Method for purifying compound

Abstract

The present invention relates to a method for purifying a compound of formula (IV-F), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, comprising a C1-4 alkyl sulfonate as a leaving group. The compounds of formula (IV-F) can be used as precursors to 18F-labeled agents for imaging and determining the amount of alpha-synuclein aggregates. The 18F-labelled agents may be used to diagnose a disease, disorder, or abnormality associated with alpha-synuclein aggregates (e.g., multi-system atrophy (MSA)), determine the susceptibility of such a disease, disorder, or abnormality, predict the prognosis of such a disease, disorder, or abnormality, monitor disease evolution in a patient suffering from such a disease, disorder, or abnormality, and, for example, to diagnose a disease, disorder, or abnormality associated with alpha-synuclein aggregates. The progression of such diseases, disorders or abnormalities is monitored, as well as the response of patients suffering from such diseases, disorders or abnormalities to their treatment is predicted. # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to a method for purifying a compound of formula (IV-F). The present invention also relates to a kit for preparing a radiopharmaceutical formulation and a method for preparing a detectable-labeled compound of formula (III-F). The compound of formula (III-F) can be used for imaging α-synuclein aggregates and determining their amount. In addition, the compound of formula (III-F) can be used for diagnosing diseases, disorders or abnormalities associated with α-synuclein (α-synuclein, A-synuclein, a-synuclein, A-syn, α-syn, aSyn, a-syn) aggregates (such as multiple system atrophy (MSA)), determining the susceptibility to such diseases, disorders or abnormalities, predicting the prognosis of such diseases, disorders or abnormalities, monitoring the disease evolution of patients suffering from such diseases, disorders or abnormalities, monitoring the progression of such diseases, conditions or abnormalities, and predicting the response of patients suffering from such diseases, disorders or abnormalities to their treatment. Background of the Invention

[0003] Many age-related diseases are based on or associated with extracellular or intracellular deposits of amyloid or amyloid-like proteins, which contribute to the pathogenesis and progression of the diseases. The most characterized amyloid protein forming extracellular aggregates is amyloid β-protein (Abeta or Aβ).

[0004] Amyloid-like proteins that mainly form intracellular aggregates include, but are not limited to, Tau, alpha-synuclein, and huntingtin (HTT). Diseases involving alpha-synuclein aggregates are generally classified as synucleinopathies (or alpha-synucleinopathies), and they include, but are not limited to, Parkinson's disease (PD) and multiple system atrophy (MSA). Synucleinopathies with mainly neuronal aggregates include, but are not limited to, Parkinson's disease (sporadic, familial SNCA (gene encoding alpha-synuclein) mutations or SNCA gene duplications or triplications, other gene mutations outside of familial SNCA, pure autonomic failure, or Lewy body dysphagia), SNCA duplication carriers, dementia with Lewy bodies (LBD), dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD), diffuse Lewy body disease (DLBD), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2, or other mutations, familial British dementia, Alzheimer's disease Lewy body variant, and normal aging in Down syndrome. Synucleinopathies with alpha-synuclein neuronal and glial aggregates include, but are not limited to, multiple system atrophy (MSA) (Shy-Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy). Other diseases that may have alpha-synuclein immunoreactive lesions include, but are not limited to, traumatic brain injury, chronic traumatic encephalopathy, pugilistic dementia, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, and Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), motor neuron disease, Huntington's disease, amyotrophic lateral sclerosis (Guam sporadic, familial, and ALS-dementia complex), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gerstmann-Sträussler-Scheinker disease, inclusion body myositis, Gaucher's disease, Krabbe's disease, and other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder (Jellinger, Mov Disord 2003, 18 Suppl 6, S2-12; Galvin et al., JAMA Neurology 2001, 58(2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol.2004, 63(4), 323 - 328; McKee et al., Brain, 2013, 136(Pt 1), 43 - 64; Puschmann et al., Parkinsonism Relat Disord 2012, 18S1, S24 - S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240 - 4246; Winder - Rhodes et al., Mov Disord. 2012, 27(2), 312 - 315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907 - 914; Smith et al., J Pathol. 2014; 232:509 - 521, Lippa et al., Ann Neurol. March 1999; 45(3):353 - 7; Schmitz et al., Mol Neurobiol. Aug 22, 2018; Charles et al., Neurosci Lett. July 28, 2000; 289(1):29 - 32; Wilhelmsen et al., Arch Neurol. March 2004; 61(3):398 - 406; Yamaguchi et al., J Neuropathol Exp Neurol. 2004, 80th Annual Meeting, Vol. 63; Askanas et al., J Neuropathol Exp Neurol. July 2000; 59(7):592 - 8).

[0005] α-Synuclein is a natural unfolded protein of 140 amino acids (Iwai et al., Biochemistry 1995, 34(32), 10139-10145). The sequence of α-synuclein can be divided into three main domains: 1) an N-terminal region composed of residues 1-60, which contains an 11-mer amphipathic imperfect repeat residue with a highly conserved hexamer (KTKEGV). This region is involved in regulating the binding of α-synuclein to membranes and its internalization; 2) a hydrophobic non-amyloid-β component (NAC) domain spanning residues 61-95; it is essential for the protofibrillation of α-synuclein; and 3) a C-terminal region spanning residues 96-140, which is highly acidic and proline-rich and has no obvious structural propensity. Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The pathogenesis of PD remains elusive. However, increasing evidence suggests that the pathogenic folding of α-synuclein leads to the formation of amyloid-like protofibrils. In fact, the hallmark of PD is the intracellular presence of α-synuclein aggregate structures called Lewy bodies and neurites (mainly present in substantia nigra neurons) and the death of dopaminergic neurons in the substantia nigra and other sites. α-Synuclein is a natural unfolded presynaptic protein that can misfold and aggregate into larger oligomeric and protofibril forms, which are related to the pathogenesis of PD. Recent studies have implicated small soluble oligomeric and oligomeric protofibril forms of α-synuclein as the most neurotoxic species (Lashuel et al., J. Mol. Biol., 2002, 322, 1089-102).

[0006] In addition to Parkinson's disease, the accumulation of aggregated α-synuclein in Lewy bodies is a feature of all Lewy body diseases, including Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) (Capouch et al., NeurolTher. 2018, 7, 249-263). In DLB, Lewy bodies are widely distributed in the cortex of the brain, and in addition to Lewy bodies and neurites, more linear and punctate structures (Lewy neurites) immunoreactive for phosphorylated a-syn at Ser-129 are found (Outeiro et al., Mol Neurodegener. 2019, 14, 5).

[0007] α - synuclein aggregates have also been found in multiple system atrophy (MSA). MSA is a rare sporadic neurodegenerative disorder characterized by rapidly progressive autonomic and motor dysfunction, as well as variable cognitive decline. Such disorders include Shy - Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy. Depending on the predominant motor phenotype, the disease is further classified clinically as the parkinsonian (MSA - P) or cerebellar (MSA - C) variant (Fanciulli et al., N Engl J Med 2015; 372, 249 - 63). It is characterized by the aggregation of α - synuclein in the cytoplasm of oligodendrocytes, forming glial cytoplasmic inclusions (GCIs). GCIs are mainly composed of the fibrillar form of α - synuclein, which is the neuropathological hallmark of MSA and is widespread in the neocortex, hippocampus, brainstem, spinal cord, and dorsal root ganglia (Galvin et al., Arch Neurol. 2001, 58, 186 - 90). GCIs are regarded as core participants in the pathogenesis of MSA. A correlation has been reported between GCI load in the striatum and olivopontocerebellar regions and the degree of neuronal loss (Stefanova et al., Neuropathol Appl Neurobiol. 2016, 42, 20 - 32).

[0008] The ability to image α - synuclein deposition in the brain could be a major achievement in α - synucleinopathy research, including studies, diagnosis, and drug development for Parkinson's disease and MSA. The accumulation of aggregated α - synuclein in the brain is regarded as a key pathological hallmark of PD and MSA and may start many years before the onset of symptoms. Therefore, α - synuclein is a priority target for drug development not only because it may contribute to neurodegeneration but also because it offers the possibility of treating the disease while it is still asymptomatic or in the prodromal stage. In - vivo imaging of α - synuclein pathology can be used as a biomarker to (i) detect the presence of underlying disease in the early stage, (ii) assess disease progression, and (iii) serve as a pharmacodynamic tool for drug development. Currently, the development of α - synuclein PET imaging agents is regarded as crucial for the accurate diagnosis of synucleinopathies and for supporting the clinical development of targeted α - synuclein therapeutics, starting with the optimal selection of the trial population (Eberling, Dave and Frasier, J. Parkinson’s Disease, 3, 565 - 567 (2013)).

[0009] Only recently, non-invasive images of pathological α-synuclein (a-syn) in the human brain have been reported for the first time, and positive clinical proof-of-concept data have been provided for the a-syn positron emission tomography (PET) tracer ACI-12589 as an imaging agent for identifying patients with MSA (Capotosti F.; Discovery of [18F]ACI-12589, a novel and promising PET-tracer for alpha-synuclein; Oral presentation; ADPD2022 International Conference; Barcelona, Spain; March 18, 2022; Smith R.; Initial scans using [18F]ACI-12589, a novel PET-tracer for alpha-synuclein; Oral presentation; ADPD 2022 International Conference; Barcelona, Spain; March 18, 2022).

[0010] Therefore, there is clearly a need to find molecular probes with high α-synuclein selectivity that recognize and bind to pathological α-synuclein. To reduce background signal interference caused by non-specific off-target binding and to lower the dosing requirements, α-synuclein imaging compounds should bind to their target with high affinity and selectivity. For imaging of α-synuclein aggregates associated with neurological diseases such as multiple system atrophy (MSA), the imaging compound needs to penetrate the blood-brain barrier and enter the relevant regions of the brain. For targeting intracellular amyloid-like inclusions, such as α-synuclein, cell permeability is a further requirement for the imaging compound. To avoid unnecessary accumulation of the compound that may lead to an increased risk of unwanted side effects, a further prerequisite is the rapid elimination of the compound from the brain (or other target organs).

[0011] WO 2021 / 224489 discloses a new class of compounds of formula (I) capable of binding to α-synuclein. Thus, when the compounds are radiolabeled with a suitable radioisotope, in particular 18 F, these compounds are eligible as PET tracers for imaging pathological α-syn aggregates in PD and other α-synucleinopathies. The precursors of the PET tracers are purified by flash chromatography. This method has a low yield and is therefore not suitable for industrial-scale production.

[0012] Since 18F has a short half-life (about 110 minutes), and thus, radiolabeled compounds are typically provided to the user, such as radiopharmaceuticals, in the form of a precursor that reacts with a 18 F-fluorinating agent shortly before use. It has been observed that the precursor is unstable under normal storage conditions. Accordingly, it is an object of the present invention to provide a method for preparing a precursor compound having improved stability. Another object of the present invention is to provide a method for producing a small amount of impurities and suitable for producing a precursor for diagnostic applications on an industrial scale. Summary of the Invention

[0014] In a first aspect, the present invention relates to a method for purifying a compound of formula (IV-F) as defined herein

[0015]

[0016] or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the method comprises

[0017] (i) dissolving the crude compound of formula (IV-F) in DMSO and ethanol to obtain a solution; and

[0018] (ii) crystallizing from the solution to obtain the purified compound of formula (IV-F).

[0019] In a second aspect, the present invention relates to a compound obtainable by the method of the first aspect.

[0020] A third aspect of the present invention relates to a kit for preparing a radiopharmaceutical formulation, wherein the kit comprises a sealed vial containing at least one compound obtainable by the method of the first aspect.

[0021] A fourth aspect of the present invention relates to a method for preparing a detectable-labeled compound of formula (III-F) as defined herein

[0022]

[0023] or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof,

[0024] wherein the method comprises:

[0025] reacting a compound obtainable by the method of the first aspect with a 18 F-fluorinating agent such that LG is replaced by 18 F.

[0026] Definitions

[0027] For the purposes of interpreting this specification, unless otherwise specified, the following definitions shall apply, and in appropriate cases, terms used in the singular shall also include the plural and vice versa.

[0028] "Alkyl" refers to a saturated straight-chain or branched-chain organic moiety consisting of carbon and hydrogen atoms. An alkyl typically contains no unsaturation and is usually attached to the remainder of the molecule by a single bond. Examples of suitable alkyls have 1-6 carbon atoms, preferably 1-4 carbon atoms. The term "C 1 -C 4 alkyl" shall be interpreted accordingly. Examples of "C 1 -C 4 alkyl" include, but are not limited to, methyl, ethyl, propyl, isopropyl, 1-methylethyl, n-butyl, tert-butyl, and isobutyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, and isobutyl.

