Pharmaceutical composition containing nitrogen-containing saturated heterocyclic derivatives

Abstract

Provided is a therapeutic and / or preventive agent for a central nervous system disease involving abnormal aggregates of brain proteins. 【Solution】A compound represented by formula (1) or a pharmaceutically acceptable salt thereof having an action of suppressing or decreasing the accumulation of abnormal aggregates of brain proteins (wherein, R 1 and R 2 represent hydrogen or the like, R 3 and R 4 represent hydrogen, C 1-6 alkyl or the like, R 5 represents halogen, C 1-6 alkyl or the like, R 6 represents hydrogen, halogen or the like, X represents oxygen or the like, Y represents carbon or the like, m and n represent integers such as 0, 1 or the like, r and s represent integers such as 0, 1, 2 or the like, Hy represents a pyridine ring or the like), as an active ingredient, a therapeutic and / or preventive agent for a central nervous system disease involving abnormal aggregates of brain proteins is provided. TIFF2026053519000070.tif4191

Description

[Technical Field]

[0001] The present invention relates to nitrogen-containing saturated heterocyclic derivatives or pharmaceutically acceptable salts thereof that have an inhibitory or reducing effect on the accumulation of abnormal aggregates of brain proteins, and to therapeutic and / or prophylactic agents for central nervous system diseases involving abnormal aggregates of brain proteins, comprising the derivative as an active ingredient. The present invention also aims to provide a method for reproducing the pathophysiology of Parkinson's disease using neuronal spheroids and a method for evaluating the amount of α-synuclein aggregates using the same. [Background technology]

[0002] In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), abnormally aggregated proteins are formed in the patient's brain, and these aggregates are thought to exhibit neurotoxicity, causing the onset and progression of the disease.

[0003] The constituent proteins of aggregates differ depending on the disease, and α-synuclein has been reported as the main component of aggregates that cause Parkinson's disease. Abnormally aggregated α-synuclein exhibits neurotoxicity, and it has been reported that aggregated α-synuclein can propagate between nerve cells.

[0004] While levodopa, a dopamine precursor, is a symptomatic treatment for Parkinson's disease, there is currently no established cure. In recent years, there has been vigorous development of disease-modifying drugs for Parkinson's disease, but so far, no drugs currently undergoing clinical trials have been reported to strongly inhibit or reduce the accumulation of α-synuclein aggregates.

[0005] Alpha-synuclein aggregates are thought to be underlying causes of Lewy body diseases, including Parkinson's disease (Lewy body dementia, multiple system atrophy, Gaucher disease, infantile neuronal axonal dystrophy, etc.). Therefore, drugs that suppress or reduce the accumulation of alpha-synuclein aggregates are expected to have a pathological effect on these diseases.

[0006] To date, no in vitro evaluation system has been reported that replicates α-synuclein aggregates that occur endogenously within nerve cells. Evaluation systems for α-synuclein-related pathologies have often been based on the increase in phosphorylated α-synuclein levels due to the addition of α-synuclein oligomers synthesized in vitro, making it impossible to evaluate the suppression of α-synuclein aggregate accumulation or the reduction of accumulated α-synuclein aggregates.

[0007] To date, drugs such as NPT200-11 (Neuropore) and Anle138b (MODAG) have been reported as having α-synuclein aggregate formation inhibitory activity, but these have been evaluated based on their inhibitory effect on the aggregation ability when α-synuclein is artificially aggregated in a test tube (Patent Documents 1 and 2).

[0008] Furthermore, it has been reported that (4aR,8aS)-hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one derivatives, such as (4aR,8aS)-6-{4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-carbonyl}hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one and (4aR,8aS)-6-[4-(5-ethylpyridine-3-yl)piperidine-1-carbonyl]hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one, have monoacylglycerol lipase (MAGL) inhibitory activity and are useful for neuroinflammation, neurodegenerative diseases, etc. (Patent Document 3). Furthermore, it has been reported that 2-chloro-3-{3-[6-(trifluoromethyl)pyridine-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile and 2-methoxy-3-{3-[6-(trifluoromethyl)pyridine-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile, which are derivatives of (azetidine-1-yl)(phenyl)methanone, have glycine transporter 1 (GlyT1) inhibitory activity and are useful for neurodegenerative diseases, etc. (Patent Document 4). However, none of these compounds are the nitrogen-containing saturated heterocyclic derivatives of the present invention. Furthermore, these documents do not disclose, nor suggest, any nitrogen-containing saturated heterocyclic derivatives of the present invention. In addition, they do not suggest any effect on suppressing or reducing the accumulation of abnormal aggregates of brain proteins. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2011 / 084642 [Patent Document 2] International Publication No. 2010 / 000372 [Patent Document 3] International Publication No. 2019 / 180185 [Patent Document 4] International Publication No. 2016 / 073420 [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof, a composition containing such compound, for use in the prevention or treatment of central nervous system diseases characterized by the suppression or reduction of the accumulation of abnormal aggregates of brain proteins. Furthermore, the invention aims to provide a method for reproducing the pathophysiology of Parkinson's disease using neuronal spheroids and a method for evaluating the amount of α-synuclein aggregates using the same. [Means for solving the problem]

[0011] As a result of diligent research, the inventors have discovered that a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof (hereinafter sometimes abbreviated as "the compound of the present invention") has the effect of suppressing or reducing the accumulation of abnormal aggregates of brain proteins. Furthermore, they have discovered a method for reproducing the pathophysiology of Parkinson's disease using neuronal spheroids and a method for evaluating the amount of α-synuclein aggregates using the same, thus completing the present invention. That is, the present invention is as follows.

[0012] [Section 1] Formula (1): [ka] [In the formula, X is oxygen or NR 7 This represents, R 7 C may be substituted with hydrogen, or 1 to 3 halogens of the same or different type. 1-3 Represents alkyl or cyclopropyl, Y represents CH or nitrogen. m represents 0, 1, or 2. n represents 0 or 1, r represents 0, 1, 2, 3, or 4. s represents 0, 1 or 2, (provided that when s = 0, Y is CH and r is 1, 2, 3 or 4, when s = 1, Y is CH and r is 0, 1, 2 or 3, when s = 2, r is 1 or 2) R 1 represents hydrogen, halogen, methyl or hydroxy, R 2 represents hydrogen, halogen, methyl or hydroxy, R 3 represents hydrogen, or C 1-3 alkyl, R 4 represents hydrogen, or C 1-3 alkyl, wherein R 3 and R 4 may together form crosslinked methylene or ethylene, R 5 represents halogen, C 1-3 alkyl optionally substituted with 1 to 3 halogen atoms of the same or different kinds, or C 1-3 alkoxy optionally substituted with 1 to 3 halogen atoms of the same or different kinds, R 6 represents hydrogen, halogen, C[[ID= forty-five]] 1-3 alkyl optionally substituted with 1 to 3 halogen atoms of the same or different kinds, or C 1-3 alkoxy optionally substituted with 1 to 3 halogen atoms of the same or different kinds, Hy represents a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring, wherein, (I) when Hy containing R 5 and R 6 is 5-fluoropyridin-2-yl and m and n are 1, r is 0 and s is 1, (II) when Hy containing R 5 and R 6 is 6-methoxypyridin-3-yl and m and n are 1 and X is oxygen, r is 0 and s is 1, (III)R 5 and R 6 Hy is 5-methoxypyridine-2-yl, m and n are 1, and X is NR 7 When this is the case, r is 0 and s is 1, (IV)R 5 is methyl and R 6 When is hydrogen, r is 0 and s is 1, however, (I')R 5 and R 6 Hy containing is not 4,6-dimethylpyrimidine-2-yl or 5-bromopyrimidine-2-yl, (II')R 5 and R 6 When Hy, which contains , is 5-chloropyridine-2-yl, then m and n are 1, and R 1 and R 2 Neither is hydrogen, However, the following compounds (III')(1-isopropylpiperidine-4-yl){3-(2-methoxypyridine-3-yl)pyrrolidine-1-yl}methanone, and (IV')(3-ethoxyoxetane-3-yl)[3-{4-(trifluoromethyl)pyrimidine-2-yl}pyrrolidine-1-yl]methanone, [excluding] A pharmaceutical composition containing a compound represented by or a pharmaceutically acceptable salt thereof.

[0013] [Section 2] m is 1, n is 1. The pharmaceutical composition described in item 1.

[0014] [Section 3] R 1 and R 2 However, each is independently hydrogen, methyl, or fluorine. A pharmaceutical composition as described in item 1 or 2.

[0015] [Section 4] R1 and R 2 However, it is hydrogen. A pharmaceutical composition as described in item 1 or 2.

[0016] [Section 5] X is oxygen, NH, or NMe. A pharmaceutical composition as described in any one of items 1 to 4.

[0017] [Section 6] R 3 However, it is hydrogen. A pharmaceutical composition as described in any one of items 1 to 5.

[0018] [Section 7] R 4 However, it is methyl or ethyl. A pharmaceutical composition as described in any one of items 1 to 6.

[0019] [Section 8] Y is CH. A pharmaceutical composition as described in any one of items 1 to 7.

[0020] [Section 9] s is 1, A pharmaceutical composition as described in any one of items 1 to 8.

[0021] [Section 10] r is 0 and s is 1. A pharmaceutical composition as described in any one of items 1 to 9.

[0022] [Section 11] Formula (2): [ka] [In the formula, X represents oxygen, NH, or NMe. R 4 represents methyl or ethyl, R 5 C may be replaced by a halogen, or one to three halogens of the same or different type. 1-3C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. however, R 5 and R 6 Hy, which contains [not 5-chloropyridine-2-yl] The pharmaceutical composition described in item 1, as represented by the above.

[0023] [Section 12] Hy is a pyridine ring. A pharmaceutical composition as described in any one of items 1 to 11.

[0024] [Section 13] R 5 However, it is trifluoromethyl. A pharmaceutical composition as described in any one of items 1 to 12.

[0025] [Section 14] Hy is pyridine-3-yl. A pharmaceutical composition as described in any one of items 1 to 13.

[0026] [Section 15] X is oxygen. A pharmaceutical composition as described in any one of items 1 to 14.

[0027] [Section 16] R 4 However, it is methyl. A pharmaceutical composition as described in any one of items 1 to 15.

[0028] [Section 17] X is NH or NMe. A pharmaceutical composition as described in any one of items 1 to 14.

[0029] [Section 18] X is NMe. A pharmaceutical composition as described in any one of items 1 to 14.

[0030] [Section 19] The pharmaceutical composition described in item 1, wherein the compound is selected from the following group of compounds: (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 1), (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 2), (3-methyloxetan-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 3), (3-methyloxetan-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 4), {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetane-3-yl)methanone (Example 29), (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 31), (1,3-dimethylazetidine-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 41), (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 42), (1,3-dimethylazetidine-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 43), and (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 44).

[0031] [Section 20] The pharmaceutical composition described in item 1, wherein the compound is selected from the following group of compounds: (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 1), (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 2), {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetane-3-yl)methanone (Example 29), (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 31), (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 42), and (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 44).

[0032] [Section 21] A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a pharmaceutical composition described in any one of items 1 to 20.

[0033] [Section 22] A therapeutic or prophylactic agent as described in paragraph 21, wherein the central nervous system disorder involving abnormal aggregates of brain proteins is a central nervous system disorder involving tau, α-synuclein, TDP-43, or polyglutamine.

[0034] [Section 23] A therapeutic or prophylactic agent as described in paragraph 21, for central nervous system disorders involving abnormal aggregates of brain proteins, such as Alzheimer's disease, frontotemporal degeneration, Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

[0035] [Section 24] A therapeutic or prophylactic agent as described in item 21, wherein a central nervous system disorder involving abnormal aggregates of brain proteins is a central nervous system disorder involving alpha-synuclein.

[0036] [Section 25] A therapeutic or prophylactic agent as described in paragraph 21, for central nervous system disorders involving abnormal aggregates of brain proteins, such as Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

[0037] [Section 26] A method for treating or preventing a central nervous system disorder involving abnormal aggregates of brain proteins, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof described in any one of items 1 to 20.

[0038] [Section 27] Use of any one of the pharmaceutical compositions described in items 1 to 20 for the manufacture of a therapeutic or prophylactic agent for a central nervous system disorder involving abnormal aggregates of brain proteins.

[0039] [Section 28] A pharmaceutical composition according to any one of claims 1 to 20, for use in the treatment or prevention of central nervous system disorders involving abnormal aggregates of brain proteins.

[0040] [Section 29] A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a pharmaceutical composition described in any one of items 1 to 20, and at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

[0041] [Section 30] A therapeutic or prophylactic agent according to item 21, for treating or preventing a central nervous system disorder involving abnormal aggregates of brain proteins, in combination with at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

[0042] [Section 31] Formula (1): [ka] [In the formula, X is oxygen or NR 7 This represents, R 7 C may be substituted with hydrogen, or 1 to 3 halogens of the same or different type. 1-3 Represents alkyl or cyclopropyl, Y represents CH or nitrogen. m represents 0, 1, or 2. n represents 0 or 1, r represents 0, 1, 2, 3, or 4. s represents 0, 1, or 2. (However, when s=0, Y is CH and r is 1, 2, 3, or 4, When s=1, Y is CH and r is 0, 1, 2, or 3. (When s=2, r is either 1 or 2.) R 1 represents hydrogen, halogen, methyl, or hydroxyl, R 2represents hydrogen, halogen, methyl, or hydroxyl, R 3 is hydrogen, or C 1-3 Represents alkyl, R 4 is hydrogen, or C 1-3 Represents alkyl, Here, R 3 and R 4 They may come together to form cross-linked methylene or ethylene. R 5 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a compound represented by or a pharmaceutically acceptable salt thereof as an active ingredient.

[0043] [Section 32] m is 1, n is 1. The therapeutic or prophylactic agent described in item 31.

[0044] [Section 33] R 1 and R 2 However, each is independently hydrogen, methyl, or fluorine. The therapeutic or prophylactic agent described in item 31 or 32.

