BR112020014949B1 - METHOD FOR PREPARING AN IMAGE FORMING COMPOUND - Google Patents

Abstract

The present invention relates to a method of preparing a compound of formula I. Diagnostic compositions and their use in the selective detection of disorders and abnormalities associated with Tau aggregates such as Alzheimer's disease (AD) and other tauopathies, for example, using Positron Emission Tomography (PET) image formation are also shown.

Description

Field of Invention

[0001] The present invention relates to a new method of preparing a compound of Formula I, which can be used in the selective detection of disorders and abnormalities associated with Tau aggregates such as Alzheimer's disease (AD) and other tauopathies, e.g. , using positron emission tomography (PET) imaging. A diagnostic composition comprising the compound obtained as well as its use in diagnostics and image formation are also the subject matter of the present patent application.

Background

[0002] Alzheimer's disease is a neurological disorder primarily thought to be caused by amyloid plaques, an extracellular accumulation of abnormal deposits of amyloid beta (Aβ) aggregates in the brain or eyes. The other main neuropathological hallmarks in AD are intracellular neurofibrillary tangles (NFT) that originate through aggregation of hyperphosphorylated Tau protein (tubulin-associated unit), phosphorylated Tau or pathological Tau and its conformers. AD shares this pathology with many neurodegenerative tauopathies, in particular with specific types of frontotemporal dementia (FTD). In AD brain, TAU pathology (tauopathy) develops after amyloid pathology, but it is still controversially discussed whether Aβ protein is the causative agent in AD which constitutes the essence of the so-called amyloid cascade hypothesis (Hardy et al., Science 1992 , 256, 184-185, and most recently, Musiek et al., Nature Neurosciences 2015, 18(6), 800-806, "Three dimensions of the amyloid hypothesis: time, space and 'wingmen'").

[0003] At present, the only defined path to diagnosing AD is to identify plaques and tangles in brain tissue through histological analyzes of biopsy or autopsy materials after the individual's death. In addition to AD, Tau plays an important role in other neurodegenerative diseases (non-AD). Such non-AD tauopathies include, for example, supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal degeneration (CBD).

[0004] The compound of Generic Formula A has been proposed to be useful in the selective detection of disorders and abnormalities associated with Tau aggregates such as Alzheimer's disease (AD) and other tauopathies, and certain methods of manufacturing this compound have been described in the prior art.

[0005] Gobbi et al. showed in WO 2015/052105 a method in which a compound of Formula A was obtained by reacting a precursor having a nitro group instead of 18F with [18F] fluoride in a microwave apparatus. After the synthesis step, the compound of Formula A was purified through a two-step procedure involving, among others:

[0006] 1) HPLC using a mobile phase of methanol and triethylamine; It is

[0007] 2) reformulation using a solid phase extraction cartridge to entrain the compound of Formula A and subsequent elution from the cartridge with ethanol diluted with saline (0.9% NaCl in water).

[0008] The method provided the compound of Formula A in a yield of 26.1%.

[0009] Gobbi et al. further showed the following two methods in J. Med. Chem. 2017, Volume 60, pages 7350 to 7370:

[0010] a) Microwave method: [18F]fluoride was drawn over a cation exchange cartridge and the activity was eluted with a mixture of Kryptofix/potassium carbonate. The mixture was dried at elevated temperature after addition of acetonitrile. The vial with the dried [18F]fluoride mixture was then transferred to a microwave apparatus and 0.5 mg of precursor molecule (unprotected nitro derivative) in 400 μL of DMSO was added. The vial was microwave irradiated at 50 W for 240 seconds. The resulting solution was diluted and purified by preparative HPLC (C-18 column, acetonitrile/trimethyl amine buffer at pH 7.2). The product spike was collected, diluted with water and passed through a C-18 Sep-Pak Plus cartridge. The cartridge was washed with water and the product labeled - 18F was eluted with ethanol and diluted with saline.

[0011] b) Thermal heating method: [18F]fluoride was drawn over a cation exchange cartridge and the activity was eluted with a mixture of Kryptofix/potassium carbonate. The mixture was dried at elevated temperature after addition of acetonitrile. 0.5 mg of precursor molecule (unprotected nitro derivative) in 400 μL of DMSO was added and the solution was heated at 160°C for 10 minutes. The resulting solution was diluted and purified by preparative HPLC (C-18 column, acetonitrile/trimethylamine buffer pH 7.2).

[0012] The product peak was collected, diluted with water and passed through a C-18 Sep-Pak Plus cartridge. The cartridge was washed with water and the product labeled 18F was eluted with ethanol and diluted with saline.

[0013] Thus, the methods for preparing the compound of Formula A in the aforementioned prior art have two purification/reformulation steps, where the method is only partially automated in the case of microwave methods as illustrated in Figure 1.

[0014] The nitro precursor (and main by-product) of the method described in the prior art has poor solubility especially in ethanol, acetonitrile and the acetonitrile/aqueous buffer mixtures used for purification (preparative HPLC), especially if aqueous buffer mixtures with neutral pH are used. Only 0.5 to 0.7 mg of the precursor was used in the prior art examples.

[0015] It is an object of the present invention to provide an improved method for preparing a compound of Formula I that is more cost and time efficient than prior art methods (as outlined in Figure 2). In addition, the yield of the method must be improved.

Description of Figures

[0016] Figure 1: Schematic overview of the prior art method chosen by Gobbi et al.

[0017] Figure 2: Schematic overview of the method of the present invention.

[0018] Figure 3: Assembly of the GE Tracerlab FX synthesizer

[0019] Figure 4: Assembling the IBA Synthera synthesizer

Summary of the Invention

[0020] The present invention refers to the following items:

[0021] 1. A method of preparing a compound of Formula I understanding the steps of

[0022] A) react a compound of Formula II with an 18F fluorinating agent where X is H or PG, LG is a leaving group, and PG is an amine protecting group, and

[0023] B) optionally, if X is PG, PG protecting group cleavage, and

[0024] C) subjecting the resulting compound of Formula I to high-performance liquid chromatography (HPLC) using a mobile phase comprising ethanol and water.

[0025] 2. The method according to item 1, where X is PG, step B is absent and the PG protecting group is cleaved in step A.

[0026] 3. The method according to item 1, where X is PG and the PG protecting group is cleaved in step B.

[0027] 4. The method according to any one of items 1 to 3, wherein the ratio of ethanol to water in the mobile phase is from about 5/95 v/v to about 80/20 v/v.

[0028] 5. The method according to any one of items 1 to 4, wherein the pH of the mobile phase is from about 0 to about 8, preferably about 1 to about 3, more preferably about 2 to about of 3.0, even more preferably about 2.2 to about 2.8.

[0029] 6. Method according to any one of items 1 to 5, wherein the mobile phase further comprises a buffer, which is preferably selected from alkali metal dihydrogen phosphates, dialkali metal hydrogen phosphates, trialkaline metal phosphates, alkali metal acetates, alkaline earth metal acetates, alkaline earth metal formates, mono/di/trialkaline metal citrates.

[0030] 7. The method according to any one of items 1 to 6, wherein high performance liquid chromatography (HPLC) is conducted under a pressure of about 50 to about 400 bar, preferably about 50 to about 250 bar.

[0031] 8. The method according to any one of items 1 to 7, wherein the method is an automated method, in which Step A, optional Step B, and Step C are performed on an automated synthesizer.

[0032] 9. The method according to any one of items 1 to 8, wherein high performance liquid chromatography (HPLC) results in a fraction comprising the compound of Formula I and this fraction is subjected to a filtration Step D) sterile.

[0033] 10. The method according to any one of items 1 to 9, where the method does not comprise solid phase extraction of the compound of Formula I after step C, preferably where the method does not comprise solid phase extraction of the compound of Formula I before or after step C.

[0034] 11. The method according to any one of items 1 to 10, wherein the compound of Formula I is not subjected to chromatography after high-performance liquid chromatography (HPLC) of step C, preferably where the compound of Formula I is not is subjected to chromatography other than step C high-performance liquid chromatography (HPLC).

[0035] 12. A diagnostic composition comprising a compound of Formula I which is obtainable by a process according to any one of items 1 to 12 and optionally a diagnostically acceptable carrier, diluent, adjuvant or excipient.

[0036] 13. The composition according to item 12 for use in diagnostics.

[0037] 14. The composition according to has 12 for use in imaging Tau aggregates, particularly for use in positron emission tomography imaging of Tau aggregates.

[0038] 15. The composition for use according to item 13 or 14, where the disorder is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann disease - Straussler - Scheinker, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick type C disease, pallido-ponto-nigral degeneration, Pick (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau ), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, traumatic stress disorder, epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical Parkinsonism, HIV-related dementia, adult-onset diabetes , senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve ganglia, optic neuropathy, optic neuritis, and regular dystrophy; preferably Alzheimer's disease.

[0039] 16. The composition for use according to item 13 or 14, where the disorder is selected from Huntington's disease, ischemic stroke and psychosis in AD.

[0040] 17. The composition for use according to any one of items 13 to 16, where the composition is to be administered by injection.

[0041] 18. A composition as defined in item 12 for use in diagnosing a disorder associated with Tau aggregates or for use in diagnosing a tauopathy, particularly where the diagnosis is conducted by positron emission tomography.

[0042] 19. A composition for use according to item 18, wherein the tauopathy is a 3R tauopathy.

[0043] 20. A composition for use according to item 18, wherein the tauopathy is a 4R tauopathy.

[0044] 21. The composition for use according to item 18, where the disorder is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann - Straussler disease - Scheinker, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease , corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick's disease ( PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, post-encephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, traumatic tension syndrome , epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical Parkinsonism, HIV-related dementia, adult-onset diabetes, amyloidosis senile heart disease, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve ganglia, optic neuropathy, optic neuritis, and regular disposition dystrophy; preferably Alzheimer's disease.

[0045] 22. The composition for use according to item 18, where the disorder is selected from Huntington's disease, ischemic stroke and psychosis in AD.

[0046] 23. The composition for use according to item 21, where the disorder is Alzheimer's disease (AD).

