2 (1H)-pyridinimine derivatives

Abstract

The present invention relates to a drug for treating or preventing central nervous system diseases caused by abnormal aggregation of proteins in the brain, which comprises, as an active ingredient, a compound represented by formula (1) (wherein R1 and R2 are hydrogen or the like, R3 and R4 are hydrogen, C1-6 alkyl or the like, X is oxygen or the like, Y is carbon or the like, Z is C1-6 alkyl or the like, and m and n are integers of 0, 1, 2 or the like) or a pharmaceutically acceptable salt thereof. The compound has the effect of inhibiting or reducing abnormal protein aggregate accumulation in the brain. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a 2(1H)-pyridine imine derivative or a pharmaceutically acceptable salt thereof having the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and a drug containing the derivative as an active ingredient for treating or preventing central nervous system diseases whose causes are related to abnormal protein aggregates in the brain. In addition, the present invention provides a method for reproducing Parkinson's disease pathology using a neurospheroid, and a method for evaluating the amount of alpha-synuclein aggregates using the reproducing method. Background Art

[0002] Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, are believed to be caused by the formation of abnormal aggregated proteins in the patient's brain. These aggregates exhibit neurotoxicity and thus contribute to the onset and progression of the disease.

[0003] The constituent proteins of aggregates vary depending on the disease, and α-synuclein has been reported to be the main constituent of aggregates that cause Parkinson's disease. It has been reported that abnormally aggregated α-synuclein exhibits toxicity and that aggregated α-synuclein spreads between cells.

[0004] As a drug for treating Parkinson's disease, administration of levodopa, a dopamine precursor, can be used as a symptomatic treatment, but a fundamental treatment method has not yet been established. In recent years, some developments of disease-modifying drugs for Parkinson's disease have been vigorously promoted, but there are currently no reports of drugs that strongly inhibit or reduce the accumulation of α-synuclein aggregates in clinical trials.

[0005] α-synuclein aggregates are considered to be the pathogenic background of Lewy body diseases including Parkinson's disease (Lewy body dementia, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, etc.). Therefore, agents that inhibit or reduce the accumulation of α-synuclein aggregates are expected to show therapeutic effects on these diseases.

[0006] So far, no in vitro evaluation system that reproduces endogenous α-synuclein aggregates in neurons has been reported, and many evaluation systems for α-synuclein pathologies are indicated by the increased amount of phosphorylated α-synuclein caused by the addition of in vitro synthesized α-synuclein oligomers. After all, it is impossible to evaluate the effect of suppressing the accumulation of α-synuclein aggregates or reducing the accumulated α-synuclein aggregates.

[0007] So far, NPT200-11 (Neuropore) and Anle138b (MODAG) have been reported as agents capable of inhibiting the formation of α-synuclein aggregates. However, the effects of these agents on the ability to inhibit the formation of aggregates when α-synuclein is artificially aggregated in vitro have been evaluated (Patent Documents 1 and 2).

[0008] It has also been reported that 2(1H)-pyridine imine compounds such as N-[(2E)-1-(cyclopropylmethyl)pyridine-2(1H)-ylidene]oxolane-3-carboxamide have an agonistic effect on cannabinoid receptors, which is effective for pain or autoimmune diseases (Patent Document 3). In addition, it has also been reported that N-[1-(6-chloropyridin-3-yl)methylpyridine-2(1H)-ylidene]-2,2,2-trifluoroacetamide can be used as a pest control agent (Patent Document 4).

[0009] However, all of these compounds are different from the 2(1H)-pyridine imine derivatives of the present invention. Moreover, these documents neither disclose nor suggest any content related to the 2(1H)-pyridine imine derivatives of the present invention. In addition, they do not suggest the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain.

[0010] Prior art

[0011] (Patent References)

[0012] [Patent Document 1] WO 2011 / 084642

[0013] [Patent Document 2] WO 2010 / 000372

[0014] [Patent Document 3] WO 2006 / 051704

[0015] [Patent Document 4] WO 2013 / 031671 Summary of the invention

[0016] (Technical Issues)

[0017] The object of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof for preventing or treating a central nervous system disease, characterized by the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and a composition comprising the compound. In addition, the object of the present invention is also to provide a method for reproducing the symptoms of Parkinson's disease using neurospheres, and a method for evaluating the amount of α-synuclein aggregates using the reproducing method.

[0018] (Solution to the problem)

[0019] To achieve the above object, the present inventors have conducted extensive research and found that the compound of the following formula (1) or a pharmaceutically acceptable salt thereof (optionally referred to as "the compound of the present invention") has an effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and found a method for reproducing Parkinson's disease symptoms using neurospheres, and a method for evaluating the amount of α-synuclein aggregates using the reproducing method. Based on these findings, the present invention has been completed. The present invention is as follows.

[0020] (Item 1)

[0021] A compound of formula (1):

[0022]

[0023] or a pharmaceutically acceptable salt thereof, wherein

[0024] X is oxygen or NR 5 ,

[0025] R 5 is hydrogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6 Cycloalkyl,

[0026] m is 0, 1, or 2,

[0027] n is 0, 1, 2, 3 or 4,

[0028] Y is CH or nitrogen,

[0029] The condition is

[0030] When m is 0, then Y is CH, and n is 1, 2, 3 or 4,

[0031] When m is 1, then Y is CH, and n is 0, 1, 2 or 3, and

[0032] When m is 2, then n is 1 or 2,

[0033] R 1 and R 2 is independently hydrogen or C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 Alkyl, or R 1 and R 2 together to form a bridged methylene or ethylene group,

[0034] R 3 and R 4 are independently hydrogen, halogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 Alkoxy, and

[0035] Z is C 1-6 alkyl.

[0036] (Item 2)

[0037] The compound of item 1 is represented by formula (2):

[0038]

[0039] in

[0040] X is oxygen or NR 5 ,

[0041] R 5 is hydrogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6 Cycloalkyl,

[0042] m is 0, 1, or 2,

[0043] n is 0, 1, 2, 3 or 4,

[0044] Y is CH or nitrogen,

[0045] The condition is

[0046] When m is 0, then Y is CH, and n is 1, 2, 3 or 4,

[0047] When m is 1, then Y is CH, and n is 0, 1, 2 or 3, and

[0048] When m is 2, then n is 1 or 2,

[0049] R 1 and R 2 is independently hydrogen or C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 Alkyl, or R 1 and R 2 together to form a bridged methylene or ethylene group,

[0050] R 3 and R 4 are independently hydrogen, halogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 Alkoxy, and

[0051] Z is C 1-6alkyl,

[0052] or a pharmaceutically acceptable salt thereof.

[0053] (Item 3)

[0054] The compound of Item 1 or 2 or a pharmaceutically acceptable salt thereof, wherein Y is CH.

[0055] (Item 4)

[0056] The compound of any one of items 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 2 For hydrogen.

[0057] (Item 5)

[0058] The compound or pharmaceutically acceptable salt thereof of any one of items 1 to 4, wherein n is 0, 1 or 2, provided that when n is 2, m is 0 or 1.

[0059] (Item 6)

[0060] The compound of any one of items 1 to 5 or a pharmaceutically acceptable salt thereof, wherein m is 1 and n is 0.

[0061] (Item 7)

[0062] The compound of item 1 is represented by formula (3):

[0063]

[0064] in

[0065] X is oxygen or NR 5 ,

[0066] R 5 is hydrogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6 Cycloalkyl,

[0067] R 1 is C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl,

[0068] R 3 and R 4 are independently hydrogen, halogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 Alkoxy, and

[0069] Z is C 1-6 alkyl,

[0070] or a pharmaceutically acceptable salt thereof.

[0071] (Item 8)

[0072] The compound of any one of items 1 to 7 or a pharmaceutically acceptable salt thereof, wherein R 1 It is methyl or ethyl.

[0073] (Item 9)

[0074] The compound of any one of items 1 to 8 or a pharmaceutically acceptable salt thereof, wherein R 5 is hydrogen, C optionally substituted by 1 to 6 identical or different halogen atoms 1-3 Alkyl, or cyclopropyl.

[0075] (Item 10)

[0076] The compound of any one of items 1 to 9 or a pharmaceutically acceptable salt thereof, wherein X is NR 5 , and R 5 It is hydrogen or methyl.

[0077] (Item 11)

[0078] The compound or pharmaceutically acceptable salt thereof according to any one of items 1 to 10, wherein Z is methyl or ethyl.

[0079] (Item 12)

[0080] The compound of any one of items 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 1 It is methyl.

[0081] (Item 13)

[0082] The compound of any one of items 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 are independently hydrogen, halogen, C optionally substituted by 1 to 6 fluorine atoms 1-3 alkyl or C optionally substituted by 1 to 6 fluorine atoms 1-3 Alkoxy.

[0083] (Item 14)

[0084] The compound of any one of items 1 to 13 or a pharmaceutically acceptable salt thereof, wherein R 3 It is methyl or methoxy.

[0085] (Item 15)

[0086] The compound or pharmaceutically acceptable salt thereof according to any one of items 1 to 8, wherein X is oxygen.

[0087] (Item 16)

[0088] The compound or pharmaceutically acceptable salt thereof according to Item 15, wherein Z is methyl or ethyl.

[0089] (Item 17)

[0090] The compound of item 15 or 16 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 are independently hydrogen, halogen, C optionally substituted by 1 to 6 fluorine atoms 1-3 alkyl or C optionally substituted by 1 to 6 fluorine atoms 1-3 Alkoxy.

[0091] (Item 18)

[0092] The compound of any one of items 1 to 13 and 15 to 17 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 are independently hydrogen, fluoro or methoxy.

[0093] (Item 19)

[0094] The compound of any one of items 1 to 13 and 15 to 17 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 All are hydrogen.

[0095] (Item 20)

[0096] The compound of item 1, which is selected from:

[0097] 3-ethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 1),

[0098] 3-methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 2),

[0099] N-[(2E)-1-ethylpyridin-2(1H)-ylidene]-3-methyloxetane-3-carboxamide (Example 3),

[0100] 3-ethyl-N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 9),

[0101] 3-ethyl-N-[(2E)-5-fluoro-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 31),

[0102] N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 51), and

[0103] N-[(2E)-5-chloro-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 60),

[0104] or a pharmaceutically acceptable salt thereof.

[0105] (Item 21)

[0106] The compound of item 1, which is selected from:

[0107] 3-ethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 1),

[0108] 3-methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 2),

[0109] N-[(2E)-1-ethylpyridin-2(1H)-ylidene]-3-methyloxetane-3-carboxamide (Example 3), and

[0110] N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 51),

[0111] or a pharmaceutically acceptable salt thereof.

[0112] (Item 22)

[0113] A medicament comprising the compound of any one of items 1 to 21 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0114] (Item 23)

[0115] A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, comprising a compound according to any one of items 1 to 21 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0116] (Item 24)

[0117] The drug of Item 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is a central nervous system disease related to tau, α-synuclein, TDP-43 or polyglutamine.

[0118] (Item 25)

[0119] The drug of Item 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease or spinocerebellar ataxia.

[0120] (Item 26)

[0121] The drug of Item 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is a central nervous system disease associated with α-synuclein.

[0122] (Item 27)

[0123] The drug of Item 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease or infantile axonal dystrophy.

[0124] (Item 28)

[0125] A method for treating and / or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, comprising administering a therapeutically effective amount of a compound of any one of items 1 to 21 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

[0126] (Item 29)

[0127] Use of the compound of any one of items 1 to 21 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating and / or preventing a central nervous system disease caused by abnormal aggregates of brain proteins.

[0128] (Item 30)

[0129] A compound according to any one of items 1 to 21 or a pharmaceutically acceptable salt thereof, for use in treating and / or preventing a central nervous system disease caused by abnormal aggregates of brain proteins.

[0130] (Item 31)

[0131] A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, comprising a compound of any one of items 1 to 21 or a pharmaceutically acceptable salt thereof in combination with at least one agent selected from L-dopa, a dopamine agonist, a MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody and a pharmaceutically acceptable salt thereof.

[0132] (Item 32)

[0133] A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, comprising a compound of any one of items 1 to 21 or a pharmaceutically acceptable salt thereof, used in combination with at least one agent selected from L-dopa, a dopamine agonist, a MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody and a pharmaceutically acceptable salt thereof.

