JP7618237B2 - Motor neuron degeneration inhibitors - Google Patents

Description

The present invention relates to an agent (medicine, pharmaceutical composition) containing a compound that has an inhibitory effect on motor neuron degeneration and is useful in the treatment of motor neuron diseases (e.g., amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy), etc.

BACKGROUND OF THEINVENTION
Neurodegenerative diseases are diseases caused by the degeneration of specific nerve cells. The degenerated nerve cells eventually become dysfunctional and eventually die. The degeneration, dysfunction, and cell death gradually spread to surrounding nerve cells, and when the overall proportion of dysfunctional nerve cells exceeds a certain threshold, the disease develops, and the pathology progresses as the proportion increases (e.g., Non-Patent Documents 1 and 2).
Therefore, it is believed that if the initial change, neuronal degeneration, can be effectively suppressed, the subsequent functional impairment will also be suppressed, and the progression of the pathology can be effectively prevented (for example, non-patent documents 3, 4 and 5).
In order to obtain candidates for therapeutic drugs for neurodegenerative diseases, many attempts have been made to search for compounds that can suppress neurodegeneration in cell models that mimic neurodegeneration (e.g., Non-Patent Documents 6 and 7). However, since most of these models are based on cell lines, differences from original human nerve cells are problematic.
In order to solve this problem, in recent years, there has been active drug discovery research in which disease model cells are prepared from induced pluripotent stem cells (hereinafter, iPS cells) obtained by initializing somatic cells collected from patients, and the cells are subjected to compound screening (for example, Non-Patent Document 8). The present inventor has previously reported that by introducing and expressing Lhx3, Ngn2, and Isl1 into iPS cells, it is possible to efficiently induce differentiation into motor neurons that have the properties of motor neurons in vivo in a short period of time (Patent Document 1). Using this method, it was revealed that motor neurons induced to differentiate from iPS cells derived from patients with amyotrophic lateral sclerosis (hereinafter, sometimes referred to as "ALS"), a representative motor neuron disease, spontaneously undergo neurodegeneration similar to that of the disease and lead to cell death, i.e., they are excellent cell models for ALS (Patent Document 1, Non-Patent Document 9). Therefore, if a compound that suppresses the spontaneous cell degeneration in motor neurons induced to differentiate from patient-derived iPS cells can be found, it is expected that a novel drug can be developed that suppresses the progression of the disease and ultimately improves symptoms (e.g., Non-Patent Documents 9, 10, and 11, and Patent Documents 2 and 3).

On the other hand, the following compounds are known from Patent Documents 4 to 8.
(1) Patent Document 4

(2) Patent Document 5

and

(3) Patent Document 6

(4) Patent Document 7

(5) Patent Document 8

The following compounds have also been reported:
(1) RN 1358409-10-5

(2) RN 1391415-15-8

(3) RN 765925-17-5

However, there is no disclosure in any of the literature that the above compounds have an inhibitory effect on motor neuron degeneration.

WO 2014/148646 WO 2016/114322 WO 2012/029994 WO 2008/016192 WO 2008/044767 WO 2009/119776 WO 2011/024872 US 5514505

K. W. Li et al, Journal of Neurochemistry 2018, 10.1111/jnc.14603 N. J. Ashton et al, Acta Neuropathologica Communications 2019, 7:5 S. Zhang et al, Cell Death and Disease 2017, 8, e2905 T. V. Berghe et al, Nature Reviews Molecular Cell Biology 2014, 15, 135-147. Z. Lia et al, European Journal of Pharmacology 2019, 844, 156-164. G. R. Smith et al, Journal of Molecular Neuroscience 2014, 52, 446-458. W. Fischer et al, Redox Biology 2019, 21, 101089 Y. Shi et al, Nature Reviews Drug Discovery 2017, 16, 115-130. K. Imamura et al, Science Translational Medicine 2017, 9, eaaf3962 N. Egawa et al, Science Translational Medicine 2012, 4, 145ra104 K. Fujimori et al, Nature Medicine 2018, 24, 1579-1589.

The object of the present invention is to provide an agent (medicine, pharmaceutical composition) which contains a compound having an inhibitory effect on motor neuron degeneration and can be used as a preventive or therapeutic agent for motor neuron diseases (e.g., amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy) and the like.

As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that the following compound has an inhibitory effect on motor neuron degeneration and therefore can be used as a preventive or therapeutic agent for motor neuron diseases (e.g., amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy), etc., and thus completed the present invention.

That is, the present invention is as follows.
[1] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof (hereinafter also referred to as "compounds used in the present invention").
[2] The agent according to [1], which is used as an agent for the prevention or treatment of a motor neuron disease.
[3] The agent according to [1], which is used as an agent for the prevention or treatment of amyotrophic lateral sclerosis.
[4] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof, comprising administering to said mammal an effective amount of one or more compounds selected from the group consisting of ribozyme, ribozyme A and salts thereof.
[5] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof, comprising administering to said mammal an effective amount of one or more compounds selected from the group consisting of ribozymes, rhabdomyolytes, erythrocytes, cerebrospinal fluid ...
[6] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof, comprising administering to said mammal an effective amount of one or more compounds selected from the group consisting of benzodiazepines, benzodiazepines, benzoyl peroxidases, and salts thereof.
[7] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide for use in the prevention or treatment of motor neuron diseases.
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof.
[8] N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide for use in the prevention or treatment of amyotrophic lateral sclerosis.
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide,
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide,
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide,
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide,
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid,
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole,
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide,
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide,
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide,
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide,
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide,
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine,
and salts thereof.

According to the present invention, it is possible to provide an agent (medicine, pharmaceutical composition) which contains a compound having excellent inhibitory activity against motor neuron degeneration and can be used as a preventive or therapeutic agent for motor neuron diseases (e.g., amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy) and the like.

FIG. 1 shows typical stained images of motor neurons differentiated from ALS10 cells (6 days after the start of culture).

