, hydrates, or pharmaceutically acceptable salts from water or an aqueous solvent.

[0077] As used herein, the term "solvent" refers to a compound or its salt containing a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Therefore, ideal solvents include those that are volatile, non-toxic, and / or suitable for human administration.

[0078] As used herein, the term "isomer" refers to a compound or its salt that has the same chemical formula or molecular formula but differs in structure or stereochemistry. These isomers include structural isomers (including tautomers) and stereoisomers; stereoisomers include all optical isomers (enantiomers) and diastereomers arising from one or more asymmetric carbon centers, as well as geometric isomers (trans, cis). All such isomers and mixtures thereof are also included within the scope of this invention.

[0079] Furthermore, the aforementioned compounds, their stereoisomers, their solvates, their hydrates, or their pharmaceutically acceptable salts can inhibit ASK1.

[0080] Furthermore, the aforementioned compounds, their stereoisomers, their solvates, their hydrates, or their pharmaceutically acceptable salts can inhibit neuronal death.

[0081] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of ASK1-related diseases, comprising a compound represented by Formula 1 or a stereoisomer thereof, a solvate, a hydrate or a pharmaceutically acceptable salt as an active ingredient.

[0082] In addition to the active ingredients described above, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0083] The ASK1-related diseases include neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, and liver diseases.

[0084] Neurodegenerative diseases include: Alzheimer's disease, hippocampal sclerosis, frontotemporal dementia (FTD), frontotemporal degeneration (FTLD), Huntington's disease, corticobasal degeneration, amyotrophic lateral sclerosis, spinal muscular atrophy, motor neuron disease, inclusion body myositis, Parkinson's disease, Lewy body dementia, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, Pick's disease, prions, traumatic brain injury, ischemic and hemorrhagic stroke, cerebral ischemia, hypoxia, and glutamate neurotoxicity.

[0085] Cardiovascular diseases include: heart failure, ischemia, recurrent ischemia, myocardial infarction, arrhythmia, acute coronary syndrome, diabetes, atherosclerosis, and intermittent claudication.

[0086] Autoimmune diseases include: rheumatoid arthritis, fibromyalgia, systemic lupus erythematosus, multiple sclerosis, diabetes, systemic sclerosis, Graves' disease, Guillain-Barré syndrome, myasthenia gravis, psoriasis, Crohn's disease, ulcerative colitis, optic neuritis, and Sjögren's syndrome.

[0087] Liver diseases include: non-alcoholic fatty liver disease (NASH), alcoholic fatty liver disease, liver fibrosis, liver cancer, hepatotoxicity, cholestasis, cirrhosis, liver ischemia, liver abscess, hepatic coma, and liver atrophy.

[0088] In clinical administration, compounds represented by Formula 1 or their pharmaceutically acceptable salts can be administered in various oral and parenteral formulations. In formulation, common diluents or excipients are used, such as fillers, expanders, binders, wetting agents, disintegrants, and surfactants. Orally administered solid dosage forms include tablets, pills, powders, granules, capsules, etc., which are prepared by mixing one or more compounds with at least one excipient (such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.). In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Orally administered liquid dosage forms include suspensions, solutions, emulsions, syrups, etc., which may contain various excipients such as lubricants, sweeteners, flavorings, and preservatives, in addition to common simple diluents such as water and liquid paraffin. Parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Permissible non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable esters (such as ethyl oleate).

[0089] Pharmaceutical compositions comprising a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration may be performed by injection, including subcutaneous, intravenous, intramuscular or intrapleural injection.

[0090] In this case, for parenteral formulations, a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof can be mixed with a stabilizer or buffer in water to prepare a solution or suspension, and can be prepared into unit dosage forms such as ampoules or vials. The composition may be sterilized and / or may contain additives such as preservatives, stabilizers, wetting agents or emulsification promoters, salts and / or buffers for adjusting osmotic pressure, and other therapeutically useful substances; and can be formulated according to conventional methods of mixing, granulation, or coating.

[0091] Oral formulations, including tablets, pills, hard / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs, and lozenges, contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine) and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone; and in some cases may contain disintegrants such as starch, agar, alginate, or their sodium salts, or effervescent mixtures and / or adsorbents, colorants, flavorings, and sweeteners.

[0092] This invention relates to compounds represented by Formula 1, their stereoisomers, their solvates, their hydrates, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them, administered at a “pharmaceuticalally effective amount.” For the purposes of this specification, the term “pharmaceuticalally effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio suitable for medical treatment or improvement; the effective dose level can be determined based on factors including: the type and severity of the disease, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and clearance, duration of treatment, and concomitant drugs, as well as other factors well known in the medical field. For example, effective amounts include 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 100 mg / kg, 0.1 to 20 mg / kg, or 0.1 to 500 mg / kg. The dosage of the compounds or pharmaceutical compositions of this invention can be selected and applied within appropriate ranges by those skilled in the art.

[0093] One aspect of the present invention provides a health food for preventing or improving ASK1-related diseases, wherein the health food comprises a compound represented by chemical formula 1, or a stereoisomer, solvate, hydrate or pharmaceutically acceptable salt thereof as an active ingredient.

[0094] The compound represented by chemical formula 1 according to the present invention exhibits high ASK1 inhibitory activity and can be prepared into a health food composition for the prevention or improvement of ASK1-related diseases according to pharmaceutical composition standards, or can be added to health supplements such as food and beverages.

[0095] The compounds represented by Chemical Formula 1 of this invention can be directly added to food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the mixed active ingredients can be appropriately determined according to their intended use (for prevention or improvement). Generally, in health functional foods, the amount of the above-mentioned compounds added can be from 0.1 to 90 parts by weight of the total weight of the food. However, in cases of long-term consumption for health and hygiene purposes or for health control purposes, the amount can be lower than the above range; and since there are no safety concerns, the active ingredients can also be used in amounts higher than the above range.

[0096] One aspect of the present invention provides a method for preventing or treating ASK1-related diseases, the method comprising administering to a subject a pharmaceutical composition comprising a compound represented by chemical formula 1, or a stereoisomer thereof, solvate, hydrate or pharmaceutically acceptable salt thereof as an active ingredient.

[0097] The above administration can be oral or parenteral. Administration can be once daily or divided into several daily doses, such that, when administered orally, the dose is 0.01 to 1000 mg per kilogram of body weight per day, or more specifically, 0.1 to 300 mg per day, based on the active ingredient; and when administered parenterally, the dose is 0.01 to 100 mg per kilogram of body weight per day, or more specifically, 0.1 to 50 mg per day, based on the active ingredient. The dose administered to a particular subject or patient should be determined based on various relevant factors, such as the patient's weight, age, sex, health status, diet, time of administration, method of administration, and disease severity, and may be appropriately increased or decreased by the practitioner.

[0098] In this specification, the term "subject" refers to an individual who requires treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle.

[0099] One aspect of the invention provides the use of a compound represented by Formula 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof, in the prevention or treatment of ASK1-related diseases.

[0100] One aspect of the invention provides the use of a compound represented by chemical formula 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of ASK1-related diseases.

[0101] The present invention will be described in detail below through examples and experimental cases.

[0102] However, the following embodiments and experimental examples are merely examples of the present invention, and the content of the present invention is not limited to the following embodiments and experimental examples.

[0103] Preparation Example 1: 6-(1H-benzo[ d [1,2,3]triazol-1-yl)pyridine-2-amine (intermediate 1)

[0104] According to the above reaction scheme, intermediate 1 was obtained.

[0105] A mixture of 6-fluoropyridin-2-amine (200 mg, 1.78 mmol), benzotriazole (255 mg, 2.14 mmol), and K₂CO₃ (740 mg, 5.34 mmol) in DMSO (6 mL) was heated at 180 °C for 12 hours. After cooling to room temperature, 1 M Na₂CO₃ solution was added. The resulting solid was filtered and dried under reduced pressure to give 6-(1H-benzo[ d [1,2,3]Triazol-1-yl)pyridine-2-amine (Intermediate 1, 230 mg, 61%). 1 H NMR (500 MHz, Chloroform- d ) δ 8.45 (d, J = 8.4Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 7.7Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 6.68 (d, J = 8.2Hz, 1H); LCMS: 212.4 [M+H + ].

[0106] Preparation Example 2: 6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-amine (intermediate 2)

[0107] Intermediate 2 was obtained according to the following reaction scheme.

