EA042728B1 - A NEW METHOD FOR OBTAINING A VISUALIZING COMPOUND - Google Patents

Description

Field of invention

The invention relates to a novel method for the preparation of a compound of formula I which can be used to selectively detect disorders and anomalies associated with tau aggregates such as Alzheimer's disease (AD) and other tauopathies, for example using positron emission tomography (PET) imaging. A diagnostic composition containing the resulting compound, as well as its use in diagnostics and imaging, is also the subject of this application.

Background of the invention

Alzheimer's disease is a neurological disorder thought to be caused by amyloid plaques, an extracellular accumulation of abnormal deposits of amyloid-beta (Ab) aggregates in the brain or eyes. Other major neuropathological features in AD are intracellular neurofibrillary tangles (NFTs), which result from the 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 some types of frontotemporal dementia (FTD). In the brains of AD patients, tau pathology (taupathy) develops later than amyloid pathology, but it is still debated whether the AV protein, which is the essence of the so-called amyloid cascade hypothesis, is the causative agent of AD (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).

Currently, the only definitive way to diagnose AD is to identify plaques and tangles in brain tissue by histological analysis of biopsy or autopsy materials after death. In addition to AD, Tau plays an important role in other (non-AD) neurodegenerative diseases. Such non-AD tauopathies include, for example, supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal degeneration (CBD).

It has been proposed that a compound of general formula A be used to selectively detect disorders and abnormalities associated with tau aggregates such as Alzheimer's disease (AD) and other tauopathies, and several methods for preparing this compound have been described in the prior art.

Gobi et al. described in WO 2015/052105 a process in which a compound of formula A was obtained by reacting a precursor having a nitro group instead of ¹⁸ F with [ ¹⁸ F]fluoride in a microwave device. After the synthesis step, the compound of formula A was purified in a two-step process including but not limited to:

1) HPLC using a mobile phase of methanol and triethylamine and

2) re-treatment using a solid phase extraction cartridge to capture the compound of formula A and subsequent elution from the cartridge with ethanol diluted with saline (0.9% NaCl in water).

The method gave the compound of formula A with a yield of 26.1%.

Gobi et al. in J. Med. Chem. 2017, volume 60, p. 7350-7370 furthermore describe the following two methods.

a) Microwave method: [ ¹⁸ F]fluoride was captured on a cation exchange cartridge and activity was eluted with Kryptofix®/potassium carbonate. After adding acetonitrile, the mixture was dried at elevated temperature. The vessel with the dried [ ¹⁸ F]fluoride mixture was then transferred to a microwave device and 0.5 mg of the precursor compound (unprotected nitro derivative) in 400 μl of DMSO was added. The vessel was irradiated with microwaves at 50 W for 240 s. The resulting solution was diluted and purified by preparative HPLC (C-18 column, acetonitrile/trimethylamine buffer pH 7.2).

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

b) Thermal heating method: [ ¹⁸ F]fluoride was captured on a cation exchange cartridge and the activity was eluted with Kryptofix®/potassium carbonate. After adding acetonitrile, the mixture was dried at elevated temperature. 0.5 mg of the precursor compound (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).

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

- 1 042728

Thus, the methods for preparing the compound of formula A in the above prior art have two purification/reworking steps, the method being only partially automated in the case of microwave methods, as shown in FIG. 1.

The nitro precursor (and major by-product) of the prior art process has poor solubility, especially in ethanol, acetonitrile, and acetonitrile/aqueous buffers used for purification (preparative HPLC), especially if neutral pH aqueous buffers are used . In the prior art examples, only 0.5-0.7 mg of the precursor was used.

The object of the present invention is to provide an improved process for the preparation of a compound of formula I that is more cost and time efficient than prior art methods (as shown in FIG. 2). In addition, the yield of the process must be improved.

Description of figures

Fig. 1 is a schematic overview of the prior art selected by Gobbi et al.

Fig. 2 is a schematic overview of the method of the present invention.

Fig. 3 - setting up the GE Tracerlab FX synthesizer.

Fig. 4 - setting up the IBA Synthera synthesizer.

The essence of the invention

The present invention relates to the following items.

1. Process for the preparation of a compound of formula I

including stages:

A) interaction of the compound of formula II with ¹⁸ F fluorinating agent

X

II where X is H or PG;

LG is a drop group and

PG is an amino protecting group, and

B) optionally, if X is PG, cleavage of the PG protecting group, and

C) subjecting the resulting compound of formula I to high performance liquid chromatography (HPLC) using a mobile phase containing ethanol and water.

2. The method according to claim 1, where X is PG, step B is absent, and the PG protecting group is cleaved off in step A.

3. The method according to claim 1, wherein X is PG and the PG protecting group is cleaved off in step B.

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

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

6. Process according to any one of claims 1 to 5, wherein the mobile phase further comprises a buffer which is preferably selected from alkali metal dihydrogen phosphates, two alkali metal hydrogen phosphates, three alkali metal phosphates, alkali metal acetates, alkaline earth metal acetates , alkaline earth metal formates, citrates with one, two or three alkali metal atoms.

7. The method according to any one of claims 1 to 6, wherein the high performance liquid chromatography (HPLC) is carried out at a pressure of from about 50 to about 400 bar, preferably from about 50 to about 250 bar.

8. The method according to any one of claims 1 to 7, wherein the method is an automated method, wherein step A, optional step B, and step C are carried out in an automatic synthesizer.

9. The method according to any one of claims 1 to 8, wherein high performance liquid chromatography (HPLC) yields a fraction containing a compound of formula I and this fraction is subjected to step D) of sterile filtration.

10. A method according to any one of claims 1 to 9, wherein the method does not include solid phase extraction of a compound of formula I after step C, preferably wherein the method does not include solid phase extraction of a compound of formula I before or after step C.

- 2 042728

11. The method according to any one of claims 1 to 10, wherein the compound of formula I is not subjected to chromatography after the high performance liquid chromatography (HPLC) in step C, preferably wherein the compound of formula I is not subjected to chromatography other than high performance liquid chromatography (HPLC) in step C. stage C.

12. Diagnostic composition containing a compound of formula I

which can be obtained by the method in accordance with any of paragraphs.1-12 and, optionally, a diagnostically acceptable carrier, diluent, adjuvant or excipient.

13. Composition according to claim 12 for use in diagnostics.

14. Composition according to claim 12 for use in imaging tau aggregates, in particular for use in positron emission tomography for imaging tau aggregates.

15. Composition for use according to claim 13 or 14, wherein the disorder is selected from the following: Alzheimer's disease (AD), familial AD disease, Creutzfeldt-Jakob disease, boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, myositis with inclusion bodies, prion protein-induced cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism-dementia-Guam complex, motor neuron disease with non-Guam-type neurofibrillary tangles, argyrophilic granule disease, corticobasal degeneration ( CBD), diffuse neurofibrillary tangles with calcification, chromosome 17-linked frontotemporal dementia with parkinsonism, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick type C disease, pallidoponto-nigral degeneration, Pick's disease (PiD), progressive subcortical gliosis , progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, dementia with a predominance of neurofibrillary tangles, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadalupe parkinsonism, brain iron storage neurodegeneration, 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-associated dementia, adult-onset diabetes, senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary degeneration retina, macular degeneration (such as age-related macular degeneration (AMD)), optic disc drusen, optic neuropathy, optic neuritis, and lattice degeneration; preferably Alzheimer's disease.

16. A composition for use according to claim 13 or 14, wherein the disorder is selected from Huntington's disease, ischemic stroke, and AD psychosis.

17. A composition for use according to any one of claims 13-16, wherein the composition is administered by injection.

18. A composition as defined in claim 12 for use in diagnosing a tau-aggregate disorder or for use in diagnosing tauopathy, in particular when diagnosing is by positron emission tomography.

19. Composition for use according to claim 18, wherein the tauopathy is 3R tauopathy.

20. Composition for use according to claim 18, wherein the tauopathy is 4R tauopathy.

21. Composition for use according to claim 18, wherein the disorder is selected from the following: Alzheimer's disease (AD), familial AD disease, Creutzfeldt-Jakob disease, boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, inclusion body myositis , prion protein-induced cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis, Parkinsonism-dementia-Guam complex, motor neuron disease with non-Guam-type neurofibrillary tangles, argyrophilic granule disease, corticobasal degeneration (CBD) , diffuse neurofibrillary tangles with calcification, chromosome 17-linked frontotemporal dementia with parkinsonism, Hallervorden-Spatz disease, multisystem atrophy, Niemann-Pick type C disease, pallido-pontonic degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurofibrillary tangle-predominant dementia, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadalupe parkinsonism, accumulation neurodegeneration

- 3 042728 iron in the brain, mental retardation associated with SLC9A6, white matter tauopathy with globular glial inclusions, traumatic stress syndrome, epilepsy, dementia with Lewy bodies (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type), moderate cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, atypical parkinsonism, HIV-associated dementia, mid-life diabetes, senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)) , optic disc drusen, optic neuropathy, optic neuritis and lattice degeneration; preferably Alzheimer's disease.

22. A composition for use according to claim 18, wherein the disorder is selected from Huntington's disease, ischemic stroke, and AD psychosis.

