Novel compound, alpha-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 or the pharmaceutically acceptable salt.

Description

[Technical field]

[0001] The present invention relates to a novel compound, an α-synuclein aggregate binding agent and uses thereof; in particular, the present invention relates to a novel compound, an α-synuclein aggregate binding agent containing the novel compound, a composition for optical imaging of α-synuclein aggregates, a composition for radiation imaging of α-synuclein aggregates, a method for optical imaging of α-synuclein aggregates in the brain, a method for radiation imaging of α-synuclein aggregates in the brain, and intermediates for synthesizing the novel compound. [Background technology]

[0002] Alpha-synuclein aggregates form the core pathology of Parkinson's disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), and are believed to have a close causal relationship with neurodegeneration. Definitive diagnosis of these diseases is performed using the presence of alpha-synuclein aggregates (also referred to as "alpha-synuclein lesions" in this specification) as an indicator in pathological analysis of autopsy brains, so the diagnosis cannot be confirmed before death. However, if alpha-synuclein aggregates can be visualized in living brains, it will be possible to obtain information close to definitive information (definitive diagnosis) regarding the diagnosis of these diseases from an early stage. In addition, if alpha-synuclein aggregates can be visualized in living brains of disease model animals, it may be useful for evaluating the efficacy of candidate substances for therapeutic or preventive drugs that target alpha-synuclein aggregates by time-lapse imaging, etc.

[0003] As a PET (positron emission tomography) probe that has been shown to bind to α-synuclein in the living brain, 11 C]BF-227 (Non-Patent Documents 1 and 2). 11 C]BF-227 does not have sufficient binding affinity to α-synuclein aggregates, and among the above diseases, α-synuclein lesions can only be detected in a portion of MSA patients. 11 C]BF-227 has the problems of nonspecific accumulation in the brain and low binding selectivity for α-synuclein aggregates because it binds to amyloid β aggregates.

[0004] Meanwhile, the inventors have developed a compound for imaging tau protein accumulated in the brain (see Patent Document 1). The compound described in Patent Document 1 can image tau protein accumulated in the brain, so the technology of Patent Document 1 is useful for treating and preventing diseases caused by the accumulation of tau protein, such as Alzheimer's disease (AD). However, Patent Document 1 does not describe binding to α-synuclein aggregates. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 097474 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] Kikuchi, A. et al., Brain, 133:1772-1778 (2010) [Non-Patent Document 2] Verdurand, M. et al., Contrast Media Mol. Imaging, 2018: 9165458 (2018) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide an α-synuclein aggregate binding agent that has high binding selectivity to α-synuclein aggregates, an imaging method using this α-synuclein aggregate binder, and a novel compound that can be used as an α-synuclein aggregate binding agent. [Means for solving the problem]

[0008] The present inventors found that a compound having a specific structure has high binding selectivity to α-synuclein aggregates, and after further investigation, completed the present invention. More specifically, the present invention provides the following.

[0009] One aspect of the present invention is a compound represented by the following formula (I) or (II), a pharma- ceutical acceptable salt thereof, or a solvate thereof.

[0010] [ka] Effect of the Invention

[0011] According to the present invention, it is possible to provide an α-synuclein aggregate-binding agent that has high binding selectivity to α-synuclein aggregates. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of fluorescence microscopy of the brains of DLB ​​patients and AD patients. [Diagram 2] FIG. 13 shows the quantitative results of fluorescence brightness in lesion and non-lesion areas. [Diagram 3] FIG. 1 shows the results of fluorescence microscopy of mice inoculated with α-synuclein fibrils. [Figure 4] FIG. 1 shows the results of two-photon laser scanning fluorescence microscopy of model mice 6 weeks after inoculation with α-synuclein fibrils. [Diagram 5] FIG. 1 shows the results of PET imaging of mice inoculated with α-synuclein fibrils. [Figure 6] FIG. 1 shows the results of PET imaging of mice inoculated with α-synuclein fibrils. [Figure 7] FIG. 1 shows the results of PET imaging of mice inoculated with α-synuclein fibrils. [Figure 8] FIG. 1 shows the results of ex vivo imaging of mouse brain. [Figure 9]FIG. 1 shows the results of PET imaging of a marmoset inoculated with α-synuclein fibrils. [Figure 10] FIG. 1 shows the results of an in vitro binding test on the brains of DLB ​​patients and AD patients. [Figure 11] FIG. 1 shows the results of autoradiography of the brains of DLB ​​and MSA patients. [Figure 12] FIG. 1 shows the results of in vitro fluorescence microscopy of the brains of DLB ​​and MSA patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, one embodiment of the present invention will be described. In this specification, the term "A and / or B" refers to at least one of A and B.

[0014] [Definition] The term "pharmaceutical acceptable salt" refers to a salt that is not harmful 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 pharmaceutical acceptable salts include metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts formed from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Pharmaceutically acceptable salts also include acid addition salts and base addition salts.

[0015] The term "pharmaceutical acceptable carrier" refers to a pharmaceutical acceptable material, composition, or vehicle, such as saline solution, liquid or solid fillers, diluents, solvents, or encapsulating materials. Examples of pharmaceutical acceptable carriers include water, saline, normal saline or phosphate buffered saline (PBS), Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose, and Lactated Ringer's Injection.

[0016] The term "effective amount" refers to an amount of a compound or composition that is capable of achieving a desired effect. For example, in some embodiments, an effective amount refers to an amount of a compound or composition that is capable of optical or radiometric imaging of substances that accumulate in the brain, such as alpha-synuclein aggregates.

[0017] The term "solvate" refers to a solvate formed by the association of one or more solvent molecules with a compound. Solvates include, for example, monosolvates, disolvates, trisolvates, and tetrasolvates. Solvates also include hydrates.

[0018] The term "hydrate" refers to a compound or a salt thereof that further contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Hydrates include, for example, monohydrates, dihydrates, trihydrates, tetrahydrates, and the like.

[0019] The term "treating" means reducing or ameliorating the progression, severity and / or duration of a disease or condition.

[0020] The term "prevention" refers to the reduction of the risk of acquiring or developing a given disease or condition, or the reduction or inhibition of the recurrence, onset, or progression of one or more symptoms of a given disease or condition.

[0021] The term "binding" refers to the binding strength of a compound to a particular protein aggregate.

[0022] The term "binding selectivity" refers to a difference between the binding ability of a compound to a specific protein aggregate and the binding ability of a compound to other protein aggregates (the binding ability to a specific protein aggregate is higher or lower than the binding ability to other protein aggregates). "High binding selectivity" means that the difference in the above-mentioned binding ability is large. For example, when a compound has "high binding selectivity to α-synuclein aggregates," it means that there is a large difference between the binding ability of the compound to α-synuclein aggregates and the binding ability of the compound to other protein aggregates, and the compound has a higher binding ability to α-synuclein aggregates.

[0023] [Compound] 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 pharma- ceutical acceptable salt thereof, or a solvate thereof.

[0024] [ka] In this specification, the compounds represented by formulas (I) and (II) are also referred to as "compound (I)" and "compound (II)", respectively.

[0025] In one embodiment, Compound (I) and Compound (II) are compounds, salts thereof, or solvates thereof, in which one or more atoms are radioisotopes of said atoms. The radioisotopes are 15 O. 13 N, 11 C, and 18 Preferably, the radioisotope is selected from the group consisting of, but not limited to, F. 11 C or 18F. Of these, 11 The half-life of C is about 20 minutes. 18 Considering that the half-life of F is about 110 minutes, 18 Compounds labeled with F are believed to have greater commercial value. Therefore, most preferably, the radioisotope is 18 It's F.

[0026] 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. Even more preferably, the fluorine atom in the 3-fluoro-2-hydroxypropoxy group is a radioisotope.

[0027] In one embodiment, the compound (I) containing a radioisotope is preferably 18 F]-(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.

[0028] In one embodiment, the compound (II) containing a radioisotope is preferably 18 F]-(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.

[0029] [Intermediate] As shown in the production method of the following compound, the compound represented by the following formula (III) is a production intermediate compound (also simply referred to as "intermediate" in this specification) of compound (I) and compound (II).

[0030] [ka] 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" refers to 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).

[0031] R1 is a hydroxy group or a group represented by the following formula (i).

[0032] [ka] Here, Ts represents a p-toluenesulfonyl group, THP represents a tetrahydro-2H-pyran-2-yl group, and * represents the bonding position to the benzothiazole ring.

[0033] R2 is a hydrogen atom or a tert-butoxycarbonyl (Boc) group.

[0034] These intermediate compounds are suitable for use in the synthesis of compound (I) and compound (II), and further for the synthesis of radioisotope-labeled compound (I) and compound (II). These intermediate compounds may be in the form of a salt.

[0035] When compound (I) or compound (II) contains various isomers such as stereoisomers (including optical isomers and rotational isomers), tautomers, or polar isomers, these isomers are also included in compound (I) or compound (II). These isomers can be obtained as single products by known synthesis and separation methods. Compound (III) can also include various isomers.

[0036] Compound (I), compound (II) or compound (III) may also be in the form of a crystal produced by a known crystallization method.

[0037] [Method for producing compound (I), compound (II) and intermediates] Compound (I) and compound (II) as well as compound (III) which is an intermediate can be produced according to a method similar to the production method shown below. In addition, if desired, compound (I), compound (II) and compound (III) can be produced by carrying out a deprotection reaction, an amidation reaction, a urea reaction, an alkylation reaction, a Mitsunobu reaction, an oxidation reaction, a reduction reaction, a halogenation reaction, a coupling reaction, a nucleophilic addition reaction by a carbanion, a Grignard reaction, a dehydration reaction, etc., either alone or in combination of two or more thereof. In addition, the reaction conditions such as the solvent, the reagent and the temperature in each reaction may be appropriately set based on the technical common sense of a person skilled in the art. The protection and deprotection reaction of the functional group are carried out according to the known reaction method, the method described in the Reference Examples or Examples, and a conventional protecting group is used.

[0038] In the following production methods, unless otherwise specified, the meanings of the symbols used are the same as those described above.

[0039] (Manufacturing method A) Compound (I), compound (II) and compound (III) in which R1 is a hydroxy group and R2 is a Boc group (compound (III-i)) can be produced by the following method shown in Production Scheme 1. Hereinafter, compounds shown as (1), (2), etc. in each scheme will be referred to as compound (1), compound (2), etc.

[0040] (Production Scheme 1)

[0041] [ka] Compound (2) can be produced by reacting compound (1) with a phosphite triester. In compound (1), TBS is a tert-butyldimethylsilyl group.

[0042] Compound (III-i) can be produced by the Horner-Wadsworth-Emmons reaction (HWE reaction) of compound (2) with compound (3) described below, followed by a deprotection reaction that proceeds during the reaction. In compound (3), one of X and Y is a nitrogen atom, and the other is an unsubstituted carbon atom (CH).

