Crystal of nitrogen-containing saturated heterocyclic derivative

Abstract

The present invention pertains to: a nitrogen-containing saturated heterocyclic derivative or a crystal thereof having an effect of reducing or suppressing accumulation of abnormal aggregates of intracerebral proteins; and a therapeutic agent and / or a prophylactic agent which is for central nervous system diseases involving such abnormal aggregates of intracerebral proteins, and which contains said derivative or a crystal thereof as an active ingredient.

Description

Crystals of nitrogen-containing saturated heterocyclic derivatives

[0001] The present invention relates to a nitrogen-containing saturated heterocyclic derivative or a crystal thereof that has the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and to a therapeutic and / or preventive agent for central nervous system diseases associated with abnormal protein aggregates in the brain, which contains the derivative or a crystal thereof as an active ingredient.

[0002] In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, abnormally aggregated proteins are formed in the brains of patients, and these aggregates are thought to be neurotoxic, causing the onset and progression of the disease.

[0003] The proteins that make up the aggregates vary depending on the disease, and α-synuclein has been reported to be the main component of the aggregates that cause Parkinson's disease. Abnormally aggregated α-synuclein is neurotoxic, and it has also been reported that aggregated α-synuclein propagates between neurons.

[0004] Although the administration of levodopa, a dopamine precursor, is a palliative treatment for Parkinson's disease, no fundamental cure has been established at present. In recent years, vigorous efforts have been made to develop disease-modifying drugs for Parkinson's disease, but no drugs currently undergoing clinical trials have been reported that can effectively inhibit or reduce the accumulation of α-synuclein aggregates.

[0005] α-Synuclein aggregates are thought to be the underlying cause of Lewy body diseases, including Parkinson's disease (dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, etc.). Therefore, drugs that inhibit or reduce the accumulation of α-synuclein aggregates are expected to be effective in ameliorating the pathology of these diseases.

[0006] To date, no in vitro evaluation system has been reported that reproduces the α-synuclein aggregates that occur endogenously in neurons. Evaluation systems for α-synuclein pathologies have often been shown by measuring the increase in the amount of phosphorylated α-synuclein upon addition of in vitro synthesized α-synuclein oligomers, making it impossible to evaluate the effect of inhibiting the accumulation of α-synuclein aggregates or reducing accumulated α-synuclein aggregates.

[0007] To date, drugs such as NPT200-11 (Neuropore) and Anle138b (MODAG) have been reported to have the ability to inhibit α-synuclein aggregate formation, but these have been evaluated based on their inhibitory effect on the aggregation ability of α-synuclein when it is artificially induced to aggregate in vitro (Patent Documents 1 and 2).

[0008] Furthermore, it has been reported that (4aR,8aS)-hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one derivatives, such as (4aR,8aS)-6-{4-[5-(trifluoromethyl)pyridin-3-yl]piperidine-1-carbonyl}hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one and (4aR,8aS)-6-[4-(5-ethylpyridin-3-yl)piperidine-1-carbonyl]hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one, have monoacylglycerol lipase (MAGL) inhibitory activity and are useful for neuroinflammation, neurodegenerative diseases, and the like (Patent Document 3). Furthermore, it has been reported that (azetidin-1-yl)(phenyl)methanone derivatives such as 2-chloro-3-{3-[6-(trifluoromethyl)pyridin-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile and 2-methoxy-3-{3-[6-(trifluoromethyl)pyridin-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile have glycine transporter 1 (GlyT1) inhibitory activity and are useful for neurodegenerative diseases, etc. (Patent Document 4). However, these compounds are all different from the nitrogen-containing saturated heterocyclic derivative of the present invention. Furthermore, these documents do not disclose or suggest the nitrogen-containing saturated heterocyclic derivative and its crystals of the present invention. Furthermore, they do not suggest any inhibitory or reducing effect on the accumulation of abnormal protein aggregates in the brain.

[0009] International Publication No. WO 2011 / 084642 International Publication No. WO 2010 / 000372 International Publication No. WO 2019 / 180185 International Publication No. WO 2016 / 073420

[0010] An object of the present invention is to provide a compound or a crystal thereof for use in the prevention or treatment of central nervous system diseases characterized by an inhibitory or reducing effect on the accumulation of abnormal protein aggregates in the brain.

[0011] As a result of extensive research, the present inventors have found that a nitrogen-containing saturated heterocyclic derivative, (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (hereinafter, sometimes abbreviated as "the compound of the present invention" as necessary), has an effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and have also found a stable crystal of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone, thereby completing the present invention. That is, the present invention is as follows.

[0012] [Item 1] A crystal of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I, having diffraction angle (2θ°) peaks at 17.8°±0.2 and 19.6°±0.2° in X-ray powder diffraction.

[0013] [Item 2] A crystal of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I, having peaks at four or more diffraction angles (2θ°) selected from 16.6°±0.2, 17.2±0.2°, 17.8°±0.2°, 18.3°±0.2°, 19.6±0.2°, 19.9°±0.2°, 22.0±0.2°, 22.8°±0.2°, 23.9°±0.2°, and 25.2°±0.2° in X-ray powder diffraction.

[0014] [Item 3] A pharmaceutical composition comprising the crystalline form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone according to Item 1 or 2 as an active ingredient.

[0015] [Item 4] A therapeutic or preventive agent for a central nervous system disease associated with abnormal brain protein aggregates, comprising (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I according to Item 1 or 2 as an active ingredient.

[0016] [Item 5] The therapeutic or prophylactic agent according to Item 4, wherein the central nervous system disease associated with abnormal aggregates of proteins in the brain is a central nervous system disease associated with tau, α-synuclein, TDP-43, or polyglutamine.

[0017] [Item 6] The therapeutic or prophylactic agent according to Item 4, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

[0018] [Item 7] The therapeutic or prophylactic agent according to Item 4, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is a central nervous system disease associated with α-synuclein.

[0019] [Item 8] The therapeutic or prophylactic agent according to Item 4, wherein the central nervous system disease involving abnormal intracerebral protein aggregates is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

[0020] [Item 9] A method for treating or preventing a central nervous system disorder associated with abnormal protein aggregation in the brain, comprising administering to a patient in need of treatment a therapeutically effective amount of the crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Form I according to Item 1 or 2.

[0021] [Item 10] Use of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in the crystalline form of Form I according to Item 1 or 2 for the manufacture of a therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of proteins in the brain.

[0022] [Item 11] The crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Form I according to Item 1 or 2, for use in the treatment or prevention of a central nervous system disorder associated with abnormal protein aggregation in the brain.

[0023] [Item 12] A therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of brain proteins, comprising a combination of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I according to Item 1 or 2, and at least one drug selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, αSyn antibodies, and pharmaceutically acceptable salts thereof.

[0024] [Item 13] A pharmaceutical agent containing as an active ingredient the crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Item 1 or 2, for treating or preventing a central nervous system disorder associated with abnormal brain protein aggregation, in combination with at least one drug selected from the group consisting of L-dopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody, and a pharmaceutically acceptable salt thereof.

[0025] The present invention provides (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone or a crystal thereof. The compound or a crystal thereof has an inhibitory or reducing effect on the accumulation of abnormal protein aggregates in the brain, and is useful as a therapeutic or preventive agent for central nervous system diseases associated with abnormal protein aggregates in the brain, particularly neurodegenerative diseases associated with α-synuclein (e.g., Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy). Furthermore, a crystal of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone Form I has a high melting point and high thermal and hygroscopic stability, making it useful as a pharmaceutical.