[0029] "C 1 -C 4 alkoxy" refers to a group of the formula -ORa, where Ra is a C 1 -C 4 alkyl as generally defined above. Examples of C 1 -C 4 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, and isobutoxy.

[0030] "Halogen C 1 -C 4 alkyl" or "halo C 1 -C 4 alkyl" refers to a C 1 -C 4 alkyl as defined above, which is substituted by one or more halogen groups as defined below. Examples of "halo C 1 -C 4 alkyl" include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl, and 1,4,4-trifluorobutan-2-yl.

[0031] "C 3 -C 6 cycloalkyl" is a stable monocyclic saturated hydrocarbon group consisting only of carbon and hydrogen atoms and having 3-6 carbon atoms. Examples of C 3 -C 6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0032] "Heterocyclic group" refers to a stable 4- to 6-membered non-aromatic monocyclic group containing 1 or 2 heteroatoms, such as selected from N, O or S. The heterocyclic group can be unsaturated or saturated. The heterocyclic group can be bonded through a carbon atom or a heteroatom. Examples include, but are not limited to, azetidinyl, oxetanyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl or perhydroazepinyl. Preferred examples of the heterocyclic group include, but are not limited to, azetidinyl, morpholinyl, piperazinyl, pyrrolidinyl or piperidinyl.

[0033] "Aryl" refers to a homoaromatic organic moiety composed of carbon and hydrogen atoms (e.g., containing 1 or 2 rings), which preferably has 5 - 12 carbon atoms, preferably 6 - 12 carbon atoms, more preferably 6 - 10 carbon atoms, more preferably 5 - 10 carbon atoms, and even more preferably 5 or 6 carbon atoms. Examples include, but are not limited to, phenyl, biphenyl and naphthyl.

[0034] "Heteroaryl" refers to an aryl as defined above, in which at least one carbon atom is replaced by a heteroatom, such as selected from N, O or S or a heteroatom-containing moiety (e.g., N, O and / or S). Generally, the heteroaryl is a 5 - 8 membered ring system, preferably a 5 - 6 membered ring system, in which at least one of the carbon atoms is replaced by a heteroatom, such as selected from N, O or S. Examples of possible heteroaryls include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl or pyridyl. Preferred examples thereof include pyridine, pyrazole, etc., and more preferably pyridine.

[0035] "Hal" or "halogen" or "halo" refers to F, Cl, Br and I. For diagnostic and pharmaceutical applications, F is particularly preferred (e.g., 19 F and 18 F).

[0036] Unless otherwise defined, the term "leaving group" (LG) as used herein is any leaving group and refers to an atom or group of atoms that can be replaced by another atom or group of atoms. Examples are given in the following literature, such as Synthesis (1982), pages 85 - 125, Table 2, Carey and Sundberg, Organische Synthese, (1995), pages 279 - 281, Table 5.8; or Netscher, Recent Res. Dev. Org. Chem., 2003, 7, 71 - 83, Schemes 1, 2, 10 and 15, etc.). (Coenen, Fluorine - 18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), Schubiger P.A., Friebe M., Lehmann L., (Eds.), PET - Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pages 15 - 50, specifically: page 25 Scheme 4, page 28 Scheme 5, page 30 Table 4, page 33 Figure 7). Preferably, the "leaving group" (LG) is selected from halogen, C 1-4 alkylsulfonates and C 6-10 arylsulfonates, where the C 6-10 aryl may optionally be substituted with -CH 3 or -NO 2 . In moiety R 3 , the leaving group LG is a C 1-4 alkylsulfonate.

[0037] Compounds of formula (III - F) and their precursors, such as compounds of formula (IV - F) having one or more chiral carbons, can exist as racemates and racemic mixtures, stereoisomers (including mixtures of diastereomers and individual diastereomers, mixtures of enantiomers and individual enantiomers, mixtures of conformational isomers and individual conformational isomers), tautomers, atropisomers and rotamers. All isomeric forms are included in the present invention. Compounds described in this specification containing an olefinic double bond include E and Z geometric isomers.

[0038] The present invention also includes all salt forms, polymorphs, hydrates and solvates (such as ethanolates).

[0039] "Pharmaceutically acceptable salts" are defined as derivatives of the disclosed compounds in which the parent compound is modified by preparing its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; base or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as those formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; and salts prepared from organic acids such as, but not limited to, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, etc. Pharmaceutically acceptable salts of the compounds of formula (III-F) and their precursors can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Organic solvents include, but are not limited to, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, PA, 1990, page 1445, the disclosure of which is incorporated herein by reference.

[0040] "Pharmaceutically acceptable" is defined as those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment, without producing excessive toxicity, irritation, allergic response or other problems or complications, and having a reasonable benefit / risk ratio.

[0041] The compounds of formula (III-F) can also be provided in the form of prodrugs, i.e., compounds that are metabolized in vivo to the active metabolite.

[0042] The patient or individual in the present invention is generally an animal, particularly a mammal, and more particularly a human.

[0043] α-Synuclein aggregates are assemblies of α-synuclein monomers that are rich in polymeric β-sheets and can form soluble oligomers or soluble / insoluble protofibrils or mature fibrils, which coalesce into intracellular deposits detected in Parkinson's disease, multiple system atrophy (MSA), and other synucleinopathies. α-Synuclein aggregates that constitute Lewy pathologies can be detected as having the following morphologies: Lewy bodies, Lewy neurites, preformed Lewy bodies or pale bodies, and perinuclear body deposits with diffuse, granular, punctate, or polymorphic patterns. In addition, α-synuclein aggregates are the major component of intracellular fibrillar inclusions detected in oligodendrocytes (also called glial cytoplasmic inclusions) and neuronal somata, axons, and nuclei (called neuronal cytoplasmic inclusions), which are histological markers of multiple system atrophy. α-Synuclein aggregates in Lewy pathologies and glial cytoplasmic inclusions typically exhibit a significant increase in post-translational modifications such as phosphorylation, ubiquitination, nitration, and truncation.

[0044] Lewy bodies are abnormal protein aggregates that develop within nerve cells in Parkinson's disease (PD), dementia with Lewy bodies, and other synucleinopathies. Lewy bodies are spherical in shape and replace other cellular components. Morphologically, Lewy bodies can be classified as brainstem type or cortical type. A typical brainstem Lewy body is an eosinophilic cytoplasmic inclusion composed of a dense core surrounded by 5- to 10-nm-wide radial fibrils, and its major structural component is α-synuclein; cortical Lewy bodies differ in that they lack a halo. The presence of Lewy bodies is a hallmark of Parkinson's disease.

[0045] Lewy neurites are abnormal neuronal processes in diseased neurons that contain granular material, abnormal α-synuclein (a-syn) filaments similar to those found in Lewy bodies, punctate varicosities, and axonal spheroids. Lewy neurites are characteristic of synucleinopathies such as dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy (MSA).

[0046] Glial cytoplasmic inclusions (GCI or Papp-Lantos bodies) are argyrophilic cytoplasmic aggregates in oligodendrocytes composed of filamentous α-synuclein. Morphologically, they are triangular, crescent-shaped, or sickle-shaped. In MSA, in addition to GCI, inclusions composed of α-synuclein filaments are detected in the cytoplasm or in neurons under the nuclear membrane and are called neuronal cytoplasmic inclusions and neuronal nuclear inclusions, respectively. GCI are considered a defining morphological feature of MSA; their widespread distribution is a criterion for establishing the neuropathological diagnosis of MSA at autopsy.

[0047] The terms "disease", "disorder", or "abnormality" are used interchangeably herein.

[0048] The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates can bind to α-synuclein aggregates. The type of bonding between the compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates has not been elucidated, and the present invention encompasses any type of bonding. The terms "compound that binds to α-synuclein aggregates", "(α-synuclein aggregate) / compound complex", "compound / α-synuclein aggregate complex", "compound / protein aggregate complex", etc. are used interchangeably herein and are not considered limited to any specific type of bonding.

[0049] Unless otherwise stated, the preferred definitions given in the "Definitions" section apply to all embodiments described below. Multiple embodiments of the present invention are described, and it should be recognized that the features specified in each embodiment can be combined with other specified features to obtain other embodiments of the present invention. Brief Description of the Drawings

[0051] Figure 1 Shows the purity profiles over time of the crude compound of Preparation Example 1 and the purified compound of Example 1. Detailed Description of the Invention

[0053] The present invention relates to a method for purifying a compound of formula (IV-F)

[0054]

[0055] or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates, wherein the method comprises:

[0056] (i) dissolving the crude compound of formula (IV-F) in dimethyl sulfoxide (DMSO) and ethanol to obtain a solution; and

[0057] (ii) crystallizing from the solution to obtain the purified compound of formula (IV-F).

[0058] The currently claimed method for recrystallizing the compound of formula (IV-F) is particularly suitable for industrial scale.

[0059] The compound of formula (IV-F) and its synthesis method are disclosed in WO 2021 / 224489.

[0060] R 3 is selected from Preferably, R 3 is selected from More preferably, R 3 is

[0061] R 3 The leaving group LG in is C 1-4 alkylsulfonate. Preferably, the leaving group (LG) is i.e., mesylate.

[0063] R 4 is aryl, or 5- or 6-membered heteroaryl, where R 4 is selected from

[0064]

[0065] wherein

[0066] R 2a , R 2a’ are independently selected from H or F;

[0067] R 2b is independently selected from F, -OH, C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, -NH 2 , -CN or C 1 -C 4 alkoxy;

[0068] R 2c , R 2c’ are independently selected from H, F, OH, OCH 3 or CH 3 ;

[0069] R 2d is selected from H, F or -OH;

[0070] R 2e is selected from H, OH, CH 3 or F;

[0071] Z is independently N, NH, N(C 1 -C 4 alkyl), N(halo C 1 -C 4 alkyl), O or S;

[0072] Z 1 is independently N, NH, O or S;

[0073] p is 0, 1 or 2;

[0074] m is 0 or 1;

[0075] Where valence allows, is a combination of single and double bonds; and

[0076] * is the bonding position.

[0077] Preferably R 4 selected from

[0078]

[0079] wherein R 2a 、R 2a’ 、R 2b 、R 2c 、R 2c’ 、R 2d 、R 2e and p are as defined above, and R z is selected from H, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl.

[0080] More preferably R 4 is selected from the following:

[0081]

[0082] wherein R 2a 、R 2a’ 、R 2b 、R 2c 、R 2c’ 、R 2d 、R 2e 、R z and p are as defined above. More preferably R 4 is selected from the following:

[0083]

[0084] wherein R 2a 、R 2a’ 、R 2b 、R 2c 、R 2c’ 、R 2e 、R z and p are as defined above.

[0085] Even more preferably R 4 is In a further preferred embodiment, R 4 is Even more preferably R 4 is The preferred compound is

[0086]

[0087] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0088] In one embodiment, the preferred compound of formula (I) is the following stereoisomers

[0089]

[0090] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0091] Even more preferred compounds are

[0092]

[0093] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0094] In one embodiment, the even more preferred compound of formula (I) is the following stereoisomers

[0095]

[0096] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0097] The crude compound of formula (IV-F) for use in the method of the present invention can be prepared by the methods exemplified in WO 2021 / 224489. In one embodiment, the crude compound of formula (IV-F) can be prepared as shown in the following scheme:

[0098] Scheme 1

[0099]

[0100] Pg is a protecting group and LG is a leaving group.

[0101] Commercially available hydrazine can be condensed with a suitable ketone to obtain the corresponding hydrazone. The crude hydrazone can be cyclized using DMF / DMA to obtain intermediate A. SNAr can be carried out with a suitable nucleophile in a suitable solvent to obtain intermediate D.

[0102] Scheme 1A

[0103]

[0104] An alternative method (Scheme 1A) involves deprotecting intermediate A and then carrying out an SNAr reaction with a suitable nucleophile, which is preferably carried out in DMSO in the presence of CsF. Intermediate D can be further functionalized preferably using copper(I) (Ullmann reaction) in the presence of a base and a solvent to obtain intermediate E. Finally, LG can be introduced into intermediate E to obtain the crude compound of formula (IV-F).