[0134] (Item 33)

[0135] A method for reproducing the symptoms of Parkinson's disease using neurospheres prepared by three-dimensional culture of human iPS cells with gene mutations associated with synucleinopathy, comprising step (I);

[0136] Step (I): Measuring the amount of α-synuclein aggregates in neurospheres.

[0137] (Item 34)

[0138] A method for evaluating an agent having an effect of inhibiting / reducing the accumulation of α-synuclein aggregates in Parkinson's disease using neurospheres prepared by three-dimensional culture of human iPS cells with gene mutations associated with synucleinopathy, comprising step (I);

[0139] Step (I): Measuring the amount of α-synuclein aggregates in neurospheres.

[0140] (Effects of the Invention)

[0141] The present invention makes it possible to provide a compound of formula (1) or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof has the effect of suppressing or reducing the accumulation of abnormal protein aggregates in the brain, and therefore can be used as a drug for treating or preventing a central nervous system disease caused by abnormal protein aggregates in the brain, the disease is particularly related to α-synuclein neurodegenerative diseases such as Parkinson's disease, Lewy body dementia, multiple system atrophy, Gaucher disease and infantile axonal dystrophy. In addition, the present invention can reproduce spontaneous α-synuclein aggregates in neurons, which are Parkinson's disease symptoms, and therefore, the present invention can be used as a method for evaluating a drug having the effect of suppressing or reducing the accumulation of α-synuclein aggregates. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 The difference in the amount of aggregates in neurospheres derived from healthy human iPS cells and in neurospheres derived from PLA2G6 mutant iPS cells is shown. The vertical axis represents the amount of aggregates in the neurospheres, and the horizontal axis represents the number of culture days. The white bars represent the amount of aggregates in neurospheres derived from healthy human iPS cells, and the black bars represent the amount of aggregates in neurospheres derived from PLA2G6 mutant iPS cells.

[0143] Figure 2 The difference in the amount of aggregates in dopamine neurospheres derived from healthy human iPS cells and in dopamine neurospheres derived from PLA2G6 mutant iPS cells is shown. The vertical axis represents the amount of aggregates in dopamine neurospheres, and the horizontal axis represents the number of culture days. The white bars represent the amount of aggregates in neurospheres derived from healthy human iPS cells, and the black bars represent the amount of aggregates in neurospheres derived from PLA2G6 mutant iPS cells.

[0144] Figure 3 The difference in the amount of tyrosine hydroxylase between dopamine neurospheres derived from healthy human iPS cells and dopamine neurospheres derived from PLA2G6 mutant iPS cells on day 26 of culture is shown. The vertical axis represents the amount of tyrosine hydroxylase in dopamine neurospheres. The white bar represents the amount of tyrosine hydroxylase in neurospheres derived from healthy human iPS cells, and the black bar represents the amount of tyrosine hydroxylase in neurospheres derived from PLA2G6 mutant iPS cells.

[0145] Figure 4 The figure shows the difference in the amount of cleaved caspase 3 in dopamine neurospheres derived from healthy human iPS cells and in dopamine neurospheres derived from PLA2G6 mutant iPS cells on culture day 40. The vertical axis represents the amount of cleaved caspase 3 in dopamine neurospheres. The white bar represents the amount of cleaved caspase 3 in neurospheres derived from healthy human iPS cells, and the black bar represents the amount of cleaved caspase 3 in neurospheres derived from PLA2G6 mutant iPS cells.

[0146] Figure 5 The difference in the amount of aggregates in dopamine neurospheres derived from healthy human iPS cells and in dopamine neurospheres derived from GBA1 gene homozygous mutation iPS cells is shown. The vertical axis represents the amount of aggregates in dopamine neurospheres, and the horizontal axis represents the number of culture days. The white bars represent the amount of aggregates in neurospheres derived from healthy human iPS cells, and the black bars represent the amount of aggregates in neurospheres derived from GBA1 gene homozygous mutation iPS cells. DETAILED DESCRIPTION

[0147] The present invention will be explained in detail below. In this specification, in the definition of "substituent", the number of carbon atoms can be expressed as, for example, "C 1-3 Specifically, the term “C 1-3 "Alkyl" is synonymous with an alkyl group having 1 to 3 carbon atoms, and other numbers have similar definitions.

[0148] The "halogen" includes, for example, fluorine, chlorine, bromine and iodine. It is preferably fluorine or chlorine, more preferably fluorine.

[0149] “C1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. 1-6 "Alkyl" preferably includes "C 1-3 Alkyl". "C 1-3 "Alkyl" includes, for example, methyl, ethyl, propyl and 1-methylethyl. "C 1-4 Alkyl" except the "C 1-3 In addition to the examples listed in "alkyl", there are also butyl, 1,1-dimethylethyl, 1-methylpropyl and 2-methylpropyl. 1-6 Alkyl" except the "C 1-4 In addition to the examples listed in "alkyl", for example, pentyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl and hexyl are also included.

[0150] “C 3-6 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 6 carbon atoms, which may have a bridge structure. 3-6 The "cycloalkyl" includes, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, preferably cyclopropyl and cyclobutyl. More preferably, it is cyclopropyl.

[0151] “C 1-6 "Alkoxy" refers to the above "C 1-6 "C 1-6 "Alkoxy" preferably includes "C 1-4 Alkoxy", more preferably "C 1-3 Alkoxy". "C 1-3 "Alkoxy" includes, for example, methoxy, ethoxy, propoxy and 1-methylethoxy. "C 1-4 Alkoxy" except the "C 1-3 In addition to the examples listed in "alkoxy", for example, butoxy, 1,1-dimethylethoxy, 1-methylpropoxy and 2-methylpropoxy are also included. "C 1-6 Alkoxy" except the "C 1-4 In addition to the examples listed in "alkoxy", for example, pentyloxy, 3-methylbutoxy, 2-methylbutoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, 1,1-dimethylpropoxy, hexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 1-methylpentyloxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy and 1,2-dimethylbutoxy are also included.

[0152] Preferred X, Y, Z, m, n, R in the compound of formula (1) of the present invention are 1 , R 2 , R3 , R 4 and R 5 It is shown below, but the technical scope of the present invention is not limited to the scope of the compounds listed below.

[0153] In the compound of formula (1), if substitutable, R 1 , R 2 , R 3 and R 4 The group may be substituted at any carbon atom, and if substitutable, R 1 and R 2 can be substituted at the same carbon atom. Also, when Y is CH, the H of CH can be replaced by R 1 or R 2 replaced.

[0154]

[0155] X preferably includes oxygen and NR 5 .

[0156] R 5 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, and C 3-6 More preferably, it comprises hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, and C 3-6 Cycloalkyl. Further preferably, it includes hydrogen, methyl, ethyl, propyl, 1-methylethyl and cyclopropyl. Even more preferably, it includes hydrogen and methyl.

[0157] Y preferably comprises CH.

[0158] Z preferably includes methyl, ethyl, propyl, 1-methylethyl and butyl. More preferably, it includes methyl, ethyl, propyl and 1-methylethyl. Further preferably, it includes methyl, ethyl and propyl. Even more preferably, it includes methyl and ethyl.

[0159] When Y is CH, m is preferably 1 or 2, more preferably 1. When Y is nitrogen, m is 2.

[0160] When Y is CH, n is preferably 0, 1, 2 or 3. More preferably, it is 0, 1 or 2. More preferably, it is 0 or 1. Further preferably, it is 0. When Y is nitrogen, n is preferably 1 or 2, more preferably 1.

[0161] R 1 Preferably, it comprises hydrogen and optionally substituted by 1 to 6 fluorine atoms. 1-6 More preferably, it comprises hydrogen and C 1-6 Further preferably, it includes C 1-6Even more preferably, it includes methyl and ethyl.

[0162] R 2 Preferably, it comprises hydrogen and optionally substituted by 1 to 6 fluorine atoms. 1-6 More preferably, it comprises hydrogen and C 1-6 More preferably, it includes hydrogen and C 1-3 Even more preferably, it comprises hydrogen.

[0163] Where R 1 and R 2 The structure of methylene or ethylene groups forming a bridge together includes the structure of the following formula (4), which is drawn together with a ring containing X and Y. In the following formula (4), the wavy line indicates the bonding position to the carbonyl group in formula (1). And, in the ring of formula (4), X and Y are as defined in item 1.

[0164]

[0165] R 3 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes hydrogen, methyl, methoxy and fluorine. Even more preferably, it includes hydrogen and methoxy.

[0166] R 4 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes hydrogen, methyl, methoxy, fluorine, chlorine and bromine. Even more preferably, it includes hydrogen and fluorine.

[0167] R 3 and R 4 Preferred substitution positions are those shown in the following formula (2).

[0168]

[0169] In the substitution position of formula (2), when X is NR 5 When R 3Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes methyl and methoxy. Even more preferably, it includes methoxy.

[0170] In the substitution position of formula (2), when X is NR 5 When R 4 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, C optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, C optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes hydrogen, methyl, chlorine and bromine. Even more preferably, it includes hydrogen.

[0171] In the substitution position of formula (2), when X is oxygen, R 3 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes hydrogen, methoxy and fluorine. More preferably, it includes hydrogen.

[0172] In the substitution position of formula (2), when X is oxygen, R 4 Preferably, it comprises hydrogen, optionally substituted by 1 to 6 fluorine atoms. 1-6 Alkyl, C optionally substituted by 1 to 6 fluorine atoms 1-6 More preferably, it includes hydrogen, C optionally substituted by 1 to 6 fluorine atoms. 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Preferably, it includes hydrogen, methoxy, fluorine, chlorine and bromine. More preferably, it includes hydrogen and fluorine.

[0173] Preferred compounds of formula (1) include the following compounds or pharmaceutically acceptable salts thereof.

[0174] In one embodiment, the compound of formula (1) comprises (A) below.

[0175] (A)

[0176] A compound or a pharmaceutically acceptable salt thereof, wherein

[0177] X is oxygen or NR 5 ,

[0178] R 5 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, or C 3-6 Cycloalkyl,

[0179] m is 1 or 2,

[0180] n is 0, 1, 2 or 3,

[0181] Y is CH or nitrogen,

[0182] The condition is

[0183] When m is 1, then Y is CH, and n is 0, 1, 2 or 3, and

[0184] When m is 2, then n is 1 or 2,

[0185] R 1 and R 2 is independently hydrogen or C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, or R 1 and R 2 together to form a bridged methylene or ethylene group,

[0186] R 3 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 Alkoxy, or fluorine,

[0187] R 4 is C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 alkoxy, or halogen, and

[0188] Z is C 1-4 alkyl.

[0189] In one embodiment, the compound of formula (1) includes (B) below.

[0190] (B)

[0191] A compound of formula (2):

[0192]

[0193] in

[0194] X is oxygen or NR 5 ,

[0195] R 5 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-3 Alkyl, or C 3-6 Cycloalkyl,

[0196] m is 1 or 2,

[0197] n is 0, 1, or 2,

[0198] Y is CH,

[0199] The condition is that when m is 2, then n is 1,

[0200] R 1 and R 2 are independently hydrogen or C 1-6 alkyl,

[0201] R 3 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 Alkoxy, or fluorine,

[0202] R 4 is C optionally substituted by 1 to 6 fluorine atoms 1-6 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-6 alkoxy, or halogen, and

[0203] Z is C 1-4 alkyl,

[0204] or a pharmaceutically acceptable salt thereof.

[0205] In one embodiment, the compound of formula (1) includes (C) below.

[0206] (C)

[0207] A compound of formula (2):

[0208]

[0209] in

[0210] X is oxygen or NR 5 ,

[0211] R 5For hydrogen, C 1-3 Alkyl or cyclopropyl,

[0212] m is 1,

[0213] n is 0 or 1,

[0214] Y is CH,

[0215] R 1 and R 2 are independently hydrogen or C 1-6 alkyl,

[0216] R 3 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 Alkoxy, or fluorine,

[0217] R 4 is hydrogen, C optionally substituted by 1 to 6 fluorine atoms 1-3 Alkyl, optionally substituted by 1 to 6 fluorine atoms 1-3 alkoxy, fluorine, chlorine or bromine, and

[0218] Z is C 1-3 alkyl,

[0219] or a pharmaceutically acceptable salt thereof.