[0026] Figure 1 shows the X-ray powder diffraction pattern of Compound of Example 2 (Form I) performed in Example 3. The x-axis represents 2θ values, and the y-axis represents intensity. Figure 2 shows differential scanning calorimetry (DSC) for Compound of Example 2 (Form I) performed in Example 4. The x-axis represents temperature (°C), and the y-axis represents heat flow (watts / g). Figure 3 shows thermogravimetric analysis (TGA) for Compound of Example 2 (Form I) performed in Example 5. The x-axis represents temperature (°C), and the y-axis represents weight variation (%). Figure 4 shows dynamic vapor sorption (DVS) for Compound of Example 2 (Form I) performed in Example 6. The x-axis represents relative humidity (%), and the y-axis represents mass variation (%). Figure 5 shows the difference in aggregate mass between neural spheroids derived from iPS cells of healthy individuals and neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis represents the aggregate mass within the neural spheroid, and the horizontal axis represents the number of days of culture. The white graph shows the aggregate mass of neural spheroids derived from healthy individuals, and the black graph shows the aggregate mass of neural spheroids derived from PLA2G6 mutant iPS cells. Figure 6 shows the difference in aggregate mass between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis shows the aggregate mass within the dopamine neural spheroids, and the horizontal axis shows the number of days of culture. The white graph shows the aggregate mass of neural spheroids derived from healthy individuals, and the black graph shows the aggregate mass of neural spheroids derived from PLA2G6 mutant iPS cells. Figure 7 shows the difference in tyrosine hydroxylase mass between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from PLA2G6 mutant iPS cells on day 26 of culture. The vertical axis shows the amount of tyrosine hydroxylase within the dopamine neural spheroids. The white graph shows the amount of tyrosine hydroxylase in neural spheroids derived from healthy individuals, and the black graph shows the amount of tyrosine hydroxylase in neural spheroids derived from PLA2G6 mutant iPS cells. Figure 8 shows the difference in the amount of cleaved caspase 3 in dopaminergic neural spheroids derived from iPS cells of healthy individuals and PLA2G6 mutant iPS cells after 40 days of culture. The vertical axis shows the amount of cleaved caspase 3 in dopaminergic neural spheroids. The white graph shows the amount of cleaved caspase 3 in neural spheroids derived from healthy individuals, and the black graph shows the amount of cleaved caspase 3 in neural spheroids derived from PLA2G6 mutant iPS cells.Figure 9 shows the difference in aggregate mass between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from iPS cells homozygous for the GBA1 gene mutation. The vertical axis shows the aggregate mass in the dopamine neural spheroids, and the horizontal axis shows the number of days of culture. The white graph shows the aggregate mass of neural spheroids derived from healthy individuals, and the black graph shows the aggregate mass of neural spheroids derived from iPS cells homozygous for the GBA1 gene mutation.

[0027] The present invention will be described in detail below. The compound of the present invention, (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone, has the following structure.

[0028] (3-Methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone forms crystals. It is generally known that crystalline materials can be analyzed using conventional techniques such as X-ray powder diffraction (hereinafter "XRPD") analysis, differential scanning calorimetry (hereinafter "DSC"), thermogravimetric analysis (hereinafter "TGA"), dynamic vapor sorption (hereinafter "DVS"), diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, near-infrared (NIR) spectroscopy, and liquid-phase and / or solid-phase nuclear magnetic resonance spectroscopy. Furthermore, although the crystalline form of Form I of the compound of the present invention is anhydrous, it may contain a small amount of water. The water content of the crystalline material can be measured by Karl Fischer analysis.

[0029] For example, one crystalline form (Form I) of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone is specified in this specification, but the crystalline form of the present invention is not limited to this crystalline form.

[0030] An embodiment of the present invention includes Form I crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone.

[0031] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having an X-ray powder diffraction pattern with characteristic peaks at least at 19.6°±0.2° in terms of 2θ.

[0032] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having an X-ray powder diffraction pattern with characteristic peaks at least at 17.8°±0.2° in terms of 2θ.

[0033] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having an X-ray powder diffraction pattern with characteristic peaks at least at 17.8°±0.2 and 19.6°±0.2 degrees 2θ.

[0034] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone, Form I, having an X-ray powder diffraction pattern with characteristic peaks at the following angles in degrees 2θ: 16.6°±0.2, 17.2±0.2, 17.8°±0.2, 18.3°±0.2, 19.6±0.2, 19.9°±0.2, 22.0±0.2, 22.8°±0.2, 23.9°±0.2, and 25.2°±0.2. The crystal is identified by the presence of four or five peaks selected from these ten peaks.

[0035] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in FIG.

[0036] A further aspect of the present invention includes (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in a crystalline form (Form I) having an endothermic peak associated with melting with an extrapolated onset temperature (Tim) of 120.0°C ± 5°C in differential scanning calorimetry (DSC).

[0037] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having a DSC curve substantially the same as that shown in FIG.

[0038] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having a TGA curve substantially the same as that shown in FIG.

[0039] A further aspect of the present invention includes a crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Form I) having a DVS graph substantially the same as that shown in FIG.

[0040] Crystals of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone can be prepared by adding hexane to the ethyl acetate solution of Example 1 and allowing it to stand.

[0041] In addition, crystals of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone can be obtained by eluting them in various solvents (e.g., water, methanol, ethanol, isopropanol, chloroform, acetonitrile, 1,2-dimethoxyethane, tetrahydrofuran, 1,4-dioxane, diisopropyl ether, cyclopropyl methyl ether, ethyl acetate, isopropyl acetate, isobutyl acetate, acetone, chlorobenzene, toluene, xylene, water / methanol mixture (1:1), water / isopropyl It can be produced by crystallization or drying from one or more combinations of solvents (e.g., a combination of 6 to 7 solvents) such as a 1:1 propanol mixture, a 1:1 water / acetone mixture, a 1:1 water / acetonitrile mixture, a 1:1 water / tetrahydrofuran mixture, a 1:1 water / 1.4-dioxane mixture, a 1:1 water / ethyl acetate mixture, a 1:1 heptane / acetone mixture, a 1:1 heptane / acetonitrile mixture, a 1:1 heptane / tetrahydrofuran mixture, a 1:1 heptane / toluene mixture, etc.) at room temperature or under refrigerated conditions or under rapid cooling or freezing conditions. Alternatively, it can be produced by shaking a suspension in each of the solvents of Example 9 (water, ethanol, isopropanol, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, acetone, chlorobenzene, toluene, a water / ethanol mixture (1:1), a water / isopropanol mixture (1:1), a water / tetrahydrofuran mixture (1:1), a water / acetone mixture (1:1), a water / acetonitrile mixture (1:1), etc.) at room temperature to about 50°C for up to 14 days.

[0042] The 2θ values ​​in X-ray powder diffraction patterns can vary slightly from instrument to instrument or sample to sample, so the values ​​reported herein are not absolute (see Jenkins, R & Snyder, RL 'Introduction to X-Ray Powder Diffractometry' John Wiley & Sons 1996; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; Klug, HP & Alexander, LE (1974), X-Ray Diffraction Procedures). Generally, the measurement error in diffraction angles in X-ray powder diffraction spectra is, for example, approximately ±0.2° in 2θ, and this degree of measurement error should be taken into account when examining X-ray powder diffraction data. Furthermore, intensities can vary depending on experimental conditions and sample preparation (preferred orientation). In this application, measurements are reported using copper radiation (Cu Kα1, λ = 1.5406 Å; Kα2, λ = 1.5444 Å).

