Novel compounds for diagnosis of TDP-43 proteinopathies

Abstract

The present invention relates to compounds suitable for the imaging of TDP-43 (trans-activation response (TAR) DNA binding protein 43kDa) aggregates. The compounds are useful, for example, in the diagnosis of diseases, disorders or abnormalities associated with TDP-43 aggregates or TDP-43 proteinopathies, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD) and edge system dominated age-related TDP-43 encephalopathy (LATE). # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to compounds suitable for imaging TDP-43 (transactivation response (TAR) DNA binding protein 43kDa) aggregates. The compounds can be used, for example, to diagnose diseases, disorders or abnormalities associated with TDP-43 aggregates or TDP-43 proteinopathies, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD) and limbic-dominant age-related TDP-43 encephalopathy (LATE). The present invention also relates to methods for preparing the compounds, diagnostic compositions comprising the compounds, methods of using the compounds, kits comprising the compounds and their uses. Background of the Invention

[0003] Age-related brain disorders characterized by pathological aggregation of proteins in the CNS (proteinopathies) and peripheral organs represent one of the leading causes of disability and death worldwide. The best characterized protein that forms extracellular aggregates is amyloid beta (Aβ) in Alzheimer's disease (AD) and Aβ-related disorders. Other aggregation-prone proteins associated with diseases leading to neurodegeneration include, but are not limited to, Tau, alpha-synuclein (a-syn), huntingtin, fused sarcoma (FUS), dipeptide repeat proteins (DPRs) generated by unconventional translation of C9orf72 repeat expansion, superoxide dismutase 1 (SOD1), and TDP-43. Diseases involving TDP-43 aggregates are generally referred to as TDP-43 proteinopathies, and include, but are not limited to, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), including frontotemporal lobar dementia with TDP-43 pathology (FTLD-TDP, frontotemporal lobar degeneration with TDP-43 inclusions) and limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0004] TDP-43 Introduction

[0005] Transactivation response (TAR) DNA binding protein 43kDa (TDP-43) is a 414 amino acid protein encoded by the TARDBP gene on chromosome 1p36.2 (ALS10). TARDBP consists of 6 exons (exon 1 is non-coding; exons 2-6 are protein-encoded). TDP-43 belongs to the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family (Wang et al., Trends in Molecular Medicine, Vol. 14, no. 11, 2008, 479-485; Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1R46-R64). TDP-43 contains five functional domains (Warraich et al., FIG. 1 in The International Journal of Biochemistry & Cell Biology, 42 (2010) 1606-1609): two RNA recognition motifs (RRM1 and RRM2) with two highly conserved hexameric ribonucleoprotein 2 (RNP2) and octameric ribonucleoprotein 1 (RNP1) regions, which enable it to shuttle between the nucleus and the cytoplasm, a nuclear export signal (NES) and a nuclear localization signal (NLS) that bind mRNA, and a C-terminal glycine-rich domain that mediates protein-protein interactions. TDP-43 is involved in multiple aspects of RNA processing, including transcription, splicing, transport, and stability (Buratti and Baralle, FEBS Journal, 277 (2010) 2268-2281). It is a highly conserved, ubiquitously expressed protein with tightly self-regulated expression levels that shuttles continuously between the nucleus and the cytoplasm, but is generally primarily localized in the nucleus. In 2006, TDP-43 was identified as a protein that accumulates in the vast majority of cases of frontotemporal lobar degeneration (FTLD) with tau-negative, ubiquitin-positive inclusions (later referred to as FTLD-TDP), as well as in most cases of amyotrophic lateral sclerosis (ALS) (Arai et al., Biochemical and Biophysical Research Communications, 351 (2006) 602-611; Neumann et al., Science, 314, (2006), 130-133).

[0006] 38 TDP-43 negative dominant mutations (K263E, N267S) were found in patients with sporadic and familial ALS and hereditary FTD, mainly located in the glycine-rich domain (Lagier-Tourenne and Cleveland, Cell, 136, 2009, Figure 1 of 1001-1004). TDP-43 has an inherent aggregation tendency, as shown in sedimentation assays, and this tendency is increased by some ALS-related TARDBP mutations (Ticozzi et al., CNS Neurol Disord Drug Targets, 2010, 9 (3), 285-296).

[0007] TDP-43 in neurodegeneration

[0008] TDP-43 aggregates have been identified in an increasing number of pathological conditions (Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1R46-R64), including but not limited to: frontotemporal dementia (sporadic or familial, with or without motor neuron disease (MND), with progranulin (GRN) mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive inclusions, argyrophilic grain disease, Pick's disease, etc.), amyotrophic lateral sclerosis (sporadic ALS, with TARDBP mutations, with ANG mutations), Alzheimer's disease (sporadic and familial), Down syndrome, familial British dementia, polyglutamine diseases (Huntington disease and SCA3), hippocampal sclerosis dementia, and myopathies (sporadic inclusion body myositis, inclusion body myopathy with VCP mutations, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with MYOT or DES mutations).

[0009] Aggregated TDP-43 from patient brains shows many abnormal modifications, including hyperphosphorylation, ubiquitination, acetylation, and C-terminal fragments cleaved by proteolytic cleavage (Arai et al., Biochemical and Biophysical Research Communications, 351 (2006) 602-611; Neumann et al., Science, 314, (2006), 130-133; Neumann et al., Acta Neuropathol, (2009) 117: 137-149; Hasegawa et al., Annals of Neurology, 2008, Vol 64 No 1, 60-70; Cohen et al., Nat Commun.; 2015, 6: 5845). Another feature of TDP-43 pathology is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. The hallmark lesions of FTLD-TDP are neuronal and glial cytoplasmic inclusions (neuronal cytoplasmic inclusions (NCI) and glial cytoplasmic inclusions (GCI), respectively) and dystrophic neurites (DN), which are immunoreactive for TDP-43 as well as ubiquitin and p62, but negative for other neurodegenerative disease-related proteins. Differences in inclusion morphology and their tissue distribution are associated with specific mutations and / or clinical manifestations. So far, four types of TDP-43 pathology have been described by histological methods (Mackenzie and Neumann, J. Neurochem., (2016), 138 (Suppl. 1), 54-70). Type A FTLD-TDP cases are characterized by abundant short DNs and dense oval or crescent-shaped NCIs located primarily in layer II of the neocortex (Mackenzie et al., J. Neurochem., 2016, 138 (Suppl. 1), 54-70 Figure 2f). Lens-shaped neuron intranuclear inclusions (NII) are also usually present, but in much less amounts. Cases with this pathology are usually clinically manifested as behavioral abnormalities of frontotemporal dementia (bvFTD) or non-fluent / grammatical variant primary progressive aphasia (nfvPPA) and are associated with mutations in progranulin (GRN). Neuropsychiatric phenomena are particularly common in those with potential GRN or C9orf72 mutations. Type A is the most common type in most AD cases TDP-43 neuropathology (Josephs et al., Acta Neuropathol. 2014, 127 (3), 441-50, Arai et al., Actaneuropathologica. 2009, 117, 125-136).The TDP-43 neuropathology in most cases of LATE is also similar to that of type A, which is also consistently found in FTD-GRN (Nelson et al., Brain, 2019, 142, 1503-1527). Type B cases show a moderate number of dense or granular NCIs in both the superficial and deep layers of the cortex, with relatively few DNs and NIIs (Mackenzie et al., J. Neurochem., 2016, 138 (Suppl. 1), Figure 2g in 54-70). Most NCIs have a dispersed granular morphology, sometimes referred to as "pre-inclusions". Importantly, some cases also have a background of fine small TDP-43 lines and dots. Most cases with co-occurring FTD and ALS symptoms were found to have the pathology of type B FTLD-TDP. Type C cases have a large number of long tortuous neurites, mainly in the superficial cortical layers, with few or no NCIs (Mackenzie et al., J. Neurochem., 2016, 138 (Suppl. 1), Figure 2j in 54-70). This pathology is particularly seen in cases with svPPA (semantic variant primary progressive aphasia). Type D FTLD-TDP shows abundant lenticular intranuclear inclusions (NIIs) and short DNs in the neocortex, and rare NCIs (Mackenzie et al., J. Neurochem., 2016, 138 (Suppl. 1), Figure 2k in 54-70). This pathological pattern is only found in cases with VCP associated with inclusion body myositis.

[0010] TDP-43 in FTD

[0011] Frontotemporal dementia (FTD) is a clinical term that covers a wide spectrum of disorders based on degeneration of the frontal and temporal lobes, a pathological feature known as frontotemporal lobar degeneration (FTLD). FTD is the second leading cause of early degenerative dementia in the age group under 65 years (LeBer, Revue Neurologique, 169 (2013), 811-819). FTD manifests itself in a variety of syndromes, including bvFTD characterized by changes in personality and behavior; semantic dementia (SD) and progressive nonfluent aphasia (PNFA) characterized by changes in language function; corticobasal syndrome (CBS), progressive supranuclear palsy syndrome and motor neuron disease characterized by motor dysfunction (FTD-MND). The diagnosis of these syndromes is complex and can only be definitively concluded by postmortem tissue analysis based on immunohistochemistry to detect aggregated proteins and description of affected brain areas. In terms of pathological, protein inclusions, approximately 45% of cases show pathological accumulation of misfolded Tau, 45% have pathological TDP-43, and smaller subsets have aggregates of FUS and other proteins. FTLD-TDP is the pathological term describing FTD cases with TDP-43 pathology, which is primarily found as cytoplasmic or neurite protein aggregates in neurons and glial cells containing misfolded, insoluble, phosphorylated, and truncated TDP-43.

[0012] TDP-43 in ALS

[0013] Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by premature loss of upper and lower motor neurons. Progression of ALS is marked by fatal paralysis and respiratory failure, with a course from diagnosis to death of 1 to 5 years. In most cases of sporadic ALS, neuropathology is characterized by abnormal cytoplasmic accumulation of TDP-43 in neurons and glial cells of the primary motor cortex, brainstem motor nuclei, spinal cord, and associated white matter tracts. ALS with dementia involves accumulation of TDP-43 in the extramotor neocortex and hippocampus. The role of TDP-43 phosphorylation in ALS patients has been explored with the help of phospho-specific antibodies that bind strongly to nuclear and cytoplasmic TDP-43 inclusions. Amino acids S379, S403, S404, S409 and S410 have been identified as major sites of TDP-43 phosphorylation (Hasegawa et al., Ann Neurol., 2008; 64: 60-70; Neumann et al., Acta Neuropathol., 2009, 117: 137-149).

[0014] TDP-43 in LATE

[0015] Limbic-predominant age-related TDP-43 encephalopathy (LATE) neuropathological changes (LATE-NC) are defined as typical TDP-43 proteinopathy in the elderly, with or without coexisting hippocampal sclerosis pathology. LATE-NC is a common TDP-43 proteinopathy associated with an amnestic dementia syndrome that mimics Alzheimer's dementia in retrospective autopsy studies. LATE is distinguished from frontotemporal lobar degeneration with TDP-43 pathology in its epidemiology (LATE typically affects elderly individuals) and the relatively limited neuroanatomical distribution of TDP-43 proteinopathy. There are no molecular specific biomarkers for LATE. The discovery of TDP-43 PET tracers can enable accurate and potentially early diagnosis and monitoring of disease progression to facilitate longitudinal drug efficacy measurements in patients during clinical trials (including as a potential exclusion criterion for Alzheimer's disease clinical trials) and longitudinal studies of clinical and pathological progression of LATE (Nelson et al., Brain, 2019, Vol. 142; No. 6, 1503-1527).

[0016] TDP-43 in AD and other diseases

[0017] TDP-43 pathology occurs in the brains of up to 57% of Alzheimer's patients (Josephs KA et al., Acta Neuropathol., 2014; 127(6): 811-824, Josephs KA et al., Acta Neuropathol., 2014; 127(3): 441-450; McAleese et al., Brain Pathol., 2017 Jul; 27(4): 472-479). TDP-43 aggregation is associated with cognitive decline, memory loss, and medial temporal lobe atrophy in AD. TDP-43-positive patients are 10 times more likely to die of cognitive impairment than TDP-43-negative individuals. TDP-43 appears to represent a secondary or independent pathology that has overlapping features with AD by targeting the medial temporal lobe. Pathological TDP-43 follows a typical deposition pattern captured from the staging scheme of TDP-43 in AD (TAD): TDP-43 is first deposited in the amygdala (stage I), followed by the hippocampus, limbus, temporal lobe, and finally the frontostriatum (stage V) (Josephs KA et al., Acta Neuropathol., 2014; 127(6):811-824; Josephs KA et al., Acta Neuropathol., 2014; 127(3):441-450).

[0018] Diagnosis of FTD and ALS

[0019] Diagnosing FTD based on clinical phenomena is inadequate because clinical manifestations can overlap with other diseases, especially in the early stages. Therefore, the development of sensitive and specific biomarkers to distinguish the types of pathology within the FTD spectrum is an urgent task. Such tools will help to better detect and understand the specific types of pathology that lead to neurodegeneration. Ultimately, this will lead to the development of diagnostic biomarkers, allowing more efficient and precise patient selection for longitudinal monitoring in clinical studies, supporting the development of new treatments for ALS and FTD.

[0020] Many approaches aim to develop biochemical biomarkers to distinguish different types of FTD pathology. Some studies have shown that TDP-43 concentrations are increased in the cerebrospinal fluid (CSF) of clinically defined FTD or FTD-MND populations, although there is significant overlap with controls or AD individuals, and it is unclear whether such methods will be clinically useful (Foulds et al., Acta Neuropathol., 2008, 116: 141-146; Steinacker et al., Arch. Neurol., 2008; 65 (11): 1481-1487). The levels of total Tau or Thr181 phosphorylated Tau cannot distinguish FTLD-Tau from controls. A diagnostic tool that may distinguish FTLD-Tau from FTLD-TDP is a reduced CSF p-Tau181 to Tau ratio below 0.37 (Hu et al., Neurology., 2013; 81 (22): 1945-1952). Another study showed that CSF phosphorylated Tau levels were positively correlated with Tau brain burden in FTD and may help distinguish TDP-43 proteinopathy from tau proteinopathy (Irwin et al., Ann. Neurol., 2017 Aug;82(2):247-258).

[0021] In parallel with the development of biochemical biomarkers, the development of biomarker imaging will enable early and specific detection of the pathology of FTD and ALS. The ability to image TDP-43 deposition in the brain will be an important achievement in the diagnosis and drug development of FTD, ALS and other neurodegenerative disorders. Progressive TDP-43 accumulation in the CNS is associated with disease progression and represents an obvious target for the development of new therapies and diagnostic tools to study pharmacodynamics and disease progression. Given that TDP-43 is a newer target, the development of PET tracers targeting this protein has just begun. However, most of the compounds reported to date are not specific for TDP-43, and it has not been demonstrated that any of these compounds directly bind to the target.

[0022] There are many challenges in the development of TDP-43-specific PET tracers, including the low abundance and heterogeneous distribution of the target in the patient's brain, and the lack of reference compounds. In order to reduce background signal interference caused by nonspecific off-target binding and reduce dosage requirements, TDP-43 imaging compounds should bind to the target with high affinity and selectivity. For imaging of TDP-43 aggregates associated with neurological disorders, such as FTD and ALS, imaging compounds need to penetrate the blood-brain barrier and enter relevant areas of the brain. For targeting intracellular amyloid-like inclusions (such as TDP-43 aggregates), a further requirement for imaging compounds is cell permeability. In order to avoid accumulation of compounds in tissues (which may lead to an increased risk of undesirable side effects), another necessary condition is to quickly clear the compound from the brain (or other target organs).

[0023] The object of the present invention is to provide compounds that are able to bind to TDP-43 aggregates. In particular, the compounds of the present invention should be useful for the identification and differentiation of patients or patient groups with TDP-43 proteinopathies (e.g., FTD, FTLD-TDP, LATE and ALS), as well as for the differentiation of TDP-43 proteinopathies from other proteinopathies.

[0024] The inventors have surprisingly found that compounds having the structure of formula (I) can recognize and bind to TDP-43 aggregates. In addition, the compounds of the present invention were found to exhibit high selectivity for TDP-43 aggregates relative to co-pathologies such as Aβ and tau in AD brain homogenates, and relative to a-syn in PD brain homogenates. In addition, the compounds of the present invention have been shown to have robust brain uptake and rapid clearance in non-human primates, meeting the criteria for further development of PET tracers for use in human subjects. SUMMARY OF THE INVENTION

[0026] The present invention is outlined in the appended claims. In particular, the present invention relates to compounds having the structure of formula (I)

[0027]

[0028] or a detectably labeled compound, stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof;

[0029] in

[0030] n is 1 or 2;

[0031] R 1 is H, hydroxy(C1-C4)alkyl or F; and

[0032] R 2is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S.

[0033] In another aspect, the present invention provides a diagnostic composition comprising a compound as defined herein of formula (I) or a subformula thereof, and optionally at least one physiologically acceptable carrier, diluent, adjuvant and / or excipient. The compound can be used for imaging of TDP-43 aggregates, particularly wherein the imaging is performed by positron emission tomography, or for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates, particularly wherein the diagnosis is performed by positron emission tomography.

[0034] In another aspect, the present invention provides a compound according to formula (I) or a subformula thereof, which can be used in the following methods:

[0035] A method for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates in an individual;

[0036] Methods for imaging TDP-43 aggregates in tissues of an individual using positron emission tomography (PET);

[0037] Methods for detecting and optionally quantifying TDP-43 aggregates in tissues of an individual;

[0038] Methods for diagnostic imaging of the brain of an individual;

[0039] Methods for collecting data for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or for diagnosing a TDP-43 proteinopathy;

[0040] Methods for collecting data for determining a disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition to a TDP-43 proteinopathy;

[0041] Methods for collecting data for monitoring the progression of a disease, disorder or abnormality associated with TDP-43 aggregates or for monitoring the progression of a TDP-43 proteinopathy in a patient;

[0042] - Methods of collecting data for predicting the response of a patient suffering from a disease, disorder or abnormality associated with TDP-43 aggregates to drug treatment.

[0043] In another aspect, the present invention provides a compound defined according to formula (I) or a subformula thereof, which can also be used as a biomarker for TDP-43 aggregates or a biomarker for TDP-43 proteinopathy, as a diagnostic reagent or diagnostic tool for TDP-43 proteinopathy, or as an in vitro analytical reference or in vitro screening tool.

[0044] Another aspect of the present invention provides a process for preparing a compound according to formula (I) or a subformula thereof.

[0045] In yet another aspect, the present invention relates to a kit for the preparation of a radiopharmaceutical formulation, said kit comprising a precursor of a compound of formula (I) or a subformula thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: Saturation binding curves of human FTD sarkosyl-insoluble brain extracts. Each point represents the mean ± standard error of the mean (SEM) of two independent experiments. Figure 1a-1e , human FTD type A sarcosyl sodium lauryl insoluble brain extract. Figure 1a :[ 3 H]-compound 1; Figure 1b :[ 3 H]-compound 7; Figure 1c :[ 3 H]-compound 8; Figure 1d :[ 3 H]-compound 9; Figure 1e :[ 3 H]-compound 15; Figure 1f :[ 3 H]-compound 16; Figure 1g :[ 3 H]-Compound 17. Figure 1h-1i , Sarcosyl-insoluble brain extract of human FTD type B. Figure 1h :[ 3 H]-compound 1; Figure 1i :[ 3 H]-Compound 17

[0048] Figure 2: Microautoradiography of FTLD-TDP brain tissue. Image of silver particle deposition on postmortem human brain tissue FTLD-TDP (A, black arrows) co-localized with pTDP-43 immunostaining (B, white arrows). Scale bar is 10 μm ( Figure 2a , 2b ) or 20 microns ( Figure 2c , 2e ). Figure 2a : FTLD-TDP type A on brain tissue [ 3 H]-compounds; Figure 2b : FTLD-TDP type A on brain tissue [ 3 H]-compound 9; Figure 2c : FTLD-TDP type A on brain tissue [ 3 H]-Compound 17. Figure 2d: FTLD-TDPB type brain tissue [ 3 H]-compound 1. Figure 2e : FTLD-TDP type B on brain tissue [ 3 H]-Compound 17.

[0049] Figure 3: [ 3 Representative autoradiograph of [H]-compound 1. Figure 3a : Autoradiographic images on brain tissue of control, FTLD-TDP type A, or FTLD-TDP type B. For each tissue type, images of total binding (total) and nonspecific binding (self-blocking) are shown. Figure 3b : Specific binding (total-nonspecific) in control, FTLD-TDP type A, and FTLD-TDP type B. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison correction. **p>0.01, *p<0.05. Figure 3c :The saturation binding curve of FTLD-TDP type A was determined by typical autoradiography. It shows [ 3 H]-specific binding of compound 1.

[0050] Figure 4: [ 3 Representative autoradiograph of [H]-compound 17. Figure 4a : Autoradiographic images on control, FTLD-TDP type A, or FTLD-TDP type B brain tissue. For each tissue type, images of total binding (total) and nonspecific binding (self-blocking) are shown. Figure 4b : Specific binding (total-nonspecific) in control, FTLD-TDP type A, and FTLD-TDP type B. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey correction for multiple comparisons. **p>0.01, *p<0.05. Figure 4c :The saturation binding curve of FTLD-TDP type A was determined by typical autoradiography. It shows [ 3 H]-specific binding of compound 17.

[0051] Figure 5: 3 H]-compound 1 and [ 3 H]-Binding specificity of compound 17 to human brain-derived extracts. Figure 5a-5b :[ 3 H]-compound 1 ( Figure 5a )or[ 3 H]-compound 17 ( Figure 5b )and[ 3[H]-Aβ saturation binding curves of reference compounds to AD brain homogenate. Figure 5c-5d :[ 3 H]-compound 1 ( Figure 5c )or[ 3 H]-Compound 17 ( Figure 5d Saturation binding curves of α-synuclein reference compounds to the insoluble fraction from PD brain. Figure 5e-5f :[ 3 H]-compound 1 ( Figure 5e )or[ 3 H]-Compound 17 ( Figure 5f )and[ 3 Saturation binding curves of [H]-tau reference compounds to tau PHF derived from AD brain.

[0052] Figure 6: Targeted engagement (emgagement) on AD brain tissue sections by autoradiography. 3 H]-compound 1 ( Figure 6a )and[ 3 H]-Compound 17 ( Figure 6b ) Classical autoradiography on Aβ- and Tau-enriched AD brain tissue sections. Scale bar is 2 mm. 3 H]-compound 1 ( Figure 6c )and[ 3 H]-Compound 17 ( Figure 6d ) of the microautoradiograph.

[0053] Figure 7: Non-human primate PK in whole monkey brain (iv). Figure 7a :use[ 18 F]-Compound 1. Figure 7b :use[ 18 F]-Compound 17.

[0054] definition

[0055] Unless otherwise defined, within the meaning of this application, the following definitions apply and, where appropriate, terms used in the singular also include the plural and vice versa:

[0056] The compounds of the present invention may have one or more optically active carbons, which may exist as racemates and racemic mixtures, stereoisomers (including diastereoisomer mixtures and single diastereoisomers, enantiomeric mixtures and single enantiomers, conformer mixtures and single conformers), tautomers, atropisomers and rotamers. All isomeric forms are included in the present invention. The compounds described in this specification containing olefin double bonds include E and Z geometric isomers. The present invention also includes all salt forms such as pharmaceutically acceptable salts, polymorphs, hydrates, solvates, prodrugs and mixtures thereof. Unless otherwise indicated, the term "compound of formula (I)" or "compound of the (present) invention" refers to "compound of formula (I) or its detectably labeled compound, stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate, or mixtures thereof". Unless otherwise indicated, the term "compound of formula (I)" or "compound of the present invention" refers to a compound of formula (I) or its subformulae, and isotopically labeled compounds thereof (including but not limited to 18 F and 3 H substituted). The term "compound of formula (I)" or "compound of the present invention" refers to a compound as defined in any one of the embodiments mentioned below.

[0057] The term "polymorph" refers to the various crystalline structures of the compounds of the invention. This may include, but is not limited to, crystalline forms (and amorphous materials) and all lattice forms. Salts may also be crystalline and may exist as more than one polymorph.

[0058] The present invention also includes solvates, hydrates and anhydrous forms of salts. The solvent contained in the solvate is not particularly limited and can be any pharmaceutically acceptable solvent. Examples include C 1-4 Alcohol (eg methanol or ethanol).

[0059] "Pharmaceutically acceptable salts" are defined as derivatives of the compounds of the invention wherein the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts 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. The pharmaceutically acceptable salts of the compound of formula (I) can be synthesized by conventional chemical methods from the parent compound containing an alkaline or acidic part. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two. Organic solvents include but are not limited to non-aqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, the disclosure of which is incorporated herein by reference. In general, pharmaceutically acceptable salts are salts of amine residues in the compounds of the invention.

[0060] The compounds of the invention may also be provided in the form of prodrugs, ie, compounds that are metabolized in vivo to active metabolites.

[0061] The "patient" or "individual" in the present invention is usually an animal, particularly a mammal, more particularly a human and a mouse, and even more particularly a human.

[0062] A "diagnostic composition" as defined in the present invention is a composition comprising a compound of the present invention in a form suitable for administration to a patient, wherein the patient is, for example, a mammal, such as a human.

[0063] "TDP-43 aggregates" are TDP-43-positive multimer-enriched assemblies of TDP-43. They can be found in intracellular deposits in a range of diseases called TDP-43 proteinopathies, particularly in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD), and age-related TDP-43 encephalopathy (LATE) mainly in the limbic system. TDP-43 aggregates can be found in the following morphologies: dense oval or crescent-shaped neuronal cytoplasmic inclusions (NCIs), bean-shaped neuronal nuclear inclusions (NIIs), glial cell cytoplasmic inclusions (GCIs), dystrophic neurites (DNs), and long tortuous neurites. In pathological aggregates, TDP-43 often exhibits a large increase in post-translational modifications, such as phosphorylation, ubiquitination, acetylation, SUMOylation, and proteolytic cleavage to produce C-terminal fragments.

[0064] The "preclinical state" of a disease is defined as the stage of the disease at which disease-associated changes at the molecular level do not result in overt clinical manifestations in the patient.

[0065] The “clinical state” of a disease is defined as the stage of the disease at which disease-related changes at the molecular level result in overt clinical manifestations in the patient.

[0066] The term "diagnosis" generally refers to the process or act of identifying, determining, or outlining a disease or condition in a patient based on symptoms, signs, and / or results of a diagnostic procedure.

[0067] The "normal control value" is determined by performing the respective method on a large number of healthy individuals, measuring the amount of the compound binding to the TDP-43 aggregate (if any) in each healthy individual, and calculating the average value thereof.

[0068] A "healthy control individual" or "healthy individual" is a person who does not show clinical evidence of a neurodegenerative disease. The person needs to meet the following criteria:

[0069] Healthy male and female individuals with no clinically relevant findings on physical examination.

[0070] No family history of TDP-43 proteinopathy, TDP-43 aggregate formation, or other early-onset neurological diseases associated with dementia.

[0071] No personal history of clinically significant neurological and / or psychiatric disorders.

[0072] No current clinical signs or symptoms of neurological deficits, such as cognitive impairment or motor deficits.

[0073] The "preclinical control value" is determined by performing the respective method on a large number of individuals in a preclinical state, measuring the amount of compound (if any) that binds to TDP-43 aggregates in each individual, and calculating the average value thereof.