[0045] [Section 34] R 1 and R 2 However, it is hydrogen. The therapeutic or prophylactic agent described in item 31 or 32.

[0046] [Section 35] X is oxygen, NH, or NMe. A therapeutic or prophylactic agent as described in any one of items 31 to 34.

[0047] [Section 36] R 3 However, it is hydrogen. A therapeutic or prophylactic agent as described in any one of items 31 to 35.

[0048] [Section 37] R 4 However, it is methyl or ethyl. A therapeutic or prophylactic agent as described in any one of items 31 to 36.

[0049] [Section 38] Y is CH. A therapeutic or prophylactic agent as described in any one of items 31 to 37.

[0050] [Section 39] s is 1, A therapeutic or prophylactic agent as described in any one of items 31 to 38.

[0051] [Section 40] r is 0 and s is 1. A therapeutic or prophylactic agent as described in any one of items 31 to 39.

[0052] [Section 41] Formula (2): [ka] [In the formula, X represents oxygen, NH, or NMe. R 4 represents methyl or ethyl, R 5 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. A therapeutic or prophylactic agent as described in item 31, represented by the same name.

[0053] [Section 42] Hy is a pyridine ring. A therapeutic or prophylactic agent as described in any one of items 31 to 41.

[0054] [Section 43] R 5 However, it is trifluoromethyl. A therapeutic or prophylactic agent as described in any one of items 31 to 42.

[0055] [Section 44] Hy is pyridine-3-yl. A therapeutic or prophylactic agent as described in any one of items 31 to 43.

[0056] [Section 45] X is oxygen. A therapeutic or prophylactic agent as described in any one of items 31 to 44.

[0057] [Section 46] R 4 However, it is methyl. A therapeutic or prophylactic agent as described in any one of items 31 to 45.

[0058] [Section 47] X is NH or NMe. A therapeutic or prophylactic agent as described in any one of items 31 to 44.

[0059] [Section 48] X is NMe. A therapeutic or prophylactic agent as described in any one of items 31 to 44.

[0060] [Section 49] A therapeutic or prophylactic agent as described in any one of paragraphs 31 to 48, wherein the central nervous system disorder is one in which abnormal aggregates of brain proteins are involved, and the central nervous system disorder is one in which tau, α-synuclein, TDP-43, or polyglutamine is involved.

[0061] [Section 50] A therapeutic or prophylactic agent according to any one of paragraphs 31 to 48, for a central nervous system disorder involving abnormal aggregates of brain proteins, which is Alzheimer's disease, frontotemporal degeneration, Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

[0062] [Section 51] A therapeutic or prophylactic agent according to any one of paragraphs 31 to 48, wherein a central nervous system disorder involving abnormal aggregates of brain proteins is a central nervous system disorder involving alpha-synuclein.

[0063] [Section 52] A therapeutic or prophylactic agent according to any one of paragraphs 31 to 48, wherein the central nervous system disorder involving abnormal aggregates of brain proteins is Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

[0064] [Section 53] A method for treating or preventing a central nervous system disorder involving abnormal aggregates of brain proteins, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof described in any one of items 31 to 48.

[0065] [Section 54] Use of any one of the compounds described in subsections 31 to 48 or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic or prophylactic agent for a central nervous system disorder involving abnormal aggregates of brain proteins.

[0066] [Section 55] A compound or a pharmaceutically acceptable salt thereof, as described in any one of paragraphs 31 to 48, for use in the treatment or prevention of central nervous system disorders involving abnormal aggregates of proteins in the brain.

[0067] [Section 56] A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a combination of a therapeutic or prophylactic agent described in any one of paragraphs 31 to 48 and at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

[0068] [Section 57] A therapeutic or prophylactic agent according to any one of paragraphs 31 to 48, for treating or preventing central nervous system disorders involving abnormal aggregates of brain proteins, in combination with at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

[0069] [Section 58] A method for reproducing the pathogenesis of Parkinson's disease using neuronal spheroids in three-dimensional culture of human iPS cells with synucleopathy-related gene mutations, including step (I); (I) A step of measuring the amount of α-synuclein aggregates from nerve spheroids.

[0070] [Section 59] A method for evaluating drugs that inhibit or reduce the accumulation of α-synuclein aggregates in the pathogenesis of Parkinson's disease by neuronal spheroids in three-dimensional culture using human iPS cells with synucleopathy-related gene mutations, including step (I); (I) A step of measuring the amount of α-synuclein aggregates from nerve spheroids.

[0071] [Section 60] Formula (3): [ka] [In the formula, R 4 represents methyl or ethyl, R 5 This represents trifluoromethyl, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents pyridine-3-yl. A method for producing a compound represented by or a pharmaceutically acceptable salt thereof, A manufacturing method including step 1 below; (Step 1) Formula (4): [ka] [In the formula, R 5 , R 6 , and Hy represent the same group as above. Compounds represented by formula (5) or their salts and formula (5): [ka] [In the formula, R 4 This represents the same base as above, A represents OH or a halogen. A step of condensing a compound represented by formula (3) or a salt thereof to produce a compound represented by formula (3) or a pharmaceutically acceptable salt thereof.

[0072] [Item 61] Formula (3): [Chemical formula] [In the formula, R 4 represents methyl or ethyl, R 5 represents trifluoromethyl, R 6 represents hydrogen, halogen, C 1-3 alkyl which may be substituted with one to three identical or different halogens, or C 1-3 alkoxy which may be substituted with one to three identical or different halogens, Hy represents pyridin-3-yl.] A method for producing a compound represented by the formula or a pharmaceutically acceptable salt thereof, comprising the following step 1; (Step 1) Formula (4): [Chemical formula] [In the formula, R 5 , R 6 , and Hy represent the same groups as above.] A compound represented by the formula or a salt thereof and Formula (5): [Chemical formula] [In the formula, R 4 represents the same group as above, A represents OH.] Condensing the compound represented by the formula or a salt thereof with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide and triethylamine to produce the compound represented by the formula or a pharmaceutically acceptable salt thereof.

[0073] [Item 62] Formula (6): [Chemical formula] [In the formula, R 4 This represents methyl, R 5 This represents trifluoromethyl, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents pyridine-3-yl. A method for producing a compound represented by or a pharmaceutically acceptable salt thereof, A manufacturing method including the following steps 1-3; (Step 1) Formula (4): [ka] [In the formula, R 5 , R 6 , and Hy represent the same group as above. Compounds represented by formula (7) or their salts and formula (7): [ka] [In the formula, R 4 This represents the same base as above, A represents OH or halogen, Pro represents a protecting group for an amino group. The compound represented by or a salt of the compound is condensed to form formula (8): [ka] [In the formula, R 4 , R 5 , R 6 Hy and Pro represent the same base as above. A process for producing a compound represented by or a salt thereof, (Step 2) Deprotect the protecting group of the amino group of the compound represented by formula (8) or a salt thereof, to obtain formula (9): [ka] [In the formula, R 4 , R 5 , R 6 , and Hy represent the same group as above. A process for producing a compound represented by or a salt thereof, (Step 3) A step of reacting a compound represented by formula (9) or a salt thereof with formaldehyde or an equivalent thereof in the presence of a reducing agent to produce a compound represented by formula (6) or a pharmaceutically acceptable salt thereof.

[0074] [Section 63] Formula (6): [ka] [In the formula, R 4 This represents methyl, R 5 This represents trifluoromethyl, R 6 C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 C may be substituted with alkyl groups or 1 to 3 halogens of the same or different types. 1-3 Represents alkoxy, Hy represents pyridine-3-yl. A method for producing a compound represented by or a pharmaceutically acceptable salt thereof, A manufacturing method including the following steps 1-3; (Step 1) Formula (4): [ka] [In the formula, R 5 , R 6 , and Hy represent the same group as above. Compounds represented by formula (7) or their salts and formula (7): [ka] [In the formula, R 4 This represents the same base as above, Pro represents tert-butoxycarbonyl, A represents OH. The compound represented by or a salt thereof is condensed in the presence of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide and triethylamine to obtain formula (8): [ka] [In the formula, R 4 , R 5 , R 6 Hy and Pro represent the same base as above. A process for producing a compound represented by or a salt thereof, (Step 2) The protecting group of the amino group of the compound represented by formula (8) or its salt is deprotected with trifluoroacetic acid to obtain formula (9): [ka] [In the formula, R 4 , R 5 , R 6 , and Hy represent the same group as above. A process for producing a compound represented by or a salt thereof, (Step 3) A step of reacting a compound represented by formula (9) or a salt thereof with formaldehyde in the presence of sodium triacetoxyborohydride and acetic acid to produce a compound represented by formula (6) or a pharmaceutically acceptable salt thereof. [Effects of the Invention]

[0075] According to the present invention, it has become possible to provide a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof has an action of suppressing or reducing the accumulation of abnormal aggregates of brain proteins, and is useful as a therapeutic or prophylactic agent for central nervous system diseases involving abnormal aggregates of brain proteins, particularly neurodegenerative diseases involving α-synuclein (Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher's disease, infantile neuroaxonal dystrophy, etc.). Further, it is useful as an evaluation method for evaluating a drug having an action of suppressing or reducing the accumulation of α-synuclein aggregates by reproducing spontaneous α-synuclein aggregates in nerve cells in the pathological state of Parkinson's disease.

Brief Description of the Drawings

[0076] [Figure 1] Figure 1 shows the difference in the amount of aggregates in neural spheroids derived from healthy human iPS cells and neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis indicates the amount of aggregates in the neural spheroids, and the horizontal axis indicates the number of culture days. The white graph shows the amount of aggregates in neural spheroids derived from healthy humans, and the black graph shows the amount of aggregates in neural spheroids derived from PLA2G6 mutant iPS cells. [Figure 2] Figure 2 shows the difference in the amount of aggregates in dopamine neural spheroids derived from healthy human iPS cells and dopamine neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis indicates the amount of aggregates in the dopamine neural spheroids, and the horizontal axis indicates the number of culture days. The white graph shows the amount of aggregates in neural spheroids derived from healthy humans, and the black graph shows the amount of aggregates in neural spheroids derived from PLA2G6 mutant iPS cells. [Figure 3] Figure 3 shows the difference in the amount of tyrosine hydroxylase in dopamine neural spheroids derived from healthy human iPS cells and dopamine neural spheroids derived from PLA2G6 mutant iPS cells on the 26th day of culture. The vertical axis indicates the amount of tyrosine hydroxylase in the dopamine neural spheroids. The white graph shows the amount of tyrosine hydroxylase in neural spheroids derived from healthy humans, and the black graph shows the amount of tyrosine hydroxylase in neural spheroids derived from PLA2G6 mutant iPS cells. [Figure 4]Figure 4 shows the difference in the amount of cleaved caspase3 in dopamine neural spheroids derived from healthy human iPS cells and dopamine neural spheroids derived from PLA2G6 mutant iPS cells on the 40th day of culture. The vertical axis indicates the amount of cleaved caspase3 in the dopamine neural spheroids. The white graph represents the amount of cleaved caspase3 in neural spheroids derived from healthy humans, and the black graph represents the amount of cleaved caspase3 in neural spheroids derived from PLA2G6 mutant iPS cells. [Figure 5] Figure 5 shows the difference in the amount of aggregates in dopamine neural spheroids derived from healthy human iPS cells and dopamine neural spheroids derived from homozygous GBA1 gene mutant iPS cells. The vertical axis indicates the amount of aggregates in the dopamine neural spheroids, and the horizontal axis indicates different culture days. The white graph represents the amount of aggregates in neural spheroids derived from healthy humans, and the black graph represents the amount of aggregates in neural spheroids derived from homozygous GBA1 gene mutant iPS cells.

Mode for Carrying Out the Invention

[0077] Hereinafter, the present invention will be described in detail. In this specification, the number of carbons in the definition of "substituent" may be expressed, for example, as "C 1-3 ", etc. Specifically, the notation "C 1-3 alkyl" is synonymous with an alkyl having 1 to 3 carbons.

[0078] Specific examples of "halogen" include fluorine, chlorine, bromine, or iodine. Preferably, they are fluorine or chlorine.

[0079] "C 1-3 alkyl" means a linear or branched saturated hydrocarbon group having 1 to 3 carbons. Preferably, it is "C 1-2 alkyl". Specific examples of "C 1-3 alkyl" include, for example, methyl, ethyl, propyl, isopropyl, etc.

[0080] The "C 1-3 alkyl" part of "C 1-3 alkoxy" is the above-mentioned "C1-3 This is synonymous with "alkyl". Preferably, "C 1-2 It is "alkoxy". 1-3 Specific examples of "alkoxy" include methoxy, ethoxy, propoxy, and isopropoxy.

[0081] Among the compounds of the present invention represented by formula (1), X, Y, m, n, r, s, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 The following are preferred, but the technical scope of the present invention is not limited to the compounds listed below.

[0082] In the compound represented by formula (1), R 1 , R 2 , R 3 , R 4 , R 5 and R 6 The group may be substituted on any carbon if it is substituted, R 1 and R 2 , and R 3 and R 4 In this case, substitution is permitted on the same carbon if it is possible, and R 3 and R 4 In this case, Y may be substituted with H in CH when Y is CH. In the compound represented by formula (1), the bonding site of Hy (arrow) to the nitrogen-containing saturated heterocycle is carbon. [ka]

[0083] X is preferably oxygen, NH, or NMe, and more preferably oxygen or NMe.

[0084] Preferably, CH is given as Y.

[0085] As n, preferably 1 can be mentioned.

[0086] As m, preferably 1 can be mentioned.

[0087] When Y is CH, as r, preferably 0, 1, 2 can be mentioned, more preferably 0 or 1 can be mentioned, and even more preferably 0 can be mentioned. When Y is nitrogen, r is 1, 2, 3 or 4, and preferably 1 can be mentioned.