[0105] In the first reaction step, 2-LG1-5-hydrazinopyridine can be reacted with a suitably protected 2,4-dioxopyrrolidine (e.g., tert-butyl 2,4-dioxopyrrolidine-1-carboxylate). There is no particular limitation on the leaving group LG1, and any leaving group can be used, which can be replaced in the subsequent SNAr reaction. Examples thereof include Hal (e.g., Cl, Br, I, F). For example, this reaction can be carried out at an elevated temperature in a suitable solvent such as C 1-4 alcohol (preferably ethanol). If appropriate, for example, DMF-DMA can be used to close the ring of the obtained hydrazone to obtain intermediate A.

[0106] The protecting group Pg can be removed using suitable conditions, depending on the protecting group selected. Acid cleavage can be given as an example.

[0107] As the desired group R 3 The group R* as a precursor can be introduced by an SNAr reaction (nucleophilic aromatic substitution reaction) to obtain intermediate D. Such reactions are well known in chemistry and can be carried out under any suitable conditions. If desired, a suitable catalyst such as cesium fluoride can be used.

[0108] R* can be selected from or the corresponding moiety, wherein OH is protected by a protecting group.

[0109] For example, the group R 4 can be introduced by coupling intermediate D with R 4 -Hal or R 4 -trifluoromethanesulfonate to obtain compound E. The coupling reagents include compounds containing Cu, Pd, and Ni, such as CuI.

[0110] In the final step, the crude (IV-F) compound can be prepared by replacing the OH group of intermediate E with a C 1-4 alkylsulfonate leaving group LG. For example, this reaction can be carried out by reacting intermediate E with C 1-4 alkylsulfonate-Hal (where Hal is, for example, Cl, such as methanesulfonyl chloride).

[0111] The above reaction scheme is advantageous because it allows the compound of formula (IV-F) to be prepared in gram amounts rather than milligram amounts. In addition, the crude (IV-F) compound obtained by this method generally has an initial purity of more than 90%. Therefore, it is an effective raw material for a GMP-grade purification process.

[0112] Scheme 1B

[0113]

[0114] Another general method is described in Scheme 1B, which follows the same preferred conditions as those described in General Scheme 1 or 1A.

[0115] In Scheme 1B, Lg is a leaving group and Pg is a protecting group. is R 1 is R 3 R 2 is R 4 and R 0 is H.

[0116] Optionally, 18 the F-precursor can be obtained by treating Intermediate A with hydroxypyrrolidine in a suitable solvent under heating. The R 4 group can be introduced by palladium-catalyzed amidation or Ullmann reaction. Finally, the alcohol intermediate E can be modified to a leaving group using standard conditions to obtain the crude (IV-F) compound.

[0117] Step (i)

[0118] In Step (i), the crude (IV-F) compound is dissolved in a solvent comprising DMSO and ethanol (preferably consisting of the same). There is no particular limitation on the ratio of DMSO to ethanol (v:v). The ratio of DMSO to ethanol generally ranges from about 5:1 to about 0.1:1, preferably from about 4:1 to about 0.5:1, more preferably from about 3:1 to about 0.5:1, even more preferably from about 2:1 to about 0.75:1, and most preferably about 1:1. In this case, the ratio (v:v) refers to the ratio of the unit volume of DMSO to the unit volume of ethanol, for example, in mL:mL.

[0119] There is no particular limitation on the ratio of the solvent to the crude (IV-F) compound, as long as the amount of the solvent is sufficient to dissolve the crude (IV-F) compound. If the amount of the solvent is very large, the cost will increase and crystallization will take a long time. Therefore, the ratio (v:w) of the solvent to the crude (IV-F) compound preferably ranges from about 5:1 to about 500:1, more preferably from about 25:1 to about 300:1, and even more preferably from about 40:1 to about 250:1. It should be understood that this ratio can vary depending on conditions, such as the conditions employed during the dissolution step, for example, the temperature selected. In this case, the ratio (v:w) refers to the ratio of the unit volume of the total solvent to the unit weight of the crude (IV-F) compound, for example, in mL:g.

[0120] The crude (IV-F) compound can be dissolved in the solvent in any suitable manner.

[0121] In one embodiment, ethanol and DMSO can be mixed, and then the crude compound of formula (IV-F) can be added. In another embodiment, the crude compound of formula (IV-F) can be mixed with ethanol, and then DMSO can be added. In another preferred embodiment, the crude compound of formula (IV-F) can be mixed with DMSO, and then ethanol can be added.

[0122] If desired, dissolution can be facilitated by using an elevated temperature, such as in the temperature range of about 50 °C to about 90 °C, preferably about 60 °C to about 90 °C, more preferably about 65 °C to about 85 °C. These temperatures are given for ambient pressure (about 1 atm). If elevated or reduced pressure is applied, the temperature range can be adjusted accordingly.

[0123] During the dissolution step (i), a major amount of the crude compound of formula (IV-F) should be dissolved. Generally, at least about 50 wt.%, preferably at least about 60 wt.%, even more preferably at least about 70 wt.%, further preferably at least about 80 wt.%, even further preferably at least about 90 wt.%, and most preferably 100% is dissolved.

[0124] During the dissolution step (i), conventional methods such as stirring can be employed to enhance or accelerate dissolution.

[0125] Step (ii)

[0126] After the dissolution step (i), the compound of formula (IV-F) is crystallized from the solution to obtain the purified compound of formula (IV-F).

[0127] Crystallization can be carried out by various methods, such as cooling the solution, removing the solvent, etc. For example, the solvent can be removed by reducing the pressure or heating the solution to cause the solvent to evaporate.

[0128] In a preferred embodiment, the solution is cooled to crystallize the compound of formula (IV-F). The temperature can be appropriately selected and, for example, the range can be from about 0 °C to about 50 °C, preferably from about 0 °C to about 40 °C, more preferably from about 0 °C to about 30 °C. If desired, the temperature can be varied within this range during the crystallization step. For example, crystallization can initially be carried out at about 10 °C to about 50 °C (preferably from about 0 °C to about 40 °C, more preferably from about 0 °C to about 30 °C), and then the temperature can be lowered to about 0 °C to about 10 °C to improve the yield.

[0129] Optional step (iii)

[0130] Finally, at least part (usually all) of the crystallization solution can be removed in order to obtain the purified compound of formula (IV-F). Filtration, centrifugation, etc. can be used.

[0131] If desired, the obtained crystals can be washed, for example, with a solvent such as ethanol and dried in step (iii).

[0132] Additional steps

[0133] Steps (i), (ii) and optional step (iii) can be carried out one or more times (e.g., at least 1 time, preferably at least 2 times, more preferably at least 3 times, even more preferably at least 4 times, further preferably at least 5 times) by using the purified compound of formula (IV-F) as the crude compound of formula (IV-F) in step (i) in order to further increase the purity to the desired level.

[0134] In a preferred embodiment, steps (i), (ii) and optional step (iii) are carried out at least twice, preferably at least three times.

[0135] In another preferred embodiment, steps (i), (ii) and optional step (iii) are carried out at least four times. In a further preferred embodiment, steps (i), (ii) and optional step (iii) are carried out at least five times.

[0136] In one embodiment, steps (i), (ii) and optional step (iii) are carried out twice. In another embodiment, steps (i), (ii) and optional step (iii) are carried out three times. In a preferred embodiment, steps (i), (ii) and optional step (iii) are carried out four times. In another preferred embodiment, steps (i), (ii) and optional step (iii) are carried out five times.

[0137] In another embodiment, steps (i), (ii) and optional step (iii) can be carried out at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times.

[0138] There is no particular limitation on the maximum number of repetitions. The method of the present invention can be repeated as many times as desired until the required purity is reached. However, since the total yield decreases when the method of the present invention is repeated, generally steps (i), (ii) and optional step (iii) are carried out at most 30 times, preferably at most 25 times, more preferably at most 20 times, even more preferably at most 15 times.

[0139] Optional purification steps

[0140] The method of the present invention can be combined with other conventional purification steps in the art as desired. Examples of further purification steps include recrystallization using other solvent systems or chromatography.

[0141] In one embodiment, a solvent system using DMSO and 2-butanone or ethyl acetate can be applied to dissolve the compound of formula (IV-F). The compound of formula (IV-F) can then be crystallized, and then at least part of the solution can be removed, followed by optional drying and / or washing.

[0142] Purification using DMSO and 2-butanone or ethyl acetate can be carried out at any point in time, but is generally carried out before or after the method of the present invention, more commonly after the method of the present invention. It can be carried out after the full sequence of steps (i), (ii) and optional step (iii), including any optional repetitions thereof that have been carried out, or can be carried out after one or more sequences of steps (i), (ii) and optional step (iii), but before any repetitions of steps (i), (ii) and optional step (iii) are carried out. In a preferred embodiment, purification using DMSO and 2-butanone or ethyl acetate is carried out after completion of the full sequence of steps (i), (ii) and optional step (iii), including any optional repetitions thereof (e.g., after steps (i), (ii) and optional step (iii) have been carried out at least 2 times, preferably at least 3 times), because this sequence of steps improves purity compared to purification using DMSO and 2-butanone or ethyl acetate at an earlier stage.

[0143] There is no particular limitation on the ratio of DMSO to 2-butanone (v:v). The ratio of DMSO to 2-butanone generally ranges from about 5:1 to about 0.1:1, preferably from about 4:1 to about 0.5:1, more preferably from about 3:1 to about 0.5:1, even more preferably from about 2:1 to about 0.75:1, and most preferably about 1:1. In this case, the ratio (v:v) refers to the ratio of the unit volume of DMSO to the unit volume of 2-butanone, for example, in mL:mL.

[0144] There is no particular limitation on the ratio of DMSO to ethyl acetate (v:v). The ratio of DMSO to ethyl acetate generally ranges from about 5:1 to about 0.1:1, preferably from about 4:1 to about 0.5:1, more preferably from about 3:1 to about 0.5:1, even more preferably from about 2:1 to about 0.75:1, and most preferably about 1:1. In this case, the ratio (v:v) refers to the ratio of the unit volume of DMSO to the unit volume of ethyl acetate, for example, in mL:mL.

[0145] There is no particular limitation on the ratio of the solvent to the crude formula (IV-F) compound, as long as the amount of the solvent is sufficient to dissolve the crude formula (IV-F) compound. If the amount of the solvent is very large, the cost will increase and the crystallization will take a long time. Therefore, the ratio (v:w) of the solvent to the crude formula (IV-F) compound is preferably in the range of about 5:1 - about 500:1, more preferably about 25:1 - about 300:1, and even more preferably about 40:1 - about 250:1. It should be understood that this ratio can vary depending on conditions, such as the conditions employed during the dissolution step, for example, the temperature selected. In this case, the ratio (v:w) refers to the ratio of the unit volume of the total solvent to the unit weight of the crude formula (IV-F) compound, for example, in mL:g.

[0146] The crude formula (IV-F) compound can be dissolved in the solvent in any suitable manner.

[0147] In one embodiment, 2-butanone or ethyl acetate and DMSO can be mixed, and then the crude formula (IV-F) compound can be added. In another embodiment, the crude formula (IV-F) compound can be suspended in 2-butanone or ethyl acetate, and then DMSO can be added. In a further preferred embodiment, the crude formula (IV-F) compound can be suspended in DMSO, and then 2-butanone or ethyl acetate can be added.

[0148] If desired, dissolution can be facilitated by using an elevated temperature, for example, in the temperature range of about 50°C - about 90°C, preferably about 60°C - about 90°C, and more preferably about 65°C - about 85°C. These temperatures are given for ambient pressure (about 1 atm). If elevated or reduced pressure is applied, the temperature range can be adjusted accordingly.

[0149] During the dissolution step (i), a major amount of the crude formula (IV-F) compound should be dissolved. Generally, at least about 50 wt.%, preferably at least about 60 wt.%, even more preferably at least about 70 wt.%, further preferably at least about 80 wt.%, even further preferably at least about 90 wt.%, and most preferably 100% is dissolved.

[0150] During the dissolution step, conventional methods such as stirring can be employed to enhance or accelerate dissolution.

[0151] After the dissolution step, the formula (IV-F) compound is crystallized from the solution to obtain the purified formula (IV-F) compound.

[0152] Crystallization can be carried out by various methods, such as cooling the solution, removing the solvent, etc. For example, the solvent can be removed by reducing the pressure or heating the solution to cause the solvent to evaporate.