[0043] It is also known that measurement conditions (e.g., the instrument or device used) may result in an X-ray powder diffraction pattern with one or more measurement errors. For example, crystal grains with a size greater than 30 microns or non-uniform aspect ratios may affect the relative intensities of the peaks. Furthermore, the positions of reflections may be affected by the precise height at which the sample is placed in the diffractometer and the zero calibration of the diffractometer. The planarity of the sample surface may also have some effect. Therefore, unless otherwise specified, the crystalline forms of the present invention described above are not limited to crystals that produce the X-ray powder diffraction pattern shown in FIG. 1 , and any crystals that produce X-ray powder diffraction patterns substantially the same as those shown in these figures are within the scope of the present invention.

[0044] In addition, the extrapolated onset temperature (Tim), endothermic peak temperature (Tpm), etc. of differential scanning calorimetry (DSC) are allowed to have a deviation of ±5°C. The extrapolated onset temperature (Tim) of differential scanning calorimetry (DSC) refers to the temperature at the point where the extrapolated lines of the rising part of the curve of the endothermic peak and the baseline intersect, and the endothermic peak temperature (Tpm) refers to the temperature at the top of the endothermic peak.

[0045] The process for producing (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone or a salt thereof according to the present invention will be described below with reference to examples, but the present invention is not limited thereto.

[0046] Production Method (3-Methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone is synthesized by a combination of the production method shown below and known synthesis methods. The compounds in the reaction schemes may each form a salt, and examples of such salts include salts used in ordinary organic synthesis. Note that these reactions are merely illustrative, and the compound of the present invention can also be produced by other appropriate methods based on the knowledge of those skilled in organic synthesis.

[0047] In each of the production methods described below, even if the use of a protecting group is not specifically specified, when a functional group that requires protection is present, the functional group may be protected as necessary, and the target product may be obtained by deprotecting the functional group after completion of the reaction or after performing a series of reactions.

[0048] Introduction and removal of the protecting group can be carried out by a method commonly used in organic synthetic chemistry (e.g., the method described in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis," 3rd Ed., John Wiley and Sons, Inc., New York (1999)) or a method analogous thereto. Examples of the protecting group for the amino group include tert-butoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, o-nitrobenzenesulfonyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl.

[0049] Production Method 1 The compound represented by formula (1) can be produced, for example, by the method shown below. [In the formula, A represents halogen or OH.]

[0050] (Step 1-1: Step for Producing Compound (1)) Compound (1) is produced by reacting compound (1-1) with compound (1-2) in a suitable inert solvent in the presence or absence of various condensing agents and / or bases. Compound (1-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO 2014 / 192868). Compound (1-1) may also be one produced by Production Method 2 or 3 described below. Compound (1-2) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO 2016 / 004272). The base used in this step is appropriately selected from the bases exemplified below, and examples thereof include sodium hydride, triethylamine, diisopropylethylamine, and sodium carbonate. The condensing agent used in this step can be any of a variety of condensing agents commonly used in organic synthesis reactions, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide. The solvent used in this step can be appropriately selected from the solvents exemplified below, including DMF, THF, dichloromethane, chloroform, and ethyl acetate. The reaction time in this step is typically 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature in this step is typically −78°C to 200°C, preferably −78°C to 80°C.

[0051] Production Method 2 The compound represented by formula (1-1) can be produced, for example, by the method shown below. [In the formula, W １ and W ２ represents a halogen; and Pro represents a protecting group for an amino group.

[0052] (Step 2-1: Preparation of Compound (2-3)) Compound (2-3) is prepared by reacting compound (2-1) with compound (2-2) in a suitable inert solvent in the presence of zinc and palladium catalysts. Compound (2-1) may be a commercially available compound or one prepared by a known method (e.g., International Publication No. WO2008 / 147831). Compound (2-2) may be a commercially available compound or one prepared by a known method (e.g., Bioorganic & Medicinal Chemistry Letters (2006), 16(17), 4528-4532). １ and W ２ Examples of the halogen in include chlorine, bromine, and iodine. Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. The palladium catalyst can be any of various palladium catalysts commonly used in conventional methods, including, for example, tetrakis(triphenylphosphine)palladium(0). The solvent used in this step is appropriately selected from the solvents exemplified below, including, for example, DMF and dimethylacetamide. The reaction time is usually 5 minutes to 48 hours, and preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, and preferably 0°C to 80°C.

[0053] (Step 2-2: Production step of compound (1-1)) Compound (1-1) is produced by deprotecting the protecting group Pro of the amino group of compound (2-3) by a known method (for example, the method described in Protective Groups in Organic Synthesis, 3rd Edition (Theodora W. Green, Peter G.M. Wuts, John Wiley & Sons Inc., 1999)).

[0054] Production Method 3 The compound represented by formula (1-1) can also be produced, for example, by the method shown below. [In the formula, W ２ represents a halogen; Pro represents a protecting group for an amino group; and T represents a boronic acid or a boronic ester.

[0055] (Step 3-1: Step for Producing Compound (3-2)) Compound (3-2) is produced by reacting compound (3-1) with compound (2-2) in a suitable inert solvent in the presence of a palladium catalyst. This step can be carried out in the presence of a base and / or a phosphorus ligand, as necessary. Compound (3-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO 2019 / 163865 ). Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. Examples of the palladium catalyst include various palladium catalysts commonly used in conventional methods, such as tetrakis(triphenylphosphine)palladium(0). The base used in this step is appropriately selected from the bases exemplified below, and examples include potassium carbonate and cesium carbonate. The phosphorus ligand used in this step can be any of various phosphorus ligands commonly used in organic synthesis reactions, including triphenylphosphine and bis(diphenylphosphino)methane. The solvent used in this step can be appropriately selected from the solvents exemplified below, including 1,4-dioxane, tetrahydrofuran, water, and mixed solvents thereof. The reaction temperature is usually 0°C to 200°C, preferably 20°C to 150°C, and the reaction can also be performed under microwave irradiation, if necessary. The reaction time varies depending on conditions such as the reaction temperature, the palladium catalyst used, the raw materials, and the solvent, but is usually 5 minutes to 72 hours, preferably 1 hour to 24 hours.

[0056] (Step 3-2: Step for Producing Compound (3-3)) Compound (3-3) is produced by reacting compound (3-2) in a suitable inert solvent in the presence of a catalyst under a hydrogen atmosphere. The solvent used in this step is appropriately selected from the solvents exemplified below, and examples thereof include methanol, ethanol, chloroform, and mixed solvents thereof. As the catalyst, various catalysts commonly used in catalytic reduction reactions can be used, and examples thereof include palladium carbon and palladium hydroxide. The reaction time is usually 5 minutes to 48 hours, and preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, and preferably 0°C to 40°C.

[0057] (Step 3-3: Step for Producing Compound (1-1)) Compound (1-1) is produced from compound (3-3) according to the method described in step 2-2.

[0058] Among the raw materials or intermediates used in the production methods described above, those for which the production method is not particularly described are commercially available compounds or can be synthesized from commercially available compounds by methods known to those skilled in the art or methods similar thereto.