[0088] When Y is CH, as s, preferably 0, 1, 2 can be mentioned, more preferably 1, 2 can be mentioned, and even more preferably 1 can be mentioned. When Y is nitrogen, s is 2.

[0089] R 1 As such, preferably hydrogen, methyl, fluorine can be mentioned, and more preferably hydrogen can be mentioned.

[0090] R 2 As such, preferably hydrogen, methyl, fluorine can be mentioned, and more preferably hydrogen can be mentioned.

[0091] R 3 As such, preferably hydrogen, methyl, ethyl can be mentioned, and more preferably hydrogen can be mentioned.

[0092] R 4 As such, preferably hydrogen, methyl, ethyl can be mentioned, more preferably methyl, ethyl can be mentioned, and even more preferably methyl can be mentioned.

[0093] In the ring containing X and Y, R 3 and R 4 together form a structure of crosslinked methylene or ethylene, and the structure of the following formula (3) together with the ring containing X and Y can be mentioned. In the following formula (3), the wavy line indicates the bonding site to the carbonyl in formula (1). Also, in the following formula (3), X and Y have the same meaning as in item 1.

Chemical formula

[0094] R 5 Preferably, C may be substituted with a halogen, or one to three halogens of the same or different type. 1-3 Examples include alkyl groups, more preferably halogens, methyl groups which may be substituted with 1 to 3 fluorines, even more preferably trifluoromethyl groups, methyl groups, fluorines, and most preferably trifluoromethyl groups.

[0095] R 6 Preferably, C is substituted with hydrogen, a halogen, or 1 to 3 halogens of the same or different type. 1-3 Examples include alkyl groups, more preferably hydrogen, halogens, and methyl groups which may be substituted with 1 to 3 fluorines, even more preferably hydrogen, halogens, and methyl groups, and most preferably hydrogen, fluorines, and methyl groups.

[0096] R 7 Preferably hydrogen, C 1-3 Examples include alkyl groups, more preferably hydrogen, methyl groups, and even more preferably methyl groups. 7 Another embodiment of this is a C containing 1 to 5 deuterium atoms. 1-3 Alkyl compounds are examples.

[0097] Preferably, the Hy is a pyridine ring, and more preferably pyridine-3-yl represented by the following formula (4). In the following formula (4), the arrows indicate the bond position to the nitrogen-containing saturated ring. [ka]

[0098] Examples of condensing agents used in the condensation reaction include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide. Preferably, examples include 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide.

[0099] Examples of reducing agents include sodium borohydride, sodium triacetoxyborohydride, and sodium cyanoborohydride. Preferably, sodium cyanoborohydride and sodium triacetoxyborohydride are used.

[0100] Examples of formaldehyde or its equivalents include formaldehyde, 1,3,5-trioxane, and paraformaldehyde. Formaldehyde is preferred.

[0101] Examples of protecting groups for the amino group include the tert-butoxycarbonyl group and the benzyloxycarbonyl group, with the tert-butoxycarbonyl group being preferred.

[0102] Among the compounds represented by formula (1), preferred compounds include the following compounds or their pharmaceutically acceptable salts.

[0103] One embodiment of the compound represented by formula (1) is (A) below. (A) X is oxygen or NR 7 And, R 7 However, hydrogen, C 1-3 Alkyl or cyclopropyl, Y is CH or nitrogen. m is 0, 1, or 2, n is 0 or 1, r is 0, 1, or 2, s is 0, 1, or 2, (However, when s=0, Y is CH and r is 1 or 2, When s=1, Y is CH and r is 0, 1 or 2. (When s=2, r is either 1 or 2.) R 1 However, it is hydrogen, halogen, methyl or hydroxyl, R 2 However, it is hydrogen, halogen, methyl or hydroxyl, R 3 However, hydrogen, or C 1-3 It is alkyl, R 4 However, hydrogen, or C 1-3 It is alkyl, Here, R 3 and R 4 They may come together to form cross-linked methylene or ethylene. R 5 However, C may be substituted with a halogen, or with 1 to 3 halogens of the same or different type. 1-3 It is alkyl, R 6 However, C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 It is alkyl, Hy is a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. Here, (I)R 5 and R 6 When Hy, which contains , is 5-fluoropyridine-2-yl, and m and n are 1, then r is 0 and s is 1, (II)R 5 is methyl and R 6 When is hydrogen, r is 0 and s is 1, however, (I')R5 and R 6 Hy containing is not 4,6-dimethylpyrimidine-2-yl or 5-bromopyrimidine-2-yl, (II')R 5 and R 6 When Hy, which contains , is 5-chloropyridine-2-yl, then m and n are 1, and R 1 and R 2 Neither is hydrogen, However, the following compounds (III')(3-ethoxyoxetane-3-yl)[3-{4-(trifluoromethyl)pyrimidine-2-yl}pyrrolidine-1-yl]methanone, Excluding A compound represented by or a pharmaceutically acceptable salt thereof.

[0104] One embodiment of the compound represented by formula (1) is (B) below. (B) X is oxygen or NR 7 And, R 7 However, hydrogen, C 1-3 Alkyl or cyclopropyl, Y is CH or nitrogen. m is 0, 1, or 2, n is 0 or 1, r is 0, 1, or 2, s is 0, 1, or 2, (However, when s=0, Y is CH and r is 1 or 2, When s=1, Y is CH and r is 0, 1 or 2. (When s=2, r is either 1 or 2.) R 1 However, it is hydrogen, halogen, methyl or hydroxyl, R 2 However, it is hydrogen, halogen, methyl or hydroxyl, R 3 However, hydrogen, or C 1-3 It is alkyl, R 4However, hydrogen, or C 1-3 It is alkyl, Here, R 3 and R 4 They may come together to form cross-linked methylene or ethylene. R 5 However, C may be substituted with a halogen, or with 1 to 3 halogens of the same or different type. 1-3 It is alkyl, R 6 However, C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 It is alkyl, Hy is a pyridine ring, Here, (I)R 5 and R 6 When Hy, which contains , is 5-fluoropyridine-2-yl, and m and n are 1, then r is 0 and s is 1, (II)R 5 is methyl and R 6 When is hydrogen, r is 0 and s is 1, however, (I')R 5 and R 6 When Hy, which contains , is 5-chloropyridine-2-yl, then m and n are 1, and R 1 and R 2 Neither is hydrogen. A compound represented by or a pharmaceutically acceptable salt thereof.

[0105] One embodiment of the compound represented by formula (1) is (C) below. (C) X is oxygen or NR 7 And, R 7 However, hydrogen, C 1-3 Alkyl or cyclopropyl, Y is CH, m is 1, n is 1, r is 0, 1, or 2, s is 0, 1, or 2, (However, when s=0, r is 1 or 2, When s=1, r is 0, 1, or 2. (When s=2, r is either 1 or 2.) R 1 However, it is hydrogen, methyl, or fluorine, R 2 However, it is hydrogen, methyl, or fluorine, R 3 However, it is hydrogen, methyl, or ethyl. R 4 However, it is hydrogen, methyl, or ethyl. R 5 However, C may be substituted with a halogen, or with 1 to 3 halogens of the same or different type. 1-3 It is alkyl, R 6 However, C may be substituted with hydrogen, halogens, or 1 to 3 halogens of the same or different type. 1-3 It is alkyl, Hy is a pyridine ring, Here, (I)R 5 and R 6 When Hy, which contains , is 5-fluoropyridine-2-yl, then r is 0 and s is 1, (II)R 5 is methyl and R 6 When is hydrogen, r is 0 and s is 1, however, (I')R 5 and R 6 When Hy, which contains 5-chloropyridine-2-yl, R 1 and R 2 Neither is hydrogen. A compound represented by or a pharmaceutically acceptable salt thereof.

[0106] One embodiment of the compound represented by formula (1) is (D) below. (D) X is oxygen or NR 7 And, R7 However, it is hydrogen or methyl, Y is CH, m is 1, n is 1, r is 0 or 1, s is 1 or 2, (however (When s=2, r is 1.) R 1 However, it is hydrogen, R 2 However, it is hydrogen, R 3 However, it is hydrogen, R 4 However, it is methyl or ethyl, R 5 However, it is a methyl atom which may be substituted with a halogen or 1 to 3 fluorine atoms. R 6 However, it is a methyl molecule which may be substituted with hydrogen, a halogen, or 1 to 3 fluorine atoms. Hy is pyridine-3-yl. A compound represented by or a pharmaceutically acceptable salt thereof.

[0107] One embodiment of the compound represented by formula (1) is the following (E), which has the structure of formula (2). (E) [ka] In equation (2), X is oxygen or NMe, R 4 However, it is methyl, R 5 However, it is trifluoromethyl, methyl, or fluorine, R 6 However, it is hydrogen, halogen, or methyl, Hy is pyridine-3-yl. A compound represented by or a pharmaceutically acceptable salt thereof.

[0108] One embodiment of the compound represented by formula (1) is the following compound or a pharmaceutically acceptable salt thereof. (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 1), (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 2), (3-methyloxetan-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 3), (3-methyloxetan-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 4), {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetane-3-yl)methanone (Example 29), (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 31), (1,3-dimethylazetidine-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 41), (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 42), (1,3-dimethylazetidine-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 43), or (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 44).

[0109] One embodiment of the compound represented by formula (1) is the following compound or a pharmaceutically acceptable salt thereof. (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 1), (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 2) {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetane-3-yl)methanone (Example 29), (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 31), (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 42), or (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone (Example 44).

[0110] In the present invention, "pharmaceutically acceptable salts" include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, or organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, or salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, "pharmaceutically acceptable salts" include amino acid salts with basic amino acids or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid.

[0111] Suitable salts of the starting compound and intermediates, and salts acceptable as pharmaceutical raw materials, are conventional non-toxic salts, including acid addition salts such as organic acid salts (e.g., acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, para-toluenesulfonate, etc.) and inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.), salts with amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.), metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.), ammonium salts or organic base salts (e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), and others that can be appropriately selected by those skilled in the art.

[0112] If you want to obtain a salt of the compound of the present invention, if the compound of the present invention is obtained in the form of a salt, it can be purified as is, and if it is obtained in the form of a free compound, it can be dissolved or suspended in a suitable organic solvent, and a salt can be formed by adding an acid or base using a commonly used method.

[0113] In the present invention, one or more compounds represented by formula (1) 1 H 2 Deuterium converters converted to H(D) are also included in the compounds represented by formula (1). The present invention includes compounds represented by formula (1) or pharmaceutically acceptable salts thereof. Furthermore, since the compounds of the present invention may also exist in the form of hydrates and / or solvates with various solvents (e.g., ethanol hydrates), these hydrates and / or solvates are also included in the compounds of the present invention. Moreover, the present invention includes all tautomers of compound (1) of the present invention, all stereoisomers that exist, and all forms of crystals, as well as mixtures thereof.

[0114] Some compounds of the present invention may have optical isomers based on optically active centers, atropisomers based on axial or planar chirality resulting from the constraint of intramolecular rotation, other stereoisomers, tautomers, and geometric isomers. Furthermore, all possible isomers, including these, and mixtures thereof, are encompassed within the compounds of the present invention.

[0115] In particular, optical isomers and atrop isomers can be obtained as racemates or as optically active compounds when optically active starting materials or intermediates are used. If necessary, at an appropriate stage of the manufacturing process described below, the corresponding racemates of the starting materials, intermediates, or final products can be physically or chemically separated into their optical isomers by known separation methods such as optically active column methods or fractional crystallization. Specifically, for example, in the diastereomer method, two diastereomers are formed from a racemate by a reaction using an optically active resolving agent. Since these different diastereomers generally have different physical properties, they can be separated by known methods such as fractional crystallization.

[0116] The following describes, with examples, a method for producing the compound represented by formula (1) in the present invention, but the present invention is not limited thereto.

[0117] Manufacturing method The compounds of the present invention are synthesized by a method that combines the manufacturing method described below with known synthesis methods. The reaction equations may also include cases where the compounds form salts, and examples of such salts include those similar to the salt of the compound represented by formula (1). These reactions are merely illustrative, and the compounds of the present invention can be produced by other methods as appropriate, based on the knowledge of those proficient in organic synthesis.

[0118] In each of the manufacturing methods described below, even if the use of protecting groups is not explicitly stated, if there are functional groups that require protection, those functional groups may be protected as necessary, and the target product may be obtained by deprotecting them after the reaction is complete or after a series of reactions have been carried out.

[0119] The introduction and removal of protecting groups can be carried out by methods commonly used in organic synthesis (for example, the methods described in TW Greene and PGM Wuts, "Protective Groups in Organic Synthesis", 3rd Ed., John Wiley and Sons, inc., New York (1999)) or similar methods. Examples of protecting groups for amino groups include tert-butoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, o-nitrobenzenesulfonyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl.

[0120] Manufacturing method 1 Of the compounds represented by formula (1), the compound represented by formula (1a) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 m, n, r, s, Y, and Hy are as defined in [Section 1] above; A represents a halogen or OH.

[0121] (Step 1-1: Manufacturing process of compound (1a)) Compound (1a) is prepared by reacting compound (1-1) and compound (1-2) in a suitable inert solvent, in or without the presence of various condensing agents and / or bases. Compound (1-1) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2014 / 192868). Alternatively, one prepared by the following preparation methods 3 to 6 can be used. Compound (1-2) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2016 / 004272). The base used in this step is appropriately selected from the bases exemplified below, but examples include sodium hydride, triethylamine, diisopropylethylamine, or sodium carbonate. The condensing agent used in this step can be any of the various condensing agents commonly used in organic synthesis reactions, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide, etc. The solvent used in this step is appropriately selected from the solvents exemplified later, such as DMF, THF, dichloromethane, chloroform, ethyl acetate, etc. The reaction time in this step is usually 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature in this step is usually -78°C to 200°C, preferably -78°C to 80°C.

