Compound, α-Synuclein Aggregate-Binding Agent, and Use Thereof

Abstract

The present invention relates to a compound represented by formula (I) or (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound.

Description

Technical Field

[0001] The present invention relates to novel compounds, α-synuclein aggregate binders and their uses. Specifically, it relates to novel compounds, α-synuclein aggregate binders containing such novel compounds, compositions for optical imaging of α-synuclein aggregates, compositions for radioimaging of α-synuclein aggregates, methods for optical imaging of α-synuclein aggregates in the brain, methods for radioimaging of α-synuclein aggregates in the brain, and intermediates for synthesizing novel compounds. Background Art

[0002] It is thought that α-synuclein aggregates form the core pathology of Parkinson's disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), and are closely causally related to neurodegeneration. The diagnosis of these diseases is based on the presence of α-synuclein aggregates (also referred to as "α-synucleinopathies" in this specification) in the pathological analysis of autopsy brains, and thus cannot be diagnosed during life. However, if α-synuclein aggregates can be visualized in the living brain, information similar to definite information (definitive diagnosis) related to the diagnosis of these diseases can be obtained at an early stage. In addition, if α-synuclein aggregates can be visualized in the living brain of a disease model animal, imaging over time, etc., can also contribute to the efficacy evaluation of candidate substances for therapeutics or prophylactics targeting α-synuclein aggregates.

[0003] Previously, as a PET (positron emission tomography) probe that shows binding to α-synuclein in the living brain, there is 11 C]BF-227 (Non-Patent Documents 1 and 2). However, 11 C]BF-227 has insufficient binding affinity for α-synuclein aggregates, and in the above-mentioned diseases, α-synucleinopathies can only be detected in some MSA patients. In addition, 11 C]BF-227 has problems of non-specific brain accumulation and low binding selectivity for α-synuclein aggregates due to binding to amyloid-β aggregates.

[0004] It should be noted that the inventors et al. developed a compound for imaging (visualizing) Tau protein accumulated in the brain (refer to Patent Document 1). Since the compound described in Patent Document 1 can image Tau protein accumulated in the brain, the technology of Patent Document 1 is helpful for the treatment and prevention of diseases caused by the accumulation of Tau protein, such as Alzheimer's disease (AD). However, binding to α-synuclein aggregates is not described in Patent Document 1.

[0005] Prior Art Documents

[0006] Patent Document

[0007] Patent Document 1: Pamphlet of International Publication No. WO 2014 / 097474

[0008] Non-Patent Document

[0009] Non-Patent Document 1: Kikuchi, A. et al., Brain, 133: 1772-1778 (2010).

[0010] Non-Patent Document 2: Verdurand, M. et al., Contrast Media Mol. Imaging, 2018: 9165458 (2018). Summary of the Invention

[0011] Problems to be Solved by the Invention

[0012] The present invention has been completed in view of the above circumstances, and an object thereof is to provide an α-synuclein aggregate binder having high binding selectivity to α-synuclein aggregates, an imaging method using the α-synuclein aggregate binder, and a novel compound that can be used as an α-synuclein aggregate binder.

[0013] Means for Solving the Problems

[0014] The present inventors have found that a compound having a specific structure has high binding selectivity to α-synuclein aggregates, and further research has led to the completion of the present invention. More specifically, the present invention provides the following aspects.

[0015] One aspect of the present invention is a compound represented by the following formula (I) or (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound.

[0016] Chemical Formula 1

[0017]

[0018] Effects of the Invention

[0019] According to the present invention, it is possible to provide an α-synuclein aggregate binder having high binding selectivity to α-synuclein aggregates. Brief Description of the Drawings

[0020] Figure 1 It is a figure showing the results of fluorescence microscope measurement of the brains of DLB patients and AD patients.

[0021] Figure 2 It is a figure showing the quantitative results of the fluorescence intensities of the lesioned area and the non-lesioned area.

[0022] Figure 3It is a figure showing the results of fluorescence microscopy measurement of mice inoculated with α-synuclein fibrils.

[0023] Figure 4 It is a figure showing the results of two-photon laser scanning fluorescence microscopy examination of model mice 6 weeks after inoculation with α-synuclein fibrils.

[0024] Figure 5 It is a figure showing the PET imaging results of mice inoculated with α-synuclein fibrils.

[0025] Figure 6 It is a figure showing the PET imaging results of mice inoculated with α-synuclein fibrils.

[0026] Figure 7 It is a figure showing the PET imaging results of mice inoculated with α-synuclein fibrils.

[0027] Figure 8 It is a figure showing the ex vivo imaging results of the mouse brain.

[0028] Figure 9 It is a figure showing the PET imaging results of marmosets inoculated with α-synuclein fibrils.

[0029] Figure 10 It is a figure showing the results of in vitro binding assays of the brains of DLB patients and AD patients.

[0030] Figure 11 It is a figure showing the autoradiography results of the brains of DLB patients and MSA patients.

[0031] Figure 12 It is a figure showing the results of in vitro fluorescence microscopy measurement of the brains of DLB patients and MSA patients. Detailed implementation mode

[0032] Next, an embodiment of the present invention will be described. It should be noted that in this specification, "A and / or B" means at least one of A and B.

[0033] [Definition]

[0034] The term "pharmaceutically acceptable salt" refers to salts that are non-toxic to mammals, particularly humans. Pharmaceutically acceptable salts can be formed using non-toxic acids or bases, including inorganic acids or bases, or organic acids or bases. Examples of pharmaceutically acceptable salts include metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, etc., or organic salts formed from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, glucosamine (N-methylglucosamine), procaine, etc. Additionally, pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0035] The term "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions, or carriers such as physiological saline solutions, liquid or solid fillers, diluents, solvents, or encapsulating materials. Examples of pharmaceutically acceptable carriers include water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose, and lactated Ringer's injection, etc.

[0036] The term "effective amount" refers to the amount of a compound or composition that is capable of achieving a desired effect. For example, in some embodiments, an effective amount refers to the amount of a compound or composition that is capable of performing optical imaging or radiological imaging of substances such as α-synuclein aggregates that accumulate in the brain.

[0037] The term "solvate" refers to a solvated compound formed by the association of one or more solvent molecules with a compound. Solvates include, for example, monosolvates, disolvates, trisolvates, and tetrasolvates. Additionally, solvates include hydrates.

[0038] The term "hydrate" refers to a compound or its salt that also contains a stoichiometric or non-stoichiometric amount of water held by non-covalent intermolecular forces. Hydrates include, for example, monohydrates, dihydrates, trihydrates, and tetrahydrates, etc.

[0039] The term "treatment" refers to reducing or alleviating the progression, severity, and / or duration of a disease or condition.

[0040] The term "prevention" refers to reducing the risk of acquiring a specified disease or condition or the progression of a specified disease or condition, or reducing or inhibiting the recurrence, onset, or progression of one or more symptoms of a specified disease or condition.

[0041] The term "binding affinity" refers to the strength of binding of a compound to a specific protein aggregate.

[0042] The term "binding selectivity" refers to a difference in the binding of a compound to a specific protein aggregate compared to its binding to other protein aggregates (higher or lower binding to a specific protein aggregate compared to other protein aggregates). "High binding selectivity" means a large difference in the above-mentioned binding. For example, a compound having "high binding selectivity for α-synuclein aggregates" means that there is a large difference in the binding of the compound to α-synuclein aggregates and its binding to other protein aggregates, indicating high binding due to α-synuclein aggregates.

[0043] [Compound]

[0044] In one embodiment, the present invention provides (E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol represented by the following structural formula (I) or (E)-1-fluoro-3-((2-(4-(2-(methylamino)pyrimidin-5-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol represented by the following structural formula (II), a pharmaceutically acceptable salt or a solvate thereof.

[0045] Chemical formula 2

[0046]

[0047] In the present specification, the compounds represented by formula (I) and (II) are also referred to as "compound (I)" and "compound (II)", respectively.

[0048] In one embodiment, compound (I) and compound (II) are compounds, salts or solvates thereof in which one or more atoms are radioactive isotopes of the atoms. The radioactive isotopes are selected from the group consisting of 15 O, 13 N, 11 C and 18 F, etc., and are not particularly limited. Preferably, the radioactive isotope is 11 C or 18 F. Among them, considering that 11 the half-life of 18 C is about 20 minutes and 18 the half-life of 18 F is about 110 minutes, it is considered that the commercial utilization value of the compound labeled with

[0049] Preferably, at least one of the methylamino group bonded to the pyrimidine ring or pyrazine ring and the 3-fluoro-2-hydroxypropoxy group (-O-CH2-CH(OH)-CH2F) bonded to the benzothiazole ring is a group containing a radioisotope. More preferably, the 3-fluoro-2-hydroxypropoxy group bonded to the benzothiazole ring is a group containing a radioisotope. Further preferably, the fluorine atom in the 3-fluoro-2-hydroxypropoxy group is a radioisotope.

[0050] In one embodiment, the compound (I) containing a radioisotope is preferably 18 [18F]-(E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol.

[0051] In one embodiment, the compound (II) containing a radioisotope is preferably 18 [18F]-(E)-1-fluoro-3-((2-(4-(2-(methylamino)pyrimidin-5-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol.

[0052] [Intermediate]

[0053] As shown in the method for manufacturing the following compounds, the compound represented by the following formula (III) is a manufacturing intermediate compound for the compound (I) and the compound (II) (in this specification, also simply referred to as "intermediate").

[0054] Chemical formula 3

[0055]

[0056] In formula (III), one of X and Y is a nitrogen atom and the other is an unsubstituted carbon atom. In this specification, "unsubstituted carbon atom" means CH. That is, in formula (III), when X is a nitrogen atom (N), Y is an unsubstituted carbon atom (CH), and when X is an unsubstituted carbon atom (CH), Y is a nitrogen atom (N).

[0057] R1 is a hydroxyl group or a group represented by the following formula (i).

[0058] Chemical formula 4

[0059]

[0060] Among them, Ts represents p-toluenesulfonyl, THP represents tetrahydro-2H-pyran-2-yl, and * represents the bonding position to the benzothiazole ring.

[0061] R2 is a hydrogen atom or tert-butoxycarbonyl (Boc).

[0062] These intermediate compounds are suitable for the synthesis of compound (I) and compound (II), as well as the synthesis of radioisotope-labeled compound (I) and compound (II). Additionally, these intermediate compounds can also be salts.

[0063] When compound (I) or compound (II) contains various isomers such as stereoisomers (including optical isomers and rotational isomers), tautomers, or polar forms, these isomers are also included in compound (I) or compound (II). These isomers can be obtained as monomers separately by known synthesis methods and separation methods. Compound (III) can also include various isomers.

[0064] Compound (I), compound (II), or compound (III) can also be a crystal produced by a known crystallization method.

[0065] [Method for manufacturing compound (I), compound (II), and intermediates]

[0066] Compound (I), compound (II), and compound (III) as an intermediate can be manufactured according to the manufacturing methods shown below. Additionally, as needed, compound (I), compound (II), and compound (III) can be manufactured by separately or in combination of two or more of deprotection reaction, amidation reaction, ureation reaction, alkylation reaction, Mitsunobu reaction, oxidation reaction, reduction reaction, halogenation reaction, coupling reaction, nucleophilic addition reaction using a carbanion, Grignard reaction, dehydration reaction, etc. Additionally, reaction conditions such as solvents, reagents, and temperature in each reaction can be appropriately set based on the common technical knowledge of those skilled in the art. Protection and deprotection reactions of functional groups are carried out according to known reaction methods, methods described in reference examples or examples, and conventional protecting groups are used as protecting groups.

[0067] In the following manufacturing methods, unless otherwise specified, the meanings of the respective symbols used are the same as those described above.

[0068] (Manufacturing method A)

[0069] Compound (I), compound (II), and compound (III) (compound (III-i)) in which R1 is a hydroxyl group and R2 is a Boc group can be manufactured by the following method shown in Manufacturing Scheme 1. Hereinafter, in each scheme, the compounds shown in (1), (2), etc. are referred to as compound (1), compound (2), etc.

[0070] (Manufacturing Scheme 1)

[0071] Chemical formula 5

[0072]

[0073] Compound (2) can be prepared by the reaction of compound (1) with trialkyl phosphite. In compound (1), TBS is tert-butyldimethylsilyl.

[0074] Compound (III-i) can be prepared by the Horner-Wadsworth-Emmons reaction (HWE reaction) of compound (2) with compound (3) described below and the deprotection reaction carried out during the reaction. In compound (3), one of X and Y is a nitrogen atom and the other is an unsubstituted carbon atom (CH).

[0075] Compound (5) can be prepared by an alkylation reaction such as the Mitsunobu reaction of compound (III-i) with compound (4) described below.

[0076] Compound (I) and compound (II) can be prepared by the deprotection reaction of compound (5).

[0077] Compound (3) used in Production Method A can be prepared from compound (6) by the following method shown in Production Scheme 2.

[0078] (Production Scheme 2)

[0079] Chemical formula 6

[0080]

[0081] In compound (6), Hal represents a halogen atom (for example, a chlorine atom, a bromine atom, an iodine atom). Compound (7) can be prepared by a coupling reaction of compound (6) with 2-propyn-1-ol or the like.

[0082] Compound (3) can be prepared by the oxidation reaction of compound (7).

[0083] Compound (4) used in Production Method A can be prepared from compound (8) by the following method shown in Production Scheme 3.

