Compounds for imaging tau protein aggregates

Abstract

The present invention relates to compounds for imaging TAU protein aggregates, in particular to novel compounds of formula (II) which can be used for selective Tau detection in disorders and abnormalities associated with Tau aggregates such as Alzheimer's disease and other tauopathies by positron emission tomography (PET) imaging.

Description

[0001] The applicant filed PCT application PCT / EP2017 / 068509 on July 21, 2017, entitled "Compound for Imaging TAU Protein Aggregates". This PCT application entered the Chinese national phase on January 21, 2019, with application number 201780045187.9. This application is a divisional application of that Chinese application. Technical Field

[0002] This invention relates to novel compounds of formula (II) that can be used for the selective detection of conditions and abnormalities associated with Tau aggregates, such as Alzheimer's disease (AD) and other tau lesions, for example, using positron emission tomography (PET) imaging. The invention also relates to intermediates that can be used to prepare such imaging compounds. Diagnostic compositions using the above-described compounds, as well as imaging or diagnostic methods and kits that can be used to prepare radiopharmaceutical products, are also the subject of this invention. Background Technology

[0003] Alzheimer's disease (AD) is a neurological disorder primarily thought to be caused by amyloid plaques, extracellular accumulations of amyloid-β (Aβ) aggregates that are abnormally deposited in the brain or eyes. Other major neuropathological markers in AD are intracellular neurofibrillary tangles (NFTs), which arise from the aggregation of hyperphosphorylated tau (tubulin-associated unit) protein, phosphorylated tau, or pathological tau and its conformational isoforms. AD shares this pathology with many neurodegenerative tau disorders, particularly certain types of frontotemporal dementia (FTD). In the brain of Alzheimer's disease (AD), tau pathology develops later than amyloid pathology, but whether Aβ protein is the causative factor in AD (which constitutes the essence of the so-called amyloid cascade hypothesis) remains controversial (Hardy et al., Science 1992, 256, 184-185, and more recently, Musiek et al., Nature Neurosciences 2015, 18(6), 800-806, “Three dimensions of theamyloid hypothesis: time, space and 'wingmen'”).

[0004] Currently, the only definitive method for diagnosing Alzheimer's disease (AD) is through biopsy or histological analysis of post-mortem materials to identify plaques and tangles in the brain tissue. Besides AD, tau plays a significant role in other (non-AD) neurodegenerative diseases. These non-AD tau lesions include, for example, supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal degeneration (CBD).

[0005] Therefore, there is great interest in the detection of Tau pathology in vivo. Tau PET imaging is expected to provide new insights into the deposition of Tau aggregates in the human brain and may allow for non-invasive examination of the extent of Tau pathology, quantification of changes in Tau deposition over time, assessment of its correlation with cognition, and analysis of the efficacy of anti-Tau therapies. Recent reviews can be found in Shah et al., J Nucl Med. 2014, 55(6), 871-874: “Molecular Imaging Insights into Neurodegeneration: Focus on Tau PET Radiotracers”, Jovalekic et al., EJNMMI Radiopharmacy and Chemistry 2016, 1:11, “New protein deposition tracers in the pipeline”, and Ariza et al., J Med Chem 2015, 58(11), 4365-82: “Tau PET Imaging: Past, Present and Future”. In addition, several patent applications have been published recently, including: WO 2013 / 176698, WO2009 / 102498, WO 2011 / 119565, US 8,932,557 B2 and US 8,691,187,B2 (Siemens Medical Solutions, Lilly), WO 2012 / 067863 and WO 2012 / 068072 (GE Healthcare), WO 2014 / 026881, WO 2014 / 177458, WO 2014 / 187762, WO 2015 / 044095, WO 2015 / 052105, WO 2015 / 173225 (Hoffmann-La Roche AG), WO 2015 / 188368 (Merck Sharp & Dohme), and WO 2016 / 124508 (UCBBiopharma SPRL) is a new compound claimed for Tau imaging.

[0006] To achieve high target selectivity, molecular probes that recognize and bind to pathological targets have been used. Therefore, selectivity in binding to pathological Tau protein relative to other protein deposits in the brain is a fundamental requirement for Tau imaging probes. To reduce background signal interference caused by nonspecific off-target binding (e.g., binding to Aβ or monoamine oxidases), imaging compounds should bind to pathological Tau with high affinity. Since amyloid proteins or amyloid-like deposits formed from proteins with different primary amino acid sequences share a common β-sheet quaternary conformation, molecular probes must be able to distinguish these structures to avoid detecting other pathologies (false positives) and thus misdiagnosis.

[0007] It has been reported that off-target binding to monoamine oxidases A or B is a significant limitation of Tau tracers, particularly T-807 and THK-5351 (Vermeiren, C, et al., Alzheimers & Dementia. 2015; 11(7) Supplement p1-2: "T807, a reported selective tau tracer, binds with nanomolar affinity to monoamine oxidase A"; Ng, KP, et al., Alzheimer's Research and Therapy 2017, 9:25: "Monoamine oxidase B inhibitor, selegiline, reduces..." 18 F-THK5351 uptake in the human brain”. Off-target binding to monoamine oxidase A or B confounds the interpretation of PET images of tau using T807 and THK5351. The presence of monoamine oxidase in several brain regions limits the interpretation of PET imaging results using these tracers.

[0008] Besides high selectivity, the binding of different Tau isoforms is an important aspect of tau tracers. To date, most tracers have been shown to bind to tau in Alzheimer's disease (AD). However, tau in AD is a mixture of two isoforms, so-called 3R-tau and 4R-tau. Other non-AD tau lesions are characterized by the predominant presence of one of these isoforms. In Pick's disease (PiD), the 3R tau isoform is predominant, while in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), the 4R-tau isoform is the existing pathology.

[0009] Furthermore, molecular probes must be designed so that they can be distributed throughout the body and reach their targets upon administration. For imaging Tau aggregates associated with neurological conditions such as Alzheimer's disease, imaging compounds must be able to penetrate the blood-brain barrier and enter the relevant brain regions. For targeting intracellular Tau aggregates, cellular permeability is an additional requirement for imaging compounds. To obtain a sufficient signal-to-noise ratio, another prerequisite is rapid compound flushing from non-target areas in the brain (or other target organs). Additionally, the compound should exhibit no defluorination activity, as bone resorption in the skull (due to the presence of free fluoride) will result in significant extravasation into the brain, limiting its availability (Chien DT, et al. J Alzheimers Dis. 2014; 38:171–84).

[0010] The specific disclosure and most advanced derivatives of WO2013 / 176698 are 2,5-disubstituted pyridine compounds. 18 F-1 (see also US 8,932,557 B2).

[0011]

[0012] The compound was studied in various clinical trials. 18 F-1. Despite 18 F-1 appears to be able to detect Tau in patients with Alzheimer's disease (AD) or amyloid-β-positive mild cognitive impairment (MCI), but various limitations have been reported.

[0013] Vermeiren and colleagues discovered the compound 18 F-1 binds to monoamine oxidase A (MAO A), K D The value is 1.5 nM. Their data consistently demonstrate that the compound... 18 F-1 binds to Tau aggregates and MAO-A with similar high affinity. These findings allow for more careful consideration of the compounds. 18 The F-1 clinical data are interpreted because MAO-A is widely expressed in most human brain regions (Vermeiren et al., Alzheimers & Dementia. 2015; 11(7) Supplement p1-2: T807-a reported selective Tau tracer, binds with nanomolar affinity to Monoamine oxidase A).

[0014] According to reports, regardless of the patient's diagnosis, the compound 18 F-1 has a fairly strong signal in parts of the basal ganglia of the brain, such as the striatum and substantia nigra. In the cortex... 18The F-1 signal did not reach a "steady state" (a time window during which the ratio of binding in the target region to binding in the reference tissue (i.e., the cerebellum) remains stable). Furthermore... 18 The dynamics of F-1 differ across various brain regions and never stabilized during a 150-minute scan (S. Baker, Human Amyloid Imaging Meeting, 2015).

[0015] The compound was confirmed by autoradiography. 18 F-1 binds to AD brain slices. However, the compound... 18 F-1 showed limitations in its association with brain slices with pathology of non-AD tau lesions. a) Lowe VJ, et al. An autoradiographic evaluation of AV-1451 Tau PET in dementia. Acta Neuropathologica Communications. 2016; 4:58; b) Marquie M, et al. Validating novel Tau Positron Emission Tomography Tracer [F-18]-AV-1451(T807) on postmortem Brain Tissue. Annals of Neurology. 2015; 78:787; c) Gomez F, et al. Quantitative assessment of [ 18 F] AV-1451 distribution in AD, PSP and PiD Post-Mortem BrainTissue Sections relative to that of the anti-Tau antibody AT8. Journal of Nuclear Medicine. 2016; 57, S2: 348, d) Sander K, et al. Characterization of taupositron emission tomography tracer AV1451 binding to postmortem tissue in Alzheimer's disease, primary Tauopathies, and other dementias. Alzheimers Dementia 2016,12(11):116-1124e) Smith R, et al. Increased basal ganglia binding of18 F-AV-1451in patients with progressive supranuclear palsy.Movement disorders2016.

[0016] Clinically, 18 F-1 appears to have limited value in detecting tau in PSP individuals. a) Smith R et al., Tauneuropathology correlates with FDG-PET, but not with AV-1451-PET, in progressive supranuclear palsy. Acta Neuropathologica 2017, 133:149-151; b) Smith R et al., Increased basal ganglia binding of 18 F-AV-1451 in patients with progressive supranuclear palsy. Movement disorders 2017, 32(1), 108-114.

[0017] The ultimate conclusion of these studies suggests that T807 / AV1451 may not reliably distinguish between PSP patients and controls. This is primarily attributed to increased nonspecific binding to midbrain structures such as the basal ganglia. The uptake observed in the cerebral cortex and white matter does not reflect tau pathology in PSP.

[0018] compound 18 The F-2 was revealed in WO 2015 / 052105.

[0019]

[0020] WO 2015 / 052105 only discloses one type. 18 F-labeled compounds and corresponding tritium-labeled compounds. The compound contains a 2,5-disubstituted pyridine moiety (compound). 18 F-2). WO2015 / 052105 does not provide any data on Tau-isotype binding in non-AD tau lesions, binding to MAO A (or other aspects of Tau selectivity), brain uptake, brain washout, or retention in a healthy brain, or any data on in vivo defluorination.

[0021] 18F-2 was found not to bind to brain tissue in patients with non-AD tau lesions such as Pick's disease (PiD) and progressive supranuclear palsy (PSP) (Honer M et al., In vitro binding of 3 H-RO6958948, 3 H-AV-1451, 3 H-THK5351 and 3 H-T808 to tau aggregates in non-AD tauopathies. Human AmyloidImaging 2017, Abstract 99).

[0022] In view of the above-mentioned prior art, one object of the present invention is to provide compounds with high affinity and selectivity for Tau, and thus suitable as PET imaging agents. Preferably, the compounds of the present invention exhibit high affinity for Tau aggregates, high selectivity for pathological Tau compared to other targets in the brain, and favorable pharmacokinetic properties without defluorination. The desired Tau PET imaging agent should combine 3R and 4R Tau to address AD and non-AD tau lesions, including PiD, CBD, and PSP. Summary of the Invention

[0023] Therefore, the present invention relates to the following items:

[0024] 1. Compounds of formula (II) and their pharmaceutically acceptable salts, hydrates, solvates, prodrugs and polymorphs;

[0025]

[0026] in

[0027] R 1 Selected from 18 F, F and LG;

[0028] R 2 Is it H or PG;

[0029] PG is a protecting group;

[0030] LG is a leaving group.

[0031] In equation II, any H can be H, 2 H or 3 H.

[0032] 2. The compound described in item 1, which is

[0033]

[0034] 3. The compound described in item 1, which is

[0035]

[0036] 4. The compound described in item 1, 2 or 3, wherein R 1 yes 18 F and R 2 It is H.

[0037] 5. The compound described in item 1, 2 or 3, wherein R 1 It is F and R 2 It is H.

[0038] 6. The compound described in item 1, 2 or 3, wherein R 1 It is LG and R 2 It is H or PG.

[0039] 7. The compound described in item 1, 2 or 3, wherein R 1 It is LG and R 2 It is H.

[0040] 8. The compound described in item 1, 2 or 3, wherein R 1 LG and R 2 It's PG.

[0041] 9. The compound described in item 1, 2, 3, 6, 7 or 8, wherein LG is a nitro, halogen or trimethylammonium.

[0042] 10. The compound described in item 9, wherein LG is nitro or trimethylammonium.

[0043] 11. The compound described in item 1, 2, 3, 6, 8, 9 or 10, wherein PG is tert-butoxycarbonyl (BOC), triphenylmethyl (Trityl), or dimethoxytriphenylmethyl (DMT).

[0044] 12. The compound described in item 11, wherein PG is tert-butoxycarbonyl (BOC).

[0045] 13. The compound of claim 1, 2 or 3, wherein the compound is detectably labeled.

[0046] 14. The compound of claim 13, wherein the detectable marker is selected from... 2 H, 3 H and 18 F.

[0047] 15. The compound described in item 14, wherein the detectable marker is 18 F.

[0048] 16. A diagnostic composition comprising a compound as defined in any one of items 4, 13, 14 or 15 and optionally a pharmaceutically acceptable carrier, diluent, excipient or excipient.

[0049] 17. The compounds defined in item 4 or 15, which are used for diagnosis.

[0050] 18. The compounds defined in item 4 or 15, used for imaging Tau aggregates, particularly for positron emission tomography imaging of Tau aggregates.

[0051] 19. A compound as defined in item 4 or 15, used for the diagnosis of conditions associated with Tau aggregates or for the diagnosis of Tau lesions, particularly wherein the diagnosis is performed by positron emission tomography.

[0052] 20. The compound used as described in item 19, wherein the Tau lesion is a 3R Tau lesion.

[0053] 21. The compound used as described in item 19, wherein the Tau lesion is a 4R Tau lesion.

[0054] 22. The compound used as described in item 19, wherein the condition is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Diseases including inclusion body myositis, prion amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Parkinsonism-dementia complex of Guam, non-Guam motor neuron disease with neurofibrillary tangles, aurophilic granulation disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and Parkinsonism, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, and pallido-ponto-nigral degeneration. Degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's disease, myotonic dystrophy, Tau panencephalopathy, astrocyte-like Alzheimer's disease, certain prion diseases (GSS with Tau), LRRK2 mutation, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal lobe degeneration, Guadeloupean Parkinson's disease, neuronal diseases with iron accumulation in the brain. Degeneration, SLC9A6-related intellectual disability, white matter Tau lesions with globular glial inclusions, post-traumatic stress disorder, epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes mellitus, age-related cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy; Alzheimer's disease is preferred.

[0055] 23. The compound used as described in item 22, wherein the condition is Alzheimer's disease (AD).

[0056] 24. The compound used as described in item 22, wherein the condition is Parkinson's disease or atypical Parkinson's disease.

[0057] 25. The compound used as described in item 22, wherein the condition is progressive supranuclear palsy (PSP).

[0058] 26. The compound used as described in item 22, wherein the condition is Pick's disease (PiD).

[0059] 27. The compound used as described in any one of items 18-26, wherein the Tau aggregates are imaged in the brain or in the eye, preferably wherein the detectable marker is 18 F and the imaging is positron emission tomography.

[0060] 28. An imaging method for Tau aggregates, particularly a positron emission tomography imaging method for Tau aggregates, wherein an effective amount of a compound as defined in item 4 or 15 is administered to a patient.

[0061] 29. A method for diagnosing a condition associated with Tau aggregates or Tau lesions, wherein an effective amount of a compound as defined in item 4 or 15 is administered to a patient, particularly wherein the diagnosis is performed by positron emission tomography.

[0062] 30. The method described in item 29, wherein the Tau lesion is a 3R Tau lesion.

[0063] 31. The method described in item 29, wherein the Tau lesion is a 4R Tau lesion.

[0064] 32. The method described in item 29, wherein the condition is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, boxing dementia, Down syndrome, Diseases, inclusion body myositis, prion amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Guam Parkinson's disease-dementia syndrome, non-Guam motor neuron disease with neurofibrillary tangles, aurophilic granulation disease, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and associated with Parkinson's syndrome, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, globus pallidus-pons-substantia nigra degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's disease, myotonic dystrophy, Tau panencephalopathy, astrocyte-like AD disease, certain prion diseases (with Tau GSS), LRRK2 mutation, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadeloupe Parkinson's disease, neurodegeneration with brain iron accumulation, SLC9A6-related intellectual disability, white matter Tau lesions with globular glial inclusions, post-traumatic stress disorder, epilepsy, Lewy body dementia (LBD), hereditary brain hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes mellitus, age-related cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy; Alzheimer's disease is preferred.