[0122] Manufacturing method 2 Of the compounds represented by formula (1), the compounds represented by formula (1b) and formula (1c) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R5 , R 6 ,m,n,r,s,Y, andHy are synonymous with [Section 1] above;R 8 C may be substituted with 1 to 3 halogens of the same or different types. 1-3 [A represents alkyl or cyclopropyl, A represents halogen or OH; Pro represents an amino group protecting group.]

[0123] (Step 2-1: Manufacturing process of compound (1c)) Compound (1c) is prepared from compound (1-1) and compound (2-1) in accordance with the method described in step 1-1. Compound (2-1) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2010 / 026096).

[0124] (Process 2-2: Manufacturing process for compound (2-3)) Compound (2-3) is prepared from compound (1-1) and compound (2-2) in accordance with the method described in step 1-1. Compound (2-2) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2008 / 085117).

[0125] (Step 2-3: Manufacturing process for compound (1b)) Compound (1b) is prepared by deprotecting the protecting group Pro of the amino group of compound (2-3) by a known method (for example, the method described in Protective Group in Organic Synthesis, 3rd Edition (Theodora W. Green, Peter GM Wuts, published by John Wiley & Sons Inc., 1999)). Examples of substituent Pro for the amino group include the tert-butoxycarbonyl group and the benzyloxycarbonyl group.

[0126] (Steps 2-4: Manufacturing process for compound (1c)) Compound (1c) is mixed with compound (1b) and R in a suitable inert solvent in the presence of a reducing agent. 8It is produced by reacting various aldehydes, ketones, ketone equivalents, etc. corresponding to the target substance. Various reducing agents commonly used in organic synthesis reactions can be used as reducing agents, such as sodium borohydride, sodium triacetoxyborohydride, and sodium cyanoborohydride. The solvent used in this process is appropriately selected from the solvents exemplified later, such as toluene, THF, dichloromethane, and methanol. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably 0°C to 80°C.

[0127] Furthermore, compound (1c) is mixed with compound (1b) and R in a suitable inert solvent in the presence of a base. 8 It can also be produced by reacting it with various alkyl halides and alkyl sulfonates. The base is appropriately selected from the bases exemplified later, but examples include potassium carbonate, cesium carbonate, sodium hydride, and lithium diisopropylamide. The solvent used in this process is appropriately selected from the solvents exemplified later, but examples include DMF, dimethyl sulfoxide, THF, and 1,4-dioxane. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably 0°C to 80°C.

[0128] Manufacturing method 3 The compound represented by formula (1-1) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 5 , R 6 , m, n, and Hy are synonymous with [Section 1] above; W 1 and W 2 [where represents a halogen; 'Pro' represents a protecting group for an amino group.]

[0129] (Process 3-1: Manufacturing process for compound (3-3)) Compound (3-3) is prepared by reacting compound (3-1) and compound (3-2) in a suitable inert solvent in the presence of zinc and palladium catalysts. Compound (3-1) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2008 / 147831). Compound (3-2) can be a commercially available compound or one prepared by a known method (e.g., Bioorganic & Medicinal Chemistry Letters (2006), 16(17), 4528-4532). 1 and W 2 Examples of halogens in this process include chlorine, bromine, and iodine. Examples of substituents Pro on the amino group include tert-butoxycarbonyl group and benzyloxycarbonyl group. Various palladium catalysts used in conventional methods can be used as the palladium catalyst, such as tetrakis(triphenylphosphine)palladium(O). The solvent used in this step is appropriately selected from the solvents exemplified later, such as DMF and dimethylacetamide. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, preferably 0°C to 80°C.

[0130] (Process 3-2: Manufacturing process for compound (1-1)) Compound (1-1) is produced from compound (3-3) in accordance with the method described in step 2-3.

[0131] Manufacturing method 4 Of the compounds represented by formula (1-1), the compound represented by formula (1-1a) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 5 , R6 , n, and Hy are synonymous with [Section 1] above; W 2 [where represents a halogen; 'Pro' represents a protecting group for an amino group; and 'T' represents a boronic acid or boronic acid ester.]

[0132] (Process 4-1: Manufacturing process for compound (4-2)) Compound (4-2) is prepared by reacting compound (4-1) and compound (3-2) in a suitable inert solvent in the presence of a palladium catalyst. This step can be carried out in the presence of a base and / or a phosphorus ligand as needed. Compound (4-1) can be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2019 / 163865). Examples of substituents Pro on the amino group include tert-butoxycarbonyl group and benzyloxycarbonyl group. Various palladium catalysts used in conventional methods can be used as the palladium catalyst, such as tetrakis(triphenylphosphine)palladium(0). The base used in this step is appropriately selected from the bases exemplified below, such as potassium carbonate and cesium carbonate. Various phosphorus ligands commonly used in organic synthesis reactions can be used as the phosphorus ligand, such as triphenylphosphine and bis(diphenylphosphine)methane. The solvent used in this process is appropriately selected from the solvents exemplified later, but examples include 1,4-dioxane, tetrahydrofuran, water, and mixtures thereof. The reaction temperature is usually 0°C to 200°C, preferably 20°C to 150°C, and can be carried out under microwave irradiation if necessary. The reaction time varies depending on the reaction temperature, the palladium catalyst used, the raw materials, and the solvent, but is usually 5 minutes to 72 hours, preferably 1 hour to 24 hours.

[0133] (Process 4-2: Manufacturing process for compound (4-3)) Compound (4-3) is produced by reacting compound (4-2) in a suitable inert solvent, in the presence of a catalyst, and under a hydrogen atmosphere. The solvent used in this step is appropriately selected from the solvents exemplified below, but examples include methanol, ethanol, chloroform, and mixtures thereof. Various catalysts commonly used in catalytic reduction reactions can be used as catalysts, but examples include palladium carbon and palladium hydroxide. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, preferably 0°C to 40°C.

[0134] (Step 4-3: Manufacturing process for compound (1-1a)) Compound (1-1a) is produced from compound (4-3) in accordance with the method described in step 2-3.

[0135] Manufacturing method 5 Of the compounds represented by formula (1-1), the compound represented by formula (1-1b) can be produced, for example, by the method shown below. [ka] [In the formula, R 2 , R 5 , R 6 , m, n, and Hy are synonymous with [Section 1] above; W 2 [where represents a halogen; 'Pro' represents a protecting group for an amino group.]

[0136] (Process 5-1: Manufacturing process for compound (5-2)) Compound (5-2) is produced by reacting compound (3-2) and compound (5-1) in a suitable inert solvent in the presence of alkyllithium. Compound (5-1) can be a commercially available compound or one produced by a known method (e.g., International Publication No. WO2005 / 058888). Examples of substituents Pro for the amino group include tert-butoxycarbonyl group and benzyloxycarbonyl group. Various alkyllithiums used in conventional methods can be used as alkyllithium, such as butyllithium. The solvent used in this step is appropriately selected from the solvents exemplified later, such as tetrahydrofuran and diethyl ether. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably -78°C to 40°C.

[0137] (Step 5-2: Manufacturing process for compound (1-1b)) Compound (1-1b) is produced from compound (5-2) in accordance with the method described in step 2-3.

[0138] Manufacturing method 6 Of the compounds represented by formula (1-1), the compound represented by formula (1-1c) can be produced, for example, by the method shown below. [ka] [In the formula, R 2 , R 5 , R 6 m, n, and Hy are synonymous with [Section 1] above; Q represents a halogen; and Pro represents a protecting group for an amino group.

[0139] (Process 6-1: Manufacturing process for compound (6-1)) Compound (6-1) is produced by reacting compound (5-2) in a suitable inert solvent in the presence of a halogenating agent. Examples of substituent Pro for the amino group include tert-butoxycarbonyl group and benzyloxycarbonyl group. Examples of halogens in Q include fluorine and chlorine. Various halogenating agents used in conventional methods can be used as halogenating agents, such as (diethylamino)sulfate trifluoride, bis(2-methoxyethyl)aminosulfate trifluoride, and phosphorus oxychloride. The solvent used in this step is appropriately selected from the solvents exemplified later, such as dichloromethane, chloroform, 1,4-dioxane, and toluene. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably -78°C to 40°C.

[0140] (Step 6-2: Manufacturing process for compound (1-1c)) Compound (1-1c) is produced from compound (6-1) in accordance with the method described in step 2-3.

[0141] Manufacturing method 7 Of the compounds represented by formula (1), the compound represented by formula (1d) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 m, n, r, s, and Hy are synonymous with [Section 1] above.

[0142] (Process 7-1: Manufacturing process of compound (1d)) Compound (1d) is produced by reacting compound (7-1) with compound (1-1) in a suitable inert solvent in the presence of triphosgene and a base, and then reacting it with compound (1-1). The base used in this step is appropriately selected from the bases exemplified later, but examples include pyridine and triethylamine. The solvent used in this step is appropriately selected from the solvents exemplified later, but examples include dichloromethane and chloroform. The reaction time is usually 5 minutes to 48 hours, preferably 30 minutes to 24 hours. The reaction temperature is usually -78°C to 100°C, preferably 0°C to 80°C.

[0143] Manufacturing method 8 Of the compounds represented by formula (1), the compound represented by formula (1e) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 ,m,n,r,s, and Hy are synonymous with [Section 1] above;R 8 C may be substituted with 1 to 3 halogens of the same or different types. 1-3 Represents alkyl or cyclopropyl. (Process 8-1: Manufacturing process of compound (1e)) Compound (1e) is produced from compound (8-1) and compound (1-1) in accordance with the method described in step 7-1.

[0144] Manufacturing method 9 Of the compounds represented by formula (1), the compounds represented by formula (1e) and formula (1f) can be produced, for example, by the method shown below. [ka] [In the formula, R 1 , R 2 , R 3, R 4 , R 5 , R 6 ,m,n,r,s, and Hy are synonymous with [Section 1] above;R 8 C may be substituted with 1 to 3 halogens of the same or different types. 1-3 [Represents alkyl or cyclopropyl; Pro represents the protecting group of the amino group.]

[0145] (Process 9-1: Manufacturing process for compound (9-2)) Compound (9-2) is produced from compound (9-1) and compound (1-1) according to the method described in step 7-1. Examples of substituents Pro for the amino group include tert-butoxycarbonyl group and benzyloxycarbonyl group.

[0146] (Process 9-2: Manufacturing process of compound (1f)) Compound (1f) is produced from compound (9-2) in accordance with the method described in step 2-3.

[0147] (Process 9-3: Manufacturing process of compound (1e)) Compound (1e) is produced from compound (1f) in accordance with the method described in step 2-4.

[0148] Of the raw materials or intermediates used in the manufacturing methods described above, those whose manufacturing methods are not specifically described can be commercially available compounds or synthesized from commercially available compounds by methods known to those skilled in the art, or by similar methods.

[0149] The bases used in each step of each of the above manufacturing methods should be selected appropriately depending on the reaction and the type of raw material compound, but examples include: alkali bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali carbonates such as sodium carbonate and potassium carbonate; metal hydrides such as sodium hydride and potassium hydride; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal alkoxides such as sodium methoxide and sodium t-butoxide; organometallic bases such as butyllithium and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU).

[0150] The solvents used in each step of each of the above manufacturing methods should be selected appropriately depending on the reaction and the type of raw material compound, but examples include alcohols such as methanol, ethanol, and isopropanol; ketones such as acetone and methyl ketone; halogenated hydrocarbons such as methylene chloride and chloroform; ethers such as tetrahydrofuran (THF) and dioxane; aromatic hydrocarbons such as toluene and benzene; aliphatic hydrocarbons such as hexane and heptane; esters such as ethyl acetate and propyl acetate; amides such as N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP); sulfoxides such as dimethyl sulfoxide (DMSO); and nitriles such as acetonitrile. These solvents can be used alone or in mixtures of two or more. In addition, depending on the type of reaction, organic bases such as diazabicycloundecene (DBU) may be used as solvents.

[0151] The compound of the present invention represented by formula (1) or its intermediates can be separated or purified by methods known to those skilled in the art. These methods include, for example, extraction, partitioning, reprecipitation, column chromatography (e.g., silica gel column chromatography, ion exchange column chromatography, or preparative liquid chromatography), or recrystallization. As recrystallization solvents, for example, alcoholic solvents such as methanol, ethanol, and 2-propanol; etheric solvents such as diethyl ether; esteric solvents such as ethyl acetate; aromatic hydrocarbon solvents such as benzene and toluene; ketoneic solvents such as acetone; halogenated solvents such as dichloromethane and chloroform; hydrocarbon solvents such as hexane; aprotic solvents such as dimethylformamide and acetonitrile; water; or mixtures thereof can be used. Other purification methods include those described in Volume 1 of "Experimental Chemistry Series" (edited by the Chemical Society of Japan, Maruzen). Furthermore, the molecular structure of the compound of the present invention can be easily determined by referring to the structure derived from each starting compound and using spectroscopic techniques such as nuclear magnetic resonance spectroscopy, infrared absorption spectroscopy, circular dichroism spectroscopy, and / or mass spectrometry.

[0152] Furthermore, the intermediates or final products in the above-mentioned manufacturing method can be converted to other compounds included in the present invention by appropriately changing their functional groups, and in particular by extending various side chains using amino, hydroxyl, carbonyl, halogen, etc. as starting points, and by performing the aforementioned protection and deprotection as necessary. The conversion of functional groups and the extension of side chains can be carried out by commonly used general methods (see, for example, Comprehensive Organic Transformations, RC Larock, John Wiley & Sons Inc. (1999), etc.).