[0108]

[0109] Step 1: 1-Azide-4-nitrobenzene

[0110] 4-Nitroaniline (1.00 g, 7.24 mmol) was dissolved in anhydrous acetonitrile (20 mL) and then cooled to 0 °C in an ice bath. Tert-butyl nitrite (1.03 mL, 8.69 mmol) was added to the stirred solution, and the mixture was stirred for 20 min. Subsequently, azidotrimethylsilane (1.44 mL, 10.86 mmol) was added dropwise over 10 min, and the resulting brown final solution was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. The residue was purified by rapid silica gel column chromatography (hexane / ethyl acetate 100 / 0 to 80 / 20) to give 1-azido-4-nitrobenzene (1.16 g, 97%).

[0111] 1H NMR (400 MHz, Methanol- d 4) δ 8.30 - 8.25 (m, 2H), 7.30 - 7.25 (m,2H); LCMS: 165.5 [M+H + ].

[0112] Step 2: 1,4,5,6-Tetrahydrocyclopentazo[ d [1,2,3]triazole

[0113] Cyclopentanone (1.08 mL, 12.2 mmol), NH4OAc (4.69 g, 60.9 mmol), and 1-azido-4-nitrobenzene were added to a screw-cap reaction tube equipped with a magnetic stir bar. The resulting mixture was dissolved in DMF (20 mL) and stirred at 80 °C for 12 hours. After the reaction was complete, the DMF was removed under reduced pressure, and the residue was purified by rapid silica gel column chromatography (100% DCM, followed by hexane / ethyl acetate 100 / 0 to 50 / 50) to give 1,4,5,6-tetrahydrocyclopentanone[ d [1,2,3] Triazole (164 mg, 61%).

[0114] 1 H NMR (300 MHz, Chloroform- d ) δ 10.03 (s, 1H), 2.88 - 2.78 (m, 4H), 2.66 - 2.54 (m, 2H).

[0115] Step 3: 6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-amine

[0116] 6-Fluoropyridine-2-amine (940 mg, 8.32 mmol), 1,4,5,6-tetrahydrocyclopentazo[ d A mixture of [1,2,3]triazole (1.00 g, 9.16 mmol) and K₂CO₃ (3.44 g, 25.0 mmol) in DMSO (20 mL) was heated at 160 °C for 12 hours. After cooling to room temperature, the mixture was diluted with water and extracted twice with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO₄, filtered, and concentrated. The residue was purified by rapid silica gel column chromatography (hexane / ethyl acetate 100 / 0 to 60 / 40) to give 6-(5,6-dihydrocyclopentano[ d [1,2,3]Triazol-1(4H)-yl)pyridine-2-amine (intermediate 2, 700 mg, 42%).

[0117] 1 H NMR (300 MHz, Chloroform- d ) δ 7.59 (t, J = 7.9 Hz, 1H), 7.41 (dd,J = 7.8, 0.7 Hz, 1H), 6.45 (dd, J = 8.0, 0.7 Hz, 1H), 3.18 - 3.05 (m, 2H), 2.88 - 2.76 (m, 2H), 2.73 - 2.59 (m, 2H); LCMS: 202.6 [M+H +].

[0118] Preparation Example 3: 6-(4,5,6,7-tetrahydro-1H-benzo[] d [1,2,3]triazol-1-yl)pyridine-2-amine (intermediate 3)

[0119] By performing the same method as described in Preparation Example 2, 6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]Triazol-1-yl)pyridine-2-amine (intermediate 3, 39%).

[0120] 1 H NMR (300 MHz, Chloroform- d) δ 7.60 (t, J = 7.9 Hz, 1H), 7.35 (dd,J = 7.8, 0.7 Hz, 1H), 6.48 (dd, J = 8.2, 0.7 Hz, 1H), 3.13 - 3.03 (m, 2H), 2.86 - 2.75 (m, 2H), 1.90 - 1.78 (m, 4H); LCMS: 216.6 [M+H + ].

[0121] Preparation Example 4: 6-(5,6,7,8-tetrahydrocycloheptan[] d [1,2,3]triazol-1(4H)-yl)pyridin-2-amine (intermediate 4)

[0122] By performing the same method as described in Preparation Example 2, 6-(5,6,7,8-tetrahydrocycloheptan[] d [1,2,3]Triazol-1(4H)-yl)pyridine-2-amine (intermediate 4, 22%).

[0123] 1 H NMR (400 MHz, Methanol- d 4) δ 7.65 (t, J = 7.9 Hz, 1H), 6.84 (d, J =7.5 Hz, 1H), 6.67 (d, J = 8.3 Hz, 1H), 3.07 - 2.97 (m, 2H), 2.95 - 2.86 (m,2H), 1.96 - 1.85 (m, 2H), 1.81 - 1.69 (m, 4H); LCMS: 230.6 [M+H+].

[0124] Preparation Example 5: 6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[ d [1,2,3]triazol-1-yl)pyridin-2-amine (intermediate 5)

[0125] By performing the same method as described in Preparation Example 2, 6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[ d [1,2,3]triazol-1-yl)pyridine-2-amine (intermediate 5, 23%).

[0126] 1 H NMR (500 MHz, Chloroform- d) δ 7.65 (t, J = 7.9 Hz, 1H), 7.20 -7.10 (m, 1H), 6.74 - 6.63 (m, 1H), 3.17 - 3.09 (m, 2H), 3.06 - 2.99 (m, 2H),1.94 - 1.87 (m, 2H), 1.85 - 1.76 (m, 2H), 1.57 - 1.45 (m, 4H); LCMS: 244.6 [M+H + ].

[0127] Preparation Example 6: 6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]triazol-1-yl)pyridine-2-amine (alternative preparation method for intermediate 3)

[0128] Intermediate 3 can also be prepared according to the following reaction scheme.

[0129]

[0130] Step 1: 2,2-Dimethoxycyclohexane-1-one

[0131] A 50 mL solution of cyclohexane-1,2-dione (6.0 g, 53.5 mmol) in methanol was slowly added to a 500 mL solution of trimethylchlorosilane (7.0 mL, 58.9 mmol) in methanol. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water and extracted with diethyl ether. The organic layer was washed with saturated ammonium carbonate solution, water, and brine. After drying with MgSO4, the solvent was removed under reduced pressure to give 2,2-dimethoxycyclohexane-1-one (quantitative yield).

[0132] 1 H NMR (400 MHz, DMSO- d 6 ) δ 3.14 (s, 6H), 2.39 (t, J = 6.6 Hz, 2H), 1.85 (t, J = 6.0 Hz, 2H), 1.77 - 1.68 (m, 2H), 1.68 - 1.60 (m, 2H).

[0133] Step 2: 6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]triazol-1-yl)pyridine-2-amine

[0134] 4-Methylbenzenesulfonylhydrazine (6.0 g, 32.2 mmol) was added to a solution of 2,2-dimethoxycyclohexane-1-one (5.6 g, 35.4 mmol) in isopropanol (100 ml) and stirred at room temperature for 30 minutes (at which point the 4-methylbenzenesulfonylhydrazine was observed to be completely consumed). Pyridine-2,6-diamine (3.86 g, 35.4 mmol) and triethylamine (4.9 ml, 35.4 mmol) were added to the mixture. After reacting at 140 °C for 3 hours, the mixture was cooled to room temperature. The reaction mixture was partitioned between dichloromethane and water, and the organic layers were then separated. The aqueous layer was extracted twice with dichloromethane, and the combined organic layers were washed with brine, dried over MgSO4, and concentrated. The product was purified by rapid silica gel column chromatography (hexane / ethyl acetate 100 / 0 to 50 / 50) to give 6-(4,5,6,7-tetrahydro-1H-benzo[ d] [1,2,3]Triazol-1-yl)pyridine-2-amine (Intermediate 3, 3.0 g, 43%).

[0135] 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.59 (t, J = 7.9 Hz, 1 H), 6.99 (d, J =7.5 Hz, 1 H), 6.48 (d, J = 8.2 Hz, 1 H), 6.33 (s, 2 H), 3.03 (d, J = 3.2 Hz, 2 H), 2.68 (s, 2 H), 1.84 - 1.72 (m, 4 H); LCMS [M+H]; 216.27.

[0136] Example 1: N-(6-(1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzamide

[0137] The compound of Example 1 was prepared according to the above reaction scheme.