23. Composition for use according to claim 21 wherein the disorder is Alzheimer's disease (AD).

24. Composition for use according to claim 21, wherein the disorder is Parkinson's disease or atypical parkinsonism.

25. Composition for use according to claim 21, wherein the disorder is progressive supranuclear palsy (PSP).

26. Composition for use according to claim 21 wherein the disorder is Pick's disease (P1D).

27. A composition for use according to any one of claims 18-26, wherein tau aggregates are visualized in the brain or eye.

28. A method for imaging tau aggregates, in particular, a method for imaging tau aggregates by positron emission tomography, wherein an effective amount of the composition defined in claim 12 is administered to the patient.

29. A method for diagnosing a disorder associated with tau aggregates or tauopathy, wherein the patient is administered an effective amount of the composition defined in claim 12, in particular when the diagnosis is carried out using positron emission tomography.

30. The method according to claim 29, wherein the tauopathy is 3R tauopathy.

31. The method according to claim 29, wherein the tauopathy is 4R tauopathy.

32. The method according to claim 29, wherein the disorder is selected from the following: Alzheimer's disease (AD), familial AD disease, Creutzfeldt-Jakob disease, boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, inclusion body myositis, cerebral prion protein-induced amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinsonism-dementia-Guam complex, motor neuron disease with non-Guam-type neurofibrillary tangles, disease characterized by the appearance of argyrophilic granules, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, fronto Chromosome 17-linked temporal dementia with parkinsonism, Hallervorden-Spatz disease, multisystem atrophy, Niemann-Pick type C disease, pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis , tangle-predominant dementia, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadalupe parkinsonism, neurodegeneration with iron accumulation in the brain, mental retardation associated with SLC9A6, 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-associated dementia, mid-life diabetes, senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic disc drusen, optic neuropathy, optic neuritis and lattice degeneration; preferably Alzheimer's disease.

33. The method according to claim 29, wherein the disorder is selected from Huntington's disease, ischemic stroke, and AD psychosis.

34. The method according to claim 32, wherein the disorder is Alzheimer's disease (AD).

35. The method according to claim 32, wherein the disorder is Parkinson's disease or atypical parkinsonism.

36. The method according to claim 32, wherein the disorder is progressive supranuclear palsy (PSP).

37. The method according to claim 32, wherein the disorder is Pick's disease (PID).

38. The method according to any one of claims 28-37, wherein tau aggregates are visualized in the brain or eye.

39. The use of the composition defined in claim 12 in the manufacture of a diagnostic agent for

- 4 042728 visualization of tau aggregates, in particular for visualization of tau aggregates using positron emission tomography.

40. The use of a composition as defined in claim 12 in the manufacture of a diagnostic agent for diagnosing a disorder associated with tau aggregates or for diagnosing tauopathy, in particular when the diagnosis is by positron emission tomography.

41. Use according to claim 40, wherein the tauopathy is 3R tauopathy.

42. Use according to claim 40, wherein the tauopathy is 4R tauopathy.

43. Use according to claim 40, wherein the disorder is selected from the following: Alzheimer's disease (AD), familial AD disease, Creutzfeldt-Jakob disease, Boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, inclusion body myositis, cerebral prion protein-induced amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinsonism-dementia-Guam complex, motor neuron disease with non-Guam-type neurofibrillary tangles, disease characterized by the appearance of argyrophilic granules, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, fronto Chromosome 17-linked temporal dementia with parkinsonism, Hallervorden-Spatz disease, multisystem atrophy, Niemann-Pick type C disease, pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis , tangle-predominant dementia, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, frontotemporal degeneration, Guadalupe parkinsonism, neurodegeneration with iron accumulation in the brain, mental retardation associated with SLC9A6, 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-associated dementia, mid-life diabetes, senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration (such as age-related macular degeneration (AMD)), optic disc drusen, optic neuropathy, optic neuritis and lattice degeneration; preferably Alzheimer's disease.

44. Use according to claim 40, wherein the disorder is selected from Huntington's disease, ischemic stroke, and AD psychosis.

45. Use according to claim 43, where the disorder is Alzheimer's disease (AD).

46. Use according to claim 43, where the disorder is Parkinson's disease or atypical parkinsonism.

47. Use according to claim 43, where the disorder is progressive supranuclear palsy (PSP).

48. Use according to claim 43, where the disorder is Pick's disease (PiD).

49. Use according to any one of claims 39-48, wherein tau aggregates are visualized in the brain or eye.

50. Use of the composition according to claim 12 as an analytical standard.

51. Use of the composition according to claim 12 as an in vitro screening tool.

52. A method for collecting data for diagnosing a disorder associated with tau aggregates in a sample or in a patient, including:

(a) bringing the sample or specific body part or area of the body suspected of containing a tau-aggregate into contact with a composition defined in paragraph 12, which contains a compound of formula I;

(b) allowing the compound of formula I to contact the tau-aggregate;

(c) detection of a compound of formula I associated with a tau aggregate; and (d) optionally correlating the presence or absence of a tau-aggregate-binding compound of Formula I with the presence or absence of a tau-aggregate in the sample or a particular body part or region of the body.

53. A method for determining the amount of tau aggregates in tissues and/or body fluids, including:

(a) providing a sample representative of the tissue and/or biological fluid under investigation;

(b) examining the sample for the presence of a tau aggregate with a composition defined in paragraph 12, which contains a compound of formula I;

(c) determining the amount of the compound of formula I associated with the tau-aggregate; and (d) calculating the amount of tau aggregate in the tissue and/or body fluid.

54. A method for collecting data to determine a susceptibility to a tau-aggregate disorder in a patient, comprising detecting specific binding of a composition defined in claim 12, which contains a compound of formula I, to a tau-aggregate in a sample or in situ, comprising the steps of:

- 5 042728 (a) bringing a sample or a certain part of the body or area of the body, suspected to contain a tau-aggregate, in contact with the composition defined in paragraph 12, which contains a compound of formula I, which specifically binds to the tau-aggregate;

(b) allowing a compound of formula I to bind to a tau aggregate to form a compound/tau aggregate complex;

(c) detecting compound/tau-aggregate complex formation;

(d) optionally correlating the presence or absence of a compound/tau-aggregate complex with the presence or absence of a tau-aggregate in the sample or a specific body part or region of the body; and (e) optionally comparing the amount of compound/tau aggregate with a normal control value.

55. A method for collecting data for monitoring a residual disorder in a patient suffering from a disorder associated with tau aggregates who was treated with a drug, where the method includes:

(a) bringing the sample or specific body part or area of the body suspected of containing a tau aggregate into contact with a composition defined in claim 12 that contains a compound of formula I that specifically binds to a tau aggregate;

(b) allowing a compound of formula I to bind to a tau aggregate to form a compound/tau aggregate complex;

(c) detecting compound/tau-aggregate complex formation;

(d) optionally correlating the presence or absence of a compound/tau-aggregate complex with the presence or absence of a tau-aggregate in the sample or a specific body part or region of the body; and (e) optionally comparing the amount of compound/tau aggregate with a normal control value.

56. A method for collecting data to predict the susceptibility of a patient suffering from a disorder associated with tau aggregates and receiving treatment with a drug, including:

(a) bringing a sample or a certain part of the body or area of the body suspected of containing a tau aggregate into contact with a composition defined in paragraph 12, which contains a compound of formula I that specifically binds to a tau aggregate;

(b) allowing a compound of formula I to bind to a tau aggregate to form a compound/tau aggregate complex;

(c) detecting compound/tau-aggregate complex formation;

(d) optionally correlating the presence or absence of a compound/tau-aggregate complex with the presence or absence of a tau-aggregate in the sample or a specific body part or region of the body; and (e) optionally comparing the amount of compound/tau aggregate with a normal control value.

Definitions

The term alkyl refers to a saturated straight or branched carbon chain which, unless otherwise indicated, contains from 1 to 6 carbon atoms.

Hal or halogen is F, Cl, Br and I. Preferably halogen is independently at each occurrence selected from F, Cl and Br, more preferably F and Cl, even more preferably F.

The term amino protecting group (PG), as used herein, means any protecting group that is suitable for protecting an amino group during an intended chemical reaction. Examples of suitable protecting groups are well known to those skilled in the art. Suitable protecting groups are discussed, for example, in Greene and Wuts, Protecting groups in Organic Synthesis, third edition, p. 494-653, which is incorporated herein by reference. The protecting groups may be selected from carbamates, amides, imides, N-alkylamines, N-arylamines, imines, enamines, boranes, N-P protecting groups, N-sulphenyl, N-sulfonyl and N-silyl. Specific preferred examples of protecting groups (PG) are carbobenzyloxy (Cbz), (pmethoxybenzyl)oxycarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), triphenylmethyl (trityl), methoxyphenyldiphenylmethyl (MMT) or dimethoxytrityl (DMT). More preferred examples of the PG protecting group include tert-butyloxycarbonyl (BOC), dimethoxytrityl (DMT), and triphenylmethyl (trityl). Another preferred example of a PG protecting group is tert-butyloxycarbonyl (BOC).

The term carbamate amine protecting group refers to an amine protecting group containing a *-CO-O group, where the asterisk denotes a bond to the amine. Examples are carbobenzyloxy (Cbz), (p-methoxybenzyl)oxycarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC) and 9fluorenylmethyloxycarbonyl (FMOC).