[0074] The "clinical control value" is determined by performing the respective method on a large number of individuals in a clinical state, measuring the amount of compound (if any) binding to TDP-43 aggregates in each individual, and calculating the average value thereof.

[0075] The term "prediction" generally refers to an advance statement, indication, or prediction of a disease or condition in a patient who does not already have the disease, disorder, or abnormality. For example, a prediction of a disease, disorder, or abnormality in a patient may indicate the probability, chance, or risk that the patient will develop the disease, disorder, or abnormality, such as within a certain time period or at a certain age.

[0076] Detectable labels include suitable isotopes such as radioisotopes, particularly positron emitters or gamma emitters, and include 2 H. 3 H. 18 F. 123 I. 124 I. 125 I. 131 I. 11 C. 13 N. 15 O. 99m Tc and 77 Br, preferably 2 H. 3 H. 11 C. 13 N. 15 O and 18 F, more preferably 2 H. 3 H and 18 F, even more preferably 3 H and 18 F, best choice 18 F.

[0077] The term "Hal", "halogen" or "halo" refers to F, Cl, Br or I, particularly Br or I, more particularly Br.

[0078] The term "carbocycle" refers to a 5 or 6-membered carbocycle, without particular limitation, and includes any 5 or 6-membered, saturated or unsaturated carbocycle. Unsaturated carbocycles include, but are not limited to, aromatic rings. Examples of 5 or 6-membered carbocycles include, for example, phenyl, cyclopentyl, cyclohexyl, cyclopentenyl and cyclohexenyl. Phenyl is preferred.

[0079] The term "heterocycle" refers to a stable 5 or 6-membered heterocycle, without particular limitation, including any 5 or 6-membered, saturated or unsaturated heterocycle. Unsaturated heterocycles include, but are not limited to, aromatic rings. The heterocycle contains one or more heteroatoms (e.g., one or two heteroatoms) selected from N, O and S. The heteroatom is preferably N or O, more preferably N. Examples of 5 or 6-membered heterocycles include, for example, pyridyl, pyrimidyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuranyl, thienyl, imidazolidinyl, pyrazolidinyl, imidazolyl, pyrazolyl, oxathiolanyl, isooxathiolanyl, oxathiolene, isooxathiolene, thiazolidinyl, isothiazolidinyl, thiazolyl and isothiazolyl.

[0080] 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 other atoms or groups of atoms. Examples are given, for example, in 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 and others). (Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), In: Schubiger PA, Friebe M., Lehmann L. (eds.), PET-Chemistry-The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50, specifically: Scheme 4 on p. 25, Scheme 5 on p. 28, Table 4 on p. 30, Figure 7 on p. 33). Preferably, the "leaving group" (LG) is selected from C 1-4 Alkyl sulfonate group, C 6-10 More preferably, the leaving group (LG) is a mesylate group, a toluenesulfonate group, a nosylate group or a nitro group. Even more preferably, the leaving group (LG) is a mesylate group or a nitro group.

[0081] The term "detection" as used herein includes quantitative detection and / or qualitative detection.

[0082] The compounds of the invention can be used as assay references or as in vitro screening tools.

[0083] For example, the non-labeled compounds of formula (I) of the present invention can be used as the corresponding labeled compounds of the present invention (e.g., the corresponding 18 The quality control and release of the compound of formula (IF) or (I-F') labeled with F are carried out by an in vitro method.

[0084] The compounds of the invention can be used as in vitro screening tools for characterizing tissues with tau pathology and testing compounds that target tau pathology on such tissues.

[0085] Unless otherwise stated, the preferred definitions given in the "Definitions" section apply to all embodiments described below. Various embodiments of the invention are described herein, and it should be recognized that features specified in each embodiment can be combined with other specified features to provide further embodiments of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0087] Having described various embodiments of the invention, it should be recognized that features specified within each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0088] It will be understood that all definitions given for formula (I) apply to all subformulae thereof, including formulae (IH), (IF), (II), (III) and (IV).

[0089] In a first aspect, the present invention relates to a compound having the structure of formula (I)

[0090]

[0091] or a detectably labeled compound, stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof;

[0092] in

[0093] n is 1 or 2;

[0094] R 1 is H, hydroxy(C1-C4)alkyl or F; and

[0095] R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N.

[0096] In one embodiment, the present invention relates to compounds of formula (I)

[0097]

[0098] or a detectably labeled compound, stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof;

[0099] in

[0100] n is 1 or 2;

[0101] R 1 is H or F; and

[0102] R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N.

[0103] The present invention relates to compounds of formula (I), wherein n is 1 or 2. In a preferred embodiment, n is 1. In another preferred embodiment, n is 2.

[0104] The present invention relates to compounds of formula (I), wherein R 1 is H, hydroxy(C1-C4)alkyl or F. In one embodiment, R 1 is H or F. In a preferred embodiment, R 1 is H. In another preferred embodiment, R 1 is F. In another preferred embodiment, R 1 is a hydroxy(C1-C4)alkyl group. Preferred examples of the hydroxy(C1-C4)alkyl group are a hydroxymethyl group or a hydroxyethyl group, more preferably a hydroxymethyl group.

[0105] In a preferred embodiment, R 1 Selected from F, H and -CH2-OH.

[0106] The present invention relates to compounds of formula (I), wherein R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S.

[0107] Preferably, R 2 is a 5- or 6-membered carbocyclic ring, preferably an aryl ring, which may be optionally substituted by F, NH2, CN and / or CH3,

[0108] a 5-membered heteroaryl ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the 5-membered heteroaryl ring comprises one or more heteroatoms selected from N, O and S, or

[0109] A 6-membered heteroaryl ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the 6-membered heteroaryl ring comprises one or two heteroatoms selected from O, N and S.

[0110] R 2 Preferred examples of the ring are given in the above definition section. Preferably, R 2 is phenyl, pyridyl, pyrimidinyl, pyrazolyl, pyridazinyl, thiazolyl or isothiazolyl (such as phenyl, pyridyl, pyrimidinyl, pyrazolyl), any of which may be optionally substituted by F, NH2, CN and / or CH3, for example substituted by F, NH2 and / or CH3.

[0111] R 2 The ring can be optionally substituted with F, NH2, CN and / or CH3 (e.g., F, NH2 and / or CH3) at any available position. In one embodiment, the 5- or 6-membered carbocyclic or heterocyclic ring is substituted with one or more of F, NH2, CN and / or CH3 (e.g., F, NH2 and / or CH3).

[0112] In one embodiment, the invention relates to compounds of formula (I), wherein R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2 and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S. In a preferred embodiment, R 2 It is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted by F, NH2 and / or CH3 at any available position. Preferably, the 5- or 6-membered carbocyclic or heterocyclic ring is substituted by one or more of F, NH2 and / or CH3.

[0113] In one embodiment, the invention relates to compounds of formula (I), wherein R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted with F, NH 2、 CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S. In a preferred embodiment, R 2 It is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted by F, NH2, CN and / or CH3 at any available position. In another embodiment, the 5- or 6-membered carbocyclic or heterocyclic ring is substituted by one or more of F, NH2, CN and / or CH3.

[0114] In a preferred embodiment, phenyl is optionally substituted by F, NH2, and / or CN, preferably by CN and NH2 or by F and NH2. In another preferred embodiment, pyrazolyl is optionally substituted by CH3. In another preferred embodiment, pyridyl is optionally substituted by F or NH2. In another preferred embodiment, pyridyl is unsubstituted. In another more preferred embodiment, pyrimidinyl is unsubstituted. In another more preferred embodiment, isothiazolyl or thiazolyl is unsubstituted. In another more preferred embodiment, pyrazinyl is unsubstituted.

[0115] In a preferred embodiment, the invention relates to compounds of formula (I), wherein

[0116] R 2 Yes(i) in

[0117] R 3 It's F, R 4 is NH2, R 7 is H, and R 8 It is H.

[0118] R 3 is NH2, R 4 Yes F, R 7 is H, and R 8 It is H.

[0119] R 3 It is CN, R 4 is NH2, R 7 is H, and R 8 It is H.

[0120] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN,

[0121] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is F, or

[0122] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN.

[0123] In another preferred embodiment, the invention relates to compounds of formula (I), wherein

[0124] R 2Yes(ii) Where X is N, and R 5 is H or CH3. In a preferred embodiment, R 5 It is CH3.

[0125] In another preferred embodiment, the invention relates to compounds of formula (I), wherein

[0126] R 2 yes Where R 9 Selected from H, F, CH3 and NH2; preferably H;

[0127] or R 2 yes

[0128] or R 2 yes

[0129] or R 2 yes

[0130] or R 2 yes

[0131] or R 2 yes

[0132] or R 2 yes

[0133] or R 2 yes

[0134] or R 2 yes

[0135] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is F; and R 2 yes Where X is N, and R 5 It is CH3 or H, preferably CH3.

[0136] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H; and R 2 yes Where X is N, and R 5 It is CH3 or H, preferably CH3.

[0137] In a preferred embodiment, the compound of formula (I) is defined as n is 1; R 1 is F or H; and R2 yes or R 2 yes

[0138] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H; and R 2 yes Where R 3 Yes F, R 4 Yes -NH2, R 7 Yes H, R 8 It's H.

[0139] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is F; and R 2 yes Where R 3 Yes F, R 4 Yes -NH2, R 7 Yes H, R 8 It's H.

[0140] In one embodiment, the compound of formula (I) is defined as n is 1 or 2; R 1 is F; and R 2 yes Where R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN.

[0141] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H or F; and R 2 yes Where R 9 is selected from H, F, NH2 or CH3. In a preferred embodiment, R 9 is F. In a more preferred embodiment, R 9 It's H.

[0142] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H or F; and R 2 yes

[0143]

[0144] In another embodiment, the compound of formula (I) is defined as n is 1; R 1 is F; and R 2 yes

[0145]

[0146] In another embodiment, the compound of formula (I) is defined as n is 2; R 1 is F; and R 2 yes

[0147]

[0148] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H or F; and R 2 yes

[0149]

[0150] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H or F; and R 2 yes

[0151]

[0152] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is H or F; and R 2 yes

[0153]

[0154] In one embodiment, the compound of formula (I) is defined as n is 1; R 1 is F; and R 2 yes

[0155]

[0156] In one embodiment, the compound of formula (I) is defined as n is 2; R 1 is hydroxy(C1-C4)alkyl, preferably hydroxymethyl or hydroxyethyl, more preferably hydroxyethyl; and R 2 yes Where R 9 is selected from H, F, NH2 or CH3. In a preferred embodiment, R 9 It's H.

[0157] Preferred compounds of formula (I) include:

[0158]

[0159] In one embodiment, preferred compounds of formula (I) may be selected from the following stereoisomers:

[0160]

[0161] In one embodiment, the invention relates to compounds of formula (I) comprising a detectable label. Preferably, the compounds of formula (I) comprise one or more detectable labels.

[0162] The type of detectable label is not particularly limited and will depend on the selected detection method. Examples of possible detectable labels include isotopes such as radioisotopes (i.e., radionuclides), in particular, positron emitters or gamma emitters. Detectable labels such as radioisotopes, in particular positron emitters or gamma emitters, should be present in an amount that is not equivalent to the natural amount of each isotope. In addition, the amount used should be detectable by the selected detection method.

[0163] In a preferred embodiment, the detectable label is selected from 3 H and 18 F, best choice 18 F. The detectable label may be present at any available position. Typically, the detectable label is a radioactive isotope of an atom present in the compound of formula (I). For example, any reference to "F" in the present invention includes 19 F (stable) or 18 F (detectable label). Any reference to "H" includes 1 H (stable) or 3 H (a detectable label called tritium, denoted herein as "T").

[0164] Isotopic variants of the compounds of the present invention can generally be prepared by conventional methods, for example, by illustrative methods or by the preparation methods described in the examples and preparation examples below, using appropriate isotopic variants of suitable reagents, which are commercially available or prepared by known synthesis techniques. Radionuclides, particularly positron emitters and gamma emitters, can be included in the compounds of the present invention by methods commonly used in the field of organic synthesis. Typically, they are introduced by using corresponding labeled starting materials. Exemplary methods for introducing detectable labels are described in, for example, US 8,932,557, which is incorporated herein by reference.

[0165] 18 F can be connected at any location suitable for connecting F. 18 F-labeled compounds are particularly useful in imaging applications such as positron emission tomography (PET). Contains natural fluorine isotopes 19 The corresponding compounds of F are also of particular interest because they can be used as 18 Analytical standards and references during preparation, quality control, release and clinical use of F-analogues.

[0166] In the compound having the structure of formula (I),18 F can be used as R 2 The F substituent or as R 1 Preferably, it is present as R 1 exist.

[0167] if 3 H is used as a detectable label, which is preferably -CT3 (T refers to 3 H) in any position where a CH3 group can be attached. 3 H substitution may offer certain diagnostic advantages due to greater metabolic stability by reducing, for example, defluorination, increasing in vivo half-life, or reducing dosage requirements, while maintaining or improving the original compound's efficacy.

[0168] In one embodiment, the present invention relates to a tritium having the structure of formula (I) 3 H) detectably labeled compounds, as described above, wherein at least one hydrogen (H) is selected from tritium ( 3 H) can be detected by replacing the label. 3 A detectably labeled compound is preferably defined as one in which from 1 to 3 hydrogens (H) are replaced by tritium ( 3 H) is substituted. Tritium ( 3 A compound detectably labeled with H) is more preferably defined as one in which 2 or 3 hydrogens (H) are replaced by tritium ( 3 H) is substituted. Tritium ( 3 A detectably labeled compound is even more preferably defined as one in which the three hydrogens (H) are replaced by tritium ( 3 H) Replacement.

[0169] In one embodiment, the present invention provides a compound of formula (I) having a structure of formula (IT). In particular, the present invention relates to a tritium ( 3 H) Detectably labeled compounds

[0170]

[0171] or its stereoisomers, polymorphs, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates or solvates or mixtures thereof; wherein n, R 1 and R 2 As defined herein for compounds of formula (I); R 6 is T or H, and / or where R 2 Replaced by at least one CT3 or R 2 At least one hydrogen atom in the reaction is replaced by T, and / or R 1In one embodiment, at least one hydrogen atom in is replaced by T. 6 is T or H, and / or where R 2 Replaced by at least one CT3 or R 2 At least one hydrogen atom in is replaced by T. T is 3 H.

[0172] In one embodiment, R 6 is T. In another embodiment, R 2 In yet another embodiment, R 2 In a preferred embodiment, at least one hydrogen atom in R is replaced by T. 6 is T, and R 2 In yet another embodiment, at least one hydrogen atom in is replaced by T. 1 At least one hydrogen atom is replaced by T.

[0173] In a preferred embodiment, the invention relates to compounds of formula (IT), wherein

[0174] n is 1 or 2; preferably n is 1;

[0175] R 1 is H or F;

[0176] R 6 is T or H;

[0177] T is 3 H; and

[0178] in

[0179] R 2 Yes(i) Where R 3 Yes F, R 4 is -NH2, and R 7 and R 8 At least one of them is T and, if applicable, the other is H;

[0180] Preferably, R 7 is T; and R 8 is T; or

[0181] R 2 Yes(i) Where R 8 It is CN, R 4 is -NH2, and R 7 and R 3 At least one of them is T, and, if applicable, another is H; in one embodiment, R 7 is T; and R 3is T; in another embodiment, R 7 is T; and R 3 It's H.

[0182] R 2 Yes(ii) Where X is N, and R 5 is tritiated CH3 (CT3); or

[0183] R 2 Yes(ii) or

[0184] R 2 Yes(ii) or

[0185] R 2 (iii) Selected from Where R 9 is selected from H, F, NH2 or CH3; or

[0186] R 2 yes Where R 9 Selected from H, F, NH2 or CH3, preferably NH2 or F;

[0187] or R 2 yes or

[0188] In one embodiment, the invention relates to compounds of formula (IT),

[0189]

[0190] or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof;

[0191] in

[0192] n is 1 or 2;

[0193] R 1 is H, hydroxy(C1-C4)alkyl or F;

[0194] T is 3 H; and

[0195] R 2 (i) Where R 3 Yes F, R 4 is NH2, and R 7 and R 8At least one of them is T, and if applicable, another one is H; preferably, R 7 is T, and R 8 is T; and

[0196] R 6 It is T;

[0197] or among them

[0198] R 2 Yes(i) Where R 4 is NH2, R 8 is CN, and R 3 or R 7 At least one of them is T, and if applicable, another one is H; preferably, R 7 is T, and R 3 is T; or R 7 is T, and R 3 is H; and

[0199] R 6 is T; or

[0200] in

[0201] R 2 Yes(ii) Where X is N, and R 5 is CT3; and

[0202] R 6 is H; or

[0203] R 2 Yes(iii)

[0204] Where R 12 It is T;

[0205] or among them

[0206] R 2 Yes(iii)

[0207] Where R 12 It's T.

[0208] or among them

[0209] R 2 Yes(iii)

[0210] Where R 12 It's T.

[0211] or among them

[0212] R 2 Yes(iii)

[0213] Where R 12 It's T.

[0214] In another embodiment, the invention relates to compounds of formula (IT), wherein

[0215] n is 1 or 2; preferably n is 2;

[0216] R 1 is H or F (preferably H);

[0217] R 6 is T; and

[0218] R 2 yes Where R 3 It is F; R 4 is NH2; and R 7 and R 8 At least one of them is T and, if applicable, the other is H.

[0219] Preferably R 7 It is T; R 8 It is T;

[0220] or

[0221] R 2 Yes(i) Where R 8 It is CN, R 4 is NH2, and R 7 and R 3 At least one of them is T and, if applicable, the other is H;

[0222] In one embodiment, R 7 is T; and R 3 is T; or in another embodiment, R 7 is T; and R 3 It is H;

[0223] R 2 Yes(ii) Where X is N, and R 5 is tritiated CH3 (CT3); or

[0224] R 2 Yes(ii) or

[0225] R 2 Yes(ii) or

[0226] R 2 (iii) is selected from Where R 9 Selected from H, F, NH2 or CH3; Selected from Selected from or R 2 yes or Where R 9 is selected from H, F, NH2 or CH3, preferably NH2 or F; or R 2 yes or In one embodiment, the invention relates to compounds of formula (IT), wherein

[0227] n is 1 or 2; preferably n is 2;

[0228] R 1 It is F;

[0229] R 6 is H; and

[0230] R 2 yes Where X is N, and R 5 It is tritiated CH3 (CT3).

[0231] In one embodiment, the invention relates to compounds of formula (IT), wherein

[0232] n is 1 or 2; preferably n is 2;

[0233] R 1 is H or F;

[0234] R 6 is T; and

[0235] R 2 Yes(iii) Where R 12 It's T.

[0236] In one embodiment, the invention relates to compounds of formula (IT), wherein

[0237] n is 1 or 2; preferably n is 2;

[0238] R 1 is H or F;

[0239] R 6 is T; and

[0240] R 2 Yes(iii)

[0241] In one embodiment, the invention relates to compounds of formula (IT), wherein

[0242] n is 1 or 2; preferably n is 2;

[0243] R 1 is H or F;

[0244] R 6 is T; and

[0245] R 2 Yes(iii)

[0246] In another embodiment, the invention relates to compounds of formula (IT), wherein

[0247] n is 1 or 2;

[0248] R 1 is H or F, preferably H;

[0249] R 6 is T; and

[0250] R 2 Yes(iii) or Where R 9 is selected from H, F, NH2 or CH3, preferably NH2 or F; or R 2 yes or

[0251] In the most preferred embodiment, the present invention relates to compounds of formula (IT), wherein

[0252] n is 1 or 2; preferably n is 2;

[0253] R 1 is hydroxy(C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl, wherein R 1 At least one hydrogen atom is replaced by T; preferably R 1 The two hydrogen atoms are replaced by T;

[0254] R 6 is T or H;

[0255] T is 3 H; and

[0256] in

[0257] R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N.

[0258] According to the preferred tritium of formula (IT) of the present invention3 H) detectably labeled compounds include (wherein T means 3 H):

[0259]

[0260]

[0261] In a preferred embodiment, the tritium ( 3 H) The detectably labeled compound may be a stereoisomer (where T means 3 H)

[0262]

[0263] In a more preferred embodiment, the tritium ( 3 H) The detectably labeled compound may be a stereoisomer (where T means 3 H)

[0264]

[0265] In another more preferred embodiment, the tritium ( 3 H) The detectably labeled compound may be a compound (wherein T means 3 H)

[0266]

[0267] In one embodiment, the present invention provides a compound of formula (IF) 18 F detectably labeled compound

[0268]

[0269] or its stereoisomers, polymorphs, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates or solvates or mixtures thereof; wherein n, R 1 and R 2 As defined herein for compounds of formula (I), and at least one F is 18 F.

[0270] In a preferred embodiment, the invention relates to compounds of formula (IF), wherein R 1 yes 18 F (detectable label).

[0271] In another preferred embodiment,

[0272] n is 1 or 2;

[0273] R 1 yes 18 F (detectable label); and

[0274] R 2 yes Where X is N, and R 5 is CH3 or H, preferably CH3; or

[0275] R 2 yes or R 2 yes or R 2 Yes(i) in

[0276] R 3 Yes F, R 4 is NH2, R 7 is H, and R 8 is H; or

[0277] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is CN; or

[0278] R 2 yes Where R 9 is selected from H, and F, NH2 or CH3; or

[0279] R 2 yes or

[0280] R 2 yes or

[0281] R 2 yes or

[0282] R 2 yes or

[0283] R 2 yes or

[0284] R 2 yes

[0285] In a preferred embodiment, the invention relates to compounds of formula (IF), wherein

[0286] n is 1 or 2;

[0287] R 1 yes18 F (detectable label);

[0288] R 2 yes Where R 9 It's H.

[0289] In a preferred embodiment, n is 1. In another preferred embodiment, n is 2.

[0290] In another embodiment, (IF) is the compound

[0291]

[0292] or its stereoisomers, polymorphs, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates or solvates or mixtures thereof; wherein n, R 1 and R 2 As defined herein for compounds of formula (I).

[0293] In another preferred embodiment, (IF) is the following compound

[0294]

[0295] Where n is 1 or 2;

[0296] R 1 is hydroxy(C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl; and

[0297] R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N.

[0298] In a preferred embodiment,

[0299] R 2 yes Where X is N, and R 5 is CH3 or H, preferably CH3; or

[0300] R 2 Yes(i) in

[0301] R 3 Yes F, R 4 is NH2, R 7 is H, and R 8 is H; or

[0302] R 3 Yes H, R4 is NH2, R 7 is H, and R 8 is CN; or

[0303] R 2 yes Where R 9 is selected from H and F, NH2 or CH3; or

[0304] R 2 yes or

[0305] R 2 yes or

[0306] R 2 yes or

[0307] R 2 yes or

[0308] R 2 yes or

[0309] R 2 yes

[0310] In a preferred embodiment, n is 1 or 2, preferably 2;

[0311] R 1 is hydroxy(C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl; and R 2 yes Where R 9 Selected from H and F, NH2 or CH3, preferably H.

[0312] In one embodiment, n is 1. In a preferred embodiment, n is 2.

[0313] In a preferred embodiment, the present invention relates to compounds of formula (IF),

[0314]

[0315] in

[0316] n is 1 or 2, preferably 2;

[0317] R 1 is hydroxy(C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl; and

[0318] R 2 yes Where R9 It's H.

[0319] According to the preferred formula (IF) of the present invention 18 The detectably labeled compound of F may be selected from

[0320]

[0321] More preferably, according to formula (IF) of the present invention 18 The detectably labeled compound of F may be a stereoisomer

[0322]

[0323] Other preferred compounds of formula (IF) according to the present invention are 18 F detectably labeled compounds can make

[0324]

[0325] Diagnostic composition

[0326] In a second aspect, the present invention relates to a diagnostic composition comprising a compound of formula (I) as described above and optionally at least one physiologically acceptable carrier, diluent, adjuvant and / or excipient.

[0327] The compounds of the invention are particularly suitable for imaging TDP-43 aggregates. The imaging may be performed in a mammal, preferably in a human. 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 brain imaging. The imaging may also be ocular / retinal imaging or imaging of tissues of the central nervous system.

[0328] The compounds of the invention are particularly suitable for diagnosis. Diagnosis can be performed in mammals, preferably in humans. In the diagnosis performed, the tissue of interest can be brain tissue, tissue of the central nervous system, tissue of the eye (e.g., retinal tissue) or other tissue, or body fluid, such as cerebrospinal fluid (CSF). The preferred tissue is brain tissue.

[0329] The "diagnostic composition" defined in the present invention is a composition comprising one or more compounds of the present invention in a form suitable for administration to a patient (e.g., a mammal, such as a human), and is suitable for diagnosing a specific disease, disorder or abnormality in a tissue. In one embodiment, the diagnostic composition comprises a detectably labeled compound of the present invention as described above and optionally at least one physiologically acceptable carrier, diluent, adjuvant and / or excipient.

[0330] Preferred detectably labeled compounds of the invention are compounds of formula (IT) or (IF).

[0331] As defined below, the diagnostic composition is suitable for the diagnosis of diseases, disorders or abnormalities associated with TDP-43 aggregates or TDP-4 protein diseases. Preferably, the diagnostic composition also comprises an optional physiologically acceptable excipient, carrier, diluent or adjuvant. Administration is preferably performed as defined below. More preferably, the composition is injected as an aqueous solution. The diagnostic composition may optionally comprise additional ingredients, such as a buffer; a pharmaceutically acceptable solubilizing agent (e.g., a cyclodextrin or a surfactant, such as pluronic, tween or phospholipid); and a pharmaceutically acceptable stabilizer or antioxidant (e.g., ascorbic acid, gentisic acid or p-aminobenzoic acid). The dosage of the compound of the invention will vary depending on the specific compound administered, the patient's weight and other variables apparent to a skilled clinician in the art.

[0332] Although it is possible to administer the compounds of the invention alone, it is preferred that they be formulated as diagnostic compositions according to standard pharmaceutical practice. Therefore, diagnostic compositions are part of the present invention, comprising a diagnostically effective amount of the compounds of the invention in combination with a pharmaceutically acceptable carrier, diluent, adjuvant and / or excipient. Preferred pharmaceutically acceptable carriers, diluents, adjuvants and / or excipients are physiologically compatible with the diagnostic compositions according to the invention.

[0333] Pharmaceutically acceptable excipients are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 18th edition (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990). Pharmaceutically acceptable excipients can be selected according to the intended route of administration and standard pharmaceutical practice. An excipient must be acceptable in the sense of not being harmful to the recipient thereof.

[0334] Pharmaceutically useful excipients, carriers, adjuvants and diluents that can be used in the preparation of 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 glycols, 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 point waxes and ion exchange resins.

[0335] The administration (delivery) route of the compounds of the present invention includes, but is not limited to, one or more of the following: intravenous, gastrointestinal, intraspinal, intraperitoneal, intramuscular, oral (e.g., as tablets, capsules, or as ingestible solutions), topical, mucosal (e.g., as nasal sprays or aerosols for inhalation), nasal, parenteral (e.g., by injectable form), intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ocular (including intravitreal or intracameral), transdermal, rectal, buccal, epidural, and sublingual. Preferably, the administration (delivery) route of the compounds of the present invention is parenteral.