[0138] Add 6-(1H-benzo[ dHATU (1.25 g, 3.30 mmol) was added to a DMF (3 mL) solution of [1,2,3]triazol-1-yl)pyridin-2-amine (intermediate 1, 70 mg, 0.33 mmol), DMAP (400 mg, 3.30 mmol), and 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid (130 mg, 0.50 mmol) [purchased from AA blocks (5 g), Labnetwork (10 g), Chemscene; reference: WO2013 / 112741]. The resulting mixture was stirred at room temperature for 12 hours and then diluted with ethyl acetate and saturated NaHCO3 aqueous solution. The separated organic layer was washed with brine, dried over anhydrous MgSO4, and filtered. The organic filtrate was evaporated under reduced pressure. The product was purified by rapid silica gel column chromatography (ethyl acetate / methanol 100 / 0 to 90 / 10) and C18 reversed-phase HPLC (using TFA in H2O solution and 0.1% TFA in ACN solution as buffer solvents) to obtain N-(6-(1H-benzo[ d [1,2,3]Triazol-1-yl)pyridin-2-yl)-5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzamide (1.35 mg, 23%).

[0139] 1 H NMR (300 MHz, Methanol- d 4 ) δ 9.07 (d, J = 1.7 Hz, 1H), 8.84 (dd, J = 8.4, 1.0 Hz, 1H), 8.29 (dd, J = 7.6, 1.3 Hz, 1H), 8.18 - 8.04 (m, 3H), 7.97(d, J = 6.4 Hz, 1H), 7.70 - 7.63 (m, 1H), 7.60 - 7.56 (m, 1H), 7.56 - 7.50(m, 1H), 7.47 (d, J = 10.8 Hz, 1H), 2.34 (s, 3H), 2.12 - 2.01 (m, 1H), 1.19- 1.10 (m, 2H), 0.91 (dt, J = 7.0, 4.7 Hz, 2H); LCMS: 454.6 [M+H + ].

[0140] Example 2: 5-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide

[0141] DMAP (0.36 g, 3.0 mmol) was added to a commercially available solution of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid (0.38 g, 1.3 mmol) in NMP (3.0 ml) under stirring, followed by the slow addition of T3P (50% ethyl acetate) (1.27 ml, 2.0 mmol) at room temperature. The resulting mixture was stirred for 10 minutes, and then 6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-amine (0.2 g, 1.0 mmol), and the mixture was stirred for 12 hours. The reaction mixture was quenched with ice water and alkalized with a saturated aqueous solution of NaHCO3 (pH 7-8). The resulting solid was filtered, washed with water, and dried. The target compound 5-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5,6-dihydrocyclopentano[]] was obtained as a pure white solid by grinding the solid with ethyl acetate. d [1,2,3]Triazol-1(4H)-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide (0.3 g, 68%).

[0142] 1 H NMR (400 MHz, DMSO) δ 10.96 (s, 1 H), 8.19 - 8.08 (m, 2 H), 7.78 (dd, J = 7.6, 1.0 Hz, 1 H), 7.69 (d, J = 1.4 Hz, 1 H), 7.63 (d, J = 6.6 Hz, 1H), 7.48 (d, J = 10.8 Hz, 1 H), 7.18 (d, J = 1.4 Hz, 1 H), 3.14 (t, J = 7.1Hz, 2 H), 2.78 - 2.70 (m, 2 H), 2.67 - 2.57 (m, 2 H), 2.25 (s, 3 H), 1.85(td, J= 8.3, 4.1 Hz, 1 H), 0.83 - 0.77 (m, 2 H), 0.73 - 0.67 (m, 2 H): LCMS;[M+H] + 444.6.

[0143] Example 3: 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)methylbenzamide

[0144] DMAP (4.25 g, 34.8 mmol) was added to a commercially available solution of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoic acid (3.93 g, 15.1 mmol) in NMP (30 ml) under stirring, followed by the slow addition of T3P (50% ethyl acetate) (14.7 g, 23.2 mmol) at room temperature. The resulting mixture was stirred for 10 minutes, and then 6-(4,5,6,7-tetrahydro-1H-benzo[]] was added. d [1,2,3]triazol-1-yl)pyridin-2-amine (2.5 g, 11.6 mmol) was mixed and stirred for 12 hours. The reaction mixture was quenched with ice water and alkalized with a saturated aqueous solution of NaHCO3 (pH 7-8). The resulting solid was filtered, washed with water, and dried. The target compound 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(4,5,6,7-tetrahydro-1H-benzo[]] was obtained as a white solid by grinding the solid with methanol. d [1,2,3]triazol-1-yl)pyridin-2-yl)benzamide (5.0 g, 94%).

[0145] 1 H NMR (400 MHz, DMSO) δ 11.03 (s, 1 H), 8.19 (dd, J = 8.3, 0.9 Hz, 1H), 8.12 (t, J = 8.0 Hz, 1 H), 7.75 (dd, J = 7.8, 0.8 Hz, 1 H), 7.70 (d, J =1.5 Hz, 1 H), 7.63 (d, J = 6.5 Hz, 1 H), 7.48 (d, J= 10.7 Hz, 1 H), 7.18 (d, J = 1.4 Hz, 1 H), 3.11 (d, J = 5.3 Hz, 2 H), 2.70 (d, J LCMS [M+H]; 458.57.

[0146] Example 4: 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(5,6,7,8-tetrahydrocycloheptan[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)benzamide

[0147] Except that intermediate 4 is used instead of intermediate 1, 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(5,6,7,8-tetrahydrocycloheptan[ d [1,2,3]Triazol-1(4H)-yl)pyridin-2-yl)benzamide (13%).

[0148] 1 H NMR (400 MHz, Methanol- d 4 ) δ 9.03 (d, J = 1.6 Hz, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.11 (t, J = 8.1 Hz, 1H), 7.94 (d, J = 6.4 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.57 - 7.52 (m, 1H), 7.46 (d, J= 10.9 Hz, 1H), 3.18 - 3.12(m, 2H), 2.94 - 2.89 (m, 2H), 2.32 (s, 3H), 2.08 - 2.01 (m, 1H), 1.96 - 1.88(m, 2H), 1.82 - 1.73 (m, 4H), 1.16 - 1.10 (m, 2H), 0.92 - 0.87 (m, 2H); LCMS:472.5 [M+H + ].

[0149] Example 5: 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-4-methylbenzamide

[0150] 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[]) was obtained by performing the same method as described in Example 1. d [1,2,3]triazol-1-yl)pyridin-2-yl)-4-methylbenzamide (4%).

[0151] 1 H NMR (500 MHz, Methanol- d 4 ) δ 9.10 (d, J = 1.7 Hz, 1H), 8.38 (d, J =8.2 Hz, 1H), 8.11 (t, J = 8.1 Hz, 1H), 7.99 (d, J = 6.4 Hz, 1H), 7.70 (d, J =7.8 Hz, 1H), 7.58 (d, J = 1.3 Hz, 1H), 7.48 (d, J= 11.0 Hz, 1H), 3.25 - 3.20(m, 2H), 2.97 - 2.92 (m, 2H), 2.33 (s, 3H), 2.08 - 2.02 (m, 1H), 1.96 - 1.89(m, 2H), 1.82 - 1.75 (m, 2H), 1.59 - 1.54 (m, 2H), 1.54 - 1.48 (m, 2H), 1.18- 1.11 (m, 2H), 0.94 - 0.88 (m, 2H); LCMS: 486.5 [M+H + ].

[0152] The structures of the compounds prepared in Examples 1 to 5 are summarized in Table 1 below.

[0153] Table 1

[0154] Experimental Example 1: Evaluation of ASK1 inhibitory activity

[0155] The following experiments were conducted to determine the inhibitory activity of the compound represented by chemical formula 1 of this invention against ASK.

[0156] The reaction with recombinant human ASK1 protein was initiated by adding a Mg / ATP mixture to the reaction solution (8 mM MOPS pH 7.0; 0.2 mM EDTA; 0.33 mg / mL myelin basic protein; 10 mM magnesium acetate and [γ-33P]-ATP); the reaction was terminated by adding 0.5% phosphate after 40 minutes at room temperature. Subsequently, 10 μL of the reaction solution was added dropwise into a P30 dropper, washed four times in 0.425% phosphate for 4 minutes, and dried with methanol. ASK1 activity was determined by scintillation counting. The activity value of the group without the compound was set as 100% to calculate the degree of inhibition of ASK1 by the compound; the ASK1 inhibitory activity of the compounds in each example is summarized in Table 2 below.

[0157] Table 2

[0158] Observation of Table 2 above confirms that the compound represented by Chemical Formula 1 according to the present invention is a substance with ASK1 inhibitory activity. Therefore, the compound of the present invention can be used as a composition for the prevention or treatment of neurodegenerative brain diseases caused by ASK1.

[0159] Experimental Example 2: Evaluation of MPP+-induced cytoprotective effects in human neurons

[0160] The following experiments were conducted to evaluate the effect of the compound represented by chemical formula 1 according to the present invention on protecting dopaminergic neurons from damage.