Detailed Description of the Invention
The present invention will be described in detail below.
The agent of the present invention comprises the following compound:
N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide (Example No. 1),
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide (Example No. 2),
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide (Example No. 3),
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide (Example No. 4),
3-ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (Example No. 5),
1-[(3-methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide (Example No. 6),
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid (Example No. 7),
3-(pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole (Example No. 8),
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide (Example No. 9),
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide (Example No. 10),
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide (Example No. 11),
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide (Example No. 12),
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide (Example No. 13),
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine (Example No. 14),
and salts thereof.

Among the compounds used in the present invention, the following compounds are included:
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide (Example No. 10),
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide (Example No. 11),
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide (Example No. 12),
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide (Example No. 13),
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine (Example No. 14),
and their salts are novel compounds.

When the compound used in the present invention is a salt, a pharmacologically acceptable salt is preferable. Examples of such salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic amino acids, salts with acidic amino acids, etc.
Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; and ammonium salts.
Preferable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, and the like.
Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like.
Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like.
Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc.
Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like.

When the compound used in the present invention is obtained as a free compound, it can be converted into the desired salt by a method known per se. Alternatively, when the compound used in the present invention is obtained as a salt, it can be converted into the free form or another type of desired salt by a method known per se.

As used herein, "neuron degeneration" refers to a morphological abnormality in neurons, specifically, the occurrence of one or more of the following abnormalities: neurite retraction, neurite fragmentation, neurite loss, cell body atrophy, cell body fragmentation, and cell body loss. Motor neuron degeneration means that the degeneration occurs in motor neurons. Here, motor neurons include both upper motor neurons and lower motor neurons. Furthermore, motor neurons in this specification also include motor neurons induced to differentiate from pluripotent stem cells.

Motor neuron degeneration can be detected by morphological observation. For example, morphological observation may be performed by immunostaining or the like using a method known to those skilled in the art, and measuring the length of the neurites, the presence or absence of fragmentation, or the number and size of cell bodies for cells expressing a motor neuron marker gene (e.g., HB9, ChAT, etc.), cells expressing a reporter gene controlled by the promoter of the marker gene, or cells expressing a neuron marker gene (e.g., β-III tubulin, NCAM, MAP2, etc.). These measurements can be performed visually, but may also be automatically measured using a cell image analyzer (e.g., IN Cell Analyzer (GE Healthcare), Opera Phenix (PerkinElmer), etc.). Furthermore, the neurite length and cell body size may be measured as the area on an image of the neurite or cell body.
Motor neuron degeneration may also be evaluated using cell death as an indicator. In a cell model of a motor neuron disease, cell death occurs after neurodegeneration, and the proportion of cells that have undergone cell death can be evaluated as the rate of neuron degeneration.
Furthermore, the proportion of motor neurons that have undergone cell death may be calculated as the reciprocal of the number of surviving motor neurons.

In the present specification, "inhibition of motor neuron degeneration" refers to inhibition of motor neuron degeneration, specifically, reduction of one or more abnormalities including neurite retraction, neurite fragmentation, neurite loss, cell body atrophy, cell body fragmentation, and cell body loss. Furthermore, the final phenotype of reduction of these abnormalities can be evaluated using an increase in the number of surviving motor neurons as an index.
The "motor neuron degeneration inhibitory effect" of the agent of the present invention may be a direct effect that targets motor neurons, or an indirect effect that targets other cells.

In the present invention, it may be determined that motor neuron degeneration has been inhibited when one or more of the abnormalities of neurite retraction, neurite fragmentation, cell body atrophy, and cell body fragmentation have recovered by 10%, 20%, 30%, 40%, 50% or more compared to control motor neuron cells. Alternatively, it may be determined that motor neuron degeneration has been inhibited when the viability of motor neuron cells has recovered by 10%, 20%, 30%, 40%, 50% or more.

In one embodiment, using motor neurons derived from iPS cells of an ALS patient, the rate of inhibition of cell degeneration of motor neurons by a certain compound (test compound) may be calculated using the following formula.
Motor neuron degeneration inhibitory activity of the test compound = ((XC)/(TC))x100
X: number of motor neurons in the test compound group after y days from the start of culture, C: number of motor neurons in the DMSO group after y days from the start of culture, T: number of motor neurons after x days from the start of culture, where x is selected from any day before spontaneous cell death occurs in the subject, and y is selected from any day on which spontaneous cell death occurs in the subject.

Since the compound used in the present invention has an excellent inhibitory effect on motor neuron degeneration, an agent containing the compound can be used as a preventive or therapeutic agent for motor neuron diseases in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans, etc.). Examples of motor neuron diseases include amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The preventive or therapeutic agent can be used for the diseases, regardless of whether they are sporadic or familial. In this specification, the diseases may be referred to as "motor neuron diseases".