[0047] 24. The composition for use according to item 21, where the disorder is Parkinson's disease or atypical Parkinsonism.

[0048] 25. The composition for use according to item 21, where the disorder is progressive supranuclear palsy (PSP).

[0049] 26. The composition for use according to item 21, where the disorder is Pick's disease (PiD).

[0050] 27. The composition for use according to any one of items 18 to 26, wherein the Tau aggregates are imaged in the brain or eye.

[0051] 28. A method of imaging Tau aggregates, particularly a method of imaging positron emission tomography of Tau aggregates, wherein an effective amount of a composition as defined in item 12 is administered to a patient.

[0052] 29. A method of diagnosing a disorder associated with Tau aggregates or a tauopathy, wherein an effective amount of a composition as defined in item 12 is administered to a patient, particularly where the diagnosis is conducted by positron emission tomography .

[0053] 30. A method according to item 29, wherein the tauopathy is a 3R tauopathy.

[0054] 31. A method according to item 29, wherein the tauopathy is a 4R tauopathy.

[0055] 32. The method according to item 29, where the disorder is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann - Straussler - Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2 , chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, traumatic tension syndrome, epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical Parkinsonism, HIV-related dementia, adult-onset diabetes, senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve ganglia, optic neuropathy, optic neuritis, and regular disposition dystrophy; preferably Alzheimer's disease.

[0056] 33. The method according to item 29, wherein the disorder is selected from Huntington's disease, ischemic stroke and psychosis in AD.

[0057] 34. The method according to item 32, where the disorder is Alzheimer's disease (AD).

[0058] 35. The method according to item 32, where the disorder is Parkinson's disease or atypical Parkinsonism.

[0059] 36. The method according to item 32, where the disorder is progressive supranuclear palsy (PSP).

[0060] 37. The method according to item 32, where the disorder is Pick's disease (PiD).

[0061] 38. The method according to any one of items 28 to 37, wherein Tau aggregates are imaged in the brain or eye.

[0062] 39. Use of a composition as defined in item 12 for the manufacture of a diagnostic agent for imaging Tau aggregates, particularly for positron emission tomography imaging of Tau aggregates.

[0063] 40. Use of a composition as defined in item 12 for the manufacture of a diagnostic agent for diagnosing a disorder associated with Tau aggregates or for diagnosing a tauopathy, particularly where the diagnosis is conducted by positron emission tomography.

[0064] 41. The use according to item 40, where the tauopathy is a 3R tauopathy.

[0065] 42. The use according to item 40, where the tauopathy is a 4R tauopathy.

[0066] 43. Use in accordance with item 40, where the disorder is selected from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann disease - Straussler - Scheinker , inclusion body myositis, cerebral prion protein amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease, degeneration corticobasal (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick's disease (PiD) , progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, traumatic tension syndrome, epilepsy , Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical Parkinsonism, HIV-related dementia, adult-onset diabetes, senile cardiac amyloidosis , endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic nerve ganglia, optic neuropathy, optic neuritis, and regular disposition dystrophy; preferably Alzheimer's disease.

[0067] 44. The use according to item 40, where the disorder is selected from Huntington's disease, ischemic stroke and psychosis in AD.

[0068] 45. The use according to item 43, where the disorder is Alzheimer's disease (AD).

[0069] 46. Use according to item 43, where the disorder is Parkinson's disease or atypical parkinsonism.

[0070] 47. Use in accordance with item 43, where the disorder is progressive supranuclear palsy (PSP).

[0071] 48. The use according to item 43, where the disorder is Pick's disease (PiD).

[0072] 49. The use according to any of items 39 to 48, where Tau aggregates are imaged in the brain or eye.

[0073] 50. Use of the composition according to item 12 as an analytical reference.

[0074] 51. Use of the composition according to item 12 as an in vitro selection tool.

[0075] 52. A data collection method for diagnosing a disorder associated with tau aggregates in a sample or a patient comprising:

[0076] (a) placing a sample or a specific body part or body area suspected of containing a tau aggregate in contact with a composition as defined in item 12 that contains the compound of Formula I;

[0077] (b) allowing compound of Formula I to bind to the tau aggregate;

[0078] (c) detection of compound of Formula I bound to the tau aggregate; It is

[0079] (d) optionally correlating the presence or absence of compound of Formula I binding with the tau aggregate with the presence or absence of tau aggregate in the sample or specific body part or body area.

[0080] 53. A method of determining the amount of tau aggregate in a tissue and/or a body fluid comprising:

[0081] (a) providing a representative sample of the tissue and/or body fluid under investigation;

[0082] (b) sample test for the presence of tau aggregate with the composition as defined in item 12 that contains the compound of Formula I;

[0083] (c) determining the amount of compound of Formula I bound to the tau aggregate; It is

[0084] (d) calculating the amount of tau aggregate in the tissue and/or body fluid.

[0085] 54. A data collection method for determining a predisposition to a disorder associated with tau aggregates in a patient comprising detecting specific binding of a composition as defined in item 12, which contains the compound of Formula I, to a tau aggregate in a sample or in situ comprising the steps of:

[0086] (a) placing the sample or a specific body part or body area suspected of containing the tau aggregate in contact with the composition as defined in item 12, which contains a compound of Formula I that specifically binds to the tau aggregate;

[0087] (b) allowing the compound of Formula I to bind to the tau aggregate to form a compound/tau aggregate complex;

[0088] (c) detect the formation of the tau compound/aggregate complex;

[0089] (d) optionally correlating the presence or absence of the tau compound/aggregate complex with the presence or absence of tau aggregate in the sample or specific body part or body area; It is

[0090] (e) optionally comparing the amount of tau compound/aggregate to a normal control value.

[0091] 55. A data collection method for monitoring residual disorder in a patient suffering from a disorder associated with tau aggregates who has been treated with a medication, wherein the method comprises:

[0092] (a) placing a sample or specific body part or area of the body suspected of containing tau aggregate in contact with a composition as defined in item 12, which contains a compound of Formula I that specifically binds to the tau aggregate;

[0093] (b) allowing the compound of Formula I to bind to the tau aggregate to form a compound/tau aggregate complex;

[0094] (c) detect the formation of the tau compound/aggregate complex;

[0095] (d) optionally correlating the presence or absence of the tau compound/aggregate complex with the presence or absence of tau aggregate in the sample or specific body part or body area; It is

[0096] (e) optionally comparing the amount of tau compound/aggregate to a normal control value.

[0097] 56. A data collection method for predicting responsiveness of a patient suffering from a disorder associated with tau aggregates and being treated with a medication comprising:

[0098] (a) placing a sample or specific body part or area of the body suspected of containing tau aggregate in contact with a composition as defined in item 12, which contains a compound of Formula I that specifically binds to the tau aggregate;

[0099] (b) allowing the compound of Formula I to bind to the tau aggregate to form a compound/tau aggregate complex;

[00100] (c) detect the formation of the tau compound/aggregate complex;

[00101] (d) optionally correlating the presence or absence of the tau compound/aggregate complex with the presence or absence of tau aggregate in the sample or specific body part or body area; It is

[00102] (e) optionally comparing the amount of tau compound/aggregate to a normal control value.

Definitions

[00103] The term "alkyl" refers to a saturated branched or straight carbon chain, which, unless otherwise specified, contains 1 to 6 carbon atoms.

[00104] "Hal" or "halogen" represents F, Cl, Br and I. Preferably, "halogen" is, independently in each occurrence, selected from f, Cl and Br, more preferably, from F and Cl, even more preferably F.

[00105] The term "amine protecting group" (PG) as used herein is any group that is suitable for protecting an amine group during an intended chemical reaction. Examples of suitable protecting groups are well known to those skilled in the art. Appropriate protective groups are discussed, for example, in Greene and Wuts' textbook, Protecting groups in Organic Synthesis, third edition, pages 494-653, which is incorporated herein by reference. Protecting groups can be chosen from carbamates, amides, imides, N-alkyl amines, N-aryl amines, imines, enamines, boranes, N-P, N-sulfenyl, N-sulfonyl and N-silyl protecting groups. Specific examples of protecting groups (PG) are carbo benzyloxy (Cbz), (p-methoxy benzyl) oxycarbonyl (Moz or MeOZ), t-butyloxy carbonyl (BOC), 9-fluorenyl methyloxy carbonyl (FMOC), benzyl (Bn) , p-methoxy benzyl (PMB), 3,4-dimethoxy benzyl (DMPM), p-methoxy phenyl (PMP), triphenyl methyl (trityl), methoxy phenyl diphenyl methyl (MMT), or dimethoxy trityl (DMT). More preferred examples of the protecting group (PG) include t-butyloxy carbonyl (BOC), dimethoxy trityl (DMT) and triphenyl methyl (Trityl). A more preferred example of the protecting group (PG) is t-butyloxy carbonyl (BOC).

[00106] The term "amine carbamate protecting group" refers to an amine protecting group containing a *-CO-O group where the asterisk indicates the amine bond. Examples are carbo benzyloxy (Cbz), (p-methoxy benzyl) oxycarbonyl (Moz or MeOZ), t-butyloxy carbonyl (BOC), and 9-fluorenyl methyloxy carbonyl (FMOC).

[00107] The term "leaving group" (LG) as used herein is any leaving group and means that an atom or group of atoms can be replaced by another atom or group of atoms. Examples are given in Synthesis (1982), p. 85-125, table 2, Carey and Sundberg, Organische Synthese, (1995), pages 279-281, table 5.8; or Netscher, Recent Res. Dev. Org. Chem., 2003, 7, 71-83, schemes 1, 2, 10 and 15 and others). (Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), in: Schubiger P.A., Friebe M., Lehmann L., (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp.1550, explicitly: scheme 4 pp. 25, scheme 5 pp 28, table 4 pp 30, Figure 7 pp 33). Preferably, the "leaving group" (LG) is selected from the group consisting of nitro, bromine, iodine, chlorine, trialkyl ammonium, hydroxyl, boronic acid, iodonium, sulfonic ester. More preferably, the "leaving group" (LG) is nitro or trimethyl ammonium. It is to be understood that compounds containing trialkyl ammonium or iodonium may still comprise an anion. Even more preferably, "leaving group" (LG) is nitro. Another more preferred "leaving group" (LG) is trimethyl ammonium.