[0153] The compounds of the present invention represented by formula (1) may exhibit chirality or have substituents with chiral carbons, and in such cases, optical isomers exist. The compounds of the present invention also include mixtures of these isomers and isolated compounds, which can be produced by conventional methods. Examples of production methods include using a starting material having a chiral center or introducing chirality at an intermediate stage. For example, in the case of optical isomers, optical isomers can be obtained by using an optically active starting material or by performing optical resolution at an appropriate stage in the production process. As an optical resolution method, for example, if the compound represented by formula (1) or its intermediate has a basic functional group, a diastereomer method can be used to form a salt using an inert solvent (e.g., alcoholic solvents such as methanol, ethanol, and 2-propanol; etheric solvents such as diethyl ether; esteric solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile, or a mixture of two or more solvents selected from the above) and an optically active acid (e.g., monocarboxylic acids such as mandelic acid, N-benzyloxyalanine, and lactic acid; dicarboxylic acids such as tartaric acid, o-diisopropylidenatartaric acid, and malic acid; sulfonic acids such as camphor sulfonic acid and bromocamphorsulfonic acid). If the compound of the present invention represented by formula (1) or its intermediate has an acidic functional group such as a carboxyl group, optical resolution can also be performed by forming a salt using an optically active amine (e.g., organic amines such as 1-phenylethylamine, quinine, quinidine, cinconidine, cinconine, and strychnine).

[0154] The temperature for salt formation is selected from a range of -50°C to the boiling point of the solvent, preferably from 0°C to the boiling point, and more preferably from room temperature to the boiling point of the solvent. To improve optical purity, it is desirable to raise the temperature to near the boiling point of the solvent. When filtering the precipitated salt, cooling can be performed as needed to improve the yield. The amount of optically active acid or amine used is suitable in the range of about 0.5 to about 2.0 equivalents relative to the substrate, preferably around 1 equivalent. If necessary, the crystals can be recrystallized in an inert solvent (for example, alcoholic solvents such as methanol, ethanol, 2-propanol; etheric solvents such as diethyl ether; esteric solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile, or a mixture of two or more solvents selected from the above) to obtain a high-purity optically active salt. Alternatively, if necessary, the optically resolved salt can be treated with an acid or base in a conventional manner to obtain a free form.

[0155] In Lewy body dementia, such as Parkinson's disease, abnormally aggregated α-synuclein is found in the brains of patients. Therefore, the present invention's drug, which suppresses or reduces the accumulation of α-synuclein aggregates, is expected to exert a pathological improvement effect on these diseases.

[0156] Furthermore, these aggregates are thought to exhibit neurotoxicity, inducing neuronal fragility and neuronal cell death, and contributing to the onset and progression of the disease. Therefore, the present invention's drug, which suppresses neurotoxicity and neuronal cell death associated with α-synuclein aggregates, is expected to exert a pathological improvement effect on Lewy body diseases such as Parkinson's disease.

[0157] Neurotransmitter production is one of the functions of the nervous system, and a decrease in neurotransmitters indicates neuronal vulnerability. This neuronal vulnerability is manifested, for example, in dopamine neurons by a decrease in the amount of tyrosine hydroxylase, which is involved in dopamine metabolism.

[0158] Furthermore, abnormalities in electroencephalograms (EEGs) have been reported in Lewy body dementia, such as Parkinson's disease. Electroencephalograms are a manifestation of neural synchronous activity. Therefore, the present invention's drug, which normalizes neural synchronous activity accompanied by α-synuclein aggregates, is expected to exert a pathological improvement effect on these diseases.

[0159] Neuronal spheroids used to measure the amount of α-synuclein aggregates can be created, for example, by three-dimensional culture of neural stem cells or dopamine (DA) neural progenitor cells created from synucleopathy-related gene mutation human iPS cells under neural differentiation induction. By using three-dimensionally cultured neuronal spheroids and measuring the amount of high molecular weight α-synuclein using protein analysis techniques with α-synuclein antibodies, the amount of α-synuclein aggregates can be evaluated. Furthermore, by performing imaging analysis using a fluorescent calcium probe with three-dimensionally cultured neuronal spheroids, synchronous neuronal firing can be evaluated. Furthermore, by using the steps of measuring the amount of α-synuclein aggregates and measuring synchronous neuronal firing in neuronal spheroids, it is possible to reproduce the pathophysiology of Parkinson's disease, and to evaluate drugs that have an inhibitory or reducing effect on the accumulation of α-synuclein aggregates in the pathophysiology of Parkinson's disease.

[0160] Differentiation induction of synucleopathy-related gene-mutated human iPS cells into neural stem cells can be performed, for example, by using PLA2G6 gene-mutated cells established from a healthy human iPS cell line (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University), culturing them in StemFitAK03N medium (Ajinomoto Co., Ltd., Basic03) at 37°C and 5% CO2, and inducing differentiation using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801).

[0161] For example, a culture medium with the following composition can be used for neural stem cells. <Composition of culture medium for neural stem cells> Neurobasal medium (Thermo Fisher Scientific, 2113049) Advanced DMEM / F-12 medium (Thermo Fisher Scientific, Ltd., 12634028) Neural Induction Supplement (Thermo Fisher Scientific, Ltd., A1647801)

[0162] The differentiation induction of neural stem cells into neural spheroids can be performed, for example, by seeding neural stem cells (10,000 cells / well) into a 96-well U-shaped plate (Thermo Fisher Scientific, cat#174929), culturing them in culture medium at 37°C and 5% CO2, and performing a 50% medium change on the 2nd and 4th days after differentiation induction.

[0163] For example, a culture medium with the following composition can be used for the differentiation of neural stem cell spheroids. <Composition of culture medium for neuronal spheroids> BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) NeuroCult SM1 Neuronal Supplement (manufactured by STEMCELL Technologies, cat#05711) N2 Supplement-A (manufactured by STEMCELL Technologies, cat#07152) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0164] Regarding the induction of differentiation from synucleopathy-related gene-mutated human iPS cells into dopamine neuronal progenitor cells, for example, this can be done by using a dopamine neuronal induction kit (Thermo Fisher Scientific, cat#A3147701) to induce dopamine neuronal progenitor cells from PLA2G6 gene-mutated cells or GBA1 gene-homozygous mutant cells established from iPS cell lines derived from healthy individuals.

[0165] Differentiation induction of dopamine neural progenitor cells into neuronal spheroids can be performed, for example, by culturing cryopreserved dopamine neural progenitor cells at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801), seeding dopamine neural progenitor cells (10,000 cells / well) into a 96-well U-shaped plate (Thermo Fisher Scientific, cat#174929), culturing in culture medium at 37°C and 5% CO2, and changing half of the culture medium every 3-4 days after differentiation induction.

[0166] For example, a culture medium with the following composition can be used for dopaminergic neuronal spheroids of dopaminergic neuronal progenitor cells. <Composition of culture medium for dopamine neuron spheroids> BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0167] For example, the amount of α-synuclein aggregates in neuronal spheroids can be measured by removing differentiated neuronal spheroids from the culture medium, adding a TBS solution (Nacalai, cat#12748-31) containing 1% TritionX-100 (Nacalai, cat#12967-32), extracting the protein using an ultrasonic disruptor, and quantitatively evaluating the waveform shown around 300 kD by non-reducing protein analysis using a Simple Western system with α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) (Protein Simple, cat#SM-W008).

[0168] For example, neuronal fragility in neuronal spheroids can be measured by transferring differentiated dopamine neuronal spheroids to a TBS solution (Nakkalai, cat#12748-31) supplemented with 1% TritionX-100 (Nakkalai, cat#12967-32), extracting the protein using an ultrasonic disruptor, and quantitatively evaluating the waveform shown around 60 kD by reducing-state protein analysis using a Simple Western system with tyrosine hydroxylase antibody (Millipore, cat#AB152) (Protein Simple, cat#SM-W004).

[0169] For example, neuronal cell death in neuronal spheroids can be measured by transferring differentiated dopamine neuronal spheroids to a TBS solution (Nakkalai, cat#12748-31) supplemented with 1% TritionX-100 (Nakkalai, cat#12967-32), extracting the protein using an ultrasonic disruptor, and quantitatively evaluating the waveform shown around 20 kD by reducing state protein analysis using a Simple Western system with cleaved caspase 3 antibody (Cell Singnaling Technology, cat#9664) (Protein Simple, cat#SM-W004).

[0170] Abnormal neural activity in neuronal spheroids can be measured, for example, by performing imaging analysis using a fluorescent calcium probe on neuronal spheroids cultured in three dimensions, thereby measuring synchronous neural firing. Synchronized neuronal firing in neuronal spheroids can be measured, for example, by imaging analysis using a measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191). For measurement, for example, 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and Hank's buffer containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576) (Thermo Fisher Scientific, cat#14065-056) can be used as measurement media.

[0171] The compounds of the present invention are useful as therapeutic and / or prophylactic agents for central nervous system diseases involving abnormal aggregates of brain proteins. Central nervous system diseases involving the aforementioned abnormal aggregates of brain proteins include those involving tau, α-synuclein, TDP-43, or polyglutamine. Examples of central nervous system diseases involving tau include Alzheimer's disease and frontotemporal dementia; examples of diseases involving alpha-synuclein aggregates include Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, and infantile axonal dystrophy; examples of central nervous system diseases involving TDP-43 include amyotrophic lateral sclerosis and frontotemporal dementia; and examples of central nervous system diseases involving polyglutamines include Huntington's disease and spinocerebellar ataxia. The compounds of this application are preferably useful as therapeutic and / or prophylactic agents for Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia. The compounds of this application are more useful as therapeutic and / or prophylactic agents for diseases involving α-synuclein aggregates. The compounds of the present invention are more preferably useful as therapeutic and / or prophylactic agents for Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy. In this invention, "prevention" refers to the act of administering the active ingredient of the present invention to a healthy person who has not developed a disease, for example, with the aim of preventing the onset of a disease. "Treatment" refers to the act of administering the compound of the present invention as an active ingredient to a person (patient) who has been diagnosed by a physician as having developed a disease.

[0172] The compound of the present invention and pharmaceuticals containing it can be administered orally or parenterally, either directly or in a suitable dosage form. Examples of dosage forms include, but are not limited to, tablets, capsules, powders, granules, liquids, suspensions, injections, patches, and poultices. The formulations are manufactured by known methods using pharmaceutically acceptable excipients. Depending on the purpose, additives such as excipients, disintegrants, binders, fluidizers, lubricants, coating agents, solvents, solubilizers, thickeners, dispersants, stabilizers, sweeteners, and flavorings may be used. Specifically, examples include lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, magnesium stearate, stearyl fumarate sodium, polyethylene glycol, propylene glycol, titanium dioxide, and talc.

[0173] Regarding the route of administration, it is desirable to use the one that is most effective for treatment, and this can include oral administration, intravenous administration, topical application, inhalation, and parenteral administration such as eye drops, but oral administration is preferred. Regarding the form of administration, for example, tablets and injections can be used, but tablets are preferred. The dosage and frequency of administration of these pharmaceutical compositions vary depending on the form of administration, the patient's disease and its symptoms, the patient's age and weight, etc., and cannot be specified in general terms, but usually for adults, the amount of the active ingredient per day is in the range of about 0.0001 to about 5000 mg, preferably in the range of about 0.001 to about 1000 mg, more preferably in the range of about 0.1 to about 500 mg, and particularly preferably in the range of about 1 to about 300 mg, and can be administered once or several times a day, preferably divided into 1 to 3 times a day.

[0174] The compound of the present invention and pharmaceuticals containing it may be used in combination with other drugs for the purpose of enhancing their effects and / or reducing side effects. For example, they may be used in combination with central nervous system disease treatments such as L-dopa, dopamine agonists (e.g., ropinirole hydrochloride, apomorphine hydrochloride hydrate, etc.), MAO-B inhibitors (e.g., selegiline hydrochloride, etc.), catechol-O-methyltransferase (COMT) inhibitors (e.g., entacapone, etc.), α-Syn antibodies (e.g., Prasenimab, etc.), or pharmaceutically acceptable salts thereof. Hereinafter, drugs that can be used in combination with the compound of the present invention will be abbreviated as "combination drugs."

[0175] The duration of administration of the compound of the present invention, the pharmaceutical product containing it, and the concomitant drug is not limited. They may be administered simultaneously to the target patient or with a time difference. Alternatively, the compound of the present invention and the concomitant drug may be used as a combination preparation. The dosage of the concomitant drug can be appropriately selected based on clinically used doses. Furthermore, the mixing ratio of the compound of the present invention and the concomitant drug can be appropriately selected depending on the target patient, route of administration, target disease, symptoms, and combination. For example, if the target patient is a human, 0.01 to 100 parts by weight of the concomitant drug may be used for every 1 part by weight of the compound of the present invention. Additionally, for the purpose of suppressing side effects, it can be used in combination with drugs such as antiemetics, hypnotics, and anticonvulsants (concomitant drugs). [Examples]

[0176] The present invention will be described in more detail below with reference examples, examples, and test examples, but the present invention is not limited thereto. In this specification, for example, "Example 1" may refer to "compound of Example 1," and "Reference Example 1" may refer to "compound of Reference Example 1," and so on, where the terms "example" and "reference example" may refer to a compound. Note that the compound names shown in the following reference examples and examples do not necessarily follow IUPAC nomenclature.

[0177] To simplify the description in the specification, the following abbreviations may be used in the reference examples, embodiments, and test examples. Me: Methyl Et: Ethyl Pr: n-propyl iPr: Isopropyl DMF: N,N-dimethylformamide THF: Tetrahydrofuran TFA: Trifluoroacetic acid HATU:1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0178] In NMR, the symbols used are as follows: s for single line, d for double line, dd for double double line, t for triple line, td for double triple line, q for quadruple line, m for multiline line, br for broad line, brs for broad single line, brm for broad multiline line, and J for coupling constant.

[0179] The measurement conditions for high-performance liquid chromatography-mass spectrometry (LCMS) are as follows, and the observed mass spectrometry value [MS(m / z)] is measured in MH. + The holding time is indicated by Rt (minutes). For each measured value, the measurement conditions used are indicated by A or B.