The compounds used in the present invention may also be, for example,
(1) Mental disorders (e.g., depression, major depression, bipolar depression, dysthymic disorder, affective disorder (seasonal affective disorder, etc.), recurrent depression, postpartum depression, stress disorder, depressive symptoms, mania, anxiety, generalized anxiety disorder, anxiety syndrome, panic disorder, phobia, social phobia, social anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder, post-traumatic stress disorder, Tourette syndrome, autism, fragile X syndrome, Rett syndrome, adaptation disorders, bipolar disorder, neurosis, schizophrenia (e.g., positive symptoms, negative symptoms, cognitive impairment), chronic fatigue syndrome, anxiety neurosis, obsessive-compulsive disorder, panic disorder, epilepsy, anxiety disorder, anxiety symptoms, unpleasant mental state, dysthymia, cyclothymia, nervousness, syncope, addiction, decreased libido, attention deficit hyperactivity disorder (ADHD), major psychotic depression, treatment-resistant major depression, treatment-resistant depression, depressive disorder, catalepsy, schizophrenia of the hymenal type, schizophrenia of the paranoid type],
(2) neurodegenerative diseases [e.g., progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Alzheimer's disease, Alzheimer's type senile dementia, Parkinson's disease, Huntington's disease, multiple infarction dementia, frontotemporal dementia, Parkinson's dementia, progressive supranuclear palsy, Pick's syndrome, corticobasal degeneration, Down's syndrome, vascular dementia, postencephalitic Parkinson's disease, Lewy body dementia, multiple system atrophy, HIV dementia, amyotrophic lateral sclerosis (ALS), motor neurogenic disease (MND), Creutzfeldt-Jakob disease or prion disease, cerebral palsy, progressive supranuclear palsy, traumatic brain injury, glaucoma, multiple sclerosis, neuromyelitis optica (NMO), postoperative cognitive dysfunction (POCD), postoperative delirium (POD), delirium],
(3) Age-related cognitive and memory disorders (e.g., age-related memory disorder, senile dementia),
(4) Sleep disorders [e.g., endogenous sleep disorders (e.g., psychophysiological insomnia, etc.), exogenous sleep disorders, circadian rhythm disorders (e.g., time zone shift syndrome (jet lag), shift work sleep disorder, irregular sleep-wake patterns, delayed sleep phase syndrome, advanced sleep phase syndrome, non-24-hour sleep-wake, etc.), parasomnia, sleep disorders associated with internal medicine or psychiatric disorders (e.g., chronic obstructive pulmonary disease, Alzheimer's disease, Parkinson's disease, vascular dementia, schizophrenia, depression, anxiety neurosis), stress insomnia, insomnia, insomnia neurosis, sleep apnea syndrome],
(5) Respiratory depression caused by anesthetics, trauma, or neurodegenerative diseases, etc.
(6) Traumatic brain injury, stroke, cerebral edema, cerebral ischemia, ischemia, anorexia nervosa, eating disorders, anorexia nervosa, bulimia, other eating disorders, alcoholism, alcohol abuse, alcoholic amnesia, alcohol delusion, alcohol preference, alcohol withdrawal, alcoholic psychosis, alcohol poisoning, alcoholic jealousy, alcoholic mania, alcohol-dependent mental disorder, alcoholic psychosis, drug preference, drug phobia, drug mania, drug withdrawal, migraine, stress headache, tension headache, diabetic neurosis rheumatoid arthropathy, obesity, diabetes, muscle spasms, Meniere's disease, autonomic imbalance, alopecia, glaucoma, high blood pressure, heart disease, tachycardia, congestive heart failure, hyperventilation, bronchial asthma, apnea, sudden infant death syndrome, inflammatory diseases, allergic diseases, impotence, menopausal disorders, infertility, tumors (e.g., cancer, liver cancer, colon cancer, breast cancer, prostate cancer, neuroblastoma, bone cancer, oral cancer, mast cell tumor, bile duct cancer, Lewis lung cancer), immunodeficiency syndrome due to HIV infection, immunodeficiency syndrome due to stress, cerebrospinal meningitis, acromegaly, incontinence, Metabolic syndrome, osteoporosis, peptic ulcer, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, stress-related gastrointestinal disorders, nervous vomiting, diarrhea, constipation, postoperative ileus, rheumatoid arthritis, osteoarthritis, functional dyspepsia, hyperalgesia, insulin resistance, boxer dementia, nausea, vomiting, tumor metastasis, brain injury, seizures, weight change, weight gain, weight loss, colitis, alcoholism, hypothermia, fatty liver, atherosclerosis, infection, muscle spasms, high blood pressure, stroke, early childhood malignant transitional partial seizures, diabetes mellitus, type 2 diabetes mellitus, dyslipidemia, visceral obesity, hypotony, anorexia, fibrosis, myocardial infarction, cachexia, induced psychotic disorder, ataxia, AIDS wasting syndrome, cirrhotic cardiomyopathy, uremic pruritus, neurobehavioral symptoms, tubulointerstitial nephritis and uveitis syndrome, interstitial cystitis, retinitis pigmentosa, complex regional pain syndrome, autoimmune diseases, coronary artery disease, aspirin asthma, platelet storage pool deficiency, diabetic embryo, Arthus type urticaria, asthma, toxic oil syndrome, otitis, etc.
(7) Pain (e.g., inflammatory pain, cancer pain, neuropathic pain, acute pain, pain due to peripheral neuropathy, central pain, fibromyalgia, vasoclussive painful crises in sickle cell disease, spasticity or pain mediated by multiple sclerosis, functional chest pain, complex regional pain syndrome, etc.),
(8) lysosomal diseases [e.g., Gaucher disease, Krabbe disease, Niemann-Pick syndrome],
It is expected that the above-mentioned compounds will be useful as preventive or therapeutic agents for diseases such as those mentioned above.

The compounds used in the present invention have excellent pharmacokinetics (e.g., blood drug half-life, brain transferability, metabolic stability) and low toxicity (e.g., superior as pharmaceuticals in terms of acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, etc.), and can be safely administered orally or parenterally to mammals (e.g., humans, monkeys, cows, horses, pigs, mice, rats, hamsters, rabbits, cats, dogs, sheep, goats, etc.) as pharmaceuticals or as pharmaceutical compositions mixed with pharmaceutical carriers, etc. "Parenteral" includes intravenous, intramuscular, subcutaneous, intravisceral, intranasal, intradermal, ophthalmic, intracerebral, rectal, vaginal, intraperitoneal, intratumor, proximal to tumor, etc., and administration directly to the lesion.

The dosage of the compound used in the present invention varies depending on the administration route, symptoms, etc., but for example, when orally administered to a patient with amyotrophic lateral sclerosis (adult, weighing 40 to 80 kg, e.g., 60 kg), the dosage is, for example, 0.001 to 1000 mg/kg body weight per day, preferably 0.01 to 100 mg/kg body weight per day, and more preferably 0.1 to 10 mg/kg body weight per day. This amount can be administered in one to three divided doses per day.

The agent of the present invention can be used as a pharmaceutical composition comprising the compound used in the present invention alone or the compound used in the present invention mixed with a pharma- ceutical acceptable carrier, according to a method known per se as a method for producing pharmaceutical preparations (e.g., a method described in the Japanese Pharmacopoeia, etc.). The agent of the present invention (medicine, pharmaceutical composition) can be, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), troches, syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., immediate-release preparations, sustained-release preparations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films ... [0043] The composition can be safely administered orally or parenterally (e.g., intravenous, intramuscular, subcutaneous, intravisceral, intranasal, intradermal, eye drops, intracerebral, rectal, vaginal, intraperitoneal, etc.) in the form of an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), infusion, transdermal preparation, ointment, lotion, patch, suppository (e.g., rectal suppository, vaginal suppository), pellet, nasal preparation, pulmonary preparation (inhalant), eye drop, etc.