[0065] 33. The method described in item 32, wherein the condition is Alzheimer's disease (AD).

[0066] 34. The method described in item 32, wherein the condition is Parkinson's disease or atypical Parkinson's disease.

[0067] 35. The method described in item 32, wherein the condition is progressive supranuclear palsy (PSP).

[0068] 36. The method described in item 32, wherein the condition is Pick's disease (PiD).

[0069] 37. The method of any one of items 28-36, wherein the Tau aggregates are imaged in the brain or in the eye, preferably wherein the detectable marker is 18 F and the imaging is positron emission tomography.

[0070] 38. Use of compounds as defined in item 4 or 15 in the preparation of diagnostic agents for imaging Tau aggregates, particularly for positron emission tomography imaging of Tau aggregates.

[0071] 39. Use of compounds as defined in paragraph 4 or 15 in the preparation of diagnostic agents for diagnosing conditions associated with Tau aggregates or for diagnosing Tau lesions, particularly wherein the diagnosis is performed by positron emission tomography.

[0072] 40. The use described in item 39, wherein the Tau lesion is a 3R Tau lesion.

[0073] 41. The use described in item 39, wherein the Tau lesion is a 4R Tau lesion.

[0074] 42. The use described in item 39, wherein the condition is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, boxing dementia, Down syndrome, Diseases, inclusion body myositis, prion amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Guam Parkinson's disease-dementia syndrome, non-Guam motor neuron disease with neurofibrillary tangles, aurophilic granulation disease, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and associated with Parkinson's syndrome, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, globus pallidus-pons-substantia nigra degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's disease, myotonic dystrophy, Tau panencephalopathy, astrocyte-like AD disease, certain prion diseases (with Tau GSS), LRRK2 mutation, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadeloupe Parkinson's disease, neurodegeneration with brain iron accumulation, SLC9A6-related intellectual disability, white matter Tau lesions with globular glial inclusions, post-traumatic stress disorder, epilepsy, Lewy body dementia (LBD), hereditary brain hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes mellitus, age-related cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy; Alzheimer's disease is preferred.

[0075] 43. The use described in item 42, wherein the condition is Alzheimer's disease (AD).

[0076] 44. The use described in item 42, wherein the condition is Parkinson's disease or atypical Parkinson's disease.

[0077] 45. The use described in item 42, wherein the condition is progressive supranuclear palsy (PSP).

[0078] 46. ​​The use described in item 42, wherein the condition is Pick's disease (PiD).

[0079] 47. The method of any one of items 38-46, wherein the Tau aggregates are imaged in the brain or in the eye, preferably wherein the detectable marker is 18 F and the imaging is positron emission tomography.

[0080] 48. Use of the compound in item 5 as an analytical reference.

[0081] 49. Use of the compounds in item 5 as in vitro screening tools.

[0082] 50. A method for preparing a compound as defined in item 4, comprising mixing the compound as defined in item 6 with [ 18 F] Fluoridating agent reaction, wherein the method further includes cleaving the protecting group PG (if present).

[0083] 51. The method as described in item 50, wherein [ 18 F] Fluoridants are selected from K 18 F, H 18 F, Cs 18 F, Na 18 F and 18 F's four (C) 1–6 Alkyl ammonium salts.

[0084] 52. A method for preparing a diagnostic composition as defined in item 16, comprising mixing the compound as defined in item 6 with [ 18 F] Fluoridation reaction, wherein the method further includes cleaving a protecting group PG (if present), and then optionally mixing in a pharmaceutically acceptable carrier, diluent, excipient or excipient.

[0085] 53. A kit for preparing radiopharmaceutical articles, the kit comprising a sealed vial containing a predetermined amount of a compound as defined in item 6.

[0086] 54. The kit of claim 53 further comprises at least one component selected from: a reaction solvent, a solid-phase extraction column, a reagent for cleaving protecting groups, a solvent for purification, a solvent for formulation, and a pharmaceutically acceptable carrier, diluent, excipient, or excipient for formulation.

[0087] 55. A method for collecting data for diagnosing conditions associated with tau aggregates in samples or patients, comprising:

[0088] (a) Contacting a sample or a specific body part or region suspected of containing tau aggregates with a compound as defined in items 13-15;

[0089] (b) To bind the compound to tau aggregates;

[0090] (c) Detection of compounds that bind to tau aggregates; and

[0091] (d) Optionally, the presence or absence of a compound that binds to tau aggregates may be associated with the presence or absence of tau aggregates in a sample or a particular body part or region.

[0092] 56. A method for determining the amount of tau aggregates in tissues and / or body fluids, comprising:

[0093] (a) Provide a sample representing the tissue and / or body fluid being studied;

[0094] (b) Test the sample for the presence of tau aggregates using compounds as defined in items 13-15;

[0095] (c) Determining the amount of compound bound to tau aggregates; and

[0096] (d) Calculate the amount of tau aggregates in tissues and / or body fluids.

[0097] 57. A method for collecting data to determine a patient's susceptibility to a condition associated with tau aggregates, comprising detecting, in a sample or in situ, the specific binding of a compound defined in items 13-15 to tau aggregates, comprising the following steps:

[0098] (a) Contacting a sample or a specific body part or region suspected of containing tau aggregates with a compound as defined in items 13-15, which specifically binds to tau aggregates;

[0099] (b) to bind the compound to tau aggregates to form a compound / tau aggregate complex;

[0100] (c) Detection of the formation of compound / tau aggregate complexes;

[0101] (d) Optionally, the presence or absence of the compound / tau aggregate complex is correlated with the presence or absence of tau aggregates in the sample or a specific body site or region; and

[0102] (e) Optionally compare the amount of compound / tau aggregates with the normal control value.

[0103] 58. A method for collecting data on residual symptoms in patients with tau aggregate-related conditions who have been treated with medication, wherein the method comprises:

[0104] (a) Contacting a sample or a specific body part or region suspected of containing tau aggregates with a compound as defined in items 13-15, which specifically binds to tau aggregates;

[0105] (b) to bind the compound to tau aggregates to form a compound / tau aggregate complex;

[0106] (c) Detection of the formation of compound / tau aggregate complexes;

[0107] (d) Optionally, the presence or absence of the compound / tau aggregate complex is correlated with the presence or absence of tau aggregates in the sample or a specific body site or region; and

[0108] (e) Optionally compare the amount of compound / tau aggregates with the normal control value.

[0109] 59. A method for collecting data to predict the responsiveness of patients with a condition associated with tau aggregates and who are receiving drug treatment, comprising:

[0110] (a) Contacting a sample or a specific body part or region suspected of containing tau aggregates with a compound as defined in items 13-15, which specifically binds to tau aggregates;

[0111] (b) to bind the compound to tau aggregates to form a compound / tau aggregate complex;

[0112] (c) Detection of the formation of compound / tau aggregate complexes;

[0113] (d) Optionally, the presence or absence of the compound / tau aggregate complex is correlated with the presence or absence of tau aggregates in the sample or a specific body site or region; and

[0114] (e) Optionally compare the amount of compound / tau aggregates with the normal control value.

[0115] It should be understood that the present invention includes compounds of formula (II), wherein one or more atoms are each substituted with a different isotope. For example, compounds of formula (II) include compounds in which one or more hydrogen atoms are substituted with tritium and / or one or more hydrogen atoms are substituted with deuterium.

[0116] The inventors were surprised to discover that R 1 for 18F or F and R 2 Compounds of formula (II) with H (respectively compounds F-3a, F-3b, ... 18 F-3a and 18 F-3b) and compounds of existing technology 18 F-1 or 18 It has significantly improved properties compared to the F-2.

[0117]

[0118] In this article, both the F-3a and F-3b are referred to as "F-3", and 18 F-3a and 18 The F-3b will be collectively referred to as " 18 F-3". Among them, compounds F-3a and 18 F-3a is the preferred choice. Attached Figure Description

[0119] Figure 1 : Using compounds 18 F-3a was used for autoradiography of brain sections from AD and HC. Strong punctate staining was detected in AD brain sections, which could be blocked by adding an excess of the corresponding cold compound. No specific signal was visible in healthy control (HC) sections.

[0120] Figure 2 : Describes compounds in mice 18 F-1, 18 F-2 and 18 F-3a's washout curve of activity clearance from normal brain.

[0121] Figure 3 In non-dementia human control individuals 18 Brain uptake and rinsing of F-3a.

[0122] Figure 4 a) Non-demented human control individuals with axial, sagittal, and coronal projections. 18 F-3a PET image, b) AD individual with axial, sagittal, and coronal projections. 18 F-3a PET image.

[0123] Figure 5 : PSP individual 18 F-3a PET images, axial, sagittal, and coronal projections: a) at the level of the substantia nigra, b) at the level of the globus pallidus. Invention Details

[0125] This invention relates to detectable labeled compounds of formula (II).

[0126]

[0127] The preferred compound of the present invention is

[0128]

[0129] A more preferred compound of the present invention is

[0130]

[0131] Even more preferred compounds of the present invention are

[0132]

[0133] Even more preferred compounds of the present invention are

[0134]

[0135] The detectable labeled compounds of the present invention can be used for the selective detection of conditions and abnormalities associated with Tau aggregates, such as Alzheimer's disease and other Tau lesions, for example, by using positron emission tomography (PET) imaging. The invention also relates to intermediates that can be used to prepare such imaging compounds. The compounds of the present invention have a high affinity for Tau and bind to Tau isotypes present in both Alzheimer's disease (AD) and non-AD Tau lesions such as progressive supranuclear palsy (PSP) and Pick's disease (PiD). Because they have low affinity for amyloid-β and MAO A, they can be used as highly selective molecular probes for binding pathological Tau, thereby avoiding the detection of other lesions and misdiagnosis.

[0136] This invention 18 F-labeled compounds also result in low signal intensity in a healthy brain, thus reducing background signal interference and providing a low detection limit.

[0137] Due to their good brain uptake, rapid washout from the healthy brain, low long-term retention in the healthy brain, and lack of in vivo defluorination, this invention... 18 F-labeled compounds provide a good signal-to-noise ratio.

[0138] Furthermore, the compounds of the present invention can be readily and detectably labeled in high yield, for example, using... 18 F mark.

[0139] definition

[0140] As used herein, the term "protecting group" (PG) is applicable to any protecting group that protects an amine group during a contemplated chemical reaction. Examples of suitable protecting groups are well known to those skilled in the art. Suitable protecting groups are discussed, for example, in the textbook Greene and Wuts, Protecting groups in Organic Synthesis, 3rd edition, pp. 494–653, which is incorporated herein by reference. Protecting groups may be selected from urethanes, amides, imides, N-alkylamines, N-arylamines, imides, enamines, boranes, NP protecting groups, N-sulfinyl groups, N-sulfonyl groups, and N-silyl groups. Preferred examples of protecting groups (PGs) are benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), triphenylmethyl (triphenylmethyl), methoxyphenyl diphenylmethyl (MMT), or dimethoxytriphenylmethyl (DMT). More preferred examples of protecting groups PGs include tert-butoxycarbonyl (BOC), dimethoxytriphenylmethyl (DMT), and triphenylmethyl (triphenylmethyl). A more preferred example of a protecting group PG is tert-butoxycarbonyl (BOC).

[0141] As used herein, the term “leaving group” (LG) refers to any leaving group and means that an atom or group of atoms can be replaced by another atom or group of atoms. Examples are listed below: Synthesis (1982), pp. 85–125, Table 2; Carey and Sundberg, Organische Synthese, (1995), pp. 279–281, Table 5.8; or Netscher, Recent Res. Dev. Org. Chem., 2003, 7, 71–83, Schemes 1, 2, 10, and 15, etc.). (Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), in: Schubiger P.A., Friebe M., Lehmann L., (eds), PET-Chemistry-The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50, specifying: Scheme 4 on page 25, Scheme 5 on page 28, Table 4 on page 30, Figure 7 on page 33). Preferably, the “leaving group” (LG) is nitro, halogen, or trimethylammonium. More preferably, the “leaving group” (LG) is nitro or trimethylammonium. In a preferred embodiment, the “leaving group” (LG) is nitro. In another preferred embodiment, the “leaving group” (LG) is trimethylammonium.

[0142] The term Tau used in this article refers to a highly soluble microtubule-binding protein primarily found in neurons, including the six main isoforms, cleaved or truncated forms, and other modified forms such as those resulting from phosphorylation, glycosylation, glycation, prolyl isomerization, nitration, acetylation, polyamineization, ubiquitination, SUMO (sumoylation), and oxidation. Pathological Tau or Tau aggregates (neurofibrillar tangles, NFTs), as used in this article, refer to insoluble aggregates of hyperphosphorylated Tau protein containing paired helical filaments and straight filaments. Their presence is a hallmark of Alzheimer's disease (AD) and other diseases known as Tau disorders.

[0143] The term "polymorph" refers to the various crystal structures of the compounds of this invention. This may include, but is not limited to, crystalline morphologies (and amorphous materials) and all lattice forms. The salts of this invention can be crystalline and can exist as more than one polymorph.

[0144] This invention also includes solvates, hydrates, and anhydrous forms of the compounds of this invention. The solvent contained in the solvate is not particularly limited and can be any pharmaceutically acceptable solvent. Examples include water and C.1-4 Alcohols (such as methanol or ethanol).

[0145] As used below in the specification and claims of this invention, the term "prodrug" refers to any covalently bonded compound that releases an active parent drug due to biotransformation in vivo. The references to Goodman and Gilman, who generally describe prodrugs (The Pharmacological Basis of Therapeutics, 8th edition, McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", pp. 13-15), are incorporated herein by reference.

[0146] As used below in the specification and claims of this invention, the term "medicinal salt" refers to a nontoxic derivative of the disclosed compound, wherein the parent compound is modified by preparing salts of its inorganic and organic acids. Inorganic acids include, but are not limited to, acids such as carboxylic acids, hydrochloric acid, nitric acid, or sulfuric acid. Organic acids include, but are not limited to, acids such as aliphatic acids, alicyclic acids, aromatic acids, arylate acids, heterocyclic acids, carboxylic acids, and sulfonic acids. The pharmaceutical salts of this invention can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, PA, 1990, p. 1445, the disclosure of which is incorporated herein by reference.

[0147] "Medicinal use" is defined as those compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues within a reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and with a reasonable benefit / risk ratio.

[0148] The patients or individuals in this invention are typically animals, particularly mammals, and more particularly humans.

[0149] The tau gene contains 16 exons, 11 of which encode the major tau protein isoform. Alternative splicing of exon 10 produces tau isoforms with three (exon 10 deletion) or four (exon 10 presence) repeating domains, referred to as 3R and 4R tau, respectively (A. Andreadis et al., Biochemistry 31, (1992) 10626–10633; M. Tolnay et al., IUBMB Life, 55(6): 299–305, 2003). In Alzheimer's disease, the 3R and 4R isoforms are present in similar proportions. In contrast, in some tau lesions, one of the two isoforms is predominant. In this paper, the term "3R tau lesion" refers to a tau lesion (e.g., Pick's disease (PiD)) in which the 3R isoform is predominant. In this article, the term "4R Tau lesion" refers to Tau lesions (such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD)), in which the 4R isotype is predominant.

[0150] Unless otherwise stated, the preferred definitions given in the "Definitions" section apply to all embodiments described herein.

[0151] Diagnostic methods

[0152] Detectable labeled compounds of the present invention (especially 18 F-3, especially 18 F-3a) is particularly suitable for imaging Tau protein aggregates. Regarding Tau protein, the labeled compounds of this invention (especially) can be detected. 18 F-3, especially 18 F-3a) can bind to various types of Tau aggregates, such as pathologically aggregated Tau, hyperphosphorylated Tau, neurofibrillary tangles, paired helical filaments, straight filaments, neurotoxic soluble oligomers, polymers, and fibrils.