[0059] The base used in each step of each of the above-mentioned production methods should be selected appropriately depending on the type of reaction and raw material compound, and examples thereof include alkali bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali carbonates such as sodium carbonate and potassium carbonate; metal hydrides such as sodium hydride and potassium hydride; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal alkoxides such as sodium methoxide and sodium t-butoxide; organometallic bases such as butyllithium and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU).

[0060] The solvent used in each step of the above-described production methods should be selected appropriately depending on the type of reaction and raw material compounds, and examples thereof include alcohols such as methanol, ethanol, and isopropanol; ketones such as acetone and methyl ketone; halogenated hydrocarbons such as methylene chloride and chloroform; ethers such as tetrahydrofuran (THF) and dioxane; aromatic hydrocarbons such as toluene and benzene; aliphatic hydrocarbons such as hexane and heptane; esters such as ethyl acetate and propyl acetate; amides such as N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP); sulfoxides such as dimethyl sulfoxide (DMSO); and nitriles such as acetonitrile. These solvents can be used alone or in combination. Depending on the type of reaction, organic bases such as diazabicycloundecene (DBU) can also be used as solvents.

[0061] Suitable salts of the starting compounds and intermediates, and salts acceptable as pharmaceutical raw materials, are conventional non-toxic salts, including acid addition salts such as organic acid salts (e.g., acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, para-toluenesulfonate, etc.) and inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.); salts with amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.); metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.); ammonium salts; or salts of organic bases (e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), which can be appropriately selected by those skilled in the art.

[0062] The compound of the present invention represented by formula (1) or an intermediate thereof can be separated or purified by methods known to those skilled in the art. Examples of such methods include extraction, distribution, reprecipitation, column chromatography (e.g., silica gel column chromatography, ion exchange column chromatography, or preparative liquid chromatography), and recrystallization. Examples of recrystallization solvents that can be used include alcoholic solvents such as methanol, ethanol, and 2-propanol; ether solvents such as diethyl 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; or a mixture thereof. Other purification methods include those described in Volume 1 of "Experimental Chemistry Lectures" (edited by the Chemical Society of Japan, Maruzen). The molecular structure of the compound of the present invention can be easily determined by spectroscopic techniques such as nuclear magnetic resonance, infrared absorption, and circular dichroism spectroscopy, and / or mass spectrometry, with reference to the structures derived from the respective starting compounds.

[0063] In diseases with Lewy bodies such as Parkinson's disease, abnormally aggregated α-synuclein is found in the brains of patients. Therefore, the drug of the present application, which inhibits or reduces the accumulation of α-synuclein aggregates, is expected to be effective in improving the pathology of these diseases.

[0064] Furthermore, these aggregates are thought to exhibit neurotoxicity, induce neuronal vulnerability and neuronal cell death, and cause the onset and progression of the disease. Therefore, the drug of the present application, which suppresses the neurotoxicity and neuronal cell death associated with α-synuclein aggregates, is expected to be effective in improving the pathology of Lewy body diseases such as Parkinson's disease.

[0065] The production of neurotransmitters is one of the neuronal functions, and a decrease in neurotransmitters indicates neuronal vulnerability, which is indicated, for example, by a decrease in the amount of tyrosine hydroxylase, which is involved in dopamine metabolism, in dopaminergic neurons.

[0066] Furthermore, electroencephalogram (EEG) abnormalities have been reported in diseases with Lewy bodies, such as Parkinson's disease. Electroencephalograms are a manifestation of synchronized neural activity. Therefore, the drug of the present application, which normalizes synchronized neural activity associated with α-synuclein aggregates, is expected to have an ameliorating effect on the pathology of these diseases.

[0067] Neural spheroids used to measure α-synuclein aggregate levels can be prepared, for example, by three-dimensionally culturing neural stem cells or dopamine (DA) neural progenitor cells generated from human iPS cells mutated in a synucleopathy-associated gene under neuronal differentiation induction. Using three-dimensionally cultured neural spheroids, the amount of high-molecular-weight α-synuclein can be measured by protein analysis using an α-synuclein antibody, thereby assessing the amount of α-synuclein aggregates. Furthermore, three-dimensionally cultured neural spheroids can be used to evaluate synchronized neuronal firing by performing imaging analysis using a fluorescent calcium probe. Furthermore, by combining the steps of measuring α-synuclein aggregate levels and measuring synchronized neuronal firing in neural spheroids, Parkinson's disease pathology can be reproduced, and drugs that inhibit or reduce the accumulation of α-synuclein aggregates in Parkinson's disease pathology can be evaluated.

[0068] Differentiation of human iPS cells carrying a mutation in a synucleopathy-associated gene into neural stem cells can be induced, for example, by using PLA2G6 gene mutant cells established from an iPS cell line derived from a healthy individual (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University), culturing them in StemFitAK03N medium (Basic03, Ajinomoto Co., Inc.) at 37°C and 5% CO2, and then inducing differentiation using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801).

[0069] As a culture medium for neural stem cells, for example, a medium having the following composition can be used. <Culture medium composition for neural stem cells> Neurobasal medium (manufactured by Thermo Fisher Scientific, 2113049) Advanced DMEM / F-12 medium (manufactured by Thermo Fisher Scientific, 12634028) Neural Induction Supplement (manufactured by Thermo Fisher Scientific, A1647801)

[0070] Neural stem cells can be induced to differentiate into neural spheroids, for example, by seeding neural stem cells (10,000 cells / well) into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and culturing them in culture medium at 37°C and 5% CO, with half of the medium replaced on days 2 and 4 after differentiation induction.

[0071] The differentiation medium for neural stem cell neural spheroids may have the following composition: <Culture medium composition for neural spheroids> BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) NeuroCult SM1 Neuronal Supplement (STEMCELL Technologies, cat#05711) N2 Supplement-A (STEMCELL Technologies, cat#07152) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0072] Differentiation of synucleopathy-associated gene-mutated human iPS cells into dopaminergic neural progenitor cells can be induced, for example, using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701) to induce dopaminergic neural progenitor cells from PLA2G6 gene-mutated cells or GBA1 gene-homozygous mutant cells established from an iPS cell line derived from a healthy individual.

[0073] Differentiation of dopaminergic neural progenitor cells into neural spheroids can be induced, for example, by culturing cryopreserved dopaminergic neural progenitor cells at 37°C and 5% CO using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801), seeding the resulting dopaminergic neural progenitor cells (10,000 cells / well) into a 96-well U-shaped plate (Thermo Fisher Scientific, cat#174929), culturing them in a culture medium at 37°C and 5% CO, and replacing half of the medium every 3 to 4 days after differentiation induction.

[0074] The culture medium for dopaminergic neural spheroids of dopaminergic neural progenitor cells can have, for example, the following composition: <Culture medium composition for dopaminergic neural spheroids> BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0075] The amount of α-synuclein aggregates in neural spheroids can be measured, for example, by removing the differentiation-induced neural spheroids from the culture medium, adding a TBS solution (Nacalai, cat. #12748-31) supplemented with 1% Trition X-100 (Nacalai, cat. #12967-32), extracting the protein using an ultrasonic homogenizer, and quantitatively evaluating the waveform exhibiting a molecular weight of approximately 300 kD by protein analysis under non-reducing conditions using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (Thermo Fisher Scientific, cat. #AHB0261).