[0153] In a preferred embodiment, the cooling solution causes crystallization of the purified compound of formula (IV-F). The temperature can be appropriately selected and, for example, can range from about 0 °C to about 50 °C, preferably from about 0 °C to about 40 °C, more preferably from about 0 °C to about 30 °C. For example, the crystallization can initially be carried out at about 10 °C to about 50 °C (preferably from about 0 °C to about 40 °C, more preferably from about 0 °C to about 30 °C), and then the temperature can be lowered to about 0 °C to about 10 °C to improve the yield.

[0154] Generally, the purified compound of formula (IV-F) is separated from the crystallization solution. Filtration, centrifugation, etc. can be used.

[0155] If desired, the obtained crystals can be washed, for example, with a solvent such as 2-butanone or ethyl acetate. If necessary, the crystals can also be dried.

[0156] The desired final purity depends on the intended use. For diagnostic applications, a purity of at least 97% (Ph.Eur. 2902 “Chemical Precursors for Radiopharmaceutical Preparations”) is usually required. The conditions during step (i), step (ii) and optional step (iii) can be the same or different during repetition.

[0157] Surprisingly, it has been found that the purified compound of formula (IV-F) prepared by the method of the present invention not only has a higher purity than the crude compound of formula (IV-F), but also has improved stability. For example, the purified compound of formula (IV-F) after three recrystallizations is stable under accelerated storage conditions (40 °C for 4 weeks). In contrast, the crude compound of formula (IV-F) degraded by 15.2 wt.% under these conditions.

[0158] Using those in which LG is C 1-4 The advantages of the present invention were observed with compounds that are alkyl sulfonates, especially mesylates.

[0159] Method for synthesizing a compound with a detectable label

[0160] The compounds obtainable by the method of the present invention are suitable as precursors for compounds with a detectable label, which can be used in imaging applications.

[0161] In one embodiment, the present invention relates to a method for preparing a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, by radioactively labeling a compound of formula (IV-F) with a radioisotope 18 F.

[0162] Can be used for 18The reagents, solvents and conditions for F-fluorination are well known to those skilled in the art (L. Cai, S. Lu, V. Pike, Eur. J. Org. Chem 2008, 2853-2873; J. Fluorine Chem., 27 (1985): 177-191; Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), Schubiger PA, Friebe M., Lehmann L. (eds.), PET-Chemistry-The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50). Preferably, the 18 The solvent for F-fluorination is DMF, DMSO, acetonitrile, DMA or a mixture thereof, preferably the solvent is acetonitrile or DMSO.

[0163] You can use any suitable 18 F-fluorinating agent. Typical examples include H 18 F. Alkali metal or alkaline earth metal 18 F-fluoride (e.g., K 18 F、Rb 18 F, Cs 18 F and Na 18 F). Optionally, 18 The F-fluorinating agent can be combined with a chelating agent such as a cryptand (e.g., 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane- ) or crown ether (e.g. 18-crown-6). 18 The F-fluorinating agent can be 18 Tetraalkylammonium salt of F or 18 A tetraalkylphosphonium salt of F; for example, 18 F's Four (C 1-6 Alkyl) ammonium salt or 18 F's Four (C 1-6 Preferably, 18 F-fluorinating agent is K 18 F, H 18 F、Cs 18 F. Na 18 F. 18 F's Four (C 1-6 Alkyl) ammonium salt, kryptofix

[222] 18 F or [ 18 F] Tetrabutylammonium fluoride.

[0164] Diagnostic composition

[0165] The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are particularly suitable for imaging α-synuclein aggregates. With respect to α-synuclein, these compounds are particularly suitable for binding to multiple types of α-synuclein aggregates. Imaging can be performed in mammals, preferably in humans. The imaging is preferably in vitro imaging, ex vivo imaging or in vivo imaging. More preferably, the imaging is in vivo imaging: even more preferably, the imaging is preferably brain imaging. The imaging can also be eye / retinal imaging. The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are particularly suitable for diagnosis.

[0166] Diagnosis can be carried out on mammals, preferably on humans. The tissue of interest for diagnosis can be the brain, central nervous system tissue, eye tissue (such as retinal tissue) or other tissues, peripheral organs such as but not limited to the intestine or body fluids such as cerebrospinal fluid (CSF). The tissue is preferably brain tissue.

[0167] The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are suitable for the diagnosis of diseases, disorders and abnormalities associated with α-synuclein aggregates due to their design and binding properties. The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are particularly suitable for positron emission tomography of α-synuclein aggregates. Diseases involving α-synuclein aggregates are generally classified as synucleinopathies (or α-synucleinopathies). The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are suitable for the diagnosis of diseases, disorders or abnormalities including but not limited to Parkinson's disease (sporadic, familial α-synuclein mutations, familial non-α-synuclein mutations, pure autonomic failure or Lewy body dysphagia), SNCA repeat carriers, dementia with Lewy bodies ("pure" Lewy body dementia), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Alzheimer's disease Lewy body variant and normal aging in Down syndrome). Synucleinopathies with α-synuclein neuronal and glial aggregates include multiple system atrophy (MSA) (Shy-Drager syndrome, striatonigral degeneration or olivopontocerebellar atrophy).Other diseases that may have α-synuclein-immunoreactive lesions include traumatic brain injury, chronic traumatic encephalopathy, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, and Niemann-Pick disease type C1), motor neuron diseases, amyotrophic lateral sclerosis (Guam sporadic, familial, and ALS-dementia complex), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, ataxia-telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gaucher disease, and other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), and rapid eye movement (REM) sleep behavior disorder (Jellinger, Mov Disord 2003, 18 Suppl 6, S2-12; Galvin et al., JAMA Neurology 2001, 58(2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol. 2004, 63(4), 323-328; McKee et al., Brain, 2013, 136(Pt 1), 43-64; Puschmann et al. Parkinsonism Relat Disord 2012, 18S1, S24-S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240-4246; Winder-Rhodes et al., Mov Disord. 2012, 27(2), 312-315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907-914). Preferably, the compound of formula (III-F) is suitable for diagnosing Parkinson's disease, multiple system atrophy, dementia with Lewy bodies, Parkinson's disease dementia, SNCA repeat carriers, or Alzheimer's disease, more preferably multiple system atrophy (MSA).

[0168] In a method of diagnosing a disease, disorder, or abnormality associated with α-synuclein aggregates, such as multiple system atrophy (MSA), or a predisposition thereto in an individual, the method comprises the following steps:

[0169] a) Administering to the individual a diagnostically effective amount of a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof;

[0170] b) Permit a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof to distribute into tissues of interest (such as the brain, central nervous system (CNS) tissues, eye tissues or other tissues, peripheral organs such as but not limited to the intestine or body fluids such as cerebrospinal fluid (CSF)); and

[0171] c) Image the tissues of interest, wherein an increased binding of the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof to the tissues of interest as compared to the binding level in a normal control indicates that the individual has a disease, disorder or abnormality associated with α-synuclein aggregates or is at risk of developing a disease, disorder or abnormality associated with α-synuclein aggregates.

[0172] The compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof can be used for imaging α-synuclein aggregates in any sample or a specific body part or body region of a patient suspected of containing α-synuclein aggregates. The compound is capable of crossing the blood-brain barrier. Thus, they are particularly suitable for imaging α-synuclein aggregates in the brain, central nervous system (CNS) tissues or eye tissues or peripheral organs such as but not limited to the intestine and body fluids such as cerebrospinal fluid (CSF).

[0173] In a diagnostic application, it is preferred to administer the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof in the form of a diagnostic composition, which comprises the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof. A "diagnostic composition" is defined in the present invention as a composition comprising one or more of the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, in a form suitable for administration to a patient, such as a mammal, such as a human, and suitable for diagnosing the specific disease, disorder or abnormality. Preferably, the diagnostic composition further comprises a physiologically acceptable excipient, carrier, diluent or adjuvant. Administration is preferably carried out as defined below. More preferably, it is carried out by injecting the composition as an aqueous solution. Such a composition may optionally contain additional ingredients, such as buffers; pharmaceutically acceptable solubilizers (such as cyclodextrins or surfactants such as Pluronics, Tweens or phospholipids); and pharmaceutically acceptable stabilizers or antioxidants (such as ascorbic acid, gentisic acid or p-aminobenzoic acid). The dose of the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof will vary depending on the exact compound administered, the patient weight and other variables that will be apparent to those skilled in the art.

[0174] The compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates can be administered alone, but are preferably formulated into a diagnostic composition in accordance with standard pharmaceutical practice. Accordingly, the present invention also provides a diagnostic composition comprising a diagnostically effective amount of the compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates admixed with at least one optional pharmaceutically acceptable excipient, carrier, diluent or adjuvant.

[0175] Pharmaceutically acceptable excipients are well known in the pharmaceutical art and are described, for example, in Remington’s Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., New Jersey (1975). The pharmaceutical excipients can be selected according to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable, i.e., harmless to its recipient.

[0176] Pharmaceutically useful excipients, carriers, adjuvants and diluents that can be used to formulate the diagnostic composition of the present invention can include, for example, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as diols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate, binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, colorants, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

[0177] There is no restriction on the route of administration (delivery) of the compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates, but administration by injection is preferred, and intravenous injection is more preferred.

[0178] Preferably, in diagnostic applications, a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof is administered parenterally. If a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof is administered parenterally, examples of such administration include one or more of the following: intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular or subcutaneous administration of the compound; and / or by using infusion techniques. For parenteral administration, these compounds are preferably used in the form of a sterile aqueous solution, which may contain other substances such as sufficient salts or glucose to render the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3-9). Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0179] Generally, a clinician will determine the actual dose most suitable for an individual. The specific dose level and dosing frequency for any particular individual may vary and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, age, body weight, general health status, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder, and the individual undergoing the diagnosis.

[0180] The diagnostic compositions of the present invention can be prepared in a manner well known per se to those skilled in the art, for example as described in Remington’s Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., New Jersey (1975).

[0181] A compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof can be used as an in vitro analysis reference or an in vitro screening tool. They can also be used in in vivo diagnostic methods.

[0182] A compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof can also be provided in a mixture that contains a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof and at least one imaging agent, pharmaceutically acceptable excipient, carrier, diluent or adjuvant selected from those different from the compound of formula (III-F). The imaging agent different from the compound of formula (III-F) is preferably present in a diagnostically effective amount. More preferably, the imaging agent different from the compound of formula (III-F) is an amyloid-β or Tau imaging agent.

[0183] By detecting the specific binding of a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof to α-synuclein aggregates in a sample or a specific body part or body region, a disease, disorder or abnormality associated with α-synuclein aggregates in a patient can be diagnosed, or the susceptibility to a disease, disorder and abnormality associated with α-synuclein aggregates in a patient can be diagnosed, including the following steps:

[0184] (a) Contacting a sample or a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, which binds to α-synuclein aggregates,

[0185] (b) Allowing the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof to bind to α-synuclein aggregates to form a compound / (α-synuclein aggregate) complex (hereinafter the "compound / (α-synuclein aggregate) complex", abbreviated as "compound / protein aggregate complex"),

[0186] (c) Detecting the formation of the compound / protein aggregate complex,

[0187] (d) Optionally correlating the presence or absence of the compound / protein aggregate complex with the presence or absence of α-synuclein aggregates in the sample or a specific body part or region, and

[0188] (e) Optionally comparing the amount of the compound / protein aggregate complex with a normal control value, wherein an increase in the amount of the compound / protein aggregate complex compared to the normal control value may indicate that the patient has a disease, disorder or abnormality associated with α-synuclein aggregates or is at risk of developing a disease, disorder or abnormality associated with α-synuclein aggregates.

[0189] The compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof can be contacted with a sample or a body part or body region suspected of containing α-synuclein aggregates by a suitable method. In an in vitro method, the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof and a liquid sample can be simply mixed. In an in vivo test, the compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof is usually administered to a patient by any suitable means, such as by parenteral administration, preferably by intravenous injection.

[0190] After contact of a sample or a specific body part or body region with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, the compound is allowed to bind to α-synuclein aggregates. The time period required for binding depends on the type of test (e.g., in vitro or in vivo) and can be determined by a person skilled in the art by routine experimentation.