[0153] Due to the aforementioned binding characteristics, labeled compounds of the present invention (especially) can be detected. 18 F-3, especially 18 F-3a) is suitable for diagnosing conditions associated with Tau aggregates. It can detect labeled compounds of the present invention (especially...). 18 F-3, especially 18 F-3a) is particularly suitable for positron emission tomography (PET) imaging of Tau deposits. If the compound is to be administered to a patient, it is typically used... 18 F-labeled compounds of formula (II) are considered as detectable labeled compounds.

[0154] In imaging of Tau aggregates, the application of a detectable labeled compound of formula (II) (preferably) 18 F-3, especially 18 F-3a), and detect signals originating from compounds that specifically bind to Tau aggregates. Specific binding is a result of the high binding affinity between compounds of formula (II) and Tau aggregates.

[0155] In a preferred embodiment, a detectable labeled compound of formula (II) is used (preferably) 18 F-3, especially 18 F-3a) is used to diagnose the presence of tau lesions (preferably Alzheimer's disease). In this method, a detectable labeled compound of formula (II) (preferably) is used. 18 F-3, especially 18 F-3a) is administered to patients suspected of having tau disease (preferably Alzheimer's disease) or to samples obtained from such patients, preferably by detecting signals originating from detectable markers via positron emission tomography (PET).

[0156] If no signal originating from a detectable marker is detected, this method can be used to rule out tau lesions, which indicate the presence of neurological disorders other than tau lesions.

[0157] In methods for diagnosing conditions associated with Tau protein aggregates, such as Alzheimer's disease or susceptibility in an individual, the method includes:

[0158] a) Administration to mammals of a diagnostically effective amount of a detectably labeled compound of the invention (especially... 18 F-3, especially 18 F-3a);

[0159] b) Enables the detection of labeled compounds of the present invention (especially 18 F-3, especially 18 F-3a) is distributed to tissues of interest (e.g., brain tissue, eyes, or bodily fluids such as cerebrospinal fluid (CSF)); and

[0160] c) Imaging of the tissue of interest, wherein the labeled compounds of the present invention (especially) are detectable compared to normal control binding levels. 18 F-3, especially 18 Increased binding of F-3a to tissues of interest indicates that an individual has or is at risk of developing a condition associated with Tau protein aggregates.

[0161] Detectable labeled compounds of the present invention (especially 18 F-3, especially 18F-3a) can be used to image Tau protein aggregates in any sample or specific body site or region of a patient suspected of containing Tau protein aggregates. It can detect labeled compounds of the present invention (especially...). 18 F-3, especially 18 F-3a) can cross the blood-brain barrier and enter the eye. Therefore, they are particularly suitable for imaging Tau protein aggregates in the brain, eye (ophthalmic and / or retinal imaging), and body fluids (e.g., cerebrospinal fluid (CSF)).

[0162] In diagnostic applications, labeled compounds of the present invention (especially) can be detected. 18 F-3, especially 18 F-3a) is preferably used in diagnostic compositions.

[0163] The compounds of the present invention can be detected by detecting the labeled compounds (especially) 18 F-3, especially 18 F-3a) Specific binding to Tau protein aggregates in or in situ in a sample is used to diagnose a patient's susceptibility to Tau disease or Tau-related diseases, including:

[0164] (a) Comparing a sample or specific body part or region suspected of containing Tau protein aggregates with a detectably labeled compound of the present invention (especially... 18 F-3, especially 18 F-3a) contacts, which binds to Tau protein aggregates;

[0165] (b) To enable the detection of labeled compounds of the present invention (especially) 18 F-3, especially 18 F-3a) binds to Tau protein aggregates to form a compound / Tau protein aggregate complex (hereinafter, “compound / Tau protein aggregate complex” is abbreviated as “compound / protein aggregate complex”);

[0166] (c) Detection of compound / protein complex formation,

[0167] (d) Optionally, the presence or absence of the compound / protein complex is correlated with the presence or absence of Tau protein aggregates in the sample or a specific body part or region; and

[0168] (e) Optionally compare the amount of compound / protein with normal control values, wherein an increase in the amount of compound / protein compared with normal control values ​​may indicate that the patient has Tau-related disease or is at risk of having Tau-related disease.

[0169] The compound of the present invention (especially) is detected in a sample or a specific body part or area and labeled accordingly.18 F-3, especially 18 Upon contact with F-3a), the compound binds to Tau protein aggregates. The amount of time required for binding depends on the type of assay (e.g., in vitro or in vivo) and can be determined by those skilled in the art through routine experiments.

[0170] The compound that has bound to the Tau protein aggregate can then be detected by any suitable method. A preferred method is positron emission tomography (PET).

[0171] The presence or absence of the compound / protein is then optionally correlated with the presence or absence of Tau protein aggregates in the sample or a specific body part or region. Finally, the amount of the compound / protein can be compared with a normal control value measured in a sample or a specific body part or region of a healthy individual, wherein an increase in the amount of the compound / protein compared with the normal control value may indicate that the patient has Tau-related disease or is at risk of developing Tau-related disease.

[0172] The present invention also relates to a method for determining the amount of Tau protein aggregates in tissues and / or body fluids. The method includes the following steps:

[0173] (a) Provide a sample representing the tissue and / or body fluid being studied;

[0174] (b) Compounds of the present invention with detectable labels (especially) 18 F-3, especially 18 F-3a) Test samples for the presence of Tau protein aggregates;

[0175] (c) Determination of detectable labeled compounds of the present invention that bind to Tau protein aggregates (especially 18 F-3, especially 18 The amount of F-3a); and

[0176] (d) Calculate the amount of Tau protein aggregates in tissues and / or body fluids.

[0177] The following describes the detectable labeling of the compounds of the present invention (especially 18 F-3, especially 18 F-3a) Test for the presence of Tau protein aggregates in a sample: The sample is reacted with a detectable labeled compound of the present invention (especially...). 18 F-3, especially 18 F-3a) contact, enabling the detection of the labeled compound of the present invention (especially 18 F-3, especially 18F-3a) binds to Tau protein aggregates to form a compound / protein aggregate complex, and the formation of the compound / protein complex as described above is detected.

[0178] The compounds of the present invention with detectable labels (especially) 18 F-3, especially 18 F-3a) Monitoring low-level residual disease in patients with Tau protein aggregate-related conditions who have received drug treatment can be achieved as follows:

[0179] (a) Comparing a sample or specific body part or region suspected of containing Tau protein aggregates with a detectably labeled compound of the present invention (especially... 18 F-3, especially 18 F-3a) contact;

[0180] (b) To enable the detection of labeled compounds of the present invention (especially) 18 F-3, especially 18 F-3a) binds to Tau protein aggregates to form a compound / protein aggregate complex;

[0181] (c) Detection of the formation of compound / protein aggregate complexes.

[0182] (d) Optionally, the presence or absence of the compound / protein aggregate complex is associated with the presence or absence of Tau protein aggregates in the sample or a specific body part or region; and

[0183] (e) Optionally compare the amount of compound / protein aggregates with normal control values, wherein an increase in the amount of aggregates compared with normal control values ​​may indicate that the patient may still have a low degree of residual disease.

[0184] The steps (a)-(e) have been explained above.

[0185] The response of patients with a condition associated with Tau protein aggregates and who are currently undergoing drug treatment can be predicted as follows:

[0186] (a) Comparing a sample or specific body part or region suspected of containing Tau protein aggregates with a detectably labeled compound of the present invention (especially... 18 F-3, especially 18 F-3a) contact;

[0187] (b) To enable the detection of labeled compounds of the present invention (especially) 18 F-3, especially 18 F-3a) binds to Tau protein aggregates to form a compound / protein aggregate complex;

[0188] (c) Detection of the formation of compound / protein aggregate complexes.

[0189] (d) Optionally, the presence or absence of the compound / protein aggregate complex is associated with the presence or absence of Tau protein aggregates in the sample or a specific body part or region; and

[0190] (e) Optionally compare the amount of compound / protein aggregates with normal control values.

[0191] The steps (a)-(e) have been explained above.

[0192] In methods used to predict responsiveness, the amount of the compound / protein complex can optionally be compared at different time points during treatment, such as before and after treatment initiation or at different time points after treatment initiation. Changes in the amount of the compound / protein complex, especially a decrease, can indicate that the patient has a high potential to respond to their respective treatments.

[0193] The compounds of this invention can also be incorporated into assay kits for detecting Tau protein aggregates. Assay kits typically include a container holding one or more compounds of this invention and instructions for using the compound to bind Tau protein aggregates to form a compound / protein complex and for detecting the formation of the compound / protein complex, thereby relating the presence or absence of the compound / protein complex to the presence or absence of Tau protein aggregates.

[0194] The term "test kit" generally refers to any diagnostic kit known in the art. More specifically, the latter term refers to diagnostic kits as described in Zrein et al., Clin. Diagn. Lab. Immunol., 1998, 5, 45-49.

[0195] Diagnostic Compositions

[0196] "Diagnostic composition" is defined in this invention as a compound of the invention containing a detectable marker (preferably). 18 F marking; especially 18 F-3, especially 18 The composition is of the F-3a) type. For in vivo application, the diagnostic composition should be in a form suitable for administration to mammals such as humans. Preferably, the diagnostic composition further comprises a physiologically acceptable carrier, diluent, excipient, or excipient. Administration to a patient is preferably carried out by injection of the composition as an aqueous solution. This composition may optionally contain other components such as solvents, buffers; pharmaceutically acceptable solubilizers; and pharmaceutically acceptable stabilizers or antioxidants.

[0197] Pharmaceutical excipients are well-known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., New Jersey (1975). Pharmaceutical excipients can be selected based on the intended route of administration and standard pharmaceutical practice. Excipients must be acceptable in the sense that they are harmless to the recipient.

[0198] Pharmaceutically useful excipients that can be used in formulations of the diagnostic compositions of the present invention may include, for example, carriers, solvents, diluents, solvents and edible oils, oily esters, binders, excipients, solubilizers, thickeners, stabilizers, disintegrants, flow aids, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, colorants, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers, and enhancers.

[0199] If the labeled compound of the present invention is administered parenterally (preferably) 18 F marking; especially 18 F-3, especially 18 For F-3a), examples of such administration include one or more of the following: intravenous, intra-arterial, intraperitoneal, intrathecal, intracardiac, intraurethral, ​​intrasternal, intracranial, intramuscular, or subcutaneous administration of the compound; and / or administration via infusion techniques. For parenteral administration, the compound is preferably used in the form of a sterile aqueous solution, which may contain other excipients. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3-9). The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0200] The identifiable labeled compounds of the present invention (preferably) 18 F marking; especially 18 F-3, especially 18 The dose of F-3a) will vary depending on the exact compound administered, the patient's weight, size, and sample type, as well as other variables that are obvious to those skilled in the art. Typically, the dose may preferably be in the range of 0.001 μg / kg to 10 μg / kg, more 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.

[0201] The diagnostic compositions of the present invention can be prepared in a manner known to those skilled in the art, for example, as described in Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., New Jersey (1975).

[0202] For example, the compounds of the present invention can be used in liposome compositions as described in WO2016057812A1, which include a compound of formula (II) as a ligand for the selective detection of symptoms and abnormalities associated with Tau aggregates by non-radioactive magnetic resonance imaging (MRI).

[0203] In particular, in one embodiment, detectable labeled compounds of the present invention (especially...) can be used. 18 F-3, especially 18 F-3a) The diseases or conditions detected and monitored are those related to Tau protein aggregates.

[0204] The compounds of the present invention with detectable labels (especially) 18 F-3, especially 18 F-3a) Diseases or disorders that can be detected and monitored include neurodegenerative diseases such as Tau lesions. Examples of diseases and disorders that can be detected and monitored are caused by or related to the formation of neurofibrillary lesions. This is the primary brain pathology in tau lesions. The diseases and disorders include a heterogeneous group of neurodegenerative diseases or disorders, including those showing coexistence of Tau and amyloidosis. Examples of diseases involving Tau aggregates are generally classified as Tau lesions; these include, but are not limited to, Alzheimer's disease (AD), Creutzfeldt-Jacob disease, boxing dementia, Down syndrome, etc. Diseases including inclusion body myositis, prion amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis (ALS), Guam Parkinson's disease-dementia syndrome, non-Guam motor neuron diseases with neurofibrillary tangles, aurophilic granulation disease, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and associated with Parkinson's syndrome, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, globus pallidus-pons-substantia nigra degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's disease, dystrophic myotonia, Tau panencephalopathy, Alzheimer's disease with astrocytes, and certain prion diseases (with Ta... GSS of u), LRRK2 mutation, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadeloupe Parkinson's disease, neurodegeneration with brain iron accumulation, SLC9A6-associated intellectual disability, white matter Tau lesions with globular glial inclusions, post-traumatic stress disorder, epilepsy, Lewy body dementia (LBD), hereditary brain hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-related dementia, adult-onset diabetes mellitus, age-related cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy. Preferably, the diseases and conditions that can be detected and monitored include Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, boxing dementia, Down syndrome, Diseases, inclusion body myositis, prion amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Guam Parkinson's disease-dementia syndrome, non-Guam motor neuron disease with neurofibrillary tangles, aurophilic granulation disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia of Parkinson's syndrome associated with chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, globus pallidus-pons-substantia nigra degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's disease, dystrophic myotonia, Tau panencephalopathy, astrocyte-containing disease Alzheimer's disease (AD-like disease), certain prion diseases (GSS with Tau), LRRK2 mutations, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadeloupe Parkinson's disease, neurodegeneration with iron buildup in the brain, SLC9A6-related intellectual disability, white matter Tau lesions with globular glial inclusions, more preferably Alzheimer's disease (AD), Creutzfeldt-Jacob disease, boxing dementia, amyotrophic lateral sclerosis (ALS), aerobatic granuloma, corticobasal degeneration, frontotemporal dementia associated with Parkinson's syndrome related to chromosome 17, Pick's disease, progressive supranuclear palsy (PSP), tangles-only dementia, Guam Parkinson's dementia syndrome, Hallervorden-Spatz disease, and frontotemporal degeneration. Preferably, the disease or disorder is Alzheimer's disease.

[0205] The present invention 18 General Synthesis of F-labeled Compounds

[0206] quilt 18 F-labeled compounds of formula (II) can be obtained by adding compounds of formula (II) (where R) 1 It is LG and R 2 (Is it H or PG) and 18 It is prepared by reacting with F-fluorinating agents, thereby removing the leaving group LG. 18 F substitution. This preparation involves the cleavage of the protecting group PG (if present).

[0207] Any suitable 18 F-fluorinating agents. Typical examples include H 18 F, alkali metals or alkaline earth metals 18 F-fluorides (e.g., K) 18 F, Rb 18 F, Cs 18 F and Na 18 F). Optional, 18F-fluorinating agents can be used with chelating agents such as cavitation ligands (e.g., 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane- It can be used in combination with crown ethers (e.g., 18-crown-6). Alternatively, 18 F-fluorinating agents can be 18 F tetraalkylammonium salt or 18 Tetraalkylphosphonium salts of F; for example, 18 F's four (C) 1–6 alkyl)ammonium salts or 18 F's four (C) 1–6 Alkyl)phosphonium salts. Examples include tetrabutylammonium [ 18 F] fluoride and tetrabutylphosphonium [ 18 F] Fluorides. Preferably, 18 F-fluorinating agents are K 18 F, H 18 F, Cs 18 F, Na 18 F or tetrabutylammonium [ 18 F] Fluorides.

[0208] Available 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), in: Schubiger PA, Friebe M., Lehmann L., (eds), PET-Chemistry-The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50). Preferably, 18 The solvent used in F-fluorination is DMF, DMSO, acetonitrile, DMA or a mixture thereof, with acetonitrile or DMSO being the preferred solvent.

[0209] If desired, compounds having formula (II) may have R 1 For LG and R 2 For PG, where the protective base PG is in 18 The amine is protected during the F-fluorination reaction. The protecting group can then be removed. Methods for removing the protecting group are known in the art, including but not limited to acid cleavage.

[0210] If necessary, the compound of formula (II) may be further isolated and / or purified before use. The corresponding procedures are well known in the art.

[0211] Where R 1 For LG and R 2 Precursor compounds of formula (II) for H or PG can provide properties suitable for use with 18 In a kit for preparing compound (II) by reacting an F-fluorinating agent, the kit comprises a sealed vial containing a predetermined amount of the precursor compound of the present invention. For example, the kit may contain 1.5 to 75 μmol, preferably 7.5 to 50 μmol, more preferably 10 to 30 μmol of the precursor compound (II) of the present invention. Optionally, the kit may contain other components, such as a reaction solvent, a solid-phase extraction column, and a reagent for obtaining the precursor compound (II). 18 F-fluorinating agents, reagents for cleaving protecting groups, solvents for purification, solvents for formulations, and pharmaceutically acceptable carriers, diluents, excipients, or excipients for formulations.