[0161] SH-SY5Y cells were purchased from ATCC and cultured in RPMI medium containing 10% FBS, 1% penicillin, and streptomycin at 37°C and 5% CO2. MPP+ was purchased from Sigma Aldrich and used at a concentration of 5 mM to induce cytotoxicity. Cell viability was measured at 540 nm after treatment with CCK-8 reagent for 1–3 hours, and the results are shown in Table 3 below.

[0162] Table 3

[0163] As shown in Table 3 above and Figure 1 As shown, the compounds of the present invention exhibited excellent cytoprotective activity in SH-SY5Y cells, particularly Examples 2 and 3, which showed superior ECG protection compared to Comparative Example 1 (selochrome). 50 value.

[0164] Experiment Example 3: Evaluation of Cell Viability

[0165] To evaluate cell viability after compound treatment, BV2 cells were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for one day. They were then treated with 0.1 μM, 0.3 μM, and 1 μM compounds, respectively, and incubated at 37°C and 5% CO2 for 72 hours. Cell viability was measured by adding Cell Counting Kit-8 (CCK-8; LPS solution, Seoul, South Korea) to the cultured cells, and quantification was performed by measuring absorbance at 565 nm using a Hidex sense microplate detector (Hidex, Turku, Finland).

[0166] like Figure 2 As shown, the compounds according to embodiments of the present invention exhibited similar cell viability levels to Comparative Example 1 (selochrome).

[0167] Experiment Example 4: Evaluation of Anti-inflammatory Efficacy

[0168] BV2 cells (2 x 10^4 cells per well) were incubated in 96-well plates for 24 hours. Subsequently, the cells were treated with 0.1 μM, 0.3 μM, and 1 μM of the compound from the examples for 1 hour each, followed by incubation with 100 pg / ml LPS for 24 hours. After incubation, the conditioned medium (CM) was collected and centrifuged at 2000 rpm. The supernatant was separated from the solution after centrifugation, and TNF-α secreted into the cell culture medium was measured in the supernatant using the appropriate ELISA kit (LABISKOMA, Seoul, South Korea).

[0169] like Figure 2 As shown, the compounds according to embodiments of the present invention reduced the secretion of the LPS-induced inflammatory cytokine TNF-α to a level similar to that of seloselte (Comparative Example 1). In particular, it was demonstrated that the compounds of Example 3 reduced TNF-α secretion at all concentrations compared to seloselte, confirming their superior anti-inflammatory efficacy.

[0170] Experimental Example 5: Evaluation of Neuroprotective Efficacy

[0171] The neurotoxic substances 6-OHDA (6-hydroxydopamine hydrobromide) and MPP+ (1-methyl-4-phenylpyridinium) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and prepared as 100 mM DMSO stock solutions. PC12 cells were cultured at 1 x 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for one day. Cells were pretreated for 1 hour with each concentration of the example compounds. Subsequently, 40 μM 6-OHDA and 2 mM MPP+ were added, and the cells were incubated for 24 hours. Cell viability was then measured using MTT assay (Invitrogen, Massachusetts, USA).

[0172] As shown in Tables 4 and 5, the compounds of the embodiments according to the present invention exhibited superior cell viability compared to Comparative Example 1 (selochrome), thus demonstrating their excellent neuroprotective effect in the PC-12 cell line.

[0173] Table 4

[0174] Table 5

[0175] Experimental Example 6: Evaluation of the inhibitory effect of TG synthesis

[0176] Triglyceride (TG) assay is performed by loading human hepatocellular carcinoma cell line HepG2 cells into wells at a density of 2 x 10⁻⁶ cells per well. 4Cells were seeded at a density of 1,000 cells per well in 96-well plates and incubated for 24 hours. The cultured cells were pretreated for 2 hours with each concentration of the example compounds, followed by the addition of 1 mM free fatty acids (FFA), wherein palmitic acid (PA, Sigma, #P9767) and oleic acid (OA, Sigma, #O7501) were mixed in a 1:2 ratio, and incubation was continued for 48 hours. After incubation, the supernatant was separated, and absorbance was measured at 570 nm using a triglyceride quantification kit (Sigma, #MAK266).

[0177] As shown in Table 6 below, the efficacy against NASH (non-alcoholic steatohepatitis) was verified by inhibiting TG synthesis. The experimental results show that the compound in Example 3 exhibited a stronger inhibitory effect on TG synthesis with increasing treatment concentration.

[0178] Table 6

[0179] Experimental Example 7: Evaluation of MDR-MDCK cell membrane permeability

[0180] MDCK-MDR1 cells (5x10) 5 Cells / mL were incubated in 24-well Transwell plates (Corning, NY, USA) for 4 days to measure their permeability through MDR-MDCK cell monolayers. Fresh medium was added every two days, and the interstitial electrical resistance (TEER) was measured using a Millicell® ERS-2 voltmeter (Merck KGaA, Darmstadt, Germany); the TEER was confirmed to be greater than or equal to 300 Ohm / cm². 2 Then, cell membrane permeability tests were performed.

[0181] The apical chamber and basolateral chamber were washed twice, respectively, with DPBS buffer and Hanks' balanced salt solution (HBSS) containing 10 mM hydroxyethylpiperazine thiosulfate (HEPES). During the final wash, the chambers were filled with HBSS and kept stable at 37°C for 30 minutes. After 30 minutes, the test material was added dropwise to each chamber and incubated at 37°C for 2 hours. The transport layer medium was collected every 30 minutes, and an equal volume of fresh HBSS was added.

[0182] The apparent permeability coefficient (Papp, cm / sec) is calculated using the following equation: Mathematical Equation 1 Papp = dQ / dt Х 1 / A Х 1 / C_0 Papp: Apparent permeability coefficient dQ / dt: Drug penetration rate A: Filter membrane area with cell layering C_0: Initial concentration of the drug In addition, the external discharge rate is calculated as follows: Mathematical Equation 2 Efflux rate (ER) = (Papp (base-to-apex)) / (Papp (apex-to-base)) Table 7

[0183] As shown in Table 7, Comparative Example 1 (Selochrome) exhibits an efflux rate of 27 due to a significant difference in apparent permeability coefficients in the absorption and efflux directions, confirming it as a substrate for efflux transporters. In contrast, the compounds of the embodiments according to the present invention exhibit extremely high apparent permeability values ​​in both the absorption and efflux directions, with an efflux rate close to 1, confirming their good cell membrane permeability and that they are not substrates for efflux transporters.

[0184] Experimental Example 8: Distribution in the mouse brain

[0185] Blood-brain barrier permeability in ICR mice was confirmed by oral administration of a test substance at a dose of 10 mg / kg. Male C57BL / 6 mice were fasted for 15 hours prior to administration. The test substance was administered at a dose of 10 mg / kg in a volume of 5 mL / kg, using a solvent prepared from N-methyl-2-pyrrolidone (NMP), polyethylene glycol 400 (PEG 400), and distilled water (DW) in a volume ratio of 10:40:50 (%). Following administration, mice were euthanized by carbon dioxide inhalation at 1, 4, and 8 hours, and blood and brain tissue samples were collected. Whole blood was collected from the abdominal vein, and brain samples were collected after systemic perfusion with physiological saline to remove as much blood as possible.

[0186] The distribution ratio in mouse plasma and brain was calculated by quantifying the concentration in each tissue using the AUC.

[0187] Mathematical Equation 3

[0188] Kp = (Total AUC of Brain) / (Total AUC of Plasma)

[0189] Table 8

[0190] As shown in Table 8, it was confirmed that Example 3 of the present invention has relatively high concentrations and Kp values ​​in plasma and brain compared with Comparative Example 1 (seloselte), thus exhibiting excellent pharmacokinetic properties in the treatment of brain diseases.

[0191] Experimental Example 9: Efficacy Evaluation in an MPTP-Induced Mouse Model of Parkinson's Disease

[0192] Experiment Example 9.1: Experiment Preparation

[0193] All animal experiments described in this instruction manual have been approved by the International Association for the Study of Animals (IACUC) and conducted in accordance with the guidelines of the Ministry of Food and Drug Safety regarding the management and use of laboratory animals.

[0194] Six-week-old male C57BL / 6 mice were purchased from Koatech (South Korea). The mice were housed in groups of up to four per cage in an environment with controlled light-dark cycles (08:00–20:00), temperature (22±1°C), and humidity (30–50%), and were provided with unlimited food and water. The mice were allowed one week to acclimatize before the experiment began.