[0084] (Production Scheme 3)

[0085] Chemical formula 7

[0086]

[0087] Compound (9) can be prepared by the protection reaction of compound (8).

[0088] Compound (4) can be prepared by the debenzylation reaction of compound (9).

[0089] The compound (5) used in Production Method A can also be produced from the compound (2) by the following method shown in Production Scheme 4.

[0090] (Production Scheme 4)

[0091] Chemical Formula 8

[0092]

[0093] The compound (10) can be produced by the deprotection reaction of the compound (2).

[0094] The compound (11) can be produced by an alkylation reaction such as Mitsunobu reaction of the compound (10) with the compound (4).

[0095] The compound (5) can also be produced by the HWE reaction of the compound (11) with the compound (3).

[0096] (Production Method B)

[0097] The compounds (I) and (II) can also be produced by the following method shown in Production Scheme 5.

[0098] (Production Scheme 5)

[0099] Chemical Formula 9

[0100]

[0101] The compound (13) can be produced by the alkylation reaction of the compound (III-i) based on the ring-opening reaction of ethylene oxide of the compound (12).

[0102] The compounds (I) and (II) can be produced by the deprotection reaction of the compound (13).

[0103] (Production Method C)

[0104] As shown in the following Production Scheme 6, the compound (III) (compound (III-ii)) in which R1 is a hydroxyl group and R2 is a hydrogen atom can be produced by the deprotection reaction of the compound (III-i).

[0105] (Production Scheme 6)

[0106] Chemical Formula 10

[0107]

[0108] (Production Method D)

[0109] Compound (III) of formula (i) (Compound (III-iii)) can be prepared by the following method shown in Preparation Scheme 7.

[0110] (Preparation Scheme 7)

[0111] Chemical formula 11

[0112]

[0113] Compound (15) can be prepared by an alkylation reaction such as Mitsunobu reaction of Compound (III-i) with Compound (14) described below.

[0114] Compound (16) can be prepared by a deprotection reaction of Compound (15).

[0115] Compound (III-iii) can be prepared by a protection reaction of Compound (16).

[0116] Compound (14) used in Production Method D can be prepared by the following method shown in Preparation Scheme 8.

[0117] (Preparation Scheme 8)

[0118] Chemical formula 12

[0119]

[0120] Compound (18) can be prepared by a protection reaction of Compound (17).

[0121] Compound (14) can be prepared by a debenzylation reaction of Compound (18).

[0122] [α-Synuclein aggregate binder]

[0123] In one embodiment, the present invention provides an α-synuclein aggregate binder. The α-synuclein aggregate binder of this embodiment (hereinafter, also referred to as the binder or α-synuclein aggregate binder) includes Compound (I) or Compound (II), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0124] Compounds (I) and (II) have a higher binding selectivity for α-synuclein aggregates compared to Tau protein and amyloid-β aggregates. Additionally, similarly, the pharmaceutically acceptable salts of compounds (I) and (II) and the solvates of compounds (I) and (II) also have a higher binding selectivity for α-synuclein aggregates compared to Tau protein and amyloid-β aggregates. Further, compounds (I) and (II) emit fluorescence. Additionally, as described above, in compounds (I) and (II), one or more atoms can be made into radioactive isotopes of those atoms. Therefore, the binding agent of the present embodiment can be used as a molecular probe for optical imaging or radioimaging of α-synuclein aggregates that accumulate in the brain.

[0125] It should be noted that α-synuclein is a protein that is also locally present in synapses of neurons in normal brains, and the aggregate formed by the aggregation of α-synuclein is an α-synuclein aggregate.

[0126] The α-synuclein aggregate binding agent can include a pharmaceutically acceptable carrier. Examples of such pharmaceutically acceptable carriers include water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose, and lactated Ringer's injection, etc.

[0127] The content of compound (I) or compound (II), its pharmaceutically acceptable salt or its solvate, and the pharmaceutically acceptable carrier included in the α-synuclein aggregate binding agent are not particularly limited. These contents are determined according to various main factors such as the type of compound used; the age, weight, health status, sex, and dietary content of the mammal to be administered; the number of administrations and the administration route; the treatment period; and other medicaments used simultaneously. The content of the pharmaceutically acceptable carrier can be set to an amount of 1 to 99% by weight of the α-synuclein aggregate binding agent. The α-synuclein aggregate binding agent can be prepared, for example, in such a way that the amount of the compound per unit body weight (kg) of the subject is 5 ng / kg to 5 mg / kg of compound (I) or compound (II). The lower limit of this compound amount is preferably 5 ng / kg or more, 0.01 mg / kg or more, 0.05 mg / kg or more, or 0.1 mg / kg or more. Additionally, the upper limit of this compound amount is 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, or 20 μg / kg or less.

[0128] [Composition for optical imaging of α-synuclein aggregates]

[0129] In one embodiment, the present invention provides a composition for optical imaging of α-synuclein aggregates. The composition for optical imaging of α-synuclein aggregates of the present embodiment (hereinafter, also referred to as the composition for optical imaging) includes the binder of the present embodiment. The optical imaging includes in vitro, ex vivo, and in vivo imaging.

[0130] Examples of the optical imaging include fluorescence microscopy, multiphoton imaging, two-photon imaging, and near-infrared fluorescence imaging.

[0131] The composition for optical imaging can include a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose, and lactated Ringer's injection.

[0132] The contents of the compound (I) or compound (II), its pharmaceutically acceptable salt or its solvate, and the pharmaceutically acceptable carrier included in the composition for optical imaging are not particularly limited. These contents are determined by various main factors such as the type of the compound used; the age, weight, health status, sex, and dietary content of the mammal to which the composition is administered; the number of administrations and the administration route; the treatment period; and other medicaments used simultaneously. The content of the pharmaceutically acceptable carrier can be set to an amount of 1 to 99% by weight of the composition for optical imaging. The composition for optical imaging can be prepared, for example, in such a manner that the amount of the compound (I) or compound (II) administered per unit body weight (kg) of the subject is 0.01 mg / kg to 5 mg / kg, preferably 0.05 mg / kg to 3 mg / kg, and more preferably 0.1 mg / kg to 1 mg / kg.

[0133] [Composition for Radiographic Imaging of α-Synuclein Aggregates]

[0134] In one embodiment, the present invention provides a composition for radiographic imaging of α-synuclein aggregates. The composition for radiographic imaging of α-synuclein aggregates of the present embodiment (hereinafter, also referred to as the composition for radiographic imaging) includes the binder of the present embodiment. The radiographic imaging includes in vitro, ex vivo, and in vivo imaging.

[0135] Examples of the radiographic imaging include positron emission tomography (PET), single photon emission computed tomography (SPECT), and autoradiography.

[0136] The composition for radioimaging can include a pharmaceutically acceptable carrier. Examples of such pharmaceutically acceptable carriers include water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, glucose, and lactated Ringer's injection, etc.

[0137] The content of compound (I) or compound (II), their pharmaceutically acceptable salts or their solvates, and the pharmaceutically acceptable carrier contained in the composition for radioimaging is not particularly limited. These contents are determined according to various main factors such as the type of compound used; the age, weight, health status, sex, and dietary content of the mammal to be administered; the number of administrations and the administration route; the treatment period; and other medicaments used simultaneously. The content of the pharmaceutically acceptable carrier can be set to an amount of 1 to 99% by weight of the composition for radioimaging. The composition for radioimaging can be prepared, for example, in such a way that the amount of the compound per unit body weight (kg) of the subject is 5 ng / kg to 5 mg / kg, preferably 5 ng / kg to 20 μg / kg, for administering compound (I) or compound (II).

[0138] [Diagnostic agent for α-synuclein aggregate-related diseases or companion diagnostic agent for treating or preventing such diseases]

[0139] In one embodiment, the present invention provides a diagnostic agent for α-synuclein aggregate-related diseases or a companion diagnostic agent for treating or preventing such diseases. The diagnostic agent for α-synuclein aggregate-related diseases or the companion diagnostic agent for treating or preventing such diseases (hereinafter, also referred to as the companion diagnostic agent) in this embodiment includes the binder of the present embodiment. The companion diagnostic agent for treatment refers to a diagnostic agent for determining whether treatment is promising when the disease is clear. In addition, the companion diagnostic agent for prevention refers to a preventive diagnostic agent for predicting future onset or for determining whether it is promising to suppress onset when the prodromal state of the disease is clear.

[0140] By using the diagnostic agent of this embodiment to match the data related to the amount and / or distribution amount of α-synuclein aggregates in the brain obtained from the subject with the correlation between the disease and the amount and / or distribution amount of α-synuclein aggregates previously obtained, diagnosis related to the target disease (specifically, whether suffering from the disease, severity, and likelihood of onset, etc.) can be performed.

[0141] In addition, by using a companion diagnostic agent and matching data related to the amount and / or distribution amount of α-synuclein aggregates in the brain obtained from a subject with the correlation between a disease and the amount and / or distribution amount of α-synuclein aggregates in the brain obtained in advance, the target disease state can be grasped. Therefore, based on this, a disease prevention / treatment plan (such as the type, combination, dosage, and use of a preventive drug or therapeutic drug to be administered) can be formulated.

[0142] One embodiment of the present invention also relates to a drug for treating or preventing a disease related to α-synuclein aggregates, and the drug is a drug administered according to an administration plan based on data related to the amount and / or distribution of α-synuclein aggregates in the brain obtained by companion diagnosis.

[0143] [Diagnostic Kit for Diseases Related to Substances Accumulated in the Brain]

[0144] The diagnostic kit for diseases related to substances accumulated in the brain (hereinafter, also referred to as the diagnostic kit) of the present invention includes the binding agent of the present embodiment.

[0145] As the substance accumulated in the brain, at least α-synuclein aggregates are included. In addition, Tau protein or amyloid-β aggregates can be cited.

[0146] As diseases related to substances accumulated in the brain, at least diseases related to α-synuclein aggregates are included. As such diseases, Parkinson's disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) can be cited. In addition, as diseases related to substances accumulated in the brain, Alzheimer's disease (AD) and frontotemporal lobar degeneration, which are diseases related to Tau protein or amyloid-β aggregates, can also be cited.

[0147] Compared with Tau protein and amyloid-β aggregates, the binding agent of the present embodiment has a higher binding selectivity for α-synuclein aggregates. On the other hand, for example, according to the results of research by the present inventors, it has been clarified that the compound described in Patent Document 1 has a higher binding property to Tau protein aggregates compared with α-synuclein aggregates.

[0148] In one embodiment of the present embodiment, the diagnostic kit can contain both a compound (I) or a compound (II) having a high binding selectivity for α-synuclein aggregates or a pharmaceutically acceptable salt thereof or a solvate thereof and another compound having a high binding selectivity for Tau protein aggregates (for example, the compound described in Patent Document 1). In such a diagnostic kit, by comparing the detection result of the former (the amount and / or distribution of the detected light or radiation) with the detection result of the latter, it is possible to specify which substance is present in each region of the imaging. Specifically, it is possible to classify α-synuclein aggregates and Tau protein aggregates, and further, it is also possible to quantify each amount present. Therefore, it is possible to diagnose α-synuclein aggregate-related diseases and / or Tau protein-related diseases with high accuracy. For example, for the binder of the present embodiment and a substance having a high binding selectivity for Tau protein aggregates (for example, the compound described in Patent Document 1), the ratio of the binding property to α-synuclein aggregates and the binding property to Tau protein aggregates is specified in advance. On this basis, as long as the ratio of the amount of light or radiation in each region of the brain after administration of the former and the latter in the test subject is measured, and based on the relationship between the ratio specified in advance and the measured ratio, it is possible to classify which of α-synuclein aggregates and Tau protein aggregates is present in the region, and it is possible to quantify each amount.

[0149] In addition, the above diagnostic kit of the present embodiment can also be made into a diagnostic kit further combined with an imaging agent for amyloid-β. The diagnostic kit formed by combining the diagnostic kit of the present embodiment with an imaging agent for amyloid-β can specify which substance is present in each region of the imaging. Specifically, it is possible to classify α-synuclein aggregates, Tau protein aggregates, and amyloid-β aggregates, and further, it is also possible to quantify each amount present. Therefore, it is possible to diagnose α-synuclein aggregate-related diseases, Tau protein-related diseases, and / or amyloid-β-related diseases with high accuracy.

[0150] One embodiment of the present invention also relates to a drug for treating or preventing α-synuclein aggregate-related diseases, which is a drug administered according to a dosing plan based on data related to the amount and / or distribution of the brain-accumulated substance containing α-synuclein aggregates obtained by the diagnostic kit of the present embodiment.

[0151] [Optical imaging method]

[0152] In one embodiment, the present invention provides an optical imaging method. The optical imaging method of the present embodiment includes a step of irradiating light of a first wavelength from outside the brain to the living brain of a test subject administered with the binder of the present embodiment, and then detecting light of a second wavelength different from the first wavelength emitted from the brain.

[0153] If an effective amount of a binder is administered to a subject to be detected, the binder transferred to the living body brain binds to α-synuclein aggregates present in the living body brain. By irradiating the living body brain of the subject to which the binder has been administered from outside the brain with light of a first wavelength that excites the binder and detecting light (e.g., fluorescence) of a second wavelength emitted from the binder in the brain, optical imaging (imaging) of α-synuclein aggregates can be performed.

[0154] Examples of the subject to be detected include mammals. Mammals include, for example, humans, rats, mice, rabbits, guinea pigs, hamsters, monkeys, dogs, ferrets, or mini pigs.

