EA039208B1 - Tau pet imaging ligands - Google Patents

Abstract

The invention relates to novel, selective radiolabelled tau ligands which are useful for imaging and quantifying tau aggregates, using positron-emission tomography (PET). The invention is also directed to compositions comprising such compounds, to processes for preparing such compounds and compositions, to the use of such compounds and compositions for imaging a tissue or a subject, in vitro or in vivo, and to precursors of said compounds.

Description

The field of technology to which the invention belongs

The invention relates to novel, selective, radiolabeled tau protein ligands that are suitable for imaging and quantifying tau protein aggregates using positron emission tomography (PET). The invention is also directed to compositions containing such compounds, to methods for preparing such compounds and compositions, to the use of such compounds and compositions for tissue or subject imaging, in vitro or in vivo, and to the precursors of said compounds.

State of the art invention

Alzheimer's disease (AD) is a neurodegenerative disease associated with aging. Patients with AD suffer from cognitive impairment and memory loss, as well as behavioral problems such as anxiety. More than 90% of those affected by AD have a sporadic form of the disorder, while less than 10% of cases have a familial or hereditary pattern. In the United States, approximately one in ten people at age 65 has AD, and at age 85, one out of every two individuals is affected by AD. Life expectancy from initial diagnosis is 7-10 years, and AD patients require comprehensive care, either in a nursing home or family members. Given the growing number of older people in the population, AD is a growing medical problem. Currently available therapies for AD treat only the symptoms of the disease and not the underlying pathology causing the disease.

The hallmark pathological features in the brains of AD patients are neurofibrillary tangles, which arise from aggregates of hyperphosphorylated tau protein, and amyloid plaques, which form from aggregation of beta-amyloid peptide. Although AD is the most common neurodegenerative disorder, tau aggregates are also characteristic of other neurodegenerative diseases known as taupathies, which also, but not exclusively, include tangle-only dementia (TD), a disease characterized by the appearance of argyrophilic granules. (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and chromosome 17-associated frontotemporal dementia and parkinsonism (FTDP-17). The heterogeneity of these disorders is closely related to the wide range of isoforms and post-translational modifications of tau in humans. The ultrastructure of tau protein aggregates can appear as paired helical filaments (PHF), straight filaments (SF), randomly folded filaments (RCF), or twisted filaments (TF); this variability translates into polymorphism. Neurofibrillary tangles have been correlated with the level of cognitive impairment in AD and/or the chance of developing AD. However, the diagnosis can still only be made after autopsy by biopsy/autopsy. History-based and statistical memory testing requires clear evidence of impairment or dementia and is often inaccurate or insensitive, and measurement of Avpeptides and total CSF tau by lumbar puncture is invasive and prone to adverse effects. In addition to the inherent complexity of AD, the development of treatments is hampered by a lack of reliable tools for early diagnosis, staging, and precise control of disease progression. Therefore, there is still a need to identify means for diagnosing and/or monitoring disease progression. Imaging of tau aggregates may provide such methods, particularly as tau therapies emerge.

Positron emission tomography (PET) is a non-invasive imaging technique that provides the highest spatial and temporal resolution of any radionuclide imaging technique and has the added benefit of being able to correctly quantify marker concentrations in tissues. It uses positron-active radionuclides for detection.

To date, several radioactive markers have been claimed for positron emission tomography to visualize tau protein aggregates (for a review, see, for example, Ariza et al. J. Med. Chem. 2015, 58, 4365-4382). In Preclinical Characterization of 18F-MK-6240, a promising PET Tracer for In Vivo Quantification of Human Neurofibrillary Tangles in J. Nucl. Med 2016; 57:1599-1610 discloses 6fluoro-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, which binds with high affinity to human AD cerebral cortex homogents containing excess NFT , but binds poorly to the amyloid-rich, NFT-poor brain homogenates of AD patients. Moderate defluorination is observed with 18F-MK-6240 absorbed by the cranium. In the article Radiopharmaceuticals for positron emission tomography investigations of Alzheimer's disease in Eur. J. Nucl. Med. Imaging 2010; 37(8): 1575-1593 report an isoquinoline compound [ ¹¹ C]PK11195 that binds to peripheral cortical benzodiazepine receptors in activated microglia and which, as further reported in Carbon 11-labeled Pittsburgh compound B and carbon 11-labeled (R )-PK11195 positron emission tomographic imaging in Alzheimer's disease in Arch. Neurol. 2009; 66(1): 60-67 does not show any difference in brain retention between sick and healthy volunteers. PET imaging of the human brain with a quinoline compound

18F-THK5351, which has a high affinity for PHF, has been shown to be difficult to off-target binding to the enzyme MAO-B monoamine oxidase (Alzheimer's Research &

therapy 2017; 9; 25 DOI 10.1186/s13195-017-0253-y).

There is still a need to provide selective, improved radioactive markers for positron emission tomography for imaging tau protein aggregates with the right balance of properties, including, but not limited to, high affinity and selectivity for tau protein aggregates, reversible binding, permeability, a suitable pharmacokinetic profile in the brain, i.e. fast distribution in the brain, fast clearance, minimal non-specific binding and availability for synthesis.

Brief description of the invention

Therefore, it is an object of the present invention to provide isoquinoline-6-amine compounds useful as radioactive tau PET markers. Therefore, in one aspect, the present invention relates to a compound characterized by formula (I)

where at least one atom is radioactive, and where

R ² is methyl and either R ¹ is F and R ³ is H, or R ¹ is H and R ³ is F; or

R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-F, -OC1-4alkyl-F and -NR ⁴ -C1-4alkyl-F, where R ⁴ is H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

In particular, the present invention relates to a compound of formula (I')

where R ² is methyl and either R ¹ is ¹⁸ F and R ³ is H, or R ¹ is H and R ³ is ¹⁸ F; or

R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C 1-4 alkyl- ¹⁸ F, -OC 1-4 alkyl- ¹⁸ F and -NR ⁴ -C _1-4 alkyl- ¹⁸ F, where R ⁴ is H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention relates to precursor compounds for the synthesis of compounds of formula (I) or (I') as defined above. Thus, the present invention also relates to a compound of formula (P-1)

where R ² is methyl and either R ¹ is selected from the group consisting of Br, -NO2, -[N(CH3)3] ⁺ and 4-CH3-Ph-SO ₂ -O- and R ³ is H , or R ¹ is H and R ³ is selected from the group consisting of Br, -NO2, -H(CH3)3] ⁺ and 4-CH3-Ph-SO2-O-; or

R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-Br, -C1-4alkyl-I, -C1-4alkyl-O-SO2CH3, 4-CH3-Ph-SO2- O-C1-4alkyl-, -C1-4alkyl-OH, -OC1-4alkyl-Br, -OC1-4alkyl-I, -OC1-4alkyl-O-SO2CH3, 4-CH3-Ph-SO2-O-C1-4alkyl -O-, -OC^alkyl-OH, -NR ⁴ -C1-4alkyl-Br, -NR ⁴ -C1-4alkyl-I, -NR ⁴ -C1-4alkyl-O-SO2CH3, 4-CH3-Ph-SO2 -O-C1-4alkyl-NR ⁴ - and -NR ⁴ -C1-4alkyl-OH, where R ⁴ represents H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

In compounds of formula (P-1), if either R ¹ or R ³ is -[N(CH3)3] ⁺ , then acceptable anionic counterions include, but are not limited to, trifluoroacetate (-[OC(O)CF3] ^- ), an organic sulfonate (eg C _1-4 alkylsulfonate or phenylsulfonate, where phenyl may be optionally substituted with C _1-4 alkyl, halogen or nitro group) and tartrate. Specific examples of the C _1-4 alkylsulfonate include methanesulfonate (mesylate), 4-methylbenzenesulfonate (tosylate), 4-bromo-2 039208 benzenesulfonate and 4-nitrobenzenesulfonate. In particular, the anionic counterion is selected from trifluoroacetate, tosylate and mesylate.

The present invention also relates to reference materials of compounds of formula (I) or (I'), corresponding to similar unlabeled compounds, herein referred to as compounds of formula [ ¹⁹ F]-(I)

^R1

[ ¹⁹ F]- (I) where R ² is methyl and either R ¹ is F and R ³ is H or R ¹ is H and R ³ is F; or

R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-F, -OC1-4alkyl-F and -NR ⁴ -C1-4alkyl-F, where R ⁴ is H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

The present invention also relates to a pharmaceutical composition containing a compound of formula (I), in particular a compound of formula (I'), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. In particular, said pharmaceutical composition is a diagnostic pharmaceutical composition. Said pharmaceutical composition is in particular a sterile solution. Thus, according to the present invention, illustrative is a sterile solution containing a compound of formula (I), in particular a compound of formula (I') as described herein.

The present invention further relates to the use of a compound of formula (I), in particular a compound of formula (I'), as an imaging agent. As an example, the present invention provides the use of a compound of formula (I), in particular a compound of formula (I') described herein, for in vitro or in vivo imaging of a tissue or a subject, or a method for such imaging.

In particular, the present invention relates to a compound of formula (I), in particular a compound of formula (I'), for use in binding and visualizing tau protein aggregates in patients suffering from or suspected of having tauopathy. Specific taupathies are, for example, Alzheimer's disease, tangle-only dementia (TD), argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17). In particular, taupathy is Alzheimer's disease.

The present invention further relates to a compound of formula (I), in particular a compound of formula (I'), for diagnostic imaging of tau protein aggregates in the brain of a subject, and to the use of a compound of formula (I), in particular a compound of formula (I'), for binding and imaging of tau protein aggregates in patients with or suspected of having tauopathy. Specific taupathies are, for example, Alzheimer's disease, tangle-only dementia (TD), argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17). In particular, taupathy is Alzheimer's disease.

The invention also relates to a method for imaging a tissue or a subject, which involves bringing a detectable amount of a labeled compound of formula (I), in particular a labeled compound of formula (I') described herein, into contact with, or delivery to, a tissue or a subject, or introduction in them, and identifying a compound of formula (I), in particular a compound of formula (I').

Additionally, as an example of the present invention, a method for imaging a tissue or a subject is provided, comprising bringing a compound of formula (I), in particular a compound of formula (I') described herein, into contact with or delivering it to a tissue or subject and imaging the tissue or a subject using a positron emission tomography imaging system.

In addition, the present invention relates to a process for the preparation of a compound of formula (I'-a) or (I'-b), referred to in particular below as (I'-a1), (I'-a2), (I' -b1), (I'-b2), (I'-b3), or a pharmaceutically acceptable salt or solvate thereof as described herein, providing:

(a) the step of reacting a compound of formula (P-a1), or a pharmaceutically acceptable salt or solvate thereof, as defined herein, with a source of ¹⁸ F ^- fluoride under suitable conditions, or (b) a step of reacting a compound of formula (P-a2) or a pharmaceutically acceptable salt or solvate thereof, as defined herein, with a source of ¹⁸ F fluoride ^- in acceptable

- 3 039208 conditions, or (c) the step of reacting a compound of formula (P-bl) or a pharmaceutically acceptable salt thereof as defined herein with an activating agent such as methanesulfonyl chloride or 4-toluenesulfonyl chloride in the presence of a base such as triethylamine or N,N-diisopropylethylamine (DIPEA), and then reacting the resulting methanesulfonate or 4-toluenesulfonate with a source of ^{18F -} fluoride under suitable conditions, or (d) the step of reacting a compound of formula (P-L2) or a pharmaceutically acceptable salt thereof, as defined herein, with an activating agent such as methanesulfonyl chloride or 4-toluenesulfonyl chloride in the presence of a base such as triethylamine or DIPEA and then reacting the resulting methanesulfonate or 4-toluenesulfonate with a source of ¹⁸ F fluoride ^- under suitable conditions, or ( f) the step of carrying out the reaction of a compound of formula (P-b3) or its pharmaceutical an acceptable salt as defined herein with an activating agent such as methanesulfonyl chloride or 4-toluenesulfonyl chloride in the presence of a base such as triethylamine or DIPEA and then reacting the resulting methanesulfonate or 4-toluenesulfonate with a source of ¹⁸ F fluoride ^- in acceptable conditions.

Typical conditions for activating the precursors of formulas (P-L1), (P-L2) and (P-L3) include, for example, methanesulfonyl chloride as an activating agent, dry dimethyl sulfoxide (DMSO) as a solvent, at room temperature for a period of time, sufficient to bring the reaction to completion, typically 10 min. The person skilled in the art will understand that if R ⁴ is H in (P-b3) - the preparation of a compound of formula (I'-b3) will involve the additional steps of protecting the amine functional group with a suitable protecting group, such as, for example, as tert-butyloxycarbonyl (Boc) or an alternative suitable amine protecting group, and then cleaving off such a protecting group using, as a rule, trifluoroacetic acid (TFA) when the protecting group is Boc.