[0336] If a compound of the invention (e.g., a detectably labeled compound, such as one having 3 H or 18 F) is administered parenterally, then examples of such routes of administration include one or more of the following: intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular or subcutaneous and / or using infusion techniques. For parenteral administration, the compound is preferably used in the form of a sterile aqueous solution that may contain other excipients. If necessary, the aqueous solution should be appropriately buffered (preferably to a pH of 3-9). The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0337] Typically, a physician will determine the actual dosage that will be most suitable for an individual patient. 3 H or 18 The dosage of the compound (F detectably labeled compound) will vary depending on the specific compound administered, the patient's weight, the size and type of the sample, and other variables apparent to a physician skilled in the art. Typically, the dosage preferably ranges from 0.001 μg / kg to 10 μg / kg, preferably 0.01 μg / kg to 1.0 μg / kg. The radioactive dose may be, for example, 100 to 600 MBq, more preferably 150 to 450 MBq.

[0338] Due to their design and binding properties, the compounds of the invention as defined herein may be used in the diagnosis of diseases, disorders and abnormalities associated with TDP-43 aggregates. The compounds of the invention are particularly useful for positron emission tomography imaging of TDP-43 aggregates.

[0339] As disclosed herein, the compounds of the invention are particularly useful for the diagnosis of diseases, disorders and abnormalities associated with TDP-43 aggregates or TDP-43 proteinopathies, such as diseases, disorders or abnormalities selected from, but not limited to, frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, associated with chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD ) include frontotemporal dementia TDP-43 or frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral FTD (bvFTD), nonfluent variant primary progressive aphasia (e.g., nfvPPA), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiopoietin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy, Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP) mutations; also known as Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with rimmed vacuoles, sarcomere protein Preferably, the disease, disorder or abnormality associated with TDP-43 aggregation or TDP-43 proteinopathy is selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE) and limbic system-dominant age-related TDP-43 encephalopathy (LATE).

[0340] In one embodiment, the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

[0341] In one embodiment, the diagnosis of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy is Alzheimer's disease (AD).

[0342] In one embodiment, the diagnosis of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy is frontotemporal dementia (FTD), including frontotemporal dementia TDP-43 or frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP).

[0343] In one embodiment, the diagnosis of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy is limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0344] Methods and uses

[0345] In a third aspect, the present invention relates to the methods and uses listed below

[0346] A method of imaging a disease, disorder or abnormality associated with TDP-43 aggregates in a subject;

[0347] Methods for imaging TDP-43 aggregates in tissues of an individual using positron emission tomography (PET);

[0348] Methods for detecting and optionally quantifying TDP-43 aggregates in tissues of an individual;

[0349] Methods for diagnostic imaging of the brain of an individual;

[0350] A method for determining the amount of TDP-43 aggregates in a sample or a specific body part or body region;

[0351] Methods for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or diagnosing a TDP-43 proteinopathy;

[0352] Methods of collecting data for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy;

[0353] Methods for collecting data for determining a disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition to a TDP-43 proteinopathy;

[0354] Methods for collecting data for monitoring the progression of a disease, disorder or abnormality associated with TDP-43 aggregates or for monitoring the progression of a TDP-43 proteinopathy in a patient;

[0355] Methods of collecting data for predicting the response of a patient suffering from a disease, disorder or abnormality associated with TDP-43 aggregates to a drug treatment;

[0356] Use of the compounds of the present invention as biomarkers of TDP-43 aggregates or biomarkers of TDP-43 proteinopathy,

[0357] Use of the compound of the present invention as a diagnostic reagent or diagnostic tool for TDP-43 protein disease.

[0358] - Use of the compounds of the invention as in vitro analytical references or in vitro screening tools.

[0359] Any compound of the invention (e.g., a compound of formula (I), (IT) or (IF)) may be used in the methods outlined above. Preferably, the compound is a detectably labeled compound (e.g., having 3 H or 18 F detectably labeled compound).

[0360] The methods of the present invention may include the steps of contacting a sample, a specific body part or a body region suspected of containing TDP-43 aggregates with a compound of the present invention;

[0361] The body is preferably mammalian, more preferably human, and includes the entire body or a body region / part of a patient suspected of containing TDP-43 aggregates.

[0362] The sample may be selected from a tissue or body fluid suspected of containing TDP-43 aggregates, and the sample is obtained from a patient. Preferably, the tissue is selected from a tissue of the central nervous system (CNS), an eye tissue or a brain tissue, more preferably a brain tissue. Examples of body fluids include cerebrospinal fluid (CSF) or blood. The sample may be obtained from a mammal, more preferably a human. Preferably, the sample is an in vitro sample obtained from a patient.

[0363] In vitro samples or specific body parts or regions obtained from a patient can be contacted with the compounds of the invention by direct incubation.

[0364] In the in vitro method, the compound of the invention is contacted with a specific body part or body region by administering to the patient a therapeutically effective amount of the compound of the invention. An effective amount of the compound of the invention is an amount suitable to allow determination of the presence or absence of TDP-43 aggregates in a specific body part or body region using a selected analytical technique.

[0365] The step of allowing the compounds of the invention to bind to TDP-43 aggregates includes allowing the binding to occur for a sufficient time. The amount of time required for binding will depend 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 tests. In in vitro methods, the amount of time will depend on the sample or specific body part or body region, and the range can be, for example, about 30 minutes to about 120 minutes. In in vivo methods, the amount of time will depend on the time required for the compounds of the invention to reach a specific body part or body region suspected of containing TDP-43 aggregates. The amount of time should not be too long to avoid washout and / or metabolism of the compounds of the invention. The range of duration can be, for example, from about 0 minutes to about 240 minutes (PET scan duration during initial compound characterization (NHP PET and subsequent FiH-studies)).

[0366] The method of detecting the compounds of the present invention bound to TDP-43 aggregates is not particularly limited, but depends on the detectable marker, the sample type, 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, fluorescent 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). Fluorescent imaging techniques and / or nuclear imaging techniques can be used to monitor and / or visualize the distribution of the compounds of the present invention in a sample or body. The imaging system thus provides an image of a detectable marker (e.g., a radioisotope, particularly a positron emitter or a gamma emitter) present in a test sample, a specific body part tested, or a body region tested. Preferably, the compounds of the present invention bound to TDP-43 aggregates are detected by an imaging device such as a PET or SPECT scanner. The amount of the compound bound to TDP-43 can be determined by visual or quantitative analysis, for example using a PET scan image.

[0367] In one embodiment, the presence or absence of a compound of the invention that binds to TDP-43 aggregates can be correlated with the presence or absence of TDP-43 aggregates in a sample or a specific body part or body region. The correlation can be qualitative or quantitative. In a preferred embodiment, the step comprises:

[0368] - determining the amount of the compound of the invention bound to TDP-43 aggregates;

[0369] - correlating the amount of a compound of the invention that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and

[0370] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0371] The amount of compound bound to TDP-43 aggregates can be determined by any suitable method. A preferred method is positron emission tomography (PET).

[0372] In another embodiment, the presence or absence of a compound of the invention that binds to TDP-43 aggregates can be correlated with a disease, disorder or abnormality associated with TDP-43 aggregates or with a TDP-43 proteinopathy or predisposition thereof. The correlation can be qualitative or quantitative. In a preferred embodiment, the step comprises:

[0373] - determining the amount of the compound of the invention bound to TDP-43 aggregates;

[0374] - correlating the amount of a compound of the invention that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and

[0375] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0376] In any of the methods disclosed herein, steps (a) to (c) and, if present, optional step (d) may be repeated at least once. The repetition of steps is particularly useful in methods for collecting data for monitoring progress and methods for collecting data for predicting responsiveness. 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 may be determined by a physician based on the severity of the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy.

[0377] In one embodiment, the present invention relates to a method for detecting a neurological disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition thereof in an individual, the method comprising the steps of:

[0378] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0379] (b) allowing the compound to bind to TDP-43 aggregates; and

[0380] (c) Detection of compounds binding to TDP-43 aggregates.

[0381] In one embodiment, the invention relates to a method (e.g., an in vivo or in vitro method) for detecting and optionally quantifying TDP-43 aggregates in a tissue of an individual, the method comprising the steps of:

[0382] (a) administering to a subject a compound of the invention as disclosed herein; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0383] (b) allowing the compound to bind to TDP-43 aggregates; and

[0384] (c) Detecting and optionally quantifying compounds binding to TDP-43 aggregates using positron emission tomography.

[0385] In one embodiment, the invention relates to a method of diagnostically imaging the brain of an individual, the method comprising the steps of:

[0386] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0387] (b) allowing the compound to bind to TDP-43 aggregates; and

[0388] (c) Compounds binding to TDP-43 aggregates are detected by collecting positron emission tomography (PET) images of the brain of the individual.

[0389] Imaging:

[0390] The present invention relates to methods for imaging TDP-43 aggregates using the compounds of the present invention. For example, imaging may be performed using any of the above methods, in particular by PET.

[0391] In one embodiment, the invention relates to a method for imaging TDP-43 aggregates in a sample or a patient, in particular in the brain or in a sample taken from the brain of a patient, the method comprising the following steps:

[0392] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0393] (b) allowing the compound to bind to TDP-43 aggregates; and

[0394] (c) Detection of compounds binding to TDP-43 aggregates.

[0395] In one embodiment, the invention relates to a method of imaging or diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition thereof in an individual, the method comprising the steps of:

[0396] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0397] (b) allowing the compound to bind to TDP-43 aggregates; and

[0398] (c) Detecting compounds that bind to TDP-43 aggregates in the brain of an individual.

[0399] In one embodiment, the invention relates to a method of imaging or diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition thereof in an individual, the method comprising the steps of:

[0400] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0401] (b) allowing the compound to bind to TDP-43 aggregates; and

[0402] (c) Detection of compounds binding to TDP-43 aggregates.

[0403] In one embodiment, the invention relates to a method of imaging or diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a predisposition thereof in an individual, the method comprising the steps of:

[0404] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0405] (b) allowing the compound to bind to TDP-43 aggregates;

[0406] (c) detecting compounds that bind to TDP-43 aggregates; and

[0407] (d) Generating an image representing the location and / or amount of compound bound to TDP-43 aggregates.

[0408] In one embodiment, the present invention relates to a method for imaging TDP-43 aggregates in a tissue of an individual by positron emission tomography (PET), the method comprising the steps of:

[0409] (a) administering to a subject a compound of the invention; or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0410] (b) allowing the compound to bind to TDP-43 aggregates; and

[0411] (c) Compounds binding to TDP-43 aggregates are detected by collecting positron emission tomography (PET) images of tissue from the individual.

[0412] Preferably, the tissue is tissue of the central nervous system (CNS), eye tissue or brain tissue. More preferably, the tissue is brain tissue.

[0413] In one embodiment, the present invention relates to a method of imaging TDP-43 aggregates in a sample or a patient, the method comprising the steps of:

[0414] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0415] (b) allowing the compound to bind to TDP-43 aggregates; and

[0416] (c) detecting compounds that bind to TDP-43 aggregates by imaging the sample, specific body part or body region using an imaging system.

[0417] In one embodiment, the present invention relates to a method of imaging TDP-43 aggregates in an in vitro sample of a patient, the method comprising the steps of:

[0418] (a) contacting an in vitro sample suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0419] (b) allowing the compound to bind to TDP-43 aggregates; and

[0420] (c) Compounds binding to TDP-43 aggregates were detected by imaging in vitro samples using an imaging system.

[0421] In one embodiment, the present invention relates to a method of imaging TDP-43 aggregates in a patient or a specific body part or body region of a patient, the method comprising the steps of:

[0422] (a) contacting a sample or a specific body part or body area suspected of containing TDP-43 aggregates with a compound of the invention, preferably a compound of formula (IT) or formula (IF); or a diagnostic composition as disclosed herein comprising a compound of the invention, preferably a compound of formula (IT) or formula (IF);

[0423] (b) allowing the compound to bind to TDP-43 aggregates; and

[0424] (c) detecting a compound that binds to TDP-43 aggregates by imaging a specific body part or region of a sample or a patient using an imaging system.

[0425] The step of imaging a sample, a patient, a specific body part or a body region of a patient using an imaging system comprises detecting a compound of the invention bound to TDP-43 aggregates using an imaging system as disclosed herein. The compound of the invention bound to TDP-43 aggregates is detected, and the distribution of TDP-43 aggregates in the test sample, patient, specific body part or body region is identified by imaging. PET imaging should be performed when the compound has penetrated the tissue and the compound has bound to TDP-43 aggregates.

[0426] Determine the amount of TDP-43 aggregates:

[0427] In one embodiment, the invention relates to a method of determining the amount of TDP-43 aggregates in a sample, a specific body part or a body region suspected of containing TDP-43 aggregates using the compounds of the invention.

[0428] In one embodiment, the present invention provides a method for determining the amount of TDP-43 aggregates in a sample, a specific body part or a body region suspected of containing TDP-43 aggregates, wherein the method comprises the following steps:

[0429] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0430] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0431] (c) detecting the compound of the present invention binding to TDP-43 aggregates;

[0432] (d) determining the amount of the compound of the invention bound to TDP-43 aggregates; and

[0433] (e) optionally calculating the amount of TDP-43 aggregates in the sample, a particular body part or a body region.

[0434] When comprising at least one radiolabeled atom (e.g. 3 H. 2 H or 18 F) When the detectably labeled compound of the present invention binds to TDP-43 aggregates, a radioactive signal is observed.

[0435] diagnosis:

[0436] In one embodiment, the present invention relates to a method for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy or a predisposition thereto, the method comprising the steps of:

[0437] (a) detecting a compound of the present invention binding to TDP-43 aggregates; and

[0438] (b) correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates with the presence or absence of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0439] Preferably, the method for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy or a predisposition thereto comprises the following steps:

[0440] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0441] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0442] (c) detecting the compound of the present invention binding to TDP-43 aggregates; and

[0443] (d) correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates with the presence or absence of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0444] In one embodiment, the invention relates to a method for collecting data for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy or a predisposition thereto, the method comprising the steps of:

[0445] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0446] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0447] (c) detecting the compound of the present invention binding to TDP-43 aggregates; and

[0448] (d) optionally correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or body region.

[0449] After contacting the sample or specific body part or body region with the compound of the invention, the compound is allowed to bind to the TDP-43 aggregates. The amount of time required for binding will depend on the type of test (e.g., in vitro or in vivo) and can be determined by a person skilled in the art through routine experiments. Subsequently, the compound bound to the TDP-43 aggregates can be detected by any appropriate method. The specific method selected will depend on the detectable label selected. Examples of possible methods include, but are not limited to, fluorescent 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). Fluorescent imaging techniques and / or nuclear imaging techniques can be used to monitor and / or visualize the distribution of detectably labeled compounds in samples or specific body parts or body regions.

[0450] As described above, optionally the step of correlating the presence or absence of a compound that binds to TDP-43 aggregates with the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region comprises the following steps:

[0451] - determining the amount of compound bound to TDP-43 aggregates;

[0452] - correlating the amount of the compound that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and

[0453] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0454] The amount of the compound that binds to TDP-43 aggregates can be compared to a normal control value determined in a sample or a specific body part or body region of a healthy individual, wherein an increase in the amount of the compound that binds to TDP-43 aggregates compared to the normal control value can indicate that the patient suffers from or is at risk of developing a disease, disorder or abnormality associated with TDP-43 aggregates.

[0455] If the amount of compound that binds to TDP-43 aggregates is above a normal control value, as defined herein, then the patient can be expected to suffer from or be at risk of suffering from a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0456] Determine the tendency:

[0457] Another aspect of the invention relates to a method for collecting data for determining a predisposition to a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy. The method comprises the following steps:

[0458] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0459] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0460] (c) detecting the compound of the present invention binding to TDP-43 aggregates; and

[0461] (d) optionally correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or body region.

[0462] As described above, optionally the step of correlating the presence or absence of a compound that binds to TDP-43 aggregates with the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region comprises the following steps:

[0463] - determining the amount of compound bound to TDP-43 aggregates;

[0464] - correlating the amount of the compound that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and

[0465] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0466] If the amount of the compound that binds to TDP-43 aggregates is higher than the normal control value for a healthy / reference individual, this indicates that the patient / individual suffers from or is at risk of developing a disease, disorder or abnormality associated with TDP-43 aggregates. In particular, if the amount of the compound that binds to TDP-43 aggregates is higher than the value in a person who does not show clinical evidence of a neurodegenerative disease, the patient can be considered to have a predisposition to a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0467] Monitoring disease progression:

[0468] In one embodiment, the invention relates to a method for monitoring the progression of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-4 proteinopathy in a patient. Typically, the patient is undergoing or has undergone treatment for a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy. In particular, treatment may involve the administration of an anti-TDP-43 drug.

[0469] A method of collecting data for monitoring the progression of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy in a patient, the method comprising the steps of:

[0470] (a) contacting a sample, a specific body part or a body region suspected of containing TDP-43 aggregates with a compound of the present invention;

[0471] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0472] (c) detecting the compound of the present invention binding to TDP-43 aggregates;

[0473] (d) optionally correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region; and

[0474] (e) optionally repeating at least once: steps (a) to (c) and optionally step (d) if present.

[0475] To monitor the progression of a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy over time, steps (a) to (c) and optionally step (d) (if present) may be repeated one or more times. Preferably, the steps should be repeated until no further progression of the disease is observed in the patient.

[0476] As described above, optionally the step of correlating the presence or absence of a compound that binds to TDP-43 aggregates with the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region comprises the following steps:

[0477] - determining the amount of compound bound to TDP-43 aggregates;

[0478] - correlating the amount of the compound that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region;

[0479] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0480] In methods for monitoring progression over time, the amount of the compound of the invention bound to TDP-43 aggregates can optionally be compared at multiple time points during treatment, for example, before and after the start of treatment and / or at multiple time points after the start of treatment. A change, in particular a decrease, in the amount of the compound of the invention bound to TDP-43 aggregates can indicate that the disease is not progressing.

[0481] Predicting Responsiveness:

[0482] In one embodiment, the invention relates to a method of predicting the responsiveness of a patient suffering from a disease, disorder or abnormality associated with TDP-43 aggregates or suffering from a TDP-43 proteinopathy to a treatment of said disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0483] The method can be used to predict the most appropriate treatment for a patient. In particular, the treatment can involve the administration of an anti-TDP-43 drug.

[0484] A method for predicting responsiveness of a patient suffering from a disease, disorder or abnormality associated with TDP-43 aggregates or suffering from a TDP-43 proteinopathy to a treatment for the disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy may comprise the following steps:

[0485] (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound of the invention, or a diagnostic composition comprising a compound of the invention as disclosed herein;

[0486] (b) allowing the compound of the present invention to bind to TDP-43 aggregates;

[0487] (c) detecting the compound of the present invention binding to TDP-43 aggregates;

[0488] (d) optionally correlating the presence or absence of a compound of the invention that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region; and

[0489] (e) optionally repeating at least once: steps (a) to (c) and optionally step (d) if present.

[0490] Typically, the patient is / has been treated for a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy. In particular, treatment may involve the administration of a drug suitable for treating a disease, disorder or abnormality associated with TDP-43 aggregates.

[0491] The present method can predict the responsiveness of a patient to a certain treatment. In one embodiment, for example, the responsiveness is estimated by repeating steps (a) to step (c) and, if present, the optional step (d) and monitoring the amount of the compound of the invention bound to TDP-43 aggregates over a period of time while the patient is experiencing a disease, disorder or abnormality associated with TDP-43 aggregates or a treatment for a TDP-43 proteinopathy. If the amount changes over time, a skilled physician can infer whether the patient is responsive to the treatment. Generally, if the amount of the compound of the invention bound to the TDP-43 aggregates decreases over time, the patient can be considered to be responsive to the treatment. Generally, if the compound bound to the TDP-43 aggregates is substantially constant or increases over time, the patient can be considered to be unresponsive to the treatment.

[0492] Alternatively, responsiveness can be estimated by determining the amount of the compound of the invention that binds to TDP-43 aggregates. The amount of the compound that binds to TDP-43 aggregates can be compared to a control value, such as a normal control value, a preclinical control value, or a clinical control value. The control value may refer to a control value for a healthy control individual. Alternatively, the control value may refer to a control value for an individual known to respond to a therapy, or to a control value for an individual known to be unresponsive to a therapy. The result about responsiveness may be "responsive" to a therapy, "unresponsive" to a therapy, or "undetermined response" to a therapy. For different patients, the response to treatment may be different.

[0493] As described above, optionally the step of correlating the presence or absence of a compound that binds to TDP-43 aggregates with the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region comprises the following steps:

[0494] - determining the amount of compound bound to TDP-43 aggregates;

[0495] - correlating the amount of the compound that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and

[0496] - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

[0497] A control value can be, for example, a normal control value, a preclinical control value, and / or a clinical control value.A "healthy control individual" or "healthy individual" is a person who does not show clinical evidence of a neurodegenerative disease.

[0498] If in any of the methods outlined above, the amount of compound that binds to TDP-43 aggregates is above a normal control value, then the patient can be expected to suffer from or be at risk of suffering from a disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy.

[0499] Any of the compounds of the invention may be used in any of the methods outlined above.Preferably, a detectably labeled compound of the invention as disclosed herein is used in the methods outlined above.

[0500] In a fourth aspect, the present invention relates to the use of compounds of the present invention as diagnostic agents or diagnostic tools for TDP-43 aggregates. In one embodiment, the present invention relates to the use of compounds of the present invention as in vitro analytical references or in vitro screening tools. The compounds of the present invention may also be used in in vivo diagnostic methods. In this case, the compounds of the present invention may be detectably labeled compounds or contain cold isotopes.

[0501] In another embodiment, the present invention also relates to the use of the compounds of the present invention, more particularly, the detectably labeled compounds of the present invention as defined herein as diagnostic biomarkers to enable more efficient and more accurate patient selection, such as longitudinal monitoring in clinical studies or to support the development of new therapies for the treatment of TDP-43 proteinopathy. In another embodiment, the present invention also relates to the use of the compounds of the present invention, more particularly, the detectably labeled compounds of the present invention as defined herein as biomarkers of TDP-43 aggregates or biomarkers of TDP-43 proteinopathy.

[0502] In another embodiment, the compounds of the invention can be used in research applications, in particular, as analytical tools or reference molecules. The compounds can be used for detection of TDP-43 aggregates in vitro or in vivo. The compounds of the invention can be used for staining of TDP-43 aggregates. For example, the compounds of the invention can be used for histochemical detection of autopsy tissues such as brain tissue. The compounds of the invention are preferably detectably labeled compounds and can be directly or indirectly labeled as discussed herein.

[0503] Complete kit

[0504] In a fifth aspect, the present invention also relates to a kit for one or more methods of the present invention, wherein the kit comprises a compound of the present invention as described herein. The kit typically comprises a container for holding the compound of the present invention and instructions for using the compound of the present invention. Preferably, the kit comprises a compound of formula (I) as disclosed herein. More preferably, the compound of the present invention is a detectably labeled compound (e.g., a compound of formula (IT) or (IF)).

[0505] The term "kit" generally refers to any diagnostic kit known in the art. More particularly, the latter term refers to the diagnostic kit described by Zrein et al. in Clin. Diagn. Lab. Immunol., 1998, 5, 45-49.

[0506] The dosage of the detectably labeled compound of the present invention will vary depending on the specific compound administered, the patient's weight, the size and type of the sample, and other variables apparent to a physician skilled in the art. Typically, the dosage is preferably in the range of 0.001 μg / kg to 10 μg / kg, preferably 0.01 μg / kg to 1.0 μg / kg. The radioactive dose can be, for example, 100 to 600 MBq, more preferably 150 to 450 MBq.

[0507] In particular, such kits can be used to perform the methods of the invention (which include, for example, but not limited to, imaging, diagnostic and monitoring methods), for example, for diagnosing diseases, disorders or abnormalities associated with TDP-43 aggregates or TDP-43 proteinopathy. Such kits can contain all the necessary components for performing the methods provided herein. Typically, each component is stored separately in a single integral package. Suitable additional components included in the kit are, for example, buffers, detectable dyes, test devices, reaction vessels, instructions, etc. The instructions for use can be customized according to the specific method of using the kit.

[0508] The present invention also relates to a kit for preparing a detectably labeled compound of the present invention, in particular, wherein the detectable label is a radioactive isotope. Thus, the kit comprises a precursor of a detectably labeled compound of formula (I) and a labeling agent that reacts with the precursor to introduce a detectable (e.g., radioactive) label. Preferred precursors are compounds of formula (II), (III) and (IV). The labeling agent that reacts with the precursor can be a reagent that introduces a detectable (e.g., radioactive) label, such as 18 F or 3 H reagent. The labeling reagent can be 18 F-fluorinating agent.

[0509] Methods for preparing the compounds of the present invention

[0510] In a sixth aspect, the present invention further relates to a process for preparing a compound of formula (I).

[0511] Cold isotope compounds:

[0512] In one embodiment, the present invention relates to a process for preparing a compound of formula (I), as described above, comprising the following steps:

[0513] The compound of formula (II) and R 10The reaction provides a compound of formula (I)

[0514]

[0515] in

[0516] n、R 1 and R 2 As defined above;

[0517] R 10 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring comprises one or more heteroatoms selected from N, O and S, wherein the 5- or 6-membered carbocyclic or heterocyclic ring is substituted by Br or I.

[0518] In a preferred embodiment, R 10 Select from the following:

[0519] Where X is N, and R 5 is CH3 or H; and wherein Hal is Br or I; or

[0520] Where Hal is Br;

[0521] R 10 yes wherein Hal is Br or I; and

[0522] R 3 Yes F, R 4 is NH2, R 7 is H, and R 8 It is H;

[0523] R 3 is NH2, R 4 Yes F, R 7 is H, and R 8 It is H.

[0524] R 3 It is CN, R 4 is NH2, R 7 is H, and R 8 It is H.

[0525] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is CN,

[0526] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8is F, or

[0527] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN;

[0528] Where R 9 is selected from H, F, NH2 or CH3, wherein Hal is Br or I;

[0529] Where Hal is Br;

[0530] Where Hal is Br;

[0531] Where Hal is Br;

[0532] Where Hal is Br;

[0533] where Hal is Br; and

[0534] Where Hal is Br.

[0535] The compound having the structure of formula (II) and R 10 The method of reaction can be carried out by any suitable method. In one selection, the reaction can be carried out in the presence of a diamine chelating agent such as DMEDA, a base such as potassium carbonate, a catalyst such as CuI and an aprotic solvent such as dioxane. In another selection, the reaction can be carried out under Pd-coupling conditions, under the presence of a Pd catalyst such as Pd[P(Ph) 3] 4 or Pd(OAc) 2, XantPhos.

[0536] tritium( 3 H) Detectably labeled compounds

[0537] In one embodiment, the present invention provides a method for preparing a tritium ( 3 H) A method for detecting a labeled compound, the method comprising the step of radiolabeling a precursor of a compound having the structure of formula (IT) with a radioisotope, wherein at least one leaving group in the precursor of the compound having the structure of formula (IT) is replaced by tritium ( 3 H) Replacement.