[0191] Subsequently, the compound that has bound to the α-synuclein aggregates can be detected by any suitable method. The specific method chosen depends on the detection label that has been selected. Examples of possible methods include, but are not limited to, fluorescence imaging techniques or nuclear imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) and contrast enhanced magnetic resonance imaging (MRI). Fluorescence imaging techniques and / or nuclear imaging techniques can be used to monitor and / or visualize the distribution of the detectable labeled compound within the sample or specific body part or body region.

[0192] Then, the presence or absence of the compound / protein aggregate complex is optionally correlated with the presence or absence of α-synuclein aggregates in the sample or specific body part or region. Finally, the amount of the compound / protein aggregate complex can be compared to a normal control value determined in a sample or specific body part or body region of a healthy individual, wherein an increase in the amount of the compound / protein aggregate complex compared to the normal control value can indicate that the patient has a disease, disorder or abnormality associated with α-synuclein aggregation or is at risk of developing a disease, disorder or abnormality associated with α-synuclein aggregation.

[0193] The present invention also relates to a method for determining the amount of α-synuclein aggregates in a tissue and / or a body fluid. The method comprises the following steps:

[0194] (a) providing a sample representative of the tissue and / or body fluid under investigation;

[0195] (b) testing the sample for the presence of α-synuclein aggregates using a compound of formula (III-F);

[0196] (c) determining the amount of the compound that binds to the α-synuclein aggregates; and

[0197] (d) calculating the amount of α-synuclein aggregates in the tissue and / or body fluid.

[0198] The presence of α-synuclein aggregates in a test sample can be assayed using a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, by contacting the sample with the compound of formula (III-F), allowing the compound of formula (III-F) to bind to the α-synuclein aggregates to form a compound / protein aggregate complex, and detecting the formation of the compound / protein aggregate complex as described above.

[0199] Monitoring for minimal residual disease, disorder or abnormality in a patient suffering from a disease, disorder or abnormality associated with α-synuclein aggregates and being treated with a medicament having a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof can be carried out by the following steps:

[0200] (a) contacting a sample or a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0201] (b) allowing the compound to bind to the α-synuclein aggregates to form a compound / protein aggregate complex;

[0202] (c) detecting the formation of the compound / protein aggregate complex;

[0203] (d) optionally correlating the presence or absence of the compound / protein aggregate complex with the presence or absence of α-synuclein aggregates in the sample or the specific body part or body region; and

[0204] (e) optionally comparing the amount of the compound / protein aggregate complex with a normal control value, wherein an increase in the amount of aggregates as compared to the normal control value may indicate that the patient may still be suffering from minimal residual disease, disorder or abnormality.

[0205] How to carry out steps (a)-(e) has been explained above.

[0206] In a method for monitoring minimal residual disease, disorder or abnormality, the method may further comprise steps (i)-(vi) prior to step (a):

[0207] (i) contacting a sample or a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, which specifically binds to α-synuclein aggregates;

[0208] (ii) Allow the compound to bind to α-synuclein aggregates to form a compound / (α-synuclein aggregate) complex;

[0209] (iii) Detect the formation of the compound / (α-synuclein aggregate) complex;

[0210] (iv) Correlate the presence or absence of the compound / (α-synuclein aggregate) complex with the presence or absence of α-synuclein aggregates in a sample or a specific body part or body region;

[0211] (v) Optionally, compare the amount of the compound / (α-synuclein aggregate) complex with a normal control value; and

[0212] (vi) Treat a patient with the drug.

[0213] Optionally, after step (d) or step (e), the method further comprises step (A):

[0214] (A) Compare the amount of the compound / (α-synuclein aggregate) complex determined in step (iv) with the amount of the compound / (α-synuclein aggregate) complex determined in step (d).

[0215] To monitor minimal residual disease, disorder or abnormality over time, steps (a)-(c) of the method for monitoring minimal residual disease, disorder or abnormality and optionally steps (d) and (e) can be repeated one or more times.

[0216] In the method for monitoring minimal residual disease, condition or abnormality, optionally at multiple time points during treatment, such as before and after the start of treatment or at multiple time points after the start of treatment, the amounts of the compound / protein aggregate complex can be compared. A change, particularly a decrease, in the amount of the compound / protein aggregate complex can indicate that the residual disease, disorder or abnormality is decreasing.

[0217] The response of a patient suffering from a disease, disorder or abnormality associated with α-synuclein aggregates and receiving drug treatment can be predicted by the following steps:

[0218] (a) Contact a sample or a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0219] (b) Allow the compound to bind to α-synuclein aggregates to form a compound / protein aggregate complex;

[0220] (c) Detect the formation of the compound / protein aggregate complex;

[0221] (d) Optionally, correlate the presence or absence of the compound / protein aggregate complex with the presence or absence of α-synuclein aggregates in a sample or a specific body part or body region; and

[0222] (e) Optionally, compare the amount of the compound / protein aggregate complex with a normal control value.

[0223] How to perform steps (a)-(e) has been explained above.

[0224] In a method for predicting a response, the method further includes steps (i)-(vi) before step (a):

[0225] (i) Contact a sample or a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, which specifically binds to α-synuclein aggregates;

[0226] (ii) Allow the compound to bind to α-synuclein aggregates to form a compound / (α-synuclein aggregate) complex;

[0227] (iii) Detect the formation of the compound / (α-synuclein aggregate) complex;

[0228] (iv) Correlate the presence or absence of the compound / (α-synuclein aggregate) complex with the presence or absence of α-synuclein aggregates in a sample or a specific body part or body region;

[0229] (v) Optionally, compare the amount of the compound / (α-synuclein aggregate) complex with a normal control value; and

[0230] (vi) Treat the patient with the drug.

[0231] Optionally, the method further includes step (A) after step (d) or step (e):

[0232] (A) Compare the amount of the compound / (α-synuclein aggregate) complex determined in step (iv) with the amount of the compound / (α-synuclein aggregate) complex determined in step (d).

[0233] To determine the response over time, steps (a)-(c) and optionally steps (d) and (e) of the method for predicting a response can be repeated one or more times.

[0234] In a method for predicting a response, it is optionally possible to compare the amount of the compound / protein aggregate complex at a plurality of time points during treatment, for example, before and after the start of treatment, or at a plurality of time points after the start of treatment. A change, in particular a decrease, in the amount of the compound / protein aggregate complex can indicate that the patient has a high response potential to the corresponding treatment.

[0235] Optionally, the diagnostic composition can be used before, during, and after a surgical procedure (such as deep brain stimulation (DBS)) and non-invasive brain stimulation (such as repetitive transcranial magnetic stimulation (rTMS)) for visualizing α-synuclein aggregates before, during, and after such procedures. Surgical techniques including DBS improve the advanced symptoms of PD and are superior to the best available drug therapies currently in use. During the past 20 years, rTMS has been closely examined as a possible treatment for PD (Ying-hui Chou et al., JAMA Neurol. April 1, 2015; 72(4):432-440).

[0236] In a further embodiment of the invention, the diagnostic composition can be used in a method for collecting data to monitor the residual disease, disorder, or abnormality in a patient suffering from a disease, disorder, or abnormality associated with α-synuclein aggregates. It should be understood that the term "monitoring minimal residual disease" as used herein relates to monitoring the evolution of the disease. For example, monitoring the evolution of the disease, disorder, or abnormality in a patient suffering from a disease, disorder, or abnormality associated with α-synuclein aggregates.

[0237] It is also possible to incorporate a compound of formula (III-F) or a compound of formula (IV-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof into a test kit for detecting protein aggregates of α-synuclein. The test kit generally comprises a container containing one or more compounds of formula (III-F) or one or more compounds of formula (IV-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof, and instructions for the purpose of using the compound to bind to α-synuclein aggregates to form a compound / protein aggregate complex and detecting the formation of the compound / protein aggregate complex, such that the presence or absence of the compound / protein aggregate complex correlates with the presence or absence of α-synuclein aggregates.

[0238] The term "test kit" generally refers to any diagnostic kit known in the art. More particularly, the latter term refers to a diagnostic kit as described by Zrein et al., Clin. Diagn. Lab. Immunol., 1998, 5, 45-49.

[0239] The dosage of the compound of formula (III-F) with a detectable label, or its racemic mixture, pharmaceutically acceptable salt, hydrate or solvate, varies depending on the exact compound to be administered, the weight of the patient, the size and type of the sample, and other variables that will be apparent to those skilled in the art. Generally, the dosage can preferably range from 0.001 μg / kg to 10 μg / kg, more preferably from 0.01 μg / kg to 1.0 μg / kg. The radioactive dosage can be, for example, 100 - 600 MBq, more preferably 150 - 450 MBq.

[0240] In another embodiment, the present invention provides a method for imaging a disease, disorder or abnormality associated with α-synuclein aggregates in a sample or a specific body part or body region, particularly the brain or a sample collected from a patient's brain, the method comprising the following steps:

[0241] (a) contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or its stereoisomers, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate;

[0242] (b) allowing the compound to bind to the α-synuclein aggregates; and

[0243] (c) imaging the sample, the specific body part or body region with an imaging system.

[0244] In another embodiment, the present invention provides a method for determining the amount of α-synuclein aggregates in a sample or a specific body part or body region, the method comprising the following steps:

[0245] (a) contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or its stereoisomers, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate;

[0246] (b) allowing the compound to bind to the α-synuclein aggregates;

[0247] (c) detecting the compound that binds to the α-synuclein aggregates;

[0248] (d) determining the amount of the compound that binds to the α-synuclein aggregates; and

[0249] (e) optionally calculating the amount of α-synuclein aggregates in the sample, the specific body part or body region in contact.

[0250] In another embodiment, the present invention provides a method for imaging a disease, disorder or abnormality associated with α-synuclein aggregates, the method comprising the following steps:

[0251] (a) Contact a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a Compound (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0252] (b) Allow the compound to bind to the α-synuclein aggregates;

[0253] (c) Detect the compound that binds to the α-synuclein aggregates; and

[0254] (d) Correlate the presence or absence of the compound that binds to the α-synuclein aggregates with a disease, disorder or abnormality associated with α-synuclein aggregates.

[0255] In another embodiment, the present invention provides a method for collecting data for diagnosing a disease, disorder or abnormality associated with α-synuclein aggregates, the method comprising the following steps:

[0256] (a) Contact a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a Compound (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0257] (b) Allow the compound to bind to the α-synuclein aggregates;

[0258] (c) Detect the compound that binds to the α-synuclein aggregates; and

[0259] (d) Optionally, correlate the presence or absence of the compound that binds to the α-synuclein aggregates with the presence or absence of α-synuclein aggregates in the sample or the specific body part or body region.

[0260] In another embodiment, the present invention provides a method for collecting data for determining the propensity for a disease, disorder or abnormality associated with α-synuclein aggregates, the method comprising the following steps:

[0261] (a) Contact a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a Compound (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0262] (b) Allow the compound to bind to the α-synuclein aggregates;

[0263] (c) Detect the compound that binds to the α-synuclein aggregates; and

[0264] (d) Optionally, establish a correlation between the presence or absence of a compound that binds to α-synuclein aggregates and the presence or absence of α-synuclein aggregates in a sample or a specific body part or body region.

[0265] If the amount of the compound that binds to α-synuclein aggregates is higher than the normal control value of a healthy / reference individual, this indicates that the patient has a disease, disorder or abnormality associated with α-synuclein aggregates or is at risk of developing a disease, disorder or abnormality associated with α-synuclein aggregates. In particular, if the amount of the compound that binds to α-synuclein aggregates is higher than the expected value of a person who does not show clinical evidence of a neurodegenerative disease, it can be presumed that the patient has a disease, disorder or abnormality associated with α-synuclein aggregates or a predisposition to synucleinopathy.

[0266] In another embodiment, the present invention provides a method for collecting data for predicting the prognosis of a disease, disorder or abnormality associated with α-synuclein aggregates, wherein the method comprises the following steps:

[0267] (a) Contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0268] (b) Allowing the compound to bind to α-synuclein aggregates;

[0269] (c) Detecting the compound that binds to α-synuclein aggregates;

[0270] (d) Optionally, establish a correlation between the presence or absence of the compound that binds to α-synuclein aggregates and the presence or absence of α-synuclein aggregates in the sample or the specific body part or body region; and

[0271] (e) Optionally, repeat steps (a)-(c) and (if present) optionally step (d) at least once.