[0212] Where R 1 For F and R 2 The compounds of this invention with the denoted H can be used as analytical references or in vitro screening tools.

[0213] Where R 1 For F and R 2 The compounds of the present invention with H can be used as R. 1 for 18 F and R 2 This serves as an analytical reference for the quality control and release of the compounds of the present invention, H.

[0214] Where R 1 It is F and R 2 The compounds of the present invention, H, can be used as in vitro screening tools for characterizing tissues with Tau pathology and for testing compounds that target Tau pathology in such tissues. Detailed Implementation

[0215] The invention is illustrated by the following examples, but should not be construed as limiting.

[0216] Example

[0217] All reagents and solvents were obtained from commercial sources and were ready for use without further purification. Protons ( 1The H) spectra were recorded on a Bruker DRX-400MHz NMR spectrometer or on a Bruker AV-400MHz NMR spectrometer in deuterated solvents. Mass spectra (MS) were recorded on an Advion CMS mass spectrometer. Chromatography was performed using silica gel (Fluka: silica gel 60, 0.063–0.2 mm) and suitable solvents as shown in the specific examples. Rapid purification was performed using the Biotage Isolera One rapid purification system, using an HP-Sil (Biotage) or puriFlash column (Interchim) and the solvent gradient indicated in the specific examples. Thin-layer chromatography (TLC) was performed on silica gel plates with UV detection.

[0218] Although some embodiments of the invention do not specify that various compounds are detectably labeled, it should be understood that corresponding detectably labeled compounds can be readily prepared, for example, by using detectably labeled starting materials, such as those containing... 3 Starting material for H atoms.

[0219] abbreviation

[0220]

[0221]

[0222] Preparation Example A

[0223]

[0224] Step A

[0225] Commercially available 2,6-dibromopyridine (4.12 g, 16.6 mmol) was suspended in ethanol (40 mL), and a solution of hydrazine hydrate (10 mL, 97.6 mmol) in water (~50-60%) was added. The mixture was heated in a sand bath at ~115 °C for 18 hours. The solvent was removed, and the residue was purified by silica gel chromatography by elution with ethyl acetate / n-heptane (60 / 40) to give the title compound as a grayish-white solid (3.05 g, 93%).

[0226] 1 H-NMR (400MHz, CDCl3): δ=7.33(t,1H),6.83(d,1H),6.67(d,1H),6.00(br-s,1H),3.33-3.00(br-s,2H)

[0227] Step B

[0228] The title compound (10 g, 53.2 mmol) obtained from step A above and commercially available 1-Boc-4-piperidinone (10.6 g, 53.2 mmol) were added to a 500 mL flask and mixed to form a homogeneous mixture. Then, polyphosphoric acid (80 g, 115% H3PO4 basis) was added, and the mixture was heated in a sand bath at ~160 °C. The Boc-protecting group was cleaved at ~120 °C, causing the reaction mixture to foam. After the Boc-cleavage was complete, the foam broke, and the dark-colored reaction mixture was stirred at ~160 °C for 20 hours. The reaction mixture was cooled to room temperature, and water (400 mL) was added. The reaction mixture was stirred / sonicated until the gel-like substance dissolved. The reaction mixture was then placed in an ice bath, and the pH of the solution was adjusted to ~12 by adding solid sodium hydroxide granules (exothermic). The precipitate was collected by filtration and washed with water (400 mL) to remove salts. The precipitate was dissolved in dichloromethane / methanol (9 / 1; 1500 mL) by sonication and washed with water (2 × 400 mL) to remove residual salts and insoluble substances. The organic phase was dried with Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The dark residue was treated with dichloromethane (100 mL), sonicated for 5 minutes, and the precipitate was collected by filtration. The precipitate was washed with dichloromethane (40 mL) and air-dried to give the title compound as a beige solid (3.5 g, 26%).

[0229] 1 H-NMR (400MHz, DMSO-d6): δ = 11.5 (br-s, 1H), 7.72 (d, 1H), 7.15 (d, 1H), 3.86-3.82 (m, 2H), 3.06-3.00 (m, 2H), 2.71-2.65 (m, 2H)

[0230] Step C

[0231] The title compound (1.75 g, 6.94 mmol) obtained from step B above was suspended in xylene (380 mL), and manganese oxide (IV) (6.62 g, 76.9 mmol) was added. The reaction mixture was then heated in a sand bath at ~160 °C for 36 hours. The cooled reaction mixture was evaporated under reduced pressure, and the residue was suspended in dichloromethane / methanol (1 / 1; 400 mL) and stirred at room temperature for 30 minutes. The reaction mixture was then filtered through a paper filter to remove manganese oxide (IV), and the filter was washed with methanol (50 mL). The combined filtrates were evaporated under reduced pressure, and the dark residue was purified by silica chromatography (50 g HP-SIL column) using a Biotage Isolera system, using an ethyl acetate / heptane gradient (5 / 95-100 / 0) to remove nonpolar impurities, followed by dichloromethane / methanol (9 / 1→4 / 1) to give the title compound as a dark yellow solid. The overall yield from the two runs was 1.77 g (51%).

[0232] 1 H-NMR (400MHz, DMSO-d6): δ = 12.52 (br-s, 1H), 9.42 (s, 1H), 8.61 (d, 1H), 8.53 (d, 1H), 7.56-7.52 (m, 2H)

[0233] Preparation Example B

[0234]

[0235] Step A

[0236] Triethylamine (1.86 mL, 13 mmol) and triphenylmethylchloro (2.63 g, 9.39 mmol) were added to a suspension of the title compound obtained from Preparation Example A (0.776 g, 3.13 mmol) in dichloromethane (65 mL). After adding 4-(dimethylamino)pyridine (0.074 g, 0.608 mmol), the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (150 mL) and water (50 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified by elution of an ethyl acetate / n-heptane gradient (5 / 95 → 100 / 0 → 100 / 0) on an HP-Sil SNAP column (50 g) using a Biotage Isolera One purification system to give title compound B as a pale yellow solid (0.831 g, 54%). Unreacted starting material was recovered by washing the column with ethyl acetate / methanol (90 / 10) to obtain a grayish-white solid (0.195 g, 25%).

[0237] 1 H-NMR (400MHz, CDCl3)δ=9.22(s,1H),8.23(d,1H),8.13(d,1H),7.48-7.42(m,7H),7.33-7.22(m,12H),6.41(d,1H)

[0238] MS(ESI); m / z=490.03 / 491.96[M+H] +

[0239] Preparation Example C

[0240]

[0241] Step A

[0242] Triethylamine (1.15 mL, 8 mmol) and 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene; DMTlt-Cl) (1.963 g, 5.8 mmol) were added to a suspension of the title compound (0.482 g, 1.94 mmol) obtained from Preparation Example A in dichloromethane (40 mL). After adding 4-(dimethylamino)pyridine (0.046 g, 0.377 mmol), the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was diluted with dichloromethane (100 mL) and water (40 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified on an HP-Sil SNAP column (50 g) using a Biotage Isolera One purification system with a gradient elution of ethyl acetate / n-heptane (5 / 95→100 / 0→100 / 0) to give title compound C as a pale yellow solid (0.825 g, 72%).

[0243] 1 H-NMR (400MHz, CDCl3)δ=9.23(s,1H),8.23(d,1H),8.13(d,1H),7.39-7.31(m,6H),7.29-7.25(4H),6.80(d,4H),6.41(dd,1H),3.81(s,6H)

[0244] Example 1 (ACI-2620)

[0245]

[0246] Step A

[0247] To a microwave-safe mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL), add [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride (a complex with dichloromethane) (0.0084 g, 0.01 mmol), followed by the title compound obtained from Preparation Example A (0.05 g, 0.2 mmol), (2-fluoropyridin-4-yl)boronic acid (0.035 g, 0.245 mmol), and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (60 mL) and water (20 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel (25 g HP-SIL) chromatography using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→95 / 5→90 / 10→80 / 20) to give the title compound F-3a as a grayish-white solid (0.033 g, 63%).

[0248] 1 H-NMR (400MHz, DMSO-d6) δ = 12.50 (br-s, 1H), 9.45 (s, 1H), 8.83 (d, 1H), 8.56-8. 52(m,1H),8.43-8.39(m,1H),8.19-8.14(m,2H),7.92(s,1H),7.54-7.50(m,1H)

[0249] MS(ESI): m / z = 265.04 [M+H] +

[0250] Example 2 (ACI-2698)

[0251]

[0252] Step A

[0253] A solution of the title compound (0.05 g, 0.202 mmol) obtained from Preparation Example A and (3-fluoropyridin-4-yl)boronic acid (0.0398 g, 0.282 mmol) in dimethoxyethane (ratio: 2, volume: 1.344 mL) and methanol (ratio: 1, volume: 0.672 mL) was added to a 5 mL microwave tube. Cesium fluoride (0.0306 g, 0.202 mmol) was added, and the resulting suspension was degassed with argon for 5 min. Then, tetrakis(triphenylphosphine)palladium(0) (0.0419 g, 0.036 mmol) was added, the tube was sealed, and the reaction mixture was heated at 150 °C in a Biotage Initiator microwave for 30 min (p = 12 bar). The reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel (10 g HP-SIL) chromatography using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→80 / 20) to give the title compound F-3b as a light brown solid (0.013 g, 24%).

[0254] 1 H-NMR (400MHz, DMSO-d6)δ=9.46(s,1H),8.82(d,1H),8.77(d,1H),8.63(d,1H),8.56(d,1H),8.09(dd,1H),7.91(dd,1H),7.54(d,1H)

[0255] MS(ESI); m / z = 265.16 [M+H] +

[0256] Example 3 (ACI-2690)

[0257]

[0258] Step A

[0259] To a microwave-safe mixture of 1,4-dioxane (4.3 mL) and water (1 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0084 g, 0.01 mmol) was added, followed by the title compound (0.1 g, 0.2 mmol) obtained from Preparation Example B, 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.061 g, 0.245 mmol), and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (60 mL) and water (20 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25g PUFI Flash column, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give title compound 13 as a pale yellow solid (0.082g, 75%).

[0260] 1 H-NMR (400MHz, CDCl3) δ = 9.32 (s, 1H); 8.56 (d, 1H), 8.48 (d, 1H), 8.33 (s, 1H); 8.30 ( d,1H),7.85(d,1H),7.69(d,1H),7.58-7.54(m,5H),7.32-7.25(m,10H),6.48(d,1H)

[0261] MS(ESI): m / z = 534.28 [M+H] + .

[0262] Example 4 (ACI-2756)

[0263]

[0264] Step A

[0265] To a mixture of 1,4-dioxane (8 mL) degassed in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0266] Step B

[0267] The crude title compound obtained from step A above was dissolved in a microwave-safe mixture of degassed 1,4-dioxane (8.6 mL) and water (2 mL). Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol), 4-chloro-3-nitropyridine (0.078 g, 0.49 mmol), and cesium carbonate (0.266 g, 0.82 mmol) were added, and the reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (80 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give title compound 16 as a pale yellow solid (0.033 g, 15%).

[0268] 1 H-NMR (400MHz, CDCl3) δ = 9.30 (s, 1H), 9.02 (s, 1H), 8.68 (d, 1H), 8.42 (d, 1H), 8. 26(d,1H),7.49-7.45(m,5H),7.31(d,1H),7.27-7.22(m,10H);7.08(d,1H),6.44 8d,1H)

[0269] MS(ESI): m / z = 533.59 [M+H] + .

[0270] Example 5 (Nitro / Boc Precursor) (ACI-2799)

[0271] Method a:

[0272]

[0273] Step A

[0274] Trifluoroacetic acid (1.2 mL) was added to a solution of the title compound (0.0396 g, 0.074 mmol) obtained from Example 3 in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 6 hours, and methanol (2 mL) was added. The solvent was evaporated under vacuum, and the residue was dissolved / suspended in methanol (5 mL). The solvent was evaporated under vacuum, and the residue was again dissolved / suspended in methanol (5 mL). The solvent was evaporated under vacuum, and the residue was suspended in dichloromethane (2 mL). After adding triethylamine (1 mL, 7.2 mmol), di-tert-butyl dicarbonate (0.098 g, 0.43 mmol) and 4-(dimethylamino)-pyridine (0.0018 g, 0.014 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and water (20 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to elute nonpolar byproducts, followed by elution with ethyl acetate / methanol (95 / 5) to give title compound 5 as a pale yellow solid (0.0184 g, 63%).

[0275] 1 H-NMR (400MHz, CDCl3) δ = 9.36 (s, 1H), 9.15 (s, 1H), 8.82-8.76 (m, 2H), 8.57 (d, 1H), 8.45 (d, 1H), 8.36 (d, 1H), 8.07 (d, 1H), 1.87 (s, 9H)

[0276] MS(ESI); m / z = 391.82 [M+H] +

[0277] Method b: (ACI-2799-2)

[0278]

[0279] Step A

[0280] To a microwave-safe mixture of degassed 1,4-dioxane (2.2 mL) and water (0.5 mL), add [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, a complex of [1,1′-bis(diphenylphosphino)ferrocene] with dichloromethane (0.0042 g, 0.005 mmol), followed by the title compound obtained from Preparation Example C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.0305 g, 0.12255 mmol), and cesium carbonate (0.067 g, 0.205 mmol). Heat the reaction mixture in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (20 mL), the precipitate was collected by filtration, washed with water (10 mL) and methanol (5 mL), and air-dried to give the crude title compound as a gray solid (0.0277 g, 95%).

[0281] Step B

[0282] Triethylamine (1 mL, 7.2 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol) were added to a suspension of the crude title compound (0.0277 g, 0.095 mmol) obtained from step A above in dichloromethane (4 mL). The reaction mixture was stirred at room temperature for 16 hours and diluted with ethyl acetate (50 mL) and water (20 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with a gradient elution of ethyl acetate / n-heptane (5 / 95 → 100 / 0 → 100 / 0) to elute nonpolar byproducts, followed by elution with ethyl acetate / methanol (95 / 5) to give title compound 5 as a pale yellow solid (0.0261 g, 70%).

[0283] 1 H-NMR (400MHz, CDCl3)δ=9.38(s,1H),9.16(s,1H),8.83-8.78(m,2H),8.58(d,1H),8.46(d,1H),8.38(d,1H),8.09(d,1H),1.88(s,9H)

[0284] MS(ESI); m / z = 391.85 [M+H] + 291.74 [M+H-Boc] +

[0285] Example 5a (Nitro precursor) (ACI-2776)

[0286]

[0287] Step A

[0288] To a microwave-safe mixture of degassed 1,4-dioxane (8 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol) obtained from Preparation Example B. The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0289] Step B

[0290] The crude title compound obtained from step A above was dissolved in a microwave-safe mixture of degassed 1,4-dioxane (8.6 mL) and water (2 mL). Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol), 4-bromo-2-nitropyridine (0.1 g, 0.49 mmol), and cesium carbonate (0.266 g, 0.82 mmol) were added, and the reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give the highly polar title compound as a pale yellow solid (0.0437 g, 20%).

[0291] Title compounds with higher polarity:

[0292] 1H-NMR (400MHz, CDCl3) δ = 9.32 (s, 1H); 8.56 (d, 1H), 8.48 (d, 1H), 8.33 (s, 1H); 8.30 ( d,1H),7.85(d,1H),7.69(d,1H),7.58-7.54(m,5H),7.32-7.25(m,10H),6.48(d,1H)

[0293] MS(ESI): m / z = 534.28 [M+H] + .

[0294] Byproducts with lower polarity:

[0295] 1 H-NMR (400MHz, CDCl3) δ = 9.26 (s, 1H), 8.31 (dd, 1H), 8.23-8.19 (m, 2H), 7.52-7.46 (m, 5H), 7.28-7.22 (m, 10H), 7.14 (dd, 1H), 6.19 (d, 1H)

[0296] Step C

[0297] The title compound (0.0437 g, 0.082 mmol) obtained from step B above was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.2 mL) was added. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with dichloromethane (50 mL) and water (20 mL). The pH of the aqueous phase was adjusted to approximately 12 by adding 1 M sodium hydroxide aqueous solution. The aqueous layer was discarded, and the precipitate of the organic layer was collected by filtration, washed with methanol (10 mL), and air-dried to give title compound 5a as a yellow solid (0.015 g, 63%).