[0043] Compound (5) can be produced by subjecting compound (III-i) to an alkylation reaction such as the Mitsunobu reaction between compound (4) described below.

[0044] Compound (I) and compound (II) can be produced by a deprotection reaction of compound (5).

[0045] Compound (3) used in Production Method A can be produced from compound (6) by the following method shown in Production Scheme 2.

[0046] (Production Scheme 2)

[0047] [ka] In compound (6), Hal represents a halogen atom (e.g., a chlorine atom, a bromine atom, an iodine atom). Compound (7) can be produced by, for example, a coupling reaction between compound (6) and 2-propyn-1-ol.

[0048] Compound (3) can be produced by oxidation reaction of compound (7).

[0049] Compound (4) used in Production Method A can be produced from compound (8) by the following method shown in Production Scheme 3.

[0050] (Production Scheme 3)

[0051] [ka] Compound (9) can be prepared by a protection reaction of compound (8).

[0052] Compound (4) can be produced by subjecting compound (9) to a debenzylation reaction.

[0053] Compound (5) used in Production Method A can also be produced from compound (2) by the following method shown in Production Scheme 4.

[0054] (Production Scheme 4)

[0055] [ka] Compound (10) can be produced by a deprotection reaction of compound (2).

[0056] Compound (11) can be produced by subjecting compound (10) to an alkylation reaction such as the Mitsunobu reaction between compound (4).

[0057] Compound (5) can also be produced by subjecting compound (11) to an HWE reaction with compound (3).

[0058] (Manufacturing method B) Compound (I) and compound (II) can also be produced by the following method shown in Production Scheme 5.

[0059] (Production Scheme 5)

[0060] [ka] Compound (13) can be produced by subjecting compound (12) to an alkylation reaction via an oxirane ring-opening reaction to give compound (III-i).

[0061] Compound (I) and compound (II) can be produced by a deprotection reaction of compound (13).

[0062] (Manufacturing method C) Compound (III) in which R1 is a hydroxy group and R2 is a hydrogen atom (compound (III-ii)) can be produced by a deprotection reaction of compound (III-i), as shown in the following Production Scheme 6.

[0063] (Production Scheme 6)

[0064] [ka] (Manufacturing method D) Compound (III) in which R1 is formula (i) (compound (III-iii)) can be prepared by the following method shown in Preparation Scheme 7.

[0065] (Production Scheme 7)

[0066] [ka] Compound (15) can be produced by subjecting compound (III-i) to an alkylation reaction such as the Mitsunobu reaction between compound (14) described below.

[0067] Compound (16) can be produced by a deprotection reaction of compound (15).

[0068] Compound (III-iii) can be produced by subjecting compound (16) to a protection reaction.

[0069] Compound (14) used in Production Method D can be produced by the following method shown in Production Scheme 8.

[0070] (Production Scheme 8)

[0071] [ka] Compound (18) can be prepared by a protection reaction of compound (17).

[0072] Compound (14) can be produced by subjecting compound (18) to a debenzylation reaction.

[0073] [α-synuclein aggregate-binding agent] In one embodiment, the present invention provides an α-synuclein aggregate-binding agent, which contains Compound (I) or Compound (II), a medicamentously acceptable salt thereof, or a solvate thereof.

[0074] Compound (I) and compound (II) have higher binding selectivity to α-synuclein aggregates than to aggregates of tau protein and amyloid β. Similarly, pharma- ceutical acceptable salts of compound (I) and compound (II), and solvates of compound (I) and compound (II) also have higher binding selectivity to α-synuclein aggregates than to aggregates of tau protein and amyloid β. Compound (I) and compound (II) also emit fluorescence. As described above, in compound (I) and compound (II), one or more atoms can be radioisotopes of the atoms. Therefore, the binding agent of this embodiment can be used as a molecular probe for optical imaging or radiation imaging of α-synuclein aggregates accumulated in the brain.

[0075] Incidentally, α-synuclein is a protein that is localized in neuronal synapses even in normal brains, and α-synuclein aggregates are formed when α-synuclein aggregates.

[0076] The alpha-synuclein aggregate binding agent can include a pharma- ceutically acceptable carrier, examples of which include water, saline, normal saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection.

[0077] The content of compound (I) or compound (II), a medicamentously acceptable salt thereof, or a solvate thereof, and a medicamentously acceptable carrier contained in the α-synuclein aggregate-binding agent is not particularly limited. The content of these is determined by various factors such as the type of compound used; the age, weight, health condition, sex, and diet of the mammal to be administered; the number of administrations and the administration route; the treatment period; and other drugs used simultaneously. The content of the medicamentously acceptable carrier can be 1 to 99% by weight of the α-synuclein aggregate-binding agent. The α-synuclein aggregate-binding agent is prepared so that compound (I) or compound (II) can be administered in an amount of, for example, 5 ng / kg to 5 mg / kg per body weight (kg) of the subject. Preferably, the lower limit of the amount of the compound is 5 ng / kg or more, 0.01 mg / kg or more, 0.05 mg / kg or more, or 0.1 mg / kg or more. The upper limit of the amount of the compound is 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, or 20 μg / kg or less.

[0078] [Composition for optical imaging of α-synuclein aggregates] 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 this embodiment (hereinafter also referred to as optical imaging composition) comprises the binder according to this embodiment described above. The optical imaging includes in vitro, ex vivo, and in vivo imaging.

[0079] Optical imaging includes, for example, fluorescence microscopy, multiphoton imaging, two-photon imaging, and near-infrared fluorescence imaging.

[0080] The optical imaging composition may include a pharma- ceutically acceptable carrier, examples of which include water, saline, normal saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection.

[0081] The content of the compound (I) or compound (II), its medicamentously acceptable salt, or its solvate, and the medicamentously acceptable carrier contained in the optical imaging composition is not particularly limited. The content of these is determined by various factors such as the type of compound used; the age, weight, health condition, sex, and diet of the mammal to be administered; the number of administrations and the administration route; the treatment period; and other drugs used at the same time. The content of the medicamentously acceptable carrier can be 1 to 99% by weight of the optical imaging composition. The optical imaging composition is prepared so that the compound (I) or compound (II) can be administered in an amount of, for example, 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 per subject's body weight (kg).

[0082] [Composition for radioimaging of α-synuclein aggregates] In one embodiment, the present invention provides a composition for radiation imaging of alpha-synuclein aggregates. The composition for radiation imaging of alpha-synuclein aggregates of this embodiment (hereinafter also referred to as radiation imaging composition) comprises the binding agent according to this embodiment. The radiation imaging includes in vitro, ex vivo, and in vivo imaging.

[0083] Emission imaging includes, for example, Positron Emission Tomography (PET), Single photon emission computed tomography (SPECT), and autoradiography.

[0084] The radioimaging composition may include a pharma- ceutically acceptable carrier, examples of which include water, saline, normal saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection.

[0085] The content of compound (I) or compound (II), a medicamentously acceptable salt thereof, or a solvate thereof, and a medicamentously acceptable carrier contained in the radioimaging composition is not particularly limited. The content of these is determined by various factors such as the type of compound used; the age, weight, health condition, sex, and diet of the mammal to be administered; the number of administrations and the administration route; the treatment period; and other drugs used simultaneously. The content of the medicamentously acceptable carrier can be 1 to 99% by weight of the radioimaging composition. The radioimaging composition is prepared so that compound (I) or compound (II) can be administered in an amount of, for example, 5 ng / kg to 5 mg / kg, preferably 5 ng / kg to 20 μg / kg per body weight (kg) of the subject.

[0086] [Diagnostic agent for diseases associated with α-synuclein aggregates, or companion diagnostic agent for treating or preventing said diseases] In one embodiment, the present invention provides a diagnostic agent for a disease associated with α-synuclein aggregates, or a companion diagnostic agent for treating or preventing the disease. The diagnostic agent for a disease associated with α-synuclein aggregates, or a companion diagnostic agent for treating or preventing the disease (hereinafter also referred to as a companion diagnostic agent) of this embodiment comprises the binding agent according to this embodiment described above. A companion diagnostic agent for treatment is a diagnostic agent for determining whether or not a treatment is possible when a disease is found to be present. A companion diagnostic agent for prevention is a diagnostic agent for determining whether or not a prevention is possible to predict future onset or suppress onset when a precursor state of a disease is found to be present.

[0087] By using the diagnostic agent of this embodiment, data regarding the amount and / or distribution of alpha-synuclein aggregates in the brain obtained from a subject can be compared with a previously obtained correlation between the disease and the amount and / or distribution of alpha-synuclein aggregates, making it possible to diagnose the disease in the subject (specifically, the presence or absence of the disease, the severity, the possibility of seizures, etc.).

[0088] In addition, by using the companion diagnostic agent to compare data on the amount and / or distribution of α-synuclein aggregates in the brain obtained from a subject with a correlation between a disease and the amount and / or distribution of α-synuclein aggregates in the brain obtained in advance, the disease state of the subject can be understood, and therefore a disease prevention / treatment plan (type, combination, dose, use, etc. of prophylactic or therapeutic drugs to be administered) can be formulated based on this.

[0089] One embodiment of the present invention also relates to a pharmaceutical for treating or preventing a disease associated with alpha-synuclein aggregates, which is administered in a dosing regimen based on data regarding the amount and / or distribution of alpha-synuclein aggregates in the brain obtained by a companion diagnostic.

[0090] [Diagnostic kit for diseases related to substances that accumulate in the brain] A diagnostic kit for a disease associated with a substance that accumulates in the brain (hereinafter, also referred to as a diagnostic kit) of the present invention contains the binding agent according to this embodiment described above.

[0091] Substances that accumulate in the brain include at least α-synuclein aggregates, and also include aggregates of tau protein or amyloid β.

[0092] Diseases associated with substances that accumulate in the brain include at least diseases associated with α-synuclein aggregates, such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Other diseases associated with substances that accumulate in the brain include Alzheimer's disease (AD) and frontotemporal lobar degeneration, which are diseases associated with tau protein or amyloid β aggregates.

[0093] The binding agent of the present embodiment has higher binding selectivity to α-synuclein aggregates than to aggregates of tau protein and amyloid β. Meanwhile, the results of studies by the present inventors have revealed that, for example, the compound described in Patent Document 1 has higher binding affinity to tau protein aggregates than to α-synuclein aggregates.

[0094] In one embodiment of this embodiment, the diagnostic kit can include both compound (I) or compound (II) or a medicamentously acceptable salt or solvate thereof having high binding selectivity to α-synuclein aggregates, and another compound having high binding selectivity to tau protein aggregates (e.g., the compound described in Patent Document 1). In such a diagnostic kit, the detection result of the former (amount and / or distribution of detected light or radiation) can be compared with the detection result of the latter, thereby identifying which substance is present in each imaged region. Specifically, α-synuclein aggregates and tau protein aggregates can be classified, and the amount of each can be quantified. Therefore, diseases associated with α-synuclein aggregates and / or diseases associated with tau protein can be diagnosed with high accuracy. For example, the ratio of the binding ability to α-synuclein aggregates to the binding ability to tau protein aggregates is previously identified for each of the binder of this embodiment and the substance having high binding selectivity to tau protein aggregates (e.g., the compound described in Patent Document 1). Then, by measuring the ratio of the amounts of light or radiation in each brain region of a subject after administration of the former and the latter, it is possible to classify whether α-synuclein aggregates or tau protein aggregates are present in the region based on the relationship between the pre-specified ratio and the measured amount ratio, and to quantify the amount of each.