[0180] Measurement condition A Detection equipment: MS detector:Waters ACQUITY SQ Detector HPLC: Waters ACQUITY UPLC Column: ACQUITY UPLC BEH C18 1.7μm 2.1×30mm Flow rate: 0.8mL / min Oven temperature: 40℃ Measurement wavelength: 254, 220nm Mobile phase: Solution A 0.06% formic acid aqueous solution Solution B: 0.06% Formic Acid, Acetonitrile Time program: Step Time (minutes) 1 0.0-1.3 A liquid: B liquid = 98:2~4:96 2 1.3-1.5 A liquid:B liquid=4:96~98;2

[0181] Measurement condition B Detection equipment: Agilent 1200 Series, Agilent 6110 Quadrupole LCMS Column: Xbridge C18 3.5μm 4.6×50mm Flow rate: 1.8mL / min Measurement wavelength: 254, 214nm Mobile phase: Solution A 10 mM ammonium bicarbonate aqueous solution Solution B: Acetonitrile Time program: Step Time (minutes) 1 0.0-1.5 A liquid: B liquid = 90:10~5:95 Oven temperature: 50℃

[0182] Reference example 1 5-(piperidine-4-yl)-2-(trifluoromethyl)pyridine [ka] To a solution of 1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (6.7 g) in cyclopentyl methyl ether / water (4 / 1) (100 mL), 5-bromo-2-(trifluoromethyl)pyridine (4.1 g), 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (0.66 g), and cesium carbonate (12 g) were added, and the mixture was stirred at 100°C for 30 minutes. After cooling to room temperature, water was added to the reaction mixture, and it was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine and dried over magnesium sulfate. After removing the solvent from the dried organic layer under reduced pressure, it was purified by aminosilica gel chromatography (eluent: hexane / ethyl acetate). The crude product obtained was dissolved in methanol (50 mL), 10% palladium carbon (55% wet) (0.30 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered once through Celite, washed with methanol, and the solvent of the resulting filtrate was removed under reduced pressure. The residue was again dissolved in methanol (50 mL), 10% palladium carbon (55% wet) (0.30 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered through Celite, washed with methanol, and the solvent of the resulting filtrate was removed under reduced pressure. The resulting residue was dissolved in chloroform (10 mL), and trifluoroacetic acid (20 mL) was added. After stirring at room temperature for 1 minute, the solvent and trifluoroacetic acid were removed under reduced pressure. The resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Reference Example 1 (3.8 g). LC / MS ([M+H]+ / Rt(min)): 231.2 / 0.50 (Measurement conditions A)

[0183] Reference examples 2~11 The compounds shown in Table 1 were obtained using the corresponding raw material compounds in accordance with the method described in Reference Example 1. [Table 1]

[0184] Reference example 12 5-(azetidine-3-yl)-2-(trifluoromethyl)pyridine [ka] 0.33 g of zinc powder was added to a dry Schlenk tube, and the inside was purged with nitrogen. Under a nitrogen atmosphere, 1.0 mL of DMF and 51 mg of iodine were added, and the mixture was stirred at room temperature for 5 minutes. To the activated zinc solution, a 1.0 mL solution of tert-butyl 3-iodoazetidine-1-carboxylate (0.19 mL) in DMF was added, and the mixture was stirred at 40°C for 1 hour. To the prepared alkylzinc solution, a 1.0 mL solution of DMF containing 0.23 g of 5-bromo-2-(trifluoromethyl)pyridine and 58 mg of tetrakis(triphenylphosphine)palladium (0) was added, and the mixture was stirred at 80°C for 2 hours. The reaction solution was cooled to 0°C, and the reaction was stopped by slowly adding an aqueous saturated ammonium chloride solution dropwise. The precipitate was removed by suction filtration and washed with ethyl acetate. The filtrate was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. After removing the solvent from the organic layer under reduced pressure, chloroform (1.0 mL) and trifluoroacetic acid (3.0 mL) were added to the crude product and refluxed for 20 minutes. The mixture was cooled to room temperature, and the solvent and trifluoroacetic acid were removed under reduced pressure. The resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate) to obtain Reference Example 12 (75 mg). LC / MS ([M+H]+ / Rt(min)): 203.1 / 0.47 (Measurement conditions A)

[0185] Reference example 13 4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-4-ol [ka] 5-bromo-2-(trifluoromethyl)pyridine (2.0 g) was added to a dry two-necked flask, and the flask was purged with nitrogen. Under a nitrogen atmosphere, dry THF (20 mL) was added and the mixture was cooled to -78°C. 2.6 mol / L n-butyllithium hexane solution (5.1 mL) was added dropwise, and the mixture was stirred at -78°C for 1 hour. A solution of 1-Boc-4-piperidone (1.8 g) in THF (10 mL) was prepared and added dropwise to the reaction solution. After the addition was complete, the mixture was heated to room temperature. After 1 hour, the reaction solution was cooled to 0°C, and saturated ammonium chloride aqueous solution was slowly added dropwise to stop the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. After removing the solvent from the organic layer under reduced pressure, the mixture was purified by aminosilica gel chromatography (eluent: hexane / ethyl acetate 95:5-0:100). Chloroform (2.0 mL) and trifluoroacetic acid (4.0 mL) were added to the crude product obtained and the mixture was stirred at room temperature for 30 minutes. The solvent and trifluoroacetic acid were removed by distillation under reduced pressure, and the resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Reference Example 13 (0.23 g). LC / MS ([M+H]+ / Rt(min)): 247.1 / 0.46 (Measurement conditions A)

[0186] Reference example 14 5-(4-fluoropiperidine-4-yl)-2-(trifluoromethyl)pyridine [ka] a) Preparation of tert-butyl 4-hydroxy-4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-carboxylate (compound W1) 5-bromo-2-(trifluoromethyl)pyridine (2.0 g) was added to a dry two-necked flask, and the flask was purged with nitrogen. Under a nitrogen atmosphere, dry THF (20 mL) was added and the mixture was cooled to -78°C. 1.6 mol / L n-butyllithium hexane solution (6.8 mL) was added dropwise, and the mixture was stirred at -78°C for 20 minutes. A solution of 1-Boc-4-piperidone (1.76 g) in THF (20 mL) was prepared and added dropwise to the reaction solution. After addition was complete, the mixture was heated to room temperature. After 1 hour, the reaction solution was cooled to 0°C, and saturated ammonium chloride aqueous solution was slowly added dropwise to stop the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. After removing the solvent from the organic layer under reduced pressure, compound W1 (1.18 g) was obtained by purification using aminosilica gel chromatography (eluent: hexane / ethyl acetate). LC / MS ([M+H]+ / Rt(min)): 347.2 / 0.93 (Measurement conditions A)

[0187] b) Preparation of 5-(4-fluoropiperidine-4-yl)-2-(trifluoromethyl)pyridine (Reference Example 14) Compound W1 (0.12 g) was dissolved in chloroform (1.5 mL), bis(2-methoxyethyl)aminosulfate trifluoride (0.24 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated and briefly purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate). The obtained compound was dissolved in chloroform (0.50 mL), reacted with TFA (1.0 mL), and stirred at room temperature for 5 minutes. The reaction solution was concentrated, and excess TFA was removed by azeotropic distillation with toluene. The resulting crude product was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Reference Example 14 (18 mg). LC / MS ([M+H]+ / Rt(min)): 249.1 / 0.60 (Measurement conditions A)

[0188] Reference examples 15~21 The compounds shown in Table 2 were obtained using the corresponding raw material compounds in accordance with the method described in Reference Example 1. [Table 2]

[0189] Example 1 (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] To a chloroform (1.0 mL) solution of Reference Example 1 (30 mg), 3-methyloxetane-3-carboxylic acid (18 mg), triethylamine (27 μL), and HATU (60 mg) were added and the mixture was stirred at room temperature for 30 minutes. Then, cesium carbonate (42 mg) was added and the mixture was stirred again at room temperature for 30 minutes. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Example 1 (41 mg). LC / MS ([M+H]+ / Rt(min)): 329.2 / 0.75 (Measurement conditions A) 1 H-NMR (400 MHz, DMSO-d6)δ: 8.72 (1H, d, J = 1.6 Hz), 8.02 (1H, dd, J = 2.0, 8.4 Hz), 7.84 (1H, d, J = 8.8 Hz), 4.83 (2H, dd, J = 6.4, 8.8 Hz), 4.55-4.52 (1H, m), 4.28 (2H, t, 6.8 Hz), 3.18-3.05 (2H, m), 3.01-2.93 (1H, m), 2.67 (1H, t, J = 12 Hz), 1.85-1.81 (2H, brs), 1.72-1.57 (5H, m).

[0190] The compound of Example 1 can also be synthesized by the following method.

[0191] To a solution of the hydrochloride salt (100 mg) of Reference Example 1 in ethyl acetate (1.0 mL), 3-methyloxetane-3-carboxylic acid (52 mg), triethylamine (0.17 mL), and a 50% 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide ethyl acetate solution (0.40 mL) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by silica gel chromatography (elution solvent: ethyl acetate / methanol) to obtain Example 1 (93 mg).

[0192] Examples 2-32 Using the corresponding starting compounds, the compounds shown in Table 3 were obtained by a method similar to the synthesis method of Example 1. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0193] Example 33 (3-methylazetidine-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] To a chloroform (1.0 mL) solution of Reference Example 1 (50 mg), 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid (56 mg), triethylamine (45 μL), HATU (99 mg), and cesium carbonate (71 mg) were added. After stirring at room temperature for 1 hour, the reaction solution was briefly purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain the crude product. The obtained crude product was dissolved in chloroform (1.0 mL), TFA (1.0 mL) was added, and the mixture was stirred at room temperature for 5 minutes. The reaction solution was concentrated and purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Example 33 (65 mg). LC / MS ([M+H]+ / Rt(min)): 328.2 / 0.60 (Measurement conditions A) 1 H-NMR (400 MHz, DMSO-d6)δ: 8.71 (1H, d, J = 2.0 Hz), 8.00 (1H, dd, J = 8.4, 2.0 Hz), 7.83 (1H, d, J = 8.4 Hz), 4.53 (1H, d, J = 12.8 Hz), 3.87 (2H, t, J = 8.4 Hz), 3.35-3.30 (1H, m), 3.16-3.07 (3H, m), 2.97 (1H, tt, J = 12, 3.6 Hz), 2.63 (1H, t, J = 12 Hz), 1.84 (2H, d, J = 12.8 Hz), 1.65-1.47 (5H, m).

[0194] The compound of Example 33 can also be synthesized by the following method.

[0195] To a solution of the hydrochloride salt (100 mg) of Reference Example 1 in ethyl acetate (1.0 mL), 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid (97 mg), triethylamine (0.17 mL), and a 50% 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide ethyl acetate solution (0.40 mL) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was briefly purified by silica gel chromatography (elution solvent: ethyl acetate / methanol) to obtain the crude product. The obtained crude product was dissolved in chloroform (1.0 mL), TFA (1.0 mL) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by aminosilica gel chromatography (elution solvent: ethyl acetate / methanol) to obtain Example 33 (90 mg).

[0196] Examples 34-40 Using the corresponding raw material compounds, the compounds shown in Table 4 were obtained by a method similar to the synthesis method of Example 33. However, if the example involved a salt, the chlorination step was included. [Table 4-1] [Table 4-2]

[0197] Example 41 (1,3-dimethylazetidine-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone hydrochloride [ka] To a solution of Example 33 (20 mg) in THF (0.50 mL), 19 μL of 36% formaldehyde aqueous solution and 5.2 μL of acetic acid were added. Sodium triacetoxyborohydride (39 mg) was added while stirring the solution at room temperature and stirred for 30 minutes. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol). The resulting oily compound was dissolved in ethyl acetate (1 mL) and treated with a 4 mol / L hydrochloric acid solution of ethyl acetate at room temperature to obtain Example 41 (15 mg). LC / MS ([M+H]+ / Rt(min)): 342.3 / 0.63 (Measurement conditions A) 1 H-NMR (400 MHz, DMSO-d6)δ: 10.6 (1H, brs), 8.72 (1H, d, J = 1.6 Hz), 7.99 (1H, dd, J = 8.0, 1.6 Hz), 7.86 (1H, d, J = 8.8 Hz), 4.52 (1H, d, J = 12.8 Hz), 4.13-4.12 (2H, m), 3.92-3.90 (2H, m), 3.32-3.29 (1H, m), 3.15 (1H, t, J = 12.8 Hz), 3.00 (1H, tt, J = 12, 3.2 Hz), 2.72 (3H, s), 1.91-1.83 (2H, m), 1.68-1.50 (5H, m).

[0198] Examples 42-48 Using the corresponding raw material compounds, the compounds shown in Table 5 were obtained by a method similar to the synthesis method of Example 41. However, if the example is not a salt, the chlorination step is unnecessary. [Table 5-1] [Table 5-2]

[0199] Example 49 (Morpholin-4-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] To a chloroform (1.0 mL) solution of Reference Example 1 (30 mg), morpholine-4-carbonyl chloride (18 μL) and N-ethyl-N-isopropylpropan-2-amine (34 μL) were added and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Example 49 (44 mg). LC / MS ([M+H]+ / Rt(min)): 344.2 / 0.79 (Measurement conditions A) 1 H-NMR (400 MHz, DMSO-d6)δ: 8.70 (1H, d, J = 2.0 Hz), 7.98 (1H, dd, J = 8.0, 2.0 Hz), 7.83 (1H, d, J = 8.0 Hz), 3.74 (2H, d, J = 13.6 Hz), 3.58 (4H, m), 3.15 (4H, m), 2.93-2.83 (3H, m), 1.82-1.79 (2H, m), 1.70-1.59 (2H, m).