[0298] 1 H-NMR (400MHz, DMSO-d6)δ=12.75-12.5(br-s,1H),9.45-9.40(br-s,1H),9.10-9.05(br-s,1H),8.85-8 .80(br-s,2H),8.68-8.63(br-s,1H),8.53-8.48(br-s,1H),8.27-8.22(br-s,1H),7.53-7.48(br-s,1H)

[0299] MS(ESI): m / z = 292.03 [M+H] + .

[0300] Example 6 (Nitro / DMTr precursor) (ACI-2916)

[0301]

[0302] Step A

[0303] To a microwave-safe mixture of degassed 1,4-dioxane (2.2 mL) and water (0.5 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0042 g, 0.005 mmol) was added, followed by the title compound obtained from Preparation Example C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.0305 g, 0.12255 mmol), and cesium carbonate (0.067 g, 0.205 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (20 mL), the precipitate was collected by filtration, washed with water (10 mL) and methanol (5 mL), and air-dried to give the crude title compound as a gray solid (0.0277 g, 95%).

[0304] Step B

[0305] Triethylamine (1 mL, 7.2 mmol), 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (0.081 g, 0.29 mmol), and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol) were added to a dichloromethane (4 mL) suspension of the crude title compound (0.0277 g, 0.095 mmol) obtained from step A above. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (50 mL) and water (20 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to give title compound 6 as a pale yellow solid (0.0261 g, 44%).

[0306] 1 H-NMR (400MHz, CDCl3) δ = 9.32 (s, 1H), 8.58 (d, 1H), 8.50 (d, 1h), 8.36 (s, 1H), 8.30 (d, 1H), 7.85 ( d,1H),7.74(d,1H),7.52-7.42(m,6H),7.27-7.23(m,4H),6.80(d,4H),6.49(d,1H),3.78(s,6H)

[0307] Example 7 (Iodine / Boc precursor) (ACI-3145)

[0308]

[0309] Step A

[0310] Trifluoroacetic acid (3.01 mL, 39.1 mmol) was added to a solution of the title compound (0.12 g, 0.195 mmol) obtained from Example 8 in dichloromethane (3.8 mL) at 0 °C. The reaction mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 1 M aqueous sodium hydroxide solution (20 mL) and extracted with dichloromethane (3 x 50 mL). The organic matter was collected, dried over Na2SO4, and purified on an HP-Sil column by elution with a dichloromethane / methanol gradient (100 / 0 → 90 / 10) to give the title compound (0.025 g, 34%).

[0311] 1 H-NMR (400MHz, DMSO-d6) δ = 12.46 (s, 1H), 9.43 (d, 1H), 8.79 (d, 1H), 8.60 (d, 1H), 8.52 (dd, 2H), 8.20 (dd, 1H), 8.14 (d, 1H), 7.50 (m, 2H).

[0312] MS(ESI): m / z = 373.03 [M+H] + .

[0313] Step B

[0314] To a solution of the title compound (0.02 g, 0.067 mmol) obtained from step A above in tetrahydrofuran (5 mL), di-tert-butyl dicarbonate (0.078 g, 0.336 mmol) and 4-(dimethylamino)-pyridine (0.0082 g, 0.0672 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours, and the solvent was removed under vacuum. The residue was purified on an HP-SilSNAP column using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (100 / 0 → 50 / 50) to give title compound 7 (0.018 g, 56%).

[0315] 1H-NMR (400MHz, CDCl3) δ = 9.31 (d, 1H), 8.73 (d, 1H), 8.63 (d, 1H), 8.52-8.44 (m, 2H), 8.34 (dd, 1H), 7.99 (dd, 1H), 7.91 (d, 1H), 1.84 (s, 9H).

[0316] MS(ESI): m / z = 473.03 [M+H] + .

[0317] Example 8 (Iodine / Tr precursor) (ACI-3143)

[0318]

[0319] Step A

[0320] To a mixture of degassed 1,4-dioxane (30 mL) and water (7 mL) in a dry pressure tube, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.043 g, 0.053 mmol) was added, followed by the title compound obtained from Preparation Example B (0.517 g, 1.054 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine-2-amine (0.278 g, 1.265 mmol), and cesium carbonate (0.687 g, 2.109 mmol). The reaction mixture was then heated at 100 °C for 4 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (40 mL) and 1 M sodium hydroxide aqueous solution (40 mL). The phases were separated, and the organic phase was washed with water (2 x 50 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The solvent was removed under reduced pressure, and the residue was purified on an HP-Sil SNAP column (50 g) using a Biotage Isolera One purification system with a dichloromethane / methanol gradient elution (100 / 0→90 / 10) to give the title compound (0.47 g, 89%).

[0321] 1 H-NMR (400MHz, DMSO-d6) δ = 9.39 (s, 1H), 8.68 (d, 1H), 8.23 ​​(d, 1H), 7.92-7.75 (m, 2H) ,7.67-7.51(m,6H),7.38-7.18(m,9H),6.65(d,1H),6.59-6.44(m,2H),5.72(s,2H).

[0322] Step B

[0323] To a suspension of the title compound (0.47 g, 0.933 mmol) obtained from step A above in dimethoxyethane (40 mL), tert-butyl nitrite (0.134 mL, 1.12 mmol) and iodine (0.308 g, 1.213 mmol) were added. The reaction mixture was stirred at 50 °C (internal temperature) for 3 hours. The reaction mixture was cooled to room temperature, and another batch of tert-butyl nitrite (0.134 mL, 1.120 mmol) and iodine (0.2 g, 0.788 mmol) was added. The reaction mixture was stirred at 60 °C (internal temperature) for 3 hours. The solvent was removed under reduced pressure, and the residue was purified twice on an HP-Sil column (100 g) by elution with a dichloromethane / methanol gradient (100 / 0 -> 95 / 5, eluting at a flow rate of 15 mL / min with a slowly increasing methanol concentration) to give title compound 8 (0.12 g, 21%).

[0324] 1 H-NMR (400MHz, CDCl3) δ = 9.28 (s, 1H), 8.41 (d, 1H), 8.29 (dd, 2H), 7.76-7.67 (m, 2H), 7.62-7.52 (m, 6H), 7.38 (dd, 1H), 7.28 (m, 9H), 6.56 (d, 1H).

[0325] Example 9 (Chlorine / Boc Precursor) (ACI-2997)

[0326]

[0327] Step A

[0328] To a mixture of degassed 1,4-dioxane (30 mL) and water (7 mL) in a dry pressure tube, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0225 g, 0.027 mmol) was added, followed by the title compound obtained from Preparation Example C (0.3 g, 0.545 mmol), (2-chloropyridin-4-yl)boronic acid (0.103 g, 0.654 mmol), and cesium carbonate (0.355 g, 1.09 mmol). The reaction mixture was then heated at 100 °C for 4 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (40 mL) and water (50 mL). The phases were separated, and the aqueous phase was extracted again with ethyl acetate (50 mL). The combined organic matter was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified on an HP-Sil SNAP column using a Biotage Isolera One purification system with a dichloromethane / methanol gradient elution (100 / 0→90 / 10) to give the title compound (0.153 g, 26%).

[0329] 1 H-NMR (400MHz, DMSO-d6) δ = 12.48 (s, 1H), 9.45 (s, 1H), 8.83 (d, 1H), 8.57 (dd, 2H), 8.31-8.22 (m, 1H), 8.22-8.13 (m, 2H), 7.53 (d, 1H).

[0330] Step B

[0331] To a suspension of the title compound (0.03 g, 0.107 mmol) obtained from step A above in tetrahydrofuran (5 mL), di-tert-butyl dicarbonate (0.037 g, 0.16 mmol) and 4-(dimethylamino)pyridine (0.0065 g, 0.053 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours, and the solvent was removed under vacuum. The residue was purified on an HP-SilSNAP column using a Biotage Isolera One purification system with a dichloromethane / methanol gradient elution (100 / 0 → 90 / 10) to give title compound 9 (0.033 g, 81%).

[0332] 1 H-NMR (400MHz, DMSO-d6) δ=9.51(d,1H),8.87(d,1H),8.71(d,1H),8.59(dd,1H),8.39(d,1H),8.26(dd,1H),8.22(dd,1H),1.77(s,9H).

[0333] MS(ESI): m / z = 280.81 [M+H] +

[0334] Examples 10 (trifluoromethanesulfonate / Trityl) and 11 (trifluoromethanesulfonate / Boc precursor) (ACI-3538; ACI- 3539)

[0335]

[0336] Step A

[0337] To a microwave-safe mixture of 1,4-dioxane (8.7 mL) and water (2 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, followed by the title compound (0.2 g, 0.4 mmol) obtained from Preparation Example B, 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine-2-amine (0.110 g, 0.5 mmol), and cesium carbonate (0.272 g, 0.84 mmol). The reaction mixture was then heated in a sand bath at ~120 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (40 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→97 / 3→95 / 5→95 / 5) to give the title compound as a grayish-white solid (0.2 g, 97%).

[0338] 1 H-NMR (400MHz, CDCl3) δ = 9.26 (s, 1H); 8.40 (d, 1H), 8.30 (d, 1H), 8.01 (d, 1H), 7.73 (d, 1H), 7. 65-7.59(m,5H),7.31-7.24(m,10H),6.98(dd,1H),6.67(d,1H),6.43(d,1H),4.80(br-s,2H)

[0339] Step B

[0340] The title compound obtained from step A above (0.2 g, 0.397 mmol) was suspended in N,N'-dimethylformamide (1.2 mL), and trifluoromethanesulfonic acid (0.6 mL) was slowly added at room temperature (exothermic). After adding sodium nitrite (0.055 g, 0.795 mmol), the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with dichloromethane (40 mL) and water (20 mL); then 2 M sodium hydroxide aqueous solution was added until pH ~12. The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was suspended in dichloromethane (15 mL), and triethylamine (2.7 mL) and di-tert-butyl dicarbonate (0.621 g, 3.13 mmol) were added. After adding 4-(dimethylamino)-pyridine (0.013 g, 0.1 mmol), the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and a 1 / 1-salt / water mixture (20 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of title compounds 10 and 11. The mixture of title compounds was separated by preparative TLC (20 x 20 cm; 1000 μm, Analtech) using ethyl acetate as the mobile phase to give the less polar title compound 10 as a pale yellow solid (0.0357 g, 14%) and the more polar title compound 11 as a pale yellow solid (0.0237 g, 12%).

[0341] Title compound 10, which has lower polarity:

[0342] 1 H-NMR (400MHz, CDCl3) δ = 9.32 (s, 1H), 8.47 (d, 1H), 8.35 (d, 1H), 8.31 (d, 1H), 7.80(d,1H),7.61-7.57(m.6H),7.32-7.26(m,10H),7.07(s,1H);6.90(d,1H)

[0343] MS(ESI): m / z = 637.25 [M+H] + .

[0344] Title compound 11, which has higher polarity:

[0345] 1H-NMR (400MHz, CDCl3)δ=9.38(s,1H),8.78(d,1H),8.57-8.50(m,2H),8.38(d,1H),8.15(d,1H),8.10(s,1H),8.00(d,1H),1.86(s,9H)

[0346] MS(ESI): m / z = 495.01 [M+H] + .

[0347] Example 12 (Trifluoromethanesulfonate / NH precursor) (ACI-3545)

[0348]

[0349] Step A

[0350] The title compound (0.272 g, 0.54 mmol) obtained from step A of Examples 10 and 11 was suspended in N,N'-dimethylformamide (1.8 mL). The reaction mixture was cooled to 0 °C, and trifluoromethanesulfonic acid (0.9 mL) was slowly added (exothermic). The reaction mixture was heated to room temperature, sodium nitrite (0.0825 g, 1.19 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with dichloromethane (60 mL) and water (25 mL); then 2 M sodium hydroxide aqueous solution was added until pH ~12. The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (25 g HP-SIL) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give the lower polar compound 10 as a pale yellow solid (0.0497 g, 14.5%). The gradient was then changed to dichloromethane / methanol (100 / 0→95 / 5→90 / 10→80 / 20) to give the title compound 12 as a gray solid (0.0425 g).

[0351] Step B

[0352] Compound 10, a lower polarity compound obtained from step A above (0.0497 g, 0.0078 mmol), was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.5 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (30 mL) and water (10 mL). The pH of the aqueous phase was adjusted to pH ~12 by adding 2 M sodium hydroxide aqueous solution. The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (10 g HP-SIL) using a Biotage Isolera One purification system with a dichloromethane / methanol gradient elution (100 / 0→95 / 5→90 / 10→80 / 20) to give the additional title compound 12 as a gray solid (0.0182 g), with a combined yield of 0.0607 g (28.5%).

[0353] 1 H-NMR (400MHz, DMSO-d6) δ = 12.54 (br-s, 1H), 9.46 (s, 1H), 8.87 (d, 1H), 8.6 4(d,1H),8.56(d,1H),8.42(dd,1H),8.30(d,1H),8.24(d,1H),7.55(dd,1H)

[0354] MS(ESI): m / z = 395.12 [M+H] + .

[0355] Example 14 (Trimethylammonium / Triphenylmethyl precursor)(ACI-3591)

[0356]

[0357] Step A

[0358] Commercially available N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine-2-amine (0.25 g, 1 mmol) was dissolved in dichloromethane (5 mL). Methyl trifluoromethanesulfonate (0.124 mL, 1.1 mmol) was added dropwise to the resulting stirred solution at room temperature. The solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated to remove dichloromethane, and the residue was dried under vacuum to give a yellow glassy / foamy substance, which was used directly in the next step.

[0359] Step B

[0360] To a microwave-safe solution of 1,4-dioxane (12 mL) and water (3 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with the title compound obtained from Preparation Example B (0.4 g, 0.816 mmol), the crude title compound obtained from step A above (~1 mmol), and cesium carbonate (0.544 g, 1.68 mmol). The reaction mixture was heated in a sand bath at ~120 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (150 mL) and water (50 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g HP-Ultra) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to elute unreacted starting materials and nonpolar byproducts. This gradient was then changed to dichloromethane / methanol (100 / 0 → 95 / 5 → 90 / 10) to give a dimethylamine derivative as a pale yellow glassy substance (0.127 g, 29%; MS (ESI): m / z = 532.27 [M+H]+) and a methylamine derivative as a gray solid (0.0547 g, 13%; MS (ESI): m / z = 519.18 [M+H]+). The gradient was again changed to dichloromethane / methanol (90 / 10 → 80 / 20) and held at (80 / 20) to give title compound 14 as a brown solid (0.104 g, 18%).

[0361] 1 H-NMR (400MHz, DMSO-d6) δ = 9.47 (s, 1H); 8.89 (d, 1H), 8.55 (d, 1H), 8-35-8.32 (m, 2H), 8. 29(d,1H),7.63-7.57(m,5H),7.48(d,1H),7.34-7.25(m,10H),6.48(d,1H),3.60(s,9H)

[0362] MS(ESI): m / z = 546.26 [M+H] +

[0363] Example 14a (Trimethylammonium / NH- precursor)(ACI-3613)

[0364]

[0365] Step A

[0366] The title compound (0.199 g, 0.364 mmol) obtained from Example 14 was suspended in dichloromethane (10 mL). After adding trifluoroacetic acid (10 mL), the reaction mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the residue was dissolved in methanol (10 mL), and the solvent was removed under reduced pressure. The treatment of the residue with methanol was repeated twice. The residue was then suspended in dichloromethane (20 mL) and sonicated for approximately 5 minutes. The precipitate was collected by filtration, washed with dichloromethane (10 mL), and air-dried to give title compound 14a as a gray solid (0.127 g, 83%).

[0367] 1 H-NMR (400MHz, DMSO-d6) δ = 13.76 (br-s, 1H), 9.84 (s, 1H); 8.12 (d, 1H), 8.89 ( d,1H),8.80(d,1H),8.75(s,1H),8.54-8.50(m,2H),8.04(d,1H),3.72(s,9H)

[0368] MS(ESI): m / z = 303.91 [M+H] +

[0369] Example 15 (Trifluoromethanesulfonate / DMTr precursor) (ACI-3546)

[0370]

[0371] Step A

[0372] Triethylamine (0.046 mL, 0.926 mmol), 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (0.062 g, 0.222 mmol), and 4-(dimethylamino)pyridine (0.00175 g, 0.014 mmol) were added to a suspension of the title compound (0.0291 g, 0.0739 mmol) obtained from Example 12 in dichloromethane (3 mL). The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (40 mL) and water (15 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to give the title compound as a semi-solid. The compound was treated with n-heptane (5 mL), sonicated for 5 minutes, and the solvent was evaporated under reduced pressure to give title compound 15, a grayish-white solid (0.0348 g, 67%).