[0155] There is no particular limitation on the administration method. For example, there are oral administration and parenteral administrations such as intravenous administration or intraperitoneal administration. Intravenous administration or intraperitoneal administration is preferred. Intravenous administration is most preferred. The administration amount is preferably 0.01 mg / kg to 5 mg / kg, 0.05 mg / kg to 3 mg / kg, or 0.1 mg / kg to 1 mg / kg, and most preferably 0.1 mg / kg to 1 mg / kg.

[0156] [Radiographic imaging method]

[0157] In one embodiment, the present invention provides a radiographic imaging method. The radiographic imaging method of this embodiment includes a step of detecting radiation emitted from the living body brain of a subject to which a binder of this embodiment, which is a compound (I) or a compound (II) containing one or more atoms as radioactive isotopes of the atoms, a pharmaceutically acceptable salt thereof, or a solvate thereof, has been administered.

[0158] If an effective amount of a binder is administered to a subject to be detected, the binder transferred to the living body brain binds to α-synuclein aggregates present in the living body brain. By detecting the radiation emitted from the binder in the brain, radiographic imaging (imaging) of α-synuclein aggregates can be performed.

[0159] Examples of the subject to be detected include mammals. Mammals include, for example, humans, rats, mice, rabbits, guinea pigs, hamsters, monkeys, dogs, ferrets, or mini pigs. Mammals are preferably humans.

[0160] There is no particular limitation on the administration method. For example, there are oral administration and parenteral administrations such as intravenous administration or intraperitoneal administration. Intravenous administration or intraperitoneal administration is preferred. Intravenous administration is most preferred. The administration amount is preferably 5 ng / kg to 5 mg / kg, more preferably 5 ng to 20 μg / kg. The administered radiation energy is preferably 37 MBq to 7.4 GBq per subject, more preferably 370 MBq to 3700 MBq.

[0161] [Screening method for therapeutic or prophylactic drugs for diseases related to α-synuclein aggregates in the brain]

[0162] In one embodiment, the present invention provides a method for screening a therapeutic or prophylactic drug for a disease associated with α-synuclein aggregates in the brain. The method for screening a therapeutic or prophylactic drug for a disease associated with α-synuclein aggregates in the brain of the present embodiment (hereinafter, also referred to as the screening method) has a step of selecting a candidate substance based on the difference in the amount and / or distribution of light or radiation detected by the optical imaging method or the radiological imaging method of the present embodiment before and after administering the candidate substance to the subject to be detected.

[0163] The disease associated with α-synuclein aggregates in the brain is the same as the disease described in the above [Diagnostic Kit for a Disease Associated with a Substance Accumulating in the Brain].

[0164] In addition, the subject to be detected and the administration method are the same as those described in the above [Optical Imaging Method] and [Radiological Imaging Method].

[0165] For example, after administering the candidate substance, when the amount (intensity) of light or radiation such as the fluorescence of the binding agent decreases compared to before administering the candidate substance, the candidate substance can be useful as a therapeutic compound for the disease or symptom.

[0166] In addition, when the amount and / or distribution of light or radiation detected in the subject to be detected after administering the candidate compound is closer to that of a normal mammal compared to before administration, the candidate compound can be useful as a therapeutic compound for the disease or symptom.

[0167] For example, the data on the amount (intensity) and / or distribution of light or radiation such as the fluorescence of the binding agent before and after administering the candidate substance obtained from the subject administered with the candidate substance is compared with the increase amount and / or distribution change of the fluorescence of the binding agent before and after the onset of the disease associated with α-synuclein aggregates in the brain in a mammal that has not been administered the candidate substance in advance. Moreover, when the increase in the amount of such fluorescence or radiation observed after the onset of the disease is suppressed by administering the candidate substance and / or the amount of such fluorescence or radiation shows a value close to that of a normal mammal after administering the candidate substance as compared to before administration, the candidate substance can be useful as a prophylactic compound for the disease or symptom. Similarly, when the change in the distribution of such fluorescence or radiation observed after the onset of the disease is suppressed by administering the candidate substance and / or the change in the distribution of such fluorescence or radiation is close to the distribution in a normal mammal after administering the candidate substance as compared to before administration, the candidate substance can also be useful as a prophylactic compound for the disease or symptom.

[0168] [Method for Quantifying or Determining the Accumulation of α-Synuclein Aggregates in the Brain]

[0169] In one embodiment, the present invention provides a method for quantifying or determining the accumulation of α-synuclein aggregates in the brain. The method for quantifying or determining the accumulation of α-synuclein aggregates in the brain according to this embodiment includes, after irradiating the living body brain of a subject administered with the above-mentioned binder including compound (I) or compound (II), a pharmaceutically acceptable salt thereof or a solvate thereof, with light of a first wavelength from outside the brain, the step of detecting light of a second wavelength different from the first wavelength emitted from the brain, and quantifying or determining the accumulation of α-synuclein aggregates in the brain based on the amount and / or distribution of the detected light. This method is a method for quantifying or determining the accumulation of α-synuclein aggregates in the brain by optical imaging.

[0170] The subject and the administration method are the same as those described in the above [optical imaging method].

[0171] By obtaining the difference between the amount and / or distribution of the detected light of the subject and that of other normal mammals, it is possible to quantify the accumulation of α-synuclein aggregates in the brain and determine whether there is accumulation of α-synuclein aggregates in the brain.

[0172] In another aspect of the method for quantifying or determining the accumulation of α-synuclein aggregates in the brain according to this embodiment, it includes the step of detecting radiation emitted from the living body brain of a subject administered with the binder according to this embodiment including compound (I) or compound (II), a pharmaceutically acceptable salt thereof or a solvate thereof, in which one or more atoms are radioactive isotopes of the atom, and quantifying or determining the accumulation of α-synuclein aggregates in the brain based on the amount and / or distribution of the detected radiation. This method is a method for quantifying or determining the accumulation of α-synuclein aggregates in the brain by radioimaging.

[0173] The subject and the administration method are the same as those described in the above [radioimaging method].

[0174] By obtaining the difference between the amount and / or distribution of the detected light or radiation of the subject and that of other normal mammals, it is possible to quantify the accumulation of α-synuclein aggregates in the brain and determine whether there is accumulation of α-synuclein aggregates in the brain.

[0175] [Method for determining the classification and accumulation of substances accumulated in the brain]

[0176] In one embodiment, the present invention provides a method for determining the classification and accumulation of substances accumulated in the brain. The method for determining the classification and accumulation of substances accumulated in the brain according to the present invention includes: a first step of irradiating light of a first wavelength from outside the brain to the living body brain of a subject administered with a binder of the present embodiment including compound (I) or compound (II), a pharmaceutically acceptable salt thereof, or a solvate thereof, and then detecting light of a second wavelength different from the first wavelength emitted from the brain; and a second step of irradiating light of a third wavelength from outside the brain to the living body brain of the subject administered with a substance having a high binding selectivity to Tau protein aggregates (for example, the compound described in Patent Document 1) at a time different from the first step, and then detecting light of a fourth wavelength different from the third wavelength emitted from the brain, and determining the classification and accumulation of substances accumulated in the brain based on the amount and / or distribution data of the light detected in the first step and the amount and / or distribution data of the light detected in the second step. This method is a method for determining the classification and accumulation of substances accumulated in the brain by optical imaging.

[0177] The subject and the administration method are the same as those described in the above [optical imaging method].

[0178] Since it includes both a first step of detecting light emitted from the brain due to the administration of a binder having a high binding selectivity to α-synuclein aggregates and a second step of detecting light emitted from the brain due to the administration of a substance having a high binding selectivity to Tau protein aggregates in the same subject, the detection result (the amount and / or distribution of the detected light) of the first step and the detection result of the second step are compared, whereby it is possible to determine the classification and accumulation of whether the substance accumulated in the brain is α-synuclein aggregates and / or Tau protein aggregates.

[0179] In another aspect of the method for determining the classification and accumulation of substances accumulated in the brain according to the present embodiment, it includes: a first step of detecting radiation emitted from the living body brain of a subject administered with compound (I) or compound (II), a pharmaceutically acceptable salt thereof, or a solvate thereof, in which one or more atoms are radioactive isotopes of the atoms; and a second step of detecting radiation emitted from the brain of the subject administered with a substance having a high binding selectivity to Tau protein aggregates (for example, the compound described in Patent Document 1) at a time different from the first step, and determining the classification and accumulation of substances accumulated in the brain based on the amount and / or distribution data of the radiation detected in the first step and the amount and / or distribution data of the light detected in the second step. This method is a method for determining the classification and accumulation of substances accumulated in the brain by radioimaging.

[0180] The subject and the administration method are the same as those described in the above [radioimaging method].

[0181] Both the first step of detecting radiation emitted from the brain due to the administration of a binder having a high binding selectivity for α-synuclein aggregates and the second step of detecting radiation emitted from the same subject due to the administration of a substance having a high binding selectivity for Tau protein aggregates. Therefore, by comparing the detection result (the amount and / or distribution of the detected radiation) of the first step with the detection result of the second step, it is possible to determine the classification and accumulation of the substance accumulated in the brain as α-synuclein aggregates and / or Tau protein aggregates.

[0182] (Summary)

[0183] Hereinafter, the embodiments of the present invention will be summarized.

[0184] One aspect of the present invention is a compound represented by the following formula (I) or (II), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0185] Chemical formula 13

[0186]

[0187] One aspect of the present invention is an α-synuclein aggregate binder containing the compound, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0188] One aspect of the present invention is an optical imaging composition for α-synuclein aggregates containing the binder.

[0189] One aspect of the present invention is a radioimaging composition for α-synuclein aggregates containing the binder.

[0190] One aspect of the present invention is an optical imaging method for α-synuclein aggregates in the brain, which includes the step of irradiating the living brain of a subject administered with the binder with light of a first wavelength from outside the brain and then detecting light of a second wavelength different from the first wavelength emitted from the brain.

[0191] One aspect of the present invention is a radioimaging method for α-synuclein aggregates in the brain, which includes the step of detecting radiation emitted from the living brain of a subject administered with the binder.

[0192] One aspect of the present invention is an intermediate for synthesizing the compound represented by the following formula (III).

[0193] Chemical formula 14

[0194]

[0195] (In formula (III), one of X and Y is a nitrogen atom (N), and the other is an unsubstituted carbon atom (CH) (that is, when X is a nitrogen atom, Y is an unsubstituted carbon atom (CH); when X is an unsubstituted carbon atom (CH), Y is a nitrogen atom).

[0196] R1 is a hydroxyl group or a group represented by the following formula (i).

[0197] Chemical formula 15

[0198]

[0199] Wherein, Ts represents p-toluenesulfonyl, THP represents tetrahydro-2H-pyran-2-yl, and * represents the bonding position to the benzothiazole ring.

[0200] R2 is a hydrogen atom or tert-butoxycarbonyl (Boc).

[0201] One aspect of the present invention is a diagnostic kit comprising the compound (I) or compound (II) having a high binding selectivity to α-synuclein aggregates.

[0202] The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0203] Examples

[0204] (Production Example)

[0205] In the following production examples (reference examples and examples), "room temperature" generally means about 10°C to about 35°C. Unless otherwise specified, % represents weight %. In addition, regarding the description of the compounds used in each production example, "produced by reference example (example) X" also includes the case of "produced in the same manner as reference example (example) X".

[0206] Unless otherwise specified, the elution in column chromatography of the production examples is carried out under the observation of TLC (Thin Layer Chromatography). In the TLC observation, 60F254 manufactured by Merck & Co., Inc. is used as the TLC plate, and the solvent used as the elution solvent in column chromatography is used as the developing solvent. In addition, UV detector is used for detection.

[0207] 1 The analysis of 1H NMR is carried out using ACD / SpecManager (trade name) software, etc., and the obtained analysis values are recorded. For peaks with very flat proton peaks such as hydroxyl groups and amino groups, they may not be recorded sometimes.

[0208] The following simplified notations are used in the examples below.

[0209] M: Molar concentration.

[0210] DMSO-d6: Deuterated dimethyl sulfoxide.

[0211] 1 1H NMR: Proton nuclear magnetic resonance.

[0212] DIEA: N-Ethyl-N-isopropylpropan-2-amine.

[0213] DMF: N,N-Dimethylformamide (also denoted as "dimethylformamide" in this specification).

[0214] THF: Tetrahydrofuran.

[0215] MeOH: Methanol.

[0216] EtOH: Ethanol.

[0217] DMSO: Dimethyl sulfoxide.

[0218] TEA: Triethylamine.

[0219] TFA: Trifluoroacetic acid.

[0220] Reference Example 1: Preparation of Di-tert-butyl (5-iodopyrazin-2-yl)-2-imidodicarbonate

[0221] Chemical Formula 16

[0222]

[0223] At 0 °C, a solution of di-tert-butyl dicarbonate (25.9 g) in THF (50 mL) and N,N-dimethylpyridin-4-amine (1.45 g) were added to a solution of 5-iodopyrazin-2-amine (CAS [886860-50-0]) (10.5 g) in THF (100 mL). The mixture was stirred overnight from 0 °C to room temperature, diluted with water and ethyl acetate, the insoluble matter was separated by filtration, and then extracted with ethyl acetate. After washing the extract with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure through a short silica gel column to obtain the title compound (18.8 g) as a brown solid.