[0200] Examples 50-59 Using the corresponding starting compounds, the compounds shown in Table 6 were obtained by a method similar to the synthesis method of Example 1. [Table 6-1] [Table 6-2]

[0201] Example 60 (1,4-Oxazepan-4-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] Pyridine (95 mg) was added to a solution of 1,4-oxazepane (61 mg) in dichloromethane (2.0 mL) and cooled to 0°C. Triphosgene (72 mg) was added and the mixture was stirred at 0°C for 30 minutes. Diiropropylethylamine (0.23 g) and Reference Example 1 (69 mg) were added and the mixture was stirred at room temperature for 1 hour. The solution was concentrated and purified by preparative HPLC to obtain Example 60 (31 mg). LC / MS ([M+H]+ / Rt(min)): 357.9 / 1.54 (Measurement conditions B) 1 H-NMR (500 MHz, CDCl3)δ: 8.63 (1H, s), 7.74-7.72 (1H, m), 7.66 (1H, d, J = 8.0 Hz), 3.83-3.79 (6H, m), 3.55-3.52 (4H, m), 2.95-2.90 (2H, m), 2.86-2.81 (1H, m), 2.02-2.00 (2H, m), 1.91 (2H, d, J = 11.0 Hz), 1.80-1.72 (2H, m).

[0202] Examples 61-63 Using the corresponding starting compounds, the compounds shown in Table 7 were obtained by a method similar to the synthesis method of Example 60. [Table 7]

[0203] Examples 64-65 Using the corresponding raw material compounds, the compounds shown in Table 8 were obtained by a method similar to the synthesis method of Example 41. However, if the example was not a salt, the chlorination step was omitted. [Table 8]

[0204] Example 66 [3-methyl-1-(2,2,2-trifluoroethyl)azetidine-3-yl]{4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] To a solution of Example 34 (46 mg) in THF (3.0 mL), 2,2,2-trifluoroethyltrifluoromethanesulfonate (42 mg) and triethylamine (71 mg) were added and stirred overnight at room temperature. The reaction mixture was concentrated and purified by preparative HPLC to obtain Example 66 (19 mg). LC / MS ([M+H]+ / Rt(min)): 410.1 / 1.78 (Measurement condition B) 1 H-NMR (400 MHz, CDCl3)δ: 8.59 (1H, d, J = 4.8 Hz), 7.79 (1H, d, J = 8.0 Hz), 7.51 (1H, dd, J = 7.6, 4.8 Hz), 4.79 (1H, d, J = 13.2 Hz), 3.54-3.50 (4H, m), 3.41 (1H, d, J = 12.4 Hz), 3.26-3.17 (2H, m), 3.01 (2H, q, J = 9.2 Hz), 2.69 (1H, t, J = 12.8 Hz), 1.90 (2H, d, J = 12.8 Hz), 1.81-1.77 (1H, m), 1.69 (3H, s), 1.67-1.57 (1H, m).

[0205] Example 67 (1-Cyclopropyl-3-methylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone [ka] To a solution of Example 34 (46 mg) in ethanol (4.0 mL), (1-ethoxycyclopropoxy)trimethylsilane (73 mg), acetic acid (13 mg), and sodium borohydride (44 mg) were added and the mixture was stirred overnight at 60°C. The reaction solution was cooled to room temperature, concentrated, and then purified by preparative HPLC to obtain Example 67 (22 mg). LC / MS ([M+H]+ / Rt(min)): 368.2 / 1.68 (Measurement condition B) 1 H-NMR (400 MHz, CDCl3)δ: 8.58 (1H, dd, J = 4.4, 0.8 Hz), 7.79 (1H, d, J = 8.0 Hz), 7.50 (1H, dd, J = 8.0, 4.8 Hz), 4.81 (1H, d, J = 12.8 Hz), 3.53-3.50 (3H, m), 3.38-3.31 (2H, m), 3.25-3.12 (2H, m), 2.76-2.65 (1H, m), 1.89-1.84 (3H, m), 1.71-1.61 (2H, m), 1.59 (3H, s), 0.38-0.35 (4H, m).

[0206] Test Example 1: Reproduction study of Parkinson's disease pathology (α-synuclein aggregate accumulation) using neuronal spheroids in three-dimensional culture of human iPS cells with PLA2G6 gene mutation. PLA2G6 gene mutant cells established from a healthy human-derived iPS cell line (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University) were cultured in StemFitAK03N medium (Ajinomoto Co., Inc., Basic03) at 37°C and 5% CO2.

[0207] We induced neural stem cells from iPS cells using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801) and created a cell stock.

[0208] Cryopreserved neural stem cells were cultured in culture medium at 37°C and 5% CO2. The culture medium used for the neural stem cells had the following composition.

[0209] Culture medium composition of neural stem cells Neurobasal medium (Thermo Fisher Scientific, 2113049) Advanced DMEM / F-12 medium (Thermo Fisher Scientific, Ltd., 12634028) Neural Induction Supplement (Thermo Fisher Scientific, Ltd., A1647801)

[0210] Neural stem cells (10,000 cells / well) were seeded in 96-well U-shaped plates (Thermo Fisher Scientific, CAT#174929) and cultured in culture medium at 37°C and 5% CO2. The culture medium was changed by half every 3-4 days. The culture medium for neural spheroids was composed of the following:

[0211] BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) NeuroCult SM1 Neuronal Supplement (manufactured by STEMCELL Technologies, cat#05711) N2 Supplement-A (manufactured by STEMCELL Technologies, cat#07152) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0212] Differentiated neuronal spheroids were removed from the culture medium, and TBS solution (Nakkalai, cat#12748-31) supplemented with 1% TritionX-100 (Nakkalai, cat#12967-32) was added. Proteins were then extracted using an ultrasonic disruptor.

[0213] The extracted proteins were analyzed in a non-reducing state using a Simple Western system with an α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of α-synuclein aggregates was measured, and the waveform shown around a molecular weight of approximately 300 kD was quantitatively evaluated.

[0214] α-synuclein aggregates increased rapidly from day 7 to day 9 of culture. On day 9 of culture, neuronal spheroids created from PLA2G6 mutant iPS cells showed more than five times the amount of α-synuclein aggregates compared to neuronal spheroids derived from healthy human iPS cells. After day 9 of culture, a gradual increase was observed. The results are shown in Figure 1.

[0215] Test Example 2: Evaluation of α-synuclein aggregate accumulation suppression using neuronal spheroids created from human iPS cells with PLA2G6 gene mutation. (1) Differentiation induction from human iPS cells into nerve cells Neural stem cells were induced from PLA2G6 gene mutant cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neuronal spheroids were created from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was halved on days 2 and 4 after differentiation induction. The test compound was diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution was added to each well during the halving of the culture medium 4 days after differentiation induction.

[0216] (2) Evaluation of α-synuclein aggregate content Proteins were extracted from neuronal spheroids 9 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system with an α-synuclein antibody (ThermoFisher, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of aggregates in nerve spheroids treated with DMSO solution was set to 100%, and the amount of aggregates in nerve spheroids treated with each test compound was measured. Table 9 shows the amount of aggregates when a representative compound is added. [Table 9-1] [Table 9-2]

[0217] Test Example 3: Evaluation of reduced α-synuclein aggregate accumulation using neuronal spheroids created from human iPS cells with PLA2G6 gene mutation. (1) Differentiation induction from human iPS cells into nerve cells Neural stem cells were induced from PLA2G6 gene mutant cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neuronal spheroids were created from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was changed by half every 3-4 days. The test compound was diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution was added to each well during the halving of the culture medium 10 days after differentiation induction.

[0218] (2) Evaluation of α-synuclein aggregate content Proteins were extracted from neuronal spheroids 15 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100. The amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system with an α-synuclein antibody (ThermoFisher, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of aggregates in nerve spheroids to which each test compound was added was measured. Table 10 shows the data (%) for the amount of aggregates when a representative compound was added, with the amount of aggregates in nerve spheroids to which DMSO solution was added set to 100%. [Table 10]

[0219] Test Example 4: Reproduction study of Parkinson's disease pathology (α-synuclein aggregate accumulation) using dopamine neuron spheroids created from human iPS cells with PLA2G6 gene mutation. Dopamine neuron progenitor cells were induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (Thermo Fisher Scientific, cat#A3147701), and a cell stock was created.

[0220] Cryopreserved dopamine neural progenitor cells were cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0221] First, dopamine neuronal progenitor cells (10,000 cells / well) were seeded into 96-well U-shaped plates (Thermo Fisher Scientific, cat#174929) and cultured in culture medium at 37°C and 5% CO2. The culture medium was changed by half every 3-4 days after differentiation induction. The culture medium used for dopamine neuronal spheroids had the following composition.

[0222] BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0223] Differentiated dopamine neuron spheroids were removed from the culture medium, and TBS solution (Nakkalai, cat#12748-31) supplemented with 1% TritionX-100 (Nakkalai, cat#12967-32) was added. Proteins were then extracted using an ultrasonic disruptor.

[0224] The extracted proteins were analyzed in a non-reducing state using a Simple Western system with an α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of α-synuclein aggregates was measured, and the waveform shown around a molecular weight of approximately 300 kD was quantitatively evaluated.

[0225] α-synuclein aggregates increased rapidly from day 10 to day 21 of culture. On day 21 of culture, dopamine neuron spheroids created from PLA2G6 mutant iPS cells showed more than five times the amount of α-synuclein aggregates compared to dopamine neuron spheroids derived from healthy human iPS cells. After day 21 of culture, a gradual increase was observed. The results are shown in Figure 2.

[0226] Test Example 5: Evaluation of α-synuclein aggregate accumulation suppression using dopamine neuron spheroids created from human iPS cells with PLA2G6 gene mutation. (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells were induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids were prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was changed by half every 3-4 days. The test compound was diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution was added to each well during the halving of the culture medium 21 days after differentiation induction.

[0227] (2) Evaluation of α-synuclein aggregate content Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100. The amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system with an α-synuclein antibody (ThermoFisher, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of aggregates in nerve spheroids to which each test compound was added was measured. Table 11 shows the data (%) for the amount of aggregates when a representative compound was added, with the amount of aggregates in nerve spheroids to which DMSO solution was added set to 100%. [Table 11]

[0228] Test Example 6: Method for reproducing neuronal vulnerability using dopamine neuron spheroids created from human iPS cells with PLA2G6 gene mutation (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells were induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids were prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was changed by half every 3-4 days.

[0229] (2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100. The amount of tyrosine hydroxylase was measured by protein analysis using a Simple Western system with a tyrosine hydroxylase antibody (Millipore, cat#AB152) (Protein Simple, cat#SM-W008). The results are shown in Figure 3.

[0230] Test Example 7: Evaluation of improvement in neuronal vulnerability using dopamine neuron spheroids created from human iPS cells with PLA2G6 gene mutation. (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells are induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids are prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium is changed by half every 3-4 days. The test compound is diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution is added to each well during the halving of the culture medium 21 days after differentiation induction.

[0231] (2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100, and the amount of tyrosine hydroxylase was measured by protein analysis using a Simple Western system with a tyrosine hydroxylase antibody (Millipore, cat#AB152) (Protein Simple, cat#SM-W008).

[0232] Test Example 8: Method for reproducing neuronal cell death using neuronal spheroids created from human iPS cells with PLA2G6 gene mutation (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells were induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids were prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was changed by half every 3-4 days.

[0233] (2) Evaluation of neuronal cell death From day 35 of differentiation induction, 10 μM dopamine was added to the culture medium. After 40 days of differentiation induction, proteins were extracted from dopamine neuronal spheroids using a TBS solution containing 1% TritionX-100. Neuronal cell death was measured by protein analysis using a Simple Western system with cleaved caspase 3 antibody (Cell Singnaling Technology, cat#9664) (Protein Simple, cat#SM-W008). The results are shown in Figure 4.

[0234] Test Example 9: Evaluation of neuronal cell death suppression using neuronal spheroids created from human iPS cells with PLA2G6 gene mutations. (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells are induced from PLA2G6 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids are prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium is changed by half every 3-4 days. The test compound is diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution is added to each well along with 10 μM dopamine during the halving of the culture medium 35 days after differentiation induction.

[0235] (2) Evaluation of neuronal cell death Proteins were extracted from dopamine neuronal spheroids 40 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100, and the amount of neuronal cell death was measured by protein analysis using a Simple Western system with cleaved caspase 3 antibody (Cell Singnaling Technology, cat#9664) (Protein Simple, cat#SM-W008).

[0236] Test Example 10: Method for reproducing Parkinson's disease pathology (α-synuclein aggregate accumulation) using neuronal spheroids in three-dimensional culture of human iPS cells with GBA1 gene mutation. GBA1 gene homozygous mutant cells established from iPS cell lines derived from healthy individuals were cultured in StemFitAK03N medium (Ajinomoto Co., Ltd., Basic03) at 37°C and 5% CO2. Dopamine neuron progenitor cells were induced from GBA1 gene homozygous mutant iPS cells using a dopamine neuron induction kit (Thermo Fisher Scientific, cat#A3147701), and a cell stock was created.

[0237] Cryopreserved dopamine neural progenitor cells were cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0238] Dopamine neuronal progenitor cells (10,000 cells / well) were seeded in 96-well U-shaped plates (Thermo Fisher Scientific, cat#174929) and cultured in culture medium at 37°C and 5% CO2. Half of the culture medium was changed every 3-4 days after differentiation induction. The culture medium for dopamine neuronal spheroids had the following composition:

[0239] BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0240] Differentiated dopamine neuron spheroids were removed from the culture medium, and TBS solution (Nakkalai, cat#12748-31) supplemented with 1% TritionX-100 (Nakkalai, cat#12967-32) was added. Proteins were then extracted using an ultrasonic disruptor.

[0241] The extracted proteins were analyzed in a non-reducing state using a Simple Western system with an α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of α-synuclein aggregates was measured, and the waveform shown around a molecular weight of approximately 300 kD was quantitatively evaluated.

[0242] The number of α-synuclein aggregates increased rapidly from day 21 to day 40 of culture, reaching saturation on day 40, and no further changes in the amount were observed thereafter. The results are shown in Figure 5.