As the "pharmaceutical acceptable carrier", various organic or inorganic carriers commonly used as starting materials are used. For example, excipients, lubricants, binders, disintegrants, etc. are used in solid preparations, while solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. are used in liquid preparations. Furthermore, preparation additives such as preservatives, antioxidants, colorants, sweeteners, etc. can also be used as necessary.
Examples of excipients include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, and the like.
Lubricants include, for example, magnesium stearate, calcium stearate, talc, colloidal silica, and the like.
Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methyl cellulose, and sodium carboxymethyl cellulose.
Examples of disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylstarch, and L-hydroxypropylcellulose.
Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil, and the like.
Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.
Examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
Examples of the isotonic agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, and the like.
Examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.
An example of the soothing agent is benzyl alcohol.
Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, and sorbic acid.
Examples of antioxidants include sulfites, ascorbic acid, and α-tocopherol.

Pharmaceutical compositions (formulations) vary depending on the dosage form, method of administration, carrier, etc., but can be prepared in accordance with conventional methods by adding the compound used in the present invention in a proportion of typically 0.01 to 100% (w/w), preferably 0.1 to 95% (w/w), based on the total amount of the formulation.

The compound used in the present invention may be used alone as an agent (medicament, pharmaceutical composition) or in combination of two or more kinds. In this specification, unless otherwise specified, the term "compound used in the present invention" also includes a combination of multiple kinds of compounds.
The compound used in the present invention may be used in combination with other active ingredients (hereinafter abbreviated as concomitant drugs).

Concomitant medications include, for example:
Benzodiazepines (chlordiazepoxide, diazepam, potassium clorazepate, lorazepam, clonazepam, alprazolam, etc.), L-type calcium channel blockers (pregabalin, etc.), tricyclic or tetracyclic antidepressants (imipramine hydrochloride, amitriptyline hydrochloride, desipramine hydrochloride, clomipramine hydrochloride, etc.), selective serotonin reuptake inhibitors (fluvoxamine maleate, fluoxetine hydrochloride, citalopram hydrochloride, sertraline hydrochloride, etc.) , paroxetine hydrochloride, escitalopram oxalate, etc.), serotonin-noradrenaline reuptake inhibitors (venlafaxine hydrochloride, duloxetine hydrochloride, desvenlafaxine hydrochloride, etc.), noradrenaline reuptake inhibitors (reboxetine mesylate, etc.), noradrenaline-dopamine reuptake inhibitors (bupropion hydrochloride, etc.), mirtazapine, trazodone hydrochloride, nefazodone hydrochloride, bupropion hydrochloride, setiptiline maleate, 5-HT _1A agonists (buspirone hydrochloride, tandospirone citrate, osemozotan hydrochloride, etc.), 5-HT ₃ antagonists (cyamemazine, etc.), non-cardiac selective β-blockers (propranolol hydrochloride, oxyprenolol hydrochloride, etc.), histamine H ₁ antagonists (hydroxyzine hydrochloride, etc.), schizophrenia treatment drugs (chlorpromazine, haloperidol, sulpride, clozapine, trifluoperazine hydrochloride, fluphenazine hydrochloride, olanzapine, quetiapine fumarate, risperidone, aripiprazole, etc.), CRF antagonists, other anxiolytics (meprobamate, etc.), tachykinin antagonists (MK-869, saredutant, etc.), drugs acting on metabotropic glutamate receptors, CCK antagonists, β3 adrenergic antagonists (amibegron hydrochloride, etc.), GAT-1 inhibitors (tiagabine hydrochloride, etc. ), N-type calcium channel inhibitors, type 2 carbonic anhydrase inhibitors, NMDA glycine site agonists, NMDA antagonists (memantine, etc.), peripheral benzodiazepine receptor agonists, vasopressin antagonists, vasopressin V1b antagonists, vasopressin V1a antagonists, phosphodiesterase inhibitors, opioid antagonists, opioid agonists, uridine, nicotinic acid receptor agonists, thyroid hormones (T3, T4), TSH, TRH, MAO inhibitors (phenelzine sulfate, tranylcypromine sulfate, moclobemide, etc.), 5-HT _2A antagonists, 5-HT _2A inverse agonists, COMT inhibitors (entacapone, etc.), drugs for treating bipolar disorder (lithium carbonate, sodium valproate, lamotrigine, riluzole, felbamate, etc.), cannabinoid CB1 antagonists (rimonabant, etc.), FAAH inhibitors, sodium channel blockers, anti-ADHD drugs (methylphenidate hydrochloride, methamphetamine hydrochloride, etc.), drugs for treating alcoholism, drugs for treating autism, drugs for treating chronic fatigue syndrome, drugs for treating convulsions, drugs for treating fibromyalgia, drugs for treating headaches, drugs for treating insomnia (etizolam, zopiclone, triazolam, zolpidem, ramelteon, indiplon, etc.), drugs for smoking cessation, drugs for treating myasthenia gravis, drugs for treating cerebral infarction, drugs for treating mania, drugs for treating hypersomnia, drugs for treating pain, drugs for treating dysthymia, drugs for treating autonomic nervous system disorders, drugs for treating male and female sexual functions drugs for functional impairment, drugs for migraine, drugs for pathological gambling, drugs for restless legs syndrome, drugs for substance dependence, drugs for alcohol-related disorders, drugs for irritable bowel syndrome, drugs for Alzheimer's disease (donepezil, galantamine, memantine, rivastigmine, etc.), drugs for Parkinson's disease (levodopa, carbidopa, benserazide, selegiline, zonisamide, endacavone, amantadine, talipexole, bramipexole, apomorphine, capergoline, bromocriptine, istradefylline, trihexyphenidyl, promethazine, pergolide, etc.), drugs for Huntington's disease (chlorpromazine hydrochloride, haloperidol, reserpine, etc.), drugs for Gaucher disease (imiglucerase, taliglucerase alfa, velaglucerase alfa, estrogen, liglustat, miglustat, etc.), ALS drugs (riluzole, etc., neurotrophic factors, etc.), multiple sclerosis drugs (fingolimod, interferon beta-1b, natalizumab and other molecular targeted drugs, etc.), antiepileptic drugs (phenytoin, carbamazepine, phenobarbital, primidone, zonizamide, sodium valproate, ethosuximide, diazepam, nitrazepam, clonazepam, clobazam, gabapentin, topiramate, lamotrigine, levetiracetam, stiripentol, rufinamide, etc.), dyslipidemia drugs such as cholesterol-lowering drugs (statin series (pravastatin sodium, atorvastatin, simvastatin, rosuvastatin, etc.), fibrates (clofibrate, etc.), squalene synthetic growth inhibitors), abnormal behavior treatment drugs or drugs to suppress wandering caused by dementia (sedatives, anti-anxiety drugs, etc.), apoptosis inhibitors, anti-obesity drugs, diabetes treatment drugs, high blood pressure treatment drugs, low blood pressure treatment drugs, rheumatism treatment drugs (DMARDs), anti-cancer drugs, parathyroid treatment drugs (PTH), calcium receptor antagonists, sex hormones or derivatives thereof (progesterone, estradiol, estradiol benzoate, etc.), neuronal differentiation promoters, neuronal regeneration promoters, non-steroidal anti-inflammatory drugs (meloxicam, tenoxicam, indomethacin, ibuprofen, celecoxib, rofecoxib, aspirin, etc.), steroids (dexamethasone, cortisone acetate, etc.), anti-cytokine drugs (TNF inhibitors, MAP kinase inhibitors, etc.), antibody drugs, nucleic acids or nucleic acid derivatives, aptamer drugs, etc.