[0224] 1 1H NMR (300 MHz, DMSO-d6) δ 1.37 - 1.41 (18H, m), 8.65 (1H, d, J = 1.3 Hz), 8.90 (1H, d, J = 1.5 Hz).

[0225] Reference Example 2: Preparation of tert-butyl (5-iodopyrazin-2-yl)carbamate

[0226] Chemical formula 17

[0227]

[0228] At room temperature, potassium carbonate (7.40 g) was added to a solution of di-tert-butyl (5-iodopyrazin-2-yl)-2-imide dicarbonate (18.8 g) prepared in Reference Example 1 in MeOH (200 mL). After stirring the mixture at room temperature for 2 hours, it was concentrated to about one-fourth under reduced pressure, cooled to 0 °C, neutralized with 5% aqueous citric acid solution and then diluted with water. The precipitated solid was collected by filtration, washed with water and dried to obtain the title compound (13.7 g) as a brown solid.

[0229] 1 H NMR (300 MHz, DMSO-d6) δ 1.47 (9H, s), 8.55 (1H, d, J = 1.3 Hz), 8.87 (1H, d, J = 1.5 Hz), 10.29 (1H, brs).

[0230] Reference Example 3: Preparation of tert-butyl (5-iodopyrazin-2-yl)(methyl)carbamate

[0231] Chemical formula 18

[0232]

[0233] At 0 °C, iodomethane (3.45 mL) was added to a solution of tert-butyl (5-iodopyrazin-2-yl)carbamate (13.7 g) and cesium carbonate (20.9 g) in DMF (85 mL) prepared in Reference Example 2. The mixture was stirred from 0 °C to room temperature overnight, cooled to 0 °C and diluted with water. The precipitated solid was collected by filtration, washed with water and dried to obtain the title compound (13.0 g) as a beige solid.

[0234] 1 H NMR (300 MHz, DMSO-d6) δ 1.49 (9H, s), 3.27 (3H, s), 8.72 (1H, d, J = 1.5 Hz), 8.83 (1H, d, J = 1.5 Hz).

[0235] Reference Example 4: Preparation of tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0236] Chemical formula 19

[0237]

[0238] Under room temperature conditions, in a nitrogen atmosphere, dichlorobis(triphenylphosphine)palladium(II) (545 mg) was added to a mixed solution of tert-butyl (5-iodopyrazin-2-yl)(methyl)carbamate (13.0 g), 2-propyn-1-ol (3.43 mL), and copper(I) iodide (739 mg) in TEA (27 mL) and THF (27 mL) prepared in Reference Example 3. After stirring the mixture at room temperature for 2 hours, it was diluted with ethyl acetate and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure, the residue was diluted with ethyl acetate, acidified with a 5% aqueous citric acid solution, filtered through diatomaceous earth, and the filtrate was extracted with ethyl acetate. After washing the extract with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure through a silica gel pad to obtain the title compound (10.2 g) as a brown solid.

[0239] 1 H NMR (300 MHz, DMSO-d6) δ 1.50 (9H, s), 3.31 (3H, s), 4.35 (2H, d, J = 6.0 Hz), 5.48 (1H, t, J = 6.0 Hz), 8.52 (1H, d, J = 1.5 Hz), 8.98 (1H, d, J = 1.5 Hz).

[0240] Reference Example 5: Preparation of tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate

[0241] Chemical Formula 20

[0242]

[0243] Using tert-butyl (5-iodopyrimidin-2-yl)(methyl)carbamate (CAS [1578264-18-2]) (250 mg), the title compound (195 mg) as a brown solid was obtained by the same method as in Reference Example 4.

[0244] 1 H NMR (300 MHz, DMSO-d6) δ 1.44 (9H, s), 3.31 (3H, s), 4.34 (2H, d, J = 5.7 Hz), 5.44 (1H, t, J = 5.8 Hz), 8.77 (2H, s).

[0245] Reference Example 6: Preparation of tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate

[0246] Chemical Formula 21

[0247]

[0248] At room temperature, 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (20.1 g) was added to a solution of tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (10.4 g) in acetonitrile (200 mL) prepared in Reference Example 4. After stirring the mixture at room temperature for 2 hours, it was cooled to 0 °C, saturated aqueous sodium thiosulfate solution and 5% aqueous sodium hydrogen carbonate solution were added, and after stirring for 5 minutes, it was extracted with ethyl acetate. After washing the extract with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure through a short silica gel column. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (6.20 g) as a yellow solid.

[0249] 1 H NMR (300 MHz, DMSO-d6) δ 1.52 (9H, s), 3.35 (3H, s), 8.82 (1H, d, J = 1.3 Hz), 9.17 (1H, d, J = 1.5 Hz), 9.48 (1H, s).

[0250] Reference Example 7: Preparation of tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrimidin-2-yl)carbamate

[0251] Chemical formula 22

[0252]

[0253] Using tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate (190 mg) prepared in Reference Example 5, the title compound (149 mg) as a white solid was obtained by the same method as in Reference Example 6.

[0254] 1 H NMR (300 MHz, DMSO-d6) δ 1.47 (9H, s), 3.36 (3H, s), 9.00 (2H, s), 9.46 (1H, s).

[0255] Reference Example 8: Preparation of 2-((1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0256] Chemical formula 23

[0257]

[0258] At room temperature, p-toluenesulfonic acid monohydrate (132 mg) was added to a solution of 1-(benzyloxy)-3-fluoropropan-2-ol (CAS [112482-36-7]) (6.37 g) and 3,4-dihydro-2H-pyran (3.75 mL) in THF (67 mL). The mixture was stirred overnight at room temperature, then DIEA (0.242 mL) was added and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (8.37 g) as a colorless liquid.

[0259] 1 H NMR (300 MHz, DMSO-d6) δ 1.35 - 1.54 (4H, m), 1.56 - 1.80 (2H, m), 3.36 - 3.47 (1H, m), 3.48 - 3.63 (2H, m), 3.80 (1H, ddd, J = 11.2, 7.9, 3.4 Hz), 3.90 - 4.06 (1H, m), 4.35 - 4.70 (2H, m), 4.51 (2H, d, J = 2.1 Hz), 4.74 - 4.81 (1H, m), 7.24 - 7.41 (5H, m).

[0260] Reference Example 9: Preparation of 2-(((R)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0261] Chemical Formula 24

[0262]

[0263] Using (R)-1-(benzyloxy)-3-fluoropropan-2-ol (CAS [147332-34-1]) (7.85 g), the title compound (10.36 g) as a colorless liquid was obtained by the same method as in Reference Example 8.

[0264] 1 H NMR (300 MHz, DMSO-d6) δ 1.47 (4H, brdd, J = 7.4, 3.9 Hz), 1.56 - 1.80 (2H, m), 3.35 - 3.47 (1H, m), 3.47 - 3.63 (2H, m), 3.80 (1H, ddd, J = 11.3, 8.0, 3.4 Hz), 3.89 - 4.11 (1H, m), 4.34 - 4.69 (4H, m), 4.73 - 4.83 (1H, m), 7.19 - 7.49 (5H, m).

[0265] Reference Example 10: Preparation of 2-(((S)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0266] Chemical formula 25

[0267]

[0268] Using (S)-1-(benzyloxy)-3-fluoropropan-2-ol (CAS [1707146-21-1]) (7.55 g), the title compound as a colorless liquid (10.48 g) was obtained in the same manner as in Reference Example 8.

[0269] 1 H NMR (300 MHz, DMSO-d6) δ 1.47 (4H, brdd, J = 7.4, 3.9 Hz), 1.56 - 1.79 (2H, m), 3.35 - 3.47 (1H, m), 3.48 - 3.62 (2H, m), 3.80 (1H, ddd, J = 11.2, 7.9, 3.3 Hz), 3.90 - 4.08 (1H, m), 4.35 - 4.70 (4H, m), 4.73 - 4.81 (1H, m), 7.24 - 7.42 (5H, m).

[0270] Reference Example 11: Preparation of 3-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate

[0271] Chemical formula 26

[0272]

[0273] At 0 °C, tert-butyldimethylchlorosilane (1.51 g) was added to a solution of 3-(benzyloxy)-2-hydroxypropyl 4-methylbenzenesulfonate (CAS [99881-48-8]) (2.80 g) and 1H-imidazole (737 mg) in DMF (30 mL). The mixture was stirred overnight at room temperature, cooled to 0 °C, diluted with ethyl acetate and water, adjusted to pH about 5 with 5% aqueous citric acid solution, and then extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound as a colorless liquid (3.37 g).

[0274] 1 H NMR (300 MHz, DMSO-d6) δ -0.02 (6H, d, J = 1.1 Hz), 0.79 (9H, s), 2.41 (3H, s), 3.36 (2H, d, J = 5.3 Hz), 3.86 - 4.05 (3H, m), 4.43 (2H, s), 7.20 - 7.39 (5H, m), 7.47 (2H, d, J = 8.1 Hz), 7.77 (2H, d, J = 8.3 Hz).

[0275] Reference Example 12: Preparation of 3-Fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0276] Chemical Formula 27

[0277]

[0278] Under room temperature conditions and in a hydrogen environment at normal pressure, a mixture of 2-((1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran (8.30 g) prepared in Reference Example 8, 10% palladium-carbon (50% water-containing, 1.98 g), and EtOH (85 mL) was stirred overnight. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the title compound (5.45 g) as a colorless liquid.

[0279] 1 H NMR (300 MHz, DMSO-d6) δ 1.33 - 1.55 (4H, m), 1.56 - 1.86 (2H, m), 3.36 - 3.58 (3H, m), 3.68 - 3.88 (2H, m), 4.31 - 4.70 (2H, m), 4.71 - 4.86 (2H, m).

[0280] Reference Example 13: Preparation of (2R)-3-Fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0281] Chemical Formula 28

[0282]

[0283] Using 2-(((R)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran (10.35 g) prepared in Reference Example 9, the title compound (6.85 g) as a colorless liquid was obtained by the same method as in Reference Example 12.

[0284] 1 H NMR (300 MHz, DMSO-d6) δ 1.35 - 1.53 (4H, m), 1.55 - 1.81 (2H, m), 3.37 - 3.62 (3H, m), 3.67 - 3.91 (2H, m), 4.29 - 4.69 (2H, m), 4.71 - 4.87 (2H, m).

[0285] Reference Example 14: Preparation of (2S)-3-Fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0286] Chemical Formula 29

[0287]

[0288] Using 2-(((S)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydropyran (10.45 g) prepared in Reference Example 10, the title compound as a colorless liquid (6.90 g) was obtained by the same method as in Reference Example 12.

[0289] 1 H NMR (300 MHz, DMSO-d6) δ 1.33 - 1.56 (4H, m), 1.57 - 1.81 (2H, m), 3.35 - 3.59 (3H, m), 3.69 - 3.90 (2H, m), 4.31 - 4.68 (2H, m), 4.71 - 4.87 (2H, m).

[0290] Reference Example 15: Preparation of 2-((tert-butyldimethylsilyl)oxy)-3-hydroxypropyl 4-methylbenzenesulfonate

[0291] Chemical Formula 30

[0292]

[0293] Using 3-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate (3.36 g) prepared in Reference Example 11, the title compound as a colorless liquid (2.66 g) was obtained by the same method as in Reference Example 12.

[0294] 1 H NMR (300 MHz, DMSO-d6) δ -0.01 (3H, s), 0.01 (3H, s), 0.80 (9H, s), 2.42 (3H, s), 3.20 - 3.38 (2H, m), 3.75 - 3.91 (2H, m), 4.02 - 4.10 (1H, m), 4.81 (1H, t, J = 5.6 Hz), 7.49 (2H, dd, J = 8.6, 0.7 Hz), 7.77 (2H, d, J = 8.3 Hz).

[0295] Reference Example 16: Preparation of a mixture of (2S)-3-fluoro-2-((tetrahydropyran-2-yl)oxy)propan-1-ol and (2S)-1-fluoro-3-((tetrahydropyran-2-yl)oxy)propan-2-ol

[0296] Chemical Formula 31

[0297]

[0298] The (2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol (6.90 g) prepared in Reference Example 14 was stored at room temperature to obtain the title compound (6.90 g) as a colorless liquid.

[0299] 1 H NMR (300 MHz, DMSO-d6) δ 1.33 - 1.54 (4H, m), 1.56 - 1.85 (2H, m), 3.28 - 3.96 (5H, m), 4.18 - 4.92 (3H, m), 4.92 - 5.20 (1H, m).

[0300] Reference Example 17: Preparation of Diethyl ((6-((tert-butyldimethylsilyl)oxy)benzothiazol-2-yl)methyl)phosphonate

[0301] Chemical Formula 32

[0302]

[0303] A mixture of 2-(bromomethyl)-6-((tert-butyldimethylsilyl)oxy)benzothiazole (CAS [1638685-65-0]) (3.06 g) and triethyl phosphite (1.78 mL) was stirred at 100 °C for 3 hours, then cooled to room temperature and purified by silica gel column chromatography (ethyl acetate / methanol) to obtain the title compound (2.39 g) as a yellow liquid.

[0304] 1 H NMR (300 MHz, DMSO-d6) δ 0.22 (6H, s), 0.97 (9H, s), 1.23 (6H, t, J = 7.0 Hz), 3.80 - 3.92 (2H, m), 4.01 - 4.13 (4H, m), 7.00 (1H, dd, J = 8.7, 2.5 Hz), 7.56 (1H, d, J = 2.4 Hz), 7.81 (1H, d, J = 8.8 Hz).