[0095] The diagnostic kit of this embodiment can also be combined with an imaging agent for amyloid β to form a diagnostic kit. The diagnostic kit of this embodiment combined with an imaging agent for amyloid β can identify the substance present in each imaged region. Specifically, it can classify α-synuclein aggregates, tau protein aggregates, and amyloid β aggregates, and can also quantify the amount of each. Therefore, it is possible to diagnose diseases associated with α-synuclein aggregates, diseases associated with tau protein, and / or diseases associated with amyloid β with high accuracy.

[0096] One embodiment of the present invention relates to a pharmaceutical for treating or preventing a disease associated with alpha-synuclein aggregates, which is administered according to a dosing regimen based on data regarding the amount and / or distribution of brain accumulated substances, including alpha-synuclein aggregates, obtained by a diagnostic kit according to this embodiment.

[0097] [Optical imaging method] In one embodiment, the present invention provides an optical imaging method, which includes the steps of irradiating a living brain of a subject administered with the above-described binding agent according to the present embodiment 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.

[0098] When an effective amount of the binding agent is administered to a subject, the binding agent that has migrated to the living brain binds to α-synuclein aggregates present in the living brain. By irradiating the living brain of the subject to which the binding agent has been administered with light of a first wavelength that excites the binding agent from outside the brain and detecting light of a second wavelength (e.g., fluorescence) emitted from the binding agent in the brain, optical imaging of α-synuclein aggregates can be performed.

[0099] The subject may be a mammal, such as a human, a rat, a mouse, a rabbit, a guinea pig, a hamster, a monkey, a dog, a ferret, or a miniature pig.

[0100] The administration method is not particularly limited, and may be, for example, oral administration or parenteral administration such as intravenous administration or intraperitoneal administration. Intravenous administration or intraperitoneal administration is preferred. Intravenous administration is most preferred. The dosage 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.

[0101] [Radiation imaging method] In one embodiment, the present invention provides a radiation imaging method, which comprises detecting radiation emitted from the living brain of a subject administered a binding agent according to the present embodiment, comprising Compound (I) or Compound (II), a pharma- ceutical acceptable salt thereof, or a solvate thereof, in which one or more atoms are radioisotopes of said atoms.

[0102] When an effective amount of the binding agent is administered to a subject, the binding agent that has been transferred to the brain of the living subject binds to α-synuclein aggregates present in the brain of the living subject. By detecting radiation emitted from the binding agent in the brain, radiation imaging of α-synuclein aggregates can be performed.

[0103] The subject may be a mammal. Mammals include, for example, humans, rats, mice, rabbits, guinea pigs, hamsters, monkeys, dogs, ferrets, minipigs, etc. Preferably, the mammal is a human.

[0104] The administration method is not particularly limited, and examples thereof include oral administration and parenteral administration such as intravenous administration or intraperitoneal administration. Intravenous administration or intraperitoneal administration is preferable. Intravenous administration is most preferable. The dosage is preferably 5 ng / kg to 5 mg / kg, and more preferably 5 ng to 20 μg / kg. The amount of radioactivity administered is preferably 37 MBq to 7.4 GBq per individual, and more preferably 370 MBq to 3,700 MBq.

[0105] [Method for screening therapeutic or preventive agents for diseases associated with brain α-synuclein aggregates] In one embodiment, the present invention provides a method for screening for a therapeutic or prophylactic agent for a disease associated with intracerebral α-synuclein aggregates. The screening method for a therapeutic or prophylactic agent for a disease associated with intracerebral α-synuclein aggregates (hereinafter also referred to as a screening method) of this embodiment includes a step of selecting a candidate substance based on a difference in the amount and / or distribution of light or radiation detected by the optical imaging method or radiation imaging method of this embodiment before and after administration of the candidate substance to a subject.

[0106] Diseases associated with intracerebral α-synuclein aggregates are the same as those described above in the section "Diagnostic kit for diseases associated with substances that accumulate in the brain."

[0107] The subject and administration method are the same as those described in the above [Optical imaging method] and [Radiation imaging method].

[0108] For example, if the amount (intensity) of light or radiation, such as fluorescence, of the binding agent is reduced after administration of the candidate substance compared to that before administration of the candidate substance, the candidate substance may be useful as a compound for treating the disease or condition.

[0109] Furthermore, the amount and / or distribution of light or radiation detected in the subject is compared with that of another normal mammal, and if it approaches that of a normal mammal after administration of the candidate compound more than before administration, the candidate compound may be useful as a compound for treating the disease or condition.

[0110] For example, data on the amount (intensity) and / or distribution of light or radiation such as fluorescence of the binding agent before and after administration of the candidate substance obtained from a subject administered with the candidate substance is compared with the increase in the amount and / or change in distribution of light or radiation such as fluorescence of the binding agent before and after the onset of a disease associated with intracerebral α-synuclein aggregates in a mammal not administered the candidate substance, which has been previously obtained. If the increase in the amount of light or radiation such as fluorescence observed after the onset of the disease is suppressed by administration of the candidate substance and / or the amount of light or radiation such as fluorescence after administration of the candidate substance is similar to that of a normal mammal before administration, the candidate substance may be useful as a compound for preventing the disease or symptom. Similarly, if the change in the distribution of light or radiation such as fluorescence observed after the onset of the disease is suppressed by administration of the candidate substance and / or the change in the distribution of light or radiation such as fluorescence after administration of the candidate substance is similar to that of a normal mammal before administration, the candidate substance may also be useful as a compound for preventing the disease or symptom.

[0111] [Method for quantifying or assessing accumulation of α-synuclein aggregates in the brain] In one embodiment, the present invention provides a method for quantifying or determining accumulation of α-synuclein aggregates in the brain. The method for quantifying or determining accumulation of α-synuclein aggregates in the brain of the subject administered with the binding agent containing compound (I) or compound (II), a medicamentously acceptable salt thereof, or a solvate thereof includes a step of irradiating a living brain of the subject 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, and quantifying or determining 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 accumulation of α-synuclein aggregates in the brain by optical imaging.

[0112] The subject and administration method were the same as those described in the above [Optical imaging method].

[0113] By determining the difference between the amount and / or distribution of light detected in the subject and that of other normal mammals, it is possible to quantify the accumulation of alpha-synuclein aggregates in the brain and to determine the presence or absence of accumulation of alpha-synuclein aggregates in the brain.

[0114] Another aspect of the method for quantifying or determining accumulation of α-synuclein aggregates in the brain of the present embodiment includes a step of detecting radiation emitted from the living brain of a subject administered with a binding agent according to the present embodiment, comprising Compound (I) or Compound (II), a medicamentously acceptable salt thereof, or a solvate thereof, in which one or more atoms are radioisotopes of the atoms, and quantifying or determining 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 accumulation of α-synuclein aggregates in the brain by radiation imaging.

[0115] The subject and administration method are the same as those described in the above [Radiation imaging method].

[0116] By determining the difference between the amount and / or distribution of light or radiation detected in the subject and that of other normal mammals, it is possible to quantify the accumulation of alpha-synuclein aggregates in the brain and to determine the presence or absence of accumulation of alpha-synuclein aggregates in the brain.

[0117] [Classification of substances that accumulate in the brain and methods for assessing accumulation] In one embodiment, the present invention provides a method for determining the classification and accumulation of a substance that accumulates in the brain. The method for determining the classification and accumulation of a substance that accumulates in the brain of the present invention includes a first step of detecting light of a second wavelength different from the first wavelength emitted from a living brain of a subject administered with a binding agent according to this embodiment, which includes compound (I) or compound (II), a medicamentously acceptable salt thereof, or a solvate thereof, after irradiating light of a first wavelength from outside the brain to the living brain of the subject administered with a substance having high binding selectivity to tau protein aggregates (e.g., a compound described in Patent Document 1) at a time different from the first step, after irradiating light of a third wavelength from outside the brain to the living brain of the subject administered with the substance at a time different from the first step, and detecting light of a fourth wavelength different from the third wavelength emitted from the brain, and the classification and accumulation of the substance that accumulates in the brain are determined 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 a substance that accumulates in the brain by optical imaging.

[0118] The subject and administration method were the same as those described in the above [Optical imaging method].

[0119] Since the method includes both a first step of detecting light emitted from the brain resulting from administration of a binding agent with high binding selectivity for α-synuclein aggregates, and a second step of detecting light emitted from the brain resulting from administration of a substance with high binding selectivity for tau protein aggregates in the same subject, by comparing the detection result of the first step (the amount and / or distribution of detected light) with the detection result of the second step, it is possible to classify whether the substance accumulating in the brain is an α-synuclein aggregate and / or a tau protein aggregate, and to determine the accumulation.

[0120] Another aspect of the method for determining classification and accumulation of a substance that accumulates in the brain of this embodiment includes a first step of detecting radiation emitted from the living brain of a subject administered with a binding agent according to this embodiment, which includes compound (I) or compound (II), a medicamentously acceptable salt thereof, or a solvate thereof, in which one or more atoms are radioisotopes of the atom, and a second step of detecting radiation emitted from the brain of the subject administered with a substance having high binding selectivity to tau protein aggregates (e.g., a compound described in Patent Document 1) at a time different from the first step, and the classification and accumulation of the substance that accumulates in the brain are determined 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 classification and accumulation of a substance that accumulates in the brain by radiation imaging.

[0121] The subject and administration method are the same as those described in the above [Radiation imaging method].

[0122] Since the method includes both a first step of detecting radiation emitted from the brain due to administration of a binding agent with high binding selectivity for α-synuclein aggregates, and a second step of detecting radiation emitted from the brain due to administration of a substance with high binding selectivity for tau protein aggregates in the same subject, by comparing the detection result of the first step (the amount and / or distribution of the detected radiation) with the detection result of the second step, it is possible to classify whether the substance accumulating in the brain is an α-synuclein aggregate and / or a tau protein aggregate, and to determine the accumulation.

[0123] (summary) The embodiments of the present invention are summarized below.

[0124] One aspect of the present invention is a compound represented by the following formula (I) or (II), a pharma- ceutical acceptable salt thereof, or a solvate thereof.

[0125] [ka] One aspect of the present invention is an α-synuclein aggregate binding agent comprising the above-mentioned compound, a pharma- ceutical acceptable salt thereof, or a solvate thereof.

[0126] One aspect of the present invention is a composition for optical imaging of α-synuclein aggregates, comprising the binding agent.