The term leaving group (LG) as used herein means 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, for example, in the reviews Synthesis (1982), p. 85-125, tab. 2, Carey and Sundberg, Organische Synthese, (1995), p. 279-281, tab. 5.8; or Netscher, Recent Res. dev. Org. Chem., 2003, 7, 71-6 042728

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. 15-50, in detail: Scheme 4, S. 25, Scheme 5, S. 28, Table 4, S. 30, Fig. 7, S. 33). Preferably the leaving group (LG) is selected from the group consisting of nitro, bromine, iodine, chlorine, trialkylammonium, hydroxyl, boronic acid, iodonium, sulfonic acid ester. More preferably the leaving group (LG) is nitro or trimethylammonium. It should be understood that compounds containing trialkylammonium or iodonium may additionally contain an anion. Even more preferably the leaving group (LG) is nitro. Another more preferred leaving group (LG) is trimethylammonium.

The term crown ether, as used herein, means chemical compounds that consist of a ring containing several ether groups. More specifically, the term crown ether preferably refers to monocyclic organic groups which may 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 one or more optional substituents may be independently selected from any organic group containing from 1 to 15 carbon atoms and optionally from 1 to 6 heteroatoms selected from N, O and S. Preferred examples of crown ether are optionally substituted monocyclic rings containing from 10 to 14 carbon atoms and 5 to 7 heteroatoms selected from N, O and S in the ring. Examples of crown ethers 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. Preferred examples of crown ether are optionally substituted monocyclic rings containing 10 to 14 carbon atoms and 5 to 7 heteroatoms selected from N, O and S in the ring. Examples of crown ether are optionally substituted monocyclic rings containing 12 carbon atoms and 6 heteroatoms selected from N and O in the ring, and diaza-18-crown-6.

The term cryptand as used herein refers to a class of crown ether polycyclic compounds having three chains attached to two nitrogen atoms. A well-known cryptand is 4,7,13,16,21,24-hexaoxy-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix®).

The term tau, as used herein, refers to a highly soluble microtubule-binding protein primarily found in neurons and includes the main 6 isoforms, cleaved or truncated forms, and other modified forms such as those resulting from phosphorylation, glycosylation, glycation, prolyl -isomerization, nitration, acetylation, polyamination, ubiquitination, sumoylation and oxidation. Pathological tau or tau aggregates (neurofibrillary tangles, NFT) as used herein refers to insoluble aggregates of hyperphosphorylated tau protein refers to insoluble aggregates of hyperphosphorylated tau protein containing paired helical filaments and straight filaments. Their presence is a sign of AD and other diseases known as tauopathy.

The tau gene contains 16 exons, with the main isoforms of the tau protein encoded by 11 of them. Alternative splicing of exon 10 generates tau isoforms with three (exon 10 absent) or four (exon 10 present) repeat domains known as 3R and 4R tau, respectively (A. Andreadis et al., Biochemistry 31, (1992) 10626-10633; M Tolnay et al., IUBMB Life, 55(6): 299-305, 2003). In Alzheimer's disease, the ratio of 3R and 4R isoforms is similar. In contrast, some tauopathies predominantly present one of the two isoforms. As used herein, the term 3R tauopathy refers to tauopathies (such as Pick's disease (PiD)) in which the 3R isoform is predominantly present. As used herein, the term 4R tauopathy refers to tauopathies (such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD)) in which the 4R isoform is predominantly present.

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 described compounds, where the parent compound is modified by obtaining its salts of 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 a parent compound that contains a basic or acidic group by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or mixtures thereof. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, which is incorporated herein by reference.

Pharmaceutically acceptable or diagnostically acceptable are defined as those compounds, substances, compositions and/or dosage forms which, within the limits of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The patients or subjects of the present invention are typically animals, especially mammals, more specifically humans.

Chromatography or liquid chromatography means a method of separating a mixture of compounds. The mixture is dissolved in a liquid and transported via the mobile phase through the stationary phase. The separation is based on the interaction of the compounds contained in the mobile phase with the stationary phases. These different interactions lead to differential confinement in 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 performed on a small sample of material and is used to determine the proportions of compounds in a mixture.

High performance liquid chromatography (HPLC) is a type of liquid chromatography for separating compounds using very fine stationary phase particles (<10 µm) and applying fairly high pressures. An HPLC system typically consists of a reservoir of mobile phase(s), a pump, an injector, a separator column (containing the stationary phase), and detectors. For the 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.

Solid phase extraction (SPE) is a process for obtaining and/or purifying a sample in 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 the stationary (solid) phase. Some compounds remain in the stationary phase while others pass through. In the second step, the remaining compounds are eluted with a suitable solvent. Optionally, the stationary phase is washed with another solution before the elution step. In contrast to the HPLC method, the particle size used is much larger (eg >25 µm compared to HPLC with a typical particle size of <10 µm) and therefore the applied pressure is much lower (for HPLC the pressure is typically >50 bar).

A solid phase extraction cartridge (SPE cartridge) is a syringe or container (eg Sep Pak®) pre-filled with SPE stationary phase.

Sterile filtration is a method of sterilizing a solution by filtering through a microfilter. A microfilter is a filter having, for example, a pore size of about 0.25 µm or less, preferably from about 20 nm to about 0.22 µm, which is commonly used to remove microorganisms. Membrane filters used in microfiltration in industrial processes are typically made from materials such as mixed cellulose ester, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), or polyethersulfone (PES).

The term automated as used herein means carrying out the steps of synthesis and/or purification on a suitable device (synthesizer).

The term radio absorber refers to a compound that reduces the rate of degradation due to radiolysis. Preferred radioabsorbents include ascorbic acid and its salts and gentisic acid and its salts. More preferred radio absorbers are ascorbic acid, sodium ascorbate and mixtures thereof.

Suitable F-labeling synthesizers 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, Comecer Taddeo, Raytest Synchrom, Sofie Elixys, Eckert&Ziegler Modular Lab, Sumitomo Heavy Industries F100 F200 F300, Siemens Explora.

Radiochemical purity refers to the proportion of the total activity of a radionuclide present in its identified chemical form. Typically, radiochemical purity is determined by thin layer chromatography or HPLC.

The preferred definitions given in the Definitions section apply to all embodiments described herein unless otherwise noted.

Methods of the invention

In a first aspect, the present invention relates to a process for the preparation of a compound of formula I

- 8 042728

This method includes the steps:

A) interaction of the compound of formula II with ¹⁸ F fluorinating agent

where X is H or PG;

LG is a drop group; And

PG is an amino protecting group, and

B) optionally, if X is PG, cleavage of the PG protecting group, and

C) subjecting the resulting compound of formula I to high performance liquid chromatography (HPLC) using a mobile phase containing ethanol and water.

Preferred compounds of formula I are selected from the group consisting of

In these compounds, PG and LG have the meanings defined in the Definitions section. Even more preferred compounds of formula II are selected from the group consisting of

where X ^- represents a counterion, such as a counterion selected from the group including halogen, CF3SO3 ^- and CF3CO2 ^- .

- 9 042728

Stage A.

Step A involves reacting a compound of formula II with a ¹⁸ F fluorinating agent

/

X

II where X is H or PG;

LG is a drop group and

PG is an amino protecting group.

If X is H, a compound having formula I will be obtained. If X is PG, an intermediate compound having formula III will be obtained

PG

III

F fluorinating agents are well known to the person skilled in the art. Any suitable ¹⁸ F-fluorinating agent may be used. Representative examples include H ¹⁸ F, alkaline or alkaline earth ¹⁸ F-fluorides (eg K ¹⁸ F, Rb ¹⁸ F, Cs ¹⁸ F and Na ¹⁸ F). Optionally, ^{a 18} F-fluorinating agent can be used in combination with a chelating agent such as cryptand (eg: 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosan - Kryptofix ®) or crown ether (for example: 18-crown-6). Alternatively, ^{the 18} F-fluorinating agent may be a ¹⁸ F tetraalkylammonium salt or a ¹⁸ F tetraalkylphosphonium salt; for example, tetra(€1-6 alkyl)ammonium salt ¹⁸ F or tetra(€1-6 alkyl)phosphonium salt ¹⁸ F. Examples thereof include tetrabutylammonium [ ¹⁸ F]fluoride and tetrabutylphosphonium [ ¹⁸ F]fluoride. Preferably ^{the 18} F-fluorinating agent is K ¹⁸ F, H ¹⁸ F, Cs ¹⁸ F, Na ¹⁸ F or tetrabutylammonium [ ¹⁸ F]fluoride. In an even more preferred embodiment, ^{the 18} F-fluorinating agent is K ¹⁸ F. In another more preferred embodiment, ^{the 18} F-fluorinating agent is tetrabutylammonium [ ¹⁸ F]fluoride.

¹⁸ F-fluorination is usually carried out in a solvent which is preferably selected from acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, amyl alcohol, t-butyl alcohol or mixtures thereof, preferably the solvent contains or is acetonitrile or DMSO. But other solvents which are well known to the person skilled in the art can also be used. The solvent may additionally contain water and/or other alcohols such as C _1-6 linear, branched or cyclic alkanols as a co-solvent. In one preferred embodiment, the ¹⁸ F radioactive labeling solvent comprises dimethyl sulfoxide. In another preferred embodiment, the ¹⁸ F radioactive labeling solvent comprises acetonitrile. In one preferred embodiment, the ¹⁸ F radiolabelling solvent is dimethyl sulfoxide. In another preferred embodiment, the ¹⁸ F radiolabelling solvent is acetonitrile.