[0305] Reference Example 18: Preparation of Diethyl ((6-hydroxybenzothiazol-2-yl)methyl)phosphonate

[0306] Chemical Formula 33

[0307]

[0308] At room temperature, tetrabutylammonium fluoride (1 M THF solution, 12.9 mL) was added to a solution of diethyl ((6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazol-2-yl)methyl)phosphonate (4.87 g) in THF (50 mL) prepared in Reference Example 17. After the mixture was stirred at room temperature for 1 hour, it was diluted with water and extracted with ethyl acetate. After the extract was washed with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solid residue was suspended in hexane / ethyl acetate, filtered, collected, washed with hexane / ethyl acetate, and dried to obtain the title compound (2.85 g) as a yellow solid.

[0309] 1 1H NMR (300 MHz, DMSO-d6) δ 1.22 (6H, t, J = 7.1 Hz), 3.81 (2H, d, J = 21.4 Hz), 4.05 (4H, dq, J = 8.3, 7.1 Hz), 6.93 (1H, dd, J = 8.8, 2.5 Hz), 7.34 (1H, d, J = 2.4 Hz), 7.73 (1H, d, J = 8.8 Hz), 9.78 (1H, brs).

[0310] Reference Example 19: Preparation of Diethyl ((6-(3-Fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)methyl)phosphonate

[0311] Chemical Formula 34

[0312]

[0313] At room temperature, diisopropyl azodicarboxylate (3.15 mL) was added to a solution of diethyl ((6-hydroxybenzo[d]thiazol-2-yl)methyl)phosphonate (2.84 g) prepared in Reference Example 18, 3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol (2.52 g) prepared in Reference Example 12, and triphenylphosphine (4.20 g) in THF (50 mL). The mixture was stirred at room temperature for 1 day, 3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol (0.504 g), triphenylphosphine (0.742 g), and diisopropyl azodicarboxylate (0.555 mL) prepared in Reference Example 12 were added at room temperature, and after stirring overnight at room temperature, it was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (3.78 g) as a yellow liquid.

[0314] 11H NMR (300 MHz, DMSO-d6) δ 1.22 (6H, t, J = 7.0 Hz), 1.37 - 1.56 (4H, m), 1.59 - 1.80 (2H, m), 3.41 - 3.53 (1H, m), 3.78 - 3.94 (3H, m), 4.01 - 4.11 (4H, m), 4.12 - 4.29 (3H, m), 4.48 - 4.65 (1H, m), 4.65 - 4.81 (1H, m), 4.83 - 4.94 (1H, m), 7.12 (1H, dt, J = 9.0, 2.1 Hz), 7.70 (1H, d, J = 2.4 Hz), 7.84 (1H, d, J = 8.9 Hz).

[0315] Reference Example 20: Preparation of tert-butyl (E)-(5-(4-(6-(3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0316] Chemical Formula 35

[0317]

[0318] Under the condition of 0 °C, sodium hydride (60% oily, 392 mg) was added to a solution of diethyl ((6-(3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)methyl)phosphonate (3.77 g) prepared in Reference Example 19 and tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate (2.56 g) prepared in Reference Example 6 in THF (30 mL). After stirring the mixture at 0 °C for 10 minutes, DMF (30 mL) was added at 0 °C, and the mixture was stirred at 0 °C to room temperature for 3 hours. At room temperature, tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate (256 mg) prepared in Reference Example 6 was added to the mixture and stirred at room temperature for 1 hour. Then, the mixture was diluted with ethyl acetate and water, neutralized with 5% aqueous citric acid solution, and extracted with ethyl acetate. After washing the extract with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate), and the fraction containing the title compound was concentrated under reduced pressure. The solid residue was suspended in hexane, and the solid obtained by filtration was washed with hexane and then dried to obtain the title compound (2.06 g) as a yellow solid.

[0319] 11H NMR (300 MHz, DMSO-d6) δ 1.39 - 1.58 (13H, m), 1.59 - 1.81 (2H, m), 3.33 (3H, s), 3.41 - 3.52 (1H, m), 3.78 - 3.95 (1H, m), 4.13 - 4.31 (3H, m), 4.48 - 4.66 (1H, m), 4.66 - 4.83 (1H, m), 4.84 - 4.93 (1H, m), 7.01 (1H, d, J = 16.2 Hz), 7.17 (1H, dt, J = 9.0, 2.2 Hz), 7.48 (1H, d, J = 16.0 Hz), 7.77 (1H, d, J = 2.4 Hz), 7.92 (1H, d, J = 8.9 Hz), 8.65 (1H, d, J = 1.5 Hz), 9.06 (1H, d, J = 1.5 Hz).

[0320] Example 1: Preparation of tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0321] Chemical Formula 36

[0322]

[0323] Under the condition of 0 °C, sodium hydride (60% oily, 322 mg) was added to a THF (25 mL) solution of diethyl ((6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazol-2-yl)methyl)phosphonate (2.39 g) prepared in Reference Example 17 and tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate (1.65 g) prepared in Reference Example 6. After stirring the mixture at 0 °C for 5 minutes, DMF (25 mL) was added at 0 °C, and the mixture was stirred at 0 °C for 2 hours. Under the condition of 0 °C, sodium hydride (60% oily, 138 mg) and tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate (601 mg) prepared in Reference Example 6 were added to the mixture. After stirring at 0 °C to room temperature for 2 hours, it was cooled to 0 °C, adjusted to pH about 5 with 5% aqueous citric acid solution, diluted with water, and then extracted with ethyl acetate. After washing the extract with water and brine, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure by short silica gel column chromatography. The solid residue was suspended in ethyl acetate / hexane, and the solid collected by filtration was washed with ethyl acetate / hexane and then dried to obtain the title compound (1.85 g) as a yellow solid.

[0324] 11H NMR (300 MHz, DMSO-d6) δ 1.51 (9H, s), 3.33 (3H, s), 6.94 (1H, d, J = 16.0 Hz), 7.00 (1H, dd, J = 8.9, 2.4 Hz), 7.39 (1H, d, J = 2.3 Hz), 7.44 (1H, d, J = 16.0 Hz), 7.82 (1H, d, J = 8.9 Hz), 8.64 (1H, d, J = 1.5 Hz), 9.05 (1H, d, J = 1.5 Hz), 10.14 (1H, brs).

[0325] Example 2: Preparation of tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate

[0326] Chemical Formula 37

[0327]

[0328] Using diethyl ((6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazol-2-yl)methyl)phosphonate (254 mg) prepared in Reference Example 17 and tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrimidin-2-yl)carbamate (145 mg) prepared in Reference Example 7, the title compound (101 mg) as a yellow solid was obtained by the same method as in Example 1.

[0329] 1 1H NMR (300 MHz, DMSO-d6) δ 1.46 (9H, s), 3.34 (3H, s), 6.92 (1H, d, J = 16.0 Hz), 7.00 (1H, dd, J = 8.9, 2.4 Hz), 7.38 (1H, d, J = 16.0 Hz), 7.39 (1H, d, J = 2.4 Hz), 7.82 (1H, d, J = 8.9 Hz), 8.87 (2H, s), 10.01 (1H, s).

[0330] Reference Example 21: Preparation of tert-butyl (5-((E)-4-(6-((2R)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0331] Chemical Formula 38

[0332]

[0333] Using the tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (100 mg) prepared in Production Example 1, (2R)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol (114 mg) prepared in Reference Example 13, and bis(2-methoxyethyl)azodicarboxylate (172 mg), the title compound (100 mg) as a pale yellow solid was obtained by the same method as in Reference Example 19.

[0334] 1 H NMR (300 MHz, DMSO-d6) δ 1.51 (13H, s), 1.60 - 1.79 (2H, m), 3.34 (3H, s), 3.42 - 3.54 (1H, m), 3.79 - 3.96 (1H, m), 4.14 - 4.31 (3H, m), 4.49 - 4.82 (2H, m), 4.84 - 4.96 (1H, m), 7.02 (1H, d, J = 16.1 Hz), 7.17 (1H, dt, J = 9.0, 2.1 Hz), 7.48 (1H, d, J = 16.1 Hz), 7.77 (1H, d, J = 2.5 Hz), 7.92 (1H, d, J = 9.0 Hz), 8.65 (1H, d, J = 1.4 Hz), 9.06 (1H, d, J = 1.5 Hz).

[0335] Reference Example 22: Preparation of tert-butyl (5-((E)-4-(6-((2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0336] Chemical Formula 39

[0337]

[0338] Using the tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (100 mg) prepared in Production Example 1 and a mixture of (2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol and (2S)-1-fluoro-3-((tetrahydro-2H-pyran-2-yl)oxy)propan-2-ol (114 mg) prepared in Reference Example 16, and bis(2-methoxyethyl)azodicarboxylate (172 mg), the title compound (22 mg) as a pale yellow solid was obtained by the same method as in Reference Example 19.

[0339] 11H NMR (300 MHz, DMSO-d6) δ 1.51 (13H, s), 1.60 - 1.79 (2H, m), 3.34 (3H, s), 3.42 - 3.54 (1H, m), 3.79 - 3.95 (1H, m), 4.13 - 4.30 (3H, m), 4.49 - 4.82 (2H, m), 4.84 - 4.96 (1H, m), 7.02 (1H, d, J = 16.2 Hz), 7.17 (1H, dd, J = 8.8, 2.4 Hz), 7.48 (1H, d, J = 16.0 Hz), 7.77 (1H, d, J = 2.3 Hz), 7.92 (1H, d, J = 9.0 Hz), 8.65 (1H, d, J = 1.3 Hz), 9.06 (1H, d, J = 1.3 Hz).

[0340] Reference Example 23: Preparation of (E)-3-((2-(4-(5-((tert-Butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate

[0341] Chemical Formula 40

[0342]

[0343] Using the tert-Butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (971 mg) prepared in Example 1, 2-((tert-butyldimethylsilyl)oxy)-3-hydroxypropyl 4-methylbenzenesulfonate (1.11 g) prepared in Reference Example 15, and bis(2-methoxyethyl)azodicarboxylate (835 mg), the title compound (1.47 g) was obtained as a pale yellow solid by the same method as in Reference Example 19.

[0344] 11H NMR (300 MHz, DMSO-d6) δ 0.04 (3H, s), 0.04 (3H, s), 0.81 (9H, s), 1.51 (9H, s), 2.38 (3H, s), 3.34 (3H, s), 3.90 - 4.19 (4H, m), 4.20 - 4.32 (1H, m), 7.02 (1H, d, J = 16.0 Hz), 7.02 - 7.08 (1H, m), 7.45 (2H, brd, J = 8.1 Hz), 7.48 (1H, brd, J = 16.0 Hz), 7.66 (1H, d, J = 2.3 Hz), 7.79 (2H, d, J = 8.3 Hz), 7.91 (1H, d, J = 9.0 Hz), 8.65 (1H, d, J = 1.5 Hz), 9.06 (1H, d, J = 1.5 Hz).

[0345] Reference Example 24: Preparation of tert-Butyl (E)-(5-(4-(6-(3-Fluoro-2-hydroxypropoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate

[0346] Chemical Formula 41

[0347]

[0348] At room temperature, 2-(fluoromethyl)oxirane (CAS [503-09-3]) (0.060 mL) was added to a solution of tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate (99 mg) and potassium carbonate (66 mg) in DMF (3 mL) prepared in Example 2. The mixture was heated at 80 °C for 4 hours, cooled to room temperature, diluted with ethyl acetate and water, neutralized with 5% aqueous citric acid solution, and then extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (36 mg) as a yellow solid.

[0349] 11H NMR (300 MHz, DMSO-d6) δ 1.46 (9H, s), 3.34 (3H, s), 4.02 - 4.16 (3H, m), 4.39 - 4.46 (1H, m), 4.56 - 4.63 (1H, m), 5.49 (1H, d, J = 5.3 Hz), 6.99 (1H, d, J = 16.2 Hz), 7.16 (1H, dd, J = 9.0, 2.6 Hz), 7.41 (1H, d, J = 16.2 Hz), 7.73 (1H, d, J = 2.3 Hz), 7.91 (1H, d, J = 9.0 Hz), 8.87 (2H, s).

[0350] Reference Example 25: Preparation of (E)-3-((2-(4-(5-((tert-Butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-hydroxypropyl 4-methylbenzenesulfonate

[0351] Chemical Formula 42

[0352]

[0353] At 0 °C, tetrabutylammonium fluoride (1 M THF solution, 0.533 mL) was added to a solution of (E)-3-((2-(4-(5-((tert-butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate (100 mg) and acetic acid (0.023 mL) in THF (10 mL). After stirring the mixture at 0 °C for 0.5 hour, it was diluted with water and extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (37 mg) as a yellow solid.

[0354] 11H NMR (300 MHz, DMSO-d6) δ 1.51 (9H, s), 2.35 (3H, s), 3.34 (3H, s), 3.91 - 4.19 (5H, m), 5.59 (1H, d, J = 4.9 Hz), 7.02 (1H, d, J = 16.1 Hz), 7.00 - 7.07 (1H, m), 7.35 - 7.43 (2H, m), 7.48 (1H, d, J = 16.1 Hz), 7.62 (1H, d, J = 2.5 Hz), 7.77 (2H, d, J = 8.3 Hz), 7.89 (1H, d, J = 9.0 Hz), 8.65 (1H, d, J = 1.5 Hz), 9.06 (1H, d, J = 1.5 Hz).