[0127] One aspect of the present invention is a composition for radiation imaging of α-synuclein aggregates, comprising the binding agent.

[0128] One aspect of the present invention is a method for optical imaging of alpha-synuclein aggregates in the brain, comprising the steps of irradiating light of a first wavelength from outside the brain to the living brain of a subject to which the binding agent has been administered, and then detecting light of a second wavelength, different from the first wavelength, emitted from the brain.

[0129] One aspect of the present invention is a method for radiation imaging of α-synuclein aggregates in the brain, comprising the step of detecting radiation emitted from the living brain of a subject to which the binding agent has been administered.

[0130] One aspect of the present invention is an intermediate for synthesizing the compound, which is represented by the following formula (III):

[0131] [ka] (In formula (III), One of X and Y is a nitrogen atom (N) and the other is an unsubstituted carbon atom (CH) (i.e., when X is a nitrogen atom, Y is an unsubstituted carbon atom (CH) and when X is an unsubstituted carbon atom (CH), Y is a nitrogen atom); R1 is a hydroxy group or a group represented by the following formula (i):

[0132] [ka] Here, Ts represents a p-toluenesulfonyl group. THP represents a tetrahydro-2H-pyran-2-yl group; * indicates the bonding position to the benzothiazole ring, R2 is a hydrogen atom or a tert-butoxycarbonyl (Boc) group.

[0133] One aspect of the present invention is a diagnostic kit comprising compound (I) or compound (II) having high binding selectivity for α-synuclein aggregates.

[0134] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of 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. EXAMPLES

[0135] (Production example) In the following Production Examples (Reference Examples and Examples), "room temperature" generally refers to about 10°C to about 35°C. Unless otherwise specified, % refers to weight %. In addition, with regard to the explanation of the compounds used in each Production Example, "produced in Reference Example (Example) X" is intended to include the case where "produced in the same manner as Reference Example (Example) X".

[0136] Unless otherwise specified, elution in column chromatography in the production examples was performed under observation by TLC (Thin Layer Chromatography). In the TLC observation, 60 F254 manufactured by Merck was used as the TLC plate, and the solvent used as the elution solvent in the column chromatography was used as the developing solvent. In addition, a UV detector was used for detection.

[0137] 1 H NMR analysis was performed using ACD / SpecManager (trade name) software, and the analytical values ​​obtained are listed. Peaks with very gentle proton peaks, such as those of hydroxyl and amino groups, may not be listed.

[0138] The following abbreviations are used in the following examples: M: Molar concentration DMSO-d6: deuterated dimethyl sulfoxide 1 H NMR: Proton nuclear magnetic resonance DIEA: N-ethyl-N-isopropylpropan-2-amine DMF: N,N-dimethylformamide (also referred to as "dimethylformamide" in this specification) THF: tetrahydrofuran MeOH: Methanol EtOH: Ethanol DMSO: Dimethyl sulfoxide TEA: Triethylamine TFA: trifluoroacetic acid.

[0139] Reference Example 1: Preparation of di-tert-butyl (5-iodopyrazin-2-yl)-2-imidodicarbonate

[0140] [ka] A solution of 5-iodopyrazine-2-amine (CAS[886860-50-0]) (10.5 g) in THF (100 ml) was added with a solution of di-tert-butyl dicarbonate (25.9 g) in THF (50 ml) and N,N-dimethylpyridin-4-amine (1.45 g) at 0°C. The mixture was stirred at 0°C to room temperature overnight, diluted with water and ethyl acetate, filtered to remove insoluble matter, and extracted with ethyl acetate. The extract was washed with water and brine, 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. 1 H NMR (300MHz, DMSO-d6) δ1.37-1.41 (18H, m), 8.65 (1H, d, J = 1.3 Hz), 8.90 (1 H, d, J = 1.5 Hz).

[0141] Reference Example 2: Preparation of tert-butyl (5-iodopyrazin-2-yl)carbamate

[0142] [ka] Potassium carbonate (7.40 g) was added at room temperature to a solution of di-tert-butyl (5-iodopyrazin-2-yl)-2-imidodicarbonate (18.8 g) prepared in Reference Example 1 in MeOH (200 ml). The mixture was stirred at room temperature for 2 hours, concentrated to about one-fourth the original volume under reduced pressure, cooled to 0°C, neutralized with a 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. 1 H NMR (300MHz, DMSO-d6) δ1.47 (9H, s), 8.55 (1H, d, J = 1.3Hz), 8.87 (1H, d, J = 1.5Hz), 10.29 (1H, brs).

[0143] Reference Example 3: Preparation of tert-butyl (5-iodopyrazin-2-yl)(methyl)carbamate

[0144] [ka] Methyl iodide (3.45 ml) was added to a solution of tert-butyl (5-iodopyrazin-2-yl)carbamate (13.7 g) prepared in Reference Example 2 and cesium carbonate (20.9 g) in DMF (85 ml) at 0° C. The mixture was stirred overnight at 0° C. to room temperature, 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. 1 H NMR (300MHz, DMSO-d6) δ1.49(9H,s), 3.27(3H,s), 8.72(1H,d,J=1.5Hz),8.83(1H,d,J=1.5Hz).

[0145] Reference Example 4: Preparation of tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrazin-2-yl)(methyl)carbamate

[0146] [ka] To a mixture of tert-butyl (5-iodopyrazin-2-yl)(methyl)carbamate (13.0 g) prepared in Reference Example 3, 2-propyn-1-ol (3.43 ml) and copper (I) iodide (739 mg) in TEA (27 ml) and THF (27 ml), dichlorobis(triphenylphosphine)palladium (II) (545 mg) was added at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 2 hours, diluted with ethyl acetate and filtered through Celite. The filtrate was concentrated under reduced pressure, the residue was diluted with ethyl acetate, acidified with 5% aqueous citric acid solution, filtered through Celite, and the filtrate was extracted with ethyl acetate. The extract was washed with water and brine, 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. 1 H NMR(300MHz,DMSO-d6)δ1.50(9H,s),3.31(3H,s),4.35(2H,d,J=6.0Hz),5.48(1H,t,J=6.0Hz),8.52(1H,d,J=1.5Hz),8.98(1H,d,J=1.5Hz).

[0147] Reference Example 5: Preparation of tert-butyl (5-(3-hydroxyprop-1-yn-1-yl)pyrimidin-2-yl)(methyl)carbamate

[0148] [ka] The title compound (195 mg) was obtained as a brown solid by the same method as in Reference Example 4 using tert-butyl (5-iodopyrimidin-2-yl)(methyl)carbamate (CAS[1578264-18-2]) (250 mg). 1 H NMR (300MHz, DMSO-d6) δ1.44 (9H, s), 3.31 (3H, s), 4.34 (2H, d, J = 5.7Hz), 5.44 (1H, t, J = 5.8Hz), 8.77 (2H, s).

[0149] Reference Example 6: Preparation of tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate

[0150] [ka] 1,1,1-triacetoxy-1,1-dihydro-1,2-benzoiodoxol-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) prepared in Reference Example 4 in acetonitrile (200 ml) at room temperature. The mixture was stirred at room temperature for 2 hours, cooled to 0°C, and a saturated aqueous solution of sodium thiosulfate and a 5% aqueous solution of sodium hydrogen carbonate were added, stirred for 5 minutes, and extracted with ethyl acetate. The extract was washed with water and brine, 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. 1 H NMR (300MHz, DMSO-d6) δ1.52 (9H, s), 3.35 (3H, s), 8.82 (1H, d, J = 1.3 Hz), 9.17 (1 H, d, J = 1.5 Hz), 9.48 (1 H, s).

[0151] Reference Example 7: Preparation of tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrimidin-2-yl)carbamate

[0152] [ka] 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) was obtained as a white solid in the same manner as in Reference Example 6. 1H NMR (300MHz, DMSO-d6) δ1.47(9H,s), 3.36(3H,s), 9.00(2H,s), 9.46(1H,s).

[0153] Reference Example 8: Preparation of 2-((1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0154] [ka] To a solution of 1-(benzyloxy)-3-fluoropropan-2-ol (CAS[112482-36-7]) (6.37g) and 3,4-dihydro-2H-pyran (3.75ml) in THF (67ml), p-toluenesulfonic acid monohydrate (132mg) was added at room temperature. The mixture was stirred overnight at room temperature, and then DIEA (0.242ml) was added and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (8.37g) as a colorless liquid. 1 H NMR(300MHz,DMSO-d6)δ1.35-1.54(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= 11.2,7.9,3.4Hz),3.90-4.06(1H,m),4.35-4.70(2H,m),4.51(2H,d,J=2.1Hz),4.74-4.81(1H,m),7.24-7.41(5H,m).

[0155] Reference Example 9: Preparation of 2-(((R)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0156] [ka] The title compound (10.36 g) was obtained as a colorless liquid by a method similar to that of Reference Example 8 using (R)-1-(benzyloxy)-3-fluoropropan-2-ol (CAS[147332-34-1]) (7.85 g). 1 H NMR(300MHz,DMSO-d6)δ1.47(4H,br dd,J=7.4,3.9Hz),1.56-1.80(2H,m),3.35-3.47(1H,m),3.47-3.63(2H,m),3.80(1H,ddd,J=1 1.3,8.0,3.4Hz),3.89-4.11(1H,m),4.34-4.69(4H,m),4.73-4.83(1H,m),7.19-7.49(5H,m).

[0157] Reference Example 10: Preparation of 2-(((S)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran

[0158] [ka] The title compound (10.48 g) was obtained as a colorless liquid in the same manner as in Reference Example 8 using (S)-1-(benzyloxy)-3-fluoropropan-2-ol (CAS[1707146-21-1]) (7.55 g). 1 H NMR(300MHz,DMSO-d6)δ1.47(4H,br dd,J=7.4,3.9Hz),1.56-1.79(2H,m),3.35-3.47(1H,m),3.48-3.62(2H,m),3.80(1H,ddd,J=1 1.2,7.9,3.3Hz),3.90-4.08(1H,m),4.35-4.70(4H,m),4.73-4.81(1H,m),7.24-7.42(5H,m).

[0159] Reference Example 11: Preparation of 3-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate

[0160] [ka] 3-(benzyloxy)-2-hydroxypropyl 4-methylbenzenesulfonate (CAS[99881-48-8]) (2.80g) and 1H-imidazole (737mg) in DMF (30ml) were added to tert-butyldimethylchlorosilane (1.51g) at 0℃. The mixture was stirred overnight at room temperature, cooled to 0℃, diluted with ethyl acetate and water, adjusted to pH 5 with 5% aqueous citric acid, 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 obtain the title compound (3.37g) as a colorless liquid. 1 H NMR(300MHz,DMSO-d6)δ-0.02(6H,d,J=1.1Hz),0.79(9H,s),2.41(3H,s),3.36(2H,d,J=5.3Hz), 3.86-4.05(3H,m),4.43(2H,s),7.20-7.39(5H,m),7.47(2H,d,J=8.1Hz),7.77(2H,d,J=8.3Hz).