[00108] The term "crown ether" as used herein means chemical compounds consisting of a ring containing several ether groups. More specifically, the term "crown ether" preferably refers to monocyclic organic groups that can be substituted and contain from 8 to 16 carbon atoms and from 4 to 8 heteroatoms selected from N, O and S in the ring. Each of the one or more optional substituents may be independently selected from any organic group containing from 1 to 15 carbon atoms and optionally 1 to 6 heteroatoms selected from N, O, and S. Preferred examples of the "crown ether" are optionally monocyclic rings. substituted containing 10 to 14 carbon atoms and 5 to 7 heteroatoms selected from N, O and S in the ring. Examples of the "crown ether" are optionally substituted monocyclic rings containing 12 carbon atoms and 6 heteroatoms selected from N and O in the ring. Specific examples include 18-crown-6, dibenzo-18-crown-6, and diaza-18-crown-6.

[00109] The term "cryptand" as used here refers to a class of polycyclic compounds related to crown ethers, having three chains linked to two nitrogen atoms. A well-known "cryptand" is 4, 7, 13, 16, 21, 24-hexaoxa-1,10-diaza bicyclo[8.8.8]hexacosane (Kryptofix).

[00110] Tau as used herein refers to a highly soluble microtubule-binding protein found in neurons and includes the 6 main isoforms, cleaved or truncated forms, and other modified forms such as arising from phosphorylation, glycosylation, glycation, prolyl isomerization, nitration, acetylation, polyamination, ubiquitination, sumoylation, and oxidation. Pathological Tau or Tau aggregates (Neurofibrillary Tangles, NFTs) as used herein refer to insoluble aggregates of hyperphosphorylated Tau protein containing paired helical filaments and straight filaments. Its presence is a hallmark of AD and other diseases known as tauopathies.

[00111] The tau gene contains 16 exons with the main tau protein isoforms being encoded by 11 of them. The alternative exon 10 splice generates tau isoforms with either three (missing exon 10) or four (exon 10 present) repeat domains, known as tau 3R and 4R, respectively (A. Andreadis et al., Biochemistry 31, (1992 ) 10626 - 10633; M. Tolnay et al., IUBMB Life, 55(6): 299305). In Alzheimer's disease, the ratio of 3R and 4R isoforms is similar. In contrast, in some tauopathies one of the two isoforms is predominantly present. Here, the term "3R tauopathy" refers to tauopathies (such as Pick's disease (PiD)) in which the 3R isoform is predominantly present. Here, the term "4R tauopathy" refers to tauopathies (such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD)) where the 4R isoform is predominantly present.

[00112] As used hereinafter in the description of the invention and in the claims, the term "pharmaceutically acceptable salt" refers to non-toxic derivatives of the compounds shown where the parent compound is modified by obtaining salts of its inorganic and organic acids. Inorganic acids include, but are not limited to, acids such as hydrochloric, nitric or sulfuric acid. Organic acids include, but are not limited to, carboxylic and sulfonic acids such as aliphatic, cycloaliphatic, aromatic, araliphatic and heterocyclic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two. Lists of appropriate salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, the exposition of which is incorporated herein by reference.

[00113] "Pharmaceutically acceptable" or "diagnostically acceptable" are defined as referring to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, appropriate for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problem or complication compatible with a reasonable benefit/risk ratio.

[00114] Patients or subjects in the present invention are typically animals, particularly mammals, more particularly humans.

[00115] "Chromatography" or "liquid chromatography" means a method for separating a mixture of compounds. The mixture is dissolved in a fluid and transported via a "mobile phase" through a "stationary phase". Separation is based on the interaction of compounds in the mobile phase with the stationary phases. Such different interactions result in differential retention on the stationary phase and thus affect separation. Chromatography can be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture, and is thus a form of purification. Analytical chromatography is done with a small sample of material and is used to measure the proportions of compounds in a mixture.

[00116] "High-performance liquid chromatography (HPLC)" is a form of liquid chromatography for separating compounds through the use of very small stationary phase particles (< 10 μm) and application of sufficiently high pressures. An HPLC system typically consists of a reservoir of mobile phase(s), a pump, an injector, a separation column (containing the stationary phase), and detectors. For separation of radioactive compounds, suitable HPLC systems are equipped with a radioactivity detector. Optionally, the HPLC system has additional detectors, such as, for example, UV, photodiode array, refractive index, conductivity, fluorescence, mass spectrometer.

[00117] "Solid phase extraction (SPE)" is a sample preparation and/or purification process with two or more separate steps. First, the compounds are dissolved or suspended in a liquid mixture of solvents and the liquid sample is passed through a stationary (solid) phase. Some compounds are retained on the stationary phase while others pass through. In the second step, the retained compounds are eluted with an appropriate solvent. Optionally, the stationary phase is washed with another solution before the elution step. In contrast to the HPLC technique, the particle size used is much larger (e.g. > 25 μm compared to HPLC with a typical particle size of < 10 μm) and therefore the applied pressure is much lower (for HPLC a pressure is typically > 50 bar).

[00118] "Solid phase extraction cartridge (SPE cartridge)" is a syringe or container (e.g. Sep Pak) pre-filled with the stationary phase for SPE.

[00119] "Sterile filtration" is a method for sterilizing a solution through filtration via a microfilter. A microfilter is a filter having, for example, a pore size of about 0.25 μm or less, preferably about 20 nm to about 0.22 μm, which is commonly used for removing microorganisms. Membrane filters used in microfiltration in production processes are commonly manufactured from materials such as blended cellulose ester, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), or polyether sulfone (PES).

[00120] "Automated" used here means the conduction of synthesis or purification steps using appropriate equipment (synthesizer).

[00121] The term "radiocleaner" refers to a compound that decreases the rate of decomposition due to radiolysis. Preferred radiocleaners include ascorbic acid and its salts and gentisic acid and its salts. Most preferred radiocleaners are ascorbic acid, sodium ascorbate and mixtures thereof.

[00122] Suitable "synthesizers" for 18F-radiolabeling are well known to those skilled in the art including but not limited to IBA Synthera, GE Fastlab, GE Tracerlab MX, GE Tracerlab FX, GE Tracerlab FX, Trasis AllinOne, ORA Neptis Perform, ORA Neptis Mosaic, ORA Neptis Plug, Scintomics GPR, Synthera, Starter Taddeo, Raytest Synchrom, Sofie Elixys, Eckert&Ziegler Modular Lab, Sumitomo Heavy Industries F100 F200 F300, Siemens Explora.

[00123] "Radiochemical purity" means what proportion of the total activity of the radionuclide present is in its established chemical form. Typically, radiochemical purity is determined by thin layer chromatography or HPLC.

[00124] The preferred definitions given in section - "Definitions" are applied to all embodiments described herein unless otherwise stated.

The Methods of the Present Invention

[00125] In a first aspect, the present invention is directed to a method for preparing a compound of Formula I

[00126] This method comprises the steps of:

[00127] A) react a compound of Formula II with an 18F fluorinating agent where X is H or PG, LG is a leaving group, and PG is an amine protecting group, and

[00128] B) optionally, if X is PG, PG protecting group cleavage, and

[00129] C) subjecting the resulting compound of Formula I to high-performance liquid chromatography (HPLC) using a mobile phase comprising a mixture of ethanol and water.

[00130] Preferred compounds of Formula I are selected from the group consisting of

[00131] A more preferred compound of Formula I is

[00132] Preferred compounds of Formula II are selected from the group consisting of

[00133] Most preferred compounds of Formula II are selected from the group consisting of

[00134] In these compounds, PG and LG are as defined in the "Definitions" section.

[00135] Even more preferred compounds of Formula II are selected from the group consisting of

[00136] With X being a counterion such as a counterion selected from the group consisting of halogen, CF3SO3-, and CF3CO2-. Step A

[00137] Step A comprises reaction of a compound of Formula II with an 18F fluorinating agent where X is H or PG, LG is a leaving group, and PG is an amine protecting group

[00138] If X is H a compound having Formula I will result. If X is PG an intermediate product having Formula III will be obtained.

[00139] 18F fluorinating agents are well known to those skilled in the art. Any appropriate 18F-fluorinating agent may be employed. Typical examples include H18F, alkaline fluorides-18F, or alkaline earth fluorides (e.g., K18F, Rb18F, Cs18F, and Na18F). Optionally, the 18F-fluorinating agent can be used in combination with a chelating agent such as a cryptand (e.g., 4,7,13,16,21,24-hexaoxa-1,10-diaza bicyclo[8.8.8] -hexacosane - Kryptofix) or a crown ether (e.g. 18-crown-6). Alternatively, the 18F-fluorinating agent may be a 18F tetra alkyl ammonium salt or an 18F tetra alkyl phosphonium salt; for example, tetra (C1-6 alkyl) ammonium salt of 18F or a tetra (C1-6 alkyl) phosphonium salt of 18F. Its examples include [18F}tetrabutylammonium fluoride and [18F}tetrabutylphosphoniumfluoride. Preferably, the 18F-fluorinating agent is K18F, H18F, Cs18F, Na18F or [18F]tetrabutylammonium fluoride. Even in a more preferred embodiment, the 18F-fluorinating agent is K18F. In another more preferred embodiment, the 18F-fluorinating agent is [18F]tetrabutylammonium fluoride.

[00140] 18F-fluorination is typically carried out in a solvent that is preferably selected from acetonitrile, dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide, amyl alcohol, t-butyl alcohol, or mixtures thereof, preferably the solvent contains or is acetonitrile or DMSO. But also other solvents can be used which are well known to those skilled in the art. The solvent may further comprise water and/or other alcohols, such as linear, branched or cyclic C1-10 alkanols, as a co-solvent. In a preferred embodiment, the solvent for carrying out 18F radiolabeling contains dimethyl sulfoxide. In another preferred embodiment, the solvent for carrying out 18F radiolabeling contains acetonitrile. In a preferred embodiment, the solvent for carrying out 18F radiolabeling is dimethyl sulfoxide. In another preferred embodiment, the solvent for carrying out 18F radiolabeling is acetonitrile.