¹⁸ F-fluorination is typically carried out for about 60 minutes. The preferred reaction time is a maximum of about 30 minutes. A further preferred reaction time is at most about 15 minutes. A typical reaction time is about 1-15 minutes, preferably 5-15 minutes, more preferably 10-15 minutes.

¹⁸ F-fluorination is typically carried out at a temperature of from about 60 to about 200°C with conventional heating or heating using microwave radiation. In a preferred embodiment, ¹⁸ F-fluorination is carried out at a temperature of from about 100 to about 180°C. In a more preferred embodiment, ¹⁸ F-fluorination is carried out at a temperature of from about 100 to about 160°C. Preferably, ^{the 18} F-fluorination is carried out under normal heating. Conventional heating refers to any heating without the use of microwaves.

The amount of starting material is not particularly limited. For example, from about 0.5 to about 50 μmol of the compound of formula II can be used in one batch to prepare a compound of formula I. In a preferred embodiment of the invention, about 2 to about 25 µmol of a compound of formula II is used. In a more preferred embodiment of the invention, about 2.5 to about 15 µmol of a compound of formula II is used. In one embodiment of the invention, at least about 2 μmol of a compound of formula II is used. In a preferred embodiment of the invention, at least about 2.5 µmol of a compound of formula II is used. In a more preferred embodiment of the invention, at least about 3 μmol of a compound of formula II is used.

Typically, from about 0.5 to about 10 mg of a compound of formula II can be used to prepare a compound of formula I in one batch. In the preferred embodiment of the invention

- 10 042728 are used from about 0.5 to about 5 mg of the compounds of formula II. In a more preferred embodiment of the invention, about 1 to about 3 mg of a compound of formula II is used.

If X is PG, an intermediate having formula III will be obtained.

The PG protecting group may be cleaved off in step A or in an optional subsequent step B.

Preferred compounds of formula III are selected from the group consisting of

In these compounds, PG has the meanings defined in the Definitions section.

Stage B.

Step B. is an optional step that involves cleavage of the PG protecting group from a compound of formula III to give a compound of formula I.

As will be apparent to one skilled in the art, this step is not applicable if step A is carried out with a compound of formula II in which X is hydrogen, or if the PG protecting group has already been cleaved off in step A.

Cleavage reaction conditions for a wide variety of protecting groups are well known to those skilled in the art and may be selected, but not limited to, from those described in Greene and Wuts, Protecting groups in Organic Synthesis, third edition, p. 494-653, and a review by P. J. Kocienski, Protecting Groups, 3rd Edition 2003, both of which are incorporated herein by reference.

The conditions used in Step B will depend on the protecting group to be cleaved off, and thus are not specifically limited.

Possible reaction conditions include: i) heating at about 60 to about 160°C, ii) adding acid and heating at about 0 to about 160°C, or iii) adding base and heating from about 0 to about 160°C .

Preferred acids are hydrochloric acid, sulfuric acid and phosphoric acid. One preferred acid is sulfuric acid. Another preferred acid is phosphoric acid. Another preferred acid is hydrochloric acid. Preferred bases are sodium hydroxide, potassium hydroxide.

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

If desired, steps A and B can be performed in the same or different reaction vessels. Preferably, steps A and B are carried out in the same reaction vessel.

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 to be more suitable for HPLC. For example, a buffer or diluent may be added prior to step C.

Preferred diluents are ethanol, water, or a combination thereof.

In addition, the pH of the solution can be adapted. In a preferred embodiment of the invention, before step C, the pH is adjusted to about 6 or less, more preferably about 4 or less, or even more preferably about 3 or less. In a preferred embodiment, before step C, 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.

Stage C.

Stage C is a stage in which the compound of formula I obtained in stage A or, if used, stage B, is subjected to HPLC using a mobile phase containing ethanol and water.

In the prior art, it has been suggested that two chromatographic steps must be used to obtain an injectable indicator, i.e., first purification by HPLC and then re-treatment with SPE of the compound of formula I. The inventors of the present invention unexpectedly found that if the mobile phase is mixture of ethanol and water, subsequent trapping of the compound by solid phase extraction can be avoided. Therefore, a single chromatographic step can be used for purification and preparation. This is especially important in the present case of radiofluorination, since the half-life of ¹⁸ F is only about 110 minutes, so the speed of the process for obtaining an injectable composition of compound I is of paramount importance.

Because the currently claimed method is faster and less complicated than

- 11 042728 previous methods, the compound of formula I can be obtained in higher yield without correction for radioactive decay.

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, which are used in the prior art) they can remain in the composition, which is administered to the patient. Therefore, the choice of ethanol and water as the mobile phase significantly reduces the time and cost required to prepare the compound of formula I and/or gives higher yields and higher radiochemical purity than prior art methods.

The method of the present invention preferably does not include solid phase extraction of the compound of formula I after step C, more preferably the method of the present invention does not include solid phase extraction of the compound of formula I before or after step C.

The compound of formula I is preferably not chromatographed after the high performance liquid chromatography (HPLC) step C, more preferably the compound of formula I is not chromatographed other than the high performance liquid chromatography (HPLC) step C.

The mobile phase used in the HPLC process contains ethanol and water. In addition, an acid, a base, a buffer, a salt and/or a radio absorber, and possibly mixtures thereof, may be included.

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

The pH of the mobile phase is not limited, 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.

Possible buffers may include salts which may be selected from alkali metal dihydrogen phosphates, two alkali hydrogen phosphates, three alkali metal phosphates, alkali metal acetates, alkaline earth metal acetates, alkaline earth metal formates, one, two or three alkali citrate. metal, where the preferred alkali and alkaline earth metals are sodium and potassium.

Possible bases may be sodium hydroxide and/or potassium hydroxide.

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

Radioabsorbers are compounds which reduce the degradation of a compound of formula I by radiolysis. Examples include ascorbic acid and ascorbic acid salts, gentisic acid and gentisic acid salts. Additional examples include citric acid and salts of citric acid. More preferred radio absorbers are ascorbic acid, sodium ascorbate and mixtures thereof.

Preferably, the mobile phase contains from about 50 to about 500 mM buffer (eg, alkali metal dihydrogen phosphate) with a pH of about 1 to about 3, more preferably from about 50 to about 250 mM buffer (eg, alkali metal dihydrogen phosphate) with a pH of about 1 up to about 3, even more preferably from about 50 to about 150 mM buffer (for example, alkali metal dihydrogen phosphate) with a pH of from about 1 to about 3.

The preferred mobile phase contains from about 5 to about 20% vol./about. ethanol, from about 95 to about 80% vol./about. water, about 50 to about 150 mM buffer (eg, alkali metal dihydrogen phosphate) with a pH of about 1 to about 3, and optionally a radio absorber. A more preferred mobile phase contains from about 5 to about 20% vol./about. ethanol, from about 95 to about 80% vol./about. water, about 50 to about 150 mM buffer (eg, alkali metal dihydrogen phosphate) with a pH of about 2.2 to about 2.8, and optionally a radio absorbent.

Stationary phases for use in HPLC methods are well known and may be appropriately selected by one skilled in the art. In a preferred embodiment, the stationary phase is a reverse phase (RP) stationary phase.

Examples of RP-HPLC stationary phases include C18, C8, phenyl, cyano (eg, cyanopropyl), pentafluorophenyl, amino (eg, aminopropyl), amide (eg, C ₁₀ .2 ₄ -alkanoaminopropyl), phenylhexyl-functionalized resins, or resins with mixed phase.

In one embodiment of the invention, the particle size of the HPLC stationary phase is from about 1.6 to about 15 microns. In a preferred embodiment of the invention, the particle size of the HPLC stationary phase is from about 5 to about 10 microns. In another embodiment of the invention, the particle size of the HPLC stationary phase is about 10 microns.

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. More preferably

- 12 042728 implementation of the invention HPLC column has a size of 10x250 mm.

The flow rate used in high performance liquid chromatography is not limited and may be from about 1 to about 20 ml/min, more typically from about 2 to about 15 ml/min, even more typically from about 2 to about 7 ml/min.

The pressure used in high performance liquid chromatography is not particularly limited and may be in the range of about 50 to about 400 bar, typically about 50 to about 250 bar, more typically about 50 to 200 bar.

In one embodiment of the invention, from about 1 to about 500 GBq of [ ¹⁸ F] fluoride are used to prepare a compound of formula I. In a preferred embodiment of the invention, about 50 to about 500 GBq of [ ¹⁸ F]fluoride are used to prepare a compound of formula I. In a more preferred embodiment of the invention, about 100 to about 500 GBq of [ ¹⁸ F]fluoride are used to prepare a compound of formula I. In an even more preferred embodiment of the invention, about 200 to about 500 GBq of [ ¹⁸ F]fluoride are used to prepare a compound of formula I.

In one embodiment of the invention, about 10 GBq or more of [ ¹⁸ F]fluoride is used to prepare a compound of formula I. In a preferred embodiment of the invention, about 50 GBq or more of [ ¹⁸ F]fluoride is used to prepare a compound of formula I. In a more preferred embodiment of the invention, about 100 GBq or more of [ ¹⁸ F]fluoride is used to prepare a compound of formula I. In an even more preferred embodiment of the invention, about 200 GBq or more of [ ¹⁸ F]fluoride is used to prepare a compound of formula I.