[0076] Measurement of neural vulnerability in neural spheroids can be carried out, for example, by transferring differentiation-induced dopamine neural spheroids to a TBS solution (Nacalai, cat#12748-31) supplemented with 1% Trition X-100 (Nacalai, cat#12967-32), extracting proteins using an ultrasonic homogenizer, and quantitatively evaluating the waveform exhibited at a molecular weight of approximately 60 kD by protein analysis under reduced conditions using a Simple Western system (Protein Simple, cat#SM-W004) with a tyrosine hydroxylase antibody (Millipore, cat#AB152).

[0077] Neuronal cell death in neural spheroids can be measured, for example, by transferring differentiation-induced dopamine neural spheroids to a TBS solution (Nacalai, cat#12748-31) supplemented with 1% Trition X-100 (Nacalai, cat#12967-32), extracting proteins using an ultrasonic homogenizer, and quantitatively evaluating the waveform exhibited at a molecular weight of approximately 20 kD by protein analysis under reduced conditions using a Simple Western system (Protein Simple, cat#SM-W004) with a cleaved caspase 3 antibody (Cell Signaling Technology, cat#9664).

[0078] Abnormal neural activity in neural spheroids can be measured, for example, by measuring synchronized neural firing in three-dimensionally cultured neural spheroids using imaging analysis with a fluorescent calcium probe. Synchronized neural firing in neural spheroids can be measured, for example, by imaging analysis using an assay medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191). Examples of assay medium that can be used include Hank's buffer (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576).

[0079] The compounds of the present invention are useful as therapeutic and / or preventive agents for central nervous system diseases associated with abnormal brain protein aggregates. Examples of central nervous system diseases associated with abnormal brain protein aggregates include central nervous system diseases associated with tau, α-synuclein, TDP-43, or polyglutamine. Examples of central nervous system diseases associated with tau include Alzheimer's disease and frontotemporal lobar degeneration. Examples of diseases associated with α-synuclein aggregates include Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, and infantile neuroaxonal dystrophy. Examples of central nervous system diseases associated with TDP-43 include amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Examples of central nervous system diseases associated with polyglutamine include Huntington's disease and spinocerebellar ataxia. The compounds of the present application are preferably useful as therapeutic and / or preventive agents for Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia. The compounds of the present application are more preferably useful as therapeutic and / or preventive agents for diseases involving α-synuclein aggregates. The compounds of the present application are even more preferably useful as therapeutic and / or preventive agents for Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy. In the present invention, "prevention" refers to the administration of an active ingredient of the present invention to a healthy person who has not developed a disease, for example, with the aim of preventing the onset of a disease. "Treatment" refers to the administration of a compound of the present invention as an active ingredient to a person (patient) who has been diagnosed by a physician as having the disease.

[0080] The compound of the present invention and pharmaceuticals containing the compound can be administered orally or parenterally, either directly or after formulation using an appropriate dosage form. Examples of dosage forms include, but are not limited to, tablets, capsules, powders, granules, liquids, suspensions, injections, patches, and poultices. Pharmaceutical preparations are prepared by known methods using pharmaceutically acceptable additives. Depending on the purpose, additives that can be used include excipients, disintegrants, binders, flow agents, lubricants, coating agents, solubilizers, solubilizers, thickeners, dispersants, stabilizers, sweeteners, and flavors. Specific examples of additives include lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, magnesium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, and talc.

[0081] The route of administration should be the most effective for the treatment, and can be oral or parenteral, such as intravenous, topical, inhalation, or ophthalmic administration, with oral administration being preferred. Dosage forms can include, for example, tablets and injections, with tablets being preferred. The dosage and frequency of administration of these pharmaceutical compositions vary depending on the dosage form, the patient's disease and symptoms, the patient's age and weight, and other factors, and cannot be generally defined. However, typically, the amount of active ingredient per day for an adult is in the range of about 0.0001 to about 5000 mg, preferably about 0.001 to about 1000 mg, more preferably about 0.1 to about 500 mg, and particularly preferably about 1 to about 300 mg, administered once or several times a day, preferably in divided doses one to three times a day.

[0082] The compound of the present invention and a pharmaceutical composition containing the compound can be used in combination with other drugs to enhance their effects and / or reduce their side effects. For example, they can be used in combination with drugs for treating central nervous system disorders, such as L-dopa, dopamine agonists (e.g., ropinirole hydrochloride, apomorphine hydrochloride hydrate, etc.), MAO-B inhibitors (e.g., selegiline hydrochloride, etc.), catechol-O-methyltransferase (COMT) inhibitors (e.g., entacapone, etc.), α-Syn antibodies (e.g., prasenimab, etc.), or pharmaceutically acceptable salts thereof. Hereinafter, drugs that can be used in combination with the compound of the present invention will be abbreviated as "concomitant drugs."

[0083] The administration period of the compound of the present invention, the pharmaceutical preparation containing the compound, and the concomitant drug is not limited, and they may be administered to a subject simultaneously or at staggered times. The compound of the present invention and the concomitant drug may also be used as a combination drug. The dose of the concomitant drug can be appropriately selected based on clinically used doses. The compounding ratio of the compound of the present invention to the concomitant drug can be appropriately selected depending on the subject, administration route, target disease, symptoms, combination, etc. For example, when the subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the compound of the present invention. Furthermore, for the purpose of suppressing side effects, the compound of the present invention may be used in combination with drugs (concomitant drugs) such as antiemetics, hypnotics, and anticonvulsants.

[0084] The present invention will be explained in more detail below with reference to examples, examples, and test examples, but the present invention is not limited thereto. In this specification, the terms "Example" and "Reference Example" may refer to compounds, such as "Example 1" being the "compound of Example 1" and "Reference Example 1" being the "compound of Reference Example 1." Note that the compound names shown in the following Reference Examples and Examples do not necessarily conform to the IUPAC nomenclature.

[0085] Symbols used in NMR include s for singlet, d for doublet, dd for doublet of doublets, t for triplet, td for doublet of triplets, q for quartet, m for multiplet, br for broad, brs for broad singlet, brm for broad multiplet, and J for coupling constant.

[0086] The measurement conditions for the high-performance liquid chromatograph mass spectrometer (LCMS) were as follows. The observed mass spectrometry values ​​[MS (m / z)] were calculated based on the MH ＋ The retention time is indicated as Rt (minutes). For each measured value, the measurement conditions used for the measurement are indicated as A or B.

[0087] Measurement condition A Detector: MS detector: Waters ACQUITY SQ Detector HPLC: Waters ACQUITY UPLC Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 × 30 mm Flow rate: 0.8 mL / min Oven temperature: 40 °C Measurement wavelength: 254, 220 nm Mobile phase: Solution A 0.06% formic acid aqueous solution Solution B 0.06% formic acid acetonitrile Time program: Step Time (min) 1 0.0-1.3 Solution A:Solution B = 98:2 to 4:96 2 1.3-1.5 Solution A:Solution B = 4:96 to 98:2

[0088] Reference Example 1 5-(piperidin-4-yl)-2-(trifluoromethyl)pyridine To a solution of 1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (6.7 g) in cyclopentyl methyl ether / water (4 / 1) (100 mL), 5-bromo-2-(trifluoromethyl)pyridine (4.1 g), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (0.66 g), and cesium carbonate (12 g) were added and stirred at 100°C for 30 minutes. After cooling to room temperature, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. The solvent in the dried organic layer was evaporated under reduced pressure, and the residue was then simply purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate). The resulting crude product was dissolved in methanol (50 mL), and 10% palladium-carbon (55% wet) (0.30 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered through Celite and washed with methanol, and the solvent from the resulting filtrate was evaporated under reduced pressure. The residue was again dissolved in methanol (50 mL), and 10% palladium-carbon (55% wet) (0.30 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered through Celite and washed with methanol, and the solvent from the resulting filtrate was evaporated under reduced pressure. The resulting residue was dissolved in chloroform (10 mL), and trifluoroacetic acid (20 mL) was added. After stirring at room temperature for 1 minute, the solvent and trifluoroacetic acid were evaporated under reduced pressure. The resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to give Reference Example 1 (3.8 g). LC / MS ([M+H]+ / Rt(min)): 231.2 / 0.50 (Measurement conditions A)