[00141] 18F-fluorination is typically conducted for a maximum of about 60 minutes. Preferred reaction times are at most about 30 minutes. Still preferred reaction times are a maximum of about 15 minutes. Typical reaction times are about 1-15 minutes, preferably 5-15 minutes, more preferably 1015 minutes.

[00142] 18F-fluorination is typically carried out at a temperature of about 60 to about 200°C under conventional or microwave-supported heating. In a preferred embodiment, 18F-fluorination is carried out at about 100 to about 180°C. In a more preferred embodiment, 18F-fluorination is carried out at about 100 to about 160°C. Preferably, 18F-fluorination is carried out under conventional heating. Conventional heating is understood to be any heating without the use of microwaves.

[00143] The amount of starting material is not particularly limited. For example, about 0.5 to about 50 μmoles of a compound of Formula II can be used to produce the compound of Formula I in a batch. In a preferred embodiment, about 2 to about 25 micromoles of a compound of Formula II are used. In a more preferred embodiment, about 2.5 to about 15 micromoles of a compound of Formula II are used. In one embodiment, at least about 2 micromoles of a compound of Formula II are used. In a preferred embodiment, at least about 2.5 micromoles of a compound of Formula II are used. In a more preferred embodiment, at least about 3 micromoles of a compound of Formula II are used.

[00144] Typically about 0.5 to about 10 mg of Formula II can be used to produce the compound of Formula I in a batch. In a preferred embodiment, about 0.5 to about 5 mg of a compound of Formula II is used. In a more preferred embodiment, about 1 to about 3 mg of a compound of Formula II are used.

[00145] If X is PG an intermediate compound of Formula III will be obtained. The PG protecting group can either be cleaved during step A or in an optional subsequent step B.

[00146] Preferred compounds of Formula III are selected from the group comprising

[00147] In these compounds PG is as defined in the "Definitions" section. Step B

[00148] Step B is an optional step comprising cleavage of a PG protecting group from a compound of Formula III to obtain a compound of Formula I. As will be apparent to those skilled in the art, this step is not applicable if step A is conducted with a compound of Formula II where X is hydrogen or if the PG protecting group is already cleaved in step A.

[00149] Reaction conditions for the cleavage of a wide variety of protecting groups are well known to those skilled in the art and can be chosen from, but are not limited to, those described in the textbook by Greene and Wuts, Protecting groups in Organic Synthesis, third edition, pages 494-653, and the textbook by P. J. Kocienski, Protecting Groups, 3rd Edition 2003, both of which are incorporated herein by reference.

[00150] The conditions that are employed in step B will depend on the protecting group that is to be cleaved and are therefore not particularly limited.

[00151] Possible reaction conditions include i) heating to about 60 to about 160°C, ii) adding an acid and heating to about 0°C to about 160°C; or iii) adding a base and heating to about 0°C to about 160°C.

[00152] Preferred acids are hydrochloric acid, sulfuric acid, and phosphoric acid. A preferred acid is sulfuric acid. Another preferred acid is phosphoric acid. Another preferred acid is hydrochloric acid. Preferred bases are sodium hydroxide, potassium hydroxide.

[00153] A preferred reaction condition is addition of an acid and heating to about 25°C to 160°C, preferably 25°C to 120°C, more preferably 90-120°C. Preferably, the reaction mixture is heated for about 1 to about 20 minutes, more preferably for about 5 to about 15 minutes.

[00154] If desired, Steps A and B can be carried out in the same or different reaction vessels. Preferably, Steps A and B are carried out in the same reaction vessel.

[00155] If desired, the solution obtained after Step B can be used as such in Step C. Alternatively, the composition of the solution can be adapted so that it is more suitable for conducting HPLC. For example, a buffer or diluent may be added before Step C.

[00156] Preferred diluents are ethanol, water; or a combination thereof.

[00157] In addition, the pH of the solution can be adapted. In a preferred embodiment, the pH is adjusted to about 6 or less, more preferably about 4 or less, or even more preferably about 3 or less before Step C. In a preferred embodiment, the pH is adjusted to about 0 to about 6, more preferably about 0 to about 4, even more preferably about 1 to about 3, before Step C. Step C

[00158] Step C is a step in which the compound of Formula I obtained in Step A or, if employed, Step B, is subjected to HPLC using a mobile phase comprising ethanol and water.

[00159] In the prior art, it was assumed that it is necessary to employ two chromatographic steps in order to arrive at an injectable tracer, that is, first purification through HPLC and then new formulation through SPE of the compound of Formula I. The present The inventors have surprisingly discovered that if a mixture of ethanol and water is used as a mobile phase subsequent to the carryover of the compound via solid phase extraction can be omitted. Therefore, it is possible to use a single chromatographic step for purification and formulation. This is particularly important in the present case of radiofluorination, as 18F has a half-life of only about 110 minutes, so the speed of the method to arrive at an injectable formulation of compound I is of utmost importance.

[00160] Since the presently claimed method is faster and less complex than previous methods, the compound of Formula I can be obtained in higher yield corrected for non-decay.

[00161] The choice of a mixture of ethanol and water as the mobile phase has the additional advantage that these two components are diagnostically acceptable, so that (unlike methanol, acetonitrile, triethylamine, and trimethylamine that are used in the prior art) they may remain in the composition that is administered to the patient. Therefore, the choice of ethanol and water as the mobile phase significantly reduces the time and costs required in preparing the compound of Formula I and/or provides greater yields and greater radiochemical purity than prior art methods.

[00162] The method of the present invention preferably does not comprise solid phase extraction of the compound of Formula I after step C, more preferably the method of the present invention does not comprise solid phase extraction of the compound of Formula I before or after step C.

[00163] The compound of Formula I is preferably not subjected to chromatography after the high-performance liquid chromatography (HPLC) of step C, more preferably the compound of Formula I is not subjected to chromatography other than high-performance liquid chromatography (HPLC ) of step C.

[00164] The mobile phase used in the HPLC method comprises ethanol and water. Furthermore, an acid, a base, a buffer, a salt and/or a radiocleaner and optionally mixtures thereof may be contained.

[00165] The ratio of ethanol to water is not particularly limited but is preferably about 5/95 v/v to about 80/20 v/v, more preferably about 5/95 v/v to about 50/50 v/v v, even more preferably about 5/95 v/v to about 20/80 v/v.

[00166] The pH of the mobile phase is not restricted, but is preferably about 0 to about 8, preferably about 0 to about 6, more preferably about 1 to about 5, even more preferably about 1 to about 3, and even more preferably about 2.2 to about 2.8.

[00167] Possible buffers may include salts that may be selected from alkali metal dihydrogen phosphates, dialkali metal hydrogen phosphates, trialkali metal phosphates, alkali metal acetates, alkaline earth metal acetates, alkaline earth metal formates, citrate mono/di/tri alkali metal, with the preferred alkali and alkaline earth metals being sodium and potassium.

[00168] Possible bases may be sodium hydroxide and/or potassium hydroxide.

[00169] If desired, the pH of the mobile phase can be adjusted using an inorganic or organic acid. Examples of organic acids include ascorbic acid, citric acid, and acetic acid. Examples of inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid, preferably phosphoric acid.

[00170] Radiocleaners are compounds that reduce the decomposition of the compound of Formula I by radiolysis. Examples include ascorbic acid and ascorbic acid salts, gentisic acid and gentisic acid salts. Further examples include citric acid and citric acid salts. Most preferred radiocleaners are ascorbic acid, sodium ascorbate and mixtures thereof.

[00171] Preferably, the mobile phase comprises about 50 to about 500 mM buffer (e.g., alkaline dihydrogen phosphate), with a pH of about 1 to about 3, more preferably about 50 to about 50 mM buffer. about 250 mM (e.g., alkaline dihydrogen phosphate), with a pH of about 1 to about 3, even more preferably buffer of about 50 to about 150 mM (e.g., alkaline dihydrogen phosphate) , with a pH of about 1 to about 3.

[00172] A preferred mobile phase comprises about 5 to about 20% v/v ethanol, about 95 to about 80% v/v water, about 50 to about 150 mM buffer (e.g. di- alkaline hydrogen phosphate), with a pH of about 1 to about 3, and optionally a radiocleaner. A more preferred mobile phase comprises about 5 to about 20% v/v ethanol, about 95 to about 80% v/v water, about 50 to about 150 mM buffer (e.g., di- alkaline hydrogen phosphate), with a pH of about 2.2 to about 2.8, and optionally a radiocleaner.

[00173] Stationary phases for use in HPLC methods are well known and can be appropriately chosen by those skilled in the art. In a preferred embodiment, the stationary phase is a "reversed phase" (RP) stationary phase.

[00174] Examples of RP-HPLC stationary phases include C18, C8, phenyl, cyano (e.g., cyano propyl), pentafluorophenyl, amino (e.g., amino propyl), amide (e.g., C10-24 alkanoic - amino propyl), phenyl hexyl functionalized resins or mixed phase resins.

[00175] In one embodiment, the particle size of the HPLC stationary phase is from about 1.6 to about 15 micrometers. In a preferred embodiment, the particle size of the HPLC stationary phase is about 5 to about 10 micrometers. In another embodiment, the particle size of the HPLC stationary phase is about 10 micrometers.

[00176] Typically, the HPLC column has a diameter of about 2.0 to about 50 mm and a length of about 50 to about 300 mm. In a preferred embodiment, the HPLC column has a diameter of about 4.6 to about 20 mm and a length of about 150 to about 250 mm. In a more preferred embodiment, the HPLC column has a dimension of 10 x 250 mm.

[00177] The flow rate employed in high performance liquid chromatography is not restricted and can be from about 1 to about 20 mL/minute, more typically from about 2 to about 15 mL/minute, even more typically from about 2 to about 7 mL/minute.