[0243] Test Example 11: Evaluation of reduced α-synuclein aggregate accumulation using dopamine neuron spheroids created from GBA1 gene mutant human iPS cells. (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells were induced from GBA1 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids were prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium was changed by half every 3-4 days. The test compound was diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution was added to each well during the halving of the culture medium 40 days after differentiation induction.

[0244] (2) Evaluation of α-synuclein aggregate content Proteins were extracted from dopamine neuron spheroids 44 days after differentiation induction using a TBS solution supplemented with 1% TritionX-100. The amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system with an α-synuclein antibody (ThermoFisher, cat#AHB0261) (Protein Simple, cat#SM-W008). The amount of aggregates in nerve spheroids to which each test compound was added was measured. Table 12 shows the data (%) for the amount of aggregates when a representative compound was added, with the amount of aggregates in nerve spheroids to which DMSO solution was added set to 100%. [Table 12]

[0245] Test Example 12: Confirmation of synchronous firing abnormalities in neuronal spheroids in three-dimensional culture using human iPS cells with GBA1 gene mutations. GBA1 gene homozygous mutant cells established from iPS cell lines derived from healthy individuals were cultured in StemFitAK03N medium (Ajinomoto Co., Ltd., Basic03) at 37°C and 5% CO2.

[0246] Dopamine neuron progenitor cells were induced from GBA1 gene homozygous mutant iPS cells using a dopamine neuron induction kit (Thermo Fisher Scientific, cat#A3147701) to create a cell stock.

[0247] Cryopreserved dopamine neuronal progenitor cells are cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0248] Dopamine neuronal progenitor cells (10,000 cells / well) are seeded into 96-well U-shaped plates (Thermo Fisher Scientific, cat#174929) and cultured in culture medium at 37°C and 5% CO2. The culture medium is changed by half every 3-4 days after differentiation induction. The culture medium for dopamine neuronal spheroids has the following composition.

[0249] BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (manufactured by Nakalai, cat#03420-52)

[0250] After 40 days of differentiation induction, remove half of the culture medium, add an equal volume of the remaining medium with a measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191), and allow to stand for 30 minutes before measurement. The measurement medium used is 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and Hank's buffer containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576) (Thermo Fisher Scientific, cat#14065-056). One frame is acquired per second.

[0251] Test Example 13: Evaluation test for improvement of synchronous firing abnormalities using dopamine neuron spheroids created from human iPS cells with GBA1 gene mutations. (1) Differentiation induction from human iPS cells into nerve cells Dopamine neuron progenitor cells are induced from GBA1 gene mutant cells using a dopamine neuron induction kit (ThermoFisher, cat#A3147701). Dopamine neuron spheroids are prepared from the induced dopamine neuron progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. The culture medium is changed by half every 3-4 days. The test compound was diluted in the culture medium to twice its final concentration, and an equal volume of the twice-concentration solution was added to each well during the halving of the culture medium 40 days after differentiation induction.

[0252] (2) Evaluation of synchronous ignition After 44 days of differentiation induction, half of the culture medium of dopamine neuronal spheroids is replaced, and an equal volume of measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) is added to the remaining medium. After standing for 30 minutes, measurement is performed. The measurement medium used is 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and Hank's buffer containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576) (Thermo Fisher Scientific, cat#14065-056). One frame is acquired per second. [Industrial applicability]

[0253] The compounds of the present invention exhibit an inhibitory or reducing effect on the accumulation of α-synuclein aggregates, and are therefore useful as therapeutic and / or prophylactic agents for central nervous system diseases characterized by the inhibition or reduction of abnormal aggregates of brain proteins. Furthermore, the present invention is useful as a method for reproducing the pathophysiology of Parkinson's disease using neuronal spheroids, and as a method for evaluating the amount of α-synuclein aggregates using the same.

[0254] As explained above, the compound represented by formula (1) or its pharmaceutically acceptable salts exhibits inhibitory or reducing effects on α-synuclein aggregates. Therefore, the compound represented by formula (1) or its pharmaceutically acceptable salts are useful as therapeutic and / or prophylactic agents for central nervous system diseases such as Parkinson's disease and Lewy body dementia, in which α-synuclein aggregates are involved.

Claims

1. Formula (1): 【Chemistry 1】 [In the formula, X is oxygen or NR ７ This represents, R ７ C may be substituted with hydrogen, or one to three halogens of the same or different type. １－３ Represents alkyl or cyclopropyl, Y represents CH or nitrogen. m represents 0, 1, or 2. n represents 0 or 1, r represents 0, 1, 2, 3, or 4. s represents 0, 1, or 2. (However, when s = 0, Y is CH and r is 1, 2, 3, or 4, When s = 1, Y is CH and r is 0, 1, 2, or 3. When s = 2, r is either 1 or 2. R １ represents hydrogen, halogen, methyl, or hydroxyl, R ２ represents hydrogen, halogen, methyl, or hydroxyl, R ３ is hydrogen, or C １－３ Represents alkyl, R ４ represents hydrogen or C １－３ alkyl, and Here, R ３ and R ４ They may come together to form cross-linked methylene or ethylene. R ５ C may be replaced by a halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, R ６ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. Here, (I)R ５ and R ６ When Hy, which contains , is 5-fluoropyridine-2-yl, and m and n are 1, then r is 0 and s is 1, (II) R ５ and R ６ When Hy is 6-methoxypyridine-3-yl, and m and n are 1, and X is oxygen, then r is 0 and s is 1. (III) R ５ and R ６ Hy is 5-methoxypyridine-2-yl, m and n are 1, and X is NR ７ When this is the case, r is 0 and s is 1, (IV)R ５ is methyl and R ６ When is hydrogen, r is 0 and s is 1, however, (I')R ５ and R ６ Hy, which contains , is not 4,6-dimethylpyrimidine-2-yl or 5-bromopyrimidine-2-yl, (II')R ５ and R ６ When Hy, which contains , is 5-chloropyridine-2-yl, then m and n are 1, and R １ and R ２ Neither is hydrogen, However, the following compounds (III') (1-isopropylpiperidine-4-yl){3-(2-methoxypyridine-3-yl)pyrrolidine-1-yl}methanone, and (IV') (3-ethoxyoxetane-3-yl) [3-{4-(trifluoromethyl)pyrimidine-2-yl}pyrrolidine-1-yl]methanone, [excluding] A pharmaceutical composition containing a compound represented by or a pharmaceutically acceptable salt thereof.

2. m is 1, n is 1, The pharmaceutical composition according to claim 1.

3. R １ and R ２ However, each is independently hydrogen, methyl, or fluorine. The pharmaceutical composition according to claim 1.

4. R １ and R ２ However, it is hydrogen. The pharmaceutical composition according to claim 1.

5. X is oxygen, NH, or NMee. The pharmaceutical composition according to claim 1.

6. R ３ However, it is hydrogen. The pharmaceutical composition according to claim 1.

7. R ４ However, it is methyl or ethyl. The pharmaceutical composition according to claim 1.

8. Y is CH. The pharmaceutical composition according to claim 1.

9. s is 1, The pharmaceutical composition according to claim 1.

10. r is 0 and s is 1, The pharmaceutical composition according to claim 1.

11. Formula (2): 【Chemistry 2】 [In the formula, X represents oxygen, NH, or NMee. R ４ represents methyl or ethyl, R ５ C may be replaced by a halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, R ６ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. however, R ５ and R ６ Hy, which contains [ ], is not 5-chloropyridine-2-yl. The pharmaceutical composition according to claim 1, as represented by [the formula].

12. Hy is a pyridine ring. The pharmaceutical composition according to claim 1.

13. R ５ However, it is trifluoromethyl. The pharmaceutical composition according to claim 1.

14. Hy is pyridine-3-yl. The pharmaceutical composition according to claim 1.

15. X is oxygen. The pharmaceutical composition according to claim 1.

16. R ４ However, it is methyl. The pharmaceutical composition according to claim 1.

17. X is NH or NMee. The pharmaceutical composition according to claim 1.

18. X is NMee. The pharmaceutical composition according to claim 1.

19. The pharmaceutical composition according to claim 1, wherein the compound is selected from the following group of compounds: (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (3-methyloxetan-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (3-methyloxetan-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetan-3-yl)methanone, (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (1,3-dimethylazetidine-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (1,3-dimethylazetidine-3-yl){4-[4-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, and (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone.

20. The pharmaceutical composition according to claim 1, wherein the compound is selected from the following group of compounds: (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, {4-[5-fluoro-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}(3-methyloxetan-3-yl)methanone, (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, (1,3-dimethylazetidine-3-yl){4-[2-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone, and (1,3-dimethylazetidine-3-yl){4-[5-(trifluoromethyl)pyridine-3-yl]piperidine-1-yl}methanone.

21. A therapeutic or prophylactic agent for central nervous system diseases involving abnormal aggregates of brain proteins, comprising the pharmaceutical composition described in any one of claims 1 to 20.

22. The therapeutic or prophylactic agent according to claim 21, wherein the central nervous system disorder involving abnormal aggregates of brain proteins is a central nervous system disorder involving tau, α-synuclein, TDP-43, or polyglutamine.

23. The therapeutic or prophylactic agent according to claim 21, wherein the central nervous system disease involving abnormal aggregates of brain proteins is Alzheimer's disease, frontotemporal degeneration, Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

24. The therapeutic or prophylactic agent according to claim 21, wherein the central nervous system disease involving abnormal aggregates of brain proteins is a central nervous system disease involving α-synuclein.

25. The therapeutic or prophylactic agent according to claim 21, wherein the central nervous system disorder involving abnormal protein aggregates in the brain is Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

26. A therapeutic or prophylactic agent for central nervous system diseases involving abnormal aggregates of brain proteins, comprising a pharmaceutical composition according to any one of claims 1 to 20, and at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

27. A therapeutic or prophylactic agent according to claim 21, for treating or preventing a central nervous system disorder involving abnormal aggregates of brain proteins, in combination with at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

28. Formula (1): 【Transformation 3】 [In the formula, X is oxygen or NR ７ This represents, R ７ C may be substituted with hydrogen, or one to three halogens of the same or different type. １－３ Represents alkyl or cyclopropyl, Y represents CH or nitrogen. m represents 0, 1, or 2. n represents 0 or 1, r represents 0, 1, 2, 3, or 4. s represents 0, 1, or 2. (However, when s = 0, Y is CH and r is 1, 2, 3, or 4, When s = 1, Y is CH and r is 0, 1, 2, or 3. When s = 2, r is either 1 or 2. R １ represents hydrogen, halogen, methyl, or hydroxyl, R ２ represents hydrogen, halogen, methyl, or hydroxyl, R ３ is hydrogen, or C １－３ Represents alkyl, R ４ is hydrogen, or C １－３ Represents alkyl, Here, R ３ and R ４ They may come together to form cross-linked methylene or ethylene. R ５ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, R ６ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a compound represented by or a pharmaceutically acceptable salt thereof as an active ingredient.

29. m is 1, n is 1, The therapeutic or preventive agent according to claim 28.

30. R １ and R ２ However, each is independently hydrogen, methyl, or fluorine. The therapeutic or preventive agent according to claim 28.

31. R １ and R ２ However, it is hydrogen. The therapeutic or preventive agent according to claim 28.

32. X is oxygen, NH, or NMee. The therapeutic or preventive agent according to claim 28.

33. R ３ However, it is hydrogen. The therapeutic or preventive agent according to claim 28.

34. R ４ However, it is methyl or ethyl. The therapeutic or preventive agent according to claim 28.

35. Y is CH. The therapeutic or preventive agent according to claim 28.

36. s is 1, The therapeutic or preventive agent according to claim 28.

37. r is 0 and s is 1, The therapeutic or preventive agent according to claim 28.

38. Formula (2): 【Chemistry 4】 [In the formula, X represents oxygen, NH, or NMee. R ４ represents methyl or ethyl, R ５ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, R ６ C may be substituted with hydrogen, halogen, or one to three halogens of the same or different type. １－３ C may be substituted with alkyl or one to three halogens of the same or different type. １－３ Represents alkoxy, Hy represents a pyridine ring, pyridazine ring, pyrimidine ring, or pyrazine ring. A therapeutic or preventive agent according to claim 28, as represented by the following:

39. Hy is a pyridine ring. The therapeutic or preventive agent according to claim 28.

40. R ５ However, it is trifluoromethyl. The therapeutic or preventive agent according to claim 28.

41. Hy is pyridine-3-yl. The therapeutic or preventive agent according to claim 28.

42. X is oxygen. The therapeutic or preventive agent according to claim 28.

43. R ４ However, it is methyl. The therapeutic or preventive agent according to claim 28.

44. X is NH or NMee. The therapeutic or preventive agent according to claim 28.

45. X is NMee. The therapeutic or preventive agent according to claim 28.

46. A therapeutic or prophylactic agent according to any one of claims 28 to 45, wherein the central nervous system disorder involving abnormal aggregates of brain proteins is a central nervous system disorder involving tau, α-synuclein, TDP-43, or polyglutamine.

47. The therapeutic or prophylactic agent according to any one of claims 28 to 45, wherein the central nervous system disease involving abnormal aggregates of brain proteins is Alzheimer's disease, frontotemporal degeneration, Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

48. A therapeutic or prophylactic agent according to any one of claims 28 to 45, wherein the central nervous system disease involving abnormal aggregates of brain proteins is a central nervous system disease involving α-synuclein.

49. A therapeutic or prophylactic agent according to any one of claims 28 to 45, wherein the central nervous system disorder involving abnormal protein aggregates in the brain is Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

50. A therapeutic or prophylactic agent for central nervous system disorders involving abnormal aggregates of brain proteins, comprising a therapeutic or prophylactic agent according to any one of claims 28 to 45, and at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

51. A therapeutic or prophylactic agent according to any one of claims 28 to 45, for treating or preventing a central nervous system disorder involving abnormal aggregates of brain proteins, in combination with at least one agent selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, α-Syn antibodies, and pharmaceutically acceptable salts thereof.

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