[0538] Tritium ( 3 A compound detectably labeled with H) is preferably defined as one in which at least 1 to 3 hydrogens (H) are each replaced by tritium ( 3H) substitution. Tritium ( 3 A compound detectably labeled with tritium (H) is more preferably defined as one in which two or three hydrogens (H) are replaced with tritium ( 3 H) is substituted. Tritium ( 3 A compound detectably labeled with tritium (H) is even more preferably defined as one in which the three hydrogens (H) are replaced with tritium ( 3 H) Replacement.

[0539] In another embodiment, the present invention provides a method for preparing a compound of formula (IT), the method comprising the steps of:

[0540] By using T2 and a suitable catalyst to exchange Br or I with T or by introducing a CT3- group, T (i.e. 3 H) Radiolabeling of a precursor compound having formula (III)

[0541]

[0542] or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, or a mixture thereof,

[0543] in

[0544] Z is selected from C-Br, CI and CH;

[0545] n is 1 or 2;

[0546] R 1 is H, hydroxy(C1-C4)alkyl or F, R 1 Preferably H or F;

[0547] R 11 is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted by Br, I, F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S;

[0548] and either Z and / or R 11 At least one of them contains Br or I;

[0549] At least one Br or I is replaced by T;

[0550] Or R 11 Contains NH structure;

[0551] wherein the NH structure is replaced by N-CT3,

[0552] And where T is 3 H.

[0553] In a preferred embodiment, Z is C-Br or CI, and Br or I is replaced by T.

[0554] In another preferred embodiment, R 11 Contains Br or I, and Br or I is replaced by T.

[0555] In a preferred embodiment, Z is C-Br or CI, and Br or I is replaced by T; and R 11 Contains Br or I, and Br or I is replaced by T.

[0556] In another embodiment, Z is CH, and R 11 yes Where R 5 is H or CH3, and R 11 At least one H is replaced by CT3.

[0557] In another embodiment, the present invention provides a method for preparing a compound of formula (IT), the method comprising using T (ie, 3 H) Radiolabeling of a precursor compound having formula (III)

[0558]

[0559] in

[0560] n is 1 or 2; preferably 2

[0561] R 1 Yes-COOR A , where R A is (C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl, wherein R 1 At least one hydrogen atom is replaced by T; preferably R 1 At least two hydrogen atoms are replaced by T;

[0562] R 6 is T or H;

[0563] T is 3 H; and

[0564] in

[0565] R 11 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N.

[0566] Preferably, the tritium ( 3 H) detectably labeled compounds include (wherein T means3 H)

[0567]

[0568] Preferably, the precursor according to the present invention having formula (III) can be selected from

[0569]

[0570] For the introduction of radioisotopes such as 3 Methods for H are well known in the art and include those described below.

[0571]

[0572] In this scheme, the substituents Br, NH2, F and CN are shown only as examples. The definitions of formula (1-T) apply in this respect.

[0573] Another example is shown in the following flow:

[0574]

[0575] Another example is shown in the following flow:

[0576]

[0577] This means that the nitrogen atom can be present at any available position in the ring.

[0578] Another example is shown in the following flow:

[0579]

[0580] For the introduction of T (tritium), 3 The H radiolabeling agent may be tritium gas. The method may be carried out in the presence of a catalyst such as palladium on carbon (Pd / C) or Lindela's catalyst, a solvent such as N,N-dimethylformamide (DMF) and a base such as N,N-diisopropylethylamine (DIEA). Alternatively, 3 The H radiolabeling agent may be LiT, which is prepared from n-butyllithium and tritium gas in the presence of TMEDA, AlCl3 and a solvent such as THF.

[0581] In one embodiment, the present invention relates to a method for preparing a precursor compound of formula (III) as described above, the method comprising the following steps:

[0582] The compound of formula (II) as defined above is reacted with R 10Reaction to obtain a compound of formula (Ia), followed by NBS bromination or acid cleavage of the trimethylsilylethoxymethyl (SEM) protecting group. The following examples are given as illustrations:

[0583]

[0584]

[0585] in

[0586] n and R 1 As defined above;

[0587] R 10 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring comprises one or more heteroatoms selected from N, O and S, wherein the 5- or 6-membered carbocyclic or heterocyclic ring is substituted by Br or I and may be optionally substituted by SEM.

[0588] In a preferred embodiment, R 10 Select from the following:

[0589] Where X is N, and R 5 is SEM; and wherein Hal is Br or I;

[0590] Where Hal is Br;

[0591] R 10 yes wherein Hal is Br or I; and

[0592] R 3 Yes F, R 4 is NH2, R 7 is H, and R 8 It is H;

[0593] R 3 is NH2, R 4 Yes F, R 7 is H, and R 8 It is H.

[0594] R 3 It is CN, R 4 is NH2, R 7 is H, and R 8 It is H.

[0595] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is CN, or

[0596] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is F;

[0597] R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN;

[0598] Where R 9 is selected from H, F, NH2 or CH3, wherein if Cl is present, Hal is Br, or if Br is present, Hal is I;

[0599] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I; and

[0600] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I.

[0601] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I;

[0602] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I;

[0603] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I; and

[0604] wherein if Cl is present, Hal is Br, or if Br is present, Hal is I.

[0605] The compound of formula (II) and R 10 The method of reaction can be carried out by any suitable method. In one selection, the reaction can be carried out in the presence of a diamine chelating agent such as DMEDA, a base such as potassium carbonate, a catalyst such as CuI and an aprotic solvent such as dioxane. In another selection, the reaction can be carried out under Pd-coupling conditions, in the presence of a Pd catalyst such as Pd[P(Ph) 3] 4 or Pd(OAc) 2, XantPhos.

[0606] fluorine( 18 F) Detectably labeled compounds:

[0607] In one embodiment, the present invention provides a method for preparing the fluoro( 18F) A method for detecting a compound by labeling it with a radioactive isotope [ 18 F] Radiolabeling a precursor having formula (IV):

[0608]

[0609] in

[0610] n and R 2 As defined above, and

[0611] R 14 was radiolabeled during the 18 F replaces the leaving group (LG).

[0612] In a preferred embodiment, the present invention provides a method for preparing the fluoro( 18 F) A method for detecting a compound by labeling it with a radioactive isotope [ 18 F] Radiolabeled precursor having formula (IV)

[0613]

[0614] in

[0615] n is 1 or 2, preferably 1;

[0616] R 2 yes Where R 9 is H; and

[0617] R 14 was radiolabeled during the 18 F replaces the leaving group.

[0618] In another embodiment, the present invention provides a method for preparing the fluoro( 18 F) A method for detecting a compound by labeling it with a radioactive isotope [ 18 F] Radiolabeling a precursor having the formula (V):

[0619]

[0620] Where n, R 1 and R 2 As defined herein with respect to the compound of formula (I), and

[0621] R 14 was radiolabeled during the 18 F replaces the leaving group (LG).

[0622] In a preferred embodiment, the present invention provides a method for preparing the fluoro(18 F) A method for detecting a compound by labeling it with a radioactive isotope [ 18 F] Radiolabeling a precursor having the formula (V):

[0623]

[0624] wherein n is 1 or 2, preferably 2;

[0625] R 1 is H or hydroxy(C1-C4)alkyl, preferably hydroxyethyl or hydroxymethyl, more preferably hydroxymethyl; and

[0626] R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S, preferably N, R 2 Preferably Where R 9 is H; and

[0627] R 14 In the radiolabeling step 18 F replaces the leaving group (LG).

[0628] Fluorination can be 18 F-fluorinating agent, 18 The F-fluorinating agent can be selected from K[ 18 F]、Cs 18 F. Na 18 F、Rb 18 F. Kryptofix

[222] K 18 F. 18 F's Four (C 1-6 Alkyl) ammonium salt and tetrabutyl[ 18 F] ammonium fluoride.

[0629] Preferably, the leaving group (LG) is C 1-4 Alkyl sulfonate group or C 6-10 More preferably, the leaving group (LG) is a mesylate group, a toluene mesylate group or a m-nitrobenzene sulfonate group or a nitro group. Even more preferably, the leaving group (LG) is a mesylate group or a nitro group.

[0630] Those skilled in the art know 18 A suitable solvent for the F-fluorination step. The solvent may be, for example, selected from DMF, DMSO, acetonitrile, DMA or a mixture thereof. Preferably, the solvent is acetonitrile or DMSO.

[0631] Preferably, the preparation of fluorine ( 18F) The method for detectably labeling compound 1 comprises a radiolabeling step in which the leaving group (LG) of precursor L1 (in this case the mesylate group) is 18 F-fluorinating agents such as K[ 18 F] or [ 18 F]TBAF was fluorinated 18 F) Replace as follows:

[0632]

[0633] Preferably, the preparation of fluorine ( 18 F) The method for detectably labeling compound 17 comprises a radiolabeling step in which the leaving group (LG) of precursor 20 (in this case the nitro group) is radiolabeled 18 F-fluorinating agents such as K[ 18 F] or [ 18 F]TBAF was fluorinated 18 F) Replace as follows:

[0634]

[0635] The compounds of the present invention can be prepared by one of the general methods shown in the following schemes. These methods are given for illustrative purposes only and should not be construed as limiting.

[0636] The precursor compounds of formula (II), (III), (IV) or (V) as defined above, or stereoisomers, polymorphs, racemic mixtures, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates or solvates thereof are part of the present invention.

[0637] abbreviation

[0638]

[0639]

[0640] General synthesis process:

[0641] For the preparation of 2-[2-fluoro-6-[(3R)-3-fluoropyrrolidin-1-yl]-3-pyridinyl]-5-(3-pyridinyl)-6,7- Synthesis process of dihydrothiazolo[5,4-c]pyridin-4-one (Compound 1)

[0642]

[0643] For the preparation of 2-(2-fluoro-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6, Synthesis process of 7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Compound 17)

[0644]

[0645] For the preparation of 3 [H] Synthesis process of precursor compounds

[0646] For the preparation of 5-(3-amino-2,6-dibromo-4-fluorophenyl)-2-(5-bromo-6-(pyrrolidin-1-yl)pyridine-3- yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one ( 3 Synthesis process of H compound 1

[0647]

[0648] For the preparation of 1-(3-bromo-5-(5-(5-bromopyridin-3-yl)-4-oxo-4,5,6,7-tetrahydrothiazolo[5,4-c] pyridin-2-yl)-6-fluoropyridin-2-yl)piperidine-4-carboxylic acid ethyl ester ( 3 H (precursor of compound 17)

[0649]

[0650] 3 H-labeled compounds can be prepared by reacting a suitable precursor compound containing a halogen atom with tritium gas via catalytic dehalogenation (M. Saljoughian Synthesis (2002), 1781-1801), or by reacting a suitable precursor compound containing an NH moiety with methyl iodide [ 3 H] by methylation, or by a 3 H] is prepared from a suitable precursor compound of a reducing agent-reduced portion (Y. Chen Chemistry 25 (2019): 3405-3439). Preferably, 3 The solvent used in H-labeling is DMF or DMA, and the preferred solvent is DMF.

[0651] Preparation for 18 Synthesis scheme of F-labeled precursors

[0652] For the preparation of methanesulfonic acid [(3S)-1-[6-fluoro-5-[4-oxo-5-(3-pyridyl)-6,7-dihydrothiazolidine [5, 4-c]pyridin-2-yl]-2-pyridinyl]pyrrolidin-3-yl] ester ( 18 F Synthesis process of the precursor of compound 1

[0653]

[0654] For the preparation of 2-(6-(4-(hydroxymethyl)piperidin-1-yl)-2-nitropyridine-3-yl)-5-(pyridin-3-yl)- 6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one ( 18 F Synthesis process of the precursor of compound 17

[0655]

[0656] The reaction takes place in the presence of a fluorinating agent and a common solvent.

[0657] 18 The F-labeled compound can be prepared by reacting a precursor compound containing LG with 18 F-fluorination reagent is used to prepare the LG 18 F replacement. 18 The F-fluorination reagent can be 18 Tetraalkylammonium salts of F (e.g. 18 F's Four (C 1-6 alkyl) ammonium salts, for example, tetrabutyl[ 18 F] ammonium fluoride), 18 Tetraalkylphosphonium salt of F (e.g. 18 F's Four (C 1-6 Alkyl)phosphonium salt), K[18 F]、Cs 18 F. Na 18 F、Rb 18 F, or Kryptofix

[222] K 18 F. Preferably, 18 F-fluorinating agent is Cs 18 F.K 18 F or tetrabutyl[ 18 F] ammonium fluoride. It can be used for 18 The 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.

[0658] Although the reaction shown above is 18 F was used as a radioactive label, and other radioactive labels can be introduced by following a similar method.

[0659] The following examples illustrate the invention but should not, however, be construed as limiting. Example

[0660] All reagents and solvents were obtained from commercial sources and used without further purification. Proton ( 1H) NMR spectra. Mass spectra (MS) were recorded using a UPLC H-Class Plus with a photodiode array detector and a QDa mass spectrometer. Chromatography was performed using silica gel (Fluka: silica gel 60, 0.063-0.2 mm) and suitable solvents as indicated in the specific examples. Rapid purification was performed using the Biotage Isolera One rapid purification system using HP-SIL or KP-NH SNAP columns (Biotage) and solvent gradients as indicated in the specific examples. Thin layer chromatography (TLC) was performed on silica gel plates with UV detection.

[0661] Although some examples do not indicate that each compound is detectably labeled, it is understood that corresponding detectably labeled compounds are contemplated and can be readily prepared, for example, by using detectably labeled starting materials, such as starting materials containing C( 3 H)3. 11 C)H3 or 18 F.

[0662] Example 1 Synthesis of 2-[2-fluoro-6-[(3R)-3-fluoropyrrolidin-1-yl]-3-pyridinyl]-5-(3-pyridinyl)-6,7-dihydrothiazolo[5,4-c]pyridin-4-one

[0663]

[0664] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0665] Tert-butyl 2,4-dioxopiperidin-1-carboxylate A (10 g, 46.9 mmol) was dissolved in carbon tetrachloride (125 mL) and cooled to 0 to 5 ° C. To the above solution, N-bromosuccinimide (8.35 g, 46.9 mmol) was added in batches and continued to stir at 28 ° C for 1 hour. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10 g, 73%).

[0666] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0667] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0668] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in ethanol (160 mL) and heated in an oil bath at 80 ° C for 2.5 hours. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting solid was recrystallized from ethanol to obtain title compound C as a white solid (6.6 g, 71%).

[0669] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0670] Step-3: Synthesis of tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (D)

[0671] The title compound C (6.6 g, 24.53 mmol) was dissolved in acetonitrile (82 mL) and cooled to -10 ° C in an ice bath with stirring. Tert-butyl nitrite (4.3 mL, 36.4 mmol) was added to the above solution and continued to stir at -10 ° C for 1 hour. Copper (II) bromide (6.5 g, 29.43 mmol) was added to the above mixture and stirred at 28 ° C for 1 hour. The reaction mixture was alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution and filtered. The collected filtrate was diluted three times with ethyl acetate (300 mL) and water (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (100 to 200 mesh) using ethyl acetate / n-hexane gradient (0 / 100->10 / 90) to obtain the title compound D as a white solid (4.73 g, 58%).

[0672] 1 H NMR (500MHz DMSO-d6): δ4.12(t,2H),3.10(t,2H),1.5(s,9H). LCMS(ESI)279m / z[M+H-C4H8] + .

[0673] Step-4: Synthesis of tert-butyl 2-(2,6-difluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (F)

[0674] Tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate D (0.3 g, 0.9 mmol) was dissolved in 1,4-dioxane (15 mL) and degassed by passing nitrogen gas through the mixture. Then, [1,1′-bis(diphenyl-phosphino)ferrocene]-dichloropalladium(II) (0.07 g, 0.09 mmol) complexed with dichloromethane, the title compound E (0.21 g, 1.35 mmol) and tripotassium phosphate (0.28 g, 1.35 mmol) were added, and the reaction mixture was heated at 100° C. in an oil bath for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (basic, 100-200 mesh) using an ethyl acetate / n-hexane gradient (0 / 100->10 / 90->15 / 85) to give the title compound F as a light yellow solid (0.2 g, 60%).

[0675] 1 H NMR (500MHz DMSO-d6): δ8.91(q,1H),7.43(dd,1H),4.1(t,2H),3.19(t,2H),1.5(s,9H). LCMS(ESI)311.95m / z[M+H-C4H8] + .

[0676] Step-5: Synthesis of (R)-tert-butyl 2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0677] Title compound F (0.3 g, 0.86 mmol), (R)-3-fluoropyrrolidine hydrochloride (0.213 g, 1.7 mmol) and triethylamine (0.33 mL, 2.36 mmol) were suspended in n-butanol (8 mL) using a microwave vial. The sealed vial was then heated at 160 ° C for 1 hour using a CEM microwave. The reaction mixture was concentrated under reduced pressure. The resulting residue was suspended in water (20 mL) and filtered through Whatmann filter paper. The solid was washed with water (20 mL), then washed with n-hexane (20 mL), and dried under reduced pressure to obtain title compound G as an off-white solid (0.27 g, 98%).

[0678] 1H NMR (500MHz, DMSO-d6): δ8.75(d,1H),8.06(dd,1H),7.81(s,1H),6.64(d,1H),5 .48(d,1H),3.9-3.7(m,3H),3.51-3.48(m,3H),2.98(t,2H),2.34.2.13(m,2H). LCMS(ESI)319.00m / z[M+H] + .

[0679] Step-6: Synthesis of (R)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0680] The title compound G (0.188g, 0.011mmol) was dissolved in DCM (4mL) and cooled to 0°C under stirring in an ice bath. 4M HCl (2mL) in 1,4-dioxane was added to the above solution and continued to stir at room temperature for 3 hours. After the reaction was completed, the solvent was removed under reduced pressure. The obtained residue was dissolved in ice-cold water and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitated solid was filtered and dried to obtain the title compound H as a light yellow solid (0.135g, 92%).

[0681] 1 H NMR (500MHz DMSO-d6): δ7.81(d,2H),7.75(s,1H),6.66(d,2H),5.48(d,1H),3.52(m,6H),2.96(t,2H),2.24(m,2H). LCMS(ESI)318[M+H]+.

[0682] Step-7: Synthesis of (R)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (1)

[0683] Compound H (0.05 g, 0.14 mmol) and 3-iodopyridine 8 (0.09 g, 0.44 mmol) were dissolved in degassed 1,4-dioxane (7.5 mL). After adding Pd[PPh3]4 (0.008 g, 0.0074 mmol), XantPhos (0.01 g, 0.014 mmol) and Cs2CO3 (0.14 g, 0.44 mmol), the reaction mixture was heated at ~100 °C in a sand bath for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give 1 as a yellow solid (0.025 g, 41%).

[0684] 1 H NMR(400MHz DMSO-d6): δ8.67(d,1H),8.43(m,2H),7.87(m,1H),7.5(dd,1H),6.64(dd,1H), 5.49(d,1H),4.17(t,2H),3.72(m,3H),3.52(m,1H),3.26(t,2H),2.22(m,2H). LCMS(ESI)414.1m / z[M+H] +

[0685] Example 2 Synthesis of 5-(3-amino-2,6-dibromo-4-fluorophenyl)-2-(5-bromo-6-(pyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0686]

[0687] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0688] Tert-butyl 2,4-dioxopiperidin-1-carboxylate A (10 g, 46.9 mmol) was dissolved in carbon tetrachloride (125 mL) and cooled to 0 to 5 ° C. To the above solution, N-bromosuccinimide (8.35 g, 46.9 mmol) was added in batches and continued to stir at 28 ° C for 1 hour. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10 g, 73%).

[0689] 1H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0690] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0691] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in ethanol (160 mL) and heated in an oil bath at 80 ° C for 2.5 hours. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting solid was recrystallized from ethanol to obtain title compound C as a white solid (6.6 g, 71%).

[0692] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0693] Step-3: Synthesis of tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (D)

[0694] The title compound C (6.6 g, 24.53 mmol) was dissolved in acetonitrile (82 mL) and cooled to -10 ° C in an ice bath with stirring. Tert-butyl nitrite (4.3 mL, 36.4 mmol) was added to the above solution and continued to stir at -10 ° C for 1 hour. Copper (II) bromide (6.5 g, 29.43 mmol) was added to the above mixture and stirred at 28 ° C for 1 hour. The reaction mixture was alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution and filtered. The collected filtrate was diluted three times with ethyl acetate (300 mL) and water (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (100 to 200 mesh) using ethyl acetate / n-hexane gradient (0 / 100->10 / 90) to obtain the title compound D as a white solid (4.73 g, 58%).

[0695] 1H NMR (500MHz DMSO-d6): δ4.12(t,2H),3.10(t,2H),1.5(s,9H). LCMS(ESI)279m / z[M+H-C4H8] + .

[0696] Step-4: Synthesis of tert-butyl 2-(2,6-difluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (3) (F)

[0697] Tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate D (0.3 g, 0.9 mmol) was dissolved in 1,4-dioxane (15 mL) and degassed by passing nitrogen gas through the mixture. Then, [1,1′-bis(diphenyl-phosphino)ferrocene]-dichloropalladium(II) (0.07 g, 0.09 mmol) complexed with dichloromethane, the title compound E (0.21 g, 1.35 mmol) and tripotassium phosphate (0.28 g, 1.35 mmol) were added, and the reaction mixture was heated at 100° C. in an oil bath for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (basic, 100-200 mesh) using an ethyl acetate / n-hexane gradient (0 / 100->10 / 90->15 / 85) to give the title compound F as a light yellow solid (0.2 g, 60%).

[0698] 1 H NMR (500MHz DMSO-d6): δ8.91(q,1H),7.43(dd,1H),4.1(t,2H),3.19(t,2H),1.5(s,9H). LCMS(ESI)311.95m / z[M+H-C4H8] + .

[0699] Step-5: Synthesis of (R)-tert-butyl 2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0700] Title compound F (0.3 g, 0.86 mmol), (R)-3-fluoropyrrolidine hydrochloride (0.213 g, 1.7 mmol) and triethylamine (0.33 mL, 2.36 mmol) were suspended in n-butanol (8 mL) using a microwave vial. The sealed vial was then heated at 160 ° C for 1 hour using a CEM microwave. The reaction mixture was concentrated under reduced pressure. The resulting residue was suspended in water (20 mL) and filtered through Whatmann filter paper. The solid was washed with water (20 mL), then washed with n-hexane (20 mL), and dried under reduced pressure to obtain title compound G as an off-white solid (0.27 g, 98%).

[0701] 1 H NMR (500MHz, DMSO-d6): δ8.75(d,1H),8.06(dd,1H),7.81(s,1H),6.64(d,1H),5 .48(d,1H),3.9-3.7(m,3H),3.51-3.48(m,3H),2.98(t,2H),2.34.2.13(m,2H). LCMS(ESI)319.00m / z[M+H] + .

[0702] Step-6: Synthesis of (R)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0703] Title compound G (0.188g, 0.011mmol) is dissolved in DCM (4mL), and cooled to 0 ℃ under stirring in an ice bath. 4M HCl (2mL) in 1,4-dioxane is added to the above solution, and stirring is continued at room temperature for 3 hours. After the reaction is completed, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water, and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution, the precipitated solid is filtered, and dried to obtain title compound H as a light yellow solid (0.135g, 92%).

[0704] 1 H NMR (500MHz DMSO-d6): δ7.81(d,2H),7.75(s,1H),6.66(d,2H),5.48(d,1H),3.52(m,6H),2.96(t,2H),2.24(m,2H). LCMS(ESI)318[M+H] + .

[0705] Step-7: Synthesis of (R)-5-(5-bromopyridin-3-yl)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (I)

[0706] (R)-2-(2-Fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one H (0.05 g, 0.14 mmol) and 3,5-dibromopyridine 7 (0.1 g, 0.44 mmol) were dissolved in degassed 1,4-dioxane (5 mL). After adding Pd[PPh3]4 (0.008 g, 0.0074 mmol), XantPhos (0.08 g, 0.014 mmol) and Cs2CO3 (0.1 g, 0.3 mmol), the reaction mixture was heated at ~100°C in an oil bath for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99 / 1->98.8 / 1.2) to afford I as a yellow solid (0.05 g, 68%).

[0707] 1 H NMR(400MHz DMSO-d6): δ8.67(d,1H),8.58(d,1H),8.41(dd,1H),6.64(dd,1H),5.48(d,1H),4.18(t,2H),3.74(m,3H),3.52(m,1H),3.25(t,2H),2.22(m,2H). LCMS(ESI)492.0m / z[M+H] +

[0708] Step-8: Synthesis of (R)-2-(5-bromo-2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(5-bromopyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (2)

[0709] The title compound I (0.14 g, 0.28 mmol) was dissolved in DMF (5 mL) and cooled to -50 °C. NBS (0.1 g, 0.56 mmol) was added in batches. The reaction mixture was allowed to warm to 20 °C over a period of 1 hour. The reaction mixture was diluted with cold water (50 mL) and the solid was filtered. The crude solid was purified on a preparative TLC plate using dichloromethane / methanol (98.5 / 1.5) as the mobile phase to give the title compound 2 as a light yellow solid (0.083 g, 52%).

[0710] 1 H NMR (400MHz DMSO-d6): δ8.67(d,1H),8.59(d,1H),8.57(d,1H),8.18(t,1H),5.44(d,1H),4.19(t,2H),3.99(m,4H),3.26(t,2H),2.17(m,2H). LCMS(ESI)571.55m / z[M+2H] + .

[0711] Example 3 Synthesis of (S)-methanesulfonic acid 1-(6-fluoro-5-(4-oxo-5-(pyridin-3-yl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)pyrrolidin-3-yl ester

[0712]

[0713] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0714] Tert-butyl 2,4-dioxopiperidin-1-carboxylate A (10 g, 46.9 mmol) was dissolved in carbon tetrachloride (125 mL) and cooled to 0 to 5 ° C. N-bromosuccinimide (8.35 g, 46.9 mmol) was added to the above solution in batches and stirred at 28 ° C for 1 hour. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10 g, 73%).

[0715] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0716] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0717] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in ethanol (160 mL) and heated in an oil bath at 80 ° C for 2.5 hours. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting solid was recrystallized from ethanol to obtain title compound C as a white solid (6.6 g, 71%).

[0718] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0719] Step-3: Synthesis of tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (D)

[0720] The title compound C (6.6 g, 24.53 mmol) was dissolved in acetonitrile (82 mL) and cooled to -10 ° C in an ice bath with stirring. Tert-butyl nitrite (4.3 mL, 36.4 mmol) was added to the above solution and continued to stir at -10 ° C for 1 hour. Copper (II) bromide (6.5 g, 29.43 mmol) was added to the above mixture and stirred at 28 ° C for 1 hour. The reaction mixture was alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution and filtered. The collected filtrate was diluted three times with ethyl acetate (300 mL) and water (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (100 to 200 mesh) using ethyl acetate / n-hexane gradient (0 / 100->10 / 90) to obtain the title compound D as a white solid (4.73 g, 58%).

[0721] 1 H NMR (500MHz DMSO-d6): δ4.12(t,2H),3.10(t,2H),1.5(s,9H). LCMS(ESI)279m / z[M+H-C4H8] + .