By combining the compound used in the present invention with a concomitant drug,
(1) The dose of the compound of the present invention or a concomitant drug can be reduced compared to when the compound or a concomitant drug is administered alone.
(2) Drugs to be used in combination with the compounds of the present invention can be selected depending on the symptoms of the patient (mild, severe, etc.);
(3) By selecting a concomitant drug having a different mechanism of action from that of the compound used in the present invention, the treatment period can be extended.
(4) By selecting a concomitant drug having a different mechanism of action from that of the compound used in the present invention, the therapeutic effect can be sustained.
(5) By using the compound of the present invention in combination with a concomitant drug, excellent effects such as a synergistic effect can be obtained.

Hereinafter, the combined use of the compound used in the present invention and a concomitant drug will be referred to as the "concomitant drug of the present invention."
When using the combination drug of the present invention, the administration time of the compound used in the present invention and the combination drug is not limited, and the compound used in the present invention or its pharmaceutical composition and the combination drug or its pharmaceutical composition may be administered to the subject at the same time or at a time lag. The dosage of the combination drug may be in accordance with the dosage used in clinical practice, and may be appropriately selected depending on the subject, administration route, disease, combination, etc.
The administration form of the combination drug of the present invention is not particularly limited, and it is sufficient that the compound used in the present invention and the concomitant drug are combined at the time of administration. Examples of such administration forms include:
(1) Administration of a single formulation obtained by simultaneously formulating the compound used in the present invention and a concomitant drug;
(2) Simultaneous administration of two types of preparations obtained by separately formulating the compound used in the present invention and a concomitant drug via the same administration route;
(3) The compound used in the present invention and the concomitant drug are separately formulated into two preparations, which are then administered via the same administration route with a time lag.
(4) Simultaneous administration of two different preparations obtained by separately formulating the compound used in the present invention and a concomitant drug via different administration routes;
(5) The compound used in the present invention and the concomitant drug are separately formulated into two preparations, which are then administered via different administration routes with a time lag (for example, the compound used in the present invention and the concomitant drug are administered in the same order, or in the reverse order).
etc.

The combination drug of the present invention has low toxicity, and can be safely administered orally or parenterally (e.g., topically, rectally, intravenously, etc.) by, for example, mixing the compound used in the present invention or/and the above-mentioned combination drug with a pharma- ceutical carrier according to a known method and preparing a pharmaceutical composition, for example, a tablet (including sugar-coated tablet, film-coated tablet), powder, granule, capsule (including soft capsule), liquid, injection, suppository, sustained-release agent, etc. Injection can be administered intravenously, intramuscularly, subcutaneously, or intraorgan, or directly to the lesion.
Examples of pharma- ceutically acceptable carriers that may be used in the production of the combination agent of the present invention include the same as those mentioned above.

The mixing ratio of the compound used in the present invention to the concomitant drug in the combination agent of the present invention can be appropriately selected depending on the subject of administration, the administration route, the disease, and the like.
For example, the content of the compound used in the present invention in the combination agent of the present invention varies depending on the form of the preparation, but is usually about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, and more preferably about 0.5 to 20% by weight, based on the total weight of the preparation.
The content of the concomitant drug in the concomitant agent of the present invention varies depending on the form of the preparation, but is usually about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, and more preferably about 0.5 to 20% by weight based on the total weight of the preparation.

The present invention will be further explained in detail by the following examples and test examples, but these are not intended to limit the present invention and may be modified without departing from the scope of the present invention.
In the following examples, "room temperature" generally refers to about 10° C. to about 35° C. Ratios shown in mixed solvents are by volume unless otherwise specified. % refers to weight % unless otherwise specified.

The elution in the column chromatography in the examples was carried out under observation by TLC (Thin Layer Chromatography) unless otherwise specified. In the TLC observation, Merck 60 F ₂₅₄ was used as the TLC plate, and the solvent used as the elution solvent in the column chromatography was used as the developing solvent. A UV detector was used for detection. In the silica gel column chromatography, aminopropylsilane-bonded silica gel was used when NH was written.
In the preparative HPLC (High Performance Liquid Chromatography) in the examples, the eluent solvent system used for purifying the compounds was any one of the following, unless otherwise specified.
Acetonitrile/water mixed solvent system containing 0.1% trifluoroacetic acid Acetonitrile/water mixed solvent system containing 10 mM ammonium bicarbonate Acetonitrile/water mixed solvent system containing 0.05% ammonia

The following abbreviations are used in the following examples:
MS: Mass spectrum M: Molar concentration
DMA: N,N-Dimethylacetamide
DMF: N,N-dimethylformamide
THF: Tetrahydrofuran
DMSO: Dimethyl sulfoxide

Example 1
N-[3-({2-[(cyclopropanecarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]cyclohexanecarboxamide

It can be produced by a method known per se, for example, the method described in Patent Document 4.