[0220] In one embodiment, the compound of formula (1) includes the following (D).

[0221] (D)

[0222] A compound of formula (3):

[0223]

[0224] in

[0225] X is NR 5 ,

[0226] R 5 is hydrogen, methyl, ethyl or propyl,

[0227] R 1 C 1-6 alkyl,

[0228] R 3 is hydrogen, methyl or methoxy,

[0229] R 4 is hydrogen, methyl, fluorine, chlorine or bromine, and

[0230] Z is C 1-3 alkyl,

[0231] or a pharmaceutically acceptable salt thereof.

[0232] In one embodiment, the compound of formula (1) includes (E) below.

[0233] (E)

[0234] A compound of formula (3):

[0235]

[0236] in

[0237] X is oxygen,

[0238] R 1 C 1-6 alkyl,

[0239] R 3 is hydrogen, methyl, methoxy or fluorine,

[0240] R 4 is hydrogen, methyl, methoxy, fluorine or chlorine, and

[0241] Z is C 1-3 alkyl,

[0242] or a pharmaceutically acceptable salt thereof.

[0243] In one embodiment, the compound of formula (1) includes the following compounds:

[0244] 3-ethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 1),

[0245] 3-methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 2),

[0246] N-[(2E)-1-ethylpyridin-2(1H)-ylidene]-3-methyloxetane-3-carboxamide (Example 3),

[0247] 3-ethyl-N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 9),

[0248] 3-ethyl-N-[(2E)-5-fluoro-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 31),

[0249] N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 51), or

[0250] N-[(2E)-5-chloro-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 60),

[0251] or a pharmaceutically acceptable salt thereof.

[0252] In one embodiment, the compound of formula (1) includes the following compounds:

[0253] 3-ethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 1),

[0254] 3-methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide (Example 2),

[0255] N-[(2E)-1-ethylpyridin-2(1H)-ylidene]-3-methyloxetane-3-carboxamide (Example 3), or

[0256] N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide (Example 51), or

[0257] or a pharmaceutically acceptable salt thereof.

[0258] "Pharmaceutically acceptable salts" include acid addition salts and base addition salts. For example, acid addition salts include inorganic 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, p-toluenesulfonate and camphorsulfonate. Base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt and aluminum salt; and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris (hydroxymethyl) methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine and N, N-dibenzylethylamine. "Pharmaceutically acceptable salts" also include amino acid salts of basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid and glutamic acid.

[0259] Suitable salts of starting materials and intermediates and acceptable salts of pharmaceutical substances are conventional non-toxic salts. Suitable salts include, for example, acid addition salts such as organic acid salts (including acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formates and p-toluenesulfonate) and inorganic acid salts (including hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate and phosphate); salts with amino acids (including arginine, aspartic acid and glutamic acid); metal salts such as alkali metal salts (including sodium and potassium salts) and alkaline earth metal salts (including calcium and magnesium salts); ammonium salts; organic base salts (including trimethylamine salts, triethylamine salts, pyridine salts, picolinate (picolinate, picolinate), dicyclohexylamine salts and N, N'-dibenzylethylenediamine salts); and other salts that can be optionally selected by those skilled in the art.

[0260] If it is desired to obtain the compound of the present invention in the form of its salt, when the compound of the present invention is obtained in the form of a salt, it can be purified as it is, and when it is obtained in a free form, it can be dissolved or suspended in an appropriate organic solvent and an acid or base is added thereto to form a salt by a conventional method.

[0261] Any one or more of 1 The H atoms are replaced by 2 The compound of formula (1) without H(D) atom is also within the scope of formula (1) of the present invention. The present invention covers the compound of formula (1) or a pharmaceutically acceptable salt thereof. Since the compound of the present invention can exist in the form of hydrates and / or solvates of various solvents, including solvates (ethanolates, etc.), these hydrates and / or solvates are included in the compound of the present invention. In addition, the present invention covers all tautomers of compound (1), all possible stereoisomers thereof, crystalline forms of various states thereof and mixtures thereof.

[0262] The compounds of formula (1) encompass optical isomers based on optically active centers, atropisomers based on axial or planar chirality due to restriction of intramolecular rotation, and all other isomers that may exist as stereoisomers, tautomers and geometric isomers, and mixtures thereof.

[0263] In particular, each optical isomer and atropisomer can be obtained as a racemate, or as an optically active substance when an optically active starting material or intermediate is used. If necessary, the racemate of the corresponding starting material, intermediate or final product can also be physically or chemically resolved into optical enantiomers at an appropriate step in the above-mentioned preparation method by known separation methods such as methods with optically active columns and fractional crystallization methods. These methods for resolving enantiomers include diastereoisomer methods, in which, for example, a racemate is reacted with an optically active resolving agent to synthesize two diastereomers, which are resolved by fractional crystallization or similar methods via different physical properties.

[0264] The method for preparing the compound of formula (1) in the present invention will be mentioned below, but the method for preparing the compound of the present invention should not be limited thereto.

[0265] Preparation method

[0266] The compound of the present invention can be synthesized according to each preparation method shown below or a combination thereof with a known synthesis method.

[0267] Each compound in the following scheme may exist as a salt thereof, wherein the salt includes, for example, the "pharmaceutically acceptable salt" mentioned above as a salt of the compound of formula (1). The following scheme is disclosed only as an example, and therefore, the compounds of the present invention can also be prepared by different methods optionally based on the knowledge of those skilled in the art of synthetic organic chemistry.

[0268] In each of the following production methods, even if the use of a protecting group is not explicitly stated, a protecting group may be used as necessary. Also, the protecting group may be deprotected after the reaction is completed or a series of reactions have been performed to obtain the desired compound.

[0269] Introduction and removal of the protecting group can be carried out by a method commonly used in organic synthetic chemistry, for example, the method described in TW Greene and PGM Wuts, "Protective Groups in Organic Synthesis", 3rd Ed., John Wiley and Sons, inc., New York (1999) or a method analogous thereto.

[0270] Examples of the protecting group for the amino group include, for example, tert-butoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, o-nitrobenzenesulfonyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl and the like.

[0271] Preparation method 1

[0272] Among the compounds of formula (1), the compound of the following formula (1a) can be produced, for example, by the following production method.

[0273]

[0274] Where R 1 , R 2 , R 3 , R 4 , m, n, Y and Z are as defined in item 1; and L is halogen or OH.

[0275] (Step 1-1: Preparation step of compound (1a))

[0276] Compound (1a) can be prepared by reacting compound (1-1) with compound (1-2) in a suitable inert solvent in the presence of any condensing agent and / or any base or in the absence of them. As compound (1-1), commercially available compounds can be used, or products prepared by known methods can be used (for example, ChemPlusChem.2020,85(7),1587-1595,Journal of Molecular Structure 2022,1253,132310, etc.). Alternatively, the product prepared by the manner described in Reference Example 1 shown below can be used as compound (1-1). Compound (1-2) can be prepared, for example, by the manner described in Organic Letters 2017,19(7),1768-1771, ACS Catalysis 2016,6(6),4010-4016, etc., or can be purchased as a commercially available product. The alkali used in this step can be suitably selected from the alkali exemplified below, including, for example, sodium hydride, triethylamine, diisopropylethylamine and cesium carbonate. The condensing agent used in this step can be selected from various condensing agents commonly used in organic synthesis reactions, including, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate. The solvent used in this step can be suitably selected from the solvent exemplified below, including, for example, DMF, THF, dichloromethane and chloroform. The reaction time of this step is generally 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature of this step is generally -78°C to 200°C, preferably -78°C to 80°C.

[0277] Preparation method 2

[0278] Among the compounds of formula (1), compounds of the following formula (1b) and formula (1c) can be prepared, for example, by the following preparation method.

[0279]

[0280] Where R 1 , R 2 , R 3 , R 4 , m, n, Y and Z are as defined in item 1; R 5a is C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6 Cycloalkyl, L is halogen or OH; and Pro is a protecting group for amino.

[0281] (Step 2-1: Preparation step of compound (1c))

[0282] Compound (1c) can be prepared by reacting compound (1-1) with compound (2-1) according to the method shown in step 1-1. Compound (2-1) can be prepared, for example, by the method described in Organic Letters 2021, 23 (17), 6648-6653, WO 2018 / 034917, etc., or can be purchased as a commercially available product.

[0283] (Step 2-2: Preparation step of compound (2-3))

[0284] Compound (2-3) can be prepared by reacting compound (1-1) with compound (2-2) according to the method shown in step 1-1. Compound (2-2) can be prepared, for example, by the method described in Organic Letters 2017, 19 (7), 1768-1771, AC Catalysis 2016, 6 (6), 4010-4016, WO 2018 / 034917, or can be purchased as a commercially available product.

[0285] (Step 2-3: Preparation step of compound (1b))

[0286] Compound (1b) can be prepared by deprotecting the amino-protecting group Pro in compound (2-3) according to a known method (e.g., the method described in Protective Group in Organic Synthesis 3rd Edition (described by Theodora W. Green, Peter GM Wuts, published by John Wiley & Sons Inc, 1999)). The amino-protecting group Pro includes, for example, a tert-butoxycarbonyl group and a benzyloxycarbonyl group.

[0287] (Step 2-4: Preparation step of compound (1c))

[0288] Compound (1c) can be prepared by reacting compound (1b) with a residue corresponding to R in the presence of a reducing agent in a suitable inert solvent. 5a The reducing agent used in this step can be selected from various reducing agents commonly used in organic synthesis reactions, including, for example, sodium borohydride, sodium triacetoxyborohydride and sodium cyanoborohydride. The solvent used in this step can be suitably selected from the solvents exemplified below, including, for example, toluene, THF, ethylene dichloride and methanol. The reaction time of this step is generally 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature of this step is generally -78°C to 100°C, preferably 0°C to 80°C.

[0289] Alternatively, compound (1c) can also be prepared by reacting compound (1b) with a residue corresponding to R 5a Any alkyl halide or alkyl sulfonate of is prepared in a suitable inert solvent in the presence of a base. The base used in this step may be suitably selected from the bases exemplified below, including, for example, potassium carbonate, cesium carbonate, sodium hydride and lithium diisopropylamide. The solvent used in this step may be suitably selected from the solvents exemplified below, including, for example, DMF, dimethyl sulfoxide, THF and 1,4-dioxane. The reaction time of this step is generally 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature of this step is generally -78°C to 100°C, preferably 0°C to 80°C.

[0290] Preparation method 3

[0291] Among the compounds of formula (1), the compound of the following formula (1d) can be produced, for example, by the following production method.

[0292]

[0293] Where R 1 , R 2 , R 3 , R 4 , m, n and Z are as defined in item 1.

[0294] (Step 3-1: Preparation step of compound (1d))

[0295] Compound (1d) can be prepared by reacting compound (3-1) in a suitable inert solvent in the presence of triphosgene, chloroformate and a base, and then reacting the product with compound (1-1). The base used in this step can be suitably selected from the bases exemplified below, including, for example, pyridine and triethylamine. The solvent used in this step can be suitably selected from the solvents exemplified below, including, for example, dichloromethane and chloroform. The reaction time of this step is generally 5 minutes to 48 hours, preferably 30 minutes to 24 hours. The reaction temperature of this step is generally -78°C to 100°C, preferably 0°C to 80°C.

[0296] Preparation method 4

[0297] Among the compounds of formula (1), the compound of the following formula (1e) can be produced, for example, by the following production method.

[0298]

[0299] Where R 1 , R 2 , R 3 , R 4 , m, n and Z are as defined in item 1; R 5a is C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6 Cycloalkyl.

[0300] (Step 4-1: Preparation step of compound (1e))

[0301] Compound (1e) can be produced by reacting compound (4-1) with compound (1-1) according to the method shown in Step 3-1.

[0302] Preparation method 5

[0303] Among the compounds of formula (1), compounds of the following formulae (1e) and (1f) can be produced, for example, by the following production methods.

[0304]

[0305] Where R 1 , R 2 , R 3 , R 4 , m, n and Z are as defined in item 1; R 5a is C optionally substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl or C optionally substituted by 1 to 6 identical or different halogen atoms 3-6Cycloalkyl; Pro is a protecting group for amino.