[0355] Reference Example 26: Preparation of (E)-2-Hydroxy-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propyl 4-Methylbenzenesulfonate

[0356] Chemical Formula 43

[0357]

[0358] At room temperature, a mixed solution of TFA (13.5 mL) and water (1.5 mL) of (E)-3-((2-(4-(5-((tert-butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-Methylbenzenesulfonate (750 mg) prepared in Reference Example 23 was stirred for 0.5 hour, then cooled to 0 °C, diluted with ethyl acetate, neutralized with saturated aqueous sodium bicarbonate, and extracted with ethyl acetate. The extract was washed with water and brine, and the solid precipitated from the extract was collected by filtration. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solid residue and the solid collected by filtration were combined and suspended in hexane. The solid collected by filtration was washed with hexane and dried to obtain the title compound (550 mg) as a yellow solid.

[0359] 11H NMR (400 MHz, DMSO-d6) δ 2.34 (3H, s), 2.84 (3H, d, J = 4.8 Hz), 3.90 - 3.98 (2H, m), 4.02 - 4.17 (3H, m), 5.60 (1H, d, J = 4.8 Hz), 6.95 (1H, d, J = 16.0 Hz), 7.00 (1H, dd, J = 8.8, 2.4 Hz), 7.29 (1H, d, J = 16.0 Hz), 7.40 (2H, d, J = 8.0 Hz), 7.59 (1H, d, J = 2.4 Hz), 7.68 (1H, brs), 7.77 (2H, d, J = 8.4 Hz), 7.86 (1H, d, J = 8.8 Hz), 7.94 (1H, d, J = 1.6 Hz), 8.21 (1H, d, J = 1.2 Hz).

[0360] Example 3: Preparation of (E)-2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-ol

[0361] Chemical Formula 44

[0362]

[0363] At room temperature, a solution of tert-butyl (E)-(5-(4-(6-hydroxybenzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (230 mg) in TFA (4 mL) was stirred for 1 hour and then concentrated under reduced pressure. Ethyl acetate was added to the residue, and after cooling to 0 °C, it was neutralized with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solid residue was suspended in ethyl acetate, and the solid collected by filtration was washed with ethyl acetate and then dried to obtain the title compound (140 mg) as a yellow solid.

[0364] 1 1H NMR (300 MHz, DMSO-d6) δ 2.84 (3H, d, J = 4.9 Hz), 6.81 - 6.92 (1H, m), 6.98 (1H, dd, J = 8.8, 2.4 Hz), 7.26 (1H, d, J = 15.8 Hz), 7.37 (1H, d, J = 2.3 Hz), 7.63 (1H, q, J = 5.0 Hz), 7.79 (1H, d, J = 8.7 Hz), 7.94 (1H, d, J = 1.5 Hz), 8.20 (1H, d, J = 1.1 Hz), 9.97 (1H, s).

[0365] Example 4: Preparation of (E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol (hereinafter referred to as "SPAL-T-06")

[0366] Chemical Formula 45

[0367]

[0368] At room temperature, a solution of tert-butyl (E)-(5-(4-(6-(3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (2.06 g) in TFA (20 mL) was stirred for 0.5 h and then concentrated under reduced pressure. Toluene was added to the residue and the mixture was concentrated under reduced pressure. Ethyl acetate and water were added to the residue and it was neutralized with 5% aqueous sodium bicarbonate solution. The precipitated solid was collected by filtration, washed with water and ethyl acetate and dried. The filtrate and washings were combined, extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The solid residue was suspended in ethyl acetate, the solid collected by filtration was washed with ethyl acetate and dried. The solid and the solid collected by filtration before extraction were combined, suspended in ethyl acetate, the solid collected by filtration was washed with ethyl acetate and dried to obtain the title compound (1.07 g) as a yellow solid.

[0369] 1 H NMR (300 MHz, DMSO-d6) δ 2.84 (3H, d, J = 4.9 Hz), 4.00 - 4.16 (3H, m), 4.37 - 4.49 (1H, m), 4.53 - 4.66 (1H, m), 5.51 (1H, d, J = 5.1 Hz), 6.95 (1H, d, J = 16.0 Hz), 7.15 (1H, dd, J = 9.0, 2.6 Hz), 7.29 (1H, d, J = 16.0 Hz), 7.67 (1H, q, J = 4.6 Hz), 7.71 (1H, d, J = 2.6 Hz), 7.89 (1H, d, J = 9.0 Hz), 7.94 (1H, d, J = 1.5 Hz), 8.21 (1H, d, J = 1.3 Hz).

[0370] Example 5: Preparation of (R,E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol

[0371] Chemical Formula 46

[0372]

[0373] The title compound (58 mg) as a pale yellow solid was obtained by the same method as in Example 4 using tert-butyl (5-((E)-4-(6-((2R)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (98 mg) prepared in Reference Example 21.

[0374] 1 H NMR (300 MHz, DMSO-d6) δ 2.84 (3H, d, J = 4.8 Hz), 4.01 - 4.17 (3H, m), 4.37 - 4.49 (1H, m), 4.53 - 4.65 (1H, m), 5.50 (1H, d, J = 5.1 Hz), 6.95 (1H, d, J = 16.1 Hz), 7.15 (1H, dd, J = 8.9, 2.6 Hz), 7.29 (1H, d, J = 16.1 Hz), 7.66 (1H, q, J = 4.9 Hz), 7.71 (1H, d, J = 2.5 Hz), 7.89 (1H, d, J = 9.0 Hz), 7.94 (1H, d, J = 1.4 Hz), 8.21 (1H, d, J = 1.3 Hz).

[0375] Example 6: Preparation of (S,E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol

[0376] Chemical formula 47

[0377]

[0378] The title compound (12 mg) as a pale yellow solid was obtained by the same method as in Example 4 using tert-butyl (5-((E)-4-(6-((2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate (20 mg) prepared in Reference Example 22.

[0379] 11H NMR (300 MHz, DMSO-d6) δ 2.84 (3H, d, J = 4.7 Hz), 4.00 - 4.17 (3H, m), 4.37 - 4.49 (1H, m), 4.53 - 4.65 (1H, m), 5.51 (1H, d, J = 4.7 Hz), 6.95 (1H, d, J = 16.0 Hz), 7.15 (1H, dd, J = 8.9, 2.5 Hz), 7.29 (1H, d, J = 16.2 Hz), 7.62 - 7.70 (1H, m), 7.71 (1H, d, J = 2.4 Hz), 7.89 (1H, d, J = 8.9 Hz), 7.94 (1H, d, J = 1.3 Hz), 8.21 (1H, d, J = 1.1 Hz).

[0380] Example 7: Preparation of (E)-1-fluoro-3-((2-(4-(2-(methylamino)pyrimidin-5-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol (hereinafter referred to as "SPAL-T-05")

[0381] Chemical formula 48

[0382]

[0383] Using tert-butyl (E)-(5-(4-(6-(3-fluoro-2-hydroxypropoxy)benzo[d]thiazol-2-yl)but-3-en-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate (34 mg) prepared in Reference Example 24, the title compound (15 mg) as a yellow solid was obtained by the same method as in Example 4.

[0384] 1 1H NMR (300 MHz, DMSO-d6) δ 2.84 (3H, d, J = 4.5 Hz), 4.00 - 4.16 (3H, m), 4.36 - 4.50 (1H, m), 4.52 - 4.66 (1H, m), 5.49 (1H, d, J = 5.3 Hz), 6.86 - 6.99 (1H, m), 7.10 - 7.18 (1H, m), 7.25 (1H, d, J = 15.8 Hz), 7.67 - 7.77 (2H, m), 7.88 (1H, d, J = 9.0 Hz), 8.38 - 8.58 (2H, m).

[0385] Example 8: Preparation of (E)-3-((2-(4-(5-((tert-butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzenesulfonate

[0386] Chemical Formula 49

[0387]

[0388] At room temperature, p-toluenesulfonic acid monohydrate (21 mg) was added to a solution of (E)-3-((2-(4-(5-((tert-butoxycarbonyl)(methyl)amino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-hydroxypropyl 4-methylbenzenesulfonate (35 mg) and 3,4-dihydro-2H-pyran (0.050 mL) in THF (15 mL). After stirring the mixture at room temperature for 4 hours, 3,4-dihydro-2H-pyran (0.249 mL) and p-toluenesulfonic acid monohydrate (21 mg) were added at room temperature. After stirring the mixture at room temperature overnight, it was diluted with ethyl acetate, neutralized with 5% aqueous sodium bicarbonate solution, diluted with water and then extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate), and the fraction containing the title compound was concentrated under reduced pressure. The solid residue was suspended in hexane, the solid obtained by filtration was washed with hexane and then dried to give the title compound (28 mg) as a yellow solid.

[0389] 1 H NMR (300 MHz, DMSO-d6) δ 1.32 - 1.72 (15H, m), 2.35 (3H, s), 3.34 - 3.46 (4H, m), 3.61 - 3.93 (1H, m), 4.01 - 4.41 (5H, m), 4.66 - 4.90 (1H, m), 6.99 - 7.05 (1H, m), 7.02 - 7.09 (1H, m), 7.41 (2H, brd, J = 8.1 Hz), 7.48 (1H, d, J = 16.2 Hz), 7.65 (1H, d, J = 2.1 Hz), 7.79 (2H, brd, J = 7.3 Hz), 7.90 (1H, d, J = 8.9 Hz), 8.65 (1H, d, J = 1.1 Hz), 9.06 (1H, d, J = 0.9 Hz).

[0390] Example 9: Preparation of (E)-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzenesulfonate

[0391] Chemical Formula 50

[0392]

[0393] At room temperature, p-toluenesulfonic acid monohydrate (12 mg) was added to a solution of (E)-2-hydroxy-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propyl 4-methylbenzenesulfonate (35 mg) and 3,4-dihydro-2H-pyran (0.018 mL) in THF (15 mL). The mixture was stirred at room temperature for 0.5 h, and then DMF (5 mL), 3,4-dihydro-2H-pyran (0.159 mL) and p-toluenesulfonic acid monohydrate (24 mg) were added at room temperature. The mixture was stirred overnight at room temperature, diluted with water, and extracted with ethyl acetate. The extract was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate), and the fraction containing the title compound was concentrated under reduced pressure. The solid residue was suspended in hexane, the solid collected by filtration was washed with hexane and then dried to give the title compound (21 mg) as a yellow solid.

[0394] 1 H NMR (300 MHz, DMSO-d6) δ 1.32 - 1.71 (6H, m), 2.35 (3H, s), 2.84 (3H, d, J = 4.9 Hz), 3.35 - 3.46 (1H, m), 3.62 - 3.87 (1H, m), 4.05 - 4.19 (3H, m), 4.20 - 4.36 (2H, m), 4.65 - 4.87 (1H, m), 6.96 (1H, d, J = 16.2 Hz), 7.02 (1H, dd, J = 8.9, 1.6 Hz), 7.30 (1H, d, J = 16.0 Hz), 7.41 (2H, d, J = 7.9 Hz), 7.62 (1H, d, J = 2.6 Hz), 7.67 (1H, q, J = 4.4 Hz), 7.79 (2H, dd, J = 8.4, 1.6 Hz), 7.86 (1H, d, J = 9.0 Hz), 7.94 (1H, d, J = 1.5 Hz), 8.21 (1H, d, J = 1.3 Hz).

[0395] Example 10: 18 Production Example 1 of F]SPAL-T-06

[0396] Fluorine atom-containing SPAL-T-06 was prepared according to the following scheme 18 F]SPAL-T-06 (hereinafter, 18 F]SPAL-T-06).

[0397] Chemical formula 51

[0398]

[0399] 18 F] The synthesis of epifluorohydrin is carried out by nucleophilic substitution of glycidyl tosylate with fluoride ions and purification using distillation. The following reaction is carried out in the dark. A solution containing 18 F] epifluorohydrin, (E)-2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-ol (2 mg) prepared in Example 3 in DMF (250 μL), and 1 M aqueous sodium hydroxide solution (6.5 μL) is added to a reaction vessel. The reaction mixture is heated at 130 °C for 20 minutes. After cooling the reaction vessel, HPLC solvent (500 μL) used in HPLC hereafter is added. The mixture is purified by HPLC (HPLC: CAPCELL PAK C18 UG8 010 mm × 250 mm, acetonitrile / water = 4 / 6 (containing 0.1% triethylamine), 5 mL / min). Components equivalent to 18 F] SPAL-T-06 are recovered into a bottle containing ethanol (300 μL), 25% ascorbic acid (100 μL), and Tween 80 (75 μL), and the solvent is distilled off under reduced pressure. The residue is dissolved in physiological saline (3 mL, pH 7.4) to obtain 18 F] SPAL-T-06. 18 F] SPAL-T-06.

[0400] Example 11: 18 F] Preparation Example 2 of SPAL-T-06

[0401] SPAL-T-06 is prepared by the following scheme 18 F] SPAL-T-06.