[0722] Step-4: Synthesis of tert-butyl 2-(2,6-difluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (3) (F)

[0723] Tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate D (0.3 g, 0.9 mmol) was dissolved in 1,4-dioxane (15 mL) and degassed by passing nitrogen gas through the mixture. Then, [1,1′-bis(diphenyl-phosphino)ferrocene]-dichloropalladium(II) (0.07 g, 0.09 mmol) complexed with dichloromethane, the title compound E (0.21 g, 1.35 mmol) and tripotassium phosphate (0.28 g, 1.35 mmol) were added, and the reaction mixture was heated at 100° C. in an oil bath for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (basic, 100-200 mesh) using an ethyl acetate / n-hexane gradient (0 / 100->10 / 90->15 / 85) to give the title compound F as a light yellow solid (0.2 g, 60%).

[0724] 1 H NMR (500MHz DMSO-d6): δ8.91(q,1H),7.43(dd,1H),4.1(t,2H),3.19(t,2H),1.5(s,9H). LCMS(ESI)311.95m / z[M+H-C4H8] + .

[0725] Step-5: Synthesis of (S)-tert-butyl 2-(2-fluoro-6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (J)

[0726] The title compound 3 (0.2 g, 0.54 mmol), (S)-pyrrolidin-3-ol hydrochloride F (0.068 g, 0.54 mmol), N,N-diisopropylethylamine (0.09 mL, 1.06 mmol) were suspended in DMSO (20 mL) and stirred at ambient temperature for 3 hours. The reaction mixture was quenched with cold water (200 mL), and the precipitated solid was filtered through Whatmann filter paper and dried to give the title compound J as a light yellow solid (0.22 g, 92%).

[0727] Step-6: Synthesis of (S)-2-(2-fluoro-6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (K)

[0728] Title compound J (0.22g, 0.5mmol) is dissolved in DCM (4.4mL) and cooled to 0°C in an ice bath under stirring. 4M HCl (2.2mL) in 1,4-dioxane is added to the above solution and continued to stir at room temperature for 2 hours. After the reaction is complete, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution, the precipitated solid is filtered, and dried to obtain title compound K as a light yellow solid (0.155g, 92%).

[0729] 1 H NMR (500MHz DMSO-d6): δ8.33(t,1H),7.8(s,1H),6.55(d,1H),5.06(s,1H),4.41(s,1H),3.54(m,6H),2.99(t,2H),1.99(d,2H). LCMS(ESI)334.9[M+H] + .

[0730] Step-7: Synthesis of (S)-2-(6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-2-fluoropyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (L)

[0731] Title compound K (0.15 g, 0.44 mmol) and imidazole (0.3 g, 4.5 mmol) dissolved in DMF (3 mL) were cooled to 0°C in an ice bath. Tert-butyldimethylsilyl chloride (0.5 g, 3.35 mmol) was added in portions. The reaction mixture was stirred at ambient temperature for 36 hours. The reaction mixture was diluted with cold water (150 mL), the precipitated solid was filtered, and dried to give title compound L as a light yellow solid (0.177 g, 88%).

[0732] 1 H NMR (400MHz, DMSO-d6): δ8.34(t,1H),7.81(s,1H),6.57(dd,1H),4.59(s,1H),3.63(dd,1H ),3.5(m,4H),3.36(m,2H),3.09(s,3H),2.99(t,2H),2.01(d,2H),0.86(s,9H),0.1(d,6H). LCMS(ESI)449.25m / z[M+H]+

[0733] Step-8: Synthesis of (S)-2-(6-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-2-fluoropyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (M)

[0734] Compound L (0.17 g, 0.37 mmol) and 3-iodopyridine 8 (0.23 g, 1.1 mmol) were dissolved in degassed 1,4-dioxane (17 mL). After adding Pd[PPh3]4 (0.043 g, 0.03 mmol), XantPhos (0.043 g, 0.075 mmol) and Cs2CO3 (0.37 g, 1.13 mmol), the reaction mixture was heated at ~100 °C in an oil bath for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99 / 1->98 / 2) to give M as a yellow solid (0.17 g, 89%).

[0735] 1 H NMR (500MHz, DMSO-d6): δ8.75(d,1H),8.54(q,1H),8.48(t,1H),7.94(m,1H),7.52(m,1H),6.7(dd,1H),4.7(s,1H), 4.25(t,2H),3.74(dd,1H),3.63(t,2H),3.35(t,2H),3.2(m,1H),2.21(s,1H),2.02(s,1H),0.96(s,9H),0.2(d,6H). LCMS(ESI)526m / z[M+H] + .

[0736] Step-9: Synthesis of (S)-2-(2-fluoro-6-(3-hydroxypyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (N)

[0737] Title compound M (0.14g, 0.26mmol) is dissolved in DCM (2.8mL), and cooled to 0 ℃ under stirring in an ice bath. 4M HCl (1.4mL) in 1,4-dioxane is added to the above solution, and stirring is continued at room temperature for 3 hours. After the reaction is completed, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water, and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution, the precipitated solid is filtered, and dried to obtain title compound N as a light yellow solid (0.08g, 72%).

[0738] 1 H NMR (500MHz, DMSO-d6): δ8.66(d,1H),8.44(q,1H),8.38(t,1H),7.84(m,1H),7.47(m,1H),6.7(d ,1H),6.58(d,1H),5.08(s,1H),4.42(s,1H),4.16(t,2H),3.55(m,4H),3.25(t,2H),2.0(d,2H). LCMS(ESI)411.9m / z[M+H] + .

[0739] Step-10: Synthesis of ((S)-1-(6-fluoro-5-(4-oxo-5-(pyridin-3-yl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)pyrrolidin-3-yl methanesulfonate (3)

[0740] The title compound N (0.07 g, 0.17 mmol) was dissolved in pyridine (2.3 mL) and cooled to -50°C in a dry ice bath with stirring. Methanesulfonyl chloride (0.46 g, 4.08 mmol) was added to the above solution and stirring was continued at 20°C for 1 hour. The reaction mixture was diluted with water (35 mL), and the precipitated solid was filtered, washed with water (2 x 15 mL), and dried to give the title compound 3 as a light yellow solid (0.06 g, 72%).

[0741] 1 H NMR (500MHz, DMSO-d6): δ8.66(d,1H),8.43(m,2H),7.84(m,1H),7.47(q,1H),6.65(dd ,1H),5.45(s,1H),4.16(t,2H),3.76(m,3H),3.55(dd,1H),3.26(m,5H),2.34(s,2H). LCMS(ESI)489.85m / z[M+H] + .

[0742] Example 4 Tritiated 2-[2-fluoro-6-[(3R)-3-fluoropyrrolidin-1-yl]-3-pyridinyl]-5-(3-pyridinyl)-6,7-dihydrothiazolo[5,4-c]pyridin-4-one 3 Synthesis of H-compound 1

[0743]

[0744] T means 3 H.

[0745] 2.49 mg of brominated precursor 2, 50 μL of DIPEA and 33.09 mg of Lindela catalyst were suspended in 0.3 ml of DMF. The suspension was degassed 3 times in a high vacuum manifold and stirred overnight at room temperature under a tritium atmosphere (772 mbar, 9.1 Ci). The solvent was removed under vacuum, and unstable tritium was exchanged by adding 0.3 ml of methanol, the solution was stirred, and the solvent was removed again under vacuum. The process was repeated twice. The dried solid was extracted with 5 ml of ethanol / DMF (4: 1), and the suspension was filtered through a 0.2 μm nylon membrane to obtain a clear solution. For the purification of the compound, the following HPLC conditions were found to be suitable: Waters Sunfire C18, 10 x 250 mm; solvent A: water + 0.1% TFA); B: acetonitrile + 0.1% TFA. The purified product was obtained 3 H-Compound 1 (SA 48.5 Ci / mmol, 99% purity).

[0746] Example 5 Fluorinated 2-[2-fluoro-6-[(3R)-3-fluoropyrrolidin-1-yl]-3-pyridinyl]-5-(3-pyridinyl)-6,7-dihydrothiazolo[5,4-c]pyridin-4-one 18 Synthesis of F compound 1

[0747]

[0748] The transport vial contains 18 F] fluoride (O-18-enriched water obtained from a commercial cyclotron facility) was transferred and trapped on a Chromafix PS-HCO column activated with KOTf (0.2 M, 6 mL) and then water (6 mL). It was then eluted with TBAOTf solution (6 mg in 0.6 mL) to The reaction vessel of the module was placed in a vacuum and helium stream at 70 ° C for 3.5 minutes, and the heat treatment was continued at 95 ° C for another 2.5 minutes. The reactor was then cooled to 60 ° C. A solution of (S)-methanesulfonic acid 1-(6-fluoro-5-(4-oxo-5-(pyridin-3-yl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)pyrrolidin-3-yl ester 3 (0.5 mg in 1 mL of anhydrous acetonitrile) was added to the reaction vial, and the reaction vessel was heated at 100 ° C for 10 minutes. The reaction vessel was cooled to 60 ° C, and the mixture was diluted with HPLC buffer (4 mL). Purification was performed by HPLC using a semi-preparative Phenomenex Gemini C18 column (5 μm, 250 x 10 mm) and eluted with a mixture of acetonitrile / ammonium acetate solution (20 mM, 42 / 58, v / v) at a flow rate of 4 mL / min. The radiolabeled product was eluted from the SPE column with 1.0 mL of ethanol into a preparation flask pre-loaded with 10 mL of the preparation matrix (ascorbic acid (10 mg) in saline). The resulting solution was passed through a 0.2 μm sterilizing membrane filter into a sterile filter vented vial. Analysis of the final product 18 F-Compound 1, take out a sample for QC test. 19 F- Reference compound samples were co-injected to confirm the identity of the product.

[0749] Example compounds 6 to 9 and 12 to 16

[0750] Following the coupling method reported in Preparative Example 1 and utilizing the building blocks of Example 1, Step 6, Example Compound 10, Step 2, Example 15, Step 2 and the halogen derivatives shown in Table 1, the following compounds were prepared.

[0751] Table 1

[0752]

[0753]

[0754]

[0755] Example compounds 11, 21, 22, 33

[0756] Following the coupling method reported in Preparative Example 15 (Step 3), and utilizing the building blocks of Example 1, Step 6, Example Compound 10, Step 2, and the halogen derivatives shown in Table 2, the following compounds were prepared.

[0757] Table 2

[0758]

[0759]

[0760]

[0761] 5-(3-amino-4-fluorophenyl)-2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolidine Synthesis of [5,4-c]pyridin-4(5H)-one (Example Compound 10)

[0762]

[0763] Step-1: Synthesis of tert-butyl 2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate

[0764] In a 20 ml microwave vial, the title compound D (0.3 g, 0.9 mmol) and commercially available (2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)boronic acid (0.38 g, 1.8 mmol) were dissolved in degassed DMA (10 mL). After adding Pd(OAc)2 (0.01 g, 0.046 mmol), dppf (0.05 g, 0.092 mmol), CuCl (0.089 g, 0.9 mmol) and Cs2CO3 (0.588 g, 1.66 mmol), the reaction mixture was heated in a sand bath at ~100°C for 5 hours. The above reaction was repeated once more. The reaction mixture of both runs was diluted with CH2Cl2 (300 mL) and water / brine (140 mL; 1 / 1). The organic phase was separated, washed with brine (80 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The dark residue was purified by silica gel chromatography (100 g, HP-Ultra) using a Biotage Isolera system using a CH2Cl2 / EtOAc gradient (100 / 0->95 / 5->90 / 10->90 / 10) to give a crude coupled product. The crude coupled product was further purified by silica gel chromatography (100 g, HP-Ultra) using a Biotage Isolera system using a CH2Cl2 / EtOAc gradient (100 / 0->95 / 5->90 / 10->90 / 10) to give the coupled product as a light yellow solid (0.473 g, 63%).

[0765] 1 H NMR (400MHz, DMSO-d6 / CDCl3): δ8.33(t,1H), 6.46(dd,1H), 4.06(t,2H), 3.08(t,2H), 2.00(br-s,4H), 1.50(s,9H). LCMS(ESI)419 / 383.15 / 319.11m / z[M+H] + .

[0766] Step-2: Synthesis of 2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0767] The raw material tert-butyl 2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (0.473 g, 1.13 mmol) was dissolved in CH2Cl2 (25 mL) and the mixture was cooled to 0°C. TFA (4.4 mL) was added at 0°C and the reaction mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and the residue was dissolved in CH2Cl2 (300 mL) and saturated NaHCO3 (80 mL). The organic phase was separated, washed with brine (80 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the deprotected product as a light yellow solid (0.349 g, 97%).

[0768] 1 H NMR (400MHz, DMSO-d6): δ8.33(dd,1H),7.80(br-s,1H),6.54(dd,1H),3.53-3.43(m,6H),2.99(t,2H),1.97(br-s,4H). LCMS(ESI)319.15m / z[M+H] + .

[0769] Step-3: Synthesis of tert-butyl (5-bromo-2-fluorophenyl)(tert-butoxycarbonyl)carbamate

[0770] Commercially available 5-bromo-2-fluoroaniline (0.5 g, 2.6 mmol) was dissolved in THF (15 mL) and Boc2O (2.29 g, 10.5 mmol) was added. After adding DMAP (0.482 g, 3.95 mmol), the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was diluted with EtOAc (150 mL) and water / brine (80 mL; 1 / 1). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The dark residue was purified by silica gel chromatography (50 g, HP-Ultra) using a Biotage Isolera system using an EtOAc / n-heptane gradient (5 / 95->10 / 90->20 / 80->20 / 80) to give the bis-Boc protected product as a colorless oil that turned into a white solid (0.452 g, 44%) when left at room temperature.

[0771] 1H NMR (400MHz, DMSO-d6): δ7.74-7.70(m,1H),7.63-7.59(m,1H),7.33(t,1H),1.39(s,18H).

[0772] Step-4: Synthesis of tert-butyl (tert-butoxycarbonyl)(2-fluoro-5-(2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridin-5(4H)-yl)phenyl)carbamate and tert-butyl (2-fluoro-5-(2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridin-5(4H)-yl)phenyl)carbamate

[0773] Pd(OAc)2 (0.002 g, 0.01 mmol) and XantPhos (0.016 g, 0.02 mmol) were dissolved in degassed 1,4-dioxane (6 mL) in a 5 ml microwave vial. The reaction mixture was heated in a sand bath at ~120 °C for about 2 minutes to form crystals. After adding 2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (0.035 g, 0.11 mmol), tert-butyl (5-bromo-2-fluorophenyl)(tert-butoxycarbonyl)carbamate (0.107 g, 0.275 mmol) and Cs2CO3 (0.163 g, 0.5 mmol), the reaction mixture was heated in a sand bath at ~120 °C for 18 hours. The reaction mixture was diluted with CH2Cl2 (50 mL) and water / brine (20 mL; 1 / 1). The organic phase was separated, washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The dark residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system using an EtOAc / n-heptane gradient (0 / 95->40 / 60->80 / 20->100 / 0) to give a mixture of bis-Boc protected and mono-Boc protected coupling products as a yellow solid (0.0081 g, 11%).

[0774] LCMS (ESI) 628.44 / 528.32 (bis-Boc) and 528.30 (mono-Boc) m / z [M+H] + .

[0775] Step-5: Synthesis of 5-(3-amino-4-fluorophenyl)-2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Compound 10)

[0776] The mixture of the bis-Boc protected and mono-Boc protected coupling products (0.0225 g, 0.036 mmol) was dissolved in CH2Cl2 (5 mL), and the mixture was cooled to 0°C. TFA (0.39 mL) was added at 0°C, and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the residue was dissolved in CH2Cl2 (100 mL) and saturated NaHCO3 (30 mL). The organic phase was separated, washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system eluting with a EtOAc / n-heptane gradient (5 / 95->40 / 60->80 / 20->80 / 20) followed by EtOAc / CH2Cl2 (50 / 50) to elute the impurities, giving compound 10 as a light amber solid (0.0084 g, 54%).

[0777] 1 H NMR (400MHz, DMSO-d6): δ8.36(t,1H),7.01(dd,1H),6.77(dd,1H),6.56(dd,1H),6.52-6 .48(m,1H),5.24(br-s,2H),3.99(t,2H),3.48(br-s,4H),3.19(t,2H),1.99(br-s,4H). LCMS(ESI)428.21m / z[M+H] + .

[0778] Example compounds 26, 28 to 32, 34

[0779] The following compounds were prepared according to the coupling method reported in Preparative Example 10 (Steps 4-5) and using the building blocks of Example 1 Step 6, Example 15 Step 2, Example 16 Step 2, Example 17 Step 2 and the halogen derivatives shown in Table 3.

[0780] Table 3

[0781]

[0782]

[0783]

[0784]

[0785] Example compounds 35-37

[0786] Following the coupling method reported in Preparative Example 1 and using the building blocks of Example 15, Step 2, Example 16, Step 2 and the halogen derivatives shown in Table 4, the following compounds were prepared.

[0787] Table 4

[0788]

[0789]

[0790]

[0791] Example 6 Synthesis of 2-(2-fluoro-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Example Compound 17)

[0792]

[0793] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0794] 2,4-dioxopiperidin-1-carboxylic acid tert-butyl ester A (10g, 46.9mmol) was dissolved in CCl4 (125mL), and the mixture was cooled to 0 to 5°C. Then NBS (8.35g, 46.9mmol) was added in batches, and stirring was continued at 28°C for 1 hour. The reaction mixture was diluted with ethyl acetate (2x 500mL) and water (200mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10g, 73%).

[0795] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0796] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0797] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in EtOH (160 mL) and heated at 80 ° C for 2.5 hours in an oil bath. The reaction mixture was diluted with ethyl acetate (2 x 500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The solid was recrystallized from EtOH to obtain title compound C as a white solid (6.6 g, 71%).

[0798] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0799] Step-3: Synthesis of tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (D)

[0800] The title compound C (6.6 g, 24.53 mmol) was dissolved in CH3CN (82 mL) and cooled to -10 °C in an ice bath with stirring. Tert-butyl nitrite (4.3 mL, 36.4 mmol) was added and stirring was continued at -10 °C for 1 hour. Copper (II) bromide (6.5 g, 29.43 mmol) was added, the ice bath was removed, and the reaction mixture was stirred at 28 °C for 1 hour. The reaction mixture was alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution and filtered. The collected filtrate was diluted with ethyl acetate (3 x 300 mL) and water (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (100 to 200 mesh) using ethyl acetate / n-hexane gradient (0 / 100->10 / 90) to obtain the title compound D as a white solid (4.73 g, 58%).

[0801] 1 H NMR (500MHz DMSO-d6): δ4.12(t,2H),3.10(t,2H),1.5(s,9H). LCMS(ESI)279m / z[M+H-C4H8] + .

[0802] Step-4: Synthesis of tert-butyl 2-(2,6-difluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (F)

[0803] The title compound D (0.3 g, 0.9 mmol) was dissolved in 1,4-dioxane (15 mL) and degassed by passing nitrogen through the mixture. Pd(dppf)Cl2 x CH2Cl2 (0.07 g, 0.09 mmol), the title compound E (0.21 g, 1.35 mmol) and tripotassium phosphate (0.28 g, 1.35 mmol) were then added and the reaction mixture was heated at 100°C in an oil bath for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (basic, 100-200 mesh) using an ethyl acetate / hexane gradient (0 / 100->10 / 90->15 / 85) to give the title compound F as a light yellow solid (0.2 g, 60%).

[0804] 1 H NMR (500MHz DMSO-d6): δ8.91(q,1H),7.43(dd,1H),4.1(t,2H),3.19(t,2H),1.5(s,9H). LCMS(ESI)311.95m / z[M+H-C4H8] +。

[0805] Step-5: Synthesis of tert-butyl 2-(2-fluoro-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0806] The title compound F (0.15 g, 0.41 mmol), piperidin-4-ylmethanol (0.045 g, 0.41 mmol) and DIPEA (0.09 g, 0.82 mmol) were dissolved in DMSO (15 mL) and stirred at ambient temperature for 2 hours. After the reaction was complete, the reaction mixture was quenched with ice-cold water, filtered, and the solid was dried under reduced pressure to give the title compound G as an off-white solid (0.18 g, 98%).

[0807] 1 H NMR (500MHz, DMSO-d6): δ8.32(t,1H),6.91(dd,1H),4.51(t,1H),4.37(d,2H),4.05(t,2H), 3.28(t,3H),3.09(t,2H),2.98(t,2H),2.54(s,3H),1.73(m,3H),1.49(s,9H),1.12(m,2H). LCMS(ESI)463.20m / z[M+H] + .

[0808] Step-6: Synthesis of 2-(2-fluoro-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0809] The title compound G (0.18g, 0.38mmol) is dissolved in DCM (4mL) and cooled to 0 ° C in an ice bath under stirring. Then 4M HCl (2mL) in 1,4-dioxane is added, and stirring is continued at room temperature for 3 hours. After the reaction is completed, the solvent is removed under reduced pressure. The residue is dissolved in ice-cold water and basified to pH 8-9 with saturated sodium bicarbonate aqueous solution. The solid is collected by filtration and dried under reduced pressure to obtain the title compound H as a light yellow solid (0.13g, 92%).

[0810] 1 H NMR (500MHz, DMSO-d6): δ8.32(t,1H),7.82(s,1H),6.91(dd,1H),4.51(t,1H) ,4.36(d,2H),3.5(m,2H),3.27(t,2H),2.97(m,4H),1.73(m,3H),1.12(m,2H). LCMS(ESI)362.90m / z[M+H] + .

[0811] Step-7: Synthesis of 2-(6-(4-(((tert-butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-2-fluoropyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (I)

[0812] The title compound H (0.13 g, 0.35 mmol) and imidazole (0.24 g, 3.6 mmol) were dissolved in DMF (3 mL), and the reaction mixture was cooled to 0 ° C in an ice bath. Tert-butyldimethylsilyl chloride (0.26 g, 1.8 mmol) was then added in batches, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with cold water (50 mL), the precipitate was collected by filtration, and dried under reduced pressure. The residue was purified by silica gel column chromatography (100-200 mesh) using CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to obtain the title compound I as a yellow solid (0.15 g, 88%).

[0813] 1H NMR (500MHz, DMSO-d6): δ8.32(t,1H),7.82(s,1H),6.90(dd,1H),4.37(d,2H), 3.48(m,4H),2.97(m,4H),1.75(d,3H),1.16(m,3H),0.86(s,9H),0.029(s,6H). LCMS(ESI)477.00m / z[M+H] + .

[0814] Step-8: Synthesis of 2-(6-(4-(((tert-butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-2-fluoropyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (J)

[0815] The title compound I (0.1 g, 0.21 mmol) and 3-iodopyridine (0.12 g, 0.63 mmol) were dissolved in degassed 1,4-dioxane (10 mL). After adding Pd[PPh3]4 (0.024 g, 0.021 mmol), XantPhos (0.024 g, 0.04 mmol) and Cs2CO3 (0.2 g, 0.63 mmol), the reaction mixture was heated in a sand bath at ~100 °C for 24 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give J as a yellow solid (0.085 g, 73%).

[0816] 1 H NMR (500MHz, DMSO-d6): δ8.32(t,1H),7.82(s,1H),6.90(dd,1H),4.37(d,2H), 3.48(m,4H),2.97(m,4H),1.75(d,3H),1.16(m,3H),0.86(s,9H),0.029(s,6H). LCMS(ESI)477.00m / z[M+H] + .

[0817] Step-9: Synthesis of 2-(2-fluoro-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Compound 17)

[0818] The title compound J (0.08g, 0.14mmol) was dissolved in DCM (2mL), and the mixture was cooled to 0°C in an ice bath under stirring. Then 4M HCl (1mL) in 1,4-dioxane was added, and stirring was continued at room temperature for 3 hours. After the reaction was completed, the solvent was removed under reduced pressure. The residue was dissolved in ice-cold water and basified to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitate was collected by filtration and dried under reduced pressure to obtain the title compound 4 as a light yellow solid (0.058g, 92%).

[0819] 1 H NMR (500MHz, DMSO-d6): δ8.66(d,1H),8.45(q,1H),8.36(dd,1H),7.84(dt,1H),7.47(q,1H),6.93(dd ,1H),4.52(t,1H),4.38(d,2H),4.16(t,2H),3.26(m,4H),2.98(m,2H),1.72(m,3H),1.13(m,2H). LCMS(ESI)439.90m / z[M+H] + .

[0820] Example 7 1-(6-fluoro-5-(4-oxo-5-(pyridin-3-yl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)piperidine-4-carboxylic acid ethyl ester (Compound 17) 3 Synthesis of H-precursor

[0821]

[0822] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0823] Tert-butyl 2,4-dioxopiperidin-1-carboxylate A (10 g, 46.9 mmol) was dissolved in carbon tetrachloride (125 mL) and cooled to 0 to 5 ° C. To the above solution, N-bromosuccinimide (8.35 g, 46.9 mmol) was added in batches and continued to stir at 28 ° C for 1 hour. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10 g, 73%).

[0824] 1H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0825] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0826] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in ethanol (160 mL) and heated in an oil bath at 80 ° C for 2.5 hours. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting solid was recrystallized from ethanol to obtain title compound C as a white solid (6.6 g, 71%).

[0827] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0828] Step-3: Synthesis of tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (D)

[0829] The title compound C (6.6 g, 24.53 mmol) was dissolved in acetonitrile (82 mL) and cooled to -10 ° C in an ice bath with stirring. Tert-butyl nitrite (4.3 mL, 36.4 mmol) was added to the above solution and continued to stir at -10 ° C for 1 hour. Copper (II) bromide (6.5 g, 29.43 mmol) was added to the above mixture and stirred at 28 ° C for 1 hour. The reaction mixture was alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution and filtered. The collected filtrate was diluted three times with ethyl acetate (300 mL) and water (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (100 to 200 mesh) using ethyl acetate / n-hexane gradient (0 / 100->10 / 90) to obtain the title compound D as a white solid (4.73 g, 58%).

[0830] 1H NMR (500MHz DMSO-d6): δ4.12(t,2H),3.10(t,2H),1.5(s,9H). LCMS(ESI)279m / z[M+H-C4H8] + .

[0831] Step-4: Synthesis of tert-butyl 2-(2,6-difluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (F)

[0832] Tert-butyl 2-bromo-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate D (0.3 g, 0.9 mmol) was dissolved in 1,4-dioxane (15 mL), and the mixture was degassed by passing nitrogen gas through the mixture. Then, [1,1′-bis(diphenyl-phosphino)ferrocene]-dichloropalladium(II) (0.07 g, 0.09 mmol) complexed with dichloromethane, the title compound E (0.21 g, 1.35 mmol) and tripotassium phosphate (0.28 g, 1.35 mmol) were added, and the reaction mixture was heated at 100° C. in an oil bath for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (basic, 100-200 mesh) using an ethyl acetate / n-hexane gradient (0 / 100->10 / 90->15 / 85) to give the title compound F as a light yellow solid (0.2 g, 60%).

[0833] 1 H NMR (500MHz DMSO-d6): δ8.91(q,1H),7.43(dd,1H),4.1(t,2H),3.19(t,2H),1.5(s,9H). LCMS(ESI)311.95m / z[M+H-C4H8]+

[0834] Step-5: Synthesis of tert-butyl 2-(6-(4-(ethoxycarbonyl)piperidin-1-yl)-2-fluoropyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (K)

[0835] The title compound F (0.3 g, 0.82 mmol), ethyl piperidine-4-carboxylate (0.13 g, 0.82 mmol) and DIPEA (0.2 g, 1.63 mmol) were dissolved in DMSO (30 mL), and the reaction mixture was stirred at ambient temperature for 2 hours. After the reaction was completed, the reaction mixture was quenched with ice-cold water, the precipitated solid was filtered and dried to give the title compound K as an off-white solid (0.37 g, 90%).