[0161] Reference Example 12: Preparation of 3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0162] [ka] 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 content, 1.98 g) and EtOH (85 ml) was stirred overnight at room temperature under a hydrogen atmosphere at normal pressure. 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. 1 H NMR (300MHz, DMSO-d6) δ1.33-1.55(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).

[0163] Reference Example 13: Preparation of (2R)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0164] [ka] 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) was obtained as a colorless liquid in the same manner as in Reference Example 12. 1 H NMR (300MHz, DMSO-d6) δ1.35-1.53(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).

[0165] Reference Example 14: Preparation of (2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol

[0166] [ka] Using 2-(((S)-1-(benzyloxy)-3-fluoropropan-2-yl)oxy)tetrahydro-2H-pyran (10.45 g) prepared in Reference Example 10 and following the procedure of Reference Example 12, the title compound (6.90 g) was obtained as a colorless liquid. 1 H NMR (300MHz, DMSO-d6) δ1.33-1.56(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).

[0167] Reference Example 15: Preparation of 2-((tert-butyldimethylsilyl)oxy)-3-hydroxypropyl 4-methylbenzenesulfonate

[0168] [ka] Using 3-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesulfonate (3.36 g) prepared in Reference Example 11 and following the procedure of Reference Example 12, the title compound (2.66 g) was obtained as a colorless liquid. 1 H NMR(300MHz,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=5.6Hz),7.49(2H,dd,J=8.6,0.7Hz),7.77(2H,d,J=8.3Hz).

[0169] Reference Example 16: Preparation of 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

[0170] [ka] (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 give the title compound (6.90 g) as a colorless liquid. 1 H NMR (300MHz, DMSO-d6) δ1.33-1.54(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).

[0171] Reference Example 17: Preparation of diethyl ((6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazol-2-yl)methyl)phosphonate

[0172] [ka] A mixture of 2-(bromomethyl)-6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazole (CAS[1638685-65-0]) (3.06 g) and triethyl phosphite (1.78 ml) was stirred at 100° C. for 3 hours, 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. 1 H NMR(300MHz,DMSO-d6)δ0.22(6H,s),0.97(9H,s),1.23(6H,t,J=7.0Hz),3.80-3.92(2H,m), 4.01-4.13(4H,m),7.00(1H,dd,J=8.7,2.5Hz),7.56(1H,d,J=2.4Hz),7.81(1H,d,J=8.8Hz).

[0173] Reference Example 18: Preparation of diethyl ((6-hydroxybenzo[d]thiazol-2-yl)methyl)phosphonate

[0174] [ka] To a solution of diethyl ((6-((tert-butyldimethylsilyl)oxy)benzo[d]thiazol-2-yl)methyl)phosphonate (4.87 g) prepared in Reference Example 17 in THF (50 ml), tetra-n-butylammonium fluoride (1M THF solution, 12.9 ml) was added at room temperature. The mixture was stirred at room temperature for 1 hour, 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 residual solid was suspended in hexane / ethyl acetate, filtered, washed with hexane / ethyl acetate, and dried to obtain the title compound (2.85 g) as a yellow solid. 1H NMR(300MHz,DMSO-d6)δ1.22(6H,t,J=7.1Hz),3.81(2H,d,J=21.4Hz),4.05(4H,dq,J=8.3,7.1 Hz),6.93(1H,dd,J=8.8,2.5Hz),7.34(1H,d,J=2.4Hz),7.73(1H,d,J=8.8Hz),9.78(1H,brs).

[0175] Reference Example 19: Preparation of diethyl ((6-(3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzo[d]thiazol-2-yl)methyl)phosphonate

[0176] [ka] 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) were added with diisopropyl azodicarboxylate (3.15 ml) at room temperature. The mixture was stirred at room temperature for one day, and 3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol (0.504 g) prepared in Reference Example 12, triphenylphosphine (0.742 g) and diisopropyl azodicarboxylate (0.555 ml) were added at room temperature, and the mixture was stirred overnight at room temperature and then 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. 1H NMR(300MHz,DMSO-d6)δ1.22(6H,t,J=7.0Hz),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),4.1 2-4.29(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=9.0,2.1Hz),7.70(1H,d,J=2.4Hz),7.84(1H,d,J=8.9Hz).

[0177] 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

[0178] [ka] Sodium hydride (60% oil, 392 mg) was added at 0° C. 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). The mixture was stirred at 0° C. for 10 minutes, and then DMF (30 ml) was added at 0° C. and stirred from 0° C. to room temperature for 3 hours. To this mixture, tert-butyl methyl (5-(3-oxoprop-1-yn-1-yl)pyrazin-2-yl)carbamate (256 mg) prepared in Reference Example 6 was added at room temperature, and the mixture was stirred at room temperature for 1 hour. The mixture was then diluted with ethyl acetate and water, neutralized with a 5% aqueous citric acid solution, 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 residual solid was suspended in hexane, and the solid obtained by filtration was washed with hexane and dried to obtain the title compound (2.06 g) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ1.39-1.58(13H,m),1.59-1.81(2H,m),3.33(3H,s),3.41-3.52(1 H,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=16.2Hz),7.17(1H,dt,J=9.0,2.2Hz),7.48(1H,d,J=16.0Hz),7 .77(1H,d,J=2.4Hz),7.92(1H,d,J=8.9Hz),8.65(1H,d,J=1.5Hz),9.06(1H,d,J=1.5Hz).

[0179] 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

[0180] [ka] To a 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 in THF (25 ml), sodium hydride (60% in oil, 322 mg) was added at 0° C. The mixture was stirred at 0° C. for 5 minutes, and then DMF (25 ml) was added at 0° C. and stirred at 0° C. for 2 hours. Sodium hydride (60% in oil, 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 at 0°C, and the mixture was stirred from 0°C to room temperature for 2 hours. The mixture was then cooled to 0°C, adjusted to pH 5 with 5% aqueous citric acid, 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 through a short silica gel column chromatography. The residual solid was suspended in ethyl acetate / hexane, and the solid obtained by filtration was washed with ethyl acetate / hexane and dried to obtain the title compound (1.85 g) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ1.51(9H,s),3.33(3H,s),6.94(1H,d,J=16.0Hz),7.00(1H,dd,J=8.9,2.4Hz),7.39(1H,d,J= 2.3Hz), 7.44(1H,d,J=16.0Hz),7.82(1H,d,J=8.9Hz),8.64(1H,d,J=1.5Hz),9.05(1H,d,J=1.5Hz),10.14(1H,brs).

[0181] 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

[0182] [ka] 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) was obtained as a yellow solid in the same manner as in Example 1. 1 H NMR(300MHz,DMSO-d6)δ1.46(9H,s),3.34(3H,s),6.92(1H,d,J=16.0Hz),7.00(1H,dd,J=8.9,2.4H z),7.38(1H,d,J=16.0Hz),7.39(1H,d,J=2.4Hz),7.82(1H,d,J=8.9Hz),8.87(2H,s),10.01(1H,s).

[0183] 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

[0184] [ka] Using 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 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) was obtained as a pale yellow solid in the same manner as in Reference Example 19. 1 H NMR(300MHz,DMSO-d6)δ1.51(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=16.1Hz),7.17(1H,dt,J=9.0,2.1Hz),7.48(1H,d,J=16.1Hz),7.77(1 H,d,J=2.5Hz),7.92(1H,d,J=9.0Hz),8.65(1H,d,J=1.4Hz),9.06(1H,d,J=1.5Hz).

[0185] 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

[0186] [ka] 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 Example 1, (2S)-3-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propan-1-ol prepared in Reference Example 16 and (2S)-1-fluoro-3-((tetrahydro-2H-pyran-2-yl)oxy)propan-2-ol mixture (114 mg), and azodicarboxylate bis(2-methoxyethyl) (172 mg) were used in the same manner as in Reference Example 19 to obtain the title compound (22 mg) as a pale yellow solid. 1 H NMR(300MHz,DMSO-d6)δ1.51(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=16.2Hz),7.17(1H,dd,J=8.8,2.4Hz),7.48(1H,d,J=16.0Hz),7.77(1 H,d,J=2.3Hz),7.92(1H,d,J=9.0Hz),8.65(1H,d,J=1.3Hz),9.06(1H,d,J=1.3Hz).

[0187] 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

[0188] [ka] Using 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 in the same manner as in Reference Example 19. 1 H NMR(300MHz,DMSO-d6)δ0.04(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=16.0Hz),7.02-7.0 8(1H,m),7.45(2H,brd,J=8.1Hz),7.48(1H,brd,J=16.0Hz),7.66(1H,d,J=2.3Hz),7.7 9(2H,d,J=8.3Hz),7.91(1H,d,J=9.0Hz),8.65(1H,d,J=1.5Hz),9.06(1H,d,J=1.5Hz).

[0189] 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

[0190] [ka] 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) prepared in Example 2 and potassium carbonate (66 mg) in DMF (3 ml) at room temperature. 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 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. 1 H NMR(300MHz,DMSO-d6)δ1.46(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=5.3Hz),6. 99(1H,d,J=16.2Hz),7.16(1H,dd,J=9.0,2.6Hz),7.41(1H,d,J=16.2Hz),7.73(1H,d,J=2.3Hz),7.91(1H,d,J=9.0Hz),8.87(2H,s).

[0191] 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

[0192] [ka] 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) prepared in Reference Example 23 and acetic acid (0.023 ml) in THF (10 ml), tetra-n-butylammonium fluoride (1M THF solution, 0.533 ml) was added at 0° C. The mixture was stirred at 0° C. for 0.5 hours, 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 obtain the title compound (37 mg) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ1.51(9H,s),2.35(3H,s),3.34(3H,s),3.91-4.19( 5H,m),5.59(1H,d,J=4.9Hz),7.02(1H,d,J=16.1Hz),7.00-7.07(1H,m),7.3 5-7.43(2H,m),7.48(1H,d,J=16.1Hz),7.62(1H,d,J=2.5Hz),7.77(2H,d,J= 8.3Hz), 7.89(1H,d,J=9.0Hz),8.65(1H,d,J=1.5Hz),9.06(1H,d,J=1.5Hz).

[0193] 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

[0194] [ka] A mixed 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 (750 mg) prepared in Reference Example 23 in TFA (13.5 ml) and water (1.5 ml) was stirred at room temperature for 0.5 hours, cooled to 0° C., diluted with ethyl acetate, neutralized with a saturated aqueous solution of sodium bicarbonate, and extracted with ethyl acetate. The extract was washed with water and brine, and the solid precipitated from the extract was filtered. The filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residual solid and the filtered solid were combined and suspended in hexane. The solid obtained by filtration was washed with hexane and then dried to obtain the title compound (550 mg) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ2.34(3H,s),2.84(3H,d,J=4.8Hz),3.90-3.98(2H,m),4.02-4. 17(3H,m),5.60(1H,d,J=4.8Hz),6.95(1H,d,J=16.0Hz),7.00(1H,dd,J=8.8,2.4Hz),7 .29(1H,d,J=16.0Hz),7.40(2H,d,J=8.0Hz),7.59(1H,d,J=2.4Hz),7.68(1H,brs),7.7 7(2H,d,J=8.4Hz),7.86(1H,d,J=8.8Hz),7.94(1H,d,J=1.6Hz),8.21(1H,d,J=1.2Hz).