[0272] A physician can estimate the prospects of progression and / or recovery (e.g., probability, duration and / or extent) of a disease, disorder or abnormality based on the presence or absence of the compound that binds to α-synuclein aggregates, the amount of the compound that binds to α-synuclein aggregates, etc. If desired, steps (a)-(c) and (if present) optionally step (d) can be repeated over time to monitor the progression of the disease, disorder or abnormality, so as to make a more reliable estimate.

[0273] In another embodiment, the present invention provides a method for collecting data for monitoring the disease progression of a patient suffering from a disease, disorder or abnormality associated with α-synuclein aggregates, the method comprising the following steps:

[0274] (a) contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a detectable-labeled compound thereof, stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates thereof;

[0275] (b) allowing the compound to bind to the α-synuclein aggregates;

[0276] (c) detecting the compound bound to the α-synuclein aggregates;

[0277] (d) optionally correlating the presence or absence of the compound bound to the α-synuclein aggregates with the presence or absence of α-synuclein aggregates in the sample or the specific body part or body region; and

[0278] (e) optionally repeating steps (a)-(c) and (if present) optionally step (d) at least once.

[0279] Typically, the patient is undergoing or has undergone treatment for a disease, disorder or abnormality associated with α-synuclein aggregates, or is undergoing / has undergone treatment for synucleinopathy. In particular, the treatment may involve administration of a drug suitable for treating a disease, disorder or abnormality associated with α-synuclein aggregates.

[0280] In another embodiment, the present invention provides a method for collecting data for monitoring the progression of a disease, disorder or abnormality associated with α-synuclein aggregates in a patient, the method comprising the following steps:

[0281] (a) contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0282] (b) allowing the compound to bind to the α-synuclein aggregates;

[0283] (c) detecting the compound bound to the α-synuclein aggregates;

[0284] (d) optionally correlating the presence or absence of the compound bound to the α-synuclein aggregates with the presence or absence of α-synuclein aggregates in the sample or the specific body part or body region; and

[0285] (e) Optionally repeat steps (a)-(c) and, if present, optionally step (d) at least once.

[0286] Typically, a patient is undergoing or has undergone treatment for a disease, disorder or abnormality associated with α-synuclein aggregates, or is undergoing / has undergone treatment for synucleinopathy. In particular, the treatment may involve administering a drug suitable for treating a disease, disorder or abnormality associated with α-synuclein aggregates.

[0287] In another embodiment, the present invention provides a method for collecting data for predicting the response of a patient suffering from a disease, disorder or abnormality associated with α-synuclein aggregates to treatment for a disease, disorder or abnormality associated with α-synuclein aggregates, the method comprising the following steps:

[0288] (a) Contacting a sample, a specific body part or body region suspected of containing α-synuclein aggregates with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof;

[0289] (b) Allowing the compound to bind to α-synuclein aggregates;

[0290] (c) Detecting the compound bound to α-synuclein aggregates;

[0291] (d) Optionally correlating the presence or absence of the compound bound to α-synuclein aggregates with the presence or absence of α-synuclein aggregates in the sample or specific body part or body region; and

[0292] (e) Optionally repeat steps (a)-(c) and, if present, optionally step (d) at least once.

[0293] Typically, a patient is undergoing or has undergone treatment for a disease, disorder or abnormality associated with α-synuclein aggregates, or is undergoing / has undergone treatment for synucleinopathy. In particular, the treatment may involve administering a drug suitable for treating a disease, disorder or abnormality associated with α-synuclein aggregates.

[0294] If the amount of the compound bound to α-synuclein aggregates decreases over time, it can be presumed that the patient responds to the treatment. If the amount of the compound bound to α-synuclein aggregates remains substantially constant or increases over time, it can be presumed that the patient does not respond to the treatment.

[0295] Alternatively, the response can be estimated by determining the amount of a compound that binds to α-synuclein aggregates. The amount of the compound that binds to α-synuclein aggregates can be compared to a control value such as a normal control value, a preclinical control value, or a clinical control value. Alternatively, the control value can refer to the control value of an individual known to respond to a certain therapy or the control value can refer to the control value of an individual known not to respond to a certain therapy. The result regarding the response can be "response" to a certain therapy, "no response" to a certain therapy, or "response undetermined" to a certain therapy. The response to a therapy may vary among patients.

[0296] In yet another embodiment, the present invention provides a method as defined herein, wherein the step of optionally correlating the presence or absence of a compound that binds to α-synuclein aggregates with the presence or absence of α-synuclein aggregates in a sample or a particular body part or body region comprises:

[0297] - determining the amount of the compound that binds to α-synuclein aggregates;

[0298] - correlating the amount of the compound that binds to α-synuclein aggregates with the amount of α-synuclein aggregates in the sample or a particular body part or body region; and

[0299] - optionally comparing the amount of α-synuclein aggregates in the sample or a particular body part or body region with a normal control value of a healthy control individual.

[0300] The control value can be, for example, a normal control value, a preclinical control value, and / or a clinical control value.

[0301] A "healthy control individual" or "healthy volunteer (HV) individual" is a person who does not show clinical evidence of a neurodegenerative disease.

[0302] If, in any of the methods outlined above, the amount of the compound that binds to α-synuclein aggregates is higher than the normal control value, it can be expected that the patient has or may have a disease, disorder, or abnormality associated with α-synuclein aggregates or has or may have a synucleinopathy.

[0303] Any of the compounds of formula (III-F) can be used in the methods outlined above.

[0304] The particular body part or body region is preferably a particular body part or body region of a mammal, more preferably a human, including the whole body or a partial body region or body part of a patient suspected of containing α-synuclein aggregates.

[0305] The sample can be selected from tissues or body fluids suspected of containing α-synuclein aggregates, and the sample is obtained from a patient. Preferably, the tissue is selected from brain tissue, central nervous system tissue, and eye tissue (such as retinal tissue), more preferably brain tissue. Examples of body fluids include cerebrospinal fluid (CSF) or blood. The sample can be from a peripheral organ, such as but not limited to the intestine. The sample can be obtained from a mammal, more preferably a human. Preferably, the sample is an in vitro sample from a patient.

[0306] In an in vivo method, by administering to a patient an effective amount of a compound of formula (III-F), a specific body part or body region can be brought into contact with the compound of formula (III-F). The effective amount of the compound of formula (III-F) is an amount suitable for enabling the determination of the presence or absence of α-synuclein aggregates in a specific body part or body region that can be determined using the selected analytical technique.

[0307] The step of allowing the compound to bind to α-synuclein aggregates includes allowing the compound of formula (III-F) to bind to α-synuclein aggregates for a sufficient time. The time period required for binding depends on the type of test (e.g., in vitro or in vivo) and can be determined by those skilled in the art through routine experimentation. In an in vivo method, the time period depends on the time required for the compound to reach a specific body part or body region suspected of containing α-synuclein aggregates. The time period should not be too long to avoid clearance and / or metabolism of the compound of formula (III-F).

[0308] There is no particular limitation on the method for detecting a compound that binds to α-synuclein aggregates, and it depends on the type of sample, the specific body part or body region, and whether the method is an in vitro or in vivo method, etc. Possible detection methods include but are not limited to fluorescence imaging techniques or nuclear imaging techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and contrast-enhanced magnetic resonance imaging. Fluorescence imaging techniques and / or nuclear imaging techniques can be used to monitor and / or visualize the distribution of the sample or the compound of formula (III-F) in vivo. The imaging system is capable of providing an image of the detectable label that binds, such as a radioisotope, especially a positron emitter or a gamma emitter, as present in the test sample, the specific body part being tested, or the body region being tested. Preferably, the compound that binds to α-synuclein aggregates is detected by an imaging device (such as a PET or SPECT scanner).

[0309] By visual or quantitative analysis, such as using PET scan images, the amount of the compound that binds to α-synuclein aggregates can be determined.

[0310] In any of the above methods, steps (a)-(c) and, if present, optional step (d) can be repeated at least once. Repeating these steps is particularly useful in methods for collecting data for prognosis, methods for collecting data for monitoring disease evolution, methods for collecting data for monitoring progression, and methods for collecting data for predicting response. In these methods, it may be advantageous to monitor the patient over time and repeat the above steps after a certain period of time. The time interval before repeating the above steps can be determined by the clinician based on the disease, disorder, or abnormality associated with α-synuclein aggregates or the severity of synucleinopathy.

[0311] In another aspect, the present invention relates to a method for imaging a disease, disorder, or abnormality associated with α-synuclein aggregates in an individual, the method comprising the following steps:

[0312] (a) Administering to the individual a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof;

[0313] (b) Allowing the compound to bind to α-synuclein aggregates; and

[0314] (c) Detecting the compound bound to α-synuclein aggregates.

[0315] In another aspect, the present invention relates to a method for imaging a disease, disorder, or abnormality associated with α-synuclein aggregates in an individual, the method comprising the following steps:

[0316] (a) Administering to the individual a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof; and

[0317] (b) Imaging the brain of the individual.

[0318] When the compound binds to α-synuclein aggregates, the brain of the individual should be imaged. Then, the compound bound to α-synuclein aggregates in the brain of the individual can be imaged.

[0319] In another aspect, the present invention relates to a method for positron emission tomography (PET) imaging of α-synuclein aggregates in the tissue of an individual, the method comprising the following steps:

[0320] (a) Administering to the individual a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof;

[0321] (b) Allowing the compound to penetrate into the individual tissue; and

[0322] (c)Collect a positron emission tomography (PET) image of an individual's tissue;

[0323] wherein the tissue is central nervous system (CNS) tissue, eye tissue, or brain tissue, preferably wherein the tissue is brain tissue.

[0324] PET imaging should be performed when the compound has penetrated into the tissue and the compound has bound to α-synuclein aggregates.

[0325] In another aspect, the present invention relates to a method for detecting a neurological disease, disorder, or abnormality associated with α-synuclein aggregates in an individual, the method comprising the following steps:

[0326] (a) Administer a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the individual;

[0327] (b) Allow the compound to bind to α-synuclein aggregates; and

[0328] (c) Determine the radioactive signal of the compound bound to α-synuclein aggregates.

[0329] When a detectable-labeled compound of formula (III-F) containing at least one radioactively labeled atom binds to α-synuclein aggregates, a radioactive signal as mentioned herein is observed.

[0330] In another aspect, the present invention relates to a method (e.g., in vivo or in vitro method) for detecting and / or quantifying α-synuclein aggregates in an individual's tissue, the method comprising the following steps:

[0331] (a) Contact the tissue with a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof administered to the individual;

[0332] (b) Allow the compound to bind to α-synuclein aggregates; and

[0333] (c) Detect and / or quantify the compound bound to α-synuclein aggregates using positron emission tomography.

[0334] In yet another aspect, the present invention relates to a method for diagnostic imaging of an individual's brain, the method comprising the following steps:

[0335] (a) Administer a compound of formula (III-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate, or solvate thereof to the individual; and

[0336] (b) Obtain a brain image of the individual using positron emission tomography.

[0337] In the method of the present invention, the compound of formula (III-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates are generally administered in a detectable amount, i.e., an amount that can be detected by an apparatus used to detect the compound in the corresponding method. There is no particular limitation on this amount, and it depends on the compound of formula (III-F), the type of detectable label, the sensitivity of the corresponding analytical method and the corresponding apparatus. This amount can be appropriately selected by those skilled in the art.

[0338] Radiopharmaceutical preparation

[0339] The compound of formula (IV-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates can also be used to prepare a kit for a radiopharmaceutical preparation. Due to radioactive decay, radiopharmaceuticals are generally prepared immediately before use. The kit generally contains the compound of formula (IV-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates, and a reagent that reacts with the compound of formula (IV-F) to introduce a radioactive label ( 18 F) into the compound of formula (IV-F) or its stereoisomers, racemic mixtures, pharmaceutically acceptable salts, hydrates or solvates.

[0340] The present invention is illustrated by the following examples. However, these examples should not be construed as limiting. Examples

[0341] All reagents and solvents were obtained from commercial sources and used without further purification.

[0342] HPLC-UV purity: A 1 mg / mL solution of the corresponding compound was prepared with an aqueous solution of 2% TFA and 5 μL was injected onto an Ascentis Express ES-C18, 5 μm, 150×4.6 mm column. Within 10 minutes, the corresponding peak was eluted at a flow rate of 1.5 mL / minute using a linear gradient of 0.1% TFA (mobile phase A) and 0.1% TFA in MeCN (mobile phase B) from 5% - 100% B. Detection was carried out by UV at a wavelength of 254 nm.