[0306] (Step 5-1: Preparation step of compound (5-2))

[0307] Compound (5-2) can be produced by reacting compound (5-1) with compound (1-1) according to the method shown in Step 3-1. The protecting group Pro of the amino group includes, for example, a tert-butoxycarbonyl group and a benzyloxycarbonyl group.

[0308] (Step 5-2: Preparation step of compound (1f))

[0309] Compound (1f) can be produced from compound (5-2) according to the method shown in Step 2-3.

[0310] (Step 5-3: Preparation step of compound (1e))

[0311] Compound (1e) can be prepared from compound (1f) according to the method shown in Step 2-4.

[0312] Among the starting materials and intermediates in each of the above-mentioned production methods, compounds not described in each method are commercially available or can be produced by a skilled person using commercially available materials in a known manner or a manner analogous thereto.

[0313] The base used in each step of the above method should be appropriately selected according to the reaction, starting compound, etc., and includes, for example, alkaline bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkaline 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 tert-butoxide; organic metal bases such as butyl lithium and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine (DMAP) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0314] The solvent used in each step of the above method should be appropriately selected according to the reaction, starting compound, etc., including, for example, alcohol solvents such as methanol, ethanol and isopropanol; ketone solvents such as acetone and methyl ketone; halogenated hydrocarbon solvents such as dichloromethane and chloroform; ether solvents such as tetrahydrofuran (THF) and dioxane; aromatic hydrocarbon solvents such as toluene and benzene; aliphatic hydrocarbon solvents such as hexane and heptane; ester solvents such as ethyl acetate and propyl acetate; amide solvents such as N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP); sulfoxide solvents such as dimethyl sulfoxide (DMSO); and nitrile solvents such as acetonitrile. The solvent used herein can be one of these solvents, or a mixture of two or more solvents selected from these solvents. And, if possible in the reaction, an organic base such as diazabicycloundecene (DBU) can be used as the solvent used herein.

[0315] The compounds of formula (1) of the present invention and their intermediates can be separated and purified in a manner known to the skilled person, including, for example, extraction, distribution, reprecipitation, column chromatography (e.g., silica gel column chromatography, ion exchange column chromatography and preparative liquid chromatography) and recrystallization.

[0316] The solvent for recrystallization used herein includes, for example, alcohol solvents such as methanol, ethanol and 2-propanol; ether solvents such as ether; ester solvents such as ethyl acetate; aromatic hydrocarbon solvents such as benzene and toluene; ketone solvents such as acetone; halogenated solvents such as dichloromethane and chloroform; hydrocarbon solvents such as hexane; aprotic solvents such as dimethylformamide and acetonitrile; water; and mixed solvents thereof. As other purification methods, for example, the method described in Volume 1 or other volumes (Jikken Kagaku Kouza, Chemical Society of Japan, edited by MARUZEN) can be used. In addition, considering the structure of each starting compound, the structure of the compound of the present invention can be easily determined by spectral analysis methods such as nuclear magnetic resonance, infrared absorption technology and circular dichroism spectroscopy and mass spectrometry.

[0317] In addition, each intermediate or each final product in the above preparation method can also be converted into another compound of the present invention by appropriately modifying its functional group, especially by extending various side chains from amine, hydroxyl, carbonyl, halogen, etc.; and optionally performing the above protection and deprotection as required. The modification of functional groups and the extension of side chains can be carried out by conventional methods (for example, see Comprehensive Organic Transformations, RC Larock, John Wiley & Sons Inc. (1999) etc.).

[0318] The compound of formula (1) of the present invention or its pharmaceutically acceptable salt is sometimes an asymmetric compound, or sometimes has a substituent including an asymmetric carbon. In such a case, the compound has optical isomers. The compound of the present invention includes a mixture of these isomers and separated isomers, which can be prepared in a conventional manner. The compound with an asymmetric structure can be prepared, for example, by using a starting material with an asymmetric center or by introducing an asymmetric structure anywhere along the method. For example, in the case of optical isomers, optical isomers can be obtained by using an optically active starting material or by splitting a mixture of optical isomers at an appropriate step. In the case where the compound of formula (1) or its intermediate has a basic functional group, its optical resolution includes, for example, a diastereoisomer method, in which the compound is converted into a salt thereof by reacting it with an optically active acid (e.g., monocarboxylic acids such as mandelic acid, N-benzyloxyaniline and lactic acid; dicarboxylic acids such as tartaric acid, o-diisopropyltartaric acid and malic acid; or sulfonic acids such as camphorsulfonic acid and bromocamphorsulfonic acid) in an inert solvent (e.g., alcohols such as methanol, ethanol and 2-propanol; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; or a mixed solvent thereof). In the case where the compound of formula (1) or its intermediate has an acidic functional group such as a carboxyl group, the compound can also be optically resolved by forming a salt thereof with an optically active amine (e.g., an organic amine such as 1-phenylethylamine, kinin, quinidine, cinchonidine, cinchonine and strychnine).

[0319] The temperature for forming the salt is selected from the range of usually -50°C to the boiling point of the solvent used herein, preferably 0°C to the boiling point, more preferably room temperature to the boiling point. In order to improve the optical purity, it is best to raise the temperature to about the boiling point of the solvent used herein. When collecting the precipitated crystals on the filter, optional cooling can improve the yield. The amount of the optically active acid or amine used herein is appropriately about 0.5 to about 2.0 equivalents relative to the amount of the substance compound, preferably about one equivalent. If appropriate, the resulting crystals can be recrystallized in an inert solvent (e.g., alcohols such as methanol, ethanol and 2-propanol; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; or mixed solvents thereof) to obtain a high-purity salt thereof. And, if appropriate, the optically resolved salt can also be treated with an acid or a base to obtain its free form.

[0320] In Lewy body diseases such as Parkinson's disease, abnormally aggregated α-synuclein is found in the brain of patients. Therefore, the drug of the present invention that inhibits or reduces the accumulation of α-synuclein aggregates is expected to play a role in improving the conditions of these diseases.

[0321] In addition, it is believed that the aggregates exhibit neurotoxicity, induce neuronal vulnerability and neuronal cell death, and lead to the onset and progression of the disease. Therefore, the drug of the present invention that suppresses neurotoxicity and neuronal cell death associated with α-synuclein aggregates is expected to play an effect of improving the pathology of Lewy body diseases such as Parkinson's disease.

[0322] Neurotransmitter production is one of the functions of neurons, and a decrease in neurotransmitters indicates neural vulnerability. For example, in dopaminergic neurons, neural vulnerability is indicated by a decrease in the amount of tyrosine hydroxylase, which is involved in dopamine metabolism.

[0323] In addition, abnormal electroencephalograms have been reported in Lewy body diseases such as Parkinson's disease. The electroencephalogram is a manifestation of neural synchronization activity. Therefore, the drug of the present invention that normalizes neural synchronization activity associated with α-synuclein aggregates is expected to play a role in improving the symptoms of these diseases.

[0324] The neurosphere for measuring the amount of α-synuclein aggregates can be prepared, for example, by three-dimensionally culturing neural stem cells prepared from human iPS cells or dopaminergic (DA) neural progenitor cells having gene mutations associated with synuclein diseases under the induction of neural differentiation. The amount of α-synuclein aggregates can be evaluated by measuring the amount of high molecular weight α-synuclein by protein analysis using neurospheres prepared by three-dimensional culture and α-synuclein antibodies.

[0325] Synchronous neural firing can be assessed by imaging analysis using fluorescent calcium probes with neurospheres prepared by three-dimensional culture.

[0326] In addition, by using the method of measuring the amount of α-synuclein aggregates and the method of measuring synchronized neural firing in neurospheres, Parkinson's disease conditions can be reproduced, and evaluation of agents for the effect of suppressing or reducing the accumulation of α-synuclein aggregates in Parkinson's disease conditions can be performed.

[0327] Inducing human iPS cells with gene mutations associated with synucleinopathy to differentiate into neural stem cells can be performed, for example, by incubating the cells in StemFitAK03N medium (Ajinomoto Co., Inc., Basic03) at 37°C and 5% CO 2 PLA2G6 gene mutant cells established from a healthy human iPS cell line (clone name: 201B7, obtained from the iPS Cell Research Institute of Kyoto University, Japan) were cultured under the same conditions and the culture product was induced with PSC neuron induction medium (Thermo Fisher Scientific Inc., cat#A1647801).

[0328] As a culture medium for neural stem cells, for example, the following composition can be used.

[0329] <Composition of the culture medium for neural stem cells>

[0330] Neurobasal medium (Thermo Fisher Scientific Inc., 2113049)

[0331] Advanced DMEM / F-12 medium (Thermo Fisher Scientific Inc., 12634028)

[0332] Neural induction supplement (Thermo Fisher Scientific Inc., A1647801)

[0333] Inducing neural stem cells to differentiate into neurospheres can be done, for example, by seeding neural stem cells (10,000 cells / well) in a 96-well round-bottom plate (Thermo Fisher Scientific Inc., cat#174929) in culture medium at 37°C and 5% CO. 2 This was done by culturing cells at 4 °C and replacing half of the culture medium on days 2 and 4 after induction of differentiation.

[0334] As a culture medium for neural stem cell neurospheres, for example, the following composition can be used.

[0335] <Medium composition of neurospheres>

[0336] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0337] NeuroCult SM1 Neuronal Supplement (STEMCELL Technologies, cat#05711)

[0338] N2 Supplement-A (STEMCELL Technologies, cat#07152)

[0339] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0340] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0341] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0342] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0343] Inducing the differentiation of human iPS cells with gene mutations associated with synucleinopathy into dopaminergic progenitor cells can be performed, for example, by inducing dopaminergic progenitor cells from GBA1 gene homozygous mutation cells established from PLA2G6 gene mutation cells or healthy human-derived iPS cell lines using a Dopaminergic Neuron Differentiation Kit (Thermo Fisher Scientific Inc., cat#A3147701).

[0344] Inducing dopaminergic progenitor cells to differentiate into neurospheres can be done, for example, by using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific Inc., cat# A3165801) at 37°C and 5% CO. 2 Cryopreserved dopaminergic progenitor cells were cultured at 37°C and 5% CO. Dopaminergic progenitor cells were seeded (10,000 cells / well) in 96-well round-bottom plates (ThermoFisher Scientific Inc., cat#174929) in culture medium. 2 This was done by culturing cells at 4 °C and replacing half of the culture every 3 or 4 days after differentiation was induced.

[0345] As a culture medium for dopamine neurospheres derived from dopaminergic progenitor cells, for example, the following composition can be used.

[0346] <Composition of culture medium for dopamine neurospheres>

[0347] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0348] Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific Inc., cat# A3147401)

[0349] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0350] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0351] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0352] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0353] The measurement of the amount of α-synuclein aggregates in neurospheres can be performed, for example, as follows:

[0354] Remove the differentiated neurospheres from the culture medium.

[0355] A TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32) was added thereto.

[0356] Extracting the protein from the mixture using an ultrasonicator, and

[0357] The extracted proteins were subjected to protein analysis (ProteinSimple, Inc., cat#SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat#AHB0261) with a Simple Western system under non-reducing conditions to evaluate the quantification of waveforms displayed at a molecular weight of approximately 300 kD.

[0358] Measurement of neural vulnerability in neurospheres can be performed, for example, as follows:

[0359] The differentiated dopamine neurospheres were transferred into a TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32).

[0360] Extracting the protein from the mixture using an ultrasonicator, and

[0361] The extracted proteins were subjected to protein analysis using a tyrosine hydroxylase antibody (Millipore, cat#AB152) with a Simple Western system (Protein Simple, Inc., cat#SM-W004) under reducing conditions to evaluate the quantification of waveforms displayed at a molecular weight of approximately 60 kD.

[0362] Measurement of neuronal cell death in neurospheres can be performed, for example, as follows:

[0363] The differentiated dopamine neurospheres were transferred into a TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32).

[0364] Extracting the protein from the mixture using an ultrasonicator, and

[0365] The extracted proteins were subjected to protein analysis using a cleaved caspase 3 antibody (Cell Signaling Technology, Inc., cat#9664) under reducing conditions with a Simple Western system (Protein Simple, Inc., cat#SM-W004) to evaluate the quantification of waveforms displayed at a molecular weight of approximately 20 kD.