[0373] 1 H-NMR (400MHz, CDCl3) δ = 9.32 (s, 1H), 8.47 (d, 1H), 8.36-8.28 (m, 2H), 7.79 (d, 1H), 7.59-7.57 (m, 1H) ,7.52-7.42(m,5H),7.33-7.27(m,4H),7.20-7.18(m,1H),6.83-6.77(m,4H),6.58(d,1H),3.80(s,6H)

[0374] MS(ESI): m / z = 697.28 [M+H] + .

[0375] Example 17 (Methanesulfonate / Triphenylmethyl Precursor) (ACI-3540)

[0376]

[0377] Step A

[0378] To a mixture of 1,4-dioxane (13 mL) and water (3 mL) degassed in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.026 g, 0.03 mmol) was added, followed by the title compound obtained from Preparation Example B (0.3 g, 0.612 mmol), (2-hydroxypyridin-4-yl)boronic acid (0.104 g, 0.75 mmol), and cesium carbonate (0.408 g, 1.15 mmol). The reaction mixture was then heated in a sand bath at ~120 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (120 mL) and water (45 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give unreacted starting material B as a grayish-white solid (0.1398 g, 47%). This gradient elution was then converted to dichloromethane / methanol (100 / 0→95 / 5→90 / 10→80 / 20→80 / 20) to give the title compound as a gray solid (0.0996 g, 32%).

[0379] 1H-NMR (400MHz, DMSO-d6) δ = 11.52 (br-s, 1H), 9.43 (s, 1H), 8.70 (d, 1H), 8.23 ​​(d, 1H), 7.9 7(d,1H),7.59-7.53(m,6H),7.32-7.21(m,10H),6.67(d,1H),6.48(d,1H),6.19(dd,1H)

[0380] MS(ESI): m / z = 505.28 [M+H] + .

[0381] Step B

[0382] Triethylamine (0.2 mL, 1.431 mmol), methanesulfonyl chloride (0.0365 mL, 0.477 mmol), and 4-(dimethylamino)pyridine (0.0054 g, 0.023 mmol) were added to a suspension of the title compound (0.080 g, 0.159 mmol) obtained from step A above in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (50 mL) and water / saline (20 mL; 1 / 1). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to give title compound 17 as a pale yellow solid (0.0438 g, 47%).

[0383] 1 H-NMR (400MHz, CDCl3) δ = 9.31 (s, 1H), 8.46 (d, 1H), 8.30-8.27 (m, 2H), 7.79 (d, 1H) ),7.59-7.55(m,5H),7.42(dd,1H),7.32-7.26(m,10H),7.08(s,1H),6.65(d,1H)

[0384] MS(ESI): m / z = 583.21 [M+H] + .

[0385] Example 17a (Methanesulfonate / NH precursor) (ACI-3572)

[0386]

[0387] Step A

[0388] The title compound (0.0388 g, 0.0067 mmol) obtained from Example 17 was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.5 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (30 mL) and water (10 mL). The pH of the aqueous phase was adjusted to pH ~12 by adding 2 M sodium hydroxide aqueous solution. The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (10 g HP-SIL) using a Biotage Isolera One purification system with a dichloromethane / methanol gradient elution (100 / 0 → 95 / 5 → 90 / 10 → 80 / 20) to give title compound 17a as a white solid (0.0059 g, 26%).

[0389] 1 H-NMR (400MHz, DMSO-d6)δ=12.51(br-s,1H),9.45(s,1H),8.84(d,1H),8.58- 8.54(m,2H);8.26(dd,1H),8.20(d,1H);8.05(d,1H),7.54(d,1H),3.68(s,3H)

[0390] MS(ESI): m / z = 341.17 [M+H] + .

[0391] Example 18 (Deuterated Compound)

[0392]

[0393] Step A

[0394] The title compound obtained from Example 1 was used as a starting material to prepare Example 18 by direct hydrogen isotope exchange with rhodium black. 2 H]F-3a).

[0395] MS(ESI): m / z = 265 (45%) [M+H] + 266 (65%) [M+H] + 267 (100%)

[0396] [M+H] + 268 (34%) [M+H] +

[0397] Example 19 (Tritium-modified compounds)

[0398]

[0399] Step A

[0400] The title compound obtained from Example 1 was used as a starting material to prepare Example 19 by direct hydrogen isotope exchange in a methanol / ethanol mixture with tritium (2.2 Ci / mL) and a Crabtree catalyst. 3 [H]F-3a). After HPLC purification (Phenomenex Prodigy ODS(2), 4.6 x 250 mm, 5 μm; solvent A: water containing 0.1% TFA; B: acetonitrile; 0-20 min 0-100% B; hold for 30 min), [ 3 H]F-3a has a radiochemical purity of 98.7% and a specific activity of 24.6 Ci / mmol.

[0401] MS(ESI): m / z=265(100%)[M+H] + ; 267 (77.5%) [M+H] + ; 269 (41.3%) [M+H] + ; 271 (11.3%) [M+H] +

[0402] Comparative Example 2 (F-2) (ACI-2448)

[0403]

[0404] Step A

[0405] Triethylamine (1.93 mL, 13.89 mmol) and di-tert-butyl dicarbonate (2.27 g, 10.02 mmol) were added to a suspension of the title compound (0.430 g, 1.73 mmol) obtained from Preparation Example A in dichloromethane (25 mL). After adding 4-(dimethylamino)pyridine (0.042 g, 0.34 mmol), the reaction mixture was stirred at room temperature for 3 days. The solvent was removed under reduced pressure, and the residue was purified on an HP-Sil SNAP column (25 g) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to give the title compound as a grayish-white solid (0.558 g, 92%).

[0406] 1 H-NMR (400MHz, CDCl3) δ = 9.28 (s, 1H), 8.73 (d, 1H), 8.22 (d, 2H), 7.59 8d, 1H), 1.80 (s, 9H)

[0407] Step B

[0408] To a microwave-safe mixture of degassed 1,4-dioxane (3 mL) and water (0.7 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0058 g, 0.007 mmol) was added, followed by the title compound from step A above (0.05 g, 0.143 mmol), (6-fluoropyridin-3-yl)boronic acid (0.024 g, 0.17 mmol), and cesium carbonate (0.092 g, 0.286 mmol). The reaction mixture was then heated in a sand bath at ~100 °C for 4 hours. The reaction mixture was diluted with ethyl acetate (80 mL) and water (35 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→98 / 2→95 / 5→90 / 10→80 / 20) to give a lower polarity Boc-protected compound (0.0255 g, 49%) and a higher polarity comparative example C2 (F-2) as a grayish-white solid (0.0116 g, 31%).

[0409] Comparative Example C2 (F-2) with higher polarity:

[0410] 1 H-NMR (400MHz, DMSO-d6)δ=12.40(br-s,1H),9.40(s,1H),9.05(s,1H),8.78-8.70(m,2H),8.51(d,1H),8.02(d,1H),7.50(d,1H),7.36(dd,1H)

[0411] MS(ESI): m / z = 265.09 [M+H] +

[0412] Lower polarity Boc-protected compounds:

[0413] 1 H-NMR (400MHz, DMSO-d6)δ=9.48(s,1H),9.13(d,1H),8.84-8.78(m,2H),8.68(d,1H),8.23(d,1H),8.19(d,1H),7.40(dd,1H),1.75 8s,9H)

[0414] The synthesis of Comparative Example C2 (F-2) was first described in WO2015 / 052105 (Example 1) by different synthesis methods.

[0415] Comparative Example 2 (F-2) Precursor (ACI-2449)

[0416]

[0417] Step A

[0418] To a microwave-safe mixture of degassed 1,4-dioxane (3 mL) and water (0.7 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0058 g, 0.007 mmol) was added, followed by the title compound (0.05 g, 0.143 mmol), 2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.0428 g, 0.17 mmol), and cesium carbonate (0.092 g, 0.286 mmol), obtained from step A of Comparative Example 2. The reaction mixture was then heated in a sand bath at ~100 °C for 4 hours. The reaction mixture was diluted with ethyl acetate (80 mL) and water (35 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→98 / 2→95 / 5→90 / 10→80 / 20) to obtain the precursor of Comparative Example C2 (F-2), which was a light yellow solid (0.0173 g, 31%).

[0419] 1 H-NMR (400MHz, CDCl3 / CD3OD)δ=9.45(d,1H),9.32(s,1H),8.93(dd,1H),8.68-8.64(m,2H),8.46(d,1H),8.35(d,1H),8.14(d,1H),1.82(s,9H)

[0420] MS(ESI): m / z = 392.13 [M+H] +

[0421] The synthesis of the precursor of Comparative Example C2 (F-2) was first described in WO2015 / 052105 (Example 3a) by different synthesis methods.

[0422] Comparative Example 5 (F-5) (ACI-2632)

[0423]

[0424] Step A

[0425] To a microwave-safe mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0084 g, 0.01 mmol) was added, followed by the title compound obtained from Preparation Example A (0.05 g, 0.2 mmol), 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.055 g, 0.246 mmol), and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (60 mL) and water (20 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g HP-SIL) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→95 / 5→90 / 10→80 / 20) to obtain Comparative Example C5 (F-5), which was a grayish-white solid (0.022 g, 43%).

[0426] 1 H-NMR (400MHz, DMSO-d6) δ = 12.45 (br-s, 1H), 9.45 (s, 1H), 9.31 (s, 1H), 8.80 ( d,1H),8.67(d,1H).8.53(d,1H),8.46-8.40(m,1H),8.11(d,1H),7.52(d,1H)

[0427] MS(ESI): m / z = 265.06 [M+H] +

[0428] Comparative Example 5 (F-5) precursor (ACI-2719)

[0429]

[0430] Step A

[0431] To a degassed mixture of 1,4-dioxane (4 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, followed by the title compound obtained from Preparation Example B (0.1 g, 0.2 mmol), bis(pinacolyl)diborane (0.056 g, 0.22 mmol), and potassium acetate (0.059 g, 0.6 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0432] Step B

[0433] The crude title compound obtained from step A above was dissolved in a microwave-safe container in a degassed mixture of 1,4-dioxane (4.3 mL) and water (1 mL). Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, along with 3-bromo-5-nitropyridine (0.05 g, 0.245 mmol) and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated in a sand bath at ~115 °C for 6 hours.

[0434] The reaction mixture was diluted with ethyl acetate (80 mL) and water (30 mL), the organic phase was separated, dried over Na2SO4, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give the C5 (F-5) precursor of Comparative Example as a pale yellow solid (0.0144 g, 13%).

[0435] 1 H-NMR (400MHz, CDCl3) δ = 9.36 (d, 1H), 9.30 (s, 1H), 9.02 (d, 1H); 8.52-8.48 (m, 2 H),8.29(d,1H),7.80(d,1H),7.60-7.55(m,5H),7.33-7.25(m,10H),6.46(d,1H)

[0436] MS(ESI): m / z = 533.67 [M+H] + .

[0437] Comparative Example 6 (F-6) (ACI-2843)

[0438]

[0439] Step A

[0440] A solution of the title compound (0.2 g, 0.408 mmol) obtained from Preparation Example B and 4-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.182 g, 0.816 mmol) in N,N'-dimethylacetamide (5.10 mL) was dissolved in a 20 mL microwave tube. Sodium carbonate (0.816 mL, 1.631 mmol) was added, and the resulting stirred solution was degassed for 5 min. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride with dichloromethane was added, and the reaction mixture was heated to 110 °C for 22 h. TLC monitoring showed that the reaction was complete. The reaction mixture was diluted with dichloromethane, filtered through diatomaceous earth to remove insoluble matter, and the filtrate was washed three times with water to remove residual N,N'-dimethylacetamide. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by Biotage Isolera One (100:0 to 90:10 dichloromethane / methanol; 25 g HP-Sil column) to give the title compound (0.1036 g; 50%).

[0441] 1 H-NMR (400MHz, DMSO-d6) δ = 9.43 (s, 1H), 8.75 (d, 1H), 8.54 (dd,, 1H), 8.26 (d, 1H), 8.17 (d,1H),7.83(dd,1H),7.61-7.52(m,6H),7.41(dd,1H),7.35-7.20(m,9H),6.46(d,1H).

[0442] MS[M+H] + =507.43,243.29

[0443] Step B

[0444] In a 25 mL round-bottom flask, the title compound (0.1 g, 0.199 mmol) obtained from step A above was dissolved in dichloromethane (1 mL). Trifluoroacetic acid (1 mL) was carefully added, and the reaction mixture was stirred at room temperature for 18 hours. After cooling to 0 °C, the reaction mixture was quenched to pH 10 with 2 M sodium hydroxide solution. The resulting suspension was filtered. The reaction mixture was washed with water and brine. The organic matter was dried over MgSO4, filtered, and concentrated. The residue was purified by Biotage Isolera One (100:0-90:10 dichloromethane / methanol; 10 g HP-Sil column) to give Comparative Example C6 (F-6) (0.026 g; 47%).

[0445] 1 H-NMR(400MHz,DMSO-d6)δ12.57(s,1H),9.46(s,1H),9.19(d,1H),8.80(d,1H),8.70(s,1H),8.59-8.52(m,1H),7.81(d,1H),7.55(d,2H)

[0446] MS[M+H] + =265.29

[0447] Comparative Example 6 (F-6) precursor (ACI-2764)

[0448]

[0449] Step A

[0450] In a 20 mL microwave-safe tube, the title compound (0.2 g, 0.408 mmol) obtained from Preparation Example B and 4-nitro-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.204 g, 0.816 mmol) were dissolved in N,N'-dimethylacetamide (5.10 mL). Sodium carbonate (0.816 mL, 1.631 mmol) was added, and the resulting stirred solution was degassed for 5 min. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, and the reaction mixture was heated to 110 °C for 22 h. TLC monitoring showed that the reaction was complete. The reaction mixture was diluted with dichloromethane, filtered through diatomaceous earth to remove insoluble matter, and the filtrate was washed three times with water to remove residual N,N'-dimethylacetamide. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by Biotage IsoleraOne and eluted with an ethyl acetate / n-heptane gradient (5 / 95→100 / 0→100 / 0) to give the C6 (F-6) precursor of Comparative Example as a pale yellow solid (0.056 g, 28%).

[0451] 1 H-NMR (400MHz, DMSO-d6) δ = 9.45 (s, 1H), 8.81 (d, 1H), 8.69 (d, 1H), 8.32-8.23 ( m,3H),8.20(d,1H),7.60(dd,6H),7.36-7.22(m,9H),6.52(d,1H),5.76(s,1H).

[0452] MS(ESI): m / z = 533.87 [M+H] + .

[0453] Comparative Example 7 (F-7) (ACI-2731)

[0454]

[0455] Step A

[0456] To a microwave-safe mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL), a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.0084 g, 0.01 mmol) was added, followed by the title compound obtained from Preparation Example A (0.05 g, 0.2 mmol), 2-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine (0.055 g, 0.246 mmol), and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (60 mL) and water (20 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g HP-SIL) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→95 / 5→90 / 10→80 / 20) to obtain Comparative Example C7 (F-7), which was a grayish-white solid (0.033 g, 63%).