[0836] 1 H NMR (500MHz, DMSO-d6): δ8.35(t,1H),6.93(dd,1H),4.26(d,2H),4.07(m,4H),3.15(m ,2H),3.10(t,2H),2.69(m,1H),1.93(dd,2H),1.58(m,2H),1.49(s,9H),1.19(t,3H). LCMS(ESI)505.35m / z[M+H] + .

[0837] Step-6: Synthesis of ethyl 1-(6-fluoro-5-(4-oxo-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)piperidine-4-carboxylate (L)

[0838] The title compound K (0.37g, 0.73mmol) is dissolved in DCM (11mL), and the mixed solution is cooled to 0°C in an ice bath under stirring. 4M HCl (4.0mL) in 1,4-dioxane is added to the above solution, and stirring is continued at room temperature for 2 hours. After the reaction is completed, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water, and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution, the precipitated solid is filtered, and dried to obtain the title compound L as a light yellow solid (0.26g, 89%).

[0839] 1 H NMR (500MHz, DMSO-d6): δ8.34(t,1H),7.83(s,1H),6.92(dd,1H),4.25(d,2H),4.08(q,2H),3 .50(m,2H),3.12(m,2H),2.99(t,2H),2.69(m,1H),1.93(m,2H),1.57(m,2H),1.21(m,3H). LCMS(ESI)405.5m / z[M+H] + .

[0840] Step-7: Synthesis of ethyl 1-(6-fluoro-5-(4-oxo-5-(pyridin-3-yl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)pyridin-2-yl)piperidine-4-carboxylate (18)

[0841] The title compound L (0.15 g, 0.37 mmol) and 3-bromopyridine (0.26 g, 1.11 mmol) were dissolved in degassed 1,4-dioxane (15 mL). After adding Pd[PPh3]4 (0.021 g, 0.018 mmol), XantPhos (0.021 g, 0.037 mmol) and Cs2CO3 (0.24 g, 0.74 mmol), the reaction mixture was heated at ~100 °C in an oil bath for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give compound 18 as a yellow solid (0.15 g, 75%).

[0842] 1 H NMR (500MHz, DMSO-d6): δ8.67(d,1H),8.58(d,1H),8.38(dd,1H),8.17(t,1H),6.95(dd,1H),4.26(d,2H), 4.18(t,2H),4.08(q,2H),3.25(t,2H),3.16(m,2H),2.72(m,1H),1.94(dd,1H),1.55(m,2H),1.19(t,3H). LCMS(ESI)560.30m / z[M+H] + .

[0843] Example 8 2-(6-(4-(Hydroxymethyl)piperidin-1-yl)-2-nitropyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Compound 17) 18 Synthesis of F-precursor

[0844]

[0845] Step-1: Synthesis of tert-butyl 3-bromo-2,4-dioxopiperidine-1-carboxylate (B)

[0846] Tert-butyl 2,4-dioxopiperidin-1-carboxylate A (10 g, 46.9 mmol) was dissolved in carbon tetrachloride (125 mL), and the mixture was cooled to 0 to 5 ° C. N-bromosuccinimide (8.35 g, 46.9 mmol) was added to the above solution in batches, and stirring was continued at 28 ° C for 1 hour. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the title compound B as a white solid (10 g, 73%).

[0847] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)292.04m / z[M+H] + .

[0848] Step-2: Synthesis of tert-butyl 2-amino-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (C)

[0849] Title compound B (10 g, 34.36 mmol), thiourea (2.61 g, 34.36 mmol) and sodium bicarbonate (2.88 g, 34.36 mmol) were dissolved in ethanol (160 mL) and heated in an oil bath at 80 ° C for 2.5 hours. The reaction mixture was diluted twice with ethyl acetate (500 mL) and water (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The resulting solid was recrystallized from ethanol to obtain title compound C as a white solid (6.6 g, 71%).

[0850] 1 H NMR (500MHz, DMSO-d6): δ8.1(s,2H),3.89(t,2H),2.76(t,2H),1.45(s,9H). LCMS(ESI)270.3m / z[M+H] + .

[0851] Step-3: Synthesis of tert-butyl 2-(dibenzylamino)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (M)

[0852] The title compound C (0.1 g, 0.37 mmol) and cesium bicarbonate (0.84 g, 2.6 mmol) were suspended in DMF (5 mL) and the mixture was cooled to 0 ° C in an ice bath. Benzyl bromide (0.26 g, 1.5 mmol) was added to the above solution and continued to stir at room temperature for 7 hours. The reaction mixture was quenched with water (50 mL) and extracted twice with ethyl acetate (25 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a hexane / EtOAc gradient (100 / 0->98 / 2->96 / 4->94 / 6->92 / 8) to obtain the title compound M as an off-white sticky solid (0.09 g, 71%).

[0853] 1 H NMR (400MHz, DMSO-d6): δ7.33(m,10H),4.8(s,4H),3.93(t,2H),2.85(t,2H),1.44(s,9H). LCMS(ESI)448m / z[MH] + .

[0854] Step-4: Synthesis of 2-(dibenzylamino)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (N)

[0855] Title compound M (0.09g, 0.2mmol) is dissolved in DCM (2mL), and is cooled to 0 ℃ under agitation in an ice bath. TFA (0.45mL) is added in the above solution, and stirring is continued at room temperature for 2 hours. After the reaction is completed, desolventizing is removed under reduced pressure. The residue obtained is dissolved in ice-cold water, and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. This mixed solution is extracted twice with ethyl acetate (25mL). The organic phase is separated, through Na SO Drying, filter, and desolventizing under reduced pressure, obtain title compound N, be rice white solid (0.065g, 93%).

[0856] 1 H NMR (400MHz, DMSO-d6): δ7.32(m,10H),4.76(s,4H),3.37(m,2H),2.73(t,2H). LCMS(ESI)350.4m / z[MH] + .

[0857] Step-5: Synthesis of 2-(dibenzylamino)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (O)

[0858] The title compound N (1.0 g, 2.86 mmol) and 3-iodopyridine (1.75 g, 8.5 mmol) were dissolved in degassed 1,4-dioxane (50 mL). After adding Pd[PPh3]4 (0.16 g, 0.14 mmol), XantPhos (0.16 g, 0.28 mmol) and Cs2CO3 (2.78 g, 8.5 mmol), the reaction mixture was heated at ~100 °C in an oil bath for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1->98 / 2) to give O as an off-white solid (0.75 g, 61%).

[0859] 1 H NMR (400MHz, DMSO-d6): δ8.57(d,1H),8.37(dd,1H),7.74(m,1H),7.39(m,5H),7.31(m,6H),4.81(s,4H),4.02(t,2H),3.0(t,2H). LCMS(ESI)424.8m / z[MH] + .

[0860] Step-6: Synthesis of 2-amino-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (P)

[0861] Title compound O (0.75g, 1.7mmol) is suspended in toluene (75mL), and is cooled to 0 ℃ under stirring in an ice bath. Methanesulfonic acid (7.5mL) is added in the above solution, and stirring is continued for 6 hours at 100 ℃. After the reaction is completed, the two layers are separated. The toluene phase is poured out to obtain a rough brown liquid. The rough liquid is cooled to 0 ℃ in an ice bath, and quenched to pH 8-9 with a saturated sodium bicarbonate aqueous solution, extracted with 10% MeOH (75mL) of two volumes in DCM. The organic phase is separated, through Na SO Drying, filtered, and desolventizing under reduced pressure to obtain title compound P, which is an off-white solid (0.22g, 51%).

[0862] 1 H NMR (400MHz, DMSO-d6): δ8.58(d,1H),8.37(d,1H),7.93(s,2H),7.74(m,1H),7.41(d,1H),3.99(t,2H),2.9(t,2H). LCMS(ESI)244.8m / z[MH] + .

[0863] Step-7: Synthesis of 2-bromo-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (Q)

[0864] Title compound P (0.5g, 2.03mmol) is dissolved in acetonitrile (25mL), and is cooled to 0 ℃ under stirring in an ice bath. Tert-butyl nitrite (0.7mL, 6.0mmol) is added to the above solution, and continues to stir for 30 minutes at 0 ℃. Copper bromide (II) (0.9g, 4.06mmol) is added to the above mixed solution, and it is stirred for 3 hours at 28 ℃. The reaction mixture is alkalized to pH 8 to 9 with saturated sodium bicarbonate aqueous solution, and filtered. The collected filtrate is diluted twice with 10% MeOH (100mL) in DCM. The organic phase is separated, dried over Na2SO4, filtered, and the solvent is removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1->98 / 2) to afford the title compound Q as an off-white solid (0.22 g, 35%).

[0865] 1 H NMR (400MHz, DMSO-d6): δ8.64(m,1H),8.46(dd,1H),7.83(m,1H),7.48(m,1H),4.13((t,2H),3.24(t,2H). LCMS(ESI)307.7m / z[MH] + .

[0866] Step-8: Synthesis of (1-(6-nitropyridin-2-yl)piperidin-4-yl)methanol (S)

[0867] Compound R (2.5 g, 31.64 mmol), piperidin-4-ylmethanol (9.4 g, 81.7 mmol) and DIPEA (12.26 mL, 94.8 mmol) were suspended in dioxane (120 mL) using a microwave vial. The sealed vial was then heated at 100 ° C for 3 hours using a CEM microwave. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a hexane / EtOAc gradient (100 / 0->90 / 10->80 / 20->70 / 30->60 / 40->50 / 50) to give the title compound S as a yellow oil (4.0 g, 53%).

[0868] 1H NMR (500MHz, DMSO-d6): δ7.83(t,1H),7.38(d,1H),7.27(d,1H),4.5(t,1H), 4.37(d,2H),3.28(t,2H),2.9(m,2H),1.75(d,2H),1.66(m,1H),1.12(m,2H). LCMS(ESI)238.1m / z[M+H] + .

[0869] Step-9: Synthesis of (1-(5-bromo-6-nitropyridin-2-yl)piperidin-4-yl)methanol (T)

[0870] The title compound S (2.1 g, 8.8 mmol) was dissolved in ACN (42 mL) and cooled to -30 ° C. NBS (2.39 g, 13.29 mmol) was added in batches. The reaction mixture was allowed to warm to 0 ° C over a period of 2 hours. The reaction mixture was quenched with water (50 mL) and extracted twice with ethyl acetate (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using hexane / EtOAc gradient (100 / 0->90 / 10->80 / 20->70 / 30->60 / 40->50 / 50) to obtain the title compound T as a light yellow oil (1.5 g, 54%).

[0871] 1 H NMR (400MHz, DMSO-d6): δ7.96(d,1H),7.11(d,1H),4.5(t,1H),4.23(d,2H),3.26(t,2H),2.88(m,2H),1.68(m,3H),1.11(m,2H). LCMS(ESI)313.9m / z[MH] + .

[0872] Step-10: Synthesis of (1-(6-nitro-5-(trimethylstannyl)pyridin-2-yl)piperidin-4-yl)methanol (U)

[0873] The title compound T (1.4 g, 4.4 mmol) was dissolved in degassed 1,4-dioxane (28 mL) in a microwave oven. After adding Pd[PPh3)2Cl2 (0.3 g, 0.44 mmol), hexamethyldistanane (1.3 mL, 1.95 g, 6.0 mmol) and triphenylarsine (0.135 g, 0.4 mmol), the vial was sealed and heated at 100 ° C for 1 hour under CEM microwave. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a hexane / EtOAc gradient (100 / 0->90 / 10->80 / 20->70 / 30->60 / 40->50 / 50) to give U as a yellow oil (0.7 g, 41%).

[0874] 1 H NMR (400MHz, DMSO-d6): δ7.8(d,1H),7.24(d,1H),4.49(t,1H),4.36(d,2H),3.26(t,2H),2.88(m,2H),1.7(m,3H),1.14(m,2H),0.26(s,9H). LCMS(ESI)402.1m / z[M+H] + .

[0875] Step-11: Synthesis of 6-(4-(((tert-butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-2-nitro-3-(trimethylstannyl)pyridine (V)

[0876] The title compound U (0.55 g, 1.37 mmol) and imidazole (1.3 g, 13.7 mmol) dissolved in DMF (11 mL) were cooled to 0 ° C in an ice bath. DMAP (0.033 g, 0.27 mmol) and tert-butyldimethylsilyl chloride (1.0 g, 6.8 mmol) were added in batches. The reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was quenched with water (100 mL) and extracted twice with ethyl acetate (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a hexane / EtOAc gradient (100 / 0->99.5 / 0.5->99 / 1->98.5 / 1.5->98 / 2) to obtain the title compound V as a yellow oil (0.32 g, 43%).

[0877] 1H NMR (400MHz, DMSO-d6): δ7.81(d,1H),7.25(d,1H),4.38(d,2H),3.45(d,2H),2.89(t,2H),1.73(d,3H),1.15(m,2H),0.85(s,9H),0.26(s,6H). LCMS(ESI)514.7m / z[M] + .; 517.3m / z[M+2H] + .

[0878] Step-12: 2-(6-(4-(((tert-butyldimethylsilyl)oxy)methyl)piperidin-1-yl)-2-nitropyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (W)

[0879] Compound Q (0.3 g, 0.96 mmol) and compound V (0.74 g, 1.4 mmol) were dissolved in degassed 1,4-dioxane (45 mL). After adding Pd(OAc)2 (0.02 g, 0.1 mmol), XantPhos (0.055 g, 0.1 mmol) and Cu(I)Cl (0.095 g, 0.96 mmol), the reaction mixture was heated in an oil bath at 80 °C for 4 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99 / 1->98 / 2) to give an impure solid. The impure solid was further purified on preparative TLC plates using dichloromethane / methanol (98 / 2) as mobile phase to afford compound W as a yellow solid (0.15 g, 26%).

[0880] 1 H NMR (400MHz, DMSO-d6): δ8.65(d,1H),8.45(dd,1H),8.17(d,1H),7.83(m,1H),7.48(dd,1H),7.23(d,1H),4.39( d,2H),4.13(t,2H),3.46(d,2H),3.18(t,2H),3.01(t,2H),1.76(d,3H),1.17(m,2H),0.86(s,9H),0.031(s,6H). LCMS(ESI)581.5m / z[M+H] + .

[0881] Step-13: 2-(6-(4-(Hydroxymethyl)piperidin-1-yl)-2-nitropyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (20)

[0882] Compound W (0.1g, 0.17mmol) is dissolved in THF (10mL), and is cooled to 0 ℃ under stirring in an ice bath. Then add 1M TBAF solution in THF (1mL), and continue stirring at room temperature for 2 hours. The reaction is quenched with saturated sodium bicarbonate aqueous solution (pH 8-9), and extracted with 5% MeOH (2x 25mL) in DCM. The organic phase is separated, through Na2SO4 drying, filtered, and desolventized under reduced pressure. Residue is purified through silica gel column chromatography (100-200 mesh) using CH2Cl2 / MeOH gradient (100 / 0->99 / 1->98 / 2) purification to obtain the desired compound containing some impurities. Impure material is further purified through preparative TLC plate using dichloromethane / ethanol (95 / 5) as mobile phase to obtain title compound 20, which is a yellow solid (0.026g, 34%). 1 H NMR (500MHz, DMSO-d6): δ8.65(d,1H),8.45(dd,1H),8.17(d,1H),7.84-7.82(m,1H),7.48(dd,1H),7.23( d,1H),4.53(t,1H),4.38(d,2H),4.14(t,2H),3.28(t,2H),3.19(t,2H),3.00(t,2H),1.78-1.67(m,3H). LCMS(ESI)467.7m / z[M+H] + .

[0883] Example 9 2-(2-Fluoro-6-(4-(hydroxymethyl-t2)piperidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one ( 3 Synthesis of H-compound 17)

[0884]

[0885] T means 3 H.

[0886] The reaction vessel was charged with 150 μL n-BuLi (0.24 mmol, 1.6 M in hexane) and 50 μL TMEDA. The solution was degassed 3 times in a high vacuum manifold and stirred at room temperature for 40 minutes under a tritium atmosphere (1343 mbar, 9.2 Ci). The process was repeated twice (2nd run: 1097 mbar, 6.6 Ci, 3rd run: 912 mbar, 4.8 Ci). A pressure of 333 mbar was obtained at the end of the reaction (LiT formation). The solvent was removed under vacuum and AlCl3 solution (6.7 mg in0.2 mL THF) was added under stirring. The mixture was stirred at room temperature for 10 minutes. A suspension of the precursor compound (3.3 mg in 0.5 mL THF) was added at -78 ° C, and the reaction mixture was stirred at -78 ° C for 3 hours. The reaction vessel was cooled to -196 ° C, and 0.2 mL H2O was added. Due to the strong pressure increase, warm to room temperature very slowly. Remove the solvent under vacuum and exchange the unstable tritium by adding 0.3 ml of methanol, stir the solution and remove the solvent again under vacuum. Repeat this process twice. Finally, the fully dried solid is dissolved in 5 ml of EtOH. The radiochemical purity of the crude material was determined to be about 44% using the following HPLC system: Waters Sunfire C18, 5 μm, 4.6 x 250 mm; solvent A: water + 0.05% TFA, B: acetonitrile + 0.05% TFA; 0 minutes 20% B; 10-14.5 minutes 95% B, 15 minutes 20% B; 254 nm; 1.0 ml / min; 30 ° C. The retention time of the product is 5.82 minutes. For purification of the compound, the following HPLC conditions were used: Waters Sunfire C18, 10 x 250 mm; solvent A: water + 0.1% TFA; B: acetonitrile + 0.1%); 27% B isoconcentration; 4.7 ml / min; 25°C. The target compound eluted in 8.8-9.6 min. The solvent was removed under vacuum and the product was dissolved in 5 ml of ethanol. The purified product was obtained. 3 H-Compound 17, specific activity 55.4 Ci / mmol, (97% purity).

[0887] Example 10 Tritiated (R)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one 3 Synthesis of H-compound 7

[0888]

[0889] T means 3 H.

[0890] 2.29 mg of brominated precursor 19 (prepared according to the method described in Example 2 above) 3 H] The synthesis process of the precursor and the corresponding structural unit for compound 7 shown in Table 1 were used to prepare it), 50 μL DIPEA and 31.7 mg Lindela catalyst were suspended in 0.3 ml DMF. The suspension was degassed 3 times in a high vacuum manifold and stirred overnight at room temperature under a tritium atmosphere (401 mbar, 11.2 Ci). The solvent was removed under vacuum, and unstable tritium was exchanged by adding 0.3 ml methanol, the solution was stirred, and the solvent was removed again under vacuum. The process was repeated twice. The dried solid was extracted with 5 ml ethanol / DMF (4: 1), and the suspension was filtered through a 0.2 μm nylon membrane to obtain a clear solution. For the purification of the compound, the following HPLC conditions were found to be suitable: Waters Sunfire C18, 10x 250 mm; Solvent A: water + 0.1% TFA); B: acetonitrile + 0.1% TFA. The purified product was obtained 3 H-Compound 7 (SA 50.8 Ci / mmol, 99% purity).

[0891] Embodiment 11: Tritiated (S)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one ( 3 Synthesis of H-compound 8)

[0892]

[0893] T means 3 H.

[0894] 2.45 mg of brominated precursor 20 (prepared according to the method described in Example 2 above) 3H] The precursor was prepared using the corresponding structural units for compound 8 shown in Table 1), 50 μL of DIPEA and 33.5 mg of Lindela catalyst were suspended in 0.3 ml of DMF. The suspension was degassed 3 times in a high vacuum manifold and stirred overnight at room temperature under a tritium atmosphere (498 mbar, 4.3 Ci). The solvent was removed under vacuum, and unstable tritium was exchanged by adding 0.3 ml of methanol, the solution was stirred, and the solvent was removed again under vacuum. The process was repeated twice. The dried solid was extracted with 5 ml of ethanol / DMF (4:1), and the suspension was filtered through a 0.2 μm nylon membrane to obtain a clear solution. For the purification of the compound, the following HPLC conditions were found to be suitable: Waters Sunfire C18, 10x 250 mm; Solvent A: water + 0.1% TFA); B: acetonitrile + 0.1% TFA. The purified product was obtained 3 H-Compound 8 (SA 48.5 Ci / mmol, 99% purity).

[0895] Example 12 Tritiated 2-(2-fluoro-6-(pyrrolidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one 3 Synthesis of H-compound 9

[0896]

[0897] T means 3 H.

[0898] 1.7 mg of brominated precursor 21 (prepared according to the method described in Example 2 above) 3 H] precursor synthesis process and using the corresponding structural unit for compound 9 shown in Table 1 to prepare), 20 μL DIPEA and 7.5 mg palladium carbon (10% metal) were suspended in 0.3 ml DMF. The suspension was degassed 3 times in a high vacuum manifold and stirred overnight at room temperature under a tritium atmosphere (670 mbar, 8.4 Ci). The solvent was removed under vacuum, and unstable tritium was exchanged by adding 0.3 ml methanol, the solution was stirred, and the solvent was removed again under vacuum. This process was repeated twice. The dried solid was extracted with 5 ml ethanol / DMF (4: 1), and the suspension was filtered through a 0.2 μm nylon membrane to obtain a clear solution. For the purification of this compound, the following HPLC conditions were found to be suitable: Waters Sunfire C18, 10x 250 mm; solvent A: water + 0.1% TFA); B: acetonitrile + 0.1% TFA. The purified product was obtained 3 H-Compound 9 (SA 57.6 Ci / mmol, 99% purity).

[0899] Example 13 Tritiated 2-[2-fluoro-6-[(3R)-3-fluoropyrrolidin-1-yl]-3-pyridinyl]-5-pyrimidin-2-yl-6,7-dihydrothiazolo[5,4-c]pyridin-4-one 3 Synthesis of H-compound 15

[0900]

[0901] T means 3 H.

[0902] The di-brominated precursor 22 (described in Example 2 above) was 3 H] The precursor was prepared using the corresponding building blocks for compound 15 shown in Table 1) (2.45 mg, provided by the user), DIPEA (50 μL, Acros A0408500) and Lindela catalyst (15.5 mg) were suspended in DMF (0.4 ml, Acros 34831000). The suspension was degassed twice in a high vacuum manifold and stirred overnight at room temperature under a tritium atmosphere (569 mbar, 7.7 Ci). At the end of the reaction, a pressure of 531 mbar was observed at room temperature. The solvent was removed under vacuum and the unstable tritium was exchanged by adding 0.3 ml of methanol, the solution was stirred, and the solvent was removed again under vacuum. The process was repeated twice. Finally, the dried solid was extracted with 5 ml of DCM and the suspension was filtered to obtain a clear solution. For purification of the compound, the following HPLC conditions were found to be suitable: Waters Sunfire C18, 10 x 250 mm; Solvent A: water (JTBaker HPLC GradientGrade4218) + 0.1% TFA (Sigma-Aldrich T6508); B: acetonitrile (MACRON ChromAR HPLC SuperGradient 2856-25) + 0.1% TFA (Sigma-Aldrich T6508); 55% B isoconcentration; 4.7 ml / min; 25° C. The purified product was obtained. 3 H-Compound 15 (SA 52 Ci / mmol, 99% purity).

[0903] Embodiment 14 Fluorinated 2-(2-(fluoro- 18 Synthesis of F)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridin-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0904] 18 F Compound 17

[0905]

[0906] The [ 18 F] fluoride was transferred and trapped on a Chromafix PS-HCO3 column (activated with KOTf (0.2 M, 6 mL) and then water (6 mL). It was then washed with K 222 / K2CO3 solution (15 / 3 mg, 0.3 mL) to The reaction vessel of the module. The solution was first heated at 70°C for 3.5 minutes under vacuum and helium flow, and the heat treatment was continued at 95°C under the same conditions for another 2.5 minutes. The reactor was then cooled to 60°C. A solution of 2-(6-(4-(hydroxymethyl)piperidin-1-yl)-2-nitropyridine-3-yl)-5-(pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one 20 (3.35 mg in 0.3 mL DMSO) was added to the reaction vial, and the reaction vessel was heated at 160°C for 10 minutes. The reaction vessel was cooled to 60°C, and the mixture was diluted with HPLC buffer (4 mL). Purification was performed by HPLC: a semi-preparative Xbridge C18 column (5 μm, 250 x 10 mm) was used and eluted with a mixture of acetonitrile / ammonium acetate solution (20 mM, 35 / 65, v / v) at a flow rate of 5 mL / min. The radiolabeled product was eluted from the SPE column with 1.0 mL of ethanol into a preparation flask pre-loaded with 10 mL of the preparation matrix (ascorbic acid (10 mg) in saline). The resulting solution was passed through a 0.2 μm sterilizing membrane filter into a sterile filter vented vial. Analysis of the final product 18 F-Compound 17, take out a sample for QC test. 19 F—Reference compound 17 sample was co-injected to confirm the identity of the product.

[0907] (S)-2-(2-Fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazol-1-yl Synthesis process of oxazolo[5,4-c]pyridin-4(5H)-one (Compound 25)

[0908]

[0909] Embodiment 15 Synthesis of (S)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0910]

[0911] Step-1: Synthesis of (S)-tert-butyl 2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0912] The title compound F (0.26 g, 0.7 mmol), (S)-3-fluoropyrrolidine hydrochloride (0.088 g, 0.7 mmol) and N, N-diisopropylethylamine (0.2 mL, 1.4 mmol) were suspended in DMSO (26 mL) and stirred at room temperature for 2 hours. After completion, the reaction mixture was suspended in water (260 mL) and the precipitated solid was filtered through Whatmann filter paper. The solid was washed with water (20 mL) and dried under reduced pressure to give the title compound G as a light yellow solid (0.27 g, 91%). 1 H NMR (500MHz, DMSO-d6): δ8.39(t,1H),6.63(d,1H),5.48(d,1H),4.06(t,2H),3.70(dd,3H), 3.54-3.49(m,1H),3.10(t,2H),2.30-2.27(m,2H),1.49(S,9H).LCMS(ESI)437.10m / z[M+H] + .

[0913] Step-2: Synthesis of (S)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0914] Title compound G (0.27g, 0.6mmol) is dissolved in DCM (6mL), and cooled to 0 ℃ under stirring in an ice bath, then 4M HCl (2.7mL) in 1,4-dioxane is added to the above solution, and stirring is continued at room temperature for 2 hours. After the reaction is completed, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water, and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitated solid is filtered, and dried to obtain title compound H as a light yellow solid (0.19g, 91%). 1H NMR(500MHz DMSO-d6): δ8.39-8.35(m,1H),7.82(s,1H),6.61-6.60(m,1H),5.48(d,1 H),3.80-3.63(m,4H),3.53-3.48(m,2H),2.99(t,2H),2.30-2.18(m,2H). LCMS(ESI)337[M+H] + .