[0195] Example 3: Preparation of (E)-2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-ol

[0196] [ka] 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) prepared in Example 1 in TFA (4 ml) was stirred at room temperature for 1 hour, and then concentrated under reduced pressure. Ethyl acetate was added to the residue, cooled to 0°C, neutralized with a saturated aqueous solution of 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 residual solid was suspended in ethyl acetate, and the solid obtained by filtration was washed with ethyl acetate and dried to obtain the title compound (140 mg) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ2.84(3H,d,J=4.9Hz),6.81-6.92(1H,m),6.98(1H,dd,J=8.8,2.4Hz),7.26(1H,d,J=15.8Hz),7.3 7(1H,d,J=2.3Hz),7.63(1H,q,J=5.0Hz),7.79(1H,d,J=8.7Hz),7.94(1H,d,J=1.5Hz),8.20(1H,d,J=1.1Hz),9.97(1H,s).

[0197] 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")

[0198] [ka] 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) prepared in Reference Example 20 in TFA (20 ml) was stirred at room temperature for 0.5 hours, 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 the mixture was neutralized with a 5% aqueous solution of sodium hydrogen carbonate. The precipitated solid was collected by filtration, washed with water and ethyl acetate, and dried. The filtrate and the washings were combined and extracted with ethyl acetate. The extract was washed with water and saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residual solid was suspended in ethyl acetate, and the solid obtained by filtration was washed with ethyl acetate and then dried. This solid was combined with the solid collected by filtration before extraction and suspended in ethyl acetate, and the solid collected by filtration was washed with ethyl acetate and then dried to obtain the title compound (1.07 g) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ2.84(3H,d,J=4.9Hz),4.00-4.16(3H,m),4.37-4.49(1 H,m),4.53-4.66(1H,m),5.51(1H,d,J=5.1Hz),6.95(1H,d,J=16.0Hz),7.15(1 H,dd,J=9.0,2.6Hz),7.29(1H,d,J=16.0Hz),7.67(1H,q,J=4.6Hz),7.71(1H,d ,J=2.6Hz),7.89(1H,d,J=9.0Hz),7.94(1H,d,J=1.5Hz),8.21(1H,d,J=1.3Hz).

[0199] 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

[0200] [ka] The title compound (58 mg) was obtained as a pale yellow solid in the same manner 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. 1 H NMR(300MHz,DMSO-d6)δ2.84(3H,d,J=4.8Hz),4.01-4.17(3H,m),4.37-4.49(1 H,m),4.53-4.65(1H,m),5.50(1H,d,J=5.1Hz),6.95(1H,d,J=16.1Hz),7.15(1 H,dd,J=8.9,2.6Hz),7.29(1H,d,J=16.1Hz),7.66(1H,q,J=4.9Hz),7.71(1H,d ,J=2.5Hz),7.89(1H,d,J=9.0Hz),7.94(1H,d,J=1.4Hz),8.21(1H,d,J=1.3Hz).

[0201] 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

[0202] [ka] 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, the title compound (12 mg) was obtained as a pale yellow solid in the same manner as in Example 4. 1H NMR(300MHz,DMSO-d6)δ2.84(3H,d,J=4.7Hz),4.00-4.17(3H,m),4.37-4.49( 1H,m),4.53-4.65(1H,m),5.51(1H,d,J=4.7Hz),6.95(1H,d,J=16.0Hz),7.15( 1H,dd,J=8.9,2.5Hz),7.29(1H,d,J=16.2Hz),7.62-7.70(1H,m),7.71(1H,d, J=2.4Hz), 7.89(1H,d,J=8.9Hz),7.94(1H,d,J=1.3Hz),8.21(1H,d,J=1.1Hz).

[0203] 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")

[0204] [ka] The title compound (15 mg) was obtained as a yellow solid in the same manner as in Example 4 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. 1 H NMR(300MHz,DMSO-d6)δ2.84(3H,d,J=4.5Hz),4.00-4.16(3H,m),4.36-4.50(1H,m),4.52-4.66(1H,m),5.49(1H,d,J=5.3Hz ),6.86-6.99(1H,m),7.10-7.18(1H,m),7.25(1H,d,J=15.8Hz),7.67-7.77(2H,m),7.88(1H,d,J=9.0Hz),8.38-8.58(2H,m).

[0205] 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

[0206] [ka] 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) prepared in Reference Example 25 and 3,4-dihydro-2H-pyran (0.050 ml) in THF (15 ml), p-toluenesulfonic acid monohydrate (21 mg) was added at room temperature. The mixture was stirred at room temperature for 4 hours, and then 3,4-dihydro-2H-pyran (0.249 ml) and p-toluenesulfonic acid monohydrate (21 mg) were added at room temperature. The mixture was stirred overnight at room temperature, diluted with ethyl acetate, neutralized with a 5% aqueous solution of sodium hydrogen carbonate, 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, and the solid obtained by filtration was washed with hexane and dried to obtain the title compound (28 mg) as a yellow solid. 1H NMR(300MHz,DMSO-d6)δ1.32-1.72(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=8.1Hz),7.48(1H,d,J=16.2Hz),7.65(1H,d,J=2.1Hz),7.79(2H ,brd,J=7.3Hz),7.90(1H,d,J=8.9Hz),8.65(1H,d,J=1.1Hz),9.06(1H,d,J=0.9Hz).

[0207] 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

[0208] [ka] 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) prepared in Reference Example 26 and 3,4-dihydro-2H-pyran (0.018 ml) in THF (15 ml) was added p-toluenesulfonic acid monohydrate (12 mg) at room temperature. The mixture was stirred at room temperature for 0.5 hours, and 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 residual solid was suspended in hexane, and the solid obtained by filtration was washed with hexane and dried to obtain the title compound (21 mg) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ1.32-1.71(6H,m),2.35(3H,s),2.84(3H,d,J=4.9Hz),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=16.2Hz),7.02(1H, dd,J=8.9,1.6Hz),7.30(1H,d,J=16.0Hz),7.41(2H,d,J=7.9Hz),7.62(1H,d,J=2.6Hz),7.67(1H,q,J=4 .4Hz),7.79(2H,dd,J=8.4,1.6Hz),7.86(1H,d,J=9.0Hz),7.94(1H,d,J=1.5Hz),8.21(1H,d,J=1.3Hz).

[0209] Example 10: 18 F]SPAL-T-06 Production Example 1 The fluorine atom is 18 SPAL-T-06 (hereinafter, 18F]SPAL-T-06) was manufactured. [ka]

[0210] [ 18 Synthesis of [F]epifluorohydrin 18 The reaction was carried out by nucleophilic substitution of glycidyl tosylate with [F] fluoride ion and purification by distillation. The following reaction was carried out in the dark. 18 [F] epifluorohydrin, a DMF (250 μl) solution containing (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, and 1M aqueous sodium hydroxide solution (6.5 μl) were added to the reaction vessel. The reaction mixture was heated at 130° C. for 20 minutes. After cooling the reaction vessel, the HPLC solvent (500 μl) used in the subsequent HPLC was added. 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). 18 The fraction corresponding to [F]SPAL-T-06 was collected in a flask containing ethanol (300 μl), 25% ascorbic acid (100 μl) and Tween 80 (75 μl), and the solvent was removed under reduced pressure. The residue was dissolved in physiological saline (3 ml, pH 7.4) and used as an injection solution, 18 F]SPAL-T-06 was obtained.

[0211] Example 11: 18 F]SPAL-T-06 Production Example 2 According to the following scheme, 18 F]SPAL-T-06 was manufactured.

[0212] [ka] [ 18[F] fluoride ion was eluted with 50% acetonitrile solution (0.4 ml) containing K.222 (Kryptofix 222) (7.5 mg) and potassium carbonate (2.77 mg), introduced into a reaction vessel, and heated under a nitrogen gas flow to dry the solvent. Then, anhydrous acetonitrile (0.1 ml) was added and azeotropically removed, and the reaction vessel was thoroughly dried. 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-yn-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 cooling the reaction vessel, TFA / water (600 μl) was added and hydrolysis was carried out at 90° C. for 10 minutes. After cooling the reaction vessel, 4N aqueous sodium acetate solution (1 ml) was added and stirred. The mixture was purified by HPLC (HPLC: CAPCELL PAK C18 UG80 10 mm x 250 mm, acetonitrile / water = 4 / 6 (containing 0.1% triethylamine), 5 ml / min). 18 The fraction corresponding to [F]SPAL-T-06 was collected in a flask containing ethanol (300 μl), 25% ascorbic acid (100 μl) and Tween 80 (75 μl), and the solvent was removed under reduced pressure. The residue was dissolved in physiological saline (3 ml, pH 7.4) and used as an injection solution, 18 F]SPAL-T-06 was obtained.

[0213] [Optical imaging of the human brain] (Dissected Brain Tissue) Postmortem human brains were obtained from autopsies performed on dementia with Lewy bodies (DLB) and Alzheimer's disease (AD) patients. Frozen DLB tissues were sliced ​​at 20 μm thickness in a cryostat (HM560, Carl Zeiss), and AD brain tissues were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and sliced ​​at 6 μm thickness.

[0214] (In vitro fluorescence microscopy measurement) We used post-fixed fresh frozen sections of amygdala tissue from DLB patients and formalin-fixed paraffin-embedded sections of deparaffinized middle frontal gyrus tissue from AD patients. 30 μM of the test compound and the brain sections were incubated in 50% ethanol solution for 30 minutes at room temperature. The sections were then washed twice, with 50% ethanol solution for 5 minutes and ultrapure water for 3 minutes. After mounting the sections using mounting medium (VECTASHIELD H-1000, Vector Laboratories), images of the lesion accumulation areas on the sections were obtained using a fluorescent microscope (DM4000, Leica, excitation wavelength 391-437 nm). The fluorescent images are shown in Figure 1. In Figure 1, the arrowhead indicates the fluorescence of the compound bound to α-synuclein aggregates in the DLB patient brain, the arrow indicates the fluorescence of the compound bound to amyloid β aggregates in the AD patient brain, and the asterisk indicates the fluorescence of the compound bound to tau aggregates in the AD patient brain. The fluorescence intensity of the lesioned area and the non-lesioned area (background) was quantified using analysis software (Image J). The results are shown in Figure 2. The test compounds used were SPAL-T-05 and SPAL-T-06. The control compounds used were BF-227 (2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole (catalog number NP039-0)) and PBB3 (2-((1E,3E)-4-(6-(methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol) (catalog number NP039-0) obtained from NARD Laboratories.