A suitable source of ¹⁸ F ^- is, for example, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane [ ¹⁸ F]-potassium fluoride (1:1) (also called [ ¹⁸ F]KF.K222). Acceptable conditions include those that correspond to nucleophilic substitution known in the art, for example using DMSO or DMF as a solvent, in particular DMSO, under conventional heating or microwave radiation (for example, 50 W), for example, at about 90-160° C or at about 120-160°C, in particular at about 90 or 160°C, for a period of time sufficient to allow the reaction to proceed to completion, for example 10 minutes, if the reaction is carried out under microwave radiation

- 4 039208

(P-b3), (G-b3).

Brief description of graphic materials

In FIG. 1 shows in vitro autoradiography (ARG) using [ ¹⁸ F]coeg. No. 1 (7.4 kBq/500 µl/section) on a section of the visual cortex of a patient with AD (Braak stage VI) (A). Adjacent sections were immunostained for tau-associated pathology (B, AT8) and for beta-amyloid pathology (C, 4G8);

in fig. 2 is a comparison between the ARG shown in FIG. 1(A) and adjacent brain sections of a human AD patient incubated with [ ¹⁸ F]comp. No. 1 (7.4 kBq/500 µl/section) in the presence of an authentic reference compound comp. No. 1 (B) or T808 (C) at 1 µmol/l;

in fig. 3 - incubation with 10 nm [ ³ H]comp. No. 1 on the tissue of the basal ganglion (striatum) of a person (A), followed by treatment with 10 μM comp. No. 1 (B) or 10 μM THK5351 (C). In FIG. 3D shows incubation with 10 nM [ ³ H]Comp. No. 1 on tissue from a human AD patient (pariental-temporal cortex, Braak stage VI) with confirmed tau-associated pathology and amyloid-associated pathology by IHC, as shown in FIG. 4. In FIG. 3E shows incubation with 10 nM [ ³ H]comp. #1 on tissue from a human AD patient (lateral occipital gyrus, Braak stage 0), with confirmed amyloid-related pathology but no tau-related pathology, by IHC, as shown in FIG. 5. In FIG. 3F shows incubation with 10 nM [ ³ H]THK5351 on human basal ganglion tissue and treatment with 10 μM Comp. No. 1 (G) or 10 μM TNK5351 (H). In FIG. 3I shows incubation with [ ³ H]TNK-5351 on tissue from a human AD patient (pariental-temporal cortex, Braak stage VI) with confirmed tau-associated pathology and amyloid-associated pathology as shown in FIG. 4. In FIG. 3J shows incubation with 10 nM [ ³ H]TNK5351 on tissue from a human patient with AD (lateral occipital gyrus, Braak stage 0), with confirmed amyloid-related pathology but no tau-related pathology, as shown in FIG. 5. In FIG. 3K shows incubation with 3 nM [ ³ H]-AV-45 on the human basal ganglion (K) of the aforementioned human AD patient tissue with both beta-amyloid and tau-related pathologies (L), and the aforementioned human AD patient tissue with beta-amyloid-related pathology but no tau-related pathology (M);

in fig. 4 - 4G8 and AT8 IHC pathology associated with amyloid and pathology associated with tauproteins, on sections of the brain of an AD patient from the region of the pariental-temporal cortex of the same patient as the sections used for images in FIG. 3;

in fig. 5 is a section of the brain of a patient with human AD (lateral occipital gyrus, female) adjacent to the section used in FIG. 3E and 3J. The presence of beta-amyloid plaques (A, B) and

- 5 039208 the absence of tau tangles (C, D) was confirmed by immunohistochemistry using 4G8 and AT 8 antibodies, respectively;

in fig. 6 - IHC confirmation of MAO-B (left) and MAO-A (right) expression in human basal ganglion tissue used for in vitro binding shown in FIG. 3;

in fig. 7 - incubation with 0.2 mCi/ml [ ¹⁸ F]comp. No. 1 on tissue sections of a patient with human AD (pariental-temporal cortex, Braak stage VI) (A), and treatment with 10 μM clorgyline (B), 10 μM deprenyl (C) and 10 μM comp. No. 1 (D);

in fig. 8 - AT8 and 4G8 IHC, respectively, tau-associated pathology and amyloid-associated pathology, in sections of the brain of an AD patient (pariental-temporal cortex, Braak stage VI) adjacent to the sections referred to in FIG. 7;

in fig. 9 are time-activity curves for cRET, expressed as standardized uptake values (SUV) for [ ¹⁸ F]comp. No. 1;

in fig. 10 - time-activity curves for pPET for [ ¹⁸ F]T807 (C) on the whole brain of three female Wistar rats. Three separate studies are shown: original scan (marker only); pre-treatment experiment (cold Comp. No. 1 or T807, 10 mg/kg, injected subcutaneously 60 min before injection of the radioactive marker), and a tracer study (cold Comp. No. 1 or T807, 1 mg/kg, injected intravenously through 30 minutes after injection of a radioactive marker);

in fig. 11 - curves %SUV _max . curves time-activity obtained using PET on a small animal, [ ¹⁸ F]Comp. No. 1 and [ ¹⁸ F]T807 in the whole brain of a Wistar rat;

in fig. 12 - time-activity curves obtained using pPET for [ ¹⁸ F] Comm. No. 1, and in Fig. 13 shows pPET activity curves for [ ¹⁸ F] T807 in the whole brain, corpus callosum, cerebellum and entorhinal cortex and skull of the rhesus monkey;

in fig. 14 - curves %SUV _max . time-activity curves obtained using pRET on a small animal, [ ¹⁸ F] Comm. No. 1 and [ ¹⁸ F]T807 in the whole rhesus monkey brain.

Detailed description of the invention

In one embodiment, the compound of formula (I) is, in particular, the compound of formula (I-a)

where at least one atom is radioactive and where R ¹ is F and R ³ is H, or R ¹ is H and R ³ is F;

or a pharmaceutically acceptable salt or solvate thereof.

More specifically, the compound of formula (I) is a compound of formula (I'-a)

or a pharmaceutically acceptable salt or solvate thereof.

In another embodiment, the compound of formula (I) is, in particular, the compound of formula (I-b)

where at least one atom is radioactive, and where R ² is selected from the group consisting of -C1.4alkyl-F, -OC1.4alkyl-F and -NR ⁴ -C1. ₄ alkyl-F, where R ⁴ is H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

More specifically, the compound of formula (I) is a compound of formula (I'-b)

- 6 039208

(I'-b) where R ² is selected from the group consisting of -C 1-4 alkyl- ¹⁹ F, -OC 1-4 alkyl- ¹⁸ F and -NR ⁴ -C _1-4 alkyl- ¹⁸ F, where R ⁴ represents H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the compound of formula (I), in particular formula (I'), is

or a pharmaceutically acceptable salt or solvate thereof.

In another specific embodiment, the precursor compound for the synthesis of a compound of formula (I) or (I') as defined above is, in particular, a compound of formula (P-1)

where R ² is methyl and either R ¹ is selected from the group consisting of Br, -NO2, -[N(CH3)3] ⁺ and 4-Me-Ph-SO2-O- and R ³ is H, or R ¹ is H and R ³ is selected from the group consisting of Br, -NO2, -[N(CH3)3] ⁺ and 4-Me-Ph-SO2-O-; or

R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-OH, -OC1-4alkyl-OH and -NR ⁴ -C1-4alkyl-OH, where R ⁴ is H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

The present invention also relates to a reference material corresponding to an unlabeled compound 1 corresponding to [ ¹⁹ F]-compound

or a pharmaceutically acceptable salt or solvate thereof.

In another specific embodiment, the compound of formula [ ¹⁹ F]-(I) is, in particular, the compound of formula [ ¹⁹ F]-(Ia)

[ ¹⁹ F] - (Ia) where R ¹ is F and R ³ is H, or R ¹ is H and R ³ is F;

or a pharmaceutically acceptable salt or solvate thereof.

In another specific embodiment, the compound of formula [ ¹⁹ F]-(I) is, in particular, the compound of formula (Ib)

[ ¹⁹ F] - (Ib) where R ² is selected from the group consisting of -C1-4alkyl-F, -OC1-4alkyl-F and -NR ⁴ -C1-4alkyl-F, where R ⁴ represents H or methyl;

or a pharmaceutically acceptable salt or solvate thereof.

[ ¹⁹ F]-Comp. No. 1 demonstrated strong binding (pIC ₅₀ 8.24) to isolated human tau aggregates in a radiolabeled assay using a proprietary tritium analog of compound T-808 (T-808 developed by Siemens, see e.g. J. Alzheim

- 7 039208 ers Dis. 2014, 38, 171-184), and which is referred to in this document as [ ³ H]-T808. In addition, Comm. No. 1 showed poor binding to isolated human beta-amyloid aggregates (pIC ₅₀ 5.18) in a radiolabeled assay using the proprietary tritium analog florbetapyr (also known as Amyvid® from Eli Lilly and Co. or AV-45, see ., for example, J. Nucl. Med. 2010, 51, 913-920), and which is referred to herein as [ ³ H]-AV-45. Protocols are described later in this document.

[ ³ H]-T808 was prepared by subjecting a solution of the bromine-containing precursor (1 eq.) in methanol to catalytic tritiation over palladium on carbon (5%) in the presence of diisopropylethylamine (5 eq.) at room temperature. The bromine-containing precursor was obtained by bromination of T808 with N-bromosuccinimide (1 eq.) in acetonitrile.

[ ³ H]-AV-45 was prepared using iridium catalyzed (Crabtree catalyst) tritium exchange AV-45 dissolved in dichloromethane

[ ³ H]-T808, [ ³ H]-AV-45.

As already mentioned, a compound of formula (I), in particular a compound of formula (I'), and compositions containing a compound of formula (I), in particular a compound of formula (I'), can be used to image a tissue or a subject in vitro or in vivo. . In particular, the present invention relates to a method for visualizing or quantifying tau protein aggregates in a tissue or subject in vitro or in vivo.

In particular, the method for visualizing tau protein aggregates involves providing a subject, in particular a patient, with a detectable amount of a compound of formula (I), in particular a compound of formula (I').

Additionally, the present invention relates to a method for visualizing deposits of tau protein aggregates, comprising the steps of providing a subject with a detectable amount of a compound of formula (I), in particular a compound of formula (I'), providing time sufficient for association of a compound of formula (I), in particular a compound formula (I'), with deposits of aggregates of tau proteins, and detection of a compound associated with deposits of aggregates of tau proteins.

If the method is carried out in vivo, then the compound of formula (I), in particular the compound of formula (I'), can be administered intravenously, for example by injection with a syringe or through a peripheral intravenous tube, such as a short catheter. A compound of formula (I), in particular a compound of formula (I'), or a sterile solution containing a compound of formula (I), in particular a compound of formula (I'), may in particular be administered by intravenous injection into the arm in any recognizable vein , in particular, from the back of the hand or into the median cubital vein in the cubital region.

Thus, in a specific embodiment, the present invention relates to a method for imaging a subject, comprising intravenously administering a compound of formula (I), in particular a compound of formula (I') as defined herein, or a composition, in particular a sterile composition containing a compound of formula ( I), in particular the compound of formula (I'), to the subject, and imaging of the subject with a system for imaging using positron emission tomography.

In a further embodiment, the present invention relates to a method for quantifying deposits of tau protein aggregates in a subject, comprising intravenously administering a compound of formula (I), in particular a compound of formula (I'), or a composition containing a compound of formula (I), in particular a compound formula (I'), the subject and imaging using a system for imaging using positron emission tomography.

The compound is provided to the subject in a detectable amount and after sufficient time has elapsed for the compound to associate with deposits of tau protein aggregates, with the labeled compound being detected in a non-invasive manner.

Definitions.

As used herein, the term composition is intended to cover a product containing certain ingredients in certain amounts, as well as any product that is obtained directly or indirectly from combinations of certain ingredients in certain amounts. The term "Cl- ₄ alkyl" means a straight or branched saturated alkyl group with 1, 2, 3 or 4 carbon atoms, respectively, eg methyl, ethyl, 1-propyl, 2-propyl, butyl, and the like.

It is understood that the addition salts of the compounds in accordance with the present invention

- 8 039208 are also covered by the scope of the present invention.

Acceptable salts of the compounds of the present invention are those in which the counterion is pharmaceutically acceptable. However, salts of acids and bases which are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are included within the scope of the present invention. Pharmaceutically acceptable salts are defined as including the therapeutically active non-toxic acid addition salt forms which may be formed by the compounds of the present invention. Said salts can be obtained by treating the compounds of the present invention in base form with suitable acids, for example inorganic acids, for example hydrohalic acid, in particular hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid; organic acids, e.g. acetic acid, hydroxyacetic acid, propanoic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid and pamoic acid.