[0195] The test drugs were administered to normal mice and to a mouse model of Parkinson's disease (PD) induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to evaluate their efficacy. For the example compounds, the test drugs were prepared using 10% NMP, 40% PEG400, and 50% DW (distilled water) as solvents.

[0196] In the evaluation results, all values ​​are expressed as mean ± standard error (SEM) and statistically analyzed by one-way ANOVA using the LSD (Least Significant Difference) post-hoc test. The significance level of the evaluation results was set according to the following criteria.

[0197] Significance level relative to G1 - p < 0.05, p < 0.01, p < 0.001

[0198] Significance levels relative to G2: #: p < 0.05, ##: p < 0.01, ###: p < 0.001

[0199] Experiment Example 9.2: Experimental Design

[0200] Figure 3 This is a schematic diagram of the efficacy experiment in an MPTP-induced PD mouse model.

[0201] As shown in Table 9 below, 7-week-old mice were divided into 7 groups to establish an acute MPTP model and administer drugs: control + solvent (G1), MPTP + solvent (G2), MPTP + nilotinib 40 mg / kg (G3), MPTP + Example 3 2 mg / kg (G4), MPTP + Example 3 10 mg / kg (G5), MPTP + Example 3 50 mg / kg (G6), and MPTP + Example 2 50 mg / kg (G7). From day 1 to day 7, the test drug was administered orally (PO) once daily (QD) at a dose of 10 ml / kg. In the acute MPTP model, all groups except the control group received MPTP·HCl (free base 20 mg / kg) via intraperitoneal injection (IP) on day 0, injected every 2 hours for a total of four times. The control group received an equal volume of 0.9% saline. A pole-climbing test was performed on day 4 (after training on days 2 and 3). On day 7, all mice were euthanized and their brains were collected.

[0202] During the study, the mice were weighed daily and examined for any abnormalities. If a mouse's general health deteriorated significantly, it was euthanized and removed from the study.

[0203] Table 9

[0204] Experiment Example 9.3: Evaluation of the efficacy in improving behavioral disorders

[0205] A vertical wooden pole (0.8 cm to 1 cm in diameter and 80 cm in length, suitable for mice to grasp) was placed at the bottom of the test cage. Mice were trained for two consecutive days (days 2 and 3), with each training session consisting of three trials. On day 3 (the actual test day, day 4), the mice were placed at the top of the pole, and the total time required for them to turn around and reach the bottom was measured. The time required to turn around (turning time, i.e., the time it takes for the mouse to turn its head downwards), the time required to descend (descent time, i.e., the time required from head-down movement to contact with the ground), and the total time required to touch the ground (total time) were recorded, and the results were obtained as follows: Figure 4 The chart shown.

[0206] Figure 4 This is a graph showing the results of the MPTP-induced behavioral disorder assessment (pole climbing test) in a mouse model of PD. In the pole climbing test, it was confirmed that the MPTP-treated group (G2) required a significantly longer turn-around time compared to the control group (G1). Figure 4 a) Fall time ( Figure 4 (b) and total contact time ( Figure 4(c) Compared to group G2, the turning time was significantly shorter in groups treated with MPTP and the compounds of the examples (G4 to G7). In particular, the turning time, descent time, and total ground contact time were significantly shorter in groups G4 and G5. These results confirm that the compounds of the examples can significantly improve behavioral disorders.

[0207] Experimental Example 9.4: Evaluation of TH staining degree

[0208] Mice were euthanized by administration of Zoltil to collect tissue samples for staining and histological section analysis. Blood was collected from the heart and then perfused with phosphate-buffered saline (PBS) to remove residual blood. For half of the mice in each group (n=6 / group), their brains were dissected into the striatum, substantia nigra, and the remainder of the brain, then rapidly frozen and stored at -80°C. For the remaining mice in each group (n=6 / group), their brains were isolated and fixed with 4% polyoxymethylene at 4°C for 24 hours, then immersed in PBS solution containing 0.02% sodium azide and 30% sucrose and stored at 4°C for 3 days. Immunohistochemical sections were prepared by embedding brain samples in optimal sectioning temperature (OCT) embedding medium. The samples were cut into 20 μm thick sections using a Leica CM1950 (Leica Microsystems, Germany) and stored in preservation solution at 4°C until antibody staining.

[0209] TH (tyrosine hydroxylase) is a catalytic enzyme that converts the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), and is one of the most important elements in the biosynthesis of catecholamines, including dopamine (DA). Changes in TH expression or activity have a significant impact on dopamine production and are therefore crucial in the pathogenesis of Parkinson's disease.

[0210] After staining the striatum and substantia nigra of the brain and mounting them on microscope slides, the slides were washed for 15 minutes at room temperature with PBS containing 0.3% Triton X-100 and peroxidase blocking solution. The slides were washed three times with PBS containing 0.3% Triton X-100, and then blocked at room temperature (RT) for 1 hour in a solution containing PBS, 5% normal goat serum, and 0.3% Triton X-100. Tyrosine hydroxylase antibody was diluted 1:1000 and incubated overnight at 4°C. The slides were washed with PBS and incubated with secondary antibody at room temperature for 1 hour. The slides were washed with PBS, and the sections were placed in solution for DAB (diaminobenzidine) reaction.

[0211] The DAB reaction was monitored under a microscope for 2 to 3 minutes, followed by washing the slides with PBS to stop the reaction. After dehydration, the slides were mounted with xylene. TH staining images were visualized using an Olympus microscope (Olympus, SZ61, BX51). The optical density (OD) values ​​of TH staining in the striatum and substantia nigra were measured and analyzed using ImageJ software.

[0212] Figure 5a and 5b This is the result of TH staining immunohistochemical analysis of the striatum. Figure 6a and 6b These are the results of immunohistochemical analysis of TH staining in the substantia nigra. In the striatum and substantia nigra, the TH level in the MPTP-treated group (G2) was significantly lower than that in the control group (G1). The striatum TH level in the nilotinib-treated group (G3) was significantly higher than that in the G2 group. Compared with the G2 group, the TH levels in the striatum and substantia nigra of the treatment groups of Example 3 or Example 2 showed a significant increase. Furthermore, the TH levels in the striatum of G5 (MPTP + 10 mg / kg Example 3) and G6 (MPTP + 50 mg / kg Example 3), as well as the TH levels in the substantia nigra of G5, G6, and G7 (MPTP + 50 mg / kg Example 2), were all significantly higher than those in the control group (G1).

[0213] Experimental Example 9.6: Evaluation of the efficacy of dopamine enhancement

[0214] The dopamine content was measured by LC-MS / MS analysis of the extracted striatum and substantia nigra of the brain tissue using an Agilent 1290 (LC) and a SCIEX QTARP 6500+ (QQQ) instrument.

[0215] Figure 7 This is a chart showing the dopamine levels in the striatum and substantia nigra of a MPTP-induced PD mouse model. (Reference) Figure 7 Compared to the group using MPTP only, the dopamine concentrations in both the striatum and substantia nigra were significantly increased in the Example 2 treatment group at a dose of 50 mg / kg; while in the Example 3 treatment groups at doses of 2 and 10 mg / kg, the dopamine concentration in the substantia nigra was significantly increased. This confirms that the compounds in the examples significantly increased dopamine levels.

[0216] Experimental Example 10: Efficacy Evaluation in an α-Syn Transgenic Parkinson's Disease Mouse Model

[0217] Experiment Example 10.1: Experiment Preparation

[0218] Female mThy-1α-syn transgenic (α-syn TG; Line No. 61) mice purchased from the University of California, San Diego (UCSD) were mated with C57Bl / 6 X DBA / 2 F1 WT (wild-type) male mice and housed at the animal center of the Osong Foundation for Advanced Medical Industry Promotion (KBIOHealth).

[0219] All mice were housed in an environment with controlled light-dark cycles (08:00–20:00), temperature (22±1°C), and humidity (30–50%), and were provided with unlimited food and water. Ear tags were used to identify ear or tail samples taken between 15 and 21 days of age for polymerase chain reaction (PCR) analysis for genotyping. Additionally, tail samples were collected at the endpoint for genotyping if necessary.

[0220] The test drugs were administered to normal mice and α-Syn (α-synuclein) transgenic Parkinson's disease mouse models, and their efficacy was evaluated. For the example compounds, the test drugs were formulated using 10% NMP, 40% PEG 400, and 50% DW as solvents.

[0221] Experimental Example 10.2: Experimental Design and Changes in Mouse Body Weight

[0222] Figure 8 This is a schematic diagram of the efficacy experiment in an α-Syn transgenic Parkinson's disease mouse model.