In one embodiment, about 10 GBq or more of a radiolabeled compound of formula I is produced. In a preferred embodiment, about 20 GBq or more of a radiolabeled compound of formula I is obtained. In a more preferred embodiment, about 50 GBq or more of a radiolabeled compound of formula I is obtained. In an even more preferred embodiment, about 100 GBq or more of the radiolabeled compound of formula I is obtained.

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

Optional steps.

Since ethanol and water are contained in the mobile phase, the HPLC fraction containing the compound of formula I can be used directly as an injection, if desired. Alternatively, the HPLC fraction containing the Formula I compound may be mixed with other diagnostically acceptable ingredients, such as a diagnostically acceptable carrier, diluent, adjuvant, or excipient, to prepare an injectable composition of the Formula I compound.

If step D is desired, sterile filtration may be performed after step C. If additional components are added, sterile filtration may be carried out before or after their addition.

In one embodiment, step A, optional step B, and step C are performed using an automated synthesis device. Examples of such automatic 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, Comecer Taddeo, Raytest Synchrom, Sofie Elixys, Eckert&Ziegler Modular Lab, Sumitomo Heavy Industries F100 F200 F300, and Siemens Explora.

One preferred method includes the following steps:

A) reaction of the compound of formula II, where LG=hutpo and PG=Boc, with [ ¹⁸ F]-fluorinating agent, preferably selected from K ¹⁸ F and tetrabutylammonium [ ¹⁸ F]fluoride, in DMSO at a temperature of from about 100 to about 180° C, preferably from about 120 to about 180°C, more preferably from about 140 to about 160°C, where the Boc protecting group is cleaved under radioactive labeling conditions,

C) HPLC purification of a compound of Formula I using an ethanol/sodium dihydrogen phosphate (about 5 to about 20% EtOH) buffer, wherein the buffer has a pH of about 1 to about 3, preferably about 2.2 to about 2.8 , And,

D) if desired, mixing the HPLC fraction containing the compound of formula I with additional diagnostically acceptable formulation components to be administered to the patient. If desired, the HPLC fraction is passed through a sterile filter before or after mixing with other diagnostically acceptable formulation components.

Another preferred method includes the following steps:

A) reaction of a compound of formula II, where LG=trimethylammonium and PG=trityl, with [ ¹⁸ F]-

- 13 042728 fluorinating agent, preferably selected from K ¹⁸ F and tetrabutylammonium [ ¹⁸ F]fluoride, in

DMSO or acetonitrile at about 80 to about 180°C, preferably from about 100 to about 180°C,

B) adding phosphoric acid and heating at about 90 to about 120° C. for about 1 to about 15 minutes,

C) HPLC purification of a compound of Formula I using an ethanol/sodium dihydrogen phosphate (about 5 to about 20% EtOH) buffer, wherein the buffer has a pH of about 1 to about 3, preferably about 2.2 to about 2.8 , And,

D) if desired, mixing the HPLC fraction containing the compound of formula I with additional diagnostically acceptable formulation components to be administered to the patient. If desired, the HPLC fraction is passed through a sterile filter before or after mixing with other diagnostically acceptable formulation components.

Unless otherwise indicated, each hydrogen in the compounds of formulas I-III may be 'll or ² H (deuterium).

The method of the present invention may provide a diagnostic composition containing a compound of formula I. In view of the rapidity and reduced complexity of the present method, the amount of N-labeled compound I may be higher than in conventional methods. This method also provides a compound of formula I with 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 (eg, at least about 20 GBq of a Formula I compound, preferably at least about 50 GBq of a Formula I compound, more preferably at least about 100 GBq of a Formula I compound).

Soluble diagnostic compositions are suitable for use in diagnostics. They are particularly suitable for diagnosing a tau-associated disorder or for imaging tau-aggregates, especially for imaging tau-aggregates using positron emission tomography (PET).

diagnostic compositions.

A diagnostic composition is defined in the present invention as a composition containing a compound of formula I. For in vivo applications, the diagnostic composition should be in a form suitable for administration to a mammal, such as a human. Preferably, the diagnostic composition further comprises a physiologically acceptable carrier, diluent, adjuvant, or excipient. Administration to a patient is preferably by injecting the composition as a solution.

Such a composition may optionally contain additional ingredients such as solvents, buffers; diagnostically acceptable solubilizers; and diagnostically acceptable stabilizers or antioxidants.

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 may be selected in accordance with standard pharmaceutical practice. The excipient must be acceptable in the sense that it is not harmful to the recipient.

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

The compounds of formula I should be administered by injection. Examples of such administration include one or more of the following: intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, or subcutaneous administration of the compounds; and/or using infusion methods. For parenteral administration, the compounds are best used as a sterile solution which may contain other excipients. Solutions should be suitably buffered (preferably pH 3 to 9, more preferably 4.0 to 8.5) if necessary. The preparation of suitable parenteral compositions under sterile conditions is readily accomplished by standard pharmaceutical methods well known to those skilled in the art.

Diagnostic compositions of the invention may be formulated in a manner known per se to one of skill in the art, as described, for example, in Remington's Pharmaceutical Sciences, ^15th Ed., Mack Publishing Co., New Jersey (1975).

The dose of detectable labeled compounds of Formula I may vary depending on the particular compound being administered, patient weight, sample size and type, and other variables, as will be apparent to those skilled in the art. Generally, the weight may preferably be in the range of about 0.001 to about 100 micrograms per patient dose, preferably about 0.01 to about 50 micrograms per patient dose. The radioactive dose may be, for example, from about 100 to about 600 MBq, more preferably from about 150 to about 450 MBq per injection, even more preferably from about 150 to about 200 MBq.

In particular, in one embodiment, the diseases or disorders that can be detected and tracked using detectable labeled compounds of formula I are diseases or conditions associated with tau protein aggregates, such as 3R or 4R tauopathies.

Diseases or conditions that can be detected and tracked using detectable labeled compounds obtained by the method of the present invention include neurodegenerative disorders such as tauopathies. Examples of diseases and conditions that can be detected and controlled are caused by or associated with the formation of neurofibrillary lesions. This is the predominant brain pathology in tauopathy. The diseases and conditions include a heterogeneous group of neurodegenerative diseases or conditions, including diseases or conditions that show the coexistence of tau and amyloid pathologies. Examples of diseases involving tau aggregates are commonly listed as tauopathies, and they include, but are not limited to, the following: Alzheimer's disease (AD), Creutzfeldt-Jakob disease, boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, myositis with inclusion bodies, prion-induced cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinsonism-dementia-Guam complex, motor neuron disease with non-Guam-type neurofibrillary tangles, argyrophilic granule disease, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with parkinsonism, chromosome 17, Hallervorden-Spatz disease, multisystem atrophy, Niemann-Pick type C disease, pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, neurofibrillary tangle-predominant dementia, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia, fronto Temporal degeneration, Guadalupe parkinsonism, neurodegeneration with iron accumulation in the brain, mental retardation associated with SLC9A6, 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-associated dementia, mid-life diabetes, senile cardiac amyloidosis, endocrine tumor, glaucoma, eye amyloidosis, primary retinal degeneration, macular degeneration ( such as age-related macular degeneration (AMD)), optic disc drusen, optic neuropathy, optic neuritis, and lattice dystrophy. Preferably diseases or conditions that can be detected and monitored include Alzheimer's disease (AD), familial AD disease, Creutzfeldt-Jakob disease, boxer's dementia, Down's syndrome, Gerstmann-Straussler-Scheinker disease, myositis with included bodies, cerebral amyloid angiopathy, prion protein-induced traumatic brain injury (TBI), amyotrophic lateral sclerosis, parkinsonism - dementia - Guam complex, motor neuron disease with non-Guamtip neurofibrillary tangles, argyrophilic granule disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, Chromosome 17-linked frontotemporal dementia with parkinsonism, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick type C disease, pallido-ponto-nigral degeneration, Pick's disease (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP) , subacute sclerosing panencephalitis, neurofibrillary tangle-dominated dementia, postencephalitic parkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like with astrocytes, some prion diseases (GSS with Tau), mutations in LRRK2, chronic traumatic encephalopathy, familial British dementia, familial Danish dementia , frontotemporal degeneration, Guadalupe parkinsonism, neurodegeneration with iron accumulation in the brain, mental retardation associated with SLC9A6 and white matter tauopathy with globular glial inclusions, more preferably Alzheimer's disease (AD), Creutzfeldt-Jakob disease, boxer's dementia, amyotrophic lateral sclerosis, argyrophilic granule disease, corticobasal degeneration, chromosome 17-linked frontotemporal dementia with parkinsonism, Pick's disease, progressive supranuclear palsy (PSP), tangle-predominant dementia, Parkinsonism-dementia-Guam complex, Hallervorden-Spatz disease, and frontotemporal degeneration. Preferably the disease or condition is Alzheimer's disease, Parkinson's disease or atypical parkinsonism, progressive supranuclear

- 15 042728 paralysis (PSP or Pick's disease (PiD), more preferably Alzheimer's disease.

Additional examples of diseases or conditions that can be detected and tracked using detectable labeled compounds obtained by the method of the present invention include Huntington's disease, ischemic stroke and psychosis in AD.