[0915] Step-3: Synthesis of (S)-2-(2-fluoro-6-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (1)

[0916] Compound H (0.05 g, 0.14 mmol) and 4-bromoisothiazole (0.073 g, 0.44 mmol) were dissolved in degassed 1,4-dioxane (5 mL). After adding CuI (0.014 g, 0.074 mmol), 1,10-phenanthroline (0.026 g, 0.014 mmol) and CsF (0.045 g, 0.29 mmol), the reaction mixture was heated in a sand bath at ~100 °C for 48 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give pure compound 25 as a light yellow solid (0.029 g, 46%). 1 H NMR(400MHz DMSO-d6): δ8.95(s,1H),8.88(s,1H),8.43-8.38(m,1H),6.65-6.62(m,1H),5.48(d,1 H),4.22(t,2H),3.85-3.72(m,3H),3.55-3.48(m,1H),3.27(t,2H),2.27-2.16(m,2H). LCMS(ESI)418.2m / z[MH] +

[0917] (S)-2-(2-Fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazole Synthesis process of 4-[5,4-c]pyridin-4(5H)-one (Compound 27)

[0918]

[0919] Example 16 Synthesis of (S)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0920]

[0921] Step-1: Synthesis of (S)-tert-butyl 2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0922] The title compound F (0.4 g, 1.0 mmol), (S)-3-fluoropiperidine hydrochloride (0.15 g, 1.0 mmol) and N, N-diisopropylethylamine (0.36 mL, 2.1 mmol) were suspended in DMSO (40 mL), and the reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction mixture was suspended in water (400 mL) and filtered through Whatmann filter paper. The solid was washed with water (20 mL) and dried under reduced pressure to obtain the title compound G as a light yellow solid (0.47 g, 96%). 1 H NMR (500MHz, DMSO-d6): δ8.37-8.33(m,1H),6.97-6.95(m,1H),4.85(d,1H),4.22-4.16(m,1H),4.06(t,2H),4.01(d,1H),3.67-3.57(m ,1H),3.38-3.35(m,1H),3.10(t,2H),1.95-1.87(m,2H),1.78-1.70(m,1H),1.61-1.56(m,1H),1.49(s,9H),LCMS(ESI)451.35m / z[M+H] + .

[0923] Step-2: Synthesis of (S)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0924] Title compound G (0.47g, 1.04mmol) is dissolved in DCM (9mL) and cooled to 0°C in an ice bath under stirring. 4M HCl (4.7mL) in 1,4-dioxane is added to the above solution and continued to stir at room temperature for 2 hours. After the reaction is complete, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitated solid is filtered and dried to obtain title compound H as a light yellow solid (0.36g, 98%). 1H NMR(500MHz DMSO-d6): δ8.35-8.31(m,1H),7.82(s,1H),6.95-6.93(m,1H),4.90-4.79(m,1H),4.18-4.13(m,1H),3.99-3.96(m,1H),3.6 6-3.60(m,1H),3.58-3.49(m,2H),3.36-3.34(m,1H),2.99(t,2H),1.94-1.87(m,2H),1.74-1.73(m,1H),1.60-1.56(m,1H). LCMS(ESI)351[M+H]+ .

[0925] Step-3: Synthesis of (S)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (28)

[0926] Compound H (0.05 g, 0.14 mmol) and 4-bromoisothiazole (0.07 g, 0.42 mmol) were dissolved in degassed 1,4-dioxane (5 mL). After adding CuI (0.013 g, 0.07 mmol), 1,10-phenanthroline (0.025 g, 0.14 mmol) and CsF (0.043 g, 0.28 mmol), the reaction mixture was heated in a sand bath at ~100 °C for 48 hours. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1->98.5 / 1.5) to give pure compound 27 as a yellow solid (0.047 g, 77%). 1 H NMR(400MHz DMSO-d6): δ8.95(s,1H),8.88(s,1H),8.39-8.35(m,1H),6.99-6.96(m,1H),4.92-4.79(m,1H),4.23-4.16(m,3 H),4.03-4.00(m,1H),3.68-3.57(m,1H),3.37-3.36(m,1H),3.27(t,2H),1.94-1.87(m,2H),1.76-1.58(m,2H). LCMS(ESI)433.9m / z[MH] +

[0927] (R)-2-(2-Fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazole Synthesis process of 4-[5,4-c]pyridin-4(5H)-one (Compound 24)

[0928]

[0929] Embodiment 17 Synthesis of (R)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0930]

[0931] Step-1: Synthesis of (R)-tert-butyl 2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0932] The title compound F (0.6 g, 1.63 mmol), (R)-3-fluoropiperidine hydrochloride (0.22 g, 1.63 mmol) and N,N-diisopropylethylamine (0.55 mL, 3.27 mmol) were suspended in DMSO (60 mL) and stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was suspended in water (600 mL) and filtered through Whatmann filter paper. The solid was washed with water (20 mL) and dried under reduced pressure to give the title compound G as a light yellow solid (0.63 g, 90%). 1 H NMR (500MHz, DMSO-d6): δ8.35(t,1H),6.97-6.95(m,1H),4.85(d,1H),4.21-4.16(m,1H),4.06-3.99(m,3H),3.67-3.57(m,1H),3.3 8-3.34(m,1H),3.11-3.08(t,2H),1.93-1.87(m,2H),1.76-1.73(m,1H),1.61-1.56(m,1H),1.49(s,9H),LCMS(ESI)451.25m / z[M+H] + .

[0933] Step-2: Synthesis of (R)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0934] Title compound G (0.63g, 1.4mmol) was dissolved in DCM (12mL) and cooled to 0°C in an ice bath under stirring. 4M HCl (6.3mL) in 1,4-dioxane was added to the above solution and continued to stir at room temperature for 2 hours. After the reaction was completed, the solvent was removed under reduced pressure. The obtained residue was dissolved in ice-cold water and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitated solid was filtered and dried to obtain title compound H as a light yellow solid (0.4g, 85%). 1HNMR(500MHz DMSO-d6): δ8.36-8.32(m,1H),7.83(s,1H),6.96-6.94(m,1H),4.89-4.79(m,1H),4.18-4.13(m,1H),3.99-3.96(m,1H),3.6 6-3.57(m,1H),3.51-3.48(m,2H),3.36-3.34(m,1H),2.99(t,2H),1.95-1.87(m,2H),1.77-1.71(m,1H),1.60-1.56(m,1H). LCMS(ESI)351[M+H] + .

[0935] Step-3: Synthesis of (R)-2-(2-fluoro-6-(3-fluoropiperidin-1-yl)pyridin-3-yl)-5-(isothiazol-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (24)

[0936] Compound H (0.05 g, 0.14 mmol) and 4-bromoisothiazole (0.07 g, 0.42 mmol) were dissolved in degassed 1,4-dioxane (5 mL). After adding CuI (0.013 g, 0.07 mmol), 1,10-phenanthroline (0.025 g, 0.14 mmol) and CsF (0.043 g, 0.28 mmol), the reaction mixture was heated in a sand bath at ~100 °C for 36 hours. The reaction mixture was concentrated under reduced pressure to give a crude product. The crude product thus obtained was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give pure compound 24 as a yellow solid (0.03 g, 50%). 1H NMR(400MHz DMSO-d6): δ8.95(s,1H),8.88(s,1H),8.39-8.35(m,1H),6.99-6.96(m,1H),4.91-4.79(m,1H),4.23-3.9 9(m,4H),3.68-3.57(m,1H),3.40-3.39(m,1H),3.28-3.24(m,2H),1.96-1.87(m,2H),1.76-1.57(m,2H). LCMS(ESI)433.8m / z[MH] +

[0937] 2-(2-Fluoro-6-(piperidin-1-yl)pyridin-3-yl)-5-(2-fluoropyridin-4-yl)-6,7-dihydrothiazolo[5,4- c] Synthesis process of pyridin-4(5H)-one (Compound 23)

[0938]

[0939] Embodiment 18 Synthesis of 2-(2-fluoro-6-(piperidin-1-yl)pyridin-3-yl)-5-(2-fluoropyridin-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one

[0940]

[0941] Step-1: Synthesis of tert-butyl 2-(2-fluoro-6-(piperidin-1-yl)pyridin-3-yl)-4-oxo-6,7-dihydrothiazolo[5,4-c]pyridine-5(4H)-carboxylate (G)

[0942] The title compound F (0.3 g, 0.81 mmol), piperidine (0.069 g (0.08 mL), 0.81 mmol) and N,N-diisopropylethylamine (0.2 mL, 1.6 mmol) were suspended in DMSO (30 mL) and stirred at room temperature for 2 hours. After completion, the reaction mixture was suspended in water (300 mL) and filtered through Whatmann filter paper. The solid was washed with water (20 mL) and dried under reduced pressure to give the title compound G as a light yellow solid (0.34 g, 98%). 1 H NMR (500MHz, DMSO-d6): δ8.32(t,1H),6.91-6.89(m,1H),4.05(t,2H),3.65(t,4H),3.09(t,2H),1.64(t,2H),1.58-1.53(m,4H),1.48(s,9H). LCMS(ESI)432.90m / z[M+H] + .

[0943] Step-2: Synthesis of 2-(2-fluoro-6-(piperidin-1-yl)pyridin-3-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (H)

[0944] Title compound G (0.34g, 0.78mmol) is dissolved in DCM (7mL) and cooled to 0°C in an ice bath under stirring. 4M HCl (3.4mL) in 1,4-dioxane is added to the above solution and continued to stir at room temperature for 2 hours. After the reaction is complete, the solvent is removed under reduced pressure. The obtained residue is dissolved in ice-cold water and alkalized to pH 8-9 with saturated sodium bicarbonate aqueous solution. The precipitated solid is filtered and dried to obtain title compound H as a yellow solid (0.255g, 98%). 1HNMR(400MHz DMSO-d6): δ8.34-8.29(m,1H),7.83(s,1H),6.90-6.87(m,1H),3.65-3.62(m ,4H),3.51-3.47(m,2H),2.99(t,2H),1.65-1.63(m,2H),1.56-1.55(m,4H). LCMS(ESI)333.2[M+H] + .

[0945] Step-3: Synthesis of 2-(2-fluoro-6-(piperidin-1-yl)pyridin-3-yl)-5-(2-fluoropyridin-4-yl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one (4)

[0946] Compound H (0.05 g, 0.15 mmol) and 4-bromo-2-fluoropyridine (0.039 g, 0.22 mmol) were dissolved in degassed 1,4-dioxane (5 mL). After adding Pd[PPh3]4 (0.008 g, 0.007 mmol), XantPhos (0.008 g, 0.015 mmol) and Cs2CO3 (0.073 g, 0.22 mmol), the reaction mixture was heated in a sand bath at ~100 °C for 24 hours. The reaction mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by silica gel column chromatography (100-200 mesh) using a CH2Cl2 / MeOH gradient (100 / 0->99.5 / 0.5->99 / 1) to give an impure compound. The impure compound was further purified by preparative TLC plate using CH2Cl2 / MeOH (98.5 / 1.5) as mobile phase to afford the title compound 23 as a light yellow solid (0.032 g, 50%). 1H NMR(400MHz DMSO-d6): δ8.37-8.32(m,1H),8.22(d,1H),7.47-7.45(m,1H),7.25(d,1H),6.93-6.9 0(m,1H),4.22(t,2H),3.66(t,4H),3.24(t,2H),1.65-1.63(m,2H),1.57-1.56(m,4H). LCMS(ESI)427.9m / z[M+H] +

[0947] Biological Assay Description

[0948] 1. General Methods

[0949] Human brain material used for these studies was obtained from Professor William Seeley at the Neurodegenerative Diseases Brain Bank UCSF (with funding from NIH grants P01AG019724 and P50AG023501, the Frontotemporal Dementia Research Consortium and the Tau Consortium), and Professor Tammaryn Lashley at the Neurodegenerative Disorders Queen Square Brain Bank UCL. All material was collected from donors, from whom the Brain Bank obtained written informed consent for brain autopsy and use of material and clinical information for research purposes.

[0950] 1.1 Radioligand

[0951] The above-described [ 3 H]-Compound 1, the above-described [ 3 H]-compound 7, the above-described [ 3 H]-compound 8, the above-described [ 3 H]-compound 9, the above-described [ 3 H]-compound 15, the above-described [ 3 H]-compound 16, with a specific activity of 55.4 Ci / mmol (1.0 mCi / mL) described above [ 3 H]-compound 17 and / or [ 3 H]-reference compound (described as [ 3H]-compound 16) was used in the following assays.

[0952] 1.2. Preparation of Sarkosyl-Insoluble Brain Extracts from Human FTD

[0953] Human brain extracts were prepared as described in Laferriere et al., 2019, Nature Neurosc. Brain tissue (frontal or temporal cortex) samples were homogenized in a 1:4 (w / v) ratio in a homogenization-dissolution (HS) buffer at 4 °C using a tissue homogenizer (Precellys) with a CKmix homogenization tube. The following sequence was used for homogenization: 3 cycles of 30 seconds at 5000 rpm (15 seconds pause between each cycle). The homogenized samples were aliquoted and stored at -80 °C in 1.5 mL low protein binding tubes.

[0954] Brain homogenate was thawed on ice and resuspended in HS buffer to obtain a final concentration of 2% sarcosyl sodium, 1 unit / μL Benzonase and 1mM MgCl2. Then, the sample was incubated at 37°C on a thermomixer with continuous shaking at 600rpm for 45 minutes. The supernatant was collected in a new tube (sarcosyl sodium-soluble fraction, S1). The precipitate was resuspended in 1000μL myelin floating buffer and centrifuged at 20,000g for 60 minutes at 4°C. The supernatant was carefully removed to remove all floating lipids. If all lipids cannot be removed in a single step, this step is repeated. The precipitate was subsequently washed with phosphate buffered saline (PBS) and centrifuged at 20,000g for 30 minutes at 4°C. The final precipitate was resuspended in 200μL PBS and stored at -80°C (sarcosyl sodium-insoluble fraction). The samples were analyzed by immunoblotting under denaturing conditions.

[0955] 2. Bioassay Description and Corresponding Results

[0956] 2.1. Microradiometric Binding Competition Assay for Determining Binding Affinity to TPD-43

[0957] Human FTD sarcosyl sodium insoluble brain extract was spotted on a microarray slide. The slides were incubated with 40 nM tritiated reference ligand and 2 μM and 250 nM of the example compounds (non-radioactive labeled). In some cases, a series of different concentrations (from 0.24 nM to 2 μM) of non-radioactive labeled example compounds were further evaluated for determination of inhibition constants (Ki). After incubation, the slides were washed and scanned with a real-time autoradiography system (BeaQuant, ai4R). Signal quantification was performed using Beamage image analysis software (AI4R). Nonspecific signals were measured with an excess of non-radioactive labeled reference compounds (2 μM), and specific binding was calculated by subtracting the nonspecific signal from the total signal. Competition was calculated as a percentage, where 0% was defined as specific binding in the presence of carrier and 100% was defined as the value obtained in the presence of an excess of non-radioactive labeled reference compounds. K was calculated in GraphPad Prism8 using a single-site specific binding model by applying nonlinear regression curve fitting. i Values. Measurements were performed with at least two technical replicates in two-concentration competition experiments and with one technical replicate in experiments covering a range of concentrations. For compounds tested in more than one experiment, the mean or K of each replicate in each independent experiment is reported. i value.

[0958] result: Evaluation of Example Compounds and [ 3 The ability of the reference ligand to compete with the binding of TDP-43 aggregates from FTD patient brain. The results of the microradioligand binding competition assay of the example compounds are shown in Table 5 below: % competition, 2 μM and 250 nM. K i The values ​​are also shown in Table 5.

[0959] Table 5

[0960]

[0961]

[0962]

[0963] a nd: not determined

[0964] 2.2 Use [ 3 [H]-Microradioligand Binding Assay for Compounds 1, 7, 8, 9, 15, 16, and 17 Kd Determination against Human FTD Sarcosyl-Insoluble Brain Extracts

[0965] Human FTD sarcosyl insoluble brain extract was spotted on microarray slides. The slides were incubated with [ 3 H]-compounds 1, 7, 8, 9, 15, 16 and 17 were incubated together. After incubation, the slides were washed and scanned with a real-time autoradiography system (BeaQuant, ai4R). The signals were quantified by using Beamage image analysis software (ai4R). Nonspecific signals were measured with excess non-radiolabeled compounds 1, 7, 8, 9, 15, 16 and 17 (1 μM), respectively, and specific binding was calculated by subtracting the nonspecific signal from the total signal.

[0966] The specific binding data were fitted by nonlinear regression analysis using a one-site specific binding model in GraphPad Prism8 to obtain Kd (dissociation constant) and R2 (the parameter range for quantifying goodness of fit was 0.0 and 1.0, and the best curve fit value obtained was 1.0).

[0967] result:

[0968] In micro-radioligand binding assays [ 3 H]-Dissociation constants (Kd) of compounds 1, 7, 8, 9, 15, 16 and 17 for human FTD type A sarcosyl sodium-insoluble brain extract. [ 3 H]-Compounds 1, 7, 8, 9, 15, 16, and 17 have high specific binding, resulting in a high dynamic range. 3 The Kd value of compound 1 for human FTD sarcosyl sodium insoluble brain extract was 69 nM ( Figure 1a ). Data from two independent experiments yielded an average Kd of 69 ± 21 nM. [ 3 H]-Compound 7 showed a Kd value of 39 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1b ). Data from three independent experiments yielded an average Kd of 39 ± 11 nM. [ 3 H]-Compound 8 showed a Kd value of 47 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1c ). Data from two independent experiments yielded an average Kd of 47±8 nM. [ 3 H]-Compound 9 showed a Kd value of 26 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1d ). [ 3 H]-compound 15 showed a Kd value of 21±7 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1e ). [ 3H]-compound 16 showed a Kd value of 24±3 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1f ). [ 3 H]-compound 17 showed a Kd value of 25±6 nM for human FTD sarcosyl sodium insoluble brain extract ( Figure 1g ). It was also determined in a microradioligand binding assay [ 3 H]-compound 1 and [ 3 H]-Dissociation constant (Kd) of compound 17 for human FTD type B sarcosyl sodium-insoluble brain extract. [ 3 H]-Compound 1 showed a Kd value of 49 nM for human FTD type B sarcosyl sodium insoluble brain extract ( Figure 1h ),and[ 3 H]-The Kd value of compound 17 was 12 nM ( Figure 1i ).

[0969] 2.3. Use in brain slices of FTD patients 3 H]-compound 1, [ 3 H]-compound 9 and [ 3 H]-microautoradiographic staining of compound 17

[0970] All tissues were collected from donors from whom each brain bank obtained written informed consent for brain autopsy and use of materials and clinical information for research purposes. All samples were anonymized and coded. Frozen brain tissue blocks confirmed to have TDP-43 pathology after autopsy were processed using a cryostat to produce sections with a thickness of 10 μm and mounted on slides. Sections were stored at -80°C until use.

[0971] Brain sections were immunostained using a commercially available antibody specific for phosphorylated serine at amino acid 409 / 410 (anti-pTDP-43pS409 / 410, Biolegend, 829901). Sections were fixed with 4% formaldehyde (Sigma, 252549) for 15 minutes at 4°C and washed 3 times with 1×PBS (Dulbecco's phosphate-buffered saline, SigmaD1408) at room temperature for 8 minutes each time. Next, sections were saturated and permeabilized in blocking buffer (PBS, 10% normal goat serum (NGS), 0.25% Triton X-100) for 1 hour at room temperature and incubated overnight with primary antibodies against pTDP-43 (1 / 500 dilution in PBS, 5% NGS, 0.25% Triton X-100) at 4°C. The next day, the sections were washed 3 times with 1×PBS for 8 minutes each time, and then incubated with secondary antibody AlexaFluor633-labeled goat-anti-rat antibody (Invitrogen, A-21094, diluted 1 / 500 in PBS) at room temperature for 45 minutes. After incubation with the primary antibody, the sections were washed three times in PBS before further processing.

[0972] For microautoradiography, 120 nM of tritiated test compounds ([ 3 H]-compound 1, [ 3 H]-compound 9 or [ 3H]-compound 17) was incubated on the slices in 50mM Tris buffer at pH 7.4 for 45 minutes. The slices were then washed as follows: once in ice-cold 50mM Tris-HCl pH 7.4 buffer for 1 minute, twice in ice-cold 70% ethanol for 1 minute, once in ice-cold 50mM Tris-HCl pH 7.4 buffer for 1 minute, and finally briefly rinsed in ice-cold distilled water. The slices were subsequently dried under air flow for one hour and then exposed to Ilford Nuclear Emulsion Type K5 (Agar Scientific, AGP9281) in a light-proof slide storage box at 4°C for 10 days. The sections were developed by sequentially soaking in the following solutions: 1.) Ilford Phenisol developer (diluted 1:5 in H2O, Agar Scientific, AGP9106) for 4 minutes, 2.) Ilfostop solution (diluted 1:20 in H2O, Agar Scientific, AGP9104) for 2 minutes, 3.) Ilford Hypam Fixer (diluted 1:5 in H2O, AgarScientific, AGP9183) for 4 minutes, and finally rinsed in H2O for 10 minutes.

[0973] For image acquisition, slides were mounted with ProLong Gold Antifade reagent (Invitrogen P36930), and bright field and fluorescence images were captured separately on a Panoramic250 slide scanner (3DHistech) equipped with a 40× objective, or on a Panoramic Scan II (3DHistech) equipped with a 20× objective. The Visiopharm image analysis software suite was used to align the fluorescence images with the bright field images.

[0974] result:

[0975] From incubation of tritiated test compounds with human brain slices ([ 3 H]-compound 1,[ 3 H]-compound 9,[ 3 The microautoradiographic signal of [H]-compound 17) was detected in the form of accumulated silver particles, which colocalized with the immunofluorescence signal from the pTDP-43-specific antibody, indicating that [ 3 H]-compound 1, [ 3 H]-compound 9 and [ 3H]-compound 17 targets TDP-43 aggregates. Brain slices from FTLD-TDP type A donors were compared with [ 3 H]-compound 1 (120 nM) ( Figure 2a ),[ 3 H]-compound 9 ( Figure 2b )and[ 3 H]-compound 17 ( Figure 2c ) were incubated with pTDP-43 aggregates, further providing evidence of targeted binding. 3 H]-compound 1 and [ 3 The same colocalization of H]-compound 17 (respectively Figure 2d and Figure 2e ).

[0976] 2.4 In brain slices from FTD patients, 3 H]-compound 1, [ 3 H]-compound 8 and [ 3 H]-Autoradiography of compound 17

[0977] All tissues were collected from donors from whom each brain bank obtained written informed consent for brain autopsy and use of materials and clinical information for research purposes. All samples were anonymized and coded. Frozen brain tissue blocks confirmed to have TDP-43 pathology after autopsy were processed using a cryostat to produce sections with a thickness of 10 μm and mounted on slides. Sections were stored at -80°C until use.

[0978] Brain slices were fixed with 4% formaldehyde on ice for 15 minutes at 4°C and washed 3 times with 1×PBS at room temperature for 8 minutes each time. The slices were blocked in assay buffer for 30 minutes (50 mM Tris-HCl, 0.9% NaCl + 0.1% BSA). The cold test compound (Compound 1, Compound 8 or Compound 17) was diluted to 4 μM in assay buffer and applied to half of the slices for 40 minutes at room temperature to determine non-specific binding. The remaining half of the slices were incubated only with assay buffer (total binding). After this incubation, an equal volume of the corresponding tritiated compound (i.e., [ 3 H]-compound 1,[ 3 H]-compound 8 or [ 3 H]-compound 17) was diluted to 20 nM (for [ 3 H]-compound 1 and [ 3 H]-compound 8) or 10 nM ([ 3 H]-compound 17), or increasing the concentration from 0.1 to 200 nM (for [3 H]-compound 1 and [ 3 H]-compound 17), added to the sections and incubated for 2 hours at room temperature, then washed as follows: once in ice-cold 50 mM Tris-HCl pH 7.4 buffer for 1 minute, twice in ice-cold PBS for 1 minute, once in ice-cold 50 mM Tris-HCl pH 7.4 buffer for 1 minute, and finally briefly rinsed in ice-cold distilled water.

[0979] The sections were then dried under a stream of air and fixed with a copper-based adhesive strip. The slides were exposed to a real-time autoradiography system (Beaquant TM The radioactivity of binding was quantified in the presence of tritium in the presence of 1% paraformaldehyde (PNA) and 1% paraformaldehyde (PNA) for 2 hours in the presence of 1% paraformaldehyde (PNA). The radioactivity of binding was quantified in the presence of 1% paraformaldehyde (PNA) and 1% paraformaldehyde (PNA) for 2 hours in the presence of 1% paraformaldehyde (PNA). The assay device was set to be used for tritium. Images were obtained using Beavacq software (AI4R) and subsequently quantified using Beamage image analysis software (AI4R). Specific binding (nonspecific binding minus total binding) and displacement (total binding divided by nonspecific) were calculated. Histograms were produced using GraphPad prism. For Kd determination of brain slices, the unit site specific binding model in GraphPad Prism was used to fit the specific binding data using nonlinear regression analysis.

[0980] result:

[0981] The Example compounds were evaluated for binding to human tissue sections from non-demented control cases, Type A FTLD-TDP, or Type B FTLD-TDP ( Figure 3a and 4a ). The displacement of the tritiated compound by the non-radiolabeled compound was calculated in the gray matter area (total binding divided by nonspecific binding) and is reported for each case in Table 6 below.

[0982] Table 6

[0983]

[0984] Compounds 1, 8, and 17 showed increased displacement on FTLD-TDP types A and B compared to non-demented controls, providing further evidence of on-target binding in pathological tissues. Figure 3b and Figure 4b As shown, 3 H]-compound 1 and [ 3 H]-compound 17 showed significantly higher specific binding in FTLD-TDP type A than in the control (larger open circles correspond to Figure 3a and 4aA and B images are shown). One-way ANOVA with Tukey correction for multiple comparisons, *p<0.05, **p<0.01). Saturation binding studies were also evaluated using FTLD-TDP A tissue sections [ 3 H]-compound 1 and [ 3 H]-compound 17, the measured Kd values ​​were 16 nM and 18 nM, respectively, suggesting that they bind to TDP-43 aggregates with high affinity ( Figure 3c and Figure 4c ).

[0985] 2.5 Selectivity of TDP-43

[0986] 2.5.1 Radioligand Binding Assay of Alzheimer's Disease (AD) Brain Homogenates

[0987] 2.5.1.1 Preparation of human AD insoluble brain tissue homogenate fraction

[0988] The method used was modified from Bagchi et al. 2013, which describes the extraction of a homogenized insoluble fraction containing protein aggregates from human brain tissue for in vitro binding and competition studies.

[0989] About 4g of frozen tissue blocks from the frontal cortex brain region from AD donors with confirmed Tau and Aβ aggregate loads were used. At 4°C, the tissue was homogenized in three volumes (1:3w / v) of high salt buffer (50mM Tris-HCl pH 7.5, 750mM NaCl, 5mMEDTA) supplemented with protease inhibitors (Complete; Roche11697498001) using a glass Dounce homogenizer. The homogenate was transferred to a polycarbonate centrifuge bottle (16×76mm; Beckman 355603) and centrifuged at 4°C for 60 minutes using a pre-cooled 70.1 rotor (Beckman, 342184) in an ultracentrifuge (Beckman, XL100K). The pellet was resuspended in a high salt buffer supplemented with 1% Triton X-100 and 1M sucrose and homogenized with a syringe at 4°C. The homogenate was centrifuged again at 100,000 x g (38,000 RPM, 70.1 rotor) for 60 minutes at 4°C. The pellet was resuspended in a high salt buffer supplemented with 1% Triton X-100 and 1 M sucrose and homogenized with a syringe at 4°C. The homogenate was centrifuged at 100,000 x g (38,000 RPM, 70.1 rotor) for 60 minutes at 4°C, and the resulting pellet containing the insoluble fraction was resuspended in PBS (1:2 w / v relative to the starting tissue weight), aliquoted, and stored at -80°C until use.