[0366] Aberrant neural activity in neurospheres can be measured, for example, by imaging neurospheres in three-dimensional culture using fluorescent calcium probes to measure synchronized neural firing.

[0367] Synchronous neural firing in neurospheres can be measured by, for example, imaging analysis using a measurement medium containing a fluorescent calcium probe (Molecular Devices, product name: FLIPR Calcium 6 Assay Bulk Kit, cat#R8191).

[0368] The measurement medium used herein includes, for example, 20 mM Hepes (Thermo Fisher Scientific Inc., cat#15630-080), and Hank's buffer solution (Thermo Fisher Scientific Inc., cat#14065-056) containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576).

[0369] The compound of the present invention is useful as a drug for treating and / or preventing central nervous system diseases the causes of which are associated with abnormal aggregation of proteins in the brain.

[0370] Central nervous system diseases whose causes are associated with abnormal aggregation of proteins in the brain include central nervous system diseases associated with tau, α-synuclein, TDP-43 or polyglutamine.

[0371] CNS diseases associated with tau include Alzheimer's disease and frontotemporal lobar degeneration; CNS diseases associated with alpha-synuclein aggregates include Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, and infantile axonal dystrophy; CNS diseases associated with TDP-43 include amyotrophic lateral sclerosis and frontotemporal lobar degeneration; and CNS diseases associated with polyglutamine include Huntington's disease and spinocerebellar ataxia.

[0372] The compounds of the present invention can be used as drugs for the treatment and / or prevention of preferably Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile axonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease or spinocerebellar ataxia.

[0373] The compounds of the present invention are useful as drugs for the treatment and / or prevention of diseases, more preferably diseases associated with α-synuclein aggregates.

[0374] The compounds of the invention can be used as medicaments for the treatment and / or prevention of even more preferably Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease or infantile axonal dystrophy.

[0375] In the present invention, "prevention" means administering the active ingredient of the present invention to a healthy subject who does not suffer from the disease, for example, the purpose of the administration is to prevent the onset of the disease. "Treatment" means administering the active ingredient of the present invention to a subject (i.e., a patient) diagnosed by a physician as suffering from the disease.

[0376] The compounds of the present invention and drugs containing the same can be administered directly or orally or parenterally as suitable pharmaceutical preparations. Formulation types include, for example, tablets, capsules, powders, granules, liquids, suspensions, injections, patches, poultices, etc., but are not limited to these. Pharmaceutical preparations are prepared by common methods using pharmaceutically acceptable additives.

[0377] As additives, excipients, disintegrants, binders, fluidizers, lubricants, coating agents, solubilizers, solubilizing aids, thickeners, dispersants, stabilizers, sweeteners, flavoring agents, etc. can be used according to the purpose. Additives used herein include, for example, lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carboxymethyl cellulose calcium, crosslinked carboxymethyl cellulose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, magnesium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium dioxide, talc, and the like.

[0378] The route of administration should be selected as the most effective route for treatment, including oral administration and parenteral administration, such as intravenous injection, wiping, inhalation and eye drops. Oral administration is preferred. Dosage forms include, for example, tablets and injections, preferably tablets. The dosage of the pharmaceutical composition and its frequency of administration may depend on the route of administration and the patient's disease, symptoms, age, body weight, etc., so it is impossible to generalize. Usually, the compound of the present invention can be applied to adults at about 0.0001-about 5000mg / day, preferably about 0.001-about 1000mg / day, more preferably about 0.1-about 500mg, and particularly preferably about 1-about 300mg, which can be applied once a day or several times a day, preferably once to three times a day.

[0379] The compound of the present invention and the medicine comprising it can be used together or in combination with different medicaments to enhance the effect and / or reduce side effects. They can be used together with medicines for the treatment of central nervous system diseases such as levodopa, dopamine agonists (for example, ropinirole hydrochloride, apomorphine hydrochloride hydrate, etc.), MAO-B inhibitors (for example, selegiline hydrochloride, etc.), catechol-O-methyltransferase (COMT) inhibitors (for example, entacapone, etc.), α-Syn antibodies (for example, Prasenimab, etc.) and pharmaceutically acceptable salts thereof. Hereinafter, the medicine that can be used together with the compound of the present invention can be abbreviated as "different medicaments (used together)".

[0380] The compound of the present invention, the medicine containing it, and the administration interval of different medicaments used together should not be limited. They can be applied to the subject in need thereof at the same time, or they can be applied at intervals. In addition, the compound of the present invention and the different medicaments can be mixed as a compound medicine. The dosage of the different medicaments used together can be appropriately specified according to the dosage used clinically. The mixing ratio of the compound of the present invention and the different medicaments used together can be appropriately specified according to the subject to be treated, the route of administration, and the disease, symptoms, combination, etc. of the subject. When the subject to be treated is human, the different medicaments used together can be used, for example, at a dosage of 0.01-100 parts by weight per portion of the compound of the present invention. In order to suppress its side effects, different medicaments used together such as antiemetic agents, sleep-inducing agents, and anticonvulsant agents can be used.

[0381] Example

[0382] The present invention is explained in more detail below by referring to reference examples, examples and tests; however, the technical scope of the present invention is not limited thereto. In this specification, the term "Example" or "Reference Example" sometimes refers to the compound itself, for example, "Example 1" means "the compound of Example 1", and "Reference Example 1" means "the compound of Reference Example 1". The compound names used in Reference Examples and Examples should not always be based on the IUPAC naming system.

[0383] To simplify description, the abbreviations shown below may sometimes be used in Reference Examples, Examples, and Tests.

[0384] Me:Methyl

[0385] Et: Ethyl

[0386] Pr:n-propyl

[0387] iPr: isopropyl

[0388] DMF: N,N-dimethylformamide

[0389] THF: Tetrahydrofuran

[0390] LDA: lithium diisopropylamide

[0391] TFA: trifluoroacetic acid

[0392] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0393] The symbols used in NMR are defined as follows, s: singlet, d: doublet, dd: doublet of doublets, t: triplet, td: triplet of doublets, q: quartet, m: multiplet, br: broad, brs: broad singlet, brm: broad multiplet, and J: coupling constant.

[0394] Liquid chromatography-mass spectrometry; LC / MS analysis conditions are as follows. Mass spectrum observation values ​​[MS (m / z)] are shown as MH+, and retention time is shown as Rt (minutes). In each measurement result, A, B, C, D, E, F, G, H, I or J is added as the analysis condition used for measurement.

[0395] Analysis Condition A

[0396] MS detector: Waters ACQUITY TM SQ Detector

[0397] HPLC: ACQUITY TM UPLC

[0398] Column: Waters ACQUITY TM UPLC BEH C18 1.7μm 2.1x 30mm

[0399] Flow rate: 0.8 mL / min

[0400] Column oven temperature: 40°C

[0401] Wavelength: 254, 220nm

[0402] Mobile Phase:

[0403] A: 0.06% formic acid / H 2 O

[0404] B: 0.06% formic acid / acetonitrile

[0405] Time program:

[0406] Step time (min)

[0407] 1 0.0-1.3 A:B=98:2->4:96

[0408] 2 1.3-1.5 A:B=4:96->98:2

[0409] 3 1.5-2.2 A:B=4:98:2

[0410] Analysis Condition B

[0411] MS detector: Waters ACQUITY TM SQ Detector

[0412] HPLC: ACQUITY TM UPLC

[0413] Column: Waters ACQUITY TM UPLC BEH C18 1.7μm 2.1x30mm

[0414] Flow rate: 0.8 mL / min

[0415] Column oven temperature: 40°C

[0416] Wavelength: 254, 220nm

[0417] Mobile Phase:

[0418] A: 0.05% formic acid / H 2 O

[0419] B: Acetonitrile

[0420] Time program:

[0421] Step time (min)

[0422] 1 0.0-1.3 A:B=98:2->4:96

[0423] 2 1.3-1.5 A:B=4:96->98:2

[0424] Analysis Conditions C

[0425] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0426] Column: Xbridge C18(2)3.5μm 4.6x50mm

[0427] Flow rate: 1.8 mL / min

[0428] Wavelength: 254, 214nm

[0429] Mobile Phase:

[0430] A: 10 mM ammonium bicarbonate / H 2 O

[0431] B: Acetonitrile

[0432] Time program:

[0433] Step time (min)

[0434] 1 0.0-1.4 A:B=95:5->10:90

[0435] Column oven temperature: 50°C

[0436] Analysis Conditions D

[0437] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0438] Column: Kinetex 2.6μm EVO C18 100A 4.6x50mm

[0439] Flow rate: 2.2mL / min

[0440] Wavelength: 254, 214nm

[0441] Mobile Phase:

[0442] A: 10 mM ammonium bicarbonate / H 2 O

[0443] B: Acetonitrile

[0444] Time program:

[0445] Step time (min)

[0446] 1 0.0-1.3 A:B=90:10->5:95

[0447] 2 1.3-2.8 A:B=5:95

[0448] Column oven temperature: 50°C

[0449] Analysis Conditions

[0450] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0451] Column: Xbridge C18(2)3.5μm 4.6x50mm

[0452] Flow rate: 1.8 mL / min

[0453] Wavelength: 254, 214nm

[0454] Mobile Phase:

[0455] A: 10 mM ammonium bicarbonate / H 2 O

[0456] B: Acetonitrile

[0457] Time program:

[0458] Step time (min)

[0459] 1 0.0-1.5 A:B=90:10->5:95

[0460] Column oven temperature: 50°C

[0461] Analysis Condition F

[0462] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0463] Column: SunFire C18 3.5μm 4.6x50mm

[0464] Flow rate: 2.0mL / min

[0465] Wavelength: 254, 214nm

[0466] Mobile Phase:

[0467] A: 0.01% TFA / H 2 O

[0468] B: 0.01% TFA / acetonitrile

[0469] Time program:

[0470] Step time (min)

[0471] 1 0.0-1.4 A:B=90:10->5:95

[0472] Column oven temperature: 50°C

[0473] Analysis Condition G

[0474] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0475] Column: Xbridge C18 3.5μm 4.6x50mm

[0476] Flow rate: 1.8 mL / min

[0477] Wavelength: 254, 214nm

[0478] Mobile Phase:

[0479] A: 10 mM ammonium bicarbonate / H 2 O

[0480] B: Acetonitrile

[0481] Time program:

[0482] Step time (min)

[0483] 1 0.0-1.4 A:B=95:5->5:95

[0484] 2 1.4-3.0 A:B=5:95

[0485] Column oven temperature: 45°C

[0486] Analysis conditions

[0487] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0488] Column: Xbridge C18 3.5μm 4.6x50mm

[0489] Flow rate: 1.8 mL / min

[0490] Wavelength: 254, 214nm

[0491] Mobile Phase:

[0492] A: 10 mM ammonium bicarbonate / H 2 O

[0493] B: Acetonitrile

[0494] Time program:

[0495] Step time (min)

[0496] 1 0.0-1.3 A:B=95:5->5:95

[0497] 2 1.3-3.0 A:B=5:95

[0498] Column oven temperature: 50°C

[0499] Analysis Condition I

[0500] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0501] Column: Poroshell 120EC-C18 4.0μm 4.6x50mm

[0502] Flow rate: 2.0mL / min

[0503] Wavelength: 254, 214nm

[0504] Mobile Phase:

[0505] A: 0.01% TFA / H 2 O

[0506] B: 0.01% TFA / acetonitrile

[0507] Time program:

[0508] Step time (min)

[0509] 1 0.0-1.5 A:B=95:5->5:95

[0510] 2 1.5-3.0 A:B=5:95

[0511] Column oven temperature: 50°C

[0512] Analysis conditions

[0513] MS detector: Agilent 1200 series, Agilent 6110Quadrupole LCMS

[0514] Column: Xbridge C18 3.5μm 4.6x50mm

[0515] Flow rate: 1.8 mL / min

[0516] Wavelength: 254, 214nm

[0517] Mobile Phase:

[0518] A: 10 mM ammonium bicarbonate / H 2 O

[0519] B: Acetonitrile

[0520] Time program:

[0521] Step time (min)

[0522] 1 0.0-1.3 A:B=95:5->5:95

[0523] 2 1.3-3.0 A:B=5:95

[0524] Column oven temperature: 45°C

[0525] Reference Example 1

[0526] 1-Methylpyridine-2(1H)-imine hydroiodide

[0527]

[0528] To a solution of pyridin-2-amine (9.41 g) in THF (50 mL) was added iodomethane (18.7 mL), and the mixture was stirred at room temperature for 3 hours. The precipitated solid was collected on a filter, washed with ethyl acetate, and dried to give Reference Example 1 (22.3 g).