The method of the present invention has a number of important advantages over prior art methods. Because only one chromatography step is required after the preparation of a compound of formula I without subsequent modification by SPE, the setup is much simpler than prior art setups that run two different chromatography steps.

The method proved to be very reliable. Due to the choice of ethanol and water as the pH controlled mobile phase, large amounts (eg >1 mg) of the precursor can be used without precipitation.

The F-labeled compound of formula I can be reliably separated from the precursor compound of formula II and possible by-products.

In addition, high yields and purity can be achieved. For example, uncorrected for radioactive decay yields of at least about 35% can be obtained. The activity of the product may be at least about 20 GBq, preferably at least about 50 GBq, more preferably at least about 100 GBq. The radiochemical purity may be at least about 95%, preferably at least about 98% at both low and high levels of radioactivity (eg, at least about 100 GBq).

The present invention is illustrated by the following examples, which should not be considered as limiting.

Examples

Abbreviations.

AD Alzheimer's disease Vos, VOS mpem-butyloxycarbonyl CBD corticobasal degeneration d.c. corrected for radioactive decay d doublet dd doublet of doublets DMF Ν,Ν-dimethylformamide DMSO dimethyl sulfoxide DMTrt-Cl 4,4 '-(chloro(phenyl)methylene)bis(methoxybenzene) dppf 1,1'-bis(diphenylphosphino)ferrocene EI electron ionization ELSD evaporative light scattering detector EOS Completion of the synthesis ESI electrospray ionization EtOH ethanol FTD frontotemporal dementia HPLC high performance liquid chromatography HC control group of healthy people GBq Gigabequerel to ₂₂₂ 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222) MBq Megabequerel MC mass spectrometry MeCN acetonitrile M multiplet me centered multiplet n.c.a. carrier-free n.d.c. without correction for radioactive decay NMR nuclear magnetic resonance spectroscopy: chemical shifts (δ) are given in ppm. PET Positron emission tomography PiD Pick's disease PSP progressive supranuclear palsy KB quadruplet (quartet) KT room temperature c singlet T triplet Tau tau protein, tau deposits, tau aggregates TBI Traumatic brain injury those thin layer chromatography

- 16 042728

Experimental data

All reagents and solvents were obtained from commercial sources and were used without further purification. Proton (1H) spectra were recorded on a Bruker DRX400 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 carried out using silica gel (Fluka: silica gel 60, 0.063-0.2 mm) and the appropriate solvents indicated in the specific examples. Flash purification was performed with a Biotage Isolera One flash purification system using HP-Sil (Biotage) or puriFlash (Interchim) columns and the solvent gradient specified in the specific examples. Thin layer chromatography (TLC) was performed on silica gel plates with UV detection.

Synthesis of the studied compounds

Preparation A

Stage A acid

Stage A.

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

Ή-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 (broad s, 2H).

Stage B.

The title compound from step A above (10 g, 53.2 mmol) and commercially available 1-Boc-4-piperidone (10.6 g, 53.2 mmol) were placed in a 500 ml flask and mixed until homogeneous. Then added polyphosphoric acid (80 g, the content of the main substance H ₃ PO ₄ 115%) and the mixture was heated at a temperature of ~160°C in a sand bath. At a temperature of ~120°C, the Boc protecting group was cleaved off, resulting in foaming of the reaction mixture. After complete recovery, 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/sonicated until the resinous 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 granules (exotherm). 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) under sonication and washed with water (2x400 ml) to remove remaining salts and insoluble matter. The organic phase was dried over Na ₂ SO ₄ , filtered and the solvents were removed under reduced pressure. The dark residue was treated with dichloromethane (100 ml), sonicated for 5 min and the precipitate was collected by filtration. The precipitate was washed with dichloromethane (40 ml) and air dried to give the title compound as a beige solid (3.5 g, 26%).

Ή-NMR (400 MHz, DMSO-d ₆ ): δ=11.5 (br s, 1H), 7.72 (d, 1H), 7.15 (d, 1H), 3.86-3, 82 (m, 2H), 3.063.00 (m, 2H), 2.71-2.65 (m, 2H).

Stage C.

The title compound from step B above (1.75 g, 6.94 mmol) was suspended in xylene (380 ml) and manganese(IV) oxide (6.62 g, 76.9 mmol) was added. The reaction mixture was then heated at ~160°C on a sand bath for 36 h. 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 min . 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 silica gel chromatography (50 g HP-SIL cartridge) using a Biotage Isolera system using an ethyl acetate/heptane (5/95-100/0) gradient to remove non-polar impurities, and then dichloromethane/methanol (9/1^4/1) to give the title compound as a dark yellow solid. The total yield from 2 runs was 1.77 g (51%).

¹ H-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).

- 17 042728

Preparation Example B

Stage A.

To a suspension of the title compound from Preparation A (0.776 g, 0.72 mmol) in dichloromethane (65 ml) were added triethylamine (1.86 ml, 13 mmol) and trityl chloride (2.63 g, 9.39 mmol). After adding 4-(dimethylamino)pyridine (0.074 g, 0.608 mmol), the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane (150 ml) and water (50 ml). The organic phase was separated, dried over Na ₂ SO ₄ , 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 using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) to give the title compound B in as a pale yellow solid (0.831 g, 54%) Unreacted starting material was recovered by washing the cartridge with ethyl acetate/methanol (90/10) to give the starting material as an off-white solid (0.195 g, 25%). 1HNMR (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), MS (ESI), m/z=490.03/491.96 [M+H]+.

Preparation C

Stage A.

Triethylamine (1.15 ml, 8 mmol) and 4,4'-(chloro(phenyl)methylene)bis (methoxybenzene) (DMTrt-Cl) (1.963 g, 5.8 mmol). After addition of 4-(dimethylamino)pyridine (0.046 g, 0.377 mmol), the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was diluted with dichloromethane (100 ml) and water (40 ml). The organic phase was separated, dried over Na2SO4, filtered and the solvents were removed in vacuo. The residue was purified on HP-Sil SNAP cartridges (50 g) using Biotage Isolera One purification systems using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) to give the title compound C as a solid pale yellow substance (0.825 g, 72%). Unreacted starting material was recovered by washing the cartridge with ethyl acetate/methanol (90/10) to give the starting material as an off-white solid (0.042 g, 8.8%).

'H-NMR (400 MHz, CDCl ₃ ) 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

-L _Λ 7 λ Pd(dppf)CI _? xCHjCI _? /)

ΓΎ Μ -------► f D x^

CsjCOjjflHQKcaH, H ₂ O

A OH 1 (lt>)

Stage A

Stage A.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.0084 g, 0.01 mmol) followed by the title compound of Preparation A (0.05 g, 0.2 mmol), (2-fluoropyridin-4-yl)boric acid (0.035 g, 0.245 mmol) and cesium carbonate ( 0.133 g, 0.41 mmol). The reaction mixture was then heated at ~115°C in a sand bath for 6 h. The reaction mixture was diluted with ethyl acetate (60 ml) and water (20 ml), the organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were evaporated in vacuum . The dark residue was purified by silica gel chromatography (25 g HP-SIL) using a Biotage Isolera system using a dichloromethane/methanol gradient (100/0^95/5^90/10^80/20) to give the title compound 1 as off-white solid (0.033 g, 63%).

'H-NMR (400 MHz, DMSO-d ₆ ) δ=12.50 (br s, 1H), 9.45 (s, 1H), 8.83 (d, 1H), 8.56-8, 52 (m, 1H), 8.438.39 (m, 1H), 8.19-8.14 (m, 2H), 7.92 (s, 1H), 7.54-7.50 (m, 1H) . MS (ESI): m/z=265.04 [M+H] ⁺ .

- 18 042728

Example 2

Stage A.

To a suspension of the title compound of Preparation A (0.430 g, 1.73 mmol) in dichloromethane (25 mL) were added triethylamine (1.93 mL, 13.89 mmol) and di-t-butyl dicarbonate (2.27 g, 10.02 mmol). After adding 4-(dimethylamino)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 Biotage Isolera One purification systems using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) to give the indicated in title compound as an off-white solid (0.558 g, 92%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ=9.28 (s, 1H), 8.73 (d, 1H), 8.22 (d, 2H), 7.59 (d, 1H), 1 .80 (s, 9H).

Stage B.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.0058 g, 0.007 mmol), then the title compound from step A above (0.05 g, 0.143 mmol), (6-fluoropyridin-3-yl)boric acid (0.024 g, 0.17 mmol) and cesium carbonate (0.092 g, 0.286 mmol). The reaction mixture was then heated at ~100°C in a sand bath for 4 h. The reaction mixture was diluted with ethyl acetate (80 ml) and water (35 ml), the organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were evaporated in vacuum . The dark residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system using a dichloromethane/methanol gradient (100/0^98/2^95/5^90/10^80/20) to give a less polar Boc-protected compound (0.0255 g, 49%) and more polar title compound 2 as an off-white solid (0.0116 g, 31%).

The more polar one listed in Compound 2 header:

¹ H-NMR (400 MHz, DMSO-d ₆ ) 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). MS (ESI): m/z=265.09 [M+H]+.

Less polar Voc-protected connection:

1H-NMR (400 MHz, DMSO-a ₆ ) 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 (s, 9H).