Example 2
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide

It can be produced by a method known per se, for example, the method described in Patent Document 5.

Example 3
N-[1-(4-fluorobutyl)-1H-pyrazol-4-yl]-4-[(2-fluorophenoxy)methyl]pyridine-2-carboxamide

It can be produced by a method known per se, for example, the method described in Patent Document 5.

Example 4
1-(3-chlorophenyl)-N-[4-(diethylcarbamoyl)phenyl]-2-methyl-1H-benzimidazole-6-carboxamide

It can be produced by a method known per se, for example, the method described in Patent Document 6.

Example 5
3-Ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-(4-methoxyphenyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide

It can be produced by a method known per se, for example, the method described in Patent Document 7.

Example 6
1-[(3-Methoxyphenyl)methyl]-N-(pyridin-3-yl)-1H-benzotriazole-5-carboxamide

This compound can be produced by combining methods known per se. In addition, a commercial product is available from ChemDiv.

Example 7
(3aS,4R,9bR)-4-[2-(trifluoromethyl)phenyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid

This compound can be produced by combining methods known per se. In addition, a commercial product is available from SPECS.

Example 8
3-(Pyridin-4-yl)-4,5-dihydro-2H-benzo[g]indazole

This compound can be produced by combining methods known per se. It is also available commercially from Alfa Chemistry.

Example 9
N-[4-(5-{[(4-chlorophenyl)methyl]sulfanyl}-4-methyl-4H-1,2,4-triazol-3-yl)phenyl]propanamide

This compound can be produced by combining methods known per se. It is also available commercially from MEDCHEM.

Example 10
4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide

A) 4-((2-chlorophenoxy)methyl)picolinate A mixture of methyl 4-(hydroxymethyl)picolinate (2.99 g), 2-chlorophenol (2.04 mL), triphenylphosphine (5.63 g), diethyl azodicarboxylate (40% toluene solution, 9.76 mL) and THF (120 ml) was heated at 70°C for 30 minutes. The reaction mixture was diluted with aqueous sodium hydrogen carbonate solution, extracted with ethyl acetate, and the organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was roughly purified by silica gel column chromatography (NH silica gel, hexane/ethyl acetate) to obtain methyl 4-((2-chlorophenoxy)methyl)picolinate. A mixture of the obtained methyl 4-((2-chlorophenoxy)methyl)picolinate (5.93 g), THF (27 mL), methanol (27 mL) and 1N aqueous sodium hydroxide solution (53.7 mL) was stirred at room temperature for 15 minutes. To the reaction mixture was added 1M hydrochloric acid (54 mL) and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and dissolved in a mixture of THF/ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound (3.57 g).
MS: [M+H] ⁺ 264.1.

B) 4-((2-chlorophenoxy)methyl)-N-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinamide
A mixture of 4-((2-chlorophenoxy)methyl)picolinic acid (1.48 g), 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-amine (1.03 g), diisopropylethylamine (2.94 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (3.21 g) and DMA (28 ml) was stirred at room temperature for 18 hours. The reaction mixture was diluted with water and saturated saline, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, hexane/ethyl acetate) to give the title compound (2.27 g).
MS: [MH] ^- 411.0.

C) 4-((2-chlorophenoxy)methyl)-N-(1H-pyrazol-4-yl)picolinamide
A mixture of 4-((2-chlorophenoxy)methyl)-N-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinamide (2.27 g), tosylic acid monohydrate (0.11 g) and methanol (27.5 mL) was stirred at room temperature for 30 minutes, at 40°C for 30 minutes, at 50°C for 1 hour, at 60°C for 1.5 hours, and then heated to reflux for 2.5 hours. Water was added to the reaction mixture, and the resulting precipitate was collected by filtration and dissolved in THF. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the title compound (1.57 g).
MS: [M+H] ⁺ 329.1.

D) 4-[(2-chlorophenoxy)methyl]-N-{1-[(2-cyanophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide
A mixture of 4-((2-chlorophenoxy)methyl)-N-(1H-pyrazol-4-yl)picolinamide (26 mg), 2-cyanobenzyl bromide (24 mg), potassium carbonate (22 mg) and DMA (1 mL) was stirred at 80° C. for 2 hours. Insoluble matter was removed by filtration, and the filtrate was purified by HPLC (Actus Triart C18, acetonitrile/10 mM ammonium carbonate aqueous solution) to obtain the title compound (20 mg).
MS: [M+H] ⁺ 444.2.

Example 11
4-[(2-chlorophenoxy)methyl]-N-[1-(2-methoxyethyl)-1H-pyrazol-4-yl]pyridine-2-carboxamide

A mixture of 4-((2-chlorophenoxy)methyl)-N-(1H-pyrazol-4-yl)picolinamide (26 mg) obtained in Step C of Example 10, 2-bromoethyl methyl ether (17 mg), potassium carbonate (22 mg) and DMA (1 mL) was stirred at 80° C. for 2 hours. Insoluble matter was removed by filtration, and the filtrate was purified by HPLC (Actus Triart C18, acetonitrile/10 mM aqueous ammonium carbonate solution) to obtain the title compound (11 mg).
MS: [M+H] ⁺ 387.1.

Example 12
1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxamide

A mixture of methyl 1-(2-chloropyrimidin-4-yl)-1H-indole-3-carboxylate, 4-methoxybenzylamine, potassium carbonate and DMF is stirred at 50° C. overnight. Saturated saline is added to the reaction mixture, and the mixture is extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (silica gel, hexane/ethyl acetate) to obtain methyl 1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxylate. A mixture of methyl 1-(2-{[(4-methoxyphenyl)methyl]amino}pyrimidin-4-yl)-1H-indole-3-carboxylate, aqueous sodium hydroxide solution and methanol is stirred at room temperature overnight. Hydrochloric acid is added to the reaction mixture, and the resulting precipitate is collected by filtration. A mixture of the obtained crude product, diisopropylethylamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, 28% aqueous ammonia, and DMA is stirred at room temperature overnight. Saturated saline is added to the reaction mixture, and the mixture is extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (silica gel, hexane/ethyl acetate) to obtain the title compound.
MS: [M+H] ⁺ 374.2.