Conversely, said salt forms can be converted to the free base form by treatment with an appropriate base.

In addition, some of the compounds of the present invention may form solvates with water (ie, hydrates) or common organic solvents, and such solvates are also intended to be within the scope of the present invention.

The term subject as used herein refers to a person who is or has been the subject of treatment, observation, or experiment. Unless otherwise indicated, the term subject includes asymptomatic humans, pre-symptomatic humans, and human patients.

Receipt.

Compounds in accordance with the present invention can usually be obtained using a sequence of stages, each of which is known to the person skilled in the art. In particular, the compounds can be obtained according to the following synthetic methods.

Compounds of formula [ ¹⁹ F]-(Ia) or [ ¹⁹ F]-(Ib) disclosed herein can be prepared by reacting 6-bromo-isoquinoline with the corresponding 2-aminopyridine compound of formula (II-a), where all variables are those described in this document for [ ¹⁹ F]-(I)

under Buchwald-Hartwig amination conditions where all variables are those described herein for [ ¹⁹ F]-(I), or alternatively by reacting a compound of formula (II-a') where R ¹ is -NO2 and R ³ is H or R ¹ is H and R ³ is -NO2 with a fluoride source such as KF in a reaction inert solvent such as DMSO under heating conditions

or, alternatively, by reacting isoquinoline-6-amine with the corresponding 2-chloropyridine compound of formula (II-b), where all variables are those described herein for [ ¹⁹ F]-(I)

under the conditions of amination according to Buchwald-Hartwig.

The compound of formula (II-a') can be obtained, for example, by reacting isoquinoline-6-9 039208 amine with 6-bromo-3-methyl-2-nitropyridine or 2-bromo-5-methyl-4-nitropyridine under the conditions amination according to Buchwald-Hartwig.

Ways of application.

The compounds of the present invention find various uses in tissue or subject imaging, both in vitro and in vivo. Thus, for example, they can be used to map the differential distribution of tau aggregate deposits in subjects of different ages and sexes. Additionally, they provide an opportunity to investigate the differential distribution of tau protein aggregate deposits in subjects suffering from various diseases or disorders, including Alzheimer's disease, as well as other diseases associated with tau protein aggregate deposits, i.e. other taupathies.

Thus, overdistribution can be useful for diagnosis, case finding, stratification of subject groups, and in tracking disease progression in individual subjects, particularly as tau therapies, such as antibodies, become available. Since the radioactive ligand is administered in trace amounts, i.e. in detectable amounts for PET imaging, then the therapeutic effect cannot be explained by the introduction of radioactive ligands in accordance with the present invention.

Experimental part.

Chemistry.

As used herein, the term ACN or MeCN means acetonitrile, aq. means aqueous, tBuOH means tert-butanol, DCM means dichloromethane, DIPE means diisopropyl ether, DIPEA means N,N-diisopropylethylamine, DMF means N,N-dimethylformamide, DMSO means dimethyl sulfoxide, Et ₂ O means diethyl ether, EtOAc means ethyl acetate, h means hours, HPLC means high performance liquid chromatography, LCMS means liquid chromatography/mass spectrometry, MeOH means methanol, min means minutes, m.p. means melting point, org. means organic, Pd ₂ (dba) ₃ means tris(dibenzylideneacetone)dipalladium(0), prep. Means preparative, r.cm. means the reaction mixture, k.t. means room temperature, Rt means retention time (in minutes), sat. means saturated, sol. means solution, TFA means trifluoroacetic acid, THF means tetrahydrofuran, XantPhos means 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.

Thin layer chromatography (TLC) was carried out on plates with a layer of silica gel 60 F254 (Merck) using chemically pure solvents. Open column chromatography was performed on silica gel with a particle size of 230-400 mesh and a pore size of 60 A (Merck) according to standard procedures.

Automated flash column chromatography was performed using ready-to-connect disposable cartridges purchased from Grace (GraceResolv™ cartridges) or Teledyne ISCO (RediSep® cartridges) on irregularly shaped silica gel with a particle size of 35-70 µm on an ISCO CombiFlash or Biotage apparatus. Isolera™ Spektra.

Nuclear Magnetic Resonance (NMR): For several compounds, 'H-NMR spectra were recorded on either a Bruker Ultrashield AV300, Bruker DPX 360 MHz NMR, or Bruker Avance III 400 MHz NMR spectrometer with standard pulse trains operating at 300, 360, and 400 MHz respectively. Samples were dissolved in DMSO-d ₆ or CDCl ₃ and transferred to 5 mm NMR tubes for measurement. Chemical shifts (δ) are given in parts per million (ppm) for the downfield shift from tetramethylsilane (TMS), which was used as an internal standard.

HPLC purification was performed on a GILSON Semi-Preparative System running with Trilution software equipped with a Phenomenex Gemini C18 100A column (100 mm length x 30 mm I.D.; 5 µm particles) at rt. °C, at a flow rate of 40 ml/min, using a 20 min gradient elution as specified in the synthesis protocols. The volume of the injected sample was 8000 µl. The acquisition frequency was set to 284 nm for the UV-Dual detector.

Some methods for preparing the compounds of the present invention are illustrated in the following examples, which are intended to illustrate, but not to limit the scope of the present invention. Unless otherwise indicated, all starting materials were obtained from commercial suppliers and used without further purification.

A. Synthesis of intermediates.

Getting Intermediate 1

Tetramethyltin (0.736 mL, 5.309 mmol) was added to a mixture of 5-bromo-4-fluoropyridine-2-amine (0.338 g, 1.77 mmol, CAS 944401-69-8), bis(triphenylphosphine)palladium(II) chloride (0.062 g, 0.0885 mmol) and LiCl (0.300 g, 7.078 mmol) in 10 ml DMF. This mixture was sealed in a test tube after degassing with nitrogen. The mixture was then agitated at 120°C for 18 hours, after which it was diluted with water. Then a saturated aqueous solution of KF and aq. layer was extracted with EtOAc. United org. the layers were dried (MgSO ₄ ), filtered and the solvents were evaporated in vacuo. The crude material was purified by flash column chromatography (silica; DCM/MeOH 100/0 to 95/5). The desired fractions were collected and concentrated in vacuo to give Intermediate 1 (0.190 g, 79%).

Preparation of Intermediate 2

O _h , O * N ⁺ n

Method 1: To a stirred solution of 6-aminoisoquinoline (18.3 g, 127.1 mmol) in toluene (400 ml) was added 2-bromo-5-methyl-4-nitropyridine (23.0 g, 105.9 mmol), Xantphos (2.45 g, 4.24 mmol), Pd ₂ (dba) ₃ (1.94 g, 2.12 mmol), tBuOK (16.6 g, 148.3 mmol) under nitrogen. Nitrogen was bubbled through the mixture for 5 minutes and then the vial was stoppered and heated at 100° C. for 2 hours. The mixture was filtered and the filter cake washed with EtOAc until all product was extracted. Water and org. the layer was separated, dried (MgSO ₄ ), filtered and concentrated. The residue was taken up in water, triturated for 1 h, filtered and dried in vacuo at 55°C. The resulting brown solid (dissolved in MeOH at pH 4) was purified by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 µm, 50x150 mm, mobile phase: 0.5% NH ₄ OAc in water+10% CH ₃ CN, MeOH). Pure fractions were combined and the organic component of the eluent was evaporated. A red precipitate formed which was filtered, washed with water and dried. The resulting precipitate was shaken in DCM/MeOH/1N. NaOH (2 l / 0.2 l / 1 l) until completely dissolved and org. layer was separated. Water the layer was extracted again with DCM. United org. the layers were dried (MgSO ₄ ), filtered and evaporated. The residue was triturated in a biphasic mixture of ether and water, filtered and dried in vacuo for 2 days at 50°C and 4 hours in a lyophilizer at rt. to give intermediate 2 as orange crystals (6.11 g, 18%). 1H-NMR (400 MHz, DMSO-d ₆ ) δ ppm 2.40 (s, 3 H) 7.50 (s, 1 H) 7.62-7.71 (m, 2 H) 8.01 (d , J=9.0 Hz, 1 H) 8.36 (d, J=5.7 Hz, 1 H) 8.45 (s, 2 H) 9.09 (s, 1 H) 9.97 (s , 1 N)

Method 2 To a stirred solution of 6-aminoisoquinoline (7.62 g, 52.85 mmol) in 200 ml of toluene was added 2-bromo-5-methyl-4-nitropyridine (11.47 g, 52.85 mmol), Xantphos ( 0.611 g, 1.057 mmol), Pd ₂ (dba) ₃ (0.484 g, 0.529 mmol) and tBuOK (8.30 g, 74.00 mmol). Nitrogen was bubbled through the mixture for 5 minutes, and then the vessel was sealed and heated at 100°C for 2 hours, and another 3 hours at 110°C. EtOAc was added to the mixture, after which it was agitated for 30 minutes. The mixture was then filtered through dicalite and the filter cake washed with EtOAc until all product was extracted. Water and org. layer was separated. Water the layer was extracted with EtOAc until no product remained (LCMS control). The combined organic layers were washed with brine, dried (MgSO ₄ ), filtered and concentrated. The residue was taken up in water/DIPE, triturated overnight, filtered and dried in vacuo at 55° C. to give Intermediate 2 (10.5 g, 71%, 57% pure) which was used as such for the next reaction step.

Preparation of Intermediate 3

Trimethylboroxine (0.256 mL, 1.83 mmol) was added to an agitated solution of 5-bromo-6fluoropyridine-2-amine (0.291 g, 1.52 mmol, CAS 944401-65-4), tetrakis(triphenylphosphine)palladium(0) (0.176 g, 0.152 mmol) and Cs ₂ CO ₃ (0.993 g, 3.047 mmol) in 1,4-dioxane (5.3 ml) under nitrogen sparging. The reaction mixture was agitated at 105° C. for 16 hours under nitrogen. Water and EtOAc were added. The phases were separated and the organic was dried over MgSO4, filtered and evaporated. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to give Intermediate 3 as a yellow solid (0.151 g, 79%).

Preparation of Intermediate 4

Deoxo-Fluor® (50% in toluene, 1.67 mL, 3.807 mmol) was added dropwise to a solution of 2-chloro-5-(2fluoroethyl)pyridine (0.400 g, 2.538 mmol, CAS 117528-28-6) in dry DCM ( 15 ml) at 0°C. After 1 min, the cold bath was removed and the reaction mixture was agitated at room temperature for 2 h.

- 11 039208

The mixture was diluted with aq. sat. NaHCO ₃ and extracted with DCM. The organic layer was washed with water, dried (MgSO ₄ ), filtered and the solvents were evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane from

0/100 to 70/30). The desired fractions were collected and concentrated in vacuo to give Intermediate 4 (0.193 g, 45%).

Preparation of Intermediate 5

NaOH (0.064 g, 1.599 mmol) was added to a mixture of [(6-chloropyridin-3-yl)oxy]acetic acid (0.300 g, 1.599 mmol, CAS 234109-28-5) in a mixture of CH3CN (4 mL) and water (4 ml) . The mixture was heated at 85° C. for 1 h. The solvent was concentrated in vacuo to give intermediate 5 (335 mg, 95%) as a white solid, which was used as such in the next step.

Preparation of Intermediate 6

Selectfluor® (1.116 g, 3.15 mmol) was added to a solution of intermediate 5 (0.314 g, 1.5 mmol) in a mixture of water (7.5 ml) and CH ₃ CN (7.5 ml), previously degassed.

Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (0.056 g, 0.075 mmol) was added. The reaction mixture was irradiated with a 500 W shipyard lamp emitting light in the visible part of the spectrum, placed at a distance of 30 cm from the reaction mixture, for 1 hour. After turning off the light, water and diethyl ether were poured into the reaction medium. The two phases were separated and the aqueous was extracted with Et ₂ O. The combined organic phases were dried over MgSO ₄ , filtered and the solvent was evaporated under reduced pressure. The crude product was filtered through silica gel with Et ₂ O as eluent and concentrated in vacuo to give intermediate 6 as a beige oily solid (0.167 g, 54%).

Preparation of Intermediate 7

OO=S=O I . nh

Cl^N^

An agitated solution of 5-amino-2-chloropyridine (1.00 g, 7.778 mmol) in THF (27 ml) was cooled to 0°C, then 2-nitrobenzenesulfonyl chloride (1.72 g, 7.778 mmol) and pyridine (0.942 ml, 11.668 mmol). The resulting mixture was allowed to heat up to rt. and agitated for 3 h. 2-Nitrobenzenesulfonyl chloride (0.52 g, 2.334 mmol) and pyridine (0.314 ml, 3.889 mmol) were added at 0°C and the mixture was agitated at rt. during the night. Water and aq. were then added to the mixture. layer was extracted with EtOAc. The extract was washed with saturated aq. NaHCO ₃ and saline. Org. the layer was dried over MgSO ₄ , filtered and evaporated. The residue was purified by flash column chromatography (silica gel, EtOAc in heptane, 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to give Intermediate 7 (2.22 g, 91%).