[0215] As shown in Figures 1 and 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 stronger binding than PBB3. It was also confirmed that the binding affinity of SPAL-T-06 and SPAL-T-05 to α-synuclein aggregates is stronger than that of tau or amyloid β aggregates formed in the brains of AD patients. In other words, it was shown that the compound of the present invention has high binding selectivity to α-synuclein aggregates. In addition, in vitro fluorescence microscopy measurement was performed on the brains of multiple system atrophy (MSA) patients in the same manner as the above-mentioned DLB patient brains, and similar results were obtained.

[0216] It was confirmed that BF-227, which has been reported as a PET probe for α-synuclein pathology, mainly binds to amyloid β aggregates in AD, and its binding to α-synuclein aggregates is weaker than that of SPAL-T-06 and SPAL-T-05. In other words, it was shown that BF-227 has low binding selectivity to α-synuclein aggregates.

[0217] [Optical imaging of mouse brain] (Creation of a mouse model inoculated with α-synuclein fibrils) Mouse α-synuclein is expressed as a recombinant protein in E. coli, extracted, and incubated in a test tube to form insoluble aggregates. When these α-synuclein aggregates are inoculated into the striatum of mice, the aggregates spread to the surrounding areas via neural circuits, and α-synuclein lesions are observed in the neocortex several months later (Masuda-Suzukake et al. Acta Neuropathol Commun 2, 88, 2014; Shimozawa et al. Acta Neuropathol Commun 5, 12, 2017). The brains of these mice are then excised, sliced, and analyzed by fluorescent staining to confirm whether the compounds of the present invention bind to lesions consisting of phosphorylated α-synuclein.

[0218] (Creation of mouse model inoculated with α-synuclein fibrils) First, mouse α-synuclein was expressed as a recombinant protein in E. coli, extracted, and incubated in a test tube to form insoluble α-synuclein aggregates. When these α-synuclein aggregates were inoculated into the striatum of mice, the aggregates spread to the surrounding areas via neural circuits, and α-synuclein lesions were observed in the neocortex several months later. The α-synuclein aggregates were inoculated into the striatum of mice as follows. First, the hair on the head of a 9-week-old C57 / BL / 6 male mouse anesthetized with 1.5% (v / v) isoflurane was removed, the scalp was disinfected with isodine, xylocaine was applied, and an incision was made in the scalp to expose the skull. A hole was then drilled into the skull at 0.05 mm Bregma and 2 mm lateral, and 3 μl of α-synuclein fibril solution (mouse α-synuclein fibrils 4 mg / ml in saline) was injected to a depth of 2 μm using a glass pipette, after which the scalp was replaced and sutured.

[0219] (In vitro fluorescence microscopy measurement) The brains of the mice inoculated with α-synuclein fibrils were removed, sliced, and analyzed by fluorescent staining. Specifically, the brain slices of the mice inoculated with α-synuclein fibrils were incubated with 30 μM of the compound in a 20% ethanol solution at room temperature for 30 minutes. The slices were then washed twice, once with a 20% ethanol solution for 5 minutes and once with ultrapure water for 3 minutes. The slices were mounted using a mounting medium (VECTASHIELD H-1000), and images of the α-synuclein aggregate accumulation areas on the slices were obtained using a fluorescence microscope (DM4000 (excitation wavelength 391-437 nm)). The same slices were washed with phosphate buffer and then autoclaved for antigen retrieval. Immunohistochemical staining was performed using anti-phosphorylated α-synuclein monoclonal antibody (pS129, Abcam, ab59264) (1:1000), and the sections were mounted using mounting medium (VECTASHIELD H-1000), after which images of the same areas as above were obtained using a fluorescence microscope (DM4000 (excitation wavelength 460-500 nm)). The results are shown in Figure 3. Figure 3 (a) and (b) show the results for SPAL-T-05 and SPAL-T-06, respectively. In Figure 3 (a) and (b), the right side shows anti-phosphorylated α-synuclein antibody staining, and the left side shows SPAL-T-05 and SPAL-T-06 fluorescent staining. In Figure 3, the arrowheads indicate lesions consisting of phosphorylated α-synuclein in the brain of a mouse inoculated with α-synuclein fibrils, and the fluorescence of compounds bound to them.

[0220] The results in Figure 3 demonstrated that SPAL-T-05 and SPAL-T-06 bound to lesions consisting of phosphorylated α-synuclein.

[0221] [In vivo two-photon laser scanning fluorescence microscopy] Model mice 6 weeks or more after injection of α-synuclein fibril solution were anesthetized with 1.5% (v / v) isoflurane, and a cranial window was placed according to the Seylaz-Tomita method (Tomita et al. J Cereb Blood Flow Metab 25, 858-67, 2005). Mice 2 weeks or more after cranial window placement were anesthetized with 1.5% (v / v) isoflurane, and 50 μl of DMSO solution containing 0.1% BF-227, PBB3, SPAL-T-05, or SPAL-T-06 was intraperitoneally administered. 30 minutes after administration, the mice were fixed under a two-photon laser fluorescence microscope, and 100 μl of saline containing 5 mM sulforhodamine 101 was intraperitoneally administered, after which in vivo two-photon fluorescence imaging was performed at an excitation wavelength of 900 nm. The detection wavelengths for BF-227, PBB3, SPAL-T-05, and SPAL-T-06 were 500 to 550 nm, and the detection wavelength for Sulforhodamine 101 was 573 to 648 nm. The results are shown in Figure 4. In Figure 4, the arrowheads indicate blood vessels, and the arrows indicate the fluorescence of the compound bound to the α-synuclein lesion.

[0222] From the results of Figure 4, when SPAL-T-05 and SPAL-T-06 were administered intraperitoneally, the compounds were transferred into the brain and further into neurons of the living body, and the binding to individual α-synuclein lesions was observed by in vivo two-photon laser fluorescence microscope. Therefore, even when the density or total number of the lesions is low and difficult to detect by PET, it can be said that the lesions can be detected by in vivo fluorescence imaging using the compounds of the present invention. On the other hand, no α-synuclein lesions were detected when BF-227 and PBB3 were administered intraperitoneally and observed.

[0223] [In vivo PET (positron emission tomography) imaging of mouse brain] PET scans were performed using a micro PET Focus 220 animal scanner (Siemens Medical Solutions), providing 95 slices at 0.851 mm thickness (center-to-center), a 19.0 cm axial field of view (FOV), and a 7.6 cm transaxial FOV. Prior to scanning, α-synuclein fibril-inoculated mice and saline-injected mice (controls) were anesthetized with 1.5% (v / v) isoflurane. Emission scans were performed using a 10-μm PET scanner. 18 After intravenous injection of [F]SPAL-T-06 (a compound in which SPAL-T-06 is labeled with a positron-emitting nuclide) (30.1 ± 0.13 MBq), 3D list mode was performed for 90 min in an energy window of 350-750 keV. Injection and scanning of the radioactive compound were performed under dim light 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, 6 × 5, 5 × 10 min). Summed images were obtained at 0-30 min, 30-60 min, and 60-90 min after injection of the radioactive compound by maximum a posteriori reconstruction. Dynamic images were also reconstructed by filtered back projection using a 0.5 mm Hanning filter. Volumes of interest (VOI) were set in the striatum, cerebral cortex, and cerebellum with reference to the MRI template using PMOD image analysis software (PMOD Technologies). The results are shown in Figures 5-7.

[0224] Figure 5 left: 18 The results are shown in Fig. 5 right, for mice inoculated with α-synuclein fibrils 30 to 60 minutes after intravenous injection of SPAL-T-06. The results are shown in Fig. 5 right, for mice injected with saline. The upper and lower images are coronal sections of the brain including the striatum and the cerebellum, respectively, and are superimposed on standard brain MRI images. Figure 6 shows the results of the [ 18 F]SPAL-T-06 intravenous injection time-radioactivity curves. (a) to (d) in Fig. 7 show 18F] Time course of standardized uptake value ratio (SUVR) in the striatum and cerebral cortex with the cerebellum as a control after intravenous injection of SPAL-T-06, and comparison of the average SUVR between α-synuclein fibril-injected mice and saline-injected mice at 30 to 60 minutes (n = 2, mean ± SEMs). 18 F] The time course of SUVR in the striatum after intravenous injection of SPAL-T-06, with the cerebellum as the control region, is shown in Fig. 7 (b), which shows the average SUVR in the striatum (control region: cerebellum) from 30 to 60 minutes. Fig. 7 (c) shows the time course of SUVR in the striatum after intravenous injection of SPAL-T-06, with the cerebellum as the control region. 18 F] The time course of SUVR in the cerebral cortex after intravenous injection of SPAL-T-06, with the cerebellum as the control region, is shown in Figure 7(d), and the average SUVR of the cerebral cortex (control region: cerebellum) from 30 to 60 minutes is shown in Figure 7(d).

[0225] SPAL-T-06 was labeled with a positron-emitting nuclide and injected intravenously into α-synuclein fibril-inoculated model mice and saline-injected mice (control) and PET scans were performed. As shown in Figures 5 to 7, it was found to have appropriate brain penetration and clearance rate from the brain, and to exhibit good properties as a probe for imaging α-synuclein aggregates with PET. In the α-synuclein fibril-inoculated model mice, the striatum and cerebral cortex, which contain abundant α-synuclein lesions, showed [ 18 F]SPAL-T-06 was clearly accumulated, and [ 18 It was confirmed that α-synuclein aggregates could be imaged by PET scanning using [F]SPAL-T-06.

[0226] [Ex vivo imaging of mouse brain] Saline-injected mice were anesthetized with 1.5% (v / v) isoflurane and [ 18 F]SPAL-T-06 (28.9MBq) was injected intravenously. 18Brains were harvested and frozen 40 min after administration of [F]SPAL-T-06, and 20 μm-thick coronal frozen sections including Bregma 0.50, -0.46, -1.94, -3.16, and -6.64 mm were prepared in a cryostat (HM560). After air-drying, the sections were placed in a cassette and contacted with an imaging plate for 15 min, after which autoradiographs were obtained using a BAS-5000 (Fuji Film). The results are shown in Figure 8.

[0227] From the results of Figure 8, the saline-injected mice 18 It was confirmed that when [F]SPAL-T-06 was administered intravenously, the compound that was transferred to the brain did not show nonspecific accumulation in the white matter region.