[0223] As shown in Table 10 below, 4-month-old mice were divided into 7 groups. From 4 to 9 months of age, the test drug was administered orally (PO) once daily (QD) at a dose of 5 mL / kg for 5 months. Motor behavior tests (balance beam test) and neurological scores (modified Irwin test) were performed at 4 months (before administration), 7 months, and 9 months of age. Tissue samples were collected after the final behavioral tests. Body weight was measured weekly on the same day from 4 months of age until the end of the study. Mice with significantly deteriorating general health were excluded from the study.

[0224] Table 10

[0225] Figure 9 This is a chart showing the changes in body weight in different groups of an α-Syn transgenic Parkinson's disease mouse model. (Reference) Figure 9 This study confirmed that the body weight of WT mice (G1) increased over time, while the body weight gain of the TG group (G2-7) stagnated. The body weight difference between WT and TG mice became apparent after approximately 21 to 22 weeks of age, and confirmed that drug administration had no significant effect on the body weight of TG mice.

[0226] Experimental Example 10.3: Neurobehavioral Improvement Effect

[0227] At 4 months of age (before administration, baseline), 7 months of age, and 9 months of age, each mouse was observed for 1 to 2 minutes, and the neurological dysfunction behaviors listed in Table 11 were recorded. Except for the startle reflex, tail pinch test, and righting reflex, other neurological dysfunction behaviors were scored by direct observation of the mice in their cages. To test the startle reflex, a loud noise was emitted using a small remote control, and the mouse was checked for behaviors such as jumping, stopping, and rapid blinking. For the righting reflex, each mouse was removed from its cage and placed supine, and its ability to correct its posture was observed. The tail pinch reflex was tested by gently squeezing the tip of the tail with tweezers.

[0228] A score of 0 is given if normal or no abnormal motor activity is observed; a score of 1 is given if a slight abnormality is observed; and a score of 2 to 3 is given if a severe abnormality is observed. The scores for all observed behaviors of each mouse are summed, and the average of the total scores for each group is calculated.

[0229] Table 11

[0230] Figure 10 This chart shows the neurobehavioral scores of each group in the α-Syn transgenic Parkinson's disease mouse model. At 4 and 7 months of age, the neurological scores of the G2 (TG + solvent) group were significantly lower than those of the G1 group. Compared with the G2 group, the groups administered Example 3 or Example 2 showed significantly lower neurological scores in TG mice. Throughout the study period, the neurological scores of the G4 to G7 groups remained at very low levels, confirming that the drug did not cause neurotoxicity.

[0231] Experiment Example 10.4: Evaluation of the efficacy in improving gait errors (balance beam test)

[0232] The balance beam test (challenge balance beam pass test) employed the method described by Fleming et al. (J Neurosci, 24:9434-40, 2004). A novel balance beam test, an improvement upon the conventional balance beam walking test, was used to measure motor ability. The balance beam, made of Perspe plexiglass, consisted of four segments (25 cm each, 1 m in total length), each with a different width. The beam was designed to start at a width of 3.5 cm and gradually narrow in increments of 0.5 cm × 1 cm. A 1 cm wide shelf was placed 1.0 cm below the top surface of the beam.

[0233] Mice were trained to cross the entire length of a balance beam, starting from the widest section and ending at the narrowest and most challenging section. The narrow end of the balance beam was designed to connect directly to the mouse's housing. Mice were trained for two days prior to testing, all without a metal mesh. On the first day, two assisted training sessions were conducted, in which the mice were placed on the balance beam with their housings positioned close to them. This induced the mice to move forward along the balance beam. After the two assisted training sessions, the mice were able to cross the entire length of the balance beam without assistance. On the first day of training, all mice were confirmed to have completed five unassisted runs across the entire length of the balance beam. On the second day of training, the mice underwent five trials. On the trial day, a mesh of appropriate width (1 cm square) was placed on the balance beam surface to increase the difficulty, with approximately 1 cm of space maintained between the mesh and the balance beam surface. A shelf underneath provided support when the mouse's limbs slipped off the mesh, allowing for evaluation without the need for compensatory movement strategies. A total of five videos were recorded of the mice crossing the mesh balance beam.

[0234] Videotape analysis was conducted by researchers whose mouse genotype / group was unknown. Errors were examined in slow motion, and the number of steps and crossing time for each mouse across five trials were evaluated. An error was counted if a limb (forelimb or hindlimb) crossed the grid during forward movement and was visible between the grid and the balance beam surface. Each mouse could slip up to four times per step. Errors were measured by scoring each slip individually. For example, a mouse with three limbs slipping off the grid in one step was rated an error score of 3, while a mouse with only one limb slipping off the grid in one step was rated an error score of 1. Slips were not counted if the mouse did not move forward, or if the mouse's head was facing the left or right side of the balance beam. The number of gait errors and the time required to cross the balance beam were measured and averaged across all five trials.

[0235] Figure 11 This is a chart showing the gait error evaluation results for each group in an α-Syn transgenic Parkinson's disease mouse model. (Reference) Figure 11 In the balance beam test, group G2 produced significantly more errors during the crossing compared to group G1. Compared to group G2, the treatment groups treated with methimazole (G3), Examples 3 (G4 to G6), or Examples 2 (G7) showed significantly fewer gait errors at 7 and 9 months. This confirms that the compounds of the examples improved behavioral errors.

[0236] Experimental Example 10.5: Evaluation of efficacy in improving dopaminergic neurodegeneration

[0237] At 9 months of age, the measurement period ended, and mice were anesthetized. The experiment was then terminated by cardiac puncture according to IACUC guidelines. For tissue staining, mice were perfused with 0.9% saline via the heart, followed by perfusion with 4% paraformaldehyde until fixation was achieved. Brains were isolated and fixed in 4% paraformaldehyde at 4°C for 24 hours, then stored in PBS containing 0.02% sodium azide and 30% sucrose at 4°C for 3 days. Brain samples embedded with OCT compounds were sectioned to a thickness of 20 μm using a Leica CM1950 (Leica Microsystems, Germany). Sections were stored in preservation solution at 4°C until antibody staining was performed.

[0238] While gently agitating the tissue, wash the brain tissue three times with PBS for 10 minutes each time. Then, allow the tissue to react at room temperature for 1 hour in blocking buffer containing PBS, 10% normal goat serum, and 0.3% Triton X-100. Incubate the tissue with primary antibody (Iba-1, TH) overnight at 4°C according to the antibody and dilution ratio specified in the table below. After primary antibody incubation, wash the tissue with PBS and react with secondary antibody at room temperature for 1 hour. Then, wash the sections three times with PBS for 10 minutes each time and mount them on slides and coverslips using DAPI mounting medium (VECTASHIELD, #H1500).

[0239] Table 12

[0240] Brain tissue stained with TH was imaged using an Olympus immunofluorescence microscope (Olympus, BZ21). The fluorescence intensity of the striatum was analyzed using ImageJ software.

[0241] Figure 12a and 12b This section presents charts and immunofluorescence analysis images showing the efficacy evaluation results of different groups in improving dopaminergic neurodegeneration in an α-Syn transgenic Parkinson's disease mouse model. (Reference) Figure 12a and Figure 12b The TH staining intensity in the striatum of group G2 tended to be lower than that of group G1. The TH staining intensity in the ferrimanin treatment groups of Example 3 or Example 2 (groups G4 to G7) was significantly higher than that in group G2. The TH staining intensity in group G7 (TG + Example 2) was also significantly higher than that in group G2. The results confirm that the compounds in the examples can significantly improve dopaminergic neurodegeneration.

[0242] Experimental Example 10.6: Evaluation of the efficacy in reducing microglia

[0243] To assess microglia proliferation in the cortex, brain tissue stained with Iba-1 in Experiment 10.5 was imaged using an Olympus immunofluorescence microscope (Olympus, BZ21). Each marker was manually counted to quantify Iba1.

[0244] Figure 13a and Figure 13b This section presents graphs and immunofluorescence images showing the evaluation results of the efficacy in reducing microglia in each group of the α-Syn transgenic Parkinson's disease mouse model. (Reference) Figure 13a and Figure 13b In the groups treated with memantine (G3), 2 mg / kg and 10 mg / kg (Examples 3, G4 and G5), and 2 mg / kg (Example 2, G7), the number of IBA-1 positive cells in the cerebral cortex was significantly reduced. This confirms that the compounds in these examples significantly reduced microglia.