[0402] Chemical formula 52

[0403]

[0404] Using a 50% acetonitrile solution (0.4 mL) containing K.222 (Kryptofix (registered trademark) 222) (7.5 mg) and potassium carbonate (2.77 mg), 18 ​After the fluoride ions were dissolved and introduced into the reaction vessel, they were heated under a nitrogen gas stream to dry the solvent. Subsequently, anhydrous acetonitrile (0.1 mL) was added and removed by azeotropic distillation to thoroughly dry the inside of the reaction vessel. The following reaction was carried out in the dark. A DMSO (300 μL) solution containing (E)-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yne-1-yl)benzo[d]thiazol-6-yl)oxy)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl 4-methylbenzenesulfonate (2 mg) prepared in Example 9 was added to the reaction vessel. The reaction mixture was heated at 120 °C for 15 minutes. After the reaction vessel was cooled, TFA / water (600 μL) was added and hydrolyzed at 90 °C for 10 minutes. After the reaction vessel was cooled, 4N aqueous sodium acetate solution (1 mL) was added and stirred. The mixture was purified by HPLC (HPLC: CAPCELL PAK C18 UG80 10 mm×250 mm, acetonitrile / water = 4 / 6 (containing 0.1% triethylamine), 5 mL / min). The components equivalent to 18 F]SPAL-T-06 were recovered into a flask containing ethanol (300 μL), 25% ascorbic acid (100 μL), and Tween 80 (75 μL), and the solvent was removed by distillation under reduced pressure. The residue was dissolved in physiological saline (3 mL, pH 7.4) to obtain an injection solution, 18 F]SPAL-T-06.

[0405] [Optical Imaging of the Human Brain]

[0406] (Dissection of Brain Tissue)

[0407] Postmortem human brains were obtained from autopsies of patients with dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). The frozen DLB tissue was cut into 20-μm-thick sections in a cryostat (HM560, Carl Zeiss). Additionally, the AD brain tissue was fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and cut into 6-μm-thick sections.

[0408] (In Vitro Fluorescence Microscopy Measurement)

[0409] Fresh frozen sections of the brain amygdala tissue of DLB patients after fixation and formalin-fixed paraffin-embedded sections of the middle frontal gyrus of the AD patient's brain after deparaffinization were used. At room temperature, 30 μM of the test compound and the brain sections were incubated in a 50% ethanol solution for 30 minutes. Subsequently, the sections were washed with a 50% ethanol solution for 5 minutes, washed with ultrapure water for 3 minutes, and washed twice. After the sections were mounted with a mounting medium (VECTASHIELD H-1000, Vector Laboratories), images of the lesion accumulation areas on the sections were obtained using a fluorescence microscope (DM4000, Leica, excitation wavelength 391 - 437 nm), and the fluorescence images are shown in Figure 1 . In Figure 1 , the triangles indicate the fluorescence of the compound that binds to α-synuclein aggregates in the DLB patient's brain, the arrows indicate the fluorescence of the compound that binds to amyloid-β aggregates in the AD patient's brain, and the asterisks indicate the fluorescence of the compound that binds to Tau aggregates in the AD patient's brain. The fluorescence intensities of the lesion areas and the non-lesion areas (background) were quantified using analysis software (Image J). The results are shown in Figure 2 . In addition, as the test compounds, SPAL-T-05 and SPAL-T-06 were used. Additionally, as control compounds, BF-227 (2-(2-[2-dimethylaminothiazol-5-yl]vinyl)-6-(2-[fluoro]ethoxy)benzoxazole (catalog number NP039-0)) and PBB3 (2-((1E,3E)-4-(6-(methylamino)pyridin-3-yl)buta-1,3-dienyl)benzothiazol-6-ol) (catalog number NP039-0) obtained from Nard Institute were used.

[0410] As shown in Figure 1 and Figure 2 , it was confirmed that SPAL-T-06 and SPAL-T-05 bind to α-synuclein aggregates formed in the brains of DLB patients with a stronger intensity than PBB3. Additionally, it was confirmed that the binding affinity of SPAL-T-06 and SPAL-T-05 to α-synuclein aggregates is stronger than their binding to Tau or amyloid-β aggregates formed in the AD patient's brain. That is, it was shown that the compounds of the present invention have a high binding selectivity for α-synuclein aggregates. In addition, after in vitro fluorescence microscopy measurements were performed on the brains of patients with multiple system atrophy (MSA) in the same manner as the DLB patient brains described above, the same results were obtained.

[0411] In addition, it was confirmed that BF-227, reported as a PET probe for α-synucleinopathy, mainly binds to amyloid-β aggregates in AD, and its binding to α-synuclein aggregates is weaker compared to SPAL-T-06 and SPAL-T-05. That is, it was shown that BF-227 has low binding selectivity for α-synuclein aggregates.

[0412] [Optical Imaging of Mouse Brain]

[0413] (Preparation of Mouse Model Inoculated with α-Synuclein Fibers)

[0414] If mouse α-synuclein is expressed and extracted as a recombinant protein in Escherichia coli and incubated in vitro, insoluble aggregates are formed. If these α-synuclein aggregates are inoculated into the striatum of a mouse, the α-synuclein aggregates spread to surrounding areas via neural circuits, and α-synucleinopathy is observed in the neocortex of the brain after several months (Masuda-Suzukake et al. Acta Neuropathol Commun 2, 88, 2014; Shimozawa et al. Acta Neuropathol Commun 5, 12, 2017). If the brain of this mouse is removed, sections are prepared and analyzed by fluorescence staining, it is possible to confirm whether the compound of the present invention binds to the lesions composed of phosphorylated α-synuclein.

[0415] (Preparation of Mouse Model Inoculated with α-Synuclein Fibers)

[0416] First, mouse α-synuclein is expressed and extracted as a recombinant protein in Escherichia coli and incubated in vitro to form insoluble α-synuclein aggregates. Then, after inoculating these α-synuclein aggregates into the striatum of a mouse, the α-synuclein aggregates spread to surrounding areas via neural circuits, and α-synucleinopathy is observed in the neocortex of the brain after several months. In addition, the α-synuclein aggregates are inoculated into the striatum of a mouse by the following method. First, the hair on the head of a 9-week-old male C57 / BL / 6 mouse anesthetized with 1.5% (v / v) isoflurane is removed, the scalp is disinfected with polyvinylpyrrolidone iodine (Isodine), lidocaine (Xylocaine) is applied, and an incision is made on the scalp to expose the skull. Then, a hole is drilled in the skull at a position 0.05 mm anterior to the bregma and 2 mm lateral, and 3 μL of an α-synuclein fiber solution (mouse α-synuclein fiber 4 mg / mL in physiological saline) is injected at a depth of 2 μm using a glass pipette, and then the scalp is returned to its original position and sutured.

[0417] (In Vitro Fluorescence Microscopy Measurement)

[0418] The brain of the mouse inoculated with α-synuclein fibrils was taken out, sectioned and analyzed by fluorescence staining. Specifically, at room temperature, the brain sections of the mouse inoculated with α-synuclein fibrils and a 30 μM compound were incubated in a 20% ethanol solution for 30 minutes. Then, the sections were washed with a 20% ethanol solution for 5 minutes, washed with ultrapure water for 3 minutes, and washed twice. After the sections were mounted with a mounting medium (VECTASHIELD H-1000), images of the α-synuclein aggregate accumulation area on the sections were obtained using a fluorescence microscope (DM4000 (excitation wavelength 391 - 437 nm)). After washing the same sections with a phosphate buffer, they were autoclaved for antigenic activation. Immunohistochemical staining was performed with an anti-phosphorylated α-synuclein monoclonal antibody (pS129, Abcam, ab59264) (1:1000). After the sections were mounted with a mounting medium (VECTASHIELD H-1000), images of the same area as above were obtained using a fluorescence microscope (DM4000 (excitation wavelength 460 - 500 nm)). The results are shown in Figure 3 . Figure 3 Panels (a) and (b) of Figure 3 show the results of SPAL-T-05 and SPAL-T-06. In Figure 3 panels (a) and (b), the right side shows staining with an anti-phosphorylated α-synuclein antibody, and the left side shows fluorescence staining of SPAL-T-05 and SPAL-T-06. In

[0419] According to Figure 3 the results, it was shown that SPAL-T-05 and SPAL-T-06 bind to the lesions formed by phosphorylated α-synuclein.

[0420] [In vivo two-photon laser scanning fluorescence microscopy]

[0421] Anesthetize the model mice 6 weeks after injection of α-synuclein fibril solution with 1.5% (v / v) isoflurane, and set up a cranial window according to the Seylaz-Tomita method (Tomita et al. J Cereb Blood Flow Metab 25, 858-67, 2005). Anesthetize the mice 2 weeks after setting up the cranial window with 1.5% (v / v) isoflurane, and intraperitoneally administer 50 μL of a DMSO solution containing 0.1% of BF-227, PBB3, SPAL-T-05 or SPAL-T-06. 30 minutes after administration, fix the mice under a two-photon laser fluorescence microscope. After intraperitoneally administering 100 μL of physiological saline containing 5 mM sulforhodamine 101, perform in vivo two-photon fluorescence imaging of the organism at an excitation wavelength of 900 nm. Set the detection wavelengths for BF-227, PBB3, SPAL-T-05 and SPAL-T-06 to 500-550 nm, and set the detection wavelength for sulforhodamine 101 to 573-648 nm. The results are shown in Figure 4 . In Figure 4 , triangles indicate blood vessels, and arrows indicate the fluorescence of the compound that binds to α-synuclein lesions.

[0422] According to Figure 4 the results, if SPAL-T-05 and SPAL-T-06 are intraperitoneally administered, the compound further transfers into neurons in the in vivo brain, and the state of binding to each α-synuclein lesion is observed through an in vivo two-photon laser fluorescence microscope. Therefore, it can be said that the density or total number of the lesions is small, and in cases where it is difficult to detect by PET, the lesions can also be detected by in vivo fluorescence imaging using the compound of the present invention. On the other hand, no α-synuclein lesions were detected when BF-227 and PBB3 were intraperitoneally administered and observed.

[0423] [In Vivo PET (Positron Emission Tomography) Imaging of Mouse Brain]

[0424] PET scans were performed using a micro PET Focus 220 animal scanner (Siemens Medical Solutions) with 95 slices that are 0.851 mm thick (center to center), providing a 19.0 cm field of view (FOV) in the body axis direction and a 7.6 cm FOV within the cross-section. Before scanning, anesthetize the mice inoculated with α-synuclein fibrils and the mice injected with physiological saline (control) with 1.5% (v / v) isoflurane. Intravenous injection 18After the injection of [F]SPAL-T-06 (compound labeled with positron-emitting nuclide SPAL-T-06) (30.1 ± 0.13 MBq), emission scanning was performed for 90 minutes in 3D list mode with an energy window of 350-750 keV. Injection and scanning of the radioactive compound were performed in dim light to avoid photoisomerization of the compound. All list mode data were sorted into 3D sinograms and then converted into 2D sinograms by Fourier reconstruction (frames: 10×1 minute, 6×5 minutes, 5×10 minutes). After the injection of the radioactive compound, the summed images of 0-30 minutes, 30-60 minutes, and 60-90 minutes were obtained by maximum a posteriori reconstruction. In addition, dynamic images were reconstructed by filtered forward and reverse projection using a 0.5 mm Hanning filter. Volumes of interest (VOI) were set for the striatum, cerebral cortex, and cerebellum using PMOD image analysis software (PMOD Technologies) with reference to the MRI template. The results are shown in Figures 5 - 7 .

[0425] Figure 5 Left is for static injection 18 F] Results of mice inoculated with α-synuclein fibers 30 to 60 minutes after SPAL-T-06, Figure 5 The right side shows the results of mice injected with saline. The upper part is a coronal section of the brain including the striatum, and the lower part is a coronal section of the brain including the cerebellum, which are superimposed on the standard brain MRI image. Figure 6 For the striatum, cerebral cortex, cerebellum [ 18 F] Time-radioactivity curve after SPAL-T-06, Figure 7 (a) to (d) are intravenous injections. 18 F] Time course of standardized uptake value ratios (SUVR) of the cerebellum to the control striatum and cerebral cortex after SPAL-T-06 and comparison of the SUVR averaged from 30 to 60 min in mice inoculated with α-synuclein fibrils and mice injected with saline (n=2, mean±SEMs). Figure 7 (a) indicates intravenous injection of 18 F] Time-lapse of SUVR in the striatum with the cerebellum as the control area after SPAL-T-06, Figure 7 (b) shows the average value of SUVR in the striatum (control area: cerebellum) for 30 to 60 minutes. Figure 7 (c) Intravenous injection 18 F] Time-lapse of SUVR in the cerebral cortex of the cerebellum as a control area after SPAL-T-06, and, Figure 7(d) shows the average value of SUVR in the cerebral cortex (control area: cerebellum) for 30 to 60 minutes.

[0426] After PET scanning, the positron-emitting radionuclide SPAL-T-06 was intravenously injected into the α-synuclein fibril-inoculated model mice and the mice injected with normal saline (control). Figures 5 - 7 As shown, it is known that it has appropriate brain migration and brain clearance rate, and exhibits good properties as a probe for imaging α-synuclein aggregates in PET. In addition, in the α-synuclein fibril inoculation model mouse, [ 18 F]SPAL-T-06 significantly accumulated in the striatum and cerebral cortex of α-synuclein-rich lesions, confirming the ability to be used with [ 18 F] PET scan of SPAL-T-06 visualizes α-synuclein aggregates.