[0529] LC / MS ([M+H]+ / Rt(min)): 109.0 / 0.20 (Analysis Condition A)

[0530] Reference Example 2-21

[0531] The compounds shown in Table 1 were prepared according to the method described in Reference Example 1 from each corresponding starting compound.

[0532] [Table 1]

[0533]

[0534]

[0535]

[0536] Example 1

[0537] 3-Ethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide

[0538]

[0539] Reference Example 1 (0.43 g) was mixed with 3-ethyloxetane-3-carboxylic acid (0.25 g), HATU (0.77 g), triethylamine (0.51 mL) and cesium carbonate (0.60 g), and chloroform (3.7 mL) was added to the mixture. The reaction solution was stirred at room temperature for one hour, and then purified by amino silica gel chromatography (elution solvent: hexane / ethyl acetate -> ethyl acetate / methanol) to obtain Example 1 (0.24 g).

[0540] LC / MS ([M+H]+ / Rt(min)): 221.1 / 0.37 (Analysis Condition A)

[0541] 1 H NMR (400MHz, DMSO-d6) δ: 8.08-8.06 (2H, m), 7.68 (1H, t, J = 7.6Hz), 6.68 (1H, t, J = 6.8Hz), 4.78 (2 H,d,J=5.6Hz), 4.27(2H,d,J=5.6Hz), 3.67(3H,s), 1.97(2H,q,J=7.6Hz), 0.80(3H,t,J=7.2Hz).

[0542] Example 2-42

[0543] The compounds shown in Table 2 were prepared according to the method described in Example 1 from the respective starting compounds.

[0544] [Table 2]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554] Embodiment 43

[0555] 3-Methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]azetidine-3-carboxamide

[0556]

[0557] Reference Example 1 (0.10 g) was mixed with 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid, HATU (0.24 g) and triethylamine (0.18 mL), and DMF (4.2 mL) was added to the mixture. The solution was stirred at 80° C. for one hour, and then cesium carbonate (0.14 g) was added to the reaction solution, and the reaction solution was stirred at 80° C. for another hour. The reaction solution was cooled to room temperature, and water was added to the reaction solution. The quenched solution was extracted with ethyl acetate, and the organic layer was dried over magnesium sulfate. Then, the solvent in the organic layer was removed, and the residue was purified by amino silica gel chromatography (eluting solvent: hexane / ethyl acetate -> ethyl acetate / methanol). TFA (1.0 mL) was added to the resulting compound in chloroform (1.0 mL), and the mixture was stirred at room temperature for one hour. TFA was removed in vacuo, and the residue was purified by amino silica gel chromatography (eluting solvent: hexane / ethyl acetate -> ethyl acetate / methanol) to obtain Example 43 (85 mg).

[0558] LC / MS ([M+H]+ / Rt(min)): 206.1 / 0.19 (Analysis Condition A)

[0559] 1 H NMR (400 MHz, CDCl 3 )δ: 8.11(1H,d,J=9.2Hz), 7.51-7.43(2H,m), 6.46-6.42(1H,m), 4.10(2H,d,J=8.4Hz), 3.67(3H,s), 3.34(2H,d,J=8.8Hz), 1.56(3H,s).

[0560] Examples 44-48

[0561] The compounds shown in Table 3 were prepared from the respective starting compounds according to the method described in Example 43. In case the example compounds were salts, a salt-forming step was also included.

[0562] [Table 3]

[0563]

[0564]

[0565] Embodiment 49

[0566] 1,3-Dimethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]azetidine-3-carboxamide

[0567]

[0568] Example 43 (50 mg) was dissolved in methanol (2.4 mL), and a 36% aqueous formaldehyde solution (0.10 g) was added thereto. The reaction mixture was stirred at room temperature for 30 minutes, and sodium borohydride (46 mg) was added thereto. The reaction mixture was stirred at room temperature for another 30 minutes, and then the reaction mixture was purified by amino silica gel chromatography (eluting solvent: ethyl acetate / methanol) to obtain Example 49 (45 mg).

[0569] LC / MS ([M+H]+ / Rt(min)): 220.2 / 0.19 (Analysis Condition A)

[0570] 1 H NMR (400 MHz, CDCl 3 )δ: 8.12 (1H, d, J = 9.2Hz), 7.48-7.43 (2H, m), 6.43 (1H, t, J = 6.8Hz), 3.69 (3H ,s),3.50(2H,d,J=6.4Hz),3.18(2H,d,J=6.4Hz),2.30(3H,s),1.55(3H,s).

[0571] Examples 50-60

[0572] The compounds shown in Table 4 were prepared according to the method described in Example 49 from each corresponding starting compound.

[0573] [Table 4]

[0574]

[0575]

[0576]

[0577] Embodiment 61

[0578] N-[(2E)-1-Methylpyridin-2(1H)-ylidene]morpholine-4-carboxamide

[0579]

[0580] Reference Example 1 (47 mg), morpholine-4-carbonyl chloride (0.028 mL) and N-ethyl-N-isopropylpropan-2-amine (0.087 mL) were dissolved in DMF (1.0 mL), and the mixture was stirred at room temperature for one hour. The reaction solution was purified by amino silica gel chromatography (eluting solvent: hexane / ethyl acetate -> ethyl acetate / methanol) to obtain Example 61 (7.5 mg).

[0581] LC / MS ([M+H]+ / Rt(min)): 222.1 / 0.13 (Analysis Condition B)

[0582] 1 H NMR (400 MHz, CDCl 3 )δ7.95(1H,d,J=8.0Hz),7.33-7.31(2H,m),6.23-6.20(1H,m),3.68(8H,brs),3.60(3H,s).

[0583] Examples 62-65

[0584] The compounds shown in Table 5 were prepared according to the method described in Example 1 from each corresponding starting compound.

[0585] [Table 5]

[0586]

[0587] Examples 66-68

[0588] The compounds shown in Table 6 were prepared according to the method described in Example 43 from each corresponding starting compound.

[0589] [Table 6]

[0590]

[0591] Examples 69-70

[0592] The compounds shown in Table 7 were prepared according to the method described in Example 49 from each corresponding starting compound.

[0593] [Table 7]

[0594]

[0595]

[0596] Embodiment 71

[0597] 3-Methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]-1-(propan-2-yl)azetidine-3-carboxamide

[0598]

[0599] Example 43 (67 mg), acetone (56.8 mg) and acetic acid (1.96 mg) were dissolved in THF (8.0 mL), and the mixture was stirred at room temperature for 30 minutes. The reaction solution was cooled to 0° C., sodium triacetoxyborohydride (345 mg) was added thereto, and the mixture was stirred for 3 hours. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC (eluting solvent: aqueous ammonium bicarbonate solution / acetonitrile) to give Example 71 (26 mg).

[0600] LC / MS ([M+H]+ / Rt(min)): 248.2 / 1.17 (Analysis condition H)

[0601] 1 H NMR (400MHz, CD 3 OD): δ8.08-8.06(2H,m),7.84-7.80(1H,m),6.87(1H,td,J=6.8,1.2Hz), 4.06(2H,d,J=9.2Hz), 3.87(3H,s), 3.59(2H,d,J=9.2Hz), 2.95(1H,sep,J=6.0Hz), 1.61(3H,s), 1.12(6H,d,J=6.0Hz).

[0602] Embodiment 72

[0603] 1-Cyclopropyl-3-methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]azetidine-3-carboxamide

[0604]

[0605] Example 43 (101 mg), (1-ethoxycyclopropyloxy)trimethylsilane (256 mg) and acetic acid (2.95 mg) were dissolved in ethanol (8.0 mL), and the solution was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (185 mg) was added to the reaction solution, and the mixture was heated and stirred at 60° C. for 3 hours. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC (eluting solvent: aqueous ammonium bicarbonate solution / acetonitrile) to give Example 72 (31 mg).

[0606] LC / MS ([M+H]+ / Rt(min)): 246.2 / 1.30 (analysis condition H)

[0607] 1 H NMR (400MHz, CD 3 OD): δ8.04-8.03(1H,m),7.98(1H,d,J=9.2Hz),7.79-7.75(1H,m),6.82(1H,td,J=6.8,1.2Hz),3.85(3 H,s),3.79(2H,d,J=8.0Hz),3.27(2H,d,J=8.0Hz),2.06-2.01(1H,m),1.56(3H,s),0.45-0.37(4H,m).

[0608] Embodiment 73

[0609] 3-Methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]-1-(2,2,2-trifluoroethyl)azetidine-3-carboxamide

[0610]

[0611] Example 43 (134 mg) and triethylamine (329 mg) were dissolved in THF (4.0 mL), and the solution was cooled to 0° C. 2,2,2-trifluoroethyl trifluoromethanesulfonate (0.10 g) was added to the reaction solution, and the mixture was stirred for 3 hours. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC (eluting solvent: aqueous ammonium bicarbonate / acetonitrile) to give Example 73 (16 mg).

[0612] LC / MS ([M+H]+ / Rt(min)): 288.1 / 1.44 (Analysis condition H)

[0613] 1 H NMR (400MHz, CD 3 OD): δ8.05(1H,d,J=6.4Hz),7.99(1H,d,J=8.8Hz),7.79(1H,t,J=7.8Hz),6.8 3(1H,m),3.85(3H,s),3.78(2H,d,J=7.6Hz),3.36-3.34(2H,m),3.15(2H,q,J H-F =5.6Hz),1.59(3H,s).

[0614] Embodiment 74

[0615] 3,3-Dimethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]morpholine-4-carboxamide

[0616]

[0617] 3,3-Dimethylmorpholine (98 mg) and pyridine (133 mg) were dissolved in dichloromethane (4.0 mL), and the solution was cooled to 0°C. Triphosgene (100 mg) was added to the reaction solution, and the mixture was stirred at 0°C for 30 minutes. Then, N-ethyl-N-isopropylpropan-2-amine (328 mg) and Reference Example 1 (100 mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC (eluting solvent: aqueous ammonium bicarbonate solution / acetonitrile) to give Example 74 (17 mg).

[0618] LC / MS ([M+H]+ / Rt(min)): 250.1 / 1.41 (Analysis Condition J)

[0619] 1 H NMR (400MHz, CD 3 OD): δ7.66(1H,d,J=6.0Hz),7.40-7.35(2H,m),6.34(1H,td,J=6.4,2.0Hz), 3.76-3.73(2H,m),3.71-3.68(2H,m),3.61(3H,s),3.38(2H,s),1.45(6H,s).

[0620] Embodiment 75

[0621] 2,2-Dimethyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]morpholine-4-carboxamide

[0622]

[0623] Reference Example 1 (47 mg) and triethylamine (64 mg) were dissolved in dichloromethane (2.0 mL), and the solution was cooled to 0°C. 4-Nitrophenyl chloroformate (27 mg) was added to the reaction solution, and the mixture was stirred for 2 hours. 2,2-Dimethylmorpholine (76 mg) was then added to the mixture, and the mixture was stirred for 2 hours. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC (eluting solvent: aqueous ammonium bicarbonate / acetonitrile) to give Example 75 (35 mg).

[0624] LC / MS ([M+H]+ / Rt(min)): 250.2 / 1.39 (analysis condition H)

[0625] 1 H NMR (400MHz, CD 3OD): δ7.74(1H,dd,J=7.2,1.2Hz),7.64(1H,d,J=8.8Hz),7.50-7.45(1H,m),6.43 (1H,td,J=6.8,1.2Hz),3.78(3H,s),3.72(3H,s),3.66-3.42(3H,m),1.23(6H,s).

[0626] Embodiment 76

[0627] The compounds shown in Table 8 were prepared according to the method described in Example 75 from each corresponding starting compound.