Example 13
N-{3-[2-(4-methoxyphenyl)-1H-benzimidazol-5-yl]phenyl}-2-phenylacetamide

A mixture of 5-bromo-2-(4-methoxyphenyl)-1H-benzimidazole, [3-(2-phenylacetamido)phenyl]boronic acid, tetrakis(triphenylphosphine)palladium, aqueous sodium carbonate and DMF is stirred at 100°C overnight. Saturated saline is added to the reaction mixture, and the mixture is extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (NH silica gel, hexane/ethyl acetate) to obtain the title compound.
MS: [M+H] ⁺ 434.2.

Example 14
N-benzyl-2-cyclopropyl-5-methylthieno[2,3-d]pyrimidin-4-amine

A mixture of 2,4-dichloro-5-methylthieno[2,3-d]pyrimidine, benzylamine, triethylamine and THF is stirred at 50°C overnight. Saturated saline is added to the reaction mixture, which is extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (silica gel, hexane/ethyl acetate) to obtain N-benzyl-2-chloro-5-methylthieno[2,3-d]pyrimidin-4-amine. A mixture of N-benzyl-2-chloro-5-methylthieno[2,3-d]pyrimidin-4-amine, cyclopropylboronic acid, palladium(II) acetate, tripotassium phosphate, water and toluene is stirred at 100°C overnight. Saturated saline is added to the reaction mixture, which is extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (silica gel, hexane/ethyl acetate) to obtain the title compound.
MS: [M+H] ⁺ 296.1.

Test Example 1
ALS is a typical motor neuron disease caused by motor neuron degeneration, and most of these diseases are sporadic. Therefore, among the iPS cell clones described in Non-Patent Document 9, the effect of the compound used in the present invention was examined using the clone ALS10 (SALS) established from a sporadic ALS patient. Specifically, the degeneration inhibitory effect on a sporadic ALS cell model was analyzed using a stable line of ALS10 (SALS) into which tetracycline-inducible Lhx3, Ngn2, and Isl1 genes were introduced (hereinafter simply referred to as ALS10 cells). ALS10 cells are an ALS cell model that rapidly differentiates (within about 7 days) into motor neurons by adding tetracycline or its derivatives to the medium, and spontaneously causes degeneration after differentiation (Non-Patent Document 9). Therefore, in ALS10 cells, significant neurodegeneration and cell death are observed from about 7 to 14 days after induction of differentiation into motor neurons (i.e., induction of expression of the Lhx3, Ngn2, and Isl1 genes) (Non-Patent Document 9).
ALS10 cells were cultured on feeder cells (mitomycin-treated SNL cells) in iPS cell maintenance medium consisting of Primate ES Cell medium (ReproCell, RCHEMD001A), 4 ng/ml hbFGF (Wako, 060-04543), 50 μg/ml G418 (Nacalai, 09380-86), and Penicillin-Streptomycin (Thermo Fisher Scientific, 15140-122).
The method for seeding ALS10 cells onto the assay plate is as follows.
DMEM/F-12 (1:1) (Thermo Fisher Scientific, 11330-057), N2 supplement (Thermo Fisher Scientific, 17502-048), Penicillin-Streptomycin (Thermo Fisher Scientific, 15140-122), 10 ng/ml recombinant human BDNF (PeproTech, 450-02), 10 ng/ml recombinant human GDNF (PeproTech, 450-10), 10 ng/ml recombinant human NT-3 (PeproTech, 450-03), 1 μM Retinoic acid (Sigma, R2625), 1 μg/ml Doxycycline (Clontech, 631311), 1 μM SAG (Enzo life sciences, ALX-270-426-M001), 10 μM Matrigel was diluted 20-fold using an assay medium containing Y-27632 (Wako, 253-00513), and then coated onto a 384-well plate.
Next, the ALS10 cells were dispersed and then suspended in an assay medium, and seeded onto a Matrigel-coated assay plate at 1×10 ⁴ cells per well.

The method for counting motor neurons differentiated from ALS10 cells was as follows.
ALS10 cells were seeded on an assay plate according to the method described above. After 4 days from seeding, assay medium not containing Y-27632 was added and the cells were cultured until 6 days after seeding. After that, the cells were fixed with PFA (Wako, 163-20145) and immunostained with βIII-tubulin. After cell fixation, membrane permeabilization, and blocking, a primary antibody solution containing anti-βIII-tubulin antibody (R&D, MAB1195) diluted 10,000 times was added and the cells were left to stand at 4°C for 16 hours. After washing and removing the primary antibody, a secondary antibody solution containing Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular Probes, A11029) diluted 1,000 times in the same solution as the primary antibody was added and the cells were left to stand at room temperature for 1 hour. Finally, the secondary antibody solution was washed and removed, and D-PBS(-) was dispensed. Most of the βIII-tubulin-positive cells obtained by this method (i.e., differentiation induction by inducing the expression of the Lhx3, Ngn2, and Isl1 genes) are motor neurons.
The plates were then analyzed using a high content analyzer to count the number of βIII-tubulin positive cells (i.e., the number of surviving motor neurons) per well. The high content analyzer used was PerkinElmer's Opera Phenix. Figure 1 shows a typical image obtained using Opera.

The method for detecting the activity of a test compound is as follows.
From 6 to 14 days after the start of culture, cells were cultured in assay medium (not containing retinoic acid, doxycycline, SAG, or Y-27632) containing a given concentration of the test compound, and the cells were immunostained according to the method described above after 6 and 14 days from the start of culture, and the number of surviving motor neurons (number of βIII-tubulin-positive cells) was counted. In motor neurons differentiated from ALS10 cells, most of the motor neurons that started to degenerate have significantly atrophied or fragmented cell bodies by 14 days, so the number of surviving motor neurons can be evaluated by counting the number of cells (more specifically, the number of cell bodies) that were βIII-tubulin-positive after 14 days. As a negative control, cells cultured in assay medium containing DMSO instead of the test substance were used.
The degree to which the test compound inhibited the decrease in the number of motor neurons in the negative control was defined as the motor neuron degeneration inhibiting activity of the test compound, and was calculated according to the following formula.
Motor neuron degeneration inhibitory activity of the test compound = ((XC)/(TC))x100
X: number of motor neurons in the test compound group 14 days after the start of culture, C: number of motor neurons in the DMSO group 14 days after the start of culture, T: number of motor neurons 6 days after the start of culture Table 1 below shows activity values at a test compound concentration of 3 μmol/l.