The synthesis of the title compound 2 was first described in WO 2015/052105 (example 1) by a different preparation method.

Synthesis of radiolabeled precursors

Stage A for(nl])

Example 3-a.

Stage A.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.0084 g, 0.01 mmol), the title compound of Preparation B (0.1 g, 0.2 mmol), 2nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)pyridine (0.061 g, 0.245 mmol) and cesium carbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated at ~115°C in a sand bath for 6 h. The reaction mixture was diluted with ethyl acetate (60 ml) and water (20 ml), the organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were evaporated in vacuum . The dark residue was purified by silica gel chromatography (25 g pufiFlash column, Interchim) using a Biotage Isolera system using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) to give the title compound 3-a as a pale yellow solid (0.082 g, 75%).

1H NMR (400 MHz, CDCl ₃ ) δ=9.32 (s, 1H); 8.56 (d, 1H), 8.48 (d, 1H), 8.33 (s, 1H); 8.30 (d, 1H), 7.85 (d, 1H), 7.69 (d, 1H), 7.58-7.54 (m, 5H), 7.32-7.25 (m, 10H), 6.48 (d, 1H). MS (ESI): m/z=534.28 [M+H] ⁺ .

- 19 042728

Example 3b.

Method a

Stage A.

Trifluoroacetic acid (1.2 ml) was added to a solution of 3-a (0.0396 g, 0.074 mmol) in dichloromethane (5 ml). The reaction mixture was stirred at room temperature for 6 h 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 vacuo and the residue was redissolved/suspended in methanol (5 ml). The solvents were evaporated in vacuo and the residue was suspended in dichloromethane (2 ml). After adding triethylamine (1 ml, 7.2 mmol), di-tert-butyl dicarbonate (0.098 g, 0.43 mmol) and 4-(dimethylamino)pyridine (0.0018 g, 0.014 mmol), the reaction mixture was stirred at room temperature within 18 hours the Reaction mixture was diluted with ethyl acetate (50 ml) and water (20 ml). The organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were removed in vacuo. The residue was purified on silica gel (25 g puriFlash, Interchim) using Biotage Isolera One purification systems using an ethyl acetate/nheptane gradient (5/95^100/0^100/0) eluting non-polar by-products, then ethyl acetate/methanol (95/ 5) to give the title compound 3-b as a pale yellow solid (0.0184 g, 63%).

¹ H-NMR (400 MHz, CDCl ₃ ) 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). MS (ESI); m/z=391.82 [M+H]+.

Method b

Pd(dppf)Cli x CH ₂ C1 _g

Stage A.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0 0042 g, 0.005 mmol) followed by the title compound of Preparation C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane -2-yl)pyridine (0.0305 g, 0.12255 mmol) and cesium carbonate (0.067 g, 0.205 mmol). The reaction mixture was then heated at ~115°C in a sand bath for 6 h. The reaction mixture was diluted with ethyl acetate (20 ml), the precipitate was collected by filtration, washed with water (10 ml) and methanol (5 ml) and air dried to give 3 -c (0.0277 g, 95%).

Stage B.

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), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol) and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol). The reaction mixture was stirred at room temperature for 16 h, diluted with ethyl acetate (50 ml) and water (20 ml). The organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were removed in vacuo. The residue was purified on silica gel (25 g puriFlash, Interchim) using Biotage Isolera One purification systems using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) eluting non-polar by-products followed by ethyl acetate/methanol ( 95/5) to give the title compound 3-b as a pale yellow solid (0.0261 g, 70%).

¹ H-NMR (400 MHz, CDCl ₃ ) 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). MS (ESI); m/z=391.85 [M+H] ⁺ ; 291.74 [M+H-Bos] ⁺ .

Example 3d.

Stage A.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0 0042 g, 0.005 mmol) followed by the title compound of Preparation C (0.055 g, 0.1 mmol), 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane -2-yl)pyridine (0.0305 g, 0.12255 mmol) and cesium carbonate (0.067 g, 0.205 mmol). The reaction mixture was then heated at ~115°C in a sand bath for 6 h. The reaction mixture was diluted with ethyl acetate (20 ml), the precipitate was collected

- 20 042728 by filtration, washed with water (10 ml) and methanol (5 ml) and air dried to give 3-c as a gray solid (0.0277 g, 95%).

Stage B.

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(methoxybenzene) (0.081 g, 0.29 mmol) and 4-(dimethylamino)pyridine (0.0036 g, 0.028 mmol). The reaction mixture was stirred at room temperature for 18 h, diluted with ethyl acetate (50 ml) and water (20 ml). The organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were removed in vacuo. The residue was purified on silica gel (25 g puriFlash, Interchim) using Biotage Isolera One purification systems using an ethyl acetate/n-heptane gradient (5/95^100/0^100/0) to give the title compound 3-d as pale yellow solid (0.0261 g, 44%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ=9.32 (s, 1H), 8.58 (d, 1H), 8.50 (d, 1h), 8.36 (s, 1H), 8 .30 (d, 1H), 7.85 (d, 1H), 7.74 (d, 1H), 7.52-7.42 (m, 6H), 7.27-7.23 (m, 4H ), 6.80 (d, 4H), 6.49 (d, 1H), 3.78 (s, 6H).

Example 3.

o l ^J d(dppf)CI; KCIhCI; CSjCO., 1,4-diaxane

Stage A.

Commercially available N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2amine (0.25 g, 1 mmol) was dissolved in dichloromethane (5 ml) . Methyl trifluoromethanesulfonate (0.124 ml, 1.1 mmol) was added dropwise to the resulting solution at room temperature. The solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated to remove dichloromethane and the residue was dried in vacuo to give a yellow glass/foam which was used directly in the next step.

Stage B.

To a solution of degassed 1,4-dioxane (12 ml) and water (3 ml) in a microwave irradiation vessel was added [1,1'-bis-(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.034 g, 0. 04 mmol), the title compound of Preparation 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 heated at ~120° C. in a sand bath for 6 h. The reaction mixture was diluted with ethyl acetate (150 ml) and water (50 ml), the organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were evaporated in vacuo. The dark residue was purified by silica gel chromatography (25 g HP-Ultra) using a Biotage Isolera system using an ethyl acetate/nheptane gradient (5/95^100/0^100/0) eluting unreacted starting material and non-polar by-products. The gradient was then changed to dichloromethane/methanol (100/0^95/5^90/10) to give the dimethylamine derivative as a pale yellow glass (0.127 g, 29%; MS (ESI): m/z=532 .27 [M+H] ⁺ ) and the methylamine derivative as a gray solid (0.0547 g, 13%; MS (ESI): m/z=519.18 [M+H] ⁺ ). The gradient was again changed to dichloromethane/methanol (90/10^80/20) and kept at (80/20) to give the title compound 3-e as a brown solid (0.104 g, 18%).

¹ H 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). MS (ESI): m/z=546.26 [M+H] ⁺ .

Example 3-f.

Stage A.

3rd (0.199 g, 0.364 mmol) were suspended in dichloromethane (10 ml). After trifluoroacetic acid (10 ml) was added, the reaction mixture was stirred at room temperature for 18 hours. The solvents were removed under reduced pressure, the residue was dissolved in methanol (10 ml) and the solvents were removed under reduced pressure. The treatment of the residue with methanol 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 air dried to give the title compound 3-f as a gray solid (0.127 g, 83%).

¹ H 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]+.

- 21 042728

Example 4-a

PdfdppfJCh s CH _g C1 ₌

Stage A.

[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.0058 g, 0.007 mmol), then 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- dioxaborolan-2-yl)pyridine (0.0428 g, 0.17 mmol) and cesium carbonate (0.092 g, 0.286 mmol). The reaction mixture was then heated at ~100°C in a sand bath for 4 h. The reaction mixture was diluted with ethyl acetate (80 ml) and water (35 ml), the organic phase was separated, dried over Na ₂ SO ₄ , filtered and the solvents were evaporated in vacuum . The dark residue was purified by silica gel chromatography (12 g, puriFlash, Interchim) using a Biotage Isolera system using a dichloromethane/methanol gradient (100/0^98/2^95/5^90/10^80/20) to give the indicated title compound 4-a as a pale yellow solid (0.0173 g, 31%).

1H NMR (400 MHz, CDCl3/CD ₂ OD) 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). MS (ESI): m/z=392.13 [M+H]+.

The synthesis of the title compound 4-a was first described in WO 2015/052105 (Example 3a) by a different synthetic route.

Precursor radio tagging with ¹⁸ F

Examples of radiolabeled precursors.

The general radio tagging method A performed using tracerlab FX as shown in FIG. 3 (radiolabeling, deprotection, HPLC and SPE) - comparative examples.

[ ¹⁸ F]fluoride was captured on a Sep-Pak Accell Plus QMA light cartridge (Waters) and eluted with a K2CO3/Kryptofix® 2.2.2 solution. Water was removed using a flow of He or N2 at 95°C and co-evaporated with MeCN (1 ml) to dryness. Thereafter, the dissolved precursor solution was added to the dried K[ ¹⁸ F]F-Kryptofix complex. The reaction vessel was sealed and heated for 15 min at 150°C. Then an acid (HCl, H2SO ₄ or H ₃ PO ₄ ) was added and the mixture was heated for 10 min at 150°C. The reaction mixture was diluted with 1 ml NaOH and 2.4 ml mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (eg Phenomenex, Gemini C18, 5 µm, 250x10 mm) at 4 ml/min. The isolated indicator was diluted with water (20 ml + 10 mg/ml sodium ascorbate), captured on 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).