[0628] [Table 8]

[0629]

[0630]

[0631] Experiment 1: Reproduction of neurospheres prepared by three-dimensional culture of human iPS cells with PLA2G6 gene mutation Trials for Parkinson's disease symptoms (α-synuclein aggregates)

[0632] PLA2G6 gene mutant cells established from a healthy human iPS cell line (clone name: 201B7, obtained from the iPS Cell Research Institute of Kyoto University, Japan) were cultured in StemFitAK03N medium (Ajinomoto Co., Inc., Basic03) at 37°C and 5% CO 2 Next cultivation.

[0633] Neural stem cells were induced from iPS cells using PSC Neuron Induction Medium (Thermo Fisher Scientific Inc., cat#A1647801) to prepare a cell stock solution thereof.

[0634] Cryopreserved neural stem cells were cultured in culture medium at 37°C and 5% CO. 2 The culture medium used for neural stem cells herein has the following composition.

[0635] Neural stem cell culture medium composition:

[0636] Neurobasal medium (Thermo Fisher Scientific Inc., 2113049)

[0637] Advanced DMEM / F-12 medium (Thermo Fisher Scientific Inc., 12634028)

[0638] Neural induction supplement (Thermo Fisher Scientific Inc., A1647801)

[0639] Neural stem cells (10,000 cells / well) were seeded in 96-well round-bottom plates (Thermo Fisher Scientific Inc., cat#174929) and incubated in culture medium at 37°C and 5% CO. 2 Half of the culture solution was replaced every 3 to 4 days. The culture medium used for neurospheres herein has the following composition.

[0640] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0641] NeuroCult SM1 Neuronal Supplement (STEMCELL Technologies, cat#05711)

[0642] N2 Supplement-A (STEMCELL Technologies, cat#07152)

[0643] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0644] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0645] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0646] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0647] The differentiated neurospheres were removed from the culture medium, a TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32) was added thereto, and proteins were extracted from the mixture using an ultrasonicator.

[0648] The extracted proteins were subjected to protein analysis (Protein Simple, Inc., cat# SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat# AHB0261) with a Simple Western system under non-reducing conditions to measure the amount of α-synuclein aggregates and evaluate the quantification of waveforms displayed at a molecular weight of approximately 300 kD.

[0649] From the 7th day to the 9th day of culture, α-synuclein aggregates increased dramatically. On the 9th day of culture, the amount of α-synuclein aggregates in neurospheres derived from PLA2G6 mutant iPS cells was 5 times or more than that in neurospheres derived from healthy iPS cells. After the 9th day of culture, it showed a trend of slow increase. Figure 1 Shown in.

[0650] Experiment 2: Evaluation of α-synuclein using neurospheres prepared from human iPS cells with PLA2G6 gene mutation Inhibition of aggregate accumulation

[0651] (1) Inducing human iPS cells to differentiate into neural cells

[0652] Neural stem cells were induced from PLA2G6 gene mutant iPS cells using PSC neuron induction medium (Thermo Fisher Scientific Inc., cat#A1647801). Neurospheres were prepared from induced neural stem cells by a three-dimensional culture system and preserved with BrainPhys neuron medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuron supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the medium was replaced on the 2nd and 4th days after induction of differentiation.

[0653] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 4 after differentiation induction.

[0654] (2) Evaluation of the amount of α-synuclein aggregates

[0655] From the neurospheres 9 days after induction of differentiation, proteins were extracted with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat#SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat#AHB0261) to measure the amount of α-synuclein aggregates.

[0656] Assuming that the amount of aggregates in the neurosphere to which the DMSO solution was added was 100%, the amount of aggregates in the neurosphere to which each test compound was added was evaluated. Table 9 shows the amount of aggregates for representative test compounds.

[0657] [Table 9]

[0658]

[0659]

[0660]

[0661] Experiment 3: Evaluation of α-synuclein using neurospheres prepared from human iPS cells with PLA2G6 gene mutation Reduction in aggregate accumulation

[0662] (1) Inducing human iPS cells to differentiate into neural cells

[0663] Neural stem cells were induced from PLA2G6 gene mutant iPS cells using PSC neuron induction medium (Thermo Fisher Scientific Inc., cat#A1647801). Neurospheres were prepared from induced neural stem cells by a three-dimensional culture system and preserved with BrainPhys neuron medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuron supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days after differentiation was induced.

[0664] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 10 after differentiation induction.

[0665] (2) Evaluation of the amount of α-synuclein aggregates

[0666] From neurospheres 15 days after induction of differentiation, proteins were extracted with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat#SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat#AHB0261) to measure the amount of α-synuclein aggregates.

[0667] The amount of aggregates in the neurospheres to which each test compound was added was measured. Assuming that the amount of aggregates in the neurospheres to which the DMSO solution was added was 100%, the amount of aggregates in the test samples was evaluated. Table 10 shows the amount of aggregates (%) for representative test compounds.

[0668] [Table 10]

[0669]

[0670] Experiment 4: Reproduction of Parkinson's disease using dopamine neurospheres prepared from human iPS cells with a mutated PLA2G6 gene Test for the pathology of α-synuclein aggregates

[0671] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701) to prepare a cell stock solution.

[0672] The cells were cultured in a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific Inc., cat# A3165801) at 37°C and 5% CO. 2 Cryopreserved dopaminergic progenitor cells were cultured under low temperature.

[0673] First, dopaminergic progenitor cells (10,000 cells / well) were seeded in 96-well round-bottom plates (Thermo Fisher Scientific Inc., cat#174929) and incubated in culture medium at 37°C and 5% CO. 2 After differentiation induction, half of the culture medium was replaced every 3 or 4 days. The culture medium used for dopamine neurospheres herein has the following composition.

[0674] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0675] Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific Inc., cat# A3147401)

[0676] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0677] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0678] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0679] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0680] The differentiated dopamine neurospheres were removed from the culture medium, a TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32) was added thereto, and proteins were extracted from the mixture using an ultrasonicator.

[0681] The extracted proteins were subjected to protein analysis (Protein Simple, Inc., cat# SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat# AHB0261) with a Simple Western system under non-reducing conditions to measure the amount of α-synuclein aggregates and evaluate the quantification of waveforms displayed at a molecular weight of approximately 300 kD.

[0682] From the 10th day to the 21st day of culture, α-synuclein aggregates increased dramatically. On the 21st day of culture, the amount of α-synuclein aggregates in dopamine neurospheres derived from PLA2G6 mutant iPS cells was 5 times or more than that in dopamine neurospheres derived from healthy iPS cells. After the 21st day of culture, it showed a trend of slow increase. Figure 2 Shown in.

[0683] Experiment 5: Evaluation of α-synapses using dopamine neurospheres prepared from human iPS cells with a PLA2G6 gene mutation Inhibition of nuclear protein aggregate accumulation

[0684] (1) Inducing human iPS cells to differentiate into neural cells

[0685] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells by a three-dimensional culture system and preserved with BrainPhys neuronal medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuronal supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days after differentiation was induced.

[0686] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 21 after differentiation induction.

[0687] (2) Evaluation of the amount of α-synuclein aggregates

[0688] From dopamine neurospheres 26 days after induction of differentiation, proteins were extracted with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat#SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat#AHB0261) to measure the amount of α-synuclein aggregates.

[0689] The amount of aggregates in the neurospheres to which each test compound was added was measured. Assuming that the amount of aggregates in the neurospheres to which the DMSO solution was added was 100%, the amount of aggregates in the test samples was evaluated. Table 11 shows the amount of aggregates (%) for representative test compounds.

[0690] [Table 11]

[0691]

[0692] Experiment 6: Reproduction of neuronal fragility using dopamine neurospheres prepared from human iPS cells with a PLA2G6 gene mutation Weak method

[0693] (1) Inducing human iPS cells to differentiate into neural cells

[0694] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells by a three-dimensional culture system and preserved with BrainPhys neuronal medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuronal supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days after differentiation was induced.

[0695] (2) Evaluation of the amount of tyrosine hydroxylase

[0696] From the dopamine neurospheres 26 days after differentiation induction, proteins were extracted with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat#SM-W008) using a tyrosine hydroxylase antibody (Millipore, cat#AB152) to measure the amount of tyrosine hydroxylase. Figure 3 Shown in.

[0697] Experiment 7: Evaluation of neuronal fragility using dopamine neurospheres prepared from human iPS cells with PLA2G6 gene mutation Improvement of weakness

[0698] (1) Inducing human iPS cells to differentiate into neural cells

[0699] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells by a three-dimensional culture system and preserved in BrainPhys neuronal medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuronal supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days.

[0700] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 21 after differentiation induction.

[0701] (2) Evaluation of the amount of tyrosine hydroxylase

[0702] From dopamine neurospheres 26 days after differentiation induction, proteins were extracted with TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat# SM-W008) using a tyrosine hydroxylase antibody (Millipore, cat# AB152) to measure the amount of tyrosine hydroxylase.

[0703] Experiment 8: Reproduction of neuronal cell death using neurospheres prepared from human iPS cells with PLA2G6 gene mutation Method of death

[0704] (1) Inducing human iPS cells to differentiate into neural cells

[0705] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells by a three-dimensional culture system and preserved in BrainPhys neuronal medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuronal supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days.

[0706] (2) Evaluation of neuronal cell death

[0707] 10 μM dopamine was added to the culture medium 35 days after differentiation induction. Proteins were extracted from dopamine neurospheres 40 days after differentiation induction with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (ProteinSimple, Inc., cat# SM-W008) using a cleaved caspase 3 antibody (Cell Signaling Technology, Inc., cat# 9664) to measure the amount of neuronal cell death. Results Figure 4 Shown in.

[0708] Experiment 9: Evaluation of neuronal cell death using neurospheres prepared from human iPS cells with PLA2G6 gene mutation Suppression of death

[0709] (1) Inducing human iPS cells to differentiate into neural cells

[0710] Dopaminergic progenitor cells were induced from PLA2G6 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells by a three-dimensional culture system and preserved in BrainPhys neuronal medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 neuronal supplement, N2 supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days.

[0711] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the twice-concentration solution and 10 μM dopamine were added to each well when half of the medium was replaced on day 35 after differentiation induction.

[0712] (2) Evaluation of neuronal cell death

[0713] From dopamine neurospheres 40 days after induction of differentiation, proteins were extracted with TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat# SM-W008) using a cleaved caspase 3 antibody (Cell Signaling Technology, Inc., cat# 9664) to measure the amount of neuronal cell death.

[0714] Experiment 10: Reproduction of neurospheres prepared by three-dimensional culture of human iPS cells with GBA1 gene mutation Methods for treating Parkinson's disease conditions (alpha-synuclein aggregate accumulation)

[0715] GBA1 gene homozygous mutant cells established from healthy human iPS cell lines were cultured in StemFitAK03N medium (Ajinomoto Co., Inc., Basic03) at 37°C and 5% CO 2 Next cultivation.

[0716] Dopaminergic progenitor cells were induced from GBA1 homozygous mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701) to prepare a cell stock solution.

[0717] The cells were cultured in a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific Inc., cat# A3165801) at 37°C and 5% CO.2 Cryopreserved dopaminergic progenitor cells were cultured under low temperature.

[0718] Dopaminergic progenitor cells (10,000 cells / well) were seeded in 96-well round-bottom plates (Thermo Fisher Scientific Inc., cat#174929) and incubated in culture medium at 37°C and 5% CO. 2 After differentiation induction, half of the culture solution was replaced every 3 to 4 days. The culture medium used for dopamine neurospheres herein has the following composition.

[0719] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0720] Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific Inc., cat# A3147401)

[0721] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0722] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0723] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0724] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0725] The differentiated DA neurospheres were removed from the culture medium, a TBS solution (Nacalai Tesque, Inc., cat#12748-31) containing 1% TritionX-100 (Nacalai Tesque, Inc., cat#12967-32) was added thereto, and proteins were extracted from the mixture using an ultrasonicator.

[0726] The extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat# SM-W008) under non-reducing conditions using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat# AHB0261) to measure the amount of α-synuclein aggregates and evaluate the quantification of waveforms displayed at a molecular weight of approximately 300 kD.