[0228] [PET (positron emission tomography) imaging of the marmoset brain] (Creation of an α-synuclein fibril-injected marmoset model) First, marmoset α-synuclein was expressed as a recombinant protein in E. coli, extracted, and incubated in a test tube to form insoluble α-synuclein aggregates. Then, when the α-synuclein aggregates were inoculated into the caudate nucleus and putamen of a marmoset, the α-synuclein aggregates spread to the surrounding areas via neural circuits, and α-synuclein lesions were observed in the substantia nigra several months later. The inoculation of the α-synuclein aggregates into the caudate nucleus and putamen of a marmoset was performed as follows. First, a marmoset immobilized with ketamine (5-10 mg / kg) and xylazine (0.2-0.5 mg / kg) was intubated with an intra-organ tube and anesthetized with 1-3% (v / v) isoflurane. The hair on the head of the marmoset was removed, the scalp was disinfected with isodine, xylocaine was applied, and an incision was made in the scalp to expose the skull. A hole (diameter: 3 mm) was drilled in the skull at the interaural 9.75 mm position, and 50 μl of α-synuclein fibril solution (4 mg / ml marmoset α-synuclein fibril in saline) was injected into the caudate nucleus and putamen of the right brain using a Hamilton syringe. In addition, 50 μl of saline was injected into the caudate nucleus and putamen of the left brain in the same manner, and the scalp was then replaced and sutured.

[0229] (In vivo PET (positron emission tomography) imaging of the marmoset brain) PET scans were performed using a micro PET Focus 220 animal scanner (Siemens Medical Solutions), which provides 95 slices at 0.851 mm thickness (center-to-center), a 19.0 cm axial field of view (FOV), and a 7.6 cm cross-sectional FOV. Prior to scanning, α-synuclein fibril-inoculated marmosets were anesthetized with 1–3% (v / v) isoflurane. Transmission scans were performed for approximately 20 min using a PET calibration source, Ge-68. Emission scans were performed using a PET calibration source, Ge-68, and a PET calibration source, Ge-68. 18 The scan was performed in 3D list mode with an energy window of 350-750 keV for 90 minutes after intravenous injection of [F]SPAL-T-06 (a compound in which SPAL-T-06 is labeled with a positron-emitting nuclide) (73.0 MBq). The injection and scan 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 to 2D sinograms by Fourier-rebinning (frames: 10 × 1, 6 × 5, 5 × 10 min). Summed images were obtained at 0-30 min, 30-60 min, and 60-90 min after injection of the radioactive compound by maximum a posteriori reconstruction. Dynamic images were also reconstructed by filtered back projection using a 0.5 mm Hanning filter. The results are shown in Figure 9. Figure 9 (a) shows the [ 18 F] Coronal slice images of the brain, including the caudate nucleus, of a marmoset inoculated with α-synuclein fibrils, 20 to 60 minutes after intravenous injection of SPAL-T-06, superimposed on a standard brain MRI image.

[0230] (In vitro fluorescence microscopy measurement) The brain of this α-synuclein fibril-inoculated marmoset was removed, sliced, and analyzed by immunohistochemical staining. Specifically, the brain slice of the α-synuclein fibril-inoculated marmoset was washed with phosphate buffer, and then autoclaved for antigenic activation. Immunohistochemical staining was performed with anti-phosphorylated α-synuclein monoclonal antibody (pS129, abcam, ab59264) (1:1000), and the slice was mounted with mounting medium (VECTASHIELD H-1000), and 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) shows an image of anti-phosphorylated α-synuclein antibody staining of a coronal slice of the brain including the caudate nucleus of the α-synuclein fibril-inoculated marmoset. 2 is a magnified image of anti-phosphorylated α-synuclein antibody staining in the caudate nucleus of the right brain after inoculation with α-synuclein fibrils, and 1 is a magnified image of anti-phosphorylated α-synuclein antibody staining in the caudate nucleus of the left brain after injection of saline.

[0231] SPAL-T-06 was labeled with a positron-emitting nuclide and injected intravenously into an α-synuclein fibril-inoculated marmoset model, and PET scans were performed. The results showed that the caudate nucleus, which contains an abundance of α-synuclein lesions, 18 F]SPAL-T-06 was clearly accumulated, and [ 18 It was confirmed that α-synuclein aggregates could be imaged by PET scanning using [F]SPAL-T-06.

[0232] [In vitro binding study in human brain] Fresh frozen tissue from the amygdala of DLB ​​patients and fresh frozen tissue from the frontal cortex of AD patients were used. Buffer and zirconia beads (ZB-20, TOMY) were added to the fresh frozen brain tissue in an amount 10 times the wet weight of the tissue, and the tissue was pulverized using a Micro Smash (MS-100R, TOMY) and then stored at -80°C. DLB patient brain homogenate or AD patient brain homogenate, [ 18F]SPAL-T-06 (final concentration 5 nM) and unlabeled SPAL-T-06 (final concentration 0.01 nM to 1 μM) were mixed. After 30 minutes of incubation at room temperature, 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 (2480WIZARD2, ParkinElmer). The test was performed in triplicate for each concentration of unlabeled SPAL-T-06. The results were analyzed using Prism 6J (GraphPad), and the displacement curves and IC 50 The results are shown in Figure 10.

[0233] SPAL-T-06 was labeled with a positron-emitting nuclide and binding tests were performed using homogenates from DLB and AD patient brains. The results showed that SPAL-T-06 was highly potent in the brain homogenates from DLB patients, which contained abundant α-synuclein lesions. 18 F]SPAL-T-06 binds with high affinity (IC 50 = 1.58 nM) was confirmed. 18 The binding affinity of [F]SPAL-T-06 was lower (IC 50 =10.86nM), [ 18 F]SPAL-T-06 showed good binding affinity as a probe for imaging α-synuclein aggregates by PET imaging, and its binding to α-synuclein aggregates was stronger than that to tau or amyloid-β aggregates, demonstrating high binding selectivity for α-synuclein aggregates.

[0234] [In vitro autoradiography of human brain] (Dissected Brain Tissue) Postmortem human brains were obtained from autopsies performed on DLB and multiple system atrophy (MSA) patients and healthy subjects. Frozen DLB and healthy subject tissues were sliced ​​at 20 μm thickness in a cryostat (HM560, Carl Zeiss). MSA brain tissues were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and sliced ​​at 6 μm thickness.

[0235] (Autoradiography test) We used post-fixed fresh frozen sections of amygdala tissue from DLB patients and frontal cortex tissue from healthy subjects, as well as formalin-fixed paraffin-embedded sections of deparaffinized cerebellar tissue from MSA patients. 18 Brain tissue sections were incubated with [F]SPAL-T-06 (final concentration 10 nM) in 50 mM Tris-HCl containing 20% ​​ethanol at room temperature for 1 h. 18 Brain tissue sections were also incubated in 50 mM Tris-HCl (containing 20% ​​ethanol) containing [F]SPAL-T-06 (final concentration 10 nM) and unlabeled SPAL-T-06 (final concentration 10 μM). The sections were then washed twice for 2 min with 50 mM Tris-HCl (containing 20% ​​EtOH) at 4°C and rinsed with MilliQ. After air drying, the sections were contacted with the imaging plate in the cassette for 5 min, and then autoradiographs were obtained using a BAS-5000 (Fuji Film). The results are shown in Figure 11. The arrowheads in Figure 11 indicate areas containing lesions consisting of phosphorylated α-synuclein in the brains of DLB ​​patients and MSA patients.

[0236] (In vitro fluorescence microscopy measurement) The sections that had been autoradiographed were analyzed by fluorescent staining. Specifically, the sections were incubated with 30 μM SPAL-T-06 in 50% ethanol solution for 30 minutes at room temperature. The sections were then washed twice, once with 50% ethanol solution for 5 minutes and once with ultrapure water for 3 minutes. The sections were mounted using mounting medium (VECTASHIELD H-1000), and images of the α-synuclein aggregate accumulation areas on the sections were obtained using a fluorescent microscope (DM4000 (excitation wavelength 391-437 nm)). Adjacent brain sections were washed with phosphate buffer and then autoclaved for antigen retrieval. Immunohistochemical staining was performed using anti-phosphorylated α-synuclein monoclonal antibody (pS129, Abcam, ab59264) (1:1000), and the sections were mounted using mounting medium (VECTASHIELD H-1000), after which 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) shows anti-phosphorylated α-synuclein antibody staining and SPAL-T-06 fluorescent staining of the brain of a DLB patient, and Figure 12(b) (indicated by "2" in the figure) shows anti-phosphorylated α-synuclein antibody staining and SPAL-T-06 fluorescent staining of the brain of an MSA patient.

[0237] SPAL-T-06 was labeled with a positron-emitting nuclide and autoradiography was performed on sections of the amygdala of DLB ​​patients, cerebellum of MSA patients, and frontal cortex of healthy subjects. The results showed that SPAL-T-06 was highly expressed in areas of the DLB and MSA brains that were rich in lesions consisting of phosphorylated α-synuclein. 18 Binding of [F]SPAL-T-06 to α-synuclein lesions in the brain sections was confirmed by fluorescent staining and immunohistochemical staining of the autoradiographic sections. In addition, in the frontal cortex sections of healthy subjects, [ 18 F]SPAL-T-06 showed almost no nonspecific binding. [Industrial Applicability]

[0238] According to the present invention, it is possible to provide an α-synuclein aggregate-binding agent having high binding selectivity to α-synuclein aggregates, and an imaging method using the α-synuclein aggregate-binding agent. It is also possible to provide a novel compound that can be used as an α-synuclein aggregate-binding agent or for other purposes.

Claims

1. A compound represented by the following formula (I) or (II), a pharma- ceutical acceptable salt thereof, or a solvate thereof: 【Chemistry 1】

2. The compound according to claim 1, wherein one or more atoms in the compound represented by formula (I) or (II) are radioisotopes of said atoms, or a pharma- ceutically acceptable salt or solvate thereof.

3. An alpha-synuclein aggregate binding agent comprising the compound according to claim 1, a pharma- ceutical acceptable salt thereof, or a solvate thereof.

4. An alpha-synuclein aggregate binding agent comprising the compound according to claim 2, a pharma- ceutical acceptable salt thereof, or a solvate thereof.

5. A composition for optical imaging of α-synuclein aggregates, comprising the α-synuclein aggregate-binding agent of claim 3.

6. A composition for radiation imaging of α-synuclein aggregates, comprising the α-synuclein aggregate-binding agent of claim 4.

7. A method for optical imaging of alpha-synuclein aggregates in the brain, comprising the steps of irradiating the living brain of a subject administered the alpha-synuclein aggregate binding agent described in claim 3 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.

8. A method for radiation imaging of α-synuclein aggregates in the brain, comprising a step of detecting radiation emitted from the living brain of a subject to which the α-synuclein aggregate binding agent according to claim 4 has been administered.

9. 3. An intermediate for synthesizing the compound according to claim 1 or 2, represented by the following formula (III): 【Chemistry 2】 (In formula (III), One of X and Y is a nitrogen atom (N) and the other is an unsubstituted carbon atom (CH); R １ is a hydroxy group or a group represented by the following formula (i): 【Chemistry 3】 Here, Ts represents a p-toluenesulfonyl group. THP represents a tetrahydro-2H-pyran-2-yl group; * indicates the bonding position to the benzothiazole ring, R ２ is a hydrogen atom or a tert-butoxycarbonyl (Boc) group.

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