[0089] Example 1 (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone To a chloroform (1.0 mL) solution of Reference Example 1 (30 mg), 3-methyloxetane-3-carboxylic acid (18 mg), triethylamine (27 μL), and HATU (60 mg) were added and stirred at room temperature for 30 minutes. Cesium carbonate (42 mg) was then added and the mixture was stirred again at room temperature for 30 minutes. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain Example 1 (41 mg). LC / MS ([M+H]+ / Rt(min)): 329.2 / 0.75 (measurement condition A). 1 H-NMR (400 MHz, DMSO-d6)δ: 8.72 (1H, d, J = 1.6 Hz), 8.02 (1H, dd, J = 2.0, 8.4 Hz), 7.84 (1H, d, J = 8.8 Hz), 4.83 (2H, dd, J = 6.4, 8.8 Hz), 4.55-4.52 (1H, m), 4.28 (2H, t, 6.8 Hz), 3.18-3.05 (2H, m), 3.01-2.93 (1H, m), 2.67 (1H, t, J = 12 Hz), 1.85-1.81 (2H, brs), 1.72-1.57 (5H, m).

[0090] The compound of Example 1 can also be synthesized by the following method.

[0091] To a solution of the hydrochloride (100 mg) of Reference Example 1 in ethyl acetate (1.0 mL) were added 3-methyloxetane-3-carboxylic acid (52 mg), triethylamine (0.17 mL), and a 50% solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide in ethyl acetate (0.40 mL), and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by silica gel chromatography (elution solvent: ethyl acetate / methanol) to give Example 1 (93 mg).

[0092] Example 2 Preparation of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone A solution of 5-(piperidin-4-yl)-2-(trifluoromethyl)pyridine hydrochloride (53 g), 3-methyloxetane-3-carboxylic acid (24 g), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (40 g), and 1-hydroxybenzotriazole (2.7 g) in chloroform (286 mL) was cooled to 0°C. Triethylamine (56 mL) was added to the reaction solution at 0°C, and the mixture was warmed to room temperature and stirred overnight. Water was added to wash the reaction solution, and the organic layer was further washed with saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. Diethyl ether (100 mL) and hexane (20 mL) were added to the resulting crude product and stirred at 0°C for 30 minutes to obtain a solid (49 g). This solid was purified by aminosilica gel chromatography (elution solvent: hexane / ethyl acetate 90:10 → 0:100), and the desired fraction was collected and concentrated to dryness. Finally, washing with diethyl ether (100 mL) yielded Example 1 (37 g). Example 1 (total 136 g) prepared in the same manner as above was dissolved in ethyl acetate (200 mL) and heated and stirred at 80°C. Hexane (300 mL) was added to the solution, which was returned to room temperature and then allowed to stand at 0°C. The precipitated solid was collected by filtration, washed with hexane / ethyl acetate (3:2, 100 mL), and dried to isolate Form I of the compound of Example 1, which was characterized as described below.

[0093] Characterization of Crystalline Forms The crystalline forms of the compounds of the invention were characterized by a variety of analytical techniques, including X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vapor sorption (DVS), using the following procedures.

[0094] Example 3: X-ray powder diffraction (XRPD) of crystals of Form I obtained in Example 2 XRPD analysis of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone was carried out under the following measurement conditions: X-ray powder diffraction: XRPD analysis was carried out on a diffractometer (Bruker AXS D8 ADVANCE, Bruker, Billerica, Massachusetts, USA) using a copper bulb (Cu Kα1, λ=1.5406 Å, Kα2, λ=1.5444 Å). The generator was operated at a voltage of 40 kV and an amperage of 40 mA. The sample was prepared for analysis by mounting it in the center of a steel holder equipped with a zero background plate. The slits used were a 2.5° Soller slit, a 0.2° divergence slit, and a 5.5 mm anti-scatter slit. The sample rotation speed was 0.25 revolutions per second. The scanning range was 2-40° in diffraction angle 2θ, with a step size of 0.015° and an exposure time of 48 seconds per step. Data analysis was performed using a DIFFRAC. EVA (Bruker, Billerica, Massachusetts, USA).

[0095] XRPD analysis of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone was carried out under the measurement conditions described above. The XRPD pattern is shown in Table 1 and Figure 1.

[0096]

[0097] Example 4: Differential scanning calorimetry (DSC) of crystals of Form I obtained in Example 2 Differential scanning calorimetry of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone was carried out by the following procedure. Differential scanning calorimetry: Thermal properties were evaluated using a differential scanning calorimetry (DSC) instrument (DSC2500, TA Instruments, New Castle, DE, USA). Approximately 1-10 mg of solid sample was placed in a T_ZERO sample vessel (TA Instruments) and heated at a rate of 10°C / min under a nitrogen purge of 50 mL / min. Data analysis was carried out using TRIOS (TA Instruments, New Castle, DE, USA).

[0098] The DSC curve of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone is shown in Figure 2. Form I has a melting point of 100°C or higher and is highly stable as a crystal.

[0099] Example 5: Thermogravimetric analysis (TGA) of crystals of Form I obtained in Example 2 Thermogravimetric analysis of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone was carried out by the following procedure. Thermogravimetric analysis: Thermogravimetric analysis (TGA) was carried out on a TGA instrument (TGA 5500, TA Instruments, New Castle, DE, USA). Approximately 1-10 mg of a solid sample was placed in an open aluminum container and heated at a rate of 10°C / min under a nitrogen purge of 25 mL / min. Data analysis was carried out using TRIOS (TA Instruments, New Castle, DE, USA).

[0100] The TGA curve of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone is shown in Figure 3. Form I has no weight loss up to the melting temperature and is suitable for formulation.

[0101] Example 6: Dynamic Vapor Sorption (DVS) of Crystals of Form I Obtained in Example 2 Dynamic vapor sorption measurements of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone were carried out by the following procedure. Dynamic Vapor Sorption: Hygroscopicity was assessed at room temperature using a Dynamic Vapor Sorption (DVS) instrument (IGAsorp, Hiden Isochema, Warrington, UK). Water sorption and desorption were studied at 25°C over the range of 0-90% relative humidity (RH) as a function of the relative humidity. The relative humidity in the chamber was increased by 10% RH and held until the solid and atmosphere reached equilibrium. At this point, the RH was increased by 10% and the process was repeated until 90% RH was reached and equilibrated. Water sorption was monitored during this period. For desorption, the relative humidity was reduced in a similar manner to measure the complete sorption / desorption cycle. Cycles were repeated as necessary. All experiments were operated in dm / dt mode (mass variation over time) to determine the equilibration endpoint. Approximately 5-20 mg of solid was used. Data analysis was performed using Hisorp (Hiden Isochema, Warrington, UK).

[0102] The DVS curve of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone is shown in FIG.