[00178] The pressure employed in high performance liquid chromatography is not particularly limited and can be in the range of about 50 to about 400 bar, typically from about 50 to about 250 bar, more typically from about 50 to 200 Pub.

[00179] In one embodiment, about 1 to about 500 GBq [18F]fluoride are used to produce the compound of Formula I. In a preferred embodiment, about 50 to about 500 GBq [18F]fluoride are used to produce the production of the compound of Formula I. In a more preferred embodiment, about 100 to about 500 GBq [18F]fluoride are used to produce the compound of Formula I. In an even more preferred embodiment, about 200 to about 500 GBq [18F]fluoride is used to produce the compound of Formula I.

[00180] In one embodiment, about 10 GBq or more of [18F]fluoride is used for the production of the compound of Formula I. In a preferred embodiment, about 50 GBq or more of [18F]fluoride is used for the production of the compound of Formula I. In a more preferred embodiment, about 100 GBq or more of [18F]fluoride is used to produce the compound of Formula I. In an even more preferred embodiment, about 200 GBq or more of [18F] }fluoride are used to produce the compound of Formula I.

[00181] In one embodiment, about 10 GBq of radiolabeled compound of Formula I are obtained. In a preferred embodiment, about 20 GBq or more of radiolabeled compound of Formula I are obtained. In a more preferred embodiment, about 50 GBq or more of radiolabeled compound of Formula I are obtained. In an even more preferred embodiment, about 100 GBq or more of radiolabeled compound of Formula I are obtained.

[00182] In one embodiment, the compound of Formula I is obtained with a radiochemical purity of at least about 90%. In a preferred embodiment, the compound of Formula I is obtained with a radiochemical purity of at least about 93%. In a preferred embodiment, the compound of Formula I is obtained with a radiochemical purity of at least about 95%. In a more preferred embodiment, the compound of Formula I is obtained with a radiochemical purity of at least about 98%.

Optional Steps

[00183] Since ethanol and water are comprised in the mobile phase, the HPLC fraction comprising the compound of Formula I can be directly used as an injectable formulation, if desired. Alternatively, the HPLC fraction comprising the compound of Formula I can be further admixed with diagnostically acceptable components such as a diagnostically acceptable carrier, diluent, adjuvant, or excipient to prepare an injectable formulation of the compound of Formula I.

[00184] If desired Step D can be conducted after Step C. If further components are added, sterile filtration can be conducted before or after their addition.

[00185] In one embodiment, Step A, optional Step B and Step C are performed using an automated synthesis device. Examples of such automated synthesis devices include, but are not limited to, IBA Synthera, GE Fastlab, GE Tracerlab MX, GE Tracerlab FX, GE Tracerlab FX, Trasis AllinOne, ORA Neptis Perform, ORA Neptis Mosaic, ORA Neptis Plug, Scintomics GPR, Synthera, Starter Taddeo, Raytest Synchrom, Sofie Elixys, Eckert&Ziegler Modular Lab, Sumitomo Heavy Industries F100 F200 F300, and Siemens Explora.

[00186] A preferred method comprises the steps of:

[00187] A) react a compound of Formula II, where LG = nitro and PG = Boc with a fluorinating agent-[18F], preferably selected from K18F and [18F]tetrabutylammonium fluoride, in DMSO at about 100 at about 180°C, preferably about 120 to about 180°C, more preferably about 140 to about 160°C, where the Boc protecting group is cleaved under the conditions of radiolabeling,

[00188] C) purify with HPLC the compound of Formula I using a buffer mixture of ethanol/sodium dihydrogen phosphate (about 5 to about 20% EtOH), where the buffer has a pH of about 1 to about 3, preferably about 2.2 to about 2.8 and

[00189] D) if desired, mixing the HPLC fraction comprising the compound of Formula I further with diagnostically acceptable components of the formulation intended for administration to a patient. If desired, the HPLC fraction is passed through a sterile filter before or after mixing with still diagnostically acceptable components of the formulation.

[00190] Another preferred method comprises the steps of:

[00191] A) react a compound of Formula II, where LG = triemthyl ammonium, and PG = trityl with a fluorinating agent-[18F], preferably selected from K18F and [18F] tetrabutyl ammonium fluoride, in DMSO or aceto nitrile at about 80 to about 180°C, preferably about 100 to about 180°C,

[00192] B) add phosphoric acid and heat to about 90 to about 120°C for about 1 to about 15 minutes,

[00193] C) purify with HPLC the compound of Formula I using an ethanol/sodium dihydrogen phosphate buffer mixture (about 5 to about 20% EtOH), where the buffer has a pH of about 1 to about of 3, preferably about 2.2 to about 2.8, and

[00194] D) if desired, mixing the HPLC fraction comprising the compound of Formula I with further diagnostically acceptable components of the formulation intended for administration to a patient. If desired, the HPLC fraction is passed through a sterile filter before or after mixing with still diagnostically acceptable components of the formulation.

[00195] Unless otherwise specified, all hydrogen in the compounds of Formulas I, II, or III can be 1H, or 2H (deuterium).

[00196] The method of the present invention can provide a diagnostic composition comprising a compound of Formula I. Due to the speed and reduced complexity of the present method, the amount of compound I labeled with 18F can be greater than in conventional methods. This method also provides compounds of Formula I with a high radiochemical purity (e.g., at least about 90%, preferably at least about 93%, more preferably at least about 95%, even more preferably at least about 98% ) at high levels of radioactivity (e.g., at least about 20 GBq of compound of Formula I, preferably at least about 50 GBq of compound of Formula I, more preferably at least about 100 GBq of compound of Formula I).

[00197] The present diagnostic compositions are suitable for use in diagnostics. They are particularly suitable for diagnosing a disorder associated with Tau aggregates or imaging Tau aggregates, particularly for imaging Tau aggregates using positron emission tomography (PET).

Diagnostic Compositions

[00198] A "diagnostic composition" is defined in the present invention as a composition comprising a compound of Formula I. For in vivo applications the diagnostic composition must be in a form suitable for administration to mammals such as humans. Preferably a diagnostic composition further comprises a physiologically acceptable carrier, diluent, adjuvant or excipient. Administration to a patient is preferably accomplished by injecting the composition as a solution. Such a composition optionally may further contain ingredients such as solvents, buffers; diagnostically acceptable solubilizers; and diagnostically acceptable stabilizers or antioxidants.

[00199] Diagnostically acceptable excipients are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey (1975). The diagnostic excipient can be selected with respect to standard pharmaceutical practice. The excipient must be acceptable in the sense of not being harmful to its recipient.

[00200] Preferably, the diagnostic composition comprises about 1% v/v to about 20% v/v ethanol, based on the total amount of ethanol and water. More preferably, the diagnostic composition comprises about 1% v/v to about 15% v/v ethanol, based on the total amount of ethanol and water. Even more preferably, the diagnostic composition comprises about 5% v/v to about 10% v/v ethanol, based on the total amount of ethanol and water. In addition to the above components the diagnostic composition comprises water. The amount of water is chosen so that the total amount of the composition is 100%.

[00201] The compounds of Formula I are to be administered via injection. Examples of such administration include one or more of: intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intraexternal, intracranial, intramuscular or subcutaneous administration of compounds; and/or through infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile solution that may contain other excipients. Solutions should be appropriately buffered (preferably to a pH of 3 to 9, more preferably 4.0 to 8.5), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[00202] The diagnostic compositions of the invention can be formulated in a manner known per se to those skilled in the art as described, for example, in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey (1975).

[00203] The dose of the detectably labeled compounds of Formula I may vary depending on the exact compound to be administered, the patient's weight, sample size and type, and other variables as may be apparent to a physician skilled in the art. Generally, the mass preferably may be in the range of about 0.001 μg to about 100 μg per patient dose, preferably about 0.01 microgram to about 50 micrograms per patient dose. The radioactive dose may be, for example, about 100 to about 600 MBq, more preferably about 150 to about 450 MBq per injection, even more preferably about 150 to about 200 MBq.

[00204] In particular, in one embodiment diseases or disorders that can be detected and monitored with the detectably labeled compounds of Formula I are diseases or conditions associated with Tau protein aggregates, such as 3R or 4R tauopathies.

[00205] Diseases or conditions that can be detected and monitored with the detectably labeled compounds obtained through the method of the present invention include neurodegenerative disorders such as tauopathies. Examples of diseases and conditions that can be detected and monitored are caused by or associated with the formation of neurofibrillary lesions. This is the predominant brain pathology in tauopathy. The diseases and conditions comprise a heterogeneous group of neurodegenerative diseases or conditions including diseases or conditions that show coexistence of Tau and amyloid pathologies. Examples of diseases involving Tau aggregates are generically listed as tauopathies and these include, but are not limited to, Alzheimer's disease (AD), Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body, cerebral prion protein amyloid angiopathy, traumatic brain damage, amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease, corticobasal degeneration, diffuse neurofibrillary tangles with calcification , frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), panencephalitis subacute sclerosing, tangle-only dementia, post-encephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia , frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, traumatic stress syndrome, epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical parkinsonism, HIV-related dementia, adult-onset diabetes, senile 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 diseases and conditions that can be detected and monitored include Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down syndrome, Gerstmann - Straussler - Shceinker disease, inclusion body myositis, cerebral amyloid angiopathy of prion protein, traumatic brain injury ((TBI), amyotrophic lateral sclerosis, Parkinsonism complex - Guam dementia, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17, Hallervorden - Spatz disease, multiple system atrophy, Niemann - Pick disease type C, pallidum - punctate - nigral degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy ( PSP), subacute sclerosing panencephalitis, tangle-only dementia, post-encephalitic Parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2, chronic ttraumatic encephalopathy, familial British dementia , familial Danish dementia, frontotemporal lobar degeneration, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, and white matter tauopathy with globular glial inclusions, most preferably Alzheimer's disease (AD), Creutzfeldt-Jacob disease, dementia pugilistica, amyotrophic lateral sclerosis, argyrophilic grain disease, corticobasal degeneration, frontotemporal dementia with Parkinsonism linked to chromosome 17, Pick's disease, progressive supranuclear palsy, (PSP), tangle-only dementia, Guam Parkinson's dementia complex, Hallervorden - Spatz and fronto-temporal lobar degeneration. Preferably the disease or condition is Alzheimer's disease, Parkinson's disease or atypical parkinsonism, progressive supranuclear palsy (PSP), or Pick's disease, more preferably Alzheimer's disease.