Preparation of Intermediate 8

Intermediate 7 (1.1 g, 3.506 mmol) and 2-fluoroethyl 4-methylbenzenesulfonate (0.918 g, 4.208 mmol, CAS 383-50-6) were dissolved in DMF (10.6 ml). Cs ₂ CO ₃ (1.942 g, 5.961 mmol) was added under nitrogen atmosphere. The mixture was heated to 85°C during the night. After cooling to ambient temperature, the dark brown suspension was diluted with water and extracted with EtOAc. Org. layer washed

- 12 039208 brine, dried over MgSO4, filtered and concentrated. The product was purified by flash column chromatography (silica, EtOAc in heptane, 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to give Intermediate 8 (0.687 g, 54%).

Preparation of Intermediate 9

Intermediate 7 (0.700 g, 2.231 mmol) and 3-fluoropropyl-4-methylbenzenesulfonate (0.674 g, 2.901 mmol, CAS 312-68-5) were dissolved in DMF (6.7 mL). Cs ₂ CO ₃ (1.45 g, 4.463 mmol) was added and the mixture was heated to 85° C. overnight under nitrogen. 3-Fluoropropyl 4-methylbenzenesulfonate (0.363 g, 1.562 mmol) and Cs ₂ CO ₃ (0.727 g, 2.231 mmol) were added and the mixture was heated at 85° C. overnight. After cooling to ambient temperature, the dark brown suspension was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. The product was purified by flash column chromatography (silica, EtOAc in heptane, 0/100 to 15/85). The desired fractions were collected and concentrated in vacuo to give Intermediate 9 (0.395 g, 42%).

Preparation of Intermediate 10

To an agitated solution of intermediate 8 (0.687 g, 1.91 mmol) in 11.6 ml DMF was added LiOH-H ₂ O (0.491 g, 11.46 mmol) and 2-mercaptoethanol (0.164 ml, 2.33 mmol) at 0°C. The reaction mixture was shaken at rt. over 2 hours. Water was added and the mixture was extracted with EtOAc. The extract was washed with water and dried over MgSO ₄ . Concentration under reduced pressure gave oily residues. The product was purified by flash chromatography (silica gel, EtOAc in heptane, 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo to give intermediate 10 as a yellow oil (0.276 g, 83%).

Preparation of Intermediate 11

Intermediate 10 (0.200 g, 1.145 mmol) was added to an agitated solution of NaH (60% dispersion in mineral oil, 0.069 g, 1.718 mmol) in DMF (9.2 ml) at 0° C. under nitrogen. After shaking for 30 minutes at this temperature, CH ₃ I (0.134 ml, 1.833 mmol) was added and shaking continued overnight at rt. NaH (60% dispersion in mineral oil, 0.032 g, 0.802 mmol) was added at 0° C. and the mixture was agitated for 30 minutes. CH ₃ I (0.032 ml, 0.573 mmol) was added and the mixture was shaken at rt. for 2 hours The mixture was diluted with water and extracted with EtOAc. Org. the layer was dried (MgSO ₄ ), filtered and evaporated in vacuo. The crude product was purified by flash column chromatography (EtOAc in heptane 0/100 to 15/85). The desired fractions were collected and concentrated in vacuo to give Intermediate 11 (0.137 g, 63%).

Preparation of Intermediate 12

NaH (60% dispersion in mineral oil, 0.178 g, 4.46 mmol) was added to an agitated solution of tert-butyl(6-chloropyridin-3-yl)carbamate (1.36 g, 75% pure, 4.46 mmol, CAS 171178-45-3) in dry DMF (7 ml) at 0°C under N ₂ . The mixture was shaken at 0°C for 15 min. Then 1-bromo-3-fluoropropane (0.491 mL, 5.353 mmol) was added at 0° C., allowing the mixture to warm to rt. and shaken overnight. More 1-bromo-3-fluoropropane (0.327 ml, 3.568 mmol) and NaH (60% dispersion in mineral oil, 0.178 g, 4.46 mmol) were added and the mixture was agitated at rt. within 4 hours.

- 13 039208 additional 1-bromo-3-fluoropropane (0.818 ml, 8.921 mmol) and NaH (60% dispersion in mineral oil, 0.178 g, 4.46 mmol) and the mixture was shaken at rt. during the night. The reaction mixture was taken up in DCM and washed with saturated NH ₄ Cl solution. The organic layer was separated, dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica; EtOAc in heptane 40 to 100%). The desired fractions were recovered and the solvents were evaporated in vacuo to give intermediate 12 as a colorless oil (1.71 g, 75% pure, quantitative yield).

Preparation of Intermediate 13

H

SG'LG

Method 1 TFA (4 ml, 53.3 mmol) was added to a mixture of intermediate 12 (1.71 g, 5.33 mmol, 75% pure) in 20 ml DCM at 0°C, and then the mixture was shaken at rt. t. within 150 min. The mixture was then diluted with DCM and washed with aq. sat. NaHCO ₃ solution. Org. the layer was separated, dried (MgSO4), filtered and concentrated in vacuo to give intermediate 13 (1.91 g, 53% pure, quantitative) which was used as such.

Method 2 To an agitated solution of intermediate 7 (0.395 g, 0.93 mmol) in DMF (5.6 ml) was added LiOH-H ₂ O (0.239 g, 5.58 mmol) and 2-mercaptoethanol (0.798 ml, 1.134 mmol) at 0°C. The reaction mixture was shaken at rt. for 2 h, after which it was extracted with EtOAc. United org. the layers were washed with water, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave an oily residue which was purified by flash chromatography (silica gel, EtOAc in heptane, 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo to give intermediate 13 as a yellow oil (0.168 g, 96%).

Preparation of Intermediate 14

Method 1 Formaldehyde (37% in water, 2.185 ml, 29.162 mmol) was added to a solution of intermediate 13 (prepared by method 1, 1.91 g, 9.721 mmol, 53% pure) in a mixture of acetic acid (0.835 ml) and MeOH ( 20 ml). NaBH ₃ CN (1.83 g, 29.162 mmol) was then added in portions. The mixture was shaken at rt. during the night (3 pm). Saturation was added. NaHCO ₃ solution and the mixture was extracted with EtOAc. Org. the layers were dried over MgSO ₄ , filtered and concentrated. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 80/20). The desired fractions were collected and concentrated in vacuo to give Intermediate 14 (0.687 g, 63%) as a colorless oil.

Method 2 Intermediate 13 (prepared by Method 2, 0.172 g, 0.912 mmol) was added to an agitated solution of NaH (60% dispersion in mineral oil, 0.055 g, 1.368 mmol) in DMF (7.3 ml) at 0° C. under atmospheric nitrogen. After shaking for 30 minutes at this temperature, CH3I (0.107 ml, 1.459 mmol) was added and shaken overnight at rt. The mixture was diluted with water and extracted with EtOAc. The organic layer was filtered, dried and evaporated in vacuo. The crude product was purified by flash column chromatography (EtOAc in heptane 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to give Intermediate 14 (0.143 g, 77%).

Preparation of Intermediate 15

2-Chloro-5-hydroxypyridine (0.300 g, 2.316 mmol) and 2-fluoroethyl-4-methylbenzenesulfonate (0.505 g, 2.316 mmol, CAS 383-50-6) were dissolved in DMF (7 ml). Cs ₂ CO ₃ (1.132 g, 3.474 mmol) was added under nitrogen atmosphere. The mixture was heated to 85° C. for 5 h. After cooling to ambient temperature, the dark brown suspension was diluted with water and extracted with EtOAc. Org. the layers were washed with brine, dried over MgSO4, filtered and concentrated. The product was purified by flash column chromatography (silica, EtOAc in heptane, 0/100 to 35/65). The desired fractions were collected and concentrated in vacuo to give Intermediate 15 (0.374 g, 92%).

Preparation of Intermediate 16

2-Chloro-5-hydroxypyridine (0.300 g, 2.316 mmol) and 3-fluoropropyl-4-methylbenzenesulfonate (0.538

- 14039208 g, 2.316 mmol, CAS 312-68-5) was dissolved in DMF (7 ml). Cs ₂ CO ₃ (1.132 g, 3.474 mmol) was added under nitrogen atmosphere. The mixture was heated to 85°C for 5 hours After cooling to ambient temperature, the dark brown suspension was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO ₄ , filtered and concentrated. The product was purified by flash column chromatography (silica, EtOAc in heptane, 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo to give Intermediate 16 (0.408 g, 93%).

Preparation of Intermediate 17

K ₃ PO ₄ (1.70 g, 7.99 mmol), Pd ₂ (dba) ₃ (0.152 g, 0.166 mmol) and Xantphos (0.161 mg, 0.277 mmol) were added to a solution of 2,5-dibromopyridine (0.738 g, 3.052 mmol, CAS 624-28-2) in dry THF (20 ml) with nitrogen bubbling through the mixture. After 10 minutes, 6-aminoisoquinoline (0.400 g, 2.774 mmol, CAS 23687-26-5) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 hours. After cooling to rt. the mixture was washed with aq. sat. NaHCO ₃ and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 75/25). The desired fractions were collected and concentrated in vacuo to give intermediate 17 (0.680 g, 80% purity, 65% yield).

Preparation of Intermediate 18

mixture of isomer E (larger) and Z (smaller)

Intermediate 17 (0.680 g, 1.812 mmol) in 1,4-dioxane (10.2 mL) was degassed for 10 min. 2-[2-ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.538 g, 2.719 mmol, CAS 1201905-61-4, prepared according to the procedure described in WO 2012010538, mixture 2:1 E and Z), Pd(OAc) ₂ (0.020 g, 0.0906 mmol), X-Phos (0.095 g, 0.199 mmol) and Cs ₂ CO ₃ (0.886 g, 2.719 mmol), followed by degassed water (1.1 ml). The mixture was degassed for another 10 minutes and agitated at 80° C. for 6 hours. The mixture was then cooled to ambient temperature and diluted with EtOAc. Org. the phase was washed with water and brine, dried over MgSO4, filtered and concentrated. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 100/0). The desired fractions were collected and concentrated in vacuo to give intermediate 18 (0.510 g, 91%, mixture of E and Z).

Preparation of Intermediate 19

To a mixture of intermediate 18 (0.510 g, 1.75 mmol) in THF (5.3 ml) was added HCl (2M in water, 3.939 ml, 7.877 mmol). The mixture was stirred at 60° C. overnight. Sat. NaHCO ₃ was added to pH 7. The mixture was extracted with EtOAc. Org. the layer was separated, dried (MgSO ₄ ), filtered and the solvents were evaporated in vacuo to give intermediate 19. The product was used as such in the next step (0.378 g, 82%).

Preparation of Intermediate 20

NaBH ₄ (0.054 g, 1.436 mmol) was added to a solution of intermediate 19 in MeOH (4.5 ml) at 0°C. The mixture was shaken at rt. within 30 min. Water was added and the mixture was extracted with EtOAc. Org. the layer was separated, dried (MgSO ₄ ), filtered and the solvents were evaporated in vacuo. The crude product was purified by flash column chromatography (silica; MeOH in DCM 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to give Intermediate 20 (0.135 g, 35%).

B. Preparation of compounds of formula [ ¹⁹ F] (I).

Get connection 1

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Method 1 K ₃ PO ₄ (0.872 g, 4.11 mmol), Pd ₂ (dba) ₃ (0.078 g, 0.0856 mmol) and Xantphos (0.083 g, 0.143 mmol) were added to a solution of 6-bromoisoquinoline (0.327 g , 1.57 mmol) in dry DMF (15 ml) with nitrogen bubbling through the mixture. After 10 min, intermediate 1 (0.180 g, 1.427 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. After cooling to rt. the mixture was washed with sat. NaHCO ₃ and extracted with EtOAc. Org. the layer was dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; DCM/MeOH 100/0 to 95/5). The desired fractions were collected and concentrated in vacuo. The product was further purified by reverse phase from 70% H ₂ O (25 mM NH4HCO3)-30% MeCN-MeOH to 27% H ₂ O (25 mM NH4HCO3)-73% MeCN-MeOH. The desired fractions were collected and concentrated in vacuo. The product was triturated with DIPE to give Compound 1 as a white solid (0.150 g, 41%).