[0427] [Ex vivo imaging of the mouse brain]

[0428] Mice injected with normal saline were anesthetized with 1.5% (v / v) isoflurane and injected intravenously with 18 F]SPAL-T-06 (28.9 MBq). 18 F] Brains were collected and frozen 40 minutes after SPAL-T-06 administration, and 20 μm thick coronal sections at bregma 0.50, -0.46, -1.94, -3.16, and -6.64 mm were prepared in a cryostat (HM560). After the sections were air-dried, they were placed in contact with an imaging plate in a box for 15 minutes, and then autoradiography images were acquired using a BAS-5000 (Fuji Film). The results are shown in Figure 8 .

[0429] according to Figure 8 The results confirmed that even if mice injected with normal saline were intravenously injected with 18 F] SPAL-T-06, a compound that translocates into the brain, also does not show nonspecific accumulation in the white matter region.

[0430] [PET (positron emission tomography) imaging of the marmoset brain]

[0431] (Preparation of a marmoset model inoculated with α-synuclein fibrils)

[0432] First, recombinant protein of marmoset α-synuclein was expressed in Escherichia coli, extracted, incubated in vitro, and insoluble α-synuclein aggregates were formed. Next, after inoculating the α-synuclein aggregates into the caudate nucleus and putamen of marmosets, the α-synuclein aggregates spread to the surrounding areas via neural circuits, and α-synuclein lesions were observed in the substantia nigra after several months. The α-synuclein aggregates were inoculated into the caudate nucleus and putamen of marmosets by the following method. First, after inserting an organ tube into a marmoset immobilized with ketamine (5 - 10 mg / kg) and xylazine (0.2 - 0.5 mg / kg), it was anesthetized with 1 - 3% (v / v) isoflurane. The hair on the head of the marmoset was removed, the scalp was disinfected with polyvinylpyrrolidone iodine, lidocaine was applied, and an incision was made on the scalp to expose the skull. A hole was drilled (diameter ~3 mm) in the skull at the position 9.75 mm between the ears, and 50 μL of α-synuclein fiber solution (marmoset α-synuclein fiber 4 mg / mL in physiological saline) was injected into the caudate nucleus and putamen of the right brain using a Hamilton syringe. Further, after injecting 50 μL of physiological saline into the caudate nucleus and putamen of the left brain by the same method, the scalp was returned to its original position and sutured.

[0433] (In vivo PET (positron emission tomography) imaging of marmoset brain)

[0434] PET scans were performed using a micro PET Focus 220 animal scanner (Siemens Medical Systems) with 95 slices (center-to-center) 0.851 mm thick, providing a 19.0 cm axial field of view (FOV) and a 7.6 cm in-plane FOV. Before scanning, the marmosets inoculated with α-synuclein fibers were anesthetized with 1 - 3% (v / v) isoflurane. Transmission scans were performed using a PET calibration radioactive source Ge-68 for approximately 20 minutes. After intravenous injection 18 [18F]SPAL-T-06 (a compound labeled with a positron-emitting radionuclide SPAL-T-06) (73.0 MBq), emission scans were performed for 90 minutes in 3D list mode with an energy window of 350 - 750 keV. The injection of the radioactive compound and scanning were performed in the dark to avoid photoisomerization of the compound. All list mode data were sorted into 3D sinograms, and then converted to 2D sinograms by Fourier rebinning (frames: 10×1 minute, 6×5 minutes, 5×10 minutes). After injecting the radioactive compound, summed images for 0 - 30 minutes, 30 - 60 minutes, and 60 - 90 minutes were obtained by reconstruction based on the maximum a posteriori estimation method (maximum a posteriori reconstruction). In addition, dynamic images were reconstructed by filtered backprojection using a 0.5 mm Hanning filter. The results are shown in Figure 9 . Figure 9(a) is a coronal cross-sectional image of the brain including the caudate nucleus of a marmoset monkey inoculated with α-synuclein fibrils 20 to 60 minutes after intravenous injection of 18 F]SPAL-T-06, superimposed and shown on a standard brain MRI image.

[0435] (In vitro fluorescence microscopy assay)

[0436] The brain of the marmoset monkey inoculated with α-synuclein fibrils was removed, sections were prepared and analyzed by immunohistochemical staining. Specifically, after washing the sections of the brain of the marmoset monkey inoculated with α-synuclein fibrils with phosphate buffer, they were treated in an autoclave for antigen activation. Immunohistochemical staining was performed with an anti-phosphorylated α-synuclein monoclonal antibody (pS129, Abcam, ab59264) (1:1000). After the sections were mounted with a mounting medium (VECTASHIELD H-1000), images were obtained using a fluorescence microscope (BZ-X710, KEYENCE (excitation wavelength 450 - 490 nm) and DM4000 (excitation wavelength 460 - 500 nm)). The results are shown in Figure 9 . Figure 9 (b) is an image of anti-phosphorylated α-synuclein antibody staining of a coronal section of the brain including the caudate nucleus of a marmoset monkey inoculated with α-synuclein fibrils. Figure 9 (b) 2 is a magnified image of anti-phosphorylated α-synuclein antibody staining of the caudate nucleus of the right brain inoculated with α-synuclein fibrils, Figure 9 (b) 1 is a magnified image of anti-phosphorylated α-synuclein antibody staining of the caudate nucleus of the left brain injected with saline.

[0437] After labeling SPAL-T-06 with a positron-emitting radionuclide, injecting it intravenously into a marmoset monkey model inoculated with α-synuclein fibrils and performing a PET scan, it was observed that 18 F]SPAL-T-06 significantly accumulated in the caudate nucleus rich in α-synuclein lesions, confirming that the α-synuclein aggregate image can be obtained by PET scan using 18 F]SPAL-T-06.

[0438] [In vitro binding assay of human brain]

[0439] Fresh frozen tissues from the amygdala of DLB patients and fresh frozen tissues from the prefrontal cortex of AD patients were used. 10 times the wet weight of the tissue was added to the fresh frozen brain tissue, along with zirconia beads (ZB-20, manufactured by TOMY), and the mixture was pulverized using a micro pulverizer (MS-100R, manufactured by TOMY), and then stored at -80°C. DLB patient brain homogenate or AD patient brain homogenate was mixed in Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) buffer (containing 20% ethanol), 18 F]SPAL-T-06 (final concentration 5 nM), and unlabeled SPAL-T-06 (final concentration 0.01 nM to 1 μM). After incubation at room temperature for 30 minutes, B / F separation (B: bound ligand, F: free ligand) was performed by suction filtration and washing, and the radioactivity captured on the glass filter was measured using a gamma counter (2480 WIZARD2, manufactured by ParkinElmer). For each concentration of unlabeled SPAL-T-06, the experiment was performed in triplicate. The results were analyzed using Prism 6J (manufactured by GraphPad), and the displacement curve and IC 50 of the test substance were calculated. The results are shown in Figure 10 .

[0440] After labeling SPAL-T-06 with a positron-emitting radionuclide and performing a binding assay using homogenates of the brains of DLB patients and AD patients, it was confirmed that 18 F]SPAL-T-06 bound to the homogenates of the brains of DLB patients with rich α-synuclein lesions with high affinity (IC 50 = 1.58 nM). 18 The binding affinity of 50 F]SPAL-T-06 for the homogenates of the brains of AD patients was lower than that for the homogenates of the brains of DLB patients (IC 18 = 10.86 nM). 18 F]SPAL-T-06 showed good binding affinity as a probe for imaging α-synuclein aggregates in PET imaging. In addition, it was confirmed that the binding to α-synuclein aggregates was stronger than the binding to Tau or amyloid-β aggregates, and it had a high binding selectivity for α-synuclein aggregates.

[0441] [In vitro autoradiography of the human brain]

[0442] (Dissected brain tissue)

[0443] Postmortem human brains were obtained from autopsies of patients with DLB, patients with multiple system atrophy (MSA), and healthy subjects. Frozen DLB tissue and frozen healthy subject tissue were cut into 20-μm-thick sections in a cryostat (HM560, Carl Zeiss). In addition, MSA brain tissue was fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and cut into 6-μm-thick sections.

[0444] (Autoradiography assay)

[0445] Fresh frozen sections of the amygdala tissue of DLB patients and the prefrontal cortex tissue of healthy subjects, and formalin-fixed paraffin-embedded sections of the cerebellum tissue of MSA patients that had been deparaffinized were used. At room temperature, 18 F]SPAL-T-06 (final concentration 10 nM) and the brain tissue sections were incubated in 50 mM Tris-HCl containing 20% ethanol for 1 hour. The brain tissue sections were also incubated in 50 mM Tris-HCl (containing 20% ethanol) containing 18 F]SPAL-T-06 (final concentration 10 nM) and unlabeled SPAL-T-06 (final concentration 10 μM) to detect specific binding. Subsequently, the sections were washed twice for 2 minutes each with 50 mM Tris-HCl (containing 20% EtOH) at 4°C and rinsed with MilliQ (trade name) water. After air-drying the sections, the sections were brought into contact with an imaging plate in a cassette for 5 minutes, and then autoradiograms were obtained using a BAS-5000 (Fuji Film). The results are shown in Figure 11 . Figure 11 The triangles in

[0446] (In vitro fluorescence microscopy measurement)

[0447] The sections subjected to autoradiography were analyzed by fluorescence staining. Specifically, at room temperature, the sections were incubated with 30 μM of SPAL-T-06 in a 50% ethanol solution for 30 minutes. Then, the sections were washed with a 50% ethanol solution for 5 minutes, washed with ultrapure water for 3 minutes, and washed twice. After enclosing the sections with a mounting medium (VECTASHIELD H-1000), images of the regions where α-synuclein aggregates accumulated on the sections were obtained using a fluorescence microscope (DM4000 (excitation wavelength 391 - 437 nm)). In addition, adjacent brain sections were washed with a phosphate buffer solution and then treated with an autoclave for antigen activation. Immunohistochemical staining was performed with an anti-phosphorylated α-synuclein monoclonal antibody (pS129, Abcam, ab59264) (1:1000). After enclosing the sections with a mounting medium (VECTASHIELD H-1000), images were obtained using a fluorescence microscope (BZ-X710 (excitation wavelength 450 - 490 nm) and DM4000 (excitation wavelength 460 - 500 nm)). The results are shown in Figure 12 . Figure 12 (a) (indicated by "1" in the figure) is the anti-phosphorylated α-synuclein antibody staining and SPAL-T-06 fluorescence staining of the brain of a DLB patient, Figure 12 (b) (indicated by "2" in the figure) is the anti-phosphorylated α-synuclein antibody staining and SPAL-T-06 fluorescence staining of the brain of an MSA patient.

[0448] After radiolabeling SPAL-T-06 with a positron-emitting radionuclide and performing autoradiography using brain tonsil sections of DLB patients, cerebellum sections of MSA patients, and prefrontal cortex sections of healthy subjects, it was confirmed that 18 F]SPAL-T-06 binds to regions rich in lesions formed by phosphorylated α-synuclein in the brains of DLB patients and MSA patients. After performing fluorescence staining and immunohistochemical staining on the sections subjected to autoradiography, it was confirmed that SPAL-T-06 binds to α-synuclein lesions contained in these brain sections. In addition, in the prefrontal cortex sections of the brains of healthy subjects, almost no 18 F]non-specific binding of SPAL-T-06 was observed in both gray matter and white matter.

[0449] Industrial Applicability

[0450] According to the present invention, it is possible to provide an α-synuclein aggregate binder having high binding selectivity to α-synuclein aggregates. Moreover, it is possible to provide an imaging method using the α-synuclein aggregate binder. In addition, it is possible to provide a new compound that can be used for an α-synuclein aggregate binder or other uses.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein, the compound is represented by the following formula (I) or (II), 2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, in the compound represented by the formula (I) or formula (II), one or more atoms are radioactive isotopes of the atom.

3. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an α-synuclein aggregate binder.

4. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 2 in the preparation of an α-synuclein aggregate binder.

5. A composition for optical imaging of α-synuclein aggregates, wherein, it contains the compound or a pharmaceutically acceptable salt thereof according to claim 1.

6. A composition for radioimaging of α-synuclein aggregates, wherein, it contains the compound or a pharmaceutically acceptable salt thereof according to claim 2.

7. A method for screening a therapeutic or prophylactic agent for a disease associated with α-synuclein aggregates in the brain, wherein, the method includes: a step of irradiating light of a first wavelength to the living body brain of a subject administered with an α-synuclein aggregate binder from outside the brain, and then detecting light of a second wavelength different from the first wavelength emitted from the brain, the α-synuclein aggregate binder contains the compound or a pharmaceutically acceptable salt thereof according to claim 1, and a step of selecting the candidate substance based on the difference in the amount and / or distribution of the detected light before and after administering the candidate substance to the subject.

8. A method for screening a therapeutic or prophylactic agent for a disease associated with α-synuclein aggregates in the brain, wherein, the method includes: a step of detecting radiation emitted from the living body brain of a subject administered with an α-synuclein aggregate binder, the α-synuclein aggregate binder contains the compound or a pharmaceutically acceptable salt thereof according to claim 2, and a step of selecting the candidate substance based on the difference in the amount and / or distribution of the detected radiation before and after administering the candidate substance to the subject.

9. An intermediate for synthesizing the compound according to claim 1 or 2, wherein, the intermediate is represented by the following formula (III), in formula (III), one of X and Y is a nitrogen atom N, the other is an unsubstituted carbon atom CH, R1 is a hydroxyl group or a group represented by the following formula (i), R2 is a hydrogen atom or tert-butoxycarbonyl Boc, in formula (i), Ts represents p-toluenesulfonyl, THP represents tetrahydro-2H-pyran-2-yl, and * represents the bonding position to the benzothiazole ring.

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