[00206] Still examples of diseases or conditions that can be detected and monitored with the detectably labeled compounds obtained by the method of the present invention include Huntington's disease, ischemic stroke, and psychosis in AD.

[00207] The method of the present invention has a number of important advantages compared to prior art methods. Since only a single chromatographic step without subsequent reformulation via SPE is required after the compound of Formula I has been prepared, the assembly is much simpler than prior art assemblies in which two different chromatographic steps are conducted.

[00208] The method proved to be very robust. Due to the choice of ethanol and water as the mobile phase with an adjusted pH, larger amounts (e.g., >1 mg) of precursor can be used without precipitation being caused.

[00209] The 18F-labeled compound of Formula I can be reliably separated from the precursor compound of Formula II and possible byproducts.

[00210] Furthermore, high yields and purities can be obtained. For example, it is possible to obtain corrected yields with no decay of at least about 35%. The product activity may be at least about 20 BGq, preferably at least about 50 GBq, more preferably at least about 100 GBq.

[00211] Radiochemical purities can be at least about 95%, preferably at least about 98% at low as well as high levels of radioactivity (at, for example, at least about 100 GBq).

[00212] The present invention is illustrated by the following examples, which should not be construed as limiting. Examples Abbreviations

Experimental data

[00213] All reagents and solvents were obtained from commercial sources and used without further purification. Proton (1H) spectra were recorded on a Bruker DRX-400 MHz NMR spectrometer or on a Bruker AV-400 MHz 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 appropriate solvents as not stated in the specific examples. Flash purification was conducted with a Biotage Isolera One flash purification system using HP-Sil (Biotage) or puriFlash (Interchim) columns and the solvent gradient indicated in the specific examples. Thin layer chromatography (TLC) was performed on silica gel plates with UV detection. Synthesis of Test Compounds Preparative Example A Step A

[00214] Commercially available 2,6-dibromo pyridine (4.12 g, 16.6 mmoles) was suspended in ethanol (40 mL) and hydrazine hydrate (10 mL, 97.6 mmoles) 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 chromatography on silica using ethyl acetate/n-heptane (60/40) to yield the title compound as an off-white solid (3.05 g, 93%).

[00215] 1H-NMR (400 MHz, 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) Step B

[00216] The title compound from Step A above (10 g, 53.2 mmoles) and commercially available 1-Boc-4-piperidone (10.6 g, 53.2 mmoles) were added to a 500 mL flask and mixed to become a homogeneous combination. Then polyphosphoric acid (80 g, base 115% H3PO4) was added and the mixture was heated to ~160°C in a sand bath. At ~120°C the Boc protecting group was cleaved resulting in foaming of the reaction mixture. After complete Boc cleavage the foam collapsed and the dark reaction mixture was stirred at ~160°C for 20 hours. The reaction mixture was allowed to cool to room temperature and water (400 mL) was added. The reaction mixture was stirred/sonified until the gummy material was dissolved. The reaction mixture was then placed in an ice bath and the pH of the solution was adjusted to pH ~12 by adding solid sodium hydroxide pellets (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) through sonication and washed with water (2x400 mL) to remove remaining salts and insoluble material. The organic phase was dried over sodium sulfate, filtered and the solvents were 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 yield the title compound as a beige solid (3.5 g, 26%).

[00217] 1H-NMR (400 MHz, 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)Step C

[00218] The title compound from Step B above (1.75 g, 6.94 mmoles) was suspended in xylene (380 mL) and manganese (IV) oxide (6.62 g, 76.9 mmoles) was added . The reaction mixture was then heated to ~160°C in a sand bath for 36 hours. The cooled reaction mixture was evaporated under reduced pressure, 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 paper filters to remove manganese (IV) oxide and the filter was washed with methanol (50 mL). The combined filtrates were evaporated under reduced pressure and the dark residue was purified by chromatography on silica (50 g cartridge-HP-SIL) using a Biotage Isolera system employing an ethyl acetate/heptane gradient (5/95-100/0). to remove non-polar impurities followed by dichloromethane/methanol (9/1 -> 4/1) to yield the title compound as a dark yellow solid. The total yield from 2 runs was 1.77 g (51%).

[00219] 1H-NMR (400 MHz, 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) Preparative Example B Step A

[00220] To a suspension of the title compound of Comparative Example A (0.776 g, 0.72 mmol) in dichloromethane (65 mL) was added triethylamine (1.86 mL, 13 mmol) and trityl chloride (2.63 g , 0.39 mmol). After adding 4-(dimethyl amino) 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 sodium sulfate, filtered and the solvents were removed in vacuo. The residue was purified on HP-Sil SNAP cartridges (50 g) using a Biotage Isolera One purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to yield the title compound B as a light yellow solid (0.831 g, 54%). Unreacted starting material was recovered by washing cartridge with ethyl acetate/methanol (90/10) to yield starting material as an off-white solid (0.195 g, 25%).

[00221] 1H-NMR (400 MHz, 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)

[00222] MS (ESI); m/z = 490.03/491.96 [M+H]+ Preparative Example C Step A

[00223] To a suspension of the title compound of Preparative Example A (0.482 g, 1.94 mmoles) in dichloromethane (40 mL) was added triethylamine (1.15 mL, 8 mmoles) and 4,4'-(chloro ( phenyl) methylene) bis (methoxy benzene) (DMTrt-Cl) (1.963 g, 5.8 mmoles). After adding 4-(dimethylk amino) 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 sodium sulfate, filtered and the solvents were removed in vacuo. The residue was purified on HP-Sil SNAP cartridges (50 g) using a Biotage Isolera purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to yield the title compound C as a light yellow solid (0.825 g, 72%). Unreacted starting material was recovered by washing cartridge with ethyl acetate/methanol (90/10) to yield starting material as an off-white solid (0.042 g, 8.8%).

[00224] 1H-NMR (400 MHz, 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) Example 1 Step A

[00225] To a degassed mixture of 1,4-dioxane (4.3 mL) and water (1 mL) in a microwave flask was added [1,1'-bis(diphenyl phosphine) ferrocene] dichloro palladium complex ( II), complex with dichloromethane (0.0084 g, 0.01 mmol), followed by the title compound of Precautionary Example A (0.05 g, 0.2 mmol), acid (2-fluoropyridin-4-yl) boronic acid (0.035g, 0.245 mmol) and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated to ~115°C in a sand bath for 6 hours. The reaction mixture was diluted with ethyl acetate (60 ml) and water (20 ml), the organic phase was separated, dried over sodium sulfate, filtered and the solvents were evaporated in vacuo. The dark residue was purified by chromatography on silica (25 g HP-SIL) using a Biotage Isolera system employing a dichloromethane/methanol gradient (100/0 -> 95/5 -> 90/10 -> 80/20) to yield the title compound 1 as an off-white solid (0.033 g, 63%).

[00226] 1H-NMR (400 MHz, 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)

[00227] MS (ESI): m/z = 265.04 [M+H]+ Example 2 Step A

[00228] To a suspension of the title compound of Preliminary Example A (0.430 g, 1.73 mmol) in dichloromethane (25 mL) was added triethylamine (1.93 mL, 13.89 mmol) and di-t dicarbonate - butyl (2.27 g, 10.02 mmoles). After adding 4-(dimethyl amino) pyridine (0.042 g, 0.34 mmol), the reaction mixture was stirred at room temperature for 3 days. Solvents were removed under reduced pressure and the residue was purified on HP-Sil SNAP cartridges (25 g) using a Biotage Isolera One purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to yield the title compound as an off-white solid (0.558, 92%).

[00229] 1H-NMR (400 MHz, CDCl3) δ = 9.28 (s, 1H), 8.73 (d, 1H), 8.22 (d, 2H), 7.59 8d, 1H), 1 .80 (s, 9H) Step B

[00230] To a mixture of degassed 1,4-dioxane (3 mL) and water (0.7 mL) in a microwave flask was added [1,1'-bis(diphenylphosphine) ferrocene] palladium dichloro (II) , complexed with dichloro methane (0.0058 g, 0.007 mmol), 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 to ~100°C in a sand bath for 4 hours. The reaction mixture was diluted with ethyl acetate (80 mL) and water (35 mL), the organic phase separated, dried over sodium sulfate, filtered and the solvents were evaporated in vacuo. The dark residue was purified by chromatography on silica (12 g, puriFlash, Interchim) using a Biotage Isolera system employing a dichloromethane/methanol gradient (100/0 -> 98/2 -> 95/5 -> 90/10 -> 80/20) to yield the less polar Boc-protected compound (0.0255 g, 49%) and the more polar title compound 2 as an off-white solid (0.0116 g, 31%).

[00231] Most polar title compound 2:

[00232] 1H-NMR (400 MHz, 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)

[00233] MS (ESI): m/z = 265.09 [M+H]+

[00234] Compound protected with less polar Boc:

[00235] 1H-NMR (400 MHz, 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)

[00236] The synthesis of compound of title 2 was first described in WO 2015/052105 (Example 1) through a different synthesis. Synthesis of Radiolabeling Precursors Example 3-a Step A

[00237] To a degassed mixture of 1,4-dioxane (4.3 mL) and water (1 mL) in a microwave flask was added [1,1'-bis(diphenylphosphine) ferrocene] palladium dichloro (II) , complexed with dichloromethane (0.0084 g, 0.01 mmol), the title compound of Preparative Example B (0.1 g, 0.2 mmol), 2-nitro-4-(4,4,5, 5-tetramethyl-1,3,2-dioxa borolan-2-yl) pyridine (0.061 g, 0.245 mmol) and cesium carbonate (0.m133 g, 0.41 mmol). The reaction mixture was then heated to ~115°C in a sand bath for 6 hours. The reaction mixture was diluted with ethyl acetate (60 mL) and water (20 mL), the organic phase was separated, dried over sodium sulfate, filtered and the solvents were evaporated in vacuo. The dark residue was purified by chromatography on silica (25 g column-pufiFlash, Interchim) using a Biotage Isolera system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to yield title compound 3-a as a light yellow solid (0.082 g, 75%).