Method 2 KF (10.2 g, 175.7 mmol) was added to intermediate 2 (prepared by method 2, 9.85 g, 35.14 mmol) in DMSO (236 ml). The resulting mixture was agitated for 16 hours at 160°C after which LCMS showed complete conversion. Water and DCM were added, the biphasic mixture was shaken and then filtered through dicalite®. Org. the layer was separated and aq. the layer was extracted with DCM. United org. the layers were dried (MgSO ₄ ), filtered and evaporated to dryness. The red-brown residue was purified by 120 g silica gel flash column chromatography using a gradient (heptane/EtOAc 1:0 to 0:1). Product fractions were evaporated to dryness to give a brown solid. Purification was performed by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 µm, 50x150 mm, mobile phase: 0.25% NH4HCO3 solution in water, MeOH) to give compound 1 as white crystals (1.38 g, 15 .5%) after evaporating the eluent, suspending in water, filtering, washing with heptane and drying in vacuo. 1H-NMR (400 MHz, DMSO-d6) δ ppm 2.17 (s, 3 H) 6.75 (d, J=11.9 Hz, 1 H) 7.62 (d, J=5.7 Hz , 1 H) 7.66 (dd, J=8.9, 2.1 Hz, 1 H) 7.97 (d, J=8.8 Hz, 1 H) 8.20 (d, J=11, 2 Hz, 1 H) 8.33 (d, J=5.7 Hz, 1 H) 8.42 (d, J=1.8 Hz, 1 H) 9.06 (s, 1 H) 9.61 (s, 1 H).

Get connection 2

K ₃ PO ₄ (0.462 g, 2.175 mmol), Pd ₂ (dba) ₃ (0.041 g, 0.0453 mmol) and Xantphos (0.044 g, 0.0755 mmol) were added to a solution of intermediate 3 (0.100 g, 0.793 mmol ) in dry THF while bubbling nitrogen through the mixture. After 10 minutes, 6-bromoisoquinoline (0.157 g, 0.755 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. After cooling to rt. the mixture was washed with aq. sat. NaHCO ₃ and extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; DCM/MeOH 100/0 to 95/5). The desired fractions were collected and concentrated in vacuo. The residue was purified by preparative HPLC ((0.1% HCOOH)/25 mM NH4HCO3); from 70/30 to 27/73). The desired fractions were collected and concentrated in vacuo to give compound 2 (0.0238 g, 12%). 1H-NMR (300 MHz, chloroform-d) δ ppm 2.24 (s, 3 H) 6.72-6.86 (m, 2 H) 7.40-7.60 (m, 3 H) 7, 89 (d, J=8.7 Hz, 1 H) 7.93 (br s, 1 H) 8.44 (d, J=5.8 Hz, 1 H) 9.10 (br s, 1 H) .

Get Compound 3

K ₃ PO ₄ (0.300 g, 1.415 mmol), Pd ₂ (dba) ₃ (0.027 g, 0.0295 mmol) and Xantphos (0.028 g, 0.0491 mmol) were added to a solution of intermediate 4 (0.080 g, 0.491 mmol ) in dry THF (5 ml) while bubbling nitrogen through the mixture. After 10 minutes, isoquinoline-6-amine (0.074 g, 0.516 mmol, CAS 23687-26-5) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 90° C. for 16 h. After cooling to rt. the mixture was washed with sat. NaHCO ₃ and extracted with EtOAc. Org. the layer was dried over MgSO4, filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; MeOH in DCM 0/100 to 3/97). The desired fractions were collected and concentrated in vacuo. The product was further purified by preparative HPLC ((0.1% HCOOH)/ACN:MeOH 1:1); from 95/5 to 63/37). The desired fractions were collected, concentrated in vacuo to give compound 3 (0.051 g, 38%). ¹ H-NMR (300 MHz, chloroform-d) δ ppm 2.98 (dt, J=24.7, 6.2 Hz, 2 H) 4.64 (dt, J=47.0, 6.3 Hz , 2 N) 6.82 (br s, 1 N) 6.98 (d, J=8.4 Hz,

H) 7.43-7.56 (t, 3 H) 7.89 (d, J=8.8 Hz, 1 H) 7.98 (s, 1 H) 8.22 (s, 1 H) 8 .43 (d, J=5.8 Hz, 1 H) 9.10 (s, 1 H).

Compound 3 could also be obtained, alternatively, from intermediate 20, for example, by formation of the corresponding methanesulfonate or 4-toluenesulfonate followed by displacement with a source of fluoride.

Get Compound 4

K ₃ PO ₄ (0.481 g, 2.264 mmol), Pd ₂ (dba) ₃ (0.043 g, 0.0472 mmol) and Xantphos (0.045 g, 0.0786 mmol) were added to a solution of intermediate 6 (0.127 g, 0.786 mmol ) in THF (6 ml) with nitrogen sparging. After 10 minutes, isoquinoline-6-amine (0.125 g, 0.865 mmol, CAS 23687-26-5) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 90° C. for 16 hours. After cooling to rt. the mixture was washed with sat. NaHCO ₃ and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 5/95 to 100/0). The desired fractions were collected and concentrated in vacuo. The product was purified by preparative HPLC (59% H ₂ O (25 mM NH4HCO3)-41% MeCN-MeOH to 17% H ₂ O (25 mM NH4HCO3)-83% MeCN-MeOH). The desired fractions were collected and concentrated in vacuo. The product was triturated with npentane to give compound 4 as a beige solid (0.075 g, 34%). ¹ H-NMR (300 MHz, chloroform-d) δ ppm 5.69 (d, J=54.6 Hz, 2 H) 6.80 (br s, 1 H) 7.00 (d, J=8, 9 Hz, 1 N) 7.39-7.47 (t, 1 N) 7.54 (d, J=5.8 Hz, 1 N) 7.89 (d, J=8.8 Hz, 1 N ) 7.97 (s, 1 H) 8.22 (d, J=2.1 Hz, 1 H) 8.43 (d, J=5.8 Hz, 1 H) 9.09 (s, 1 H ).

Get Compound 5

K ₃ PO ₄ (0.294 g, 1.386 mmol), Pd ₂ (dba) ₃ (0.026 g, 0.0289 mmol) and Xantphos (0.028 g, 0.0481 mmol) were added to a solution of intermediate 10 (0.084 g, 0.481 mmol ) in THF (4 ml) with nitrogen sparging. After 10 minutes, isoquinoline-6-amine (0.069 g, 0.481 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. Pd ₂ (dba) ₃ (0.026 g, 0.0289 mmol) and Xantphos (0.028 g, 0.0481 mmol) were added with nitrogen bubbling and the mixture was further heated at 100° C. during the night. Pd ₂ (dba) ₃ (0.026 g, 0.0289 mmol) and Xantphos (0.028 g, 0.0481 mmol) were added with nitrogen bubbling and the mixture was further heated at 100° C. overnight. The mixture was washed with aq. sat. NaHCO ₃ and extracted with EtOAc. The organic layer was dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo. The product was further purified by preparative HPLC (from 75% [25 mM NH4HCO3]-25% [ACN: MeOH 1:1] to 38% [25 mM NH4HCO3] 62% [ACN: MeOH 1:1]). The desired fractions were combined and the solvent was evaporated. The product was triturated with Et ₂ O and filtered to give compound 5 (0.042 g, 31%). ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ ppm 3.34-3.48 (m, 2 H) 4.58 (dt, J=47.6, 4.8 Hz, 2 H) 5, 63 (br t, J=5.9 Hz, 1 H) 6.86 (d, J=8.8 Hz, 1 H) 7.11 (dd, J=8.8, 2.7 Hz, 1 H ) 7.47-7.57 (m, 2 H) 7.78 (d, J=2.5 Hz, 1 H) 7.87 (d, J=8.9 Hz, 1 H) 8.21- 8.31(m, 2H) 8.96(s, 1H) 9.12(s, 1H).

Getting Compound 6

K3PO4 (0.444 g, 2.092 mmol), Pd ₂ (dba) ₃ (0.040 g, 0.0436 mmol) and Xantphos (0.042 g, 0.0726 mmol) were added to a solution of intermediate 11 (0.137 g, 0.726 mmol) in THF (6 ml) under nitrogen sparging. After 10 minutes, isoquinoline-6-amine (0.105 g, 0.726 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. Pd ₂ (dba) ₃ (0.026 g, 0.0289 mmol) and Xantphos (0.028 g, 0.0481 mmol) were further added while nitrogen was bubbled through the mixture, after which it was further heated at 100°C overnight. The mixture was washed with aq. sat. _NaHCO3

- 17 039208 and extracted with EtOAc. The combined organic layers were dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 65/35). The desired fractions were collected and concentrated in vacuo. The product was further purified by preparative HPLC (from 70% [25 mM NH ₄ HCO ₃ ]-30% [ACN: MeOH 1:1] to 27% [25 mM NH4HCO3]-73% [ACN: MeOH 1:1]). The desired fractions were combined and the solvent was evaporated. The product was triturated with Et2O and filtered to give compound 6 (0.092 g, 43%). ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ ppm 2.93 (s, 3 H) 3.62 (dt, J=26.7, 4.6 Hz, 2 H) 4.60 (dt, J=47.7, 4.7Hz, 2H) 6.93 (d, J=9.1Hz, 1H) 7.28 (dd, J=9.0, 2.7Hz, 1H) 7.46-7.60 (m, 2 H) 7.82-7.94 (m, 2 H) 8.27 (d, J=5.8 Hz, 1 H) 8.34 (s, 1 H ) 8.97 (s, 1 H) 9.21 (s, 1 H).

Obtaining Compound 7

K ₃ PO ₄ (0.544 g, 2.565 mmol), Pd ₂ (dba) ₃ (0.049 g, 0.0534 mmol) and Xantphos (0.052 g, 0.0891 mmol) were added to a solution of intermediate 13 (0.168 g, 0.891 mmol ) in THF (6 ml) with nitrogen sparging. After 10 minutes, isoquinoline-6-amine (0.128 g, 0.891 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 hours. More Pd ₂ (dba) ₃ (0.049 g, 0.0534 mmol) and Xantphos (0.052 g, 0.0891 mmol) were added under nitrogen flow and the mixture was heated again at 100° C during the night. Pd2(dba)3 (0.049 g, 0.0534 mmol) and Xantphos (0.052 g, 0.0891 mmol) were added under a stream of nitrogen and the mixture was heated again at 100° C. for 6 h. The mixture was washed with sat. NaHCO ₃ and extracted with EtOAc. Org. the layers were dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 45/55). The desired fractions were collected and concentrated in vacuo. The product was purified by preparative HPLC (from 75% [25 mM NH4HCO3] - 25% [ACN: MeOH 1:1] to 0% [25 mM NH4HCO3] - 100% [ACN: MeOH 1:1]). The desired fractions were combined and the solvent was evaporated. The residue was dissolved in DCM, HCl (5M in 2-propanol) was added and the resulting mixture was concentrated in vacuo. The residue was triturated with Et ₂ O and filtered to give compound 7 as an orange solid (0.066 g, 22% yield). ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ ppm 1.83-2.05 (m, 2 H) 3.17 (br t, J=6.8 Hz, 2 H) 4.58 (dt , J=47.4, 5.8 Hz, 2 H) 5.83 (br s, 1 H) 7.00 (d, J=8.8 Hz, 1 H) 7.12 (dd, J=8 .7, 2.7Hz, 1H) 7.77 (dd, J=9.2, 1.2Hz, 1H) 7.81 (d, J=2.5Hz, 1H) 7.99 (d, J=6.9Hz, 1H) 8.20 (d, J=9.1Hz, 1H) 8.28 (d, J=6.6Hz, 1H) 8.42 (s , 1 H) 9.30 (s, 1 H) 10.08 (s, 1 H).

Getting connection 8

K ₃ PO ₄ (0.431 g, 2.032 mmol), Pd ₂ (dba) ₃ (0.039 g, 0.0423 mmol) and Xantphos (0.041 g, 0.0706 mmol) were added to a solution of intermediate 14 (0.143 g, 0.706 mmol ) in THF (5.9 ml) while bubbling nitrogen through the mixture. After 10 minutes, isoquinoline-6-amine (0.102 g, 0.706 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100°C for 16 h. More Pd2(dba)3 (0.039 g, 0.0423 mmol) and Xantphos (0.041 g, 0.0706 mmol) were added while nitrogen was bubbled through the mixture, after which it was heated at 100 °C during the night. Additional Pd2(dba)3 (0.039 g, 0.0423 mmol) and Xantphos (0.041 g, 0.0706 mmol) were added under nitrogen sparging and the mixture was heated at 100°C for 6 h. The mixture was washed with sat. NaHCO ₃ and extracted with EtOAc. Org. the layer was dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in DCM 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo. The product was further purified by preparative HPLC (from 75% [25 mM NH4HCO3] - 25% [ACN: MeOH 1:1] to 0% [25 mM NH4HCO3] - 100% [ACN: MeOH 1:1]). The desired fractions were combined and the solvent was evaporated. The crude product was dissolved in DCM, HCl (5N in 2-propanol) was added and the mixture was concentrated in vacuo. The residue was triturated with Et ₂ O and filtered to give compound 8 as an orange solid (0.058 g, 23%). ¹ H-NMR (300 MHz, DMSO-d6) δ ppm. 1.78-2.02 (m, 2 H) 2.92 (s, 3 H) 3.45 (br t, J=7.1 Hz, 2 H) 4.52 (dt, J=47.4 , 5.7 Hz, 2 H) 7.09 (d, J=8.9 Hz, 1 H) 7.29 (dd, J=9.0, 3.0 Hz, 1 H) 7.81 (dd , J=8.8, 1.2 Hz, 1 H) 7.91 (d, J=2.6 Hz, 1 H) 7.98 (d, J=6.6 Hz, 1 H) 8.21 (d, J=9.1 Hz, 1 H) 8.29 (d, J=6.7 Hz, 1 H) 8.51 (s, 1 H) 9.32 (s, 1 H) 10.20 (s, 1 H).