[0727] From the 21st to the 40th day of culture, the amount of α-synuclein aggregates increased dramatically. On the 40th day of culture, the amount of aggregates reached saturation, and no change was observed thereafter. Figure 5 Shown in.

[0728] Experiment 11: Evaluation of α-synapses using dopamine neurospheres prepared from human iPS cells with GBA1 gene mutation Reduction of nuclear protein aggregate accumulation

[0729] (1) Inducing human iPS cells to differentiate into neural cells

[0730] Dopaminergic progenitor cells were induced from GBA1 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells using a three-dimensional culture system and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP, and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days.

[0731] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 40 after differentiation induction.

[0732] (2) Evaluation of the amount of α-synuclein aggregates

[0733] From dopamine neurospheres 44 days after induction of differentiation, proteins were extracted with a TBS solution containing 1% TritionX-100, and the extracted proteins were subjected to protein analysis using a Simple Western system (Protein Simple, Inc., cat#SM-W008) using an α-synuclein antibody (Thermo Fisher Scientific Inc., cat#AHB0261) to measure the amount of α-synuclein aggregates.

[0734] The amount of aggregates in the neurospheres to which each test compound was added was measured. Assuming that the amount of aggregates in the neurospheres to which the DMSO solution was added was 100%, the amount of aggregates in the test samples was evaluated. Table 12 shows the amount of aggregates (%) for representative test compounds.

[0735] [Table 12]

[0736]

[0737] Experiment 12: Examination of neurospheres prepared by three-dimensional culture of human iPS cells with GBA1 gene mutation Synchronous discharge abnormality

[0738] GBA1 gene homozygous mutant cells established from healthy human iPS cell lines were cultured in StemFitAK03N medium (Ajinomoto Co., Inc., Basic03) at 37°C and 5% CO 2 Next cultivation.

[0739] Dopaminergic progenitor cells were induced from GBA1 homozygous mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701) to prepare a cell stock solution.

[0740] The cells were cultured in a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific Inc., cat# A3165801) at 37°C and 5% CO. 2 Cryopreserved dopaminergic progenitor cells were cultured under low temperature.

[0741] Dopaminergic progenitor cells (10,000 cells / well) were seeded in 96-well round-bottom plates (Thermo Fisher Scientific Inc., cat#174929) and incubated in culture medium at 37°C and 5% CO. 2 After differentiation induction, half of the culture solution was replaced every 3 to 4 days. The culture medium used for dopamine neurospheres herein has the following composition.

[0742] BrainPhys Neuronal Culture Medium (STEMCELL Technologies, cat#ST-05793)

[0743] Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific Inc., cat# A3147401)

[0744] 20ng / mL BDNF (PeproTech, Inc., cat#450-02)

[0745] 20ng / mL GDNF (PeproTech, Inc., cat#450-10)

[0746] 1mM dibutyryl cAMP (Nacalai Tesque, Inc., cat#11540-74)

[0747] 200 nM ascorbic acid (Nacalai Tesque, Inc., cat#03420-52)

[0748] On the 40th day or later after induction of differentiation, half of the culture medium was removed, and a measurement culture medium containing a fluorescent calcium probe (Molecular Devices, product name: FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) equal to the remaining culture medium was added, and the mixture was left to stand for 30 minutes before measurement. The measurement culture medium used in this article is 20mM Hepes (Thermo Fisher Scientific Inc., cat#15630-080) and Hank's buffer solution (Thermo Fisher Scientific Inc., cat#14065-056) containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Shooting was performed at one frame per second.

[0749] Experiment 13: Evaluation of synchronization using dopamine neurospheres prepared from human iPS cells with GBA1 gene mutation Improvement of abnormal discharge

[0750] (1) Inducing human iPS cells to differentiate into neural cells

[0751] Dopaminergic progenitor cells were induced from GBA1 gene mutant iPS cells using a dopaminergic neuron differentiation kit (Thermo Fisher Scientific Inc., cat#A3147701). Dopamine neurospheres were prepared from induced dopaminergic progenitor cells using a three-dimensional culture system and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793), which contained NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20ng / mL BDNF, 20ng / mL GDNF, 1mM dibutyryl cAMP, and 200nM ascorbic acid. Half of the culture medium was replaced every 3 or 4 days.

[0752] The test compound was diluted with the medium to twice the final concentration, and an equal volume of the double-concentration solution was added to each well when half of the medium was replaced on day 40 after differentiation induction.

[0753] (2) Evaluation of synchronous discharge

[0754] On the 44th day after induction of differentiation, half of the culture medium of the dopamine neurosphere was removed, and a measurement culture medium containing a fluorescent calcium probe (Molecular Devices, product name: FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) equal to the remaining culture medium was added, and the mixture was left to stand for 30 minutes and then measured. The measurement culture medium used in this article is 20mM Hepes (Thermo Fisher Scientific Inc., cat#15630-080) and Hank's buffer solution (Thermo Fisher Scientific Inc., cat#14065-056) containing 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Shooting was performed at one frame per second.

[0755] Industrial Applicability

[0756] The compound of the present invention has the effect of inhibiting or reducing the accumulation of α-synuclein aggregates, and therefore, the compound of the present invention can be used as a drug for treating or preventing central nervous system diseases, characterized by the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain. In addition, the present invention can be used as a method for reproducing Parkinson's disease symptoms using neurospheres, and a method for evaluating the amount of α-synuclein aggregates using the reproducing method.

[0757] As described above, the compound of formula (1) or a pharmaceutically acceptable salt thereof has the effect of inhibiting or reducing the accumulation of α-synuclein aggregates. Therefore, the compound of formula (1) or a pharmaceutically acceptable salt thereof can be used as a drug for treating or preventing central nervous system diseases associated with α-synuclein aggregates such as Parkinson's disease and Lewy body dementia.

Claims

1. A compound of formula (1): or a pharmaceutically acceptable salt thereof, wherein X is oxygen or NR 5 , R 5 is hydrogen, C 1-6 alkyl optionally substituted with 1 to 6 identical or different halogen atoms or C 3-6 cycloalkyl, m is 0, 1 or 2, n is 0, 1, 2, 3 or 4, Y is CH or nitrogen, provided that when m is 0, then Y is CH and n is 1, 2, 3 or 4, when m is 1, then Y is CH and n is 0, 1, 2 or 3, and when m is 2, then n is 1 or 2, R 1 and R 2 are independently hydrogen or C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms, or R 1 and R 2 together form a bridged methylene or ethylene group, R 3 and R 4 are independently hydrogen, halogen, C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms or C 1-6 alkoxy optionally substituted by 1 to 6 identical or different halogen atoms, and Z is C 1-6 alkyl group.

2. The compound according to claim 1, said compound being represented by formula (2): wherein X is oxygen or NR 5 , R 5 is hydrogen, C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms or C 3-6 cycloalkyl, m is 0, 1 or 2, n is 0, 1, 2, 3 or 4, Y is CH or nitrogen, provided that when m is 0, then Y is CH and n is 1, 2, 3 or 4, when m is 1, then Y is CH and n is 0, 1, 2 or 3, and when m is 2, then n is 1 or 2, R 1 and R 2 are independently hydrogen or C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms, or R 1 and R 2 together form a bridged methylene or ethylene group, R 3 and R 4 are independently hydrogen, halogen, C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms or C 1-6 alkoxy optionally substituted by 1 to 6 identical or different halogen atoms, and Z is C 1-6 alkyl or a pharmaceutically acceptable salt thereof.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein Y is CH.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 2 is hydrogen.

5. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein n is 0, 1 or 2, provided that when n is 2, then m is 0 or 1.

6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein m is 1 and n is 0.

7. The compound according to claim 1, said compound being represented by formula (3): wherein X is oxygen or NR 5 , R 5 is hydrogen, C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms, or C 3-6 cycloalkyl optionally substituted by 1 to 6 identical or different halogen atoms, R 1 is an optionally C-substituted by 1 to 6 identical or different halogen atoms 1-6 alkyl group R 3 and R 4 are independently hydrogen, halogen, C 1-6 alkyl optionally substituted by 1 to 6 identical or different halogen atoms or C 1-6 alkoxy optionally substituted by 1 to 6 identical or different halogen atoms, and Z is C 1-6 alkyl or a pharmaceutically acceptable salt thereof.

8. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl or ethyl.

9. A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein R 5 is hydrogen, C 1-3 alkyl optionally substituted by 1 to 6 identical or different halogen atoms, or cyclopropyl.

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, wherein X is NR 5 , and R 5 is hydrogen or methyl.

11. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein Z is methyl or ethyl.

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl.

13. The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 are independently hydrogen, halogen, C 1-3 alkyl optionally substituted with 1 to 6 fluorine atoms or C 1-3 alkoxy optionally substituted with 1 to 6 fluorine atoms.

14. The compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein R 3 is methyl or methoxy.

15. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein X is oxygen.

16. The compound or a pharmaceutically acceptable salt thereof according to claim 15, wherein Z is methyl or ethyl.

17. The compound or a pharmaceutically acceptable salt thereof according to claim 15 or 16, wherein R 3 and R 4 are independently hydrogen, halogen, C 1-3 alkyl optionally substituted with 1 to 6 fluorine atoms or C 1-3 alkoxy optionally substituted with 1 to 6 fluorine atoms.

18. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 and 15 to 17, wherein R 3 and R 4 are independently hydrogen, fluorine or methoxy.

19. A compound according to any one of claims 1 to 13 and 15 to 17 or a pharmaceutically acceptable salt thereof, wherein R 3 and R 4 are both hydrogen.

20. The compound according to claim 1, said compound being selected from: 3 - ethyl - N - [(2E) - 1 - methylpyridin - 2(1H) - ylidene]oxetane - 3 - carboxamide, 3 - methyl - N - [(2E) - 1 - methylpyridin - 2(1H) - ylidene]oxetane - 3 - carboxamide, N - [(2E) - 1 - ethylpyridin - 2(1H) - ylidene] - 3 - methyloxetane - 3 - carboxamide, 3 - ethyl - N - [(2E) - 4 - methoxy - 1 - methylpyridin - 2(1H) - ylidene]oxetane - 3 - carboxamide, 3 - ethyl - N - [(2E) - 5 - fluoro - 1 - methylpyridin - 2(1H) - ylidene]oxetane - 3 - carboxamide, N - [(2E) - 4 - methoxy - 1 - methylpyridin - 2(1H) - ylidene] - 1,3 - dimethylazetidine - 3 - carboxamide, and N - [(2E) - 5 - chloro - 4 - methoxy - 1 - methylpyridin - 2(1H) - ylidene] - 1,3 - dimethylazetidine - 3 - carboxamide, or a pharmaceutically acceptable salt thereof.

21. The compound according to claim 1, said compound being selected from: 3 - ethyl - N - [(2E) - 1 - methylpyridin - 2(1H) - ylidene]oxetane - 3 - carboxamide, 3-Methyl-N-[(2E)-1-methylpyridin-2(1H)-ylidene]oxetane-3-carboxamide, N-[(2E)-1-ethylpyridin-2(1H)-ylidene]-3-methyloxetane-3-carboxamide, and N-[(2E)-4-methoxy-1-methylpyridin-2(1H)-ylidene]-1,3-dimethylazetidine-3-carboxamide, or a pharmaceutically acceptable salt thereof.

22. A drug, which comprises the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof as an active ingredient.

23. A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, which comprises the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof as an active ingredient.

24. The drug according to claim 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is a central nervous system disease related to tau, α-synuclein, TDP-43 or polyglutamine.

25. The drug according to claim 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is Alzheimer's disease, frontotemporal degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher's disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease or spinocerebellar ataxia.

26. The drug according to claim 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is a central nervous system disease related to α-synuclein.

27. The drug according to claim 23, wherein the central nervous system disease caused by abnormal aggregates of brain proteins is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher's disease or infantile neuroaxonal dystrophy.

28. A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, which comprises a combination of the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof and at least one agent selected from L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, αSyn antibodies and pharmaceutically acceptable salts thereof.

29. A drug for treating or preventing a central nervous system disease caused by abnormal aggregates of brain proteins, which comprises the compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and is used in combination with at least one agent selected from L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, αSyn antibodies and pharmaceutically acceptable salts thereof.

Images