[0103] Example 7 Preparation of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Alternative Method 1) 5 mg of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone obtained in Example 2 was dissolved in each of the solvents shown in Table 2 at 85° C. Then, crystallization was carried out under three conditions, namely, room temperature / refrigerated / drying, to obtain Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone.

[0104] Example 8: Preparation of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Alternative Method 2) 5 mg of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone obtained in Example 2 was dissolved in each solvent shown in Table 3 at 85°C. The solution was then rapidly cooled and stored in a freezer (-20°C) for crystallization, thereby obtaining Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone.

[0105] Example 9 Preparation of Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Alternative Method 3) 15 to 30 mg of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone obtained in Example 2 was suspended in 50 to 500 μL of each solvent shown in Table 4, and the suspension was shaken for 14 days under two conditions, room temperature and 50° C., to obtain Form I of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone.

[0106] Test Example 1: Reproduction of Parkinson's disease pathology (accumulation of α-synuclein aggregates) using neural spheroids in three-dimensional culture using PLA2G6 gene-mutated human iPS cells PLA2G6 gene-mutated cells established from an iPS cell line derived from a healthy individual (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University) were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% CO2.

[0107] Neural stem cells were induced from the iPS cells using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801) to create a cell stock.

[0108] The cryopreserved neural stem cells were cultured in a culture medium at 37°C in 5% CO2. The culture medium for neural stem cells had the following composition:

[0109] Neural stem cell culture medium composition: Neurobasal medium (Thermo Fisher Scientific, product number 2113049), Advanced DMEM / F-12 medium (Thermo Fisher Scientific, product number 12634028), Neural Induction Supplement (Thermo Fisher Scientific, product number A1647801).

[0110] Neural stem cells (10,000 cells / well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% CO2. Half of the culture medium was replaced every 3-4 days. The culture medium for neural spheroids had the following composition:

[0111] BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) NeuroCult SM1 Neuronal Supplement (STEMCELL Technologies, cat#05711) N2 Supplement-A (STEMCELL Technologies, cat#07152) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0112] The differentiated neural spheroids were removed from the culture medium, and TBS solution (Nacalai, cat. #12748-31) supplemented with 1% Trition X-100 (Nacalai, cat. #12967-32) was added, and proteins were extracted using an ultrasonic homogenizer.

[0113] The amount of α-synuclein aggregates in the extracted protein was measured under non-reducing conditions by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (Thermo Fisher Scientific, cat. #AHB0261), and the waveform corresponding to a molecular weight of approximately 300 kD was quantitatively evaluated.

[0114] The amount of α-synuclein aggregates rapidly increased from day 7 to day 9 of culture. On day 9 of culture, neural spheroids generated from PLA2G6 mutant iPS cells showed more than five times the amount of α-synuclein aggregates compared to neural spheroids derived from iPS cells of healthy individuals. After day 9 of culture, the amount of aggregates showed a slower increase. The results are shown in Figure 5.

[0115] Test Example 2: Evaluation of α-synuclein aggregate accumulation inhibition using neural spheroids generated from PLA2G6 gene-mutated human iPS cells (1) Differentiation induction of human iPS cells into neurons. Neural stem cells were induced from PLA2G6 gene-mutated cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neural spheroids were generated from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced on days 2 and 4 after differentiation induction. The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 4 days after differentiation induction.

[0116] (2) Evaluation of α-synuclein aggregate amount. Proteins were extracted from neural spheroids 9 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261). The amount of aggregates in neural spheroids to which each test compound was added was measured. When the amount of aggregates in neural spheroids to which DMSO solution was added was defined as 100%, the amount of aggregates in the presence of the compound of Example 1 was 14% at 100 nM and 16% at 1000 nM.

[0117] Test Example 3: Evaluation of the Reduction of α-Synuclein Aggregate Accumulation Using Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Neural stem cells were induced from PLA2G6 gene-mutant cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neural spheroids were prepared from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 10 days after differentiation induction.

[0118] (2) Evaluation of α-synuclein aggregate amount. Proteins were extracted from neural spheroids 15 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261). The amount of aggregates in neural spheroids to which each test compound was added was measured. When the amount of aggregates in neural spheroids to which DMSO solution was added was taken as 100%, the amount of aggregates in the presence of the compound of Example 1 was 20% at 100 nM and 30% at 1000 nM.

[0119] Test Example 4: Reproduction of Parkinson's disease pathology (accumulation of α-synuclein aggregates) using dopaminergic neural spheroids prepared from PLA2G6 gene-mutated human iPS cells Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701), and a cell stock was prepared.

[0120] The cryopreserved dopaminergic neural progenitor cells were cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0121] First, dopaminergic neural progenitor cells (10,000 cells / well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% CO2. Half of the medium was replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids had the following composition:

[0122] BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0123] The differentiated dopamine neural spheroids were removed from the culture medium, and a TBS solution (Nacalai, cat. #12748-31) containing 1% Trition X-100 (Nacalai, cat. #12967-32) was added, followed by extraction of proteins using an ultrasonic homogenizer.

[0124] The amount of α-synuclein aggregates in the extracted protein was measured under non-reducing conditions using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (Thermo Fisher Scientific, cat. #AHB0261), and the waveform corresponding to a molecular weight of approximately 300 kD was quantitatively evaluated.

[0125] α-Synuclein aggregates rapidly increased from day 10 to day 21 of culture. On day 21 of culture, the amount of α-synuclein aggregates in dopaminergic neural spheroids generated from PLA2G6 mutant iPS cells was more than five times higher than that in dopaminergic neural spheroids derived from iPS cells of healthy individuals. After day 21 of culture, the amount of aggregates showed a slower increase. The results are shown in Figure 6.

[0126] Test Example 5: Evaluation of α-synuclein aggregate accumulation inhibition using dopaminergic neural spheroids generated from PLA2G6 gene-mutated human iPS cells (1) Induction of differentiation from human iPS cells to neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 21 days after differentiation induction.

[0127] (2) Evaluation of α-synuclein aggregate amount. Proteins were extracted from dopamine neural spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261). The aggregate amount of neural spheroids to which each test compound was added was measured. When the aggregate amount of neural spheroids to which DMSO solution was added was taken as 100%, the aggregate amount upon addition of the compound of Example 1 was 17% at 100 nM and 21% at 1000 nM.

[0128] Test Example 6: Method for Reproducing Neurovulnerability Using Dopaminergic Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutant cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.

[0129] (2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neural spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the levels of tyrosine hydroxylase were measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a tyrosine hydroxylase antibody (Millipore, cat. #AB152). The results are shown in Figure 7.

[0130] Test Example 7: Evaluation of Improvement of Neurovulnerability Using Dopaminergic Neural Spheroids Prepared from PLA2G6 Gene-Mutated Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. The test compound is diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution is added to each well when half the volume is replaced 21 days after differentiation induction.

[0131] (2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the levels of tyrosine hydroxylase were measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a tyrosine hydroxylase antibody (Millipore, cat. #AB152).

[0132] Test Example 8: Method for Reproducing Neuronal Cell Death Using Neural Spheroids Prepared from PLA2G6 Gene-Mutated Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.

[0133] (2) Evaluation of neuronal cell death Starting from day 35 of differentiation induction, 10 μM dopamine was added to the culture medium. 40 days after differentiation induction, proteins were extracted from dopamine neuronal spheroids using a TBS solution containing 1% Trition X-100. Neuronal cell death was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a cleaved caspase 3 antibody (Cell Signaling Technology, cat. #9664). The results are shown in Figure 8.