[0343] HPLC-UV chiral purity: Using MeCN / IPA / 5 mM NH 4OAc = 3 / 4 / 1 Prepare a solution of the corresponding compound at approximately 0.5 mg / mL (filter the sample if necessary), and inject 10 μL onto a Daicel Chiralpak OX-RH, 5 μm, 150×4.6 mm column. The peak of the precursor S-enantiomer (Rt approximately 7.9 minutes) is isocratically eluted at 40 °C at a flow rate of 1 mL / minute using a mixture of MeCN:water (containing 5 mM NH 4 Oac) in a volume ratio of 75:25. Detection is carried out by UV at a wavelength of 254 nm.

[0344] Identification: The identity of the corresponding compound was confirmed based on 1 H and 13 C nuclear magnetic resonance (NMR) spectra obtained using a Bruker AV500 spectrometer, while the ESI-MS molecular ion was confirmed using a Thermo Fisher Scientific Surveyor MSQ Plus mass detector in the positive ionization mode. LC analysis was carried out on an XSelect Peptide CSH C18 column 3.5 μm, 150×2.1 mm. For sample preparation, dissolve 0.5 mg of the corresponding compound in 1 mL of MeCN / H 2 O (1:1), and filter the suspension. After injection, within 15 minutes, elute the corresponding compound at a flow rate of 0.4 mL / minute using a linear gradient of 0.1% HCO 2 H (mobile phase B) and 0.1% HCO 2 H in MeCN (mobile phase A) from 5% - 100% A.

[0345] Preparation Example 1

[0346]

[0347] Step A:

[0348] A suspension of 2-bromo-5-hydrazinopyridine (3.21 g, 17.07 mmol) and tert-butyl 2,4-dioxopyrrolidine-1-carboxylate (3.40 g, 17.07 mmol) in ethanol (150 mL) was refluxed for 3 hours and monitored by TLC. The crude product was concentrated under reduced pressure and diluted with dichloromethane and water. The layers were separated, and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried over Na 2 SO 4 4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (silica gel, ethyl acetate:hexane = 4:1) to give (E)-4-(2-(6-bromopyridin-3-yl)hydrazinylidene)-2-oxopyrrolidine-1-carboxylic acid tert-butyl ester as a brown solid (4.97 g, 79%).

[0349] Step B:

[0350] The compound from Step A (3.9 g, 10.56 mmol) was stirred in 1,1 - dimethoxy - N,N - dimethylmethanamine (80 mL) at 50 °C for 3 h. The reaction was concentrated to about 10 mL, and ethanol was added. The solid was filtered and washed with a small portion of ethanol to give tert - butyl 2 - (6 - bromopyridin - 3 - yl) - 4 - oxo - 4,6 - dihydropyrrolo[3,4 - c]pyrazole - 5(2H) - carboxylate as a light brown powder (2.30 g, 57.4%).

[0351] Step C:

[0352] The compound from Step B (1000 mg, 2.64 mmol) was stirred in 4M HCl in dioxane (37 mL) at room temperature (RT) for 4 h. The solvent was evaporated under reduced pressure, and the solid was suspended in dichloromethane. Saturated NaHCO 3 solution was added, and the aqueous phase was extracted twice. The combined organic layers were filtered to give 2 - (6 - bromopyridin - 3 - yl) - 5,6 - dihydropyrrolo[3,4 - c]pyrazol - 4(2H) - one as a light brown solid (682 mg, 93%).

[0353] Step N:

[0354] Under argon, in a round - bottom flask, 2 - (6 - bromopyridin - 3 - yl) - 5,6 - dihydropyrrolo[3,4 - c]pyrazol - 4(2H) - one (7.03 g, 25.17 mmol), (S) - pyrrolidin - ol (3.29 g, 37.76 mmol), and cesium fluoride (6.69 g, 50.35 mmol) were mixed in dry DMSO (70 mL). The resulting mixture was purged with argon and stirred at 120 °C for 3 h. The reaction mixture was cooled and poured into ice - cold water pre - cooled in an ice bath. The resulting suspension was filtered, and the solid was rinsed with ice - cold water. 3 mL of isopropanol was used to grind the solid directly in a fritted funnel to give the product as a light brown solid (6.75 g, 7.23 mmol, 94%).

[0355] Step O:

[0356] Under argon, in a flask, the compound from step N (6.26 g, 21.94 mmol), 3-bromopyridine (4.65 mL, 48.3 mmol), potassium carbonate (5.27 g, 87.75 mmol) and copper(I) iodide (1.67 g, 8.78 mmol) were mixed, and the system was purged with argon. Dioxane (772 mL) and N1,N2-dimethylethane-1,2-diamine (1.87 mL, 17.55 mmol) were added, and the mixture was stirred at 110 °C for 22 h. The crude product was concentrated under reduced pressure and suspended in water. Ammonia was added until the solution became basic (pH 13). The aqueous layer was extracted 20 times with a DCM / MeOH (9:1) solution. Over Na 2 SO 4 The combined organic layers were dried, filtered and concentrated to dryness to give the product as a light brown solid (6.36 g, 17.56 mmol, 80%).

[0357] Step P:

[0358] Under argon, in a vial cooled to 0 °C, (S)-2-(6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazol-4(2H)-one (100 mg, 0.276 mmol) and 4-dimethylaminopyridine (337 mg, 2.76 mmol) were mixed in pyridine (4 mL). Methanesulfonyl chloride (0.108 mL, 1.380 mmol) was added and the mixture was purged with argon. Pyridine (13 mL) was added and the reaction mixture was transferred to a flask. The reaction mixture was warmed to RT and stirred for 2 h, after which 4-dimethylaminopyridine (169 mg, 1.380 mmol) and methanesulfonyl chloride (0.054 mL, 0.690 mmol) were added at 0 °C. The reaction was complete after 40 min. Then 0.1 N NaOH in water (5 mL) was added to the mixture to basify it. Since the alkalinity was insufficient, 1 N NaOH in water (15 mL) was added again. The reaction mixture turned brown at pH = 14. The solution was poured into cold water and filtered. The solid residue was washed with water until the pH of the water was 7. The resulting solid was dried under high vacuum for 30 min to give the compound as an orange solid (86 mg, 0.195 mmol, 70.8%).

[0359] The product was observed to be unstable and sensitive to temperature, oxygen and light.

[0360] Example 1

[0361] The first crystallization:

[0362] The compound of Preparation Example 1 (2.97 g, 6.7 mmol, purity: 93.7%) was suspended in ethanol (148 mL) and heated to reflux using an oil bath. DMSO (233 mL) was added at 80 °C until a solution was obtained. The oil bath was then removed and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed three times with ethanol (1.8 g, 4.1 mmol, 61% yield, HPLC purity 95.5%).

[0363] The second crystallization:

[0364] The crystalline mesylate precursor from the first cycle (1.8 g, 4.1 mmol) was suspended in DMSO (60 mL) at room temperature. The suspension was heated to 80 °C (a solution formed at approximately 70 °C). At 80 °C, ethanol (60 mL) was added and the resulting slightly turbid solution was stirred at room temperature for 10 minutes. The oil bath was removed and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed three times with ethanol (1.6 g, 3.6 mmol, 89% yield, HPLC purity 96.4%).

[0365] The third crystallization:

[0366] The crystalline mesylate precursor from the second cycle (1.6 g, 3.6 mmol) was further crystallized by repeating the conditions of the second cycle (1.5 g, 3.3 mmol, 93% yield, HPLC purity 96.9%).

[0367] Example 2: Stability Test

[0368] The compound of Preparation Example 1 was transferred into glass vials and purged with argon. The vials were frozen (-20 °C ± 5 °C), refrigerated (2 - 8 °C), stored at room temperature (20 - 25 °C) with or without light protection, and at 40 °C. The material was retested by HPLC-UV weekly for the first month and then monthly thereafter. Degradation of the compound of Preparation Example 1 was noted under all test conditions. However, it was significantly reduced when the material was stored refrigerated at -20 °C and protected from light. The major degradation product initially formed was detected by HPLC-UV at RT 1.6 minutes and identified by HPLC-ESI-MS as a pyrrole decomposition product resulting from the elimination of the mesylate group and subsequent aromatization. The putative pyrrole decomposition product has the following structure

[0369]

[0370] The stability of the purified compound of Example 1 was also evaluated at 40 °C under the same stress test study conditions. As can be seen fromFigure 1 As observed, when stored at 40 °C (for one month), no degradation of the purified compound of Example 1 was observed, indicating that purification according to the method of the present invention significantly improves stability.

[0371] Example 3

[0372] The first crystallization:

[0373] 1.21 g of the compound prepared in Preparation Example 1 (HPLC purity: 93.2%) was suspended in 40 mL of DMSO and heated to 80 °C. The compound completely dissolved at 80 °C. 40 mL of ethanol was added at 80 °C, and the mixture was stirred at 80 °C for 10 minutes. The stirrer was turned off, and the mixture was cooled to RT. The flask was stored at 2 - 8 °C overnight to complete crystallization. The product was separated by suction filtration of the still cold suspension and washed 3 times with ethanol. Yield: 963 mg, 80%, HPLC purity: 93.6%.

[0374] The second crystallization:

[0375] 963 mg of the crystals were crystallized using the above method to obtain 880 mg, 92%, HPLC purity: 96.1%.

[0376] The third crystallization:

[0377] 880 mg of the crystals were crystallized using the above method to obtain 810 mg, 92%, HPLC purity: 96.7%.

[0378] The fourth crystallization:

[0379] 250 mg from the material separated from the above 3rd crystallization was suspended in DMSO (8.9 mL) at room temperature. The suspension was heated to 80 °C (a solution formed at about 70 °C). Ethyl acetate (8.9 mL) was added at 80 °C, and the resulting solution was stirred at 80 °C for 10 minutes. The oil bath was removed, and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed 3 times with ethyl acetate (200 mg, 0.45 mmol, 80% yield, HPLC purity 97.6%).

[0380] Example 4

[0381] The first crystallization:

[0382] 1.21 g of the compound prepared in Preparation Example 1 (HPLC purity: 93.2%) was suspended in 40 mL of DMSO and heated to 80 °C. The compound completely dissolved at 80 °C. 40 mL of ethanol was added at 80 °C and the mixture was stirred at 80 °C for 10 minutes. The stirrer was turned off and the mixture was cooled to RT. The flask was stored at 2 - 8 °C overnight to complete crystallization. The product was separated by suction filtration of the still cold suspension and washed 3 times with ethanol. Yield: 963 mg, 80%, HPLC purity: 93.6%.

[0383] The second crystallization:

[0384] 963 mg of the crystals were crystallized using the above method to obtain 880 mg, 92%, HPLC purity: 96.1%.

[0385] The third crystallization:

[0386] 880 mg of the crystals were crystallized using the above method to obtain 810 mg, 92%, HPLC purity: 96.7%.

[0387] The fourth crystallization:

[0388] At room temperature, 250 mg of the material separated from the above third crystallization was suspended in DMSO (8.9 mL). The suspension was heated to 80 °C (a solution formed at about 70 °C). 2-butanone (8.9 mL) was added at 80 °C and the resulting solution was stirred at 80 °C for 10 minutes. The oil bath was removed and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed 3 times with ethyl acetate (175 mg, 0.40 mmol, 70% yield, HPLC purity 97.9%).

[0389] Example 5

[0390] The first crystallization:

[0391] 14.7 g of the compound prepared in Preparation Example 1 (HPLC purity: 92.2%) was suspended in 484 mL of DMSO and heated to 80 °C. The compound completely dissolved at 80 °C. 484 mL of ethanol was added at 80 °C and the mixture was stirred at 80 °C for 10 minutes. The stirrer was turned off and the mixture was cooled to RT. The flask was stored at 2 - 8 °C overnight to complete crystallization. The product was separated by suction filtration of the still cold suspension and washed 3 times with 118 mL of ethanol. The product was transferred to a flask and dried under high vacuum for 3 hours. Yield: 12.8 g, 29.1 mmol, 87.1%, HPLC purity: 96.2%.

[0392] The second crystallization:

[0393] Using the above method, 12.8 g of crystals were obtained, yielding 11.9 g, 27.0 mmol, 93%, HPLC purity: 96.9%.

[0394] The third crystallization:

[0395] Using the above method, 11.9 g of crystals were obtained, yielding 11.1 g, 25.2 mmol, 93.3%, HPLC purity: 97.1%.