[00238] 1H NMR (400 MHz, 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), 7.587.54 (m, 5H), 7.32-7.25 (m, 10H), 6 .48 (d, 1H)

[00239] MS (ESI): m/z = 534.28 [M+H]+. Example 3-b Method a: Step A

[00240] To a solution of 3-a (0.0396 g, 0.074 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (1.2 mL). The reaction mixture was stirred at room temperature for 6 hours and methanol (2 mL) was added. The solvents were evaporated in vacuo and the residue was dissolved/suspended in methanol (5 mL). The solvents were evaporated in vacuum and the residue was redissolved/suspended in methanol (5 mL). The solvents were evaporated in vacuo and the residue suspended in dichloromethane (2 ml). After addition of triethylamine (1 mL, 7.2 mmol), di-t-butyl dicarbonate (0.098 g, 0.43 mmol), and 4-(dimethyl amino) pyridine (0.0018 g, 0.014 mmol) , 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 sodium sulfate, filtered and the solvents removed in vacuo. The residue was purified on silica (25 g puriFlash, Interchim) using a Biotage Isolera One purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to elute by-products nonpolar compounds followed by ethyl acetate/methanol (95/5) to provide the title compound 3-b as a pale yellow solid (0.0184 g, 63%).

[00241] 1H-NMR (400 MHz, 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)

[00242] MS (ESI); m/z = 391.82 [M+H]+ Method b Step A

[00243] To a degassed mixture of 1,4-dioxane (2.2 mL) and water (0.5 mL) in a microwave flask was added [1,1'-bis(diphenylphosphine) ferrocene] palladium dichloro ( II), complex with dichloromethane (0.0042 g, 0.005 mmol), followed by the title compound from Preparative Example C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5 -tetramethyl-1,3,2-dioxa borolan-2-yl) pyridine (0.0305 g, 0.12255 mmol) and cesium carbonate (0.067 g, 0.205 mmol). The reaction mixture was then heated to ~115°C in a sand bath for 6 hours. The reaction mixture was diluted with ethyl acetate (20 mL), the precipitate collected by filtration, washed with water (10 mL) and methanol (5 mL), and air-dried to yield 3-c (0.0277 g, 95 %).Step B

[00244] To a suspension of the crude title compound from Step A above (0.0277 g, 0.095 mmol) in dichloromethane (4 mL) were added triethylamine (1 mL, 7.2 mmol), di-t-butyl dicarbonate (0.2 g, 0.86 mmol), and 4-(dimethyl amino) pyridine (0.0036 g, 0.028 mmol). The reaction mixture was stirred at room temperature for 16 hours, diluted with ethyl acetate (50 mL) and water (20 mL). The organic phase was separated, dried over sodium sulfate, filtered and the solvents were removed in vacuo. The residue was purified on silica (25 g puriFlash, Interchim) using a Biotage Isolera purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to elute byproducts. non-polar compounds followed by ethyl acetate/methanol (95/5) to yield the title compound 3-b as a light yellow solid (0.0261 g, 70%).

[00245] 1H-NMR (400 MHz, 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)

[00246] MS (ESI); m/z = 391.85 [M+H]+; 291.74 [M+H-Boc]+ Example 3-d Step A

[00247] To a degassed mixture of 1,4-dioxane (2.2 mL) and water (0.5 mL) in a microwave flask was added [1,1'-bis(diphenylphosphine) ferrocene] palladium dichloro ( II), complex with dichloromethane (0.0042 g, 0.005 mmol), followed by the title compound of Comparative Example C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5- tetramethyl-1,3,2-dioxa borolan-2-yl) pyridine (0.0305 g, 0.12255 mmol) and cesium carbonate (0.067 g, 0.205 mmol). The reaction mixture was then heated to ~115°C in a sand bath for 6 hours. The reaction mixture was diluted with ethyl acetate (20 mL), the precipitate collected by filtration, washed with water (10 mL) and methanol (5 mL), and air-dried to yield 3-c as a gray solid (0. 0277 g, 95%).Step B

[00248] To a suspension of the crude title compound from Step A above (0.0277 g, 0.095 mmol) in dichloromethane (4 mL) was added triethylamine (1 mL, 7.2 mmol), 4,4'-(chloro (phenyl) methylene) bis (methoxy benzene) (0.081 g, 0.29 mmol), and 4-(dimethyl amino) pyridine (0.0036 g, 0.028 mmol). The reaction mixture was stirred at room temperature for 18 hours, diluted with ethyl acetate (50 mL) and water (20 mL). The organic phase was separated, dried over sodium sulfate, filtered and the solvents were removed in vacuo. The residue was purified on silica (25 g puriFlash, Interchim) using a Biotage Isolera One purification system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to yield the title compound 3-d as a light yellow solid (0.0261 g, 44%).

[00249] 1H-NMR (400 MHz, 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), 7.527.42 (m, 6H), 7.27-7.23 (m, 4H), 6 .80 (d, 4H), 6.49 (d, 1H), 3.78 (s, 6H) Example 3-e Step A

[00250] N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa borolan-2-yl) pyridin-2-amine (0.25 g, 1 mmol) commercially available solution was dissolved in dichloromethane (5 mL). To the resulting stirring solution was added dropwise at room temperature methyl trifluoro methane sulfonate (0.124 mL, 1.1 mmol). The solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated to remove dichloromethane and the residue was dried in vacuum to obtain a yellow glass/foam, which was used directly for the next step. Step B

[00251] To a degassed solution of 1,4-dioxane (12 mL0 and water (3 mL) in a microwave flask was added [1,1'-bis (diphenyl phosphine) ferrocene] palladium(II) dichloro, complexed with dichloromethane (0.034 g, 0.04 mmol), the title compound from Comparative Example B (0.4 g, 0.816 mmol), the crude title compound from Step A above (~1 mmol), and cesium carbonate (0.544 g , 1.68 mmol). The reaction mixture was diluted with ethyl acetate (150 ml) and water (50 ml), the organic phase separated, dried over sodium sulfate, filtered and the solvents were evaporated in vacuo. dark was purified by chromatography on silica (25 g HP-Ultra) using a Biotage Isolera system employing an ethyl acetate/n-heptane gradient (5/95 -> 100/0 -> 100/0) to elute starting material unreacted and non-polar byproducts. The gradient was then changed to dichloromethane/methanol (100/0 -> 95/5 -> 90/10) to yield the dimethyl amine derivative as a light yellow glass (0.127 g, 29%; MS (ESI): m/z = 532.27 [M+H]+) and the methyl amine derivative as a gray solid (0.0547 g, 13%; MS (ESI): m/z = 519.18 [M+H]+). The gradient was again switched to dichloro methane/methanol (90/10 -> 80/20) and maintained at (80/20) to obtain the title compound 3-e as a brown solid (0.104 g, 18%).

[00252] 1H NMR (400 MHz, 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)

[00253] MS (ESI): m/z = 546.26 [M+H]+ Example 3-f Step A

[00254] 3-e (0.199 g, 0.364 mmol) was suspended in dichloromethane (10 mL). After adding trifluoroacetic acid (10 mL), the reaction mixture was stirred at room temperature for 18 hours. The solvents were removed under reduced pressure, the residue dissolved in methanol (10 ml) and the solvents were removed under reduced pressure. The methanol treatment of the residue was repeated two more times. The residue was then suspended in dichloromethane (20 mL) and sonicated for ~5 minutes. The precipitate was collected by filtration, washed with dichloromethane (10 mL) and dried with air to yield the title compound 3-f as a gray solid (0.127 g, 83%).

[00255] 1H NMR (400 MHz, 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) MS (ESI): m/z = 303.91 [M+H]+ Example 4-a Step A

[00256] To a degassed mixture of 1,4-dioxane (3 mL) and water (0.7 mL) in a microwave flask was added [1,1'-bis(diphenylphosphine) ferrocene] palladium(II) dichlorine, complex with dichloromethane (0.0058 g, 0.007 mmol), followed by the title compound from Example 2 Step A (0.05 g, 0.143 mmol), 2-nitro-5-(4,4,5,5-tetramethyl -1,3,2-dioxa borolan-2-yl) pyridine (0.0428 g, 0.17 mmol) and cesium carbonate (0.092 g, 0.286 mmol). The reaction mixture was then heated to ~100°C in a sand bath for 4 hours. The reaction mixture was diluted with ethyl acetate (80 mL) and water (35 mL), the organic phase separated, dried over sodium sulfate, filtered and the solvents were evaporated in vacuo. The dark residue was purified by chromatography on silica (12 g, puriFlash, Interchim) using a Biotage Isolera system employing a dichloromethane/methanol gradient (100/0 -> 98/2 -> 90/10 -> 80/20) to provide title compound 4-a as a light yellow solid (0.0173 g, 31%).

[00257] 1H NMR (400 MHz, 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)

[00258] MS (ESI): m/z = 392.13 [M+H]+

[00259] The synthesis of title compound 4-a was first described in WO 2015/052105 (Example 3a) through a different synthesis.