The general radio tagging method B performed using tracerlab FX as shown in FIG. 3 (radiolabeling, HPLC and SPE) - comparative examples.

[ ¹⁸ F]fluoride was captured on a Sep-Pak Accell Plus QMA light cartridge (Waters) and eluted with a K ₂ CO ₃ /Kryptofix® 2.2.2 solution. Water was removed using a stream of He or N ₂ at 95°C and co-evaporated with MeCN (1 ml) to dryness. Thereafter, the dissolved precursor solution was added to the dried K[ ¹⁸ F]F-Kryptofix complex. The reaction vessel was sealed and heated for 15 min at 150°C. The reaction mixture was diluted with 1 ml NaOH and 2.4 ml mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (eg Phenomenex, Gemini C18, 5 µm, 250x10 mm) at 4 ml/min. The isolated indicator was diluted with water (20 ml + 10 mg/ml sodium ascorbate), captured on 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).

The general C radiolabeling method performed using IBA Synthera+Synthera HPLC as shown in FIG. 4 (radiolabeling, HPLC).

[ ¹⁸ F]fluoride was captured on a Sep-Pak Accell Plus QMA light cartridge (Waters) and eluted with a K ₂ CO ₃ /Kryptofix® 2.2.2 solution. Water was removed using a flow of He or N ₂ at a temperature of 95-110°C and evaporated to dryness. After that, the solution of the dissolved precursor was added to the

- 22 042728 K[ ¹⁸ F]F-Kryptofix. The reaction vessel was sealed and heated for 15 min at 150°C. The reaction mixture was diluted with 0.5-1 ml of 1 M H ₃ PO ₄ and 3-3.5 ml of the aqueous component of the mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (eg Waters XBridge Peptide BEN C18, 130 A, 10 µm, 10x250 mm) at 3-6 ml/min. Fractions containing product (5-10 ml) were collected and diluted with a dilution medium containing 0-2 ml EtOH, 10-20 ml water and 0-4 ml phosphate buffer concentrate (Braun, 3.05 g disodium monohydrogen phosphate dodecahydrate, 0.462 g dihydrate sodium dihydrogen phosphate in 20 ml water for injection) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or gentisic acid and/or gentisic acid (20-200 mg).

The general D radio tagging method performed using tracerlab FX as shown in FIG. 3 (radiolabeling, HPLC).

[ ¹⁸ F]fluoride was captured on a Sep-Pak Accell Plus QMA light 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 evaporated to dryness with MeCN (1 ml). Thereafter, the dissolved precursor solution was added to the dried K[ ¹⁸ F]F-Kryptofix complex. The reaction vessel was sealed and heated for 15 min at 150°C. The reaction mixture was diluted with 0.5-1 ml of 1 M H3PO ₄ and 3-3.5 ml of the aqueous component of the mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (e.g. Waters XBridge Peptide BEN C18, 130 A, 10 µm, 10x250 mm or Gemini 5 µm C18, 250x10 mm, Phenomenex: 00G-4435-N0) at 3-6 ml/ min. Fractions containing product (5-10 ml) were collected and diluted with dilution medium containing 0-2 ml EtOH, 10-20 ml water, and 0-4 ml phosphate buffer concentrate (Braun) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or sodium citrate (20-200 mg).

The general E radio tagging method performed using tracerlab FX as shown in FIG. 3 (radiolabeling, deprotection, HPLC).

[ ¹⁸ F]fluoride was captured on a Sep-Pak light cartridge. Accell Plus QMA. (Waters) and eluted with K2CO3/Kryptofix® 2.2.2 solution. Water was removed using a stream of He or N2 at 95° C. and evaporated to dryness with MeCN (1 ml). Thereafter, the dissolved precursor solution was added to the dried K[ ¹⁸ F]F-Kryptofix complex. The reaction vessel was sealed and heated for 15 min at 150°C. Then 1 ml of 0.5 M H2SO ₄ was added and the mixture was heated for 10 min at 100°C. The reaction mixture was diluted with 0.5-1 ml of 1 M NaOH and 2-3 ml of the aqueous component of the mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (e.g. Waters XBridge Peptide BEN C18, 130 A, 10 µm, 10x250 mm or Gemini 5 µm C18, 250x10 mm, Phenomenex: 00G-4435-N0) at 3-6 ml/ min. Fractions containing product (5-10 ml) were collected and diluted with dilution medium containing 0-2 ml EtOH, 10-20 ml water, and 0-4 ml phosphate buffer concentrate (Braun) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or sodium citrate (20-200 mg).

The general F radiolabeling method performed using IBA Synthera+Synthera HPLC as shown in FIG. 4 (radiolabeling, deprotection, HPLC).

[ ¹⁸ F]fluoride was captured on a Sep-Pak Accell Plus QMA light cartridge (Waters) and eluted with a K2CO3/Kryptofix® 2.2.2 solution. Water was removed using a stream of He or N2 at 95-110° C. and evaporated to dryness. Thereafter, the dissolved precursor solution was added to the dried K[ ¹⁸ F]F-Kryptofix complex. The reaction vessel was hermetically sealed and heated for 5-10 min at a temperature of 110-120°C. After fluorination, 0.5-1 ml of 1 M H3PO ₄ was added and the reaction mixture was heated for 10-15 min at a temperature of 100-110°C. The mixture was diluted with 3-3.5 ml of the aqueous component of the mobile phase prep. HPLC and the crude product was purified by semi-preparative HPLC (eg Waters XBridge Peptide BEN C18, 130 A, 10 µm, 10x250 mm) at 3-6 ml/min. Fractions containing product (5-10 ml) were collected and diluted with a dilution medium containing 0-2 ml EtOH, 10-20 ml water and 0-4 ml phosphate buffer concentrate (Braun, 3.05 g disodium monohydrogen phosphate dodecahydrate, 0.462 g dihydrate sodium dihydrogen phosphate in 20 ml water for injection) and/or sodium ascorbate (100-1000 mg) and/or sodium citrate (100-1000 mg) and/or gentisic acid (20-200 mg).

Determination of radiochemical purity

Radiochemical purity was determined by analytical HPLC, for example: Atlantis T3 column, Waters, 100x4.6 mm, 3 µm, 100; mobile phase A: 40 mM sodium acetate, finally adjusted to pH 5.0 with glacial acetic acid; mobile phase B: 35% methanol in acetonitrile; flow rate: 1.8 ml/min; gradient: 0-5 min 15-32% B, 5-8 min 32-80% B, 8-12 min 80% B, 12-13 min 80-15% B, 13-16 min 15% B.

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Claims (13)

Synthesis results ¹⁸ F-Ib # Predecessor General radiotagging method Mobile phase prey. HPLC Total assay time [min] Initial activity [GBq] Product activity [GBq] Refined yield of undecomposed product [%] Radiochemical purity at EOS [%] 1* 2 mg Za in 0.8 ml DMSO A 23% MeCN, 77% NaH ₂ PO ₄ /Na ₂ HPO ₄ buffer "74 375 36 9.6 97.6 2 2 mg 3b in 1 ml DMSO with 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2 with H3PO4), +1 mg/ml ascorbic acid 60 230 85 37.0 99.3 3 2 mg With 15% EtOH, 85% 100 mM »60 249 102 41.0 100 3b in 1 ml DMSO NaH ₂ PO ₄ (adjustment to pH 2 USING H3PO4) 4 2 mg 3b in 1 ml DMSO with 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2 WITH H3PO4) 60 236 91 38.6 99.7 5* 2.4 mg 3b in 1 ml DMSO in 23% MeCN, 77% NaH ₂ PO ₄ /Na ₂ HPO4 buffer »70 5.1 0.34 6.7 99.0 6 2.8 mg 3f in 1 ml DMSO With 25% EtOH, 75% 50 mM NaOAc (adjust to pH 2) 55 1.0 0.18 17.8 100 7 3.1 mg Ze in 1 ml DMSO E 25% EtOH, 75% 50 mM NaOAc (adjust to pH 2) 60 3.4 0.6 17.7 100 8 2 mg 3b in 1 ml DMSO with 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2.4 with H3PO4) 60 58.8 17.3 29.5 99.1 9 2 mg 3b in 1 ml DMSO with 15% EtOH, 85% 100 mM NaH ₂ P0 ₄ +400 mM NaCl (adjustment to pH 2.4 with H3PO4) 60 13.8 4.0 28.7 99.5 10 2 mg 3f in 1 ml DMSO C 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2.4 with H3PO4) 60 9.2 1.5 17 100 11 2 mg Ze in 1 ml DMSO F 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2.4 with H3PO4) 65 48 11.5 23.9 100 12 2 mg Ze in 1 ml MeCN F 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH 2.4 with H3PO4) »60 35.6 7 19.6 100 13 2 mg 3g in 1C 15% EtOH, 85% 100 mM NaH ₂ PO4 (adjustment to pH »60 1.1 0.066 5.9 99.6 ml DMSO 2.4 with H3PO4) * Comparative examples. CLAIM 1. Process for the preparation of a compound of formula I including stages: A) interaction of the compound of formula II with ¹⁸ F fluorinating agent -