[0245] Experimental Example 11: Efficacy Evaluation in the SOD1 G93A Transgenic Amyotrophic Lateral Sclerosis Mouse Model

[0246] Experiment Example 11.1: Experiment Preparation

[0247] Experimental F1 mice were generated by crossing female B6SJL strain (non-carrier, WT) mice from Jackson Labs with male G93A SOD1 mice (high-copy SOD1-G93A, with 25 TG copies of G1H, stock number 002726). All mice were housed in a controlled environment with normal light-dark cycles (08:00–20:00), temperature (22±1°C), and humidity (30–50%), and provided with unlimited food and water. Ear tags were used at 15–21 days of age, and ear or tail samples were collected for PCR analysis for genotyping. Additionally, tail samples were collected at the endpoint for genotyping if necessary.

[0248] The test drugs were administered to normal mice and SOD1 G93A transgenic amyotrophic lateral sclerosis (ALS) mouse models, and their efficacy was evaluated. For the example compounds, the test drugs were formulated using 10% NMP, 40% PEG 400, and 50% DW as solvents.

[0249] Experiment Example 11.2: Experimental Design

[0250] Figure 14 This is a schematic diagram of the efficacy experiment in the SOD1 G93A transgenic ALS mouse model.

[0251] As shown in Table 13 below, 8-week-old mice were divided into three groups. From 8 weeks of age until death, the test drug was administered orally (PO) once daily at a dose of 5 mL / kg. Body weight was measured weekly, and disease scores were measured daily from week 8 until death. Motor behavior tests (grip strength) and neurological scores (modified Irwin test) were performed at weeks 9, 11, 13, 15, 17, 19, and 21. Mice were monitored for survival daily. Mice whose general health significantly deteriorated were excluded from the study.

[0252] Table 13

[0253] Figure 15 This is a chart showing the changes in body weight in different groups of an SOD1 G93A transgenic ALS mouse model. (Reference) Figure 15 The body weight of the TG group (G2) was significantly lower than that of the WT group (G1). On the other hand, there was no significant difference between the TG group (G2) and the drug-treated group (G3), confirming that the compounds of the examples did not significantly reduce body weight.

[0254] Example 11.3: Evaluation of the efficacy in improving neurobehavioral function

[0255] In the same manner as in Experiment 10.3, each mouse was observed for 1 to 2 minutes of neurological disease behavior at 9, 11, 13, 15, 17, 19 and 21 weeks of age.

[0256] Figure 16 This is a chart displaying the neurological scores of each group in the SOD1 G93A transgenic ALS animal model. (Reference) Figure 16 The study found that the neurological scores of the WT group (G1) were very low, less than 1, throughout the study period. After 13 weeks, the neurological scores of the TG group were significantly higher than those of the G1 group. From 19 weeks of age, the group treated in Example 3 (G3) showed a significant reduction in neurological scores compared to G2. This confirms that the compounds in the examples significantly improved neurobehavioral function.

[0257] Experimental Example 11.4: Evaluation of efficacy in improving disease symptoms

[0258] Starting at 8 weeks of age, disease symptoms in mice were evaluated using a neurological scoring system developed by the ALS Treatment Development Institute (ALSTDI). The hind limbs were scored according to the criteria shown in Table 14 below.

[0259] Table 14

[0260] Figure 17This is a chart showing the changes in disease scores across different groups in the SOD1 G93A transgenic ALS mouse model. (Reference) Figure 17 Compared to the WT group (G1), the TG groups (G2 and G3) showed a significant increase in disease symptom scores, with a statistically significant difference at approximately 15 weeks of age. During the observation period, the group treated in Example 3 (G3) tended to have lower disease symptom scores compared to G2, with a significant reduction at 20 weeks. This confirms that the compounds of the examples significantly improved disease symptoms.

[0261] Experimental Example 11.5: Evaluation of the effect of improving grip strength

[0262] Grip strength was measured in mice at 9, 11, 13, 15, 17, 19, and 21 weeks of age. Mice were placed on a grip strength meter (San Diego Instruments, San Diego, USA) and allowed to grasp a small wire mesh handle with their forepaws. After placing the mouse on the platform, its tail was slowly pulled until the mouse released the handle. The device automatically measured the grip strength in grams. Five scores were recorded for each mouse during a single daily test.

[0263] Figure 18 This is a chart showing the changes in grip strength in different groups of an SOD1 G93A transgenic ALS mouse model. (Reference) Figure 18 The grip strength of the TG group generally decreased over time and was significantly lower than that of the G1 group. Compared with the G2 group, the group treated in Example 3 (G3) tended to have increased grip strength, and the grip strength increased significantly from week 19. Therefore, the compounds of the examples are confirmed to significantly improve grip strength.

[0264] Experimental Example 11.1: Evaluation of the efficacy in improving survival rate

[0265] The survival status of mice was monitored daily. The survival age of the mice was recorded and presented as Kaplan-Meier survival curves.

[0266] Figure 19 This is a chart showing the survival rates of different groups in the SOD1 G93A transgenic ALS mouse model. (Reference) Figure 19 The survival rate of the TG group began to decline between approximately 110 and 120 days of age. Although the survival rate of the group treated with Example 3 (G3) was not significantly different from that of the G2 group, it showed a trend toward improved survival. The results confirm that the compounds of the examples improved the survival rate.

[0267] Experimental Example 12: Efficacy Evaluation in a Reserpine-Induced Fibromyalgia Rat Model

[0268] Experiment Example 12.1: Experiment Preparation

[0269] Six-week-old male Sprague Dawley (SD) rats were used. All rats were housed in an environment with a normal light-dark cycle (08:00–20:00), controlled temperature (22±1°C), and controlled humidity (30–50%), and were provided with unlimited food and water.

[0270] The test drugs were administered to normal rats and a reserpine-induced fibromyalgia rat model, and their efficacy was evaluated. For the example compounds, the test drugs were formulated using 10% NMP, 40% PEG 400, and 50% DW as solvents.

[0271] Experiment Example 12.2: Experimental Design

[0272] Figure 20 This is a schematic diagram of the efficacy experiment in a rat model of reserpine-induced fibromyalgia.

[0273] As shown in Table 15 below, 6-week-old male SD rats were divided into 6 groups for the establishment and drug administration of a reserpine-induced fibromyalgia model: control group (G1), reserpine + solvent (G2), reserpine + gabapentin 50 mg / kg (G3), reserpine + Example 2 3 mg / kg (G4), 10 mg / kg (G5), and 30 mg / kg (G6). To establish the rat fibromyalgia model, reserpine (Sigma-Aldrich, 1 mg / kg) dissolved in 0.5% acetic acid was subcutaneously injected once daily for 3 days, starting 3 days before the start of the experiment. From day 1, the solvent, gabapentin, and Example 2 were administered once daily (QD) via intraperitoneal injection (IP) or oral administration (PO) 2 hours before the experiment.

[0274] Table 15

[0275] Experimental Example 12.3: Evaluation of Analgesic Efficacy

[0276] Five von Frey tests were performed on the right hindfoot (before fibromyalgia model establishment, and on days 1, 2, 4, and 6 after model establishment). Rats were allowed 30 minutes to acclimatize before all behavioral tests, and all personnel conducting the tests were unaware of the medication administration. The von Frey test was performed on all rats before model establishment for baseline measurements. Rats were placed on a metal grid within a transparent plastic box (20 x 20 x 14 cm), and the withdrawal threshold was measured by applying von Frey filaments (weight increments: 0.4, 0.6, 1.4, 2, 4, 6, 8, and 15 g, North Coast) to the center of the right hindfoot to assess mechanotropic pain.

[0277] Figure 21 This is a graph showing the analgesic efficacy evaluation results in a reserpine-induced fibromyalgia rat model. Compared with the control group (G1), the reserpine-treated group (G2) showed high sensitivity, and after model establishment, the sensitivity of the gabapentin-treated group (G3) and the Example 2-treated groups (G4 to G6) gradually improved over time. Example 2 showed a dose-dependent recovery trend and demonstrated significant efficacy at 30 mg / kg. This confirms that the compounds in the examples significantly relieve pain.

[0278] Experimental Example 13: Efficacy Evaluation in a CDAHFD-Induced Nonalcoholic Steatohepatitis Mouse Model

[0279] Experiment Example 13.1: Experiment Preparation

[0280] Three-week-old male C57BL / 6 mice (Koatech, South Korea) were purchased. Mice were housed in groups of up to five per cage in an environment with controlled light-dark cycles (08:00–20:00), temperature (22±1°C), and humidity (30–50%), and provided with unlimited food and water. Mice were allowed one week to acclimatize before the start of the experiment.