Getting Compound 9

- 18 039208

K3PO4 (0.449 g, 2.117 mmol), Pd ₂ (dba) ₃ (0.040 g, 0.0441 mmol) and Xantphos (0.043 g, 0.0735 mmol) were added to a solution of intermediate 15 (0.142 g, 0.809 mmol) in THF (4 ml) while bubbling nitrogen through the mixture. After 10 minutes, isoquinoline-6-amine (0.106 g, 0.735 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. After cooling to rt. the mixture was washed with aq. sat. NaHCO ₃ and extracted with EtOAc. The combined organic layers were dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica gel; EtOAc in heptane 0/100 to 40/60). The desired fractions were collected and concentrated in vacuo. The product was triturated with diisopropyl ether and filtered to give compound 9 as a beige solid (0.116 g, 55%). 1H-NMR (300 MHz, DMSO-d ₆ ) δ ppm 4.28 (br dt, J=30.2, 3.3 Hz, 2 H) 4.75 (br dt, J=47.9, 3, 3 Hz, 2 H) 6.99 (d, J=8.9 Hz, 1 H) 7.43 (dd, J=8.9, 2.7 Hz, 1 H) 7.52-7.68 ( m, 2 H) 7.93 (d, J=8.8 Hz, 1 H) 8.07 (d, J=2.5 Hz, 1 H) 8.30 (br d, J=5.6 Hz , 1 H) 8.42 (s, 1 H) 9.02 (s, 1 H) 9.44 (s, 1 H).

Get connection 10

K ₃ PO ₄ (0.449 g, 2.117 mmol), Pd ₂ (dba) ₃ (0.040 g, 0.0441 mmol) and Xantphos (0.043 g, 0.0735 mmol) were added to a solution of intermediate 16 (0.153 g, 0.809 mmol ) in THF (4 ml) while bubbling nitrogen through the mixture. After 10 minutes, isoquinoline-6-amine (0.106 g, 0.735 mmol) was added and the mixture was shaken at rt. within 10 min. The mixture was then heated at 100° C. for 16 h. The mixture was washed with aq. sat. NaHCO ₃ and extracted with EtOAc. United org. the layers were dried over MgSO ₄ , filtered and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 35/65). The desired fractions were collected and concentrated in vacuo. The residue was dissolved in DCM, HCl (5N in 2-propanol) was added and the mixture was concentrated in vacuo. The residue was triturated with DIPE and filtered to give compound 10 as a yellow solid (0.147 g, 47%). ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ ppm 2.00-2.23 (m, 2 H) 4.16 (t, J=6.2 Hz, 2 H) 4.63 (dt, J=47.3, 5.8Hz, 2H) 7.18 (d, J=8.9Hz, 1H) 7.51 (dd, J=8.9, 3.0Hz, 1H) 7.91 (dd, J=9.2, 1.4 Hz, 1 H) 8.07 (d, J=6.7 Hz, 1 H) 8.13 (d, J=2.7 Hz, 1 H) 8.28 (d, J=9.1 Hz, 1 H) 8.34 (d, J=6.6 Hz, 1 H) 8.63 (s, 1 H) 9.41 (s, 1 H) 10.44 (s, 1 H).

C. Radiochemical synthesis.

Materials and methods.

General information.

A non-radioactive reference material for T808 was synthesized by Janssen Research & Development (a division of Janssen Pharmaceutica NV, Beers, Belgium) according to reports in the literature (Declercq L., Celen S., Lecina J. et al. Molecular Imaging 2016, 15, 1-151 ). All chemicals and reagents were purchased from commercial sources and used without further purification. HPLC analysis was performed on a LaChrom Elite HPLC system (Hitachi, Darmstadt, Germany) connected to a UV detector set at 254 nm. For the analysis of radiolabelled compounds, the HPLC eluate after passing through a UV detector was passed through a 3 inch NaI (Tl) scintillation detector attached to a single channel analyzer (GABI box; Raytest, Straubenhardt, Germany). Data were acquired and analyzed using GINA Star data acquisition systems (Raytest).

Comm. tritiation No. 1

H(T)

Comm. No. 1 [ ³ H] conn. No. 1.

Comm. No. 1 (1.74 mg, 6.87 µmol) and (1,2,5,6-p)-1,5-cyclooctadiene (pyridine) (tricyclohexylphosphine) iridium (1) hexafluorophosphate, also called Crabtree's catalyst (7, 72 mg, 9.59 μmol) was dissolved in CH ₂ Cl ₂ (0.6 ml). The bright orange solution was degassed three times on an RC Tritec tritiation manifold and then agitated under tritium gas (8.9 Ci) for 3.5 hours at room temperature. The maximum pressure reached during the reaction was 882 mbar. After the reaction

- 19 039208 the solvent was removed under vacuum. Unstable tritium was displaced by adding methanol (0.8 ml), shaking the solution and removing the solvent again under vacuum. This method was repeated three times to obtain a dry solid crude product.

The crude product was purified by HPLC method: Macherey+Nagel Nucleodur Gravity C18 column, 5 µm, 8x150 mm; mobile phase A: water with 0.1% TFA, B: acetonitrile with 0.1% TFA; isocratic at 27% B; flow rate 3.1 ml/min, 230 and 254 nm, 20°C. After HPLC, the pH of the collected product fractions was adjusted to neutral with an aqueous solution of NaHCO ₃ (10%). The volume of the mixture was reduced on a rotary evaporator. The product was then extracted with a Phenomenex StrataX cartridge (3 ml, 100 mg) which was eluted with ethanol (5 ml). The resulting product was further purified by HPLC under basic conditions: Macherey+Nagel Nucleodur Sphinx RP, 5 µm, 8x150 mm; mobile phase A: 10 mM NH ₄ OAc with 0.05% NH ₄ OH, B: acetonitrile; isocratic at 46.5% B; flow rate 3.1 ml/min, 230 and 254 nm, 20°C. The volume of fractions of the collected product was reduced on a rotary evaporator. The product was then extracted with a Phenomenex StrataX cartridge (3 ml, 100 mg) which was eluted with ethanol (5 ml).

Radiochemical purity [ ³ H] Comm. No. 1 was defined as >99% using the following HPLC method: column: Macherey+Nagel Nucleodur Sphinx RP (5 μm), 4.6x150 mm, column temperature: 30°C, mobile phase A: 10 mM NH ₄ OAc with 0, 05% v/v NH ₄ OH in water, mobile phase B: acetonitrile, flow rate: 1.0 ml/min, injection volume: 5 µl, detection wavelength: UV at 254 nm, elution gradient: gradient from 5%B to 95%B per 0-20 min; isocratic at 95% B for 20-25 minutes; gradient from 95% B to 5% B in 25-25.5 minutes. The radioactivity flow detector was a Berthold LB 513 with a Zinsser Quickszint Flow 302 cocktail at a flow rate of 3.0 ml/min.

The specific activity was determined to 57.7 mCi/mmol using mass spectrometry. LCMS conditions: Agilent Zorbax SB C18 column (1.8 µm) 2.1x50 mm; mobile phase A: water, 0.1% formic acid, B: MeCN 0.1% formic acid; 0 min. 5% B; 0.2 min. 5% B; 5 minutes. 80% B; flow rate 0.6 ml/min; injection 1.0 μl (1.06 μCi, 39.2 kBq), UV detection 225 nm; temperature 60°C.

Radiofluorination Comm. No. 1 (n=6)

[ ¹⁸ F]KF.K222

Η

DMSO, 160 °C (microwave, 50 W), 10 min.

Intermediate, conn. 2 [ ¹⁸ P]comp. No. 1

Fluoride-18 ([ ¹⁸ F]F ^- ) was obtained using the nuclear reaction ¹⁸ O(p,n) ¹⁸ F on the Cyclone 18/9 cyclotron (Ion Beam Applications, Louvain-la-Neuve, Belgium) by irradiating 2 ml of 97% enriched in ¹⁸ О-Н2О (Rotem HYOX18, Rotem Industries, Beer Sheva, Israel) with 18-MeV protons. After irradiation, [ ¹⁸ F]F ^{- was} captured in a SepPak Light Accell plus QMA anion exchange cartridge (CO3 ^2- form, Waters, Milford, MA, USA) and eluted with Kryptofix 2.2.2 (K-222, 27.86 mg) and K2CO3 (2.46 mg) dissolved in CH3CN/H2O (0.75 ml; 95:5 v/v). After evaporating the solvent with a flow of helium at 80°C and 35 W (microwave resonator), anhydrous CH3CN (1 ml) was added and further dried with [ ¹⁸ F]F ^- under the same conditions. A solution of 0.50 mg of the nitro-containing precursor in 0.25 ml of DMSO was added to the dry residue of [ ¹⁸ F]F-/K2CO3/K-222 and the mixture was heated at 160° C. and 50 W for 10 min in a microwave resonator. The crude radiolabelling mix was diluted with 0.6 ml prep buffer (0.01 M Na2HPO ₄ , pH 7.4, and EtOH (60:40 v/v)) and purified using reverse phase HPLC (RP -HPLC) on an XBridge C ₁₈ column (5 μm, 4.6 mm x 150 mm; Waters, Milford, USA) eluting with Na ₂ HPO ₄ , pH 7.4, and EtOH (60:40 v/v). ) at a flow rate of 0.8 ml/min, and UV detection at 254 nm. [ ¹⁸ F]comp. No. 1 was eluted for 27 min. The purified radioactive marker solution was diluted with saline to give an ethanol concentration of <10% suitable for intravenous injection. The solution was successively passed through a 0.22 μm filter (Millex-GV, Millipore, Billerica, MA, USA) to obtain a sterile product. Quality control was performed using RP-HPLC on an XBridge column (Ci8, 3.5 μm, 3.0 mmxIOO mm; Waters, Milford, USA) eluting with 0.01M Na ₂ HPO ₄ , pH 7.4, and CH3N ( 68:32 v/v) at a flow rate of 0.8 ml/min. UV detection was performed at 254 nm. [ ¹⁸ F]comp. No. 1 was eluted for 8 min.

Received [ ¹⁸ F]Comp. No. 1 with an average attenuation-corrected radiochemical yield of 12%, respectively (relative to [ ¹⁸ F]F radioactivity in ^the preparative chromatogram, n=6). This radiochemical purity was examined using HPLC on a C ₁₈ analytical column and was greater than 99%. [ ¹⁸ F]comp. No. 1 was obtained over a total synthesis time of 60 minutes and collected with an average specific radioactivity of 85 GBq/μmol at the end of the synthesis (EOS, n=6).

D. Analytical part.

melting points.

- 20 039208

The values are either maximum values or melting temperature ranges, and were obtained with the experimental uncertainties that are commonly associated with this analytical method.

For several compounds, melting points were determined using a DSC823e instrument (MettlerToledo). Melting points were measured at a temperature gradient of 10° C./min. The maximum temperature was 300°C.

For several compounds, melting point values were determined in open capillary tubes on a Mettler Toledo MP50. Melting points were measured at a temperature gradient of 10° C./min. The maximum temperature was 300°C. Melting point data were read from a digital display and compared with data from a video recording system.

LC/MS methods.

High performance liquid chromatography (HPLC) measurements were performed using an LC pump, diode array detector (DAD) or UV detector and column as described in the respective methods. If necessary, additional detectors were included (see table of methods below).

The flow from the column was sent to a mass spectrometer (MS), which was equipped with an atmospheric pressure ionization source. It is within the skill of the art to set tunable parameters (eg, scan range, dwell time, etc.) to produce ions that provide a determination of the nominal monoisotopic molecular weight (MW) of the compound. Data collection was carried out using the appropriate software.

Compounds were described by their experimental retention times (Rt) and ions. Unless otherwise indicated in the data sheet, the indicated molecular ion corresponds to [M+H]+ (protonated molecule) and/or [M-H] ^- (deprotonated molecule). In case the compound was not directly ionizable, the type of adduct is indicated (i.e. [M+NH4]+, [M+HCOO] ^- etc.). For molecules with complex isotopic distributions (Br, Cl), the reported value is the value that was obtained for the smallest isotope mass. All results were obtained with experimental errors, which are usually associated with the method used.