Radiolabeling of Precursors with 18F

[00260] Examples of radiolabeling precursors

[00261] Generic radiolabeling method A, carried out on a tracerlab FX as illustrated in Figure 3 (radiolabeling, deprotection, HPLC and SPE) - Comparative Examples

[00262] [18F]fluoride was drawn over a lightweight Sep-Pak Accell Plus QMA cartridge (Waters) and eluted with a K2CO3/Kryptofix 2.2.2 solution. Water was removed using a stream of He or N2 at 95°C and co-evaporated to dryness with MeCN (1 mL). Next, a solution of the dissolved precursor was added to the dry K[18F]F-Kriptofix complex. The reaction vial was sealed and heated for 15 minutes at 150°C. Subsequently, an acid (HCl, H2SO4 or H3PO4) was added and the mixture was heated for 10 minutes at 150°C. The reaction mixture was diluted with 1 mL of NaOH and 2.4 mL of HPLC prep mobile phase. and the crude product was purified via semipreparative HPLC (e.g., Phenomenex, Gemini C18, 5 μm, 250 x 10 mm) at 4 mL/minute. The isolated tracer was diluted with water (20 mL + 10 mg/mL sodium ascorbate), drawn over a C-18 Plus cartridge (Waters), washed with water (10 mL + 10 mg/mL sodium ascorbate), eluted with ethanol (1 mL) and mixed with water (14 mL + 10 mg/mL sodium ascorbate).

Generic radiolabeling method B, carried out on a tracerlab FX as illustrated in Figure 3 (Radiolabelling, HPLC and SPE) - Comparative Examples

[00263] [18F]fluoride was drawn over a light sep-Pak Accell Plus QMA cartridge (Waters) and eluted with a K2CO3] Kriptofix 2.2.2 solution. Water was removed using a stream of He or N2 at 95°C and co-evaporated to dryness with MeCN (1 mL). Next, a solution of the dissolved precursor was added to the dried K[18F]F-Kryptoxy complex. The reaction vial was sealed and heated for 15 minutes at 150°C. The reaction mixture was diluted with 0.5-1 mL of NaOH and 2.4 mL of HPLC prep mobile phase. and the crude product was purified via semipreparative HPLC (e.g., Phenomenex, Gemini C18, 5 μm, 250 x 10 mm) at 4 mL/minute. The isolated tracer was diluted with water (20 mL + 10 mg/mL sodium ascorbate), drawn over a C-18 Plus cartridge (Waters), washed with water (10 mL + 10 mg/mL sodium ascorbate), eluted with ethanol (1 mL) and mixed with water (14 mL + 10 mg/mL sodium ascorbate).

Generic radiolabeling method C performed on an IBA Synthera + Synthera HPLC as illustrated in Figure 4 (radiolabeling, HPLC)

[00264] [18F]Fluoride was drawn over a lightweight Sep-Pak Accell Plus QMA cartridge (Waters) and eluted with a K2CO3/Kryptofix 2.2.2 solution. Water was removed using a He or N2 stream at 95-110°C and co-evaporated to dryness. Next, a solution of the dissolved precursor was added to the dry K[18F]F - Kriptofix complex. the reaction vial was sealed and heated for 15 minutes at 150°C. The reaction mixture was diluted with 0.5-1 mL of 1M H3PO4 and 3-3.5 mL of the aqueous component of the prep HPLC mobile phase. and the crude product was purified via semipreparative HPLC (e.g., Waters XBridge Peptide BEH C18, 130 angstroms, 10 micrometers, 10 mm x 250 mm) at 3-6 mL/minute. The fraction containing the product (5-10 mL) was collected and diluted with a dilution medium containing 0-2 mL of EtOH, 10-20 mL of water, and 0-4 mL of phosphate buffer concentrate (Braun, 3, 5 g of disodium monohydrogen phosphate dodeca hydrate in 20 mL of water for injection) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or gentisic acid (20-200 mg).

Generic radiolabeling method D, carried out on a tracerlab FX as illustrated in Figure 3 (Radiolabelling, HPLC)

[00265] [18F]fluoride was drawn over a lightweight Sep-Pak Accell QMA cartridge (Waters) and eluted with a K2CO3/Kriptofix 2.2.2 solution. Water was removed using a stream of He or N2 at 95°C and co-evaporated to dryness with MeCN (1 mL). Next, a solution of the dissolved precursor was added to the dry K[18F]F- Kriptofix complex. The reaction vial was sealed and heated for 15 minutes at 150°C. The reaction mixture was diluted with 0.5-1 mL of 1M H3PO4 and 33.5 mL of the aqueous component of the prep. HPLC mobile phase and the crude product was purified via semipreparative HPLC (e.g., Waters XBridge Peptide BEH C18 , 130 angstroms, 10 micrometers, 10 mm x 250 mm or Gemini 5 micrometers C18, 250x10 mm, Phenomenex: 00G-4435-N0) at 3-6 mL/minute. The fraction containing the product (5-10 mL) was collected and diluted with a dilution medium containing 0-2 mL of EtOH, 10-20 mL of water, and 0-4 mL of phosphate buffer concentrate (Braun) and/or or sodium ascorbate (1001000 mg) and/or sodium citrate (100-1000 mg) and/or gentisic acid (20200 mg).

Generic radiolabeling method E, carried out on a tracerlab FX as illustrated in Figure 3 (radiolabelling, deprotection, HPLC)

[00266] [18F]Fluoride was drawn over a lightweight Sep-Pak accell Plus QMA cartridge (Waters) and eluted with a K2CO3/Kriptofix 2.2.2 solution. Water was removed using a stream of He or N2 at 95°C and co-evaporated to dryness with MeCN (1 mL). Next, a solution of the dissolved precursor was added to the dry K[18F]F-Kriptofix complex. The reaction vial was sealed and heated for 15 minutes at 150°C. Subsequently, 1 mL of 0.5 M H2SO4 was added and the mixture was heated for 10 minutes at 100°C. The reaction mixture was diluted with 0.5-1 mL of 1M NaOH and 2-3 mL of the aqueous component of the prep HPLC mobile phase. and the crude product was purified via semipreparative HPLC (e.g., Waters mL/minute. The fraction containing the product (5-10 mL) was collected and diluted with a dilution medium containing 0-2 mL of EtOH, 10-20 mL of water, and 0-4 mL of phosphate buffer concentrate (Braun) and/or or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or gentisic acid (20-200 mg).

Generic radiolabeling method F, performed on an IBA Synthera + Synthera HPLC as illustrated in Figure 4 (Radiolabeling, deprotection, HPLC)

[00267] [18F]Fluoride was drawn over a lightweight Sep-Pak accell Plus QMA cartridge (Waters) and eluted with a K2CO3/Kriptofix 2.2.2 solution. Water was removed using a He or N2 stream at 95°C-110°C and co-evaporated to dryness. Next, a solution of the dissolved precursor was added to the dry K[18F]F- Kriptofix complex. The reaction vial was sealed and heated for 5-10 minutes at 110-120°C. After fluorination, 0.5-1 mL of 1M H3PO4 was added and the reaction mixture heated for 10-15 minutes at 100-110°C. The reaction mixture was diluted with 3-3.5 mL of the aqueous component of the HPLC prep mobile phase. and the crude product was purified via semipreparative HPLC (e.g., Waters XBridge peptide BEH C18, 130 angstroms, 10 micrometers, 10 mm x 250 mm) at 3-6 mL/minute. The fraction containing the product (5-10 mL) was collected and diluted with a dilution medium containing 0-2 mL of EtOH, 10-20 mL of water, and 0-4 mL of phosphate buffer concentrate (Braun, 3, 05 g of sodium dihydrogen phosphate dodeca hydrate, 0.462 g of sodium dihydrogen phosphate dihydrate in 20 mL of water for injection) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate sodium (100-1000 mg) and/or gentisic acid (20-200 mg).

Determination of Radiochemical Purity

[00268] Radiochemical purity was determined through analytical HPLC, for example: column: Atlantis T3, Waters, 100 x 4.6 mm, 3 micrometers, 100; mobile phase: A: 40 mM sodium acetate, finally adjusted to pH 5.0 with glacial acetic acid; mobile phase B: 35% methane in acetonitrile; flow rate: 1.8 mL/minute; gradient: 0-5 minutes 15-32% B, 5-8 minutes 32-80% B, 8-12 minutes 80% B, 12-13 minutes 80-15% B, 13-16 minutes 15% B. Results for 18F-Ib synthesis:

Claims (9)

1. Method for preparing a compound of Formula I, characterized by the fact that it comprises the steps of: (A) reacting a compound of Formula II with an 18F fluorinating agent, in which X is H or PG, LG is a leaving group, and PG is an amine protecting group; and (B) optionally, if 4-dimethoxy benzyl, p-methoxy phenyl, triphenyl methyl, methoxy phenyl diphenyl methyl, or dimethoxy trityl; and (C) subjecting the resulting compound of Formula I to high performance liquid chromatography (HPLC) using a mobile phase comprising a mixture of ethanol and water. 2. Method, according to claim 1, characterized by the fact that: X is PG; step (B) is missing; and the PG protection group is cleaved in step (A); or X is PG; and the PG protecting group is cleaved in step (B). 3. Method, according to claim 1 or 2, characterized by the fact that the ratio of ethanol to water, in the mobile phase, is from 5/95 v/v to 80/20 v/v. 4. Method according to any one of claims 1 to 3, characterized by the fact that the pH of the mobile phase is from 0 to 8, or from 1 to 3. 5. Method according to any one of claims 1 to 4, characterized in that the mobile phase further comprises a buffer, which is selected from alkali metal dihydrogen phosphates, dialkali metal hydrogen phosphates, trialkaline metal phosphates , alkali metal acetates, alkali metal formates, and mono/di/trialkali metal citrates. 6. Method according to any one of claims 1 to 5, characterized in that high performance liquid chromatography (HPLC) is conducted under a pressure of 50 to 400 bar, or 50 to 250 bar. 7. Method according to any one of claims 1 to 6, characterized by the fact that high performance liquid chromatography (HPLC) results in a fraction comprising the compound of Formula I, and that this fraction is subjected to a step ( D) sterile filtration. 8. Method according to any one of claims 1 to 7, characterized in that it does not comprise solid phase extraction of the compound of Formula I after step (C), or wherein the method does not comprise solid phase extraction of the compound of Formula I before or after step (C). 9. Method according to any one of claims 1 to 8, characterized by the fact that the compound of Formula I is not subjected to chromatography after the high performance liquid chromatography (HPLC) of step (C), or wherein the compound of Formula I is not subjected to chromatography other than the high performance liquid chromatography (HPLC) of step (C).