As shown in Table 1, the motor neuron degeneration inhibition rate was 40% or more for all the test compounds of Examples 1 to 14, and it was found that spontaneous cell death of ASL10 cells was inhibited significantly. Therefore, it was demonstrated that these compounds can be used as excellent motor neuron degeneration inhibitors for ALS, particularly sporadic ALS.
In addition, the test compound of Example 10 resulted in a motor neuron degeneration inhibition rate of over 100%, which is thought to be because some cells were still in the process of differentiating into motor neurons at the time when addition of the test compound was started (6 days after the start of differentiation induction).

Test Example 2
5-10% of ALS cases are hereditary familial cases. Therefore, among the iPS cell clones described in Non-Patent Document 9, the effect of the compound used in the present invention was examined using the clone ALS1 established from a familial ALS patient with the SOD1 gene mutation L144FVX. Specifically, the degeneration inhibitory effect on a familial ALS cell model was analyzed using a stable line of ALS1 (hereinafter simply referred to as ALS1 cells) into which tetracycline-inducible Lhx3, Ngn2, and Isl1 genes were introduced. ALS1 cells are an ALS cell model that rapidly differentiates (within about 7 days) into motor neurons by adding tetracycline or its derivatives to the medium, and spontaneously causes degeneration after differentiation (Non-Patent Document 9). Therefore, in ALS1 cells, significant neurodegeneration and cell death are observed from about 7 to 14 days after induction of differentiation into motor neurons (i.e., induction of expression of the Lhx3, Ngn2, and Isl1 genes) (Non-Patent Document 9).
ALS1 cells were seeded on an assay plate in the same manner as in Test Example 1. After 4 days from seeding, assay medium not containing Y-27632 was added and the cells were cultured for 7 days after seeding, and then fixed with PFA (Wako, 163-20145), immunostained in the same manner as in Test Example 1, and motor neuron cells were counted. Most of the βIII-tubulin-positive cells obtained by this method (i.e., differentiation induction by inducing expression of Lhx3, Ngn2, and Isl1 genes) are motor neuron cells.

The method for detecting the activity of a test compound is as follows.
From 7 to 14 days after the start of culture, cells were cultured in assay medium (not containing retinoic acid, doxycycline, SAG, or Y-27632) containing a given concentration of the test compound, and immunostaining was performed according to the method described above to count the number of surviving motor neurons (number of βIII-tubulin-positive cells). In motor neurons differentiated from ALS1 cells, most of the motor neurons that began to degenerate have significantly atrophied or fragmented cell bodies by 14 days, so the number of surviving motor neurons can be evaluated by counting the number of cells (more specifically, the number of cell bodies) that were βIII-tubulin-positive after 14 days. As a negative control, cells cultured in assay medium containing DMSO instead of the test substance were used.
The degree to which the test compound inhibited the decrease in the number of motor neurons in the negative control was defined as the motor neuron degeneration inhibiting activity of the test compound, and was calculated according to the following formula.
Motor neuron degeneration inhibitory activity of the test compound = ((XC)/(TC))x100
X: number of motor neurons in the test compound group 14 days after the start of culture, C: number of motor neurons in the DMSO group 14 days after the start of culture, T: number of motor neurons 7 days after the start of culture Table 2 below shows activity values at a test compound concentration of 3 μmol/l.

As shown in Table 2, for all the tested compounds, a motor neuron degeneration inhibition rate of 16% or more was obtained. Therefore, it was demonstrated that the compound according to the present invention has an excellent motor neuron degeneration inhibition effect even in familial ALS.
The above results indicate that an agent containing the compound used in the present invention can be an inhibitor of motor neuron degeneration.

Formulation Examples Medicines containing the compounds of the present invention as active ingredients can be produced, for example, according to the following formulations.
1. Capsules (1) Compound obtained in Example 1 10 mg
(2) Lactose 90 mg
(3) Microcrystalline cellulose 70 mg
(4) Magnesium stearate 10 mg
1 capsule: 180 mg
The total amount of (1), (2), and (3) is mixed with 5 mg of (4), and the mixture is granulated. The remaining 5 mg of (4) is added to the granules, and the whole is then enclosed in a gelatin capsule.

2. Tablet (1) Compound obtained in Example 1 10 mg
(2) Lactose 35 mg
(3) Cornstarch: 150 mg
(4) Microcrystalline cellulose 30 mg
(5) Magnesium stearate 5 mg
1 tablet 230mg
The total amount of (1), (2), and (3) is mixed with 20 mg of (4) and 2.5 mg of (5), and then granulated. The remaining 10 mg of (4) and 2.5 mg of (5) are added to the granules, and the mixture is compressed to form tablets.

According to the present invention, it is possible to provide an agent (medicine, pharmaceutical composition) containing a compound having excellent inhibitory activity against motor neuron degeneration and useful as a preventive or therapeutic agent for motor neuron diseases (e.g., amyotrophic lateral sclerosis, progressive bulbar palsy, progressive muscular atrophy, primary lateral sclerosis, progressive pseudobulbar palsy, spinal muscular atrophy, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy) and the like.

This application is based on Patent Application No. 2019-189606 filed in Japan on October 16, 2019, the contents of which are incorporated in their entirety into this specification.

Claims (6)

A motor neuron degeneration inhibitor comprising 4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide or a salt thereof . A preventive or therapeutic agent for a motor neuron disease, comprising the agent according to claim 1. A preventive or therapeutic agent for amyotrophic lateral sclerosis, comprising the agent according to claim 1. Use of 4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide or a salt thereof for producing an inhibitor of motor neuron degeneration. Use of 4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide or a salt thereof for the manufacture of an agent for the prevention or treatment of a motor neuron disease. Use of 4-[(2-chlorophenoxy)methyl]-N-{1-[(2-fluorophenyl)methyl]-1H-pyrazol-4-yl}pyridine-2-carboxamide, or a salt thereof , for the manufacture of an agent for the prevention or treatment of amyotrophic lateral sclerosis.