Throughout this document, SQD stands for Single Quadrupole Detector, MSD stands for Mass Selective Detector, k.t. stands for room temperature, BH stands for ethylsiloxane/silica bridged hybrid, DAD stands for diode array detector, HSS stands for high strength silica.

Table 1a. LCMS method codes (flow expressed in ml/min;

column temperature (T) in °С; analysis time in minutes)

Method No. device Column mobile phase Gradient Raskho D Analysis time (min. ) T count one waters: Acquity® UPLC®-DAD and SQD waters: HSS T3 (1.8um, 2.1*100mm) A: 10 mM CH3COONH4 in 95% H ₂ O+5% CH ₃ CN B: CH ₃ CN From 100% A to 5% A in 2.10 min. , up to 0% A in 0.90 min., up to 5% A in 0.5 min. 0.7 3.5 55 2 waters: Acquity® UPLC®- DAD and SQD Waters : WEN C18 (1.7um, 2.1*50mm) A: 10 mM CH3COONH ⁴ in 95% H ₂ O+5% CH ₃ CN B: CH ₃ CN From 95% A to 5% A in 1.3 min. , holding for 0.7 min. 0.8 2 55 3 Agilent: 1100-DAD and MSD YMC Pack ODS-AQ (3 MKM, 4.6x50mm) A: 0.1% HCOOH in water, B: CH ₃ CN From 95% A to 5% A in 4.8 min. , hold for 1 2.6 6

- 21 039208

MIN. , back to 95% A in 0.2 min. four Agilent 1290 Infinity DAD TOFLC/MS G6224A YMC-pack ODS-AQ C18 (50x4.6 mm, 3 µm) A: 0.1% HCOOH in H ₂ O B: CH ₃ CN ISET 2V1,O emulation system for pump Agilent G1312A VI.0 94.51% A to 5% A in 4.8 min, hold for 1.0 min, to 95% A in 0.2 min. 2.6 6.0 35 5 waters: Acquity® UPLC®- DAD and SQD Waters : WEN C18 (1.7 µm, 2.1 * 50 mm) A: 10 mM CH3COONH4 in 95% H ₂ O+5% CH ₃ CN B: CH3CN From 95% A to 5% A in 1.3 min. , retention for 0.2 min., up to 95% A in 0.2 min. , holding for 0.1 min. 0.7 1.8 70

Table 1b. LCMS analysis data (Rt means retention time (in minutes), [M+H]+ means mass of protonated compound, method refers to the method used for (LC)MS analysis)

No. conn. T. pl. (°C) K. t. [M+N] ⁺ [M—N]" Way LCMS 1-2 0.94 281.2 279, 1 2 one 182.53°C. (DSC)* 1, 84 254.2 252.1 one 2 203.3°C. 2.073 254.0 3 3 208.5°C. 1.878 268.0 3 four 173.1°C. 1.772 270.0 3 5 191.4°C. 1.413 283.2 four 6 146.3°C. 1.647 297.2 four 7 285.1°C. 1.533 297.2 four eight 1.758 311.2 four 9 104.6°C. 1.72 284.2 282.2 5 ten 249.3°C. 1.947 298.2 four

* 30 to 300°C at 10°C/min 50 ml N2.

CHN definitions and water definitions.

For several compounds, the amount of carbon, hydrogen and nitrogen (CHN) in (% wt./wt.) was determined using Dynamic Flash Combustion on an EA 1108 CHN analyzer from Fisons instruments.

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For several compounds, water (% wt./wt.) was determined using a CA-02 moisture meter (Mitsubishi), the coulometric principle applied to the Karl-Fischer titration.

Table 1c. Definitions of CHN and water

No. conn. C (% w/w) H (% w/w) N (% w/w) H ₂ O (% w/w) one 70.52 4.95 16.79 1-2 57, 63 3.96 15, 07 0.42

E. Biological part.

Materials and methods.

General information.

Quantification of radioactivity in samples for biodistribution studies and radioactive metabolites was performed using an automated γ-counter equipped with a 3-inch NaI(Tl) crystal with a channel connected to a multichannel analyzer placed in a sample changer (Wallac 2480 Wizard 3q, Wallac, Turku, Finland). The values were corrected for background radiation, physical attenuation and counter dead time. The rodents were housed in individually ventilated cages in a temperature controlled (~22°C) room, humidity controlled on a 12-12 hour light-dark cycle, with ad libitum access to food and water. All animal experiments were performed in accordance with the Belgian Code of Practice for the Care and Use of Animals, after approval by the University Animal Ethics Committee.

Isolation of aggregated tau proteins and amyloid plaques from the brain of a human with AD.

Enriched fractions of aggregated tau proteins were obtained according to a slightly modified version of the protocol described by Greenberg and Davies (Greenberg S.G., Davies P. A preparation of Alzheimer paired helical filaments that displays distinct T proteins by polyacrylamide gel electrophoresis. Proc. Natl. Acad. Sci 1990; 87: 5827-5831) using human brain tissue with AD (occipital cortex highly loaded with tau fibrils). Briefly, frozen human AD brain samples (~10 g) were homogenized with 10x cold homogenization buffer (10 mM Tris, 800 mM NaCl, 1 mM EGTA, 10% sucrose, pH 7.4, containing PhosSTOP phosphatase and complete protease inhibitor, EDTA-free (Roche, Vilvoorde, Belgium)) on ice. After centrifugation at 27,000 xg for 20 min at 4°C, the supernatant was removed and 1% (w/v) N-lauroylsarcosine and 1% (v/v) 2-mercaptoethanol were added. The N-lauroylsarcosine/2-mercaptoethanol supernatant was incubated for 2 hours at 37°C with shaking on an orbital shaker. Subsequently, by ultracentrifugation at 108,000 x g for 1.5 h at room temperature, the precipitate was enriched with aggregated tau proteins. The supernatant was removed and the pellet was gently washed twice with a small amount of TBS (50 mM Tris, 150 mM NaCl, pH 7.4). Finally, the precipitate of aggregated tau proteins was recovered in TBS and resuspended to ensure sample homogeneity. Small aliquots were stored at -80°C.

Enriched preparations of aggregated amyloid-beta were prepared from frozen human AD brain samples (10 g occipital cortex with a high loading of tau fibrils) that were homogenized with 7x cold homogenization buffer (250 mM sucrose, 20 mM Tris base). , 1 mM EDTA, 1 mM EGTA, and PhosSTOP phosphatase and complete protease inhibitor without EDTA) on ice. After centrifugation at 27,000 x g for 20 min, cell debris was removed at 4°C. The supernatant containing amyloid plaques was aliquoted and stored at -80°C.

In vitro assays for competitive binding of radioactive ligand.

Radioligand competitive binding assays measured the binding of a radiolabelled reference ligand in the presence of a range of dose-dependent test compound concentrations.

Briefly, aggregated tau protein preparations were diluted to 100 μg protein/ml in PBS buffer with 5% ethanol. In a 96-well format, 3H-T808 (specific activity; 32.97 Ci/mmol) was added to a final concentration of 10 nM to increasing amounts of test compound in the presence of 20 μg of tau aggregate preparation protein. Non-specific binding was defined as the number of units remaining in the presence of 50 μM thioflavin T (a common beta-sheet binder). After 2 h incubation at room temperature, unbound ligand was removed by filtering the binding mixtures through GF/B glass filters using a Filtermate 96 collection device (Perkin Elmer, Zaventem, Belgium). The filters were washed three times with PBS buffer containing 20% ethanol. After drying the filtration plate, Microscint O liquid (Perkin Elmer) was added overnight and the amount of radiolabeled ligand bound to the fibrils was measured by liquid scintillation detection in a Topcount instrument (Packard Instrument Company, Connecticut, USA).

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Half maximum inhibitory concentration (IC50) values were determined from displacement curves obtained from at least two independent experiments using GraphPad Prism software (GraphPad Software, San Diego, CA).

A similar assay was performed to determine compound binding to aggregated amyloid beta, but with some minor modifications. Briefly, amyloid preparations were diluted to 150 μg protein/ml in 50 mM Tris with 0.1% BSA and 5% ethanol. 3H-AV-45 (florbetapyr specific activity; 45.95 Ci/mmol) was added to a final concentration of 10 nM to increasing amounts of test compound in the presence of 30 μg amyloid plaque preparation protein. Non-specific binding was determined in the presence of 500 μM thioflavin T. After 150 min incubation at room temperature, the binding mixtures were filtered through GF/B glass filters. The filters were washed three times with PBS buffer containing 20% ethanol. The subsequent steps were identical to those described for aggregated tau protein preparations.

[ ¹⁹ F]comp. No. 1 showed strong binding (pIC50 8.24 corresponding to Ki 2.4 nM) to extracted human tau protein using [ ³ H]-T808 and weak binding to extracted human amyloid-beta aggregates (pIC50 5.18 corresponding to Kj 3.2 μM) using [ ³ H]-AV-45 in this radiolabel displacement assay.

Immunohistochemistry (IHC): M&M of the human brain.

Human brain blocks with AD (Braak stage V-VI) were flash frozen, cut on a cryostat (20 μm thick) and stored at -80° C. until used in immunohistochemistry. Sections were dried, fixed in formalin, and incubated in hydrogen peroxide (DAKO, S2023) for 5 min and in blocking reagent (PBS1x + 0.05% Triton X-100) for 1 h. Anti-amyloid or tau antibody [( 4G8, Covance, SIG-38220), 1/500 dilution in antibody diluent with background reducing components (DAKO, S3022) or (AT8 (Bierna et al., EMBO J. 1992, 11(4):1593-7), home-made, initial concentration 1 mg/ml), 0.2 μg/ml in diluent for antibodies with background reducing components (DAKO, S3022)] were applied to sections for 1 h. Striatal tissue was immunologically labeled with antibodies to MAO-B and MAO -A (1:100, Thermo Fisher) to confirm MAO expression.

After a strong wash, sections were incubated with an HRP-conjugated anti-mouse secondary antibody (Envision, DAKO, R4000) followed by DAB chromogenic labeling (DAKO, K3468). After counterstaining with hematoxylin, sections were dehydrated and embedded with organic embedding medium (Vectamount, Vector labs, H-5000). In FIG. 1c shows beta-amyloid pathology in AD brain as determined by 4G8 IHC, and FIG. 1b shows tau-associated pathology in AD brain as determined by AT8 IHC.

Autoradiographic studies.

a) Air-dried, 10-µm-thick frozen sections of the optic cortex of a patient with AD (68-year-old woman with Braak stage V-VI disease) were incubated for 60 min with [ ¹⁸ F]comp. #1 (7.4 kBq/500 µl per section) and then washed with mixtures of PBS and ethanol as described elsewhere (Xia CF, Arteaga J., Chen G. et al. Alzheimer's Dement. 2013, 1-11) . To assess binding specificity, sections were incubated with a marker in the presence of 1 μM of authentic T808 or comp. No. 1. After drying, the sections were exposed to a phosphor screen with a long afterglow (high resolution screen, Perkin Elmer). Screens were read on a Cyclone Plus system (Perkin Elmer, Waltham, MA, USA) and analyzed using Optiquant software. The results were expressed as digital light units per square mm (DLU/mm ² ). Adjacent sections with AD were immunostained with antibodies to tau protein (AT8) and Av (4G8) to establish a relationship with the binding of [ ¹⁸ F]comp. No. 1.

Digital autoradiography with [ ¹⁸ F]Comp. No. 1 on human sections of AD patient showed high and selective binding to cortical regions rich in tau proteins (Fig. 1A). Immunohistochemistry with anti-tau and anti-Ae antibodies was performed on adjacent sections and identified numerous deposits in the form of NFTs and neuritic plaques, confirming co-localization of marker binding to NFTs (FIG. 1, B and C, respectively). To evaluate the specificity of marker binding to these NFTs, blocking studies were performed with an authentic reference compound. No. 1 and structurally unrelated reference compound T808 (Fig. 2). Self-locking cold connection No. 1 resulted in 99% inhibition, which demonstrated that the binding of [ ¹⁸ F]Comp. No. 1 is specific. Binding of [ ¹⁸ F]comm. No. 1 decreased by 99% in the presence of 1 μM T808, indicating tau-specific binding, since high affinity of T808 (K _D = 22 nM) for aggregated tau and high selectivity for tau compared to Ae aggregates (27- multiple) (Zhang W., Arteaga J., Cashion DK et al. J Alzheimers Dis. 2012, 31(3), 601-612).

b) [ ³ H]-Comp. #1 bound to frozen brain sections of AD patient with tau-associated pathology and AV-associated pathology, but did not bind to tissue from AD patient without tau-related pathology containing only AV plaques (Fig. 3D, E ). The marker was tested at 10 nM and higher concentrations were not considered in this study. It is important to note,

- 24 039208 that [ ³ H]-comp. No. 1 also showed no binding to MAO-rich human tissue (the striatum) (Fig. 3A). These data confirm that the compound of the invention is selective for tau-associated pathology but not for Ae and MAO-associated pathology.