[0134] Test Example 9: Evaluation of Neuronal Cell Death Inhibition Using Neural Spheroids Prepared from PLA2G6 Gene-Mutated Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. Test compounds are diluted with culture medium to a concentration twice that of the final concentration, and at the time of half-exchange 35 days after differentiation induction, an equal volume of the double concentration solution is added to each well together with 10 μM dopamine.

[0135] (2) Evaluation of neuronal cell death. Proteins were extracted from dopamine neuronal spheroids 40 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of neuronal cell death was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with cleaved caspase 3 antibody (Cell Signaling Technology, cat. #9664).

[0136] Test Example 10: Method for reproducing Parkinson's disease pathology (accumulation of α-synuclein aggregates) using neural spheroids in three-dimensional culture using GBA1 gene-mutated human iPS cells GBA1 gene homozygous mutant cells established from an iPS cell line derived from a healthy individual were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% CO2. Dopaminergic neural progenitor cells were induced from the GBA1 gene homozygous mutant iPS cells using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701) to create a cell stock.

[0137] The cryopreserved dopaminergic neural progenitor cells were cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0138] Dopaminergic neural progenitor cells (10,000 cells / well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% CO2. Half of the medium was replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids had the following composition:

[0139] BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0140] The differentiated dopamine neural spheroids were removed from the culture medium, and a TBS solution (Nacalai, cat. #12748-31) containing 1% Trition X-100 (Nacalai, cat. #12967-32) was added, followed by extraction of proteins using an ultrasonic homogenizer.

[0141] The amount of α-synuclein aggregates in the extracted protein was measured under non-reducing conditions by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (Thermo Fisher Scientific, cat. #AHB0261), and the waveform corresponding to a molecular weight of approximately 300 kD was quantitatively evaluated.

[0142] The amount of α-synuclein aggregates increased rapidly from day 21 to day 40 of culture, reaching saturation on day 40 of culture, after which no change in the amount was observed. The results are shown in Figure 9.

[0143] Test Example 11: Evaluation of reduction in α-synuclein aggregate accumulation using dopaminergic neural spheroids generated from GBA1 gene-mutated human iPS cells (1) Induction of differentiation from human iPS cells to neurons Dopaminergic neural progenitor cells were induced from GBA1 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 40 days after differentiation induction.

[0144] (2) Evaluation of α-synuclein aggregate amount. Proteins were extracted from dopamine neural spheroids 44 days after differentiation induction using TBS solution supplemented with 1% Trition X-100. The amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261). The amount of aggregates in neural spheroids to which each test compound was added was measured. When the amount of aggregates in neural spheroids to which DMSO solution was added was defined as 100%, the amount of aggregates in the presence of the compound of Example 1 was 23% at 100 nM and 14% at 1000 nM.

[0145] Test Example 12: Confirmation of abnormal synchronous firing in neural spheroids in three-dimensional culture using GBA1 gene-mutated human iPS cells GBA1 gene homozygous mutant cells established from an iPS cell line derived from a healthy individual were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% CO2.

[0146] Dopaminergic neural progenitor cells are induced from GBA1 gene homozygous mutant iPS cells using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701) to create a cell stock.

[0147] Cryopreserved dopaminergic neural progenitor cells are cultured at 37°C and 5% CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

[0148] Dopaminergic neural progenitor cells (10,000 cells / well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% CO2. Half of the medium was replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids had the following composition:

[0149] BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401) 20 ng / mL BDNF (Peprotech, cat#450-02) 20 ng / mL GDNF (Peprotech, cat#450-10) 1 mM dibutyryl cAMP (Nacalai, cat#11540-74) 200 nM ascorbic acid (Nacalai, cat#03420-52)

[0150] After 40 days of differentiation induction, half of the culture medium was removed, and an equal volume of measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) was added to the remaining medium. After 30 minutes of incubation, the cells were measured. The measurement medium used was Hank's buffer (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Images were captured at one frame per second.

[0151] Test Example 13: Test to evaluate the improvement of synchronous firing abnormalities using dopaminergic neural spheroids created from GBA1 gene-mutated human iPS cells (1) Differentiation induction from human iPS cells to neurons Dopaminergic neural progenitor cells were induced from GBA1 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng / mL BDNF, 20 ng / mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days. The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 40 days after differentiation induction.

[0152] (2) Evaluation of synchronized firing. 44 days after differentiation induction, half of the culture medium for dopaminergic neuronal spheroids was replaced, and an equal volume of measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) was added. After 30 minutes of incubation, measurements were performed. The measurement medium used was Hank's buffer (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Images were captured at one frame per second.

[0153] The compound of the present invention and a crystal thereof exhibit an inhibitory or reducing effect on the accumulation of α-synuclein aggregates, and are therefore useful as therapeutic and / or preventive agents for central nervous system diseases characterized by an inhibitory or reducing effect on the accumulation of abnormal protein aggregates in the brain (e.g., Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, etc.). Furthermore, a crystal of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone Form I of the present invention has a high melting point and high thermal and hygroscopic stabilities, making it useful as a pharmaceutical.

Claims

1. A crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in Form I, having diffraction angle (2θ°) peaks at 17.8°±0.2 and 19.6°±0.2° in X-ray powder diffraction.

2. A crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in Form I, having peaks in X-ray powder diffraction at four or more diffraction angles (2θ°) selected from 16.6°±0.2, 17.2±0.2°, 17.8°±0.2°, 18.3°±0.2°, 19.6±0.2°, 19.9°±0.2°, 22.0±0.2°, 22.8°±0.2°, 23.9°±0.2°, and 25.2°±0.2°.

3. A medicine containing as an active ingredient the crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Form I according to claim 1 or 2.

4. A therapeutic or preventive agent for central nervous system diseases associated with abnormal aggregation of proteins in the brain, comprising (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I according to claim 1 or 2 as an active ingredient.

5. The therapeutic or prophylactic agent according to claim 4, wherein the central nervous system disease associated with abnormal aggregates of proteins in the brain is a central nervous system disease associated with tau, α-synuclein, TDP-43, or polyglutamine.

6. The therapeutic or preventive agent according to claim 4, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

7. The therapeutic or prophylactic agent according to claim 4, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is a central nervous system disease associated with α-synuclein.

8. The therapeutic or preventive agent according to claim 4, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

9. A method for treating or preventing a central nervous system disorder associated with abnormal aggregation of proteins in the brain, comprising administering to a patient in need thereof a therapeutically effective amount of the crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Form I as defined in claim 1 or 2.

10. Use of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in the crystalline form of Form I according to claim 1 or 2 for the manufacture of a therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of proteins in the brain.

11. (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form of Form I according to claim 1 or 2 for use in the treatment or prevention of a central nervous system disorder involving abnormal aggregation of proteins in the brain.

12. A therapeutic or preventive agent for a central nervous system disorder associated with abnormal aggregation of brain proteins, comprising a combination of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone in crystalline form Form I according to claim 1 or 2, and at least one drug selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, αSyn antibodies, and pharmaceutically acceptable salts thereof.

13. A pharmaceutical composition comprising as an active ingredient the crystalline form of (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone of Form I according to claim 1 or 2, for treating or preventing a central nervous system disease associated with abnormal aggregation of brain proteins in combination with at least one drug selected from the group consisting of L-dopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT) inhibitors, αSyn antibodies, and pharmaceutically acceptable salts thereof.

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