[0396] The fourth crystallization:

[0397] Using the above method, 11.1 g of crystals were obtained, yielding 10.4 g, 23.6 mmol, 93.7%, HPLC purity: 97.3%.

[0398] The fifth crystallization:

[0399] Using the above method, 10.4 g of crystals were obtained, yielding 9.9 g, 22.5 mmol, 95.2%, HPLC purity: 98.1%.

[0400] The total yield after the crystallization cycle was 67.4%.

[0401] Stability test

[0402] The stability of the purified compound prepared according to the purification method of Example 5 was evaluated after storage at -20°C ± 5°C and 5°C ± 3°C for 9 months, where the impurities were measured by HPLC every 3 months, and a two-week short-term stress test was conducted at 40°C ± 2°C and 75% ± 5 relative humidity, and the impurities were measured by HPLC at 0, 3, 7, and 14 days. It was observed that the purified compound was stable after storage at -20°C ± 5°C and 5°C ± 3°C for 9 months. At these two temperatures, the detected impurities did not change significantly over time. It was also found that the purified compound was stable after storage at 40°C ± 2°C and 75% ± 5 relative humidity for 14 days. These results indicate that the purified compound prepared according to the method of the present invention has good stability.

[0403] Example 6 (comparative)

[0404] A comparative compound of formula (IV-F) was synthesized according to the method of Preparation Example 1, where LG is nosylate or tosylate instead of mesylate. The tosylate compound could not be obtained by this method.

[0405] At 140°C, 10 mg of Kryptofix

[222] and 1.24 mg of K in 0.7 mL of DMSO2 CO 3 In the presence of [substance], 5 mg of the mesylate compound prepared in Preparation Example 1 and 5 mg of the corresponding nitrobenzenesulfonate compound were radioactively labeled for 10 minutes. The labeling rate of the mesylate compound was 60%, while the labeling rate of the comparative nitrobenzenesulfonate was only 7%.

[0406] Example 7 (comparative)

[0407] The compound prepared in Preparation Example 1 was recrystallized twice using ethyl acetate:DMSO as the solvent system.

[0408] The first crystallization:

[0409] At room temperature, the compound prepared in Preparation Example 1 (325 mg, 0.74 mmol, purity: 93.6%) was suspended in DMSO (11.6 mL). The suspension was heated to 80 °C (a solution was formed at about 70 °C). Ethyl acetate (11.6 mL) was added at 80 °C, and the resulting solution was stirred at 80 °C for 10 minutes. The oil bath was removed, and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed 3 times with ethyl acetate (156 mg, 0.35 mmol, 47% yield, HPLC purity 94.7%).

[0410] The second crystallization:

[0411] The crystalline mesylate precursor from Cycle 1 (156 mg, 0.35 mmol) was further crystallized by repeating the conditions of Cycle 1. (50 mg, 0.11 mmol, 32% yield, HPLC purity 94.1%).

[0412] As can be observed, compared with the solvent mixture used in the method of the present invention, the solvent mixture of Example 7 resulted in much lower purity and yield.

[0413] Example 8 (comparative)

[0414] The compound prepared in Preparation Example 1 was recrystallized twice using 2-butanone:DMSO as the solvent system.

[0415] The first crystallization:

[0416] At room temperature, the compound of Preparation Example 1 (325 mg, 0.74 mmol, purity: 93.6%) was suspended in DMSO (11.6 mL). The suspension was heated to 80 °C (a solution was formed at about 70 °C). 2-Butanone (11.6 mL) was added at 80 °C, and the resulting solution was stirred at 80 °C for 10 minutes. The oil bath was removed, and the solution was cooled to room temperature. The flask was sealed and stored at 2 - 8 °C overnight. The precipitate was separated by suction filtration and washed 3 times with 2-butanone (157 mg, 0.36 mmol, 48% yield, HPLC purity 94.2%).

[0417] The second crystallization:

[0418] The crystalline mesylate precursor from Cycle 1 (157 mg, 0.36 mmol) was further crystallized by repeating the conditions of Cycle 1. (50 mg, 0.11 mmol, 32% yield, HPLC purity 94.4%).

[0419] As can be observed, compared to the solvent mixture used in the method of the present invention, the solvent mixture of Example 8 results in a lower purity and a much lower yield.

Claims

1. A method for purifying a compound of formula (IV-F) or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R 3 selected from wherein LG is C 1-4 alkyl sulfonate; R 4 is an aryl group or a 5- or 6-membered heteroaryl group, where R 4 is selected from: wherein R 2a 、R 2a’ are independently selected from H or F; R 2b independently selected from F, -OH, C 1 -C 4 alkyl, halo C 1 -C 4 alkyl, -NH 2 , -CN or C 1 -C 4 alkoxy; R 2c 、R 2c’ are independently selected from H, F, OH, OCH 3 or CH 3 ; R 2d selected from H, F or -OH; R 2e selected from H, OH, CH 3 or F; Z is independently N, NH, N(C 1 -C 4 -alkyl), N(halo-C 1 -C 4 -alkyl), O or S; Z 1 independently N, NH, O or S; p is 0, 1 or 2; m is 0 or 1; When the valence allows, is a combination of single bonds and double bonds; and * is the bonding position; wherein the method comprises: (i) Dissolving the crude compound of formula (IV-F) in DMSO and ethanol to obtain a solution; and (ii) Crystallizing from the solution to obtain the purified compound of formula (IV-F).

2. The method according to claim 1, wherein the compound of formula (IV-F) is or a pharmaceutically acceptable salt, hydrate or solvate thereof.

3. The method according to claim 1 or 2, wherein LG is mesylate.

4. The method according to any one of claims 1 - 3, the method further comprising the following steps: (iii) Separating the purified compound of formula (IV-F) from the solution, and optionally washing and drying the separated purified compound of formula (IV-F).

5. The method according to any one of claims 1 - 4, wherein steps (i), (ii) and optionally step (iii) are carried out at least twice, preferably at least three times, more preferably at least four times, even more preferably at least five times.

6. The method according to any one of claims 1 - 5, wherein step (i) comprises: Mixing the crude compound of formula (IV-F) with DMSO, and then adding ethanol to the resulting mixture.

7. The method according to any one of claims 1 - 6, wherein the ratio of DMSO to ethanol (v:v) ranges from about 5:1 to about 0.1:1, preferably from about 4:1 to about 0.5:1, more preferably from about 3:1 to about 0.5:1, even more preferably from about 2:1 to about 0.75:

1.

8. The method according to any one of claims 1 - 7, wherein the crude compound of formula (IV-F) is dissolved in a temperature range of about 50°C to about 90°C, preferably about 60°C to about 90°C, more preferably about 65°C to about 85°C.

9. The method according to any one of claims 1 - 8, wherein the crystallization is carried out in a temperature range of about 0°C to about 40°C, preferably about 0°C to about 40°C, more preferably about 0°C to about 30°C.

10. The method according to any one of claims 1 - 9, the method further comprising after step (ii) or if optional step (iii) is present (iv) Dissolving the compound of formula (IV-F) in DMSO and 2-butanone or ethyl acetate to obtain a solution; and (v) Crystallizing from the solution to obtain the purified compound of formula (IV-F).

11. The method according to any one of claims 1 - 10, wherein the purified compound of formula (IV-F) has a purity of at least 97%.

12. A compound obtainable by the method according to any one of claims 1 - 11.

13. A kit for preparing a radiopharmaceutical formulation, wherein the kit comprises a sealed vial containing at least one compound as defined in claim 12.

14. The kit according to claim 13, wherein the radiopharmaceutical preparation is used for diagnosing a disease, disorder or abnormality or its susceptibility associated with α-synuclein aggregates, wherein the disease, disorder or abnormality is optionally selected from Parkinson's disease (including sporadic, familial α-synuclein mutations, familial non-α-synuclein mutations, pure autonomic failure or Lewy body dysphagia), SNCA repeat carriers, Lewy body dementia (LBD), dementia with Lewy bodies (DLB) (including "pure" Lewy body dementia), Parkinson's disease dementia (PDD), diffuse Lewy body disease (DLBD), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Alzheimer's disease Lewy body variant, Down syndrome, multiple system atrophy (MSA) (including Shy-Drager syndrome, striatonigral degeneration or olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, punch-drunk dementia, tauopathies (including Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (including Guam sporadic, familial or ALS-dementia complex), neuronal ceroid lipofuscinosis, neurodegeneration with brain iron accumulation type 1 (including Hallervorden-Spatz syndrome), prion diseases, ataxia telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gerstmann-Sträussler-Scheinker disease, inclusion body myositis, Gaucher's disease, Krabbe's disease and other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder, wherein the disease is preferably selected from Parkinson's disease, multiple system atrophy, dementia with Lewy bodies, Parkinson's disease dementia, SNCA repeat carriers and Alzheimer's disease, and more preferably wherein the disease is multiple system atrophy.

15. The kit according to claim 13, wherein the radiopharmaceutical preparation is used for imaging α-synuclein aggregates, wherein the imaging is preferably performed by positron emission tomography.

16. The kit according to claim 13, wherein the radiopharmaceutical preparation is used for in vitro imaging, ex vivo imaging or in vivo imaging, preferably for in vivo imaging, and more preferably for brain imaging.

17. A method for preparing a detectable-labeled compound of formula (III-F), or a stereoisomer, racemic mixture, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R 3 selected from R 4 is an aryl group or a 5- or 6-membered heteroaryl group, where R 4 is selected from: wherein R 2a 、R 2a’ are independently selected from H or F; R 2b independently selected from F, -OH, C 1 -C 4 alkyl, halo-C 1 -C 4 alkyl, -NH 2 , -CN or C 1 -C 4 alkoxy; R 2c 、R 2c’ are independently selected from H, F, OH, OCH 3 or CH 3 ; R 2d selected from H, F or -OH; R 2e selected from H, OH, CH 3 or F; Z is independently N, NH, N(C 1 -C 4 -alkyl), N(halo-C 1 -C 4 -alkyl), O or S; Z 1 independently N, NH, O or S; p is 0, 1 or 2; m is 0 or 1; When the valence allows, is a combination of single bonds and double bonds; and * is the bonding position; wherein the method comprises: React the compound according to claim 12 or the compound prepared by the method according to any one of claims 1-11 with 18 an F-fluorinating agent such that LG is 18 replaced by F.

18. The method according to claim 17, wherein 18 the F-fluorinating agent is selected from K 18 F, Rb 18 F, Cs 18 F, Na 18 F, Rb 18 F, Kryptofix[222]K 18 F, 18 tetra(C 1-6 alkyl)ammonium salts of F and 18 tetrabutylammonium fluoride [F].

19. The method according to claim 17 or 18, wherein the detectable-labeled compound of formula (III-F) is used for diagnosing a disease, disorder or abnormality or susceptibility thereto associated with α-synuclein aggregates, wherein the disease, disorder or abnormality is optionally selected from Parkinson's disease (including sporadic, familial α-synuclein mutations, familial non-α-synuclein mutations, pure autonomic failure or Lewy body dysphagia), SNCA repeat carriers, Lewy body dementia (LBD), dementia with Lewy bodies (DLB) (including "pure" Lewy body dementia), Parkinson's disease dementia (PDD), diffuse Lewy body disease (DLBD), Alzheimer's disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Alzheimer's disease Lewy body variant, Down syndrome, multiple system atrophy (MSA) (including Shy-Drager syndrome, striatonigral degeneration or olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, punch-drunk syndrome, tauopathies (including Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (including Guam sporadic, familial or ALS-dementia complex), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (including Hallervorden-Spatz syndrome), prion disease, ataxia telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gerstmann-Sträussler-Scheinker disease, inclusion body myositis, Gaucher's disease, Krabbe's disease and other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder, wherein the disease is preferably selected from Parkinson's disease, multiple system atrophy, dementia with Lewy bodies, Parkinson's disease dementia, SNCA repeat carriers and Alzheimer's disease, more preferably wherein the disease is multiple system atrophy.

20. The method according to claim 17 or 18, wherein the detectable-labeled compound of formula (III-F) is used for imaging of α-synuclein aggregates, wherein the imaging is preferably performed by positron emission tomography.

21. The method according to claim 17 or 18, wherein the detectable-labeled compound of formula (III-F) is used for in vitro imaging, ex vivo imaging or in vivo imaging, preferably for in vivo imaging, more preferably for brain imaging.

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