The tau marker [ ³ H]-THK-5351 and the AB marker [ ³ H]-AV-45 were used for comparison, since THK-5351 is also known to bind to MAO. These experiments show that 10 nM [ ³ H]-TNK-5351 bound to MAO-rich human tissue and this binding could be self-blocked by 10 μM TNK-5351 (cold) as well as 10 μM Comm. No. 1 (cold) (Fig. 3F-H). This indicates that the conn. No. 1 at higher concentrations can bind MAO.

[ ³ H]-THK5351 also bound to AD patient tissue containing only Ae plaques (tau-negative), thus confirming the low selectivity of THK-5351 (Fig. 3I, J).

[ ³ H]-AV-45 (selective for AV plaques) has been suggested to be associated with human tissues as both a tau-associated pathology and an AV-related pathology (Ab+/tau+, tissue no. 28391 ), and pathology associated with AB only (Ab+/tau-, tissue no. 92/050) (Fig. 3L-M). As a control tissue from a human AD patient (tissue no. 28391: Fig. 4, tissue No. 92/050: Fig. 5) and striatal tissue (Fig. 6) used in this study, performed immunohistochemistry. Tissues from a human patient with AD (#28391; #92/050) were immunologically labeled with 4G8 (anti-amyloid) and AT8 (anti-tau) and clearly demonstrated that sections from #28391 are AB+/tau +, while sections from No. 92/050 are Av+/tau- (FIGS. 4 and 5). Additionally, the expression of MAO-B and MAO-A in human striatal tissue (No. S96/037) was confirmed using immunohistochemistry (FIG. 6). These data confirm the findings from the in vitro binding observed in FIG. 3.

c) Binding of [ ¹⁸ F] Comm. No. 1 with tissue from a human patient with AD was evaluated in the presence of confirmed MAO binders. As can be seen in FIG. 7, 0.2 mCi/ml [ ¹⁸ F] comp. No. 1 showed strong binding to sections of a human AD patient with tau-related pathology (FIG. 7A). This binding pattern was maintained when the sample was incubated with 10 μM clorgyline, a strong MAO-B irreversible binder, with moderate A-subtype selectivity (Fig. 7B), or 10 μM selegiline (= deprenyl), a strong MAO-B irreversible binder (Fig. 7C). ). Linking [ ¹⁸ F] Comm. No. 1 could be completely blocked by 10 μM authentic Comm. No. 1 (Fig. 7D). Adjacent sections with AD were immunostained with antibodies to tau proteins (AT8) and antibodies to AB (4G8) to establish a relationship with the binding of [ ¹⁸ F]comp. No. 1 (Fig. 8).

Imaging studies using MicroPET Wistar rats.

Dynamic DRET scanning for 120 min with [ ¹⁸ F]Comp. No. 1 or [ ¹⁸ F]T807 was performed on a Focus 220 μΡΕΤ scanner (Concorde Microsystems, Knoxville, TN, USA) simultaneously on three female Wistar rats. Throughout the procedure, rats were kept under gas anesthesia (2.5% isoflurane in O ₂ at a flow rate of 1 l/min). Scanned images were collected in list mode and the data collection was subjected to Fourier resampling at 24 time intervals (4x15 s, 4x60 s, 5x180 s, 8x300 s, 3x600 s). Data that were reconstructed by 3D reconstruction using maximum posterior probability (3D-MAP) were manually aligned with the rat brain ¹⁸ FFDG matrix in Paxinos coordinates using affine transformation to provide analysis of predefined volume regions of interest (VOI) (Casteels C et al., J. Nucl. Med. 2006, 47, 1858-1866). Whole brain time activity (TAC) curves were generated using VOI using PMOD software (v 3.2, PMOD Technologies, Zurich, Switzerland). The concentration of radioactivity in the brain was expressed as a standardized uptake value (SUV calculated as (radioactivity in Bq in brain/ml)/(total injected dose (Bq)/body weight in g)) as a function of time after marker injection. Scans were started immediately after IV injection of approximately 50 MBq of radioactive marker (n=3/marker). For pretreatment and substitution studies cold reference compound comp. No. 1 or T807 was dissolved in a mixture of 5% DMSO, 5% Tween 80 and 40% (2-hydroxypropyl)-beta-cyclodextrin, filtered through a 0.22 μm membrane filter (Millex-GV, Millipore) before injection. Pre-treatment (n=1) was performed by subcutaneous (SC) injection of 10 mg/kg Comm. No. 1 or T807 in 60 min. before injection of a radioactive marker. Substitution (n=1) was performed using an IV injection of 1 mg/kg Comm. No. 1 or T807 30 minutes after the injection of the radioactive marker. The μRET images were compared with the original scan (n=1) obtained from an untreated rat.

Results of a 120 min baseline study and a pretreatment and tracking study of [ ¹⁸ F]comp. No. 1 and comparative compound [ ¹⁸ F]T807 are shown in FIG. 9-11 (curves time-activity, TAC and %SUV _max .). Using the TAS of the original scans [ ¹⁸ F]Comp. No. 1 (FIG. 9) showed high initial brain uptake with a high intensity brain SUV value of ~2.0 compared to ~1.8 for [ ¹⁸ F]T807 (FIG. 10). Bone uptake was observed at later time points for both compounds.

[ ¹⁸ F]comp. No. 1 had a higher clearance rate (0.2 SUV value at 60 min pi) compared to [ ¹⁸ F]T807 (0.4 SUV value at 60 min pi) as shown in the %SUV _max curves. (Fig. 11). For [ ¹⁸ F]comp. No. 1 did not observe the effects of spontaneous blocking or

- 25 039208 spontaneous tracking label, which indicates the absence of specific binding, not associated with tau proteins, in the brain (Fig. 9). Lower brain uptake was recorded during the initial [ ¹⁸ F]T807 scan compared to the pretreatment study, probably due to saturation of metabolic enzymes and/or plasma proteins (FIG. 10). A similar effect was observed after 40 min pi in the tag tracking study.

Imaging studies using MicroPET in rhesus monkeys.

Dynamic scanning μRET using [ ¹⁸ F]Comp. No. 1 or [ ¹⁸ F]T807 for 120 min was performed with a μPET Focus 220 scanner in a rhesus monkey (6-year-old male Macaca mulatta, 7.6 kg) sedated with ketamine (Ketalar®) and xylazine (Rompun®) via intramuscular (IM) injection. During the course of the scan, the monkey received an additional dose of ketamine/xylazine several times by IV injection. O ₂ blood saturation, respiratory rate, and heart rate were monitored throughout the experiment.

The head of the animal was placed in the center in the field of view of the μPET scanner. Scanned images were collected in list mode and Fourier resampled at 24 time intervals (4x15 s, 4x60 s, 5x180 s, 8x300 s, 3x600 s). The data were reconstructed by iterative 3D reconstruction (3D-MAP) using the maximum posterior probability. TAS for the whole brain was built using VOI using the PMOD software. The concentration of radioactive substances in the brain is expressed as SUV depending on the time after the injection of the marker. Scans were started immediately after IV injection of 185 MBq [ ¹⁸ F]comp. No. 1 or [ ¹⁸ F]T807 into the saphenous vein of the right leg. For research with pre-treatment cold reference connection Comm. No. 1 was dissolved in a mixture of 10% DMSO and 40% (2hydroxypropyl)-beta-cyclodextrin, filtered through a 0.22 μm membrane filter (Millex-GV, Millipore) before injection. Pre-treatment (n=1) was performed by IV co-injection of 1 mg/kg cold comp. No. 1 and a solution of a radioactive marker. The μRET images were compared with the original scan (n=1) obtained from an untreated monkey.

Results of the 120 min initial scan of [ ¹⁸ F]comp. No. 1 and [ ¹⁸ F]T807 are shown in FIG. 12-14 (TAC and %SUV _max .). With the help of the original TAC scan [ ¹⁸ F]comp. No. 1 in the brain showed high initial brain uptake (SUV value ~5.4 in whole brain) with fast clearance (FIG. 12). With the TAC baseline scan, [ ¹⁸ F]T807 in the brain showed slower initial brain uptake (SUV ~1.3) and clearance (FIG. 13). Homogeneous distribution of [ ¹⁸ F]Comp. No. 1 was recorded in all observed areas of the brain without increased uptake in the corpus callosum. TAS on the side of the skull showed no bone uptake of free ¹⁸ F-fluoride observed 120 min after pi injection, as the SUV signal did not increase with time.

Claims (15)

1. Compound of formula (I') R ¹ where R ² is methyl and either R ¹ is ¹⁸ F and R ³ is H, or R ¹ is H and R ³ is ¹⁸ F; or R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1.4alkyl- ¹⁸ F, -OC1. ₄ alkyl- ¹⁸ F and -NR ⁴ -C _1-4 alkyl- ¹⁸ F, where R ⁴ represents H or methyl; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, where the compound is or a pharmaceutically acceptable salt thereof. 3. A pharmaceutical composition containing a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. 4. Pharmaceutical composition according to claim 3, where the composition is a sterile solution. 5. Use of a compound according to claim 1 or 2 in the binding and visualization of tau protein aggregates. 6. The use of a pharmaceutical composition according to claim 3 or 4 in the binding and visualization of tau protein aggregates. 7. Use of a compound according to claim 1 or 2 for diagnostic imaging of tau protein aggregates in the brain of a subject. - 26 039208 8. The use of a pharmaceutical composition according to claim 3 or 4 for diagnostic imaging of tau protein aggregates in the brain of a subject. 9. The use of a compound according to claim 1 or 2 in the binding and visualization of tau protein aggregates in a patient suffering or suspected of suffering from tauopathy. 10. The use of a pharmaceutical composition according to claim 3 or 4 in the binding and visualization of tau protein aggregates in a patient suffering or suspected of suffering from tauopathy. 11. A process for preparing a compound according to claim 1, comprising the step of reacting (a) a compound of formula (P-a1) or (b) a compound of formula (P-a2), or a pharmaceutically acceptable salt thereof, with a source of ¹⁸ F- fluoride using DMSO or DMF as a solvent by heating or microwave irradiation at 90-160°C where R ¹ and R ³ are H. 12. A process for preparing a compound according to claim 1, comprising the step of reacting (c) a compound of formula (P-b1), or (d) a compound of formula (P-b2), or (e) a compound of formula (P-b3), or a pharmaceutically acceptable salt thereof with an activating agent of methanesulfonyl chloride or 4-toluenesulfonyl chloride in the presence of a base and then reacting the resulting methanesulfonate or 4-toluenesulfonate with a source of 18F fluoride using DMSO or DMF as a solvent, by heating or microwave irradiation at 90-160°C where R ² is methyl and either R ¹ is selected from the group consisting of Br, -NO2, -[N(CH3)3] ⁺ and 4-CH3-Ph-SO ₂ -O- and R ³ is H or R ¹ is H and R ³ is selected from the group where R ⁴ is as defined in claim 1. 13. Compound of formula (P-1) - 27 039208 consisting of Br, -NO2, -[K(CH3)3] ⁺ and 4-CH3-Ph-SO2-O-; or R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-Br, -C1-4alkyl-I, -C1-4alkyl-O-SO2CH3, 4-CH3-Ph-SO2- O-C1-4alkyl-, -C1-4alkyl-OH, -OC1-4alkyl-Br, -OC1-4alkyl-I, -OC1-4αkyl-O-SO2CH3, 4-CH3-Ph-SO2-O-C1-4alkyl -O-, -OC1-4alkyl-OH, -NR ⁴ -C1-4alkyl-Br, -NR ⁴ -C1-4alkyl-I, -NR ⁴ -C1-4alkyl-O-SO ₂ CH ₃ , 4-CH ₃ -Ph-SO ₂ -O-C _1-4 alkyl-NR ⁴ - and -NR ⁴ -C _1-4 alkyl-OH, where R ⁴ is H or methyl; or a pharmaceutically acceptable salt thereof. 14. The compound according to claim 13, where the compound is or a pharmaceutically acceptable salt thereof. 15. Compound characterized by the formula [ ¹⁹ F]-(I) where R ² is methyl and either R ¹ is F and R ³ is H or R ¹ is H and R ³ is F; or R ¹ and R ³ are both H, and R ² is selected from the group consisting of -C1-4alkyl-F, -OC1-4alkyl-F and -NR ⁴ -C1-4alkyl-F, where R ⁴ is H or methyl; or a pharmaceutically acceptable salt thereof.