[0281] The test drug was administered to normal mice and a CDAHFD (choline deficiency, L-amino acid definition, high-fat diet)-induced nonalcoholic steatohepatitis (NASH) mouse model to evaluate its efficacy. For the example compounds, the test drug was formulated using 10% NMP, 40% PEG 400, and 50% DW as solvents.

[0282] CDAHFD was formulated as a high-fat diet containing 0.1% methionine and 60 kcal of fat, but lacking choline. C57BL / 6J mouse models fed CDAHFD rapidly developed fibrotic steatohepatitis within a short period.

[0283] Experiment Example 13.2: Experimental Design

[0284] Figure 22 This is a schematic diagram of the efficacy experiment in a CDAHFD-induced non-alcoholic steatohepatitis mouse model.

[0285] As shown in Table 16 below, 3-week-old male C57BL / 6 mice were divided into 7 groups for the construction of a CDAHFD-induced NASH model and drug administration. The control group (G1) was fed a standard diet and water. Groups G2 to G7 were fed a CDAHFD (Research Diet Inc., #A06071302) diet for 4 and 8 weeks, respectively. Except for the control group, each group was orally administered the excipient, 30 mg / kg selonsertib, 10 mg / kg in Example 3, 30 mg / kg in Example 3, 10 mg / kg in Example 2, and 30 mg / kg in Example 2 once daily (QD) starting from 5 weeks of age.

[0286] Table 16

[0287] Experimental Example 13.3: Evaluation of the efficacy in improving liver fibrosis

[0288] Histological visualization of collagen fibers was performed using Sirius red staining. Histopathological analysis was conducted to evaluate the efficacy in improving liver fibrosis.

[0289] For each group of mice, three liver sections fixed with 4% PFA (paraformaldehyde) were prepared into paraffin blocks. The paraffin blocks were cut to a thickness of 5 μm (Histocore AUTOCUT R, Leica Microsystems, Germany), and the sections were mounted on silane-coated slides (MUTO, Japan) and stored until staining.

[0290] The paraffin on the slides was removed using xylene, and the slides were then moistened and washed. The slides were stained with Sirius Red solution (ab150681, Abcam, UK) and washed off with acetic acid solution. The slides were then mounted after dehydration and cleaning with alcohol and xylene. For each section, two images of different regions were taken using a microscope (Axioscan7, ZEISS, Germany). The area of ​​fibrosis relative to the total area was measured using ImageJ software.

[0291] Figure 23a and 23b These are charts and images showing the proportion of Sirius red stained areas in the liver of a CDAHFD-induced non-alcoholic steatohepatitis mouse model. (Reference) Figure 23a and 23bCompared to group G1, the CDAHFD group showed a significantly higher relative area of ​​Sirius red staining, indicating that the NASH model was adequately established. In the groups treated with the control drug siloncerti (G3), Examples 3 (G4 and G5), and Examples 2 (G6 and G7), the Sirius red staining area was significantly reduced, with the most significant improvement observed in the group treated with 30 mg / kg of Example 2 after 4 weeks of CDAHFD diet. This confirms that the compounds in these examples significantly improve liver fibrosis.

Claims

1. A compound represented by Formula 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof: Chemical Formula 1: ； in, For substituted or unsubstituted phenyl or substituted or unsubstituted C 3-8 Cycloalkenyl; Wherein, the substituted phenyl and the substituted C 3-8 Each cycloalkenyl group is independently converted by C 1-6 Alkyl, C 1-6 Alkoxy or halogen substitution; and, R1, R2, and R3 are each independently H and C. 1-6 Alkyl, halogen, C 3-6 cycloalkyl or halogenated C 1-6 alkyl.

2. The compound represented by Formula 1 as claimed in claim 1, or its stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt, wherein, For substituted or unsubstituted phenyl or substituted or unsubstituted C 5-8 Cycloalkenyl, Wherein, the substituted phenyl and the substituted C 3-8 Each cycloalkenyl group is independently converted by C 1-3 Alkyl, C 1-3 Alkoxy or halogen substitution; and, R1, R2, and R3 are each independently H and C. 1-3 Alkyl, halogen, C 3-6 cycloalkyl or halogenated C 1-3 alkyl.

3. The compound represented by Formula 1 as claimed in claim 1, or its stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt, wherein, It is a phenyl group or a C group containing a double bond. 5-8 Cycloalkenyl; in, R1 is C 3-6 cycloalkyl; R2 is C 1-3 Alkyl; and, R3 is a halogen.

4. The compound represented by Formula 1 as claimed in claim 1, or its stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt, wherein, It is a phenyl group or a C group containing a double bond. 5-8 Cycloalkenyl; Wherein, R1 is cyclopropyl; R2 is a methyl group; and, R3 stands for fluorine.

5. The compound represented by Formula 1 as claimed in claim 1, or its stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt, wherein, The compound represented by chemical formula 1 is selected from the group consisting of: <1> N-(6-(1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzamide; <2> 5-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5,6-dihydrocyclopentano[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide; <3> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(4,5,6,7-tetrahydro-1H-benzo[ d [1,2,3]triazol-1-yl)pyridin-2-yl)methylbenzamide; <4> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-(6-(5,6,7,8-tetrahydrocycloheptan[ d [1,2,3]triazol-1(4H)-yl)pyridin-2-yl)benzamide; and, <5> 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4,5,6,7,8,9-hexahydro-1H-cyclooctano[ d [1,2,3]triazol-1-yl)pyridin-2-yl)-4-methylbenzamide.

6. The compound represented by Formula 1 as claimed in claim 1, or its stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt, wherein, The compound inhibits ASK1.

7. A method for preparing the compound represented by chemical formula 1 as described in claim 1, comprising: As shown in reaction scheme 1, the compound represented by chemical formula 2 is reacted with the compound represented by chemical formula 3 to prepare the compound represented by chemical formula 1: Reaction scheme 1: ； in, The definitions of R1, R2, and R3 are the same as those in chemical formula 1 as described in claim 1.

8. A pharmaceutical composition comprising a compound represented by Formula 1 as claimed in claim 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition for the prevention or treatment of ASK1-related diseases, comprising, as an active ingredient, a compound represented by chemical formula 1 as claimed in claim 1, or a stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition of claim 9, wherein the ASK1-related disease is a neurodegenerative disease, cardiovascular disease, autoimmune disease, or liver disease.

11. The pharmaceutical composition of claim 10, wherein the neurodegenerative disease is one or more selected from the group consisting of: Alzheimer's disease, hippocampal sclerosis, frontotemporal dementia (FTD), frontotemporal degeneration (FTLD), Huntington's disease, corticobasal degeneration, amyotrophic lateral sclerosis, spinal muscular atrophy, motor neuron disease, inclusion body myositis, Parkinson's disease, Lewy body dementia, Lewy body disease, multiple system atrophy, progressive supranuclear palsy, Pick's disease, prions, traumatic brain injury, ischemic and hemorrhagic stroke, cerebral ischemia, hypoxia, and glutamate neurotoxicity.

12. The pharmaceutical composition of claim 10, wherein the cardiovascular disease is one or more selected from the group consisting of: heart failure, ischemia, recurrent ischemia, myocardial infarction, arrhythmia, acute coronary syndrome, diabetes, atherosclerosis, and intermittent claudication.

13. The pharmaceutical composition of claim 10, wherein the autoimmune disease is one or more selected from the group consisting of: rheumatoid arthritis, fibromyalgia, systemic lupus erythematosus, multiple sclerosis, diabetes, systemic sclerosis, Graves' disease, Guillain-Barré syndrome, myasthenia gravis, psoriasis, Crohn's disease, ulcerative colitis, optic neuritis, and Sjögren's syndrome.

14. The pharmaceutical composition of claim 10, wherein the liver disease is one or more selected from the group consisting of: non-alcoholic fatty liver disease (NASH), alcoholic fatty liver disease, liver fibrosis, liver cancer, hepatotoxicity, cholestasis, cirrhosis, liver ischemia, liver abscess, hepatic coma, and liver atrophy.

15. A method for preventing or treating ASK1-related diseases, comprising: The subject is given a compound as described in any one of claims 1-6, or a solvate, stereoisomer, or pharmaceutically acceptable salt thereof.

16. Use of a compound as claimed in any one of claims 1-6, or a solvate, stereoisomer, or pharmaceutically acceptable salt thereof, in the prevention or treatment of ASK1-related diseases.

17. Use of a compound as described in any one of claims 1-6, or a solvate, stereoisomer, or pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention or treatment of ASK1-related diseases.

Images