[0990] 2.5.1.2 Radioligand Binding Competition Assay Using Aβ Reference Compounds for Determination of Inhibition Constants (Ki) of AD-Insoluble Fractions of Brain Homogenates

[0991] A fixed concentration of the AD insoluble fraction was incubated with a fixed concentration of a radiolabeled Aβ reference compound (Kd = 33 nM for AD brain homogenate) and increasing concentrations of non-radiolabeled compounds (ranging from 0.41 nM to 2 μM). The reaction was performed in assay buffer (50 mM Tris pH 7.5 in 0.9% NaCl, 0.1% BSA) and incubated for two hours at room temperature (RT). The samples were then vacuum filtered in duplicate on a GF / C filter (PerkinElmer) to capture aggregates with bound radioligand and washed with 100 μL of ice-cold 50 mM Tris pH 7.5. The GF / C filter was then dried and scintillation fluid (Ultimatecold, PerkinElmer, 6013151) was added to each well. The filters were analyzed on a Microbeta2 scintillation counter (PerkinElmer). The value of the maximum signal was obtained in the absence of non-radiolabeled compound, while 100% displacement was obtained using 1 μM of non-radiolabeled reference compound. Ki values ​​were calculated by non-linear regression using a single-site fit Ki model in Prism V7 (GraphPad). The average Ki of the non-radiolabeled Aβ reference compound was 32 nM.

[0992] 2.5.1.3 Radioligand Binding Assay for Determination of the Dissociation Constant (Kd) of the AD-Insoluble Fraction of Brain Homogenates

[0993] The AD insoluble fraction was mixed with different concentrations of [ 3 H] Compound 1, [ 3 H] Compound 17 or [ 3 H]Aβ reference compound ([ 3H]Aβref). To determine nonspecific signals, 1 μM non-radiolabeled compound was added to all concentrations of tritiated compound in the control wells. The reaction was carried out in assay buffer (50 mM Tris pH 7.5 in 0.9% NaCl, 0.1% BSA) and incubated at room temperature for two hours. The samples were then filtered through a GF / C filter (PerkinElmer) under vacuum to capture / fix aggregates with bound radioligands and washed five times with 50 mM Tris pH 7.5. The GF / C filter was then dried and scintillation fluid (Ultimatecold, PerkinElmer, 6013151) was added to each well. The filters were analyzed on a Microbeta scintillation counter (PerkinElmer). Specific binding was calculated by subtracting the nonspecific signal from the total signal. Kd and R 2 Values ​​were obtained by fitting the specific binding data using a one-site specific binding model in GraphPad Prism using nonlinear regression analysis.

[0994] 2.5.2. Microradiation Binding Assay in PD Brain-Derived α-syn Aggregates and AD Case-Derived Tau PHFs

[0995] 2.5.2.1 Isolation of pathological a-syn aggregates from PD brain

[0996] The method was adapted from the protocol described in Spillantini et al., 1998. Frozen tissue blocks from PD donors were thawed on ice and homogenized using a glass Downs homogenizer. The homogenate was then centrifuged at 11,000 x g (12,700 RPM) for 20 minutes at 4°C in an ultracentrifuge (Beckman, XL100K) using a pre-cooled 70.1 rotor (Beckman, 342184). The pellet was resuspended in extraction buffer [10 mM Tris-HCl pH 7.4, 10% sucrose, 0.85 mM NaCl, 1% protease inhibitor (Calbiochem 539131), 1 mM EGTA, 1% phosphatase inhibitor (Sigma P5726 and P0044)] and centrifuged at 15,000 x g (14,800 RPM, 70.1 Ti rotor) for 20 minutes at 4 ° C, the pellet was discarded, and sodium lauryl sarcosine (20% stock solution, Sigma L7414) was added to the supernatant to a final concentration of 1% for one hour at room temperature. The solution was then centrifuged at 100,000 x g (38,000 RPM, 70.1 Ti rotor) at 4 ° C for one hour, and the pellet containing the enriched a-syn aggregates was resuspended in PBS and stored at -80 ° C until use.

[0997] 2.5.2.2 Preparation and extraction of paired helical filament (PHF)-Tau aggregates from AD brain

[0998] The enrichment procedure was modified from Jicha et al., 1997 and Rostagno and Ghiso, 2009. Briefly, approximately 15 g of AD human brain was thawed on ice and homogenized in a glass Downs homogenizer with homogenization buffer (3 ml per gram of tissue) (0.75 M NaCl in RAB buffer (100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 1 mM EGTA, 0.5 mM MgSO4, 2 mM DTT, pH 6.8) supplemented with protease inhibitors (Complete; Roche 693124001)). The homogenate was then incubated at 4°C for 20 minutes to disaggregate any residual microtubules, then transferred to a polycarbonate centrifuge bottle (16×76 mm; Beckman 355603) and centrifuged at 11,000 g (12,700 RPM) at 4°C for 20 minutes in an ultracentrifuge (Beckman, XL100K) using a pre-cooled 70.1 rotor (Beckman, 342184). The pellet was kept on ice. The supernatant was combined in a polycarbonate bottle and centrifuged again at 100,000 g (38,000 RPM) at 4°C for 1 hour in a 70.1 Ti rotor. The pellets from the first and second centrifugations were resuspended in extraction buffer (10 ml per gram of tissue) (10 mM Tris-HCl pH 7.4, 10% sucrose, 0.85 M NaCl, 1 tablet / 50 ml protease inhibitor (Roche, 4693124001), 1 mM EGTA, 1% phosphatase inhibitor (Sigma P5726 and P0044)). The solution was then transferred to a polycarbonate centrifuge bottle (16×76 mm; Beckman 355603) and centrifuged at 15,000 g (14,800 RPM) for 20 minutes at 4° C. in an ultracentrifuge (Beckman, XL100K) using a 70.1Ti rotor. The pellets were stored at −80° C. 30% Sarkosyl (Fluka Analytical, 61747) was added to the supernatant to a final concentration of 1%, and stirred at room temperature for 1 hour. The samples were then centrifuged at 100,000 g (38,000 RPM) for 1 hour at 4°C in a 70.1 Ti rotor in a polycarbonate bottle and the pellet containing PHF-enriched material was resuspended in 50 μl PBS / 1 g brain tissue. The resuspended PHF was then sonicated on ice for 2 minutes with 0.5 s-on / 0.5 s-off cycles at 30% amplitude. Aliquots were snap frozen and stored at -80°C.

[0999] 2.5.2.3 Compound incubation and readout

[1000] α-Synuclein aggregates from PD brain and Tau PHF aggregates from AD brain were spotted onto microarray slides. 3 Slides spotted with α-synuclein aggregates were incubated with [H]-α-synuclein reference and example compounds (non-radiolabeled) at 100 nM and in increasing concentrations ranging from 0.05 nM to 2 μM. In addition, slides spotted with α-synuclein aggregates or with Tau PHF samples were incubated with [H]-α-synuclein reference and example compounds (non-radiolabeled) at concentrations ranging from 0.6 to 200 nM, respectively. 3 H] Compound-1, [ 3 H] Compound-17 and [ 3 H]-a-synuclein reference or [ 3 H]Tau reference compound incubated together. After incubation, the slides were washed and scanned with a real-time autoradiography system (BeaQuant, ai4R). The signal was quantified using the image processing software Beamage (ai4R). Nonspecific signals were measured with an excess of non-radiolabeled compounds (2 μM), and specific binding was calculated by subtracting the nonspecific signal from the total signal. Competition was calculated as a percentage, where 0% was defined as specific binding in the presence of a carrier and 100% was defined as the value obtained in the presence of an excess of non-radiolabeled a-syn reference compound. A single-site specific binding model was used in GraphPad Prism7, and nonlinear regression curve fitting was applied to calculate Ki values. All measurements were repeated with at least two techniques. Kd and R 2 Values ​​were obtained by fitting the specific binding data using a one-site specific binding model in GraphPad Prism using nonlinear regression analysis.

[1001] result: The selectivity of the example compounds for TDP-43 relative to Aβ and α-synuclein was evaluated. To evaluate the selectivity relative to Aβ, the activity of the compounds in AD brain homogenate relative to [ 3 H]Aβ reference compound ([ 3 The inhibitor constant (Ki) values ​​of H]Aβref) (Table 7) are shown in Table 7. As can be seen from the high Ki values ​​in Table 7, the compounds of the present invention show excellent selectivity for TDP-43 relative to Aβ.

[1002] Table 7

[1003] Example Ki (nM), in AD brain homogenate 1 >1μM 7 >1μM 8 >1μM 9 >1μM 10 350 13 >1μM 15 >1μM 16 >1μM 17 >1μM 23 >1μM 24 >1μM 25 >1μM 26 318 27 >1μM 29 >1μM 31 >1μM 32 >1μM 34 >1μM 35 >1μM 37 >1μM

[1004] a nd: not determined

[1005] To evaluate the selectivity for α-synuclein, the potency of Example Compound 1 in competing with the binding of the [3H]-reference α-syn compound in the insoluble fraction from PD brain was determined, and Compound 1 showed a Ki>1000nM in the AD brain homogenate and a Ki>500nM in the insoluble fraction from PD brain.

[1006] In addition, by using AD brain homogenate (containing Aβ and Tau, Figure 5a-5b ), in PD brain-derived a-synuclein aggregates ( Figure 5c-5d )) and in AD brain-derived Tau PHF aggregates ( Figure 5e-5f ) are directly combined, and [ 3 H] Compound-1 and [ 3 H] Selectivity of Compound-17 for TDP-43 over Aβ, α-synuclein, and Tau. In contrast to the respective reference ligands, no [ 3 H] Compound-1 and [ 3 H] Significant binding of compound-17.

[1007] The results demonstrate that the compounds according to the present invention show excellent TDP-43 selectivity relative to Aβ, a-synuclein and Tau.

[1008] 2.5.3. Evaluation of Targeted Binding of Example 1 and Example 17 in Aβ and Tau Aggregates in AD Tissues by Classical Autoradiography and High-Resolution Microautoradiography

[1009] 2.5.3.1 Typical autoradiography

[1010] All tissues were collected from donors from whom each brain bank obtained written informed consent for brain autopsy and use of materials and clinical information for research purposes. All samples were anonymized and coded. Frozen brain tissue blocks confirmed to have TDP-43 pathology after autopsy were processed using a cryostat to produce sections with a thickness of 10 μm and mounted on slides. Sections were stored at -80°C until use.

[1011] Brain slices were fixed with 4% formaldehyde on ice at 4°C for 15 minutes and washed 3 times with 1x PBS at room temperature for 8 minutes each time. The slices were blocked in assay buffer for 30 minutes (50mM Tris-HCl, 0.9% NaCl + 0.1% BSA). The cold test compound (Compound 1) was diluted to 4 μM in assay buffer and applied to half of the slices for 40 minutes at room temperature to determine non-specific binding. The remaining half of the slices were incubated with assay buffer only (total binding). After this incubation, an equal volume of the corresponding tritiated compound (i.e., [3 H]-compound 1, [ 3 H]-compound 17, [ 3 H]-Aβ reference compound or [ 3 H]-tau reference compound) was added to the sections and incubated for 2 hours at room temperature, followed by washing as follows: once in ice-cold 50 mM Tris-HCl pH 7.4 buffer for 1 minute, twice in ice-cold PBS for 1 minute, once in ice-cold 50 mM Tris-HCl pH 7.4 buffer for 1 minute, and finally briefly rinsed in ice-cold distilled water.

[1012] The sections were then dried under a stream of air and fixed with a copper-based adhesive strip. The slides were exposed to a real-time autoradiography system (Beaquant TM The incorporated radioactivity was quantified by incubation for 2 hours in the presence of tritium in the presence of 1% paraformaldehyde (PNA). The images were acquired using Beavacq software (AI4R) and subsequently quantified using Beamage image analysis software (AI4R).

[1013] result:

[1014] Example compounds 1 and 17 were evaluated for their binding on human tissue sections from AD cases containing abundant Aβ and Tau pathology ( Figure 6a and 6b ). It shows [ 3 H]-compound 1 or 17 (first column), [ 3 H]-Aβ reference compound (second column) or [ 3 [H]-Tau reference compound (last column) total binding (first row) and non-specific binding (middle row) images. Immunofluorescence images (bottom row) of pTDP-43 (first column), Aβ (second column) or pTau (last column). Example compounds 1 and 17 showed no signal on the tissue under any of the conditions tested (radiolabeled compounds alone or in self-competition), which correlated with the lack of pTDP-43 immunolabeling on adjacent sections. While both Aβ and Tau references showed total binding signals that were displaced under self-competition, which correlated with immunolabeling of pTau and Aβ on adjacent sections of the tissue. The results further demonstrate that the compounds of the present invention show good TDP-43 selectivity relative to Aβ and Tau.

[1015] 2.5.3.2 High-resolution microautoradiography

[1016] This protocol was modified from Marquie et al., 2015. AD slices were incubated with 20 nM [ 3 H]-compound 1, [3 H]-compound 17 or [ 3 H]-Tau reference compounds were incubated for one hour at room temperature (RT). The sections were then washed as follows: once in ice-cold 50mM Tris-HCl pH 7.4 buffer for one minute, twice in 70% ice-cold ethanol for one minute, once in ice-cold 50mM Tris-HCl pH 7.4 buffer for one minute, and finally rinsed briefly in ice-cold distilled water. The sections were then dried and then exposed to Ilford Nuclear Emulsion Type K5 (AgarScientific, AGP9281) in a light-proof slide storage box. Five days later, the sections were developed by immersing them successively in the following solutions: 1.) Ilford Phenisol developer (1:5 dilution in H2O, Agar Scientific, AGP9106), 2.) Ilfostop solution (1:20 dilution in H2O, Agar Scientific, AGP9104), 3.) Ilford Hypam fixative (1:5 dilution in H2O, Agar Scientific, AGP9183), and finally rinsed with H2O.

[1017] Thioflavin S staining was performed on adjacent sections. To acquire images, sections were mounted using Prolong Gold Antifade reagent (Invitrogen P36930) and imaged on a Panoramic150 slide scanner (3DHistech) using a 20x objective to capture bright field and fluorescent images, respectively.

[1018] result:

[1019] The accumulation of silver particles in regions rich in tau tangles was assessed in human brain slices incubated with 3 H]-compound 1, [ 3 H]-compound 17 and [ 3 H]-Tau reference microautoradiography signal. Figure 6c ([[ 3 H]-compound 1) and Figure 6d ([ 3 H]-compound 17), left, thioflavin S staining on the same tissue to mark Tau aggregates. Right, on the same tissue [ 3 H]-compound 1 or [ 3 H]-compound 17 and [ 3 H]-Tau reference compound silver particle deposition image. 3 Compared with the [H]-Tau control,3 H]-compound 1 or [ 3 H]-Compound 17 does not accumulate silver particles on Tau tangles. The bottom row of images is a magnified image of the area indicated by the square on the top row of images. *NFT: neurofibrillary tangle. Scale bars are 200 μm (top row) and 50 μm (bottom row, Figure 6c ) or 100 μm (bottom row, Figure 6d ).like Figure 6c and Figure 6d As shown, brain sections of AD cases were compared with [ 3 H]-compound 1 or [ 3 H]-compound 17 showed no accumulation of silver particles, whereas on adjacent slices [ 3 The [H]-Tau reference compound showed targeted binding to Tau tangle regions, as determined by Thioflavin S staining. The results further demonstrated that the compounds of the present invention showed excellent TDP-43 selectivity relative to Tau.

[1020] 2.6 PK studies in healthy monkeys

[1021] Will[ 18 F]-labeled compound 1 ([ 18 F] Compound 1) (4.2 mCi) or [ 18 F]-labeled compound 17 ([ 18 F]-Compound 17) (5.3 mCi) was injected intravenously (iv) into non-human primates (NHPs). Monkey PET scans were performed using a Siemens Focus 220. PET acquisition was started immediately before the injection of the radioactive dose. Images were generated by dynamic scanning for 120 minutes with the head focused. [ 18 F]-compound 1 and [ 18 F]-Compound 17 rapidly entered the brain (5.5 and 4.5 min after injection, respectively) and showed robust uptake of 2.7 and 1.1 SUVmax in the whole brain, respectively ( Figure 7a-7b ).also,[ 18 F]-compound 1 and [ 18 F]-compound 17 has a fast elution rate, with peak to half peak values ​​of <17 min and <13 min, respectively. These data demonstrate that the [ 18 F]-compound 1 and [ 18 F] - PK profile of compound 17 in non-human primates, which is suitable for use as a brain PET agent in humans.

Claims

1. Compounds having the structure of formula (I) or a detectably labeled compound, stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 1 is H, hydroxy(C1-C4)alkyl or F; and R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S.

2. A compound according to claim 1 having the structure of formula (I), wherein R 1 It is H or F.

3. A compound according to claim 1 or 2, wherein R 2 yes in R 3 It's F, R 4 is NH2, R 7 is H, and R 8 It is H; R 3 is NH2, R 4 It's F, R 7 is H, and R 8 It is H. R 3 It is CN, R 4 is NH2, R 7 is H, and R 8 It is H. R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is CN, R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 is F, or R 3 Yes H, R 4 is NH2, R 7 is H, and R 8 It is CN; or R 2 yes Where X is N, and R 5 is CH3 or H; or R 2 yes Where R 9 is selected from H, F, CH3 and NH2, preferably H; or R 2 yes or R 2 yes or R 2 yes or R 2 yes or R 2 yes or R 2 yes or R 2 yes or R 2 yes 4. A compound according to any one of claims 1 to 3, wherein R 2 yes Where R 3 It's F, R 4 is NH2, R 7 is H, and R 8 It is H; Where X is N, and R 5 It is CH3; Where R 9 Selected from H and F; 5. A compound according to any one of claims 1 to 3, selected from 6. A compound according to any one of claims 1 to 5, comprising a detectable label.

7. A compound according to claim 6, wherein the detectable label is 3 H or 18 F.

8. The compound according to claim 7, which has the structure of formula (IT) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 1 is H, hydroxy(C1-C4)alkyl or F; T is 3 H; and in R 2 Yes(i) Where R 3 It's F, R 4 is -NH2, and R 7 and R 8 At least one of them is T, and if applicable, another one is H; preferably, R 7 is T, and R 8 is T; and R 6 It is T; or among them R 2 Yes(i) Where R 4 Yes -NH2, R 8 It is CN, R 3 or R 7 At least one of them is T, and if applicable, another one is H; preferably, R 7 is T, and R 3 is T; or R 7 is T, and R 3 is H, and R 6 is T; or in R 2 Yes(ii) Where X is N, and R 5 is CT3; and R 6 is H; or R 2 Yes(iii) Where R 12 It is T; or among them R 2 Yes(iii) Where R 12 It is T; or among them R 2 Yes(iii) Where R 12 It is T; or among them R 2 Yes(iii) Where R 12 It's T.

9. The compound according to claim 7, which has the formula (I-T') or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 1 is hydroxy(C1-C4)alkyl, which is substituted by one or two, preferably two, T, Where T is 3 H; and R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S.

10. The compound according to claim 8, wherein the compound is selected from Where T is 3 H.

11. A compound according to claim 9, wherein the compound is Where T is 3 H.

12. A compound according to claim 7 having the formula (IF) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 1 yes 18 F; R 2 yes Where X is N, and R 5 is CH3; or Where R 9 It's H.

13. A compound according to claim 7 having the formula (IF) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 1 is H or hydroxy(C1-C4)alkyl, and R 2 yes Where R 9 It's H.

14. The compound according to claim 12, which is 15. The compound according to claim 13, which is 16. A diagnostic composition comprising a compound according to any one of claims 1 to 15 and optionally at least one physiologically acceptable carrier, diluent, adjuvant and / or excipient.

17. A compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 for use in diagnostics.

18. A compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 for use in the imaging of TDP-43 aggregates, in particular, wherein the imaging is performed by positron emission tomography.

19. A compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 for use in diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates, in particular, wherein the diagnosis is performed by positron emission tomography.

20. A method for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates, or a predisposition thereto, in an individual, the method comprising the steps of: (a) administering to an individual a compound according to any one of claims 1 to 15; or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; and (c) Detection of compounds binding to TDP-43 aggregates.

21. The method for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregation or a predisposition thereof according to claim 20, further comprising the following steps: (d) generating an image representing the location and / or amount of the compound bound to TDP-43 aggregates, (e) optionally comparing the generated image to a control image of a healthy control individual, wherein an increase in the binding signal indicates that the individual is suffering from or is at risk of developing a disease, disorder or abnormality associated with TDP-43 aggregates.

22. A method for positron emission tomography (PET) imaging of TDP-43 aggregates in a tissue of an individual, the method comprising the steps of: (a) administering to an individual a compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; and (c) Compounds binding to TDP-43 aggregates were detected by collecting positron emission tomography (PET) images of individual tissues.

23. The method for positron emission tomography (PET) imaging of TDP-43 aggregates in a tissue of an individual according to claim 22, wherein the tissue is a tissue of the central nervous system (CNS), an eye tissue or a brain tissue, preferably, wherein the tissue is a brain tissue.

24. A method for the detection and optional quantification of TDP-43 aggregates in a tissue of an individual, the method comprising the following steps: (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; (c) detecting the compound binding to TDP-43 aggregates using positron emission tomography; and (d) optionally quantifying the amount of compound bound to TDP-43 aggregates.

25. A method of collecting data for diagnosing a disease, disorder or abnormality associated with TDP-43 aggregates, or a predisposition thereto, the method comprising the steps of: (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; (c) detecting compounds that bind to TDP-43 aggregates; and (d) optionally correlating the presence or absence of a compound that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region.

26. A method of collecting data for monitoring the progression of a disease, disorder or abnormality associated with TDP-43 aggregates in a patient, the method comprising the steps of: (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; (c) detecting compounds that bind to TDP-43 aggregates; (d) optionally correlating the presence or absence of a compound that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region; and (e) optionally repeating at least once: steps (a) to (c) and optionally step (d) if present.

27. A method of collecting data for predicting the response of a patient suffering from a disease, disorder or abnormality associated with TDP-43 aggregates to a drug treatment, the method comprising the steps of: (a) contacting a sample or a specific body part or body region suspected of containing TDP-43 aggregates with a compound according to any one of claims 1 to 15 or a diagnostic composition according to claim 16 comprising a compound according to any one of claims 1 to 15; (b) allowing the compound to bind to TDP-43 aggregates; (c) detecting compounds that bind to TDP-43 aggregates; (d) optionally correlating the presence or absence of a compound that binds to TDP-43 aggregates to the presence or absence of TDP-43 aggregates in said sample or in a particular body part or region; and (e) optionally repeating at least once: steps (a) to (c) and optionally step (d) if present.

28. The method of any one of claims 25 to 27, wherein the step of optionally correlating the presence or absence of a compound that binds to TDP-43 aggregates with the presence or absence of TDP-43 aggregates in the sample or in a specific body part or region comprises - determining the amount of compound bound to TDP-43 aggregates; - correlating the amount of the compound that binds to TDP-43 aggregates to the amount of TDP-43 aggregates in a sample or a specific body part or body region; and - Optionally comparing the amount of compound binding to TDP-43 aggregates in the sample or in a specific body part or body region with a normal control value in a healthy control individual.

29. Use of a compound according to any one of claims 1 to 15 as a biomarker for TDP-43 aggregates or a biomarker for a TDP-43 proteinopathy.

30. Use of a compound according to any one of claims 1 to 15 as a diagnostic agent or diagnostic tool for TDP-43 proteinopathy.

31. Compounds according to any one of claims 1 to 15 for use as in vitro analytical references or in vitro screening tools.

32. A compound or diagnostic composition for use according to claim 18 or 19, or a method according to any one of claims 20 to 28, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy is selected from frontotemporal dementia (FTD, e.g. sporadic or familial, with or without motor neuron disease (MND), with a progranulin (GRN) mutation, with a C9orf72 mutation, with a TARDBP mutation, with a valosin-containing protein (VCP) mutation, linked to chromosome 9p, corticobasal degeneration, Frontotemporal lobar degeneration (FTLD) includes frontotemporal dementia TDP-43 or frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral FTD (bvFTD), nonfluent variant primary progressive aphasia (e.g., nfvPPA), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiopoietin (ANG) mutations), Alexander disease (AxD), limbic system The main age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy, Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP) mutations; also known as Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with rimmed vacuoles, Myofibrillar myopathy with mutations in the sarcomeric protein (MYOT) gene or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB) and Parkinson's disease (PD), preferably, the disease, disorder or abnormality associated with TDP-43 aggregation or TDP-43 proteinopathy is selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE) and limbic system-dominant age-related TDP-43 encephalopathy (LATE).

33. A compound or diagnostic composition or method for use according to claim 32, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

34. A compound or diagnostic composition or method for use according to claim 32, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or a TDP-43 proteinopathy is Alzheimer's disease (AD).

35. A compound or diagnostic composition or method for use according to claim 32, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy is frontotemporal dementia (FTD), including frontotemporal dementia TDP-43 or frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP).

36. A compound or diagnostic composition or method for use according to claim 32, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy is limbic-predominant age-associated TDP-43 encephalopathy (LATE).

37. A compound for use, or a diagnostic composition, or a method according to claim 32, wherein the disease, disorder or abnormality associated with TDP-43 aggregates or TDP-43 proteinopathy is frontotemporal dementia (FTD) with progranulin (GRN) mutation, or frontotemporal dementia (FTD) with C9orf72 mutation.

38. Compounds having the structure of formula (II) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, or a mixture thereof, in n is 1 or 2; and R 1 is H, hydroxy(C1-C4)alkyl or F.

39. Compounds having formula (III) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, or a mixture thereof, in Z is selected from C-Br, CI and CH, wherein n is 1 or 2; R 1 is H, hydroxy(C1-C4)alkyl or F; R 11 is a 5- or 6-membered carbocyclic or heterocyclic ring, which may be optionally substituted by Br, I, F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S; and Z and / or R 11 At least one of them contains Br or I.

40. Compounds having formula (IV) in n is 1 or 2; R 2 yes Where R 9 is H; and R 14 is a leaving group (LG).

41. Compounds having formula (V) in n is 1 or 2; R 1 is H or hydroxy(C1-C4)alkyl; R 2 yes Where R 9 is H; and R 14 is a leaving group (LG).

42. Compounds having formula (III) or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof; in n is 1 or 2; R 15 is selected from -COOR' or -C1-C3 alkylCOOR'; wherein R' is C1-C3 alkyl; and R 2 is a 5- or 6-membered carbocyclic or heterocyclic ring which may be optionally substituted by F, NH2, CN and / or CH3, wherein the heterocyclic ring contains one or more heteroatoms selected from N, O and S.

43. A kit for the preparation of a radiopharmaceutical formulation comprising a precursor of a detectably labeled compound according to any one of claims 6 to 15, wherein the precursor is a compound of formula (II) as defined in claim 38, a compound of formula (III) as defined in claim 39 or claim 42, or a compound of formula (IV) as defined in claim 40, or a compound of formula (V) as defined in claim 41.

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