[0457] 1 H-NMR (400MHz, DMSO-d6)δ=12.42(s,1H),9.41(s,1H),8.77(d,1H),8.52(d,1H),8.40(dd,1H),8.27(d,1H),8.18(q,1H),7.51(d,1H),7.26(dd,1H)

[0458] MS(ESI): m / z = 265.09 [M+H] +

[0459] Comparative Example 7 (F-7) Precursor (ACI-2778)

[0460]

[0461] Step A

[0462] To a degassed mixture of N,N'-dimethylacetamide (4 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, followed by the title compound obtained from Preparation Example B (0.1 g, 0.2 mmol), bis(pinacolyl)diborane (0.056 g, 0.22 mmol), and potassium acetate (0.059 g, 0.6 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0463] Step B

[0464] In a 20 mL microwave-safe tube, the crude title compound obtained from step A above, 2-bromo-6-nitropyridine (0.05 g, 0.245 mmol), was dissolved in N,N'-dimethylacetamide (5.10 mL). Sodium carbonate (0.408 mL, 0.816 mmol) was added, and the resulting stirred solution was degassed for 5 minutes. Then, a complex of [1,1′-bis(diphenylphosphine)ferrocene]palladium(II) dichloride and dichloromethane (0.017 g, 0.02 mmol) was added, and the reaction mixture was heated to 110 °C for 22 hours. TLC monitoring showed that the reaction was complete. The reaction mixture was diluted with dichloromethane, and the insoluble matter was filtered through diatomaceous earth. The filtrate was washed three times with water to remove residual N,N'-dimethylacetamide. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by Biotage Isolera One and eluted with an ethyl acetate / n-heptane gradient (5 / 95→100 / 0→100 / 0) to give the precursor of Comparative Example C7 (F-7) as a pale yellow solid (0.0174 g, 16%).

[0465] 1 H-NMR (400MHz, DMSO-d6)δ=9.43(s,1H),9.38(s,1H),8.81(d,1H),8.60(dd,1H),8.33(d,1H),8.28-8.24(m,2 H),8.18(d,1H),8.10(t,1H),7.61(d,7H),7.47(d,4H),7.42(d,1H),7.28(tt,18H),6.58(d,1H),6.19(d,1H)

[0466] MS(ESI): m / z = 533.62 [M+H] + .

[0467] Comparative Example 8 (F-8) (ACI-2876)

[0468]

[0469] Step A

[0470] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0471] Step B

[0472] The crude title compound obtained from step A above was dissolved in a degassed mixture of 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave-safe flask. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-5-fluoropyridine (0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.064 g).

[0473] Step C

[0474] The mixture (0.064 g) of the title compound and byproduct obtained from step B above was purified by preparative TLC at a loading of ~0.03 g mixture / 1000 μM Snaltech Uniplate (20 x 20 cm) with dichloromethane / acetone (90 / 10) as the mobile phase to give the highly polar title compound as a grayish-white solid (0.0385 g, 18.5% from 3 steps).

[0475] 1 ¹H-NMR (400MHz, CDCl₃) δ = 9.26 (s, 1H), 8.45 (d, 1H), 8.38 (AB-system, 2H), 8.25 (d, 1H), 7.62–7.58 (m, 5H), 7.30–7.18 (m, 12H), 6.56 (d, 1H)

[0476] Step D

[0477] The title compound (0.0385 g, 0.076 mmol) obtained from step C above was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.2 mL) was added. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with dichloromethane (50 mL) and water (20 mL). The pH of the aqueous phase was adjusted to pH ~12 by adding 1 M sodium hydroxide aqueous solution. The aqueous layer was separated, extracted with dichloromethane (25 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (10 g HP-SIL column) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0 → 95 / 5 → 90 / 10) to give Comparative Example C8 (F-8) as a white solid (0.0079 g, 39.3%).

[0478] 1 H-NMR (400MHz, DMSO-d6)δ=12.40(br-s,1H),9.40(s,1H),8.77(d,1H),8.72(d,1H),8.55-8.50(m,2H),8.35(d,1H),7.95-7.90(m,1H),7.51(d,1H)

[0479] MS(ESI): m / z = 265.06 [M+H] + .

[0480] The synthesis of the precursor of Comparative Example C8 (F-8) was first described in WO2016 / 124508 (Example 18) by different synthesis methods.

[0481] Comparative Example 8 (F-8) Precursor (ACI-2877)

[0482]

[0483] Step A

[0484] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0485] Step B

[0486] The crude title compound obtained from step A above was dissolved in a degassed mixture of 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave-safe flask. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-5-nitropyridine (0.1 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.0788 g).

[0487] Step C

[0488] The title compound and byproduct (0.0788 g) obtained from step B above were dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2.4 mL) was added. The reaction mixture was stirred at room temperature for 6 hours, followed by the addition of methanol (10 mL). The solvent was evaporated under vacuum, and the residue was suspended in methanol (10 mL). The solvent was evaporated under vacuum again, and the residue was suspended in dichloromethane (4 mL). Triethylamine (2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (40 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give the C8 (F-8) precursor of Comparative Example as a pale yellow solid (0.0149 g, 25.7%).

[0489] 1 H-NMR (400MHz, CDCl3)δ=9.55(d,1H),9.36(s,1H),8.88(d,1H),8.77(d,1H),8.72(d,1H),8.65(dd,1H),8.56(d,1H),8.30(d,1H),1.87(s,9H)

[0490] MS(ESI): m / z = 391.93 [M+H] + .

[0491] Comparative Example 9 (F-9) (ACI-2930)

[0492]

[0493] Step A

[0494] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0495] Step B

[0496] The crude title compound obtained from step A above was dissolved in a degassed mixture of 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave-safe flask. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-4-fluoropyridine (0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.0489 g).

[0497] Step C

[0498] The mixture (0.0489 g) of the title compound and byproduct obtained from step B above was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.5 mL) was added. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with dichloromethane (50 mL) and water (20 mL). The pH of the aqueous phase was adjusted to approximately 12 by adding 1 M sodium hydroxide aqueous solution. The aqueous layer was separated, extracted with dichloromethane (25 mL), and the combined organic layers were dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by preparative TLC, with approximately 0.03 g of the mixture loaded per 1000 μM Analtech Uniplate (20 x 20 cm) using dichloromethane / methanol (90 / 10) as the mobile phase, to give the lower polarity title compound as a grayish-white solid (0.0145 g, 7%, 3 steps) and a mixture of the two higher polarity compounds.

[0499] 1 H-NMR (400MHz, DMSO-d6) δ = 9.42 (s, 1H), 8.76 (d, 1H), 8.67 (dd, 1H), 8.35 (d, 1H) ,8.27(d,1H),7.67-7.60(m,5H),7.35-7.22(m,11H),6.81(dd,1H),6.60(d,1H)

[0500] Step D

[0501] The title compound of lower polarity obtained from step C above (0.0145 g, 0.027 mmol) was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (2 mL) was added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with dichloromethane (50 mL) and water (20 mL). The pH of the aqueous phase was adjusted to pH ~12 by adding 1 M sodium hydroxide aqueous solution. The aqueous layer was separated, extracted with dichloromethane (25 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (10 g HP-SIL) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0 → 95 / 5 → 90 / 10) to give Comparative Example C9 (F-9) as a grayish-white solid (0.0025 g, 33%).

[0502] 1H-NMR (400MHz, DMSO-d6)δ=12.43(br-s,1H),9.45(s,1H),8.82-8.77(m,2H),8.54(d,1H),8.44(d,1H),8.22(dd,1H),7.53(d,1H),7.46-7.42(m,1H)

[0503] MS(ESI): m / z = 264.63 [M+H] + .

[0504] Comparative Example 9 (F-9) precursor (ACI-2915)

[0505]

[0506] Step A

[0507] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0508] Step B

[0509] The crude title compound obtained from step A above was dissolved in a microwave-safe mixture of degassed 1,4-dioxane (8.6 mL) and water (2 mL). Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-4-nitropyridine (0.1 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.076 g).

[0510] Step C

[0511] A mixture (0.076 g) of the title compound and byproduct obtained from step B above was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2.4 mL) was added. The reaction mixture was stirred at room temperature for 6 hours, followed by the addition of methanol (10 mL). The solvent was evaporated under vacuum, and the residue was suspended in methanol (10 mL). The solvent was evaporated under vacuum again, and the residue was suspended in dichloromethane (4 mL). Triethylamine (2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (40 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give the C9 (F-9) precursor and byproduct of Comparative Example as a ~1.1- mixture (0.0231 g, pale yellow solid).

[0512] 1H-NMR (400MHz, CDCl3)δ=9.38(d,1H),9.35(d,1H),9,31(s,2H),9.02(d,1H),8.76-8.70(m,5H),8.68(d, 1H),8.55(d,1H),8.43-8.37(m,3H),8.12(dd,1H),8.07(dd,1H),7.43(d,1H),7.41(d,1H),1.82(s,18H)

[0513] MS (ESI): m / z = 291.94 [MH-Boc of the title compound] + 170.04 [MH of byproducts] + -Boc] +

[0514] Comparative Example 10 (F-10) (ACI-2931)

[0515]

[0516] Step A

[0517] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0518] Step B

[0519] The crude title compound obtained from step A above was dissolved in a degassed mixture of 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave-safe flask. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-3-fluoropyridine (0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.0586 g).

[0520] Step C

[0521] The mixture (0.0586 g) of the title compound and byproduct obtained from step B above was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.8 mL) was added. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with dichloromethane (50 mL) and water (20 mL). The pH of the aqueous phase was adjusted to pH ~12 by adding 1 M sodium hydroxide aqueous solution. The aqueous layer was separated, extracted with dichloromethane (25 mL), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (10 g HP-SIL) using a Biotage Isolera system with a dichloromethane / methanol gradient elution (100 / 0→95 / 5→90 / 10) to give Comparative Example C10 (F-10) as a grayish-white solid (0.0067 g, 5.7% in 3 steps).

[0522] 1 H-NMR (400MHz, DMSO-d6) δ = 12.47 (br-s, 1H), 9.45 (s, 1H), 8.80 (d, 1H), 8.63-8.61 (m, 1H),8.55-8.53(m,1H),8.00(d,1H),7.94-7.88(m,1H),7.63-7.58(m,1H),7.52(d,1H)

[0523] MS(ESI): m / z = 264.84 [M+H] + .

[0524] Comparative Example 10 (F-10) Precursor (ACI-2941)

[0525]

[0526] Step A

[0527] To a degassed mixture of 1,4-dioxane (8 mL) in a microwave flask, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, followed by the title compound obtained from Preparation Example B (0.2 g, 0.4 mmol), bis(pinacolyl)diborane (0.112 g, 0.44 mmol), and potassium acetate (0.118 g, 1.2 mmol). The reaction mixture was then heated in a sand bath at ~95 °C for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum to give the crude title compound, which was used directly in the next step.

[0528] Step B

[0529] The crude title compound obtained from step A above was dissolved in a degassed mixture of 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave-safe flask. Then, a complex of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride and dichloromethane (0.034 g, 0.04 mmol) was added, along with 2-bromo-3-nitropyridine (0.1 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol). The reaction mixture was heated in a sand bath at ~115 °C for 6 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (30 mL), the organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was evaporated under vacuum. The dark residue was purified by silica gel chromatography (25 g puriFlash, Interchim) using a Biotage Isolera system with an ethyl acetate / n-heptane gradient elution (5 / 95→100 / 0→100 / 0) to give a mixture of the title compound and byproducts (0.0538 g).

[0530] Step C

[0531] The mixture (0.0538 g) of the title compound and byproduct obtained from step B above was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (2.5 mL) was added. The reaction mixture was stirred at room temperature for 16 hours, and then methanol (10 mL) was added. The solvent was evaporated under vacuum, and the residue was suspended in methanol (10 mL). The solvent was removed again under vacuum, and the residue was suspended in dichloromethane (4 mL). Triethylamine (2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and 4-(dimethylamino)-pyridine (0.0036 g, 0.028 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (40 mL). The organic phase was separated, dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The residue was purified on silica gel (25 g puriFlash, Interchim) using a Biotage Isolera One purification system with an ethyl acetate / n-heptane gradient elution (5 / 95 → 100 / 0 → 100 / 0) to give the C10 (F-10) precursor of Comparative Example as a pale yellow solid (0.0194 g, 12.1% in 3 steps).

[0532] 1 H-NMR (400MHz, CDCl3)δ=9.35(d,1H),8.90(d,1H),8.73(d,1H),8.58(d,1H),8.24-8.17(m,3H),7.57-7.53(m,1H),1.73(s,9H)

[0533] MS(ESI): m / z = 391.92 [MH] + ],291.90[MH + -Boc]

[0534] 18 Synthesis of F-labeled compounds

[0535] General 18 F-fluorination method A (direct aromaticity) 18 F-fluorination)

[0536] NCA[18F] fluoride (2-5 GBq) was captured on a Sep-Pak Accell Plus QMA column (Waters) and treated with a solution of K2CO3 / 2.2.2 Elution. Water was removed using a nitrogen stream at 120°C, and the solution was co-evaporated to dryness with MeCN (3 × 1 mL). The dissolved precursor solution was then added to dry K[ 18 F]FK 222In the complex. The reaction vial was sealed and heated at 130 °C for 15 min under normal heating conditions. Subsequently, the reaction mixture was quenched with water, and the crude product was purified by semi-preparative HPLC. The separated tracer was diluted with water (35 mL), captured on a C-18 Plus column (Waters), washed with water (5 mL), eluted with ethanol (1 mL), and prepared in brine.

[0537] General 18 F-fluorination method B (direct) 18 F-marker with deprotection)

[0538] tracer from nca[ 18 F] Fluoride (1-10 GBq) begins to pass 18 F-direct fluorination synthesis. [ 18 F] Fluoride aqueous solution was captured on the Sep-Pak Accell Plus QMA light column (Waters) and treated with K2CO3 solution / 2.2.2 Elution. Water was removed using a nitrogen stream at 120°C, and the mixture was co-evaporated to dryness with MeCN (3 × 1 mL). The corresponding dissolved precursor was then added to dry K[ 18 F]FK 222 In the complex. The reaction vial was sealed and heated at 120–160 °C for 15 minutes (heating block). To deprotect, hydrochloric acid was added and the mixture was stirred at 110 °C for another 10 minutes. After neutralization with sodium hydroxide solution, the reaction mixture was quenched with ammonium formate buffer and captured on a C-18 Plus column (Waters). The column was washed with water (5 mL), eluted with acetonitrile, and the crude product was purified by semi-preparative HPLC. The separated tracer was diluted with water (25 mL), captured on a C-18 Plus column (Waters), washed with water (5 mL), eluted with ethanol (1 mL), and prepared in brine.

[0539] Comparative Examples 18 F-1

[0540] 18 The F-1 (680MBq) is based on General Motors. 18 F-fluorination method A synthesizes the corresponding nitro precursor molecule (M. Timothy et al., J. Labelled Comp. Radiopharm. (2013), 56(14), 736-740) (2.8 mg, 7.1 μmol) in dimethyl sulfoxide (0.6 mL).

[0541] Analytical reversed-phase HPLC (t) R (RAD-trace = 3.19 min) The radiochemical purity was determined to be 100%. This was confirmed by comparing the retention time with that of the non-radioactive reference F-1. 18 F-1.

[0542] Comparative Examples 18 F-2

[0543] 18 F-2 (680MBq) according to General Motors 18 F-fluorination method A was synthesized using the precursor (WO2015 / 052105) (3.4 mg, 8.7 μmol) from Comparative Example 2 (F-2) in dimethyl sulfoxide (0.6 mL). The synthesis was performed by analytical reversed-phase HPLC (t...). R (RAD-trace = 3.27 min) The radiochemical purity was determined to be 98%. This was confirmed by comparing the retention time with that of the non-radioactive reference F-2. 18 F-2.

[0544] Example 18 F-3a[ 18 F]PI-2620

[0545]

[0546] 18 F-3a (450MBq) according to General 18 F-fluorination method B synthesized using precursor compound 13 (2.6 mg, 4.8 μmol) in dimethyl sulfoxide (0.6 mL). The synthesis was performed by analytical reversed-phase HPLC (t...). R (RAD-trace = 3.31 min) The radiochemical purity was determined to be 100%. This was confirmed by comparing the retention time with the non-radioactive reference F-3a. 18 F-3a.

[0547] Examples of radioactive labeling 18 F-3b and Comparative Examples 18 F-5, 18 F-6 18 F-7 18 F-8 18 F-9 18 F-10 is synthesized from the corresponding precursor molecules as described above, according to method B.

[0548] Determination of binding in brain homogenates of AD and healthy controls

[0549] At 800Bq 18In the presence of F-labeled Tau binder, 20 μg of human Alzheimer's disease brain homogenate was incubated with serial dilutions (1000 to 0.06 nM) of each test compound. The sample was shaken at 110 rpm at 37 °C for 45 min. The sample was then filtered through a GF / B 96-well plate and washed twice with 300 μL of assay buffer (PBS containing 0.1% BSA and 2% DMSO). The plate was then sealed and the Fuji Film Imaging Plate (BAS-SR2025) was placed on top. The Fuji Film BAS-5000 analysis imaging plate was used after overnight exposure. The sample was then analyzed with assay buffer containing […] in the presence of a brain substrate-free and competitor-free buffer. 18 Non-specific signals were measured for F-labeled Tau reference binders in the samples. Specific binding was calculated by subtracting the non-specific signal from the measured sample signal. Unblocked binding... 18 The F-labeled Tau-binding agent signal is defined as the total binding. The IC50 value is calculated by setting the total binding to 100% using Prism V6 (GraphPad).

[0550] result:

[0551] High tau affinity of compounds F-1, F-2, and F-3a was found in a competitive assay using human AD brain homogenate. The IC50 values ​​for tau binding of all compounds were measured to be <2 nM.

[0552] Using compounds 18 F-3a achieved a high signal-to-noise ratio of 6.7 between AD brain homogenate and healthy control brain homogenate. For compounds... 18 F-1 yielded a low signal-to-noise ratio of 1.3 between AD brain homogenate and healthy control brain homogenate.

[0553] Different data was generated using different human brain tissue.

[0554] Identify compounds 18 The signal-to-noise ratios between F-3a AD brain homogenate and healthy control brain homogenate were 14.0, 17.9, and 33.8, respectively.

[0555] compound 18 F-1 values ​​were significantly lower, with those ratios being only 1.7, 1.8, and 2.5.

[0556] compound 18 F-2 showed a significantly lower signal ratio in AD brain homogenate compared to that in healthy control brain homogenate (3.3, 4.5, 6.9).

[0557] Autoradiography in human brain slices

[0558] 18-micron thick frozen human brain sections and 6-micron thick human FFPE brain sections were examined by autoradiography. Brain sections were equilibrated in 1xPBS solution for at least 1 hour before use in experiments. Each brain section was... 18 Cover with a solution of F-labeled tracer (200 Bq / μl, 500 μl) in 1xPBS. 19 The blocking experiment conducted using the F-compound involved mixing an excess of the blocking compound (10 μM) with... 18 F-compounds were mixed. Brain slices were incubated with the tracer solution at room temperature for 1 hour, then drained and placed in a slide holder. The slides were then washed sequentially with 1×PBS for 1 minute; 70% EtOH in 1×PBS for 2 minutes; 30% EtOH in 1×PBS for 2 minutes; and 1×PBS for 1 minute. The slides were air-dried and then placed on a Fuji imaging plate for 30 minutes, followed by overnight exposure. The imaging plate was scanned and the signal was measured using Fuji software to produce autoradiographic images of the brain slices.

[0559] result:

[0560] Compounds were tested in an autoradiography study using human brain slices (AD, PSP, PiD, HC). 18 F-3a. Strong punctate staining was detected using sections from AD brains, which could be blocked by adding an excess of the corresponding cold compound. No specific signal was visible in healthy control (HC) sections. Figure 1 ). For compounds on PSP and PiD brain slices 18 The F-3a achieved similar results.

[0561] Determining the binding affinity of amyloid-β to AD brain homogenate.

[0562] At 800Bq 18 In the presence of F-labeled β-amyloid binding agent, 20 μg of human Alzheimer's disease brain homogenate was incubated with serial dilutions (1000 to 0.06 nM) of each test compound. The sample was shaken at 37°C for 45 min at 110 rpm. The sample was then filtered through a GF / B 96-well plate and washed twice with 300 μL of assay buffer (PBS containing 0.1% BSA and 2% DMSO). The plate was then sealed and the Fuji Film Imaging Plate (BAS-SR2025) was placed on top. An overnight post-exposure analysis imaging plate was performed using a Fuji Film BAS-5000. The sample was incubated with assay buffer containing […] in the absence of brain substrates and competitors. 18Non-specific signals were measured in samples of F-labeled β-amyloid binding agents. Specific binding was calculated by subtracting the non-specific signal from the measured sample signal. Unblocked binding... 18 The F-labeled β-amyloid binding signal was defined as total binding. IC was calculated by setting total binding to 100% using Prism V6 (GraphPad). 50 value.

[0563] result:

[0564] In a competitive assay using human AD brain homogenate, compounds F-1, F-2, and F-3a were found to have low affinity for β-amyloid protein. The IC50 values ​​for β-amyloid binding of all compounds were >1 μM.

[0565] Determination of binding affinity for MAO A in HC brain homogenate

[0566] At 800Bq 18 F-labeled MAO-A binder ([ 18 In the presence of fluoroethylharmine (FEH), 20 μg of human brain homogenate (without AD pathology) was incubated with serial dilutions (1000 to 0.06 nM) of each test compound. The sample was shaken at 37°C for 45 min at 110 rpm. The sample was then filtered through a GF / B 96-well plate and washed twice with 300 μL of assay buffer (PBS containing 0.1% BSA and 2% DMSO). The plate was then sealed and the Fuji Film Imaging Plate (BAS-SR2025) was placed on top. The imaging plate was analyzed using a Fuji Film BAS-5000 after overnight exposure. The sample was incubated with assay buffer containing […] in the absence of brain substrates and competitors. 18 Non-specific signals were measured in F-labeled FEH samples. Specific binding was calculated by subtracting the non-specific signal from the measured sample signal. Unblocked binding... 18 The FEH signal marked with F is defined as the total binding. The IC50 value is calculated by setting the total binding to 100% using Prism V6 (GraphPad).

[0567] result:

[0568] In mouse brain homogenate, compound F-1... 18 The F-FEH competitive assay showed a high off-target affinity for MAO A at 22 nM. The affinity of compound F-2 decreased to 475 nM, while the off-target affinity of compound F-3a for MAO A further decreased, IC50... 50The value was 1400 nM. Using human control brain homogenate (healthy control), compound F-1 showed a high off-target affinity for MAO A of 5 nM in the FEH competitive assay. The affinity of compound F-2 decreased to 100 nM, while the off-target affinity of compound F-3a for MAO A further decreased, IC50 value 1400 nM. 50 The values ​​are 1100 nM and 530 nM, respectively.

[0569] Determination of the binding affinity of MAO B in HC brain homogenate

[0570] At 800Bq 18 F-labeled MAO-B binder ([ 18 In the presence of fluorodeprenyl (F), 20 μg of human brain homogenate (without AD pathology) was incubated with serial dilutions (1000 to 0.06 nM) of each test compound. The sample was shaken at 110 rpm at 37 °C for 45 min. The sample was then filtered through a GF / B 96-well plate and washed twice with 300 μL of assay buffer (PBS containing 0.1% BSA and 2% DMSO). The plate was then sealed and the Fuji Film Imaging Plate (BAS-SR2025) was placed on top. The imaging plate was analyzed using a Fuji Film BAS-5000 after overnight exposure. The sample was then used with assay buffer containing […] in the absence of brain substrates and competitors. 18 Nonspecific signals were measured in F-labeled fluoroselegiline samples. Specific binding was calculated by subtracting the nonspecific signal from the measured sample signal. Unblocked binding... 18 The F-labeled fluoroselegiline signal is defined as the total binding. The IC is calculated by setting the total binding to 100% using Prism V6 (GraphPad). 50 value.

[0571] result:

[0572] In human HC brain homogenate, compound F-1... 18 F-labeled fluoroselegiline showed a high off-target affinity of 170 nM for MAO B in competitive assays. The affinity of compound F-3 decreased to >1000 nM.

[0573] PK study in healthy mice

[0574] use 18 F-labeled compounds were intravenously injected into NMRI mice (weight range 25-35g). Injections of up to 150 μL containing [the compound name is missing]. 18F-labeled compound (2-10 MBq) in 1xPBS solution containing 10%-15% EtOH or diluent (57% water for injection, 18% polyethylene glycol 400, 15% ethanol, 10% water). Anesthesia was induced with isoflurane before tracer injection and maintained during image acquisition. PET scans were performed using a SIEMENS INVEON small animal PET / CT scanner (Siemens, Knoxville, TN). PET acquisition began immediately before the radioactive dose was injected into the animal via the tail vein. Images were generated after 60 minutes of dynamic scanning.

[0575] result:

[0576] compound 18 F-1: Peak uptake: 5.3% ID / g, peak uptake ratio / 30 minutes: 6.8, brain retention at 60 minutes: 0.8% ID / g, shoulder bone uptake at 60 minutes: 4.0% ID / g.

[0577] compound 18 F-2: Peak uptake: 5.7% ID / g, peak uptake ratio / 30 minutes: 10.9, brain retention at 60 minutes: 0.6% ID / g, shoulder bone uptake at 60 minutes: 6.2% ID / g.

[0578] compound 18 F-3a: Peak uptake: 4.4% ID / g, peak uptake ratio / 30 min: 11.2, brain retention at 60 min: 0.3% ID / g, shoulder bone uptake at 60 min: not detected.

[0579] Peak uptake in the brain was set to 100%, and washout curves were generated to assess the clearance rate of active substances from normal brain. Figure 2 ).

[0580] Human imaging research

[0581] In clinical trials, individuals with AD or PSP, as well as non-dementia controls (NDC), received a 370 MBq bolus injection. 18 Dynamic PET imaging of F-3a lasted 3 hours.

[0582] result

[0583] Initial imaging data showed strong brain uptake and rapid washout in non-target areas. No increased uptake was observed in the NDC in the choroid plexus, basal ganglia, striatum, amygdala, meninges, or other areas noted with other tau reagents. Figure 4 a). 18 F-3a demonstrates good brain uptake and rapid washout from non-target areas (see...). Figure 3In AD, focal asymmetric uptake is evident in the temporal, parietal, and frontal lobes. Figure 4 b). Finally, PSP individuals exhibited focal increases in uptake in the globus pallidus and substantia nigra (b). Figure 5 a&b).

[0584] Table 1: Summary of Preclinical Characteristics

[0585]

[0586] -Poor, ○Moderate, +Good, ++Excellent, +++Outstanding

[0587] a) In-house data, see the experimental section above;

[0588] b) Determined using non-radioactive fluorine-19 derivatives F-1, F-2, and F-3a;

[0589] c) Using radioactive fluorine-18 derivatives 18 F-1, 18 F-2 and 18 F-3a determination;

[0590] d) Marquie et al. 2015;

[0591] e)WO2015 / 052105;

[0592] f) No defluorination detected

[0593] e)Honer et al., Human Amyloid Imaging Meeting 2017;

[0594] NA: Not obtained.

[0595] As can be seen from Table 1, the compounds in the prior art 18 F-1 and 18 The F-2 has limitations, particularly in the following aspects:

[0596] • Low binding to Tau isotypes in non-AD Tau lesions

[0597] • High affinity for MAO A, and therefore low selectivity for Tau.

[0598] • It does not have a low signal in a healthy brain.

[0599] • In a healthy brain, there is no rapid cleansing.

[0600] • Long-term retention in a healthy brain, and / or

[0601] • Defluorination in vivo.

[0602] On the other hand, compounds 18 F-3a display:

[0603] ·Specific binding to AD and Non-AD tau lesion brain slices (Example: with compounds) 18 F-1 and 18 Compared to the F-2 report, there are stronger signals for the PSP and PiD.

[0604] • The affinity for MAO A in whole mouse brain homogenate is low (IC50). 50 Compared to compounds 18 F-1 High 64 times IC 50 Compared to compounds 18 F-2 High 3 times ),

[0605] • Low affinity for MAO A in HC brain homogenate (IC50) 50 Compared to compounds 18 F-1 High 220 times IC 50 Compared to compounds 18 F-2 11 times higher ),

[0606] • HC brain homogenate has a low affinity for MAO B (IC50). 50 Compared to compounds 18 F-1 High 5 times ),

[0607] • Higher signal-to-noise ratio, determined by comparing the binding in AD brain homogenate and HC brain homogenate (compared to...). 18 The F-1 ratio is high 5.2 times ,

[0608] • Higher signal-to-noise ratio, determined by further comparison of binding in AD brain homogenate and HC brain homogenate (compared to...). 18 The F-1 ratio is 8.2- 13.5 times, compared to 18 The F-2 ratio is high 4.0 times to 4.9 times ),

[0609] • Higher signal-to-noise ratio, determined by comparing the binding in AD brain homogenate with that in whole mouse brain homogenate (compared to...). 18 The F-1 ratio is high 4.2 times,

[0610] • Faster removal from a healthy brain (than compounds) 18 F-1 Fast 1.6 times ),

[0611] • Lower long-term retention in the healthy brains of mice (compared to the compound) 18 F-1 Low 2.7 times And more than compounds 18 F-2 Low 2 times ),

[0612] • No defluorination (with compound) in mice 18 F-1 has 4.0% ID / g and compounds 18 Compared to F-2, which has an ID / g of 6.2%, none Bone uptake).

[0613] At least due to its high affinity for Tau, its rapid brain washout, lower long-term retention in the healthy brain, and / or lower binding affinity to other brain targets, compared with compounds in the prior art... 18 F-1 and 18 Compared to F-2, Tau deposits and compounds in the brain were identified and quantified using positron emission tomography. 18 F-3a exhibits significantly better properties. In addition to the detection and quantification of Tau sediments in AD, the compounds... 18 F-3a can be used for clinical assessment of non-AD Tau lesions.

[0614] It has already been confirmed in human individuals. 18 Favorable preclinical characteristics of F-3a. 18 F-3a demonstrates good brain uptake and rapid washout from non-target brain regions (see [link]). Figure 3 ).

[0615] The uptake patterns observed in individuals with AD and PSP are consistent with the expected patterns in Tau pathology. Figure 4 and Figure 5 ).

[0616] Surprisingly, 3a / 18 F-3a shows significant advantages over its regional isomers in terms of key characteristics of Tau PET imaging tracers (Table 2).

[0617] For comparative example 6 / 18 F-6 and 10 / 18 F-10 has a weak affinity for Tau, compared to Comparative Example 5 / 18 F-5, 7 / 18 F-7 and 9 / 18 F-9 has poor binding affinity for Tau (IC50) as measured in AD brain homogenate. 50 ).

[0618] For comparative example 2 / 18 F-2, 7 / 18F-7 and 8 / 18 F discovered selectivity for the difference between MAO and A.

[0619] Comparative Examples under Standard Conditions 18 F-5, 18 F-6 18 F-8 18 F-9 and 18 The radiolabeling of F-10 is poor (or failed).

[0620] For comparative embodiments 18 F-10 mice were found to have poor brain uptake.

[0621] For comparative embodiments 18 F-5, 18 F-7 and 18 F-10 was poorly washed out of the brains of healthy mice.

[0622] For comparative embodiments 18 F-2, 18 F-5, 18 F-7 and 18 F-10 was found to be defluorinated in mice.

[0623] Table 2: Comparison of regioisomers

[0624]

[0625] -Poor, ○Moderate, +Good, ++Excellent, +++Outstanding

[0626] a) Internal data, see the experimental section above;

[0627] b) Determined using the corresponding non-radioactive fluorine-19 derivative;

[0628] c) Using radioactive fluorine-18 derivatives 18 F-1, 18 F-2 and 18 F-3a determination;

[0629] d) IC50 in mouse brain homogenate 50

[0630] f) No defluorination detected

[0631] NA: Not obtained.

Claims

1. Compounds of formula (II) and their pharmaceutically usable salts; in R 1 It's LG; R 2 Is it H or PG; PG is tert-butoxycarbonyl (BOC), triphenylmethyl (Trityl), or dimethoxytriphenylmethyl (DMT); LG stands for nitro, halogen, or trimethylammonium.

2. The compound of claim 1, wherein it is 3. The compound of claim 1, wherein it is 4. The compound according to any one of claims 1-3, wherein R 1 It is LG and R 2 It is H or PG.

5. The compound according to any one of claims 1-3, wherein R 1 It is LG and R 2 It is H.

6. The compound according to any one of claims 1-3, wherein R 1 It is LG and R 2 It's PG.

7. The compound according to any one of claims 1-3, wherein LG is nitro or trimethylammonium.

8. The compound according to any one of claims 1-3, wherein PG is tert-butoxycarbonyl (BOC).

9. A method for preparing a compound having formula (II-1) and its pharmaceutically acceptable salt, in R 1 yes 18 F; R 2 It is H; It includes making the compound defined in any one of claims 1-8 with [ 18 The reaction of the fluorinating agent, wherein the method further includes the cleavage of the protecting group PG, if present.

10. A kit for preparing radiopharmaceutical articles, the kit comprising a sealed vial containing a predetermined amount of a compound as defined in any one of claims 1-8.

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