WO2011110488A1

WO2011110488A1 - A novel pet imaging agent of amyloid plaques and its precursor

Info

Publication number: WO2011110488A1
Application number: PCT/EP2011/053289
Authority: WO
Inventors: Heribert Schmitt-Willich; Tobias Heinrich; Damian Brockschnieder
Original assignee: Bayer Pharma Aktiengesellschaft
Priority date: 2010-03-09
Filing date: 2011-03-04
Publication date: 2011-09-15

Abstract

The invention relates to a [¹⁸F]-labeled PET tracer for imaging of Alzheimer's Disease, a suitable precursor for that PET tracer, and the process for preparing the respective [¹⁸F]-labeled PET tracer of the fol lowing Formula, wherein X is selected from the group consisting of: F, CI, Br, I, ¹⁸F, F, leaving groups, such as nitro and trimethyl ammonium, or a pharmaceutically acceptable salt thereof.

Description

A novel PET imaging agent of amyloid plaques and its precursor

The present invention is directed to a novel Positron Emitting Tomography (PET) imaging agent for binding and imaging amyloid deposits, to respective novel precursors for the syn- thesis of such agent, and the process for producing said imaging agent.

Background of the Invention

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by loss of memory, cognition, and behavioral stability. AD is defined pathologically by extracellular senile plaques comprised of fibrillar deposits of the beta-amyloid peptide (Αβ) and neurofibrillary tangles comprised of paired helical filaments of hyperphosphorylated tau. The 39 to 43 amino acids comprising Αβ peptides are derived from the larger amyloid precursor protein (APP). In the amyloidogenic pathway, Αβ peptides are cleaved from APP by the sequential proteolysis of β- and γ-secretases. Αβ peptides are released as soluble proteins and can be detected at low levels in the cerebrospinal fluid (CSF) in normal aging brains. During the progress of AD the Αβ peptides aggregate and form amyloid deposits in the parenchyma and vasculature of the brain, which can be detected post mortem as diffuse and senile plaques and vascular amyloid during histological examination (for a recent review see: Blennow et al. Lancet. 2006 Jul 29;368(9533):387-403).

Alzheimer's disease is becoming a great health and social economical problem all over the world. There are great efforts being made to develop techniques and methods for the early detection and effective treatment of the disease. Currently, diagnosis of AD in an academic setting of memory-disorder clinics is approximately 85-90% accurate (Petrella JR et al. Radi- ology. 2003 226:315-36). It is based on the exclusion of a variety of diseases causing similar symptoms and the careful neurological and psychiatric examination, as well as neuropsychological testing. However, post mortem histological examination of the brain is still the only definite diagnosis of this disease. Thus the in vivo detection of one pathological feature of the disease - the deposition of amyloid aggregates in the brain - is thought to have a big impact on the early detection of AD and differentiation from other dementias. Additionally, most disease modifying therapies that are under development are aiming at lowering the amyloid load in the brain. Thus imaging the amyloid load in the brain may provide an essential tool for patient stratification and treatment monitoring. In addition, amyloid deposits are also known to play a role in amyloidoses, in which amyloidogenic proteins are abnormally deposited in different organs and/or tissues, causing disease. For a recent review see Chiti et al. Annu Rev Biochem. 2006;75:333-66. Potential ligands for visualizing amyloid aggregates in the brain must show a high binding affinity to amyloid and must cross the blood brain barrier. PET tracers that have been already investigated in humans regarding their binding patterns in brains of AD patients are [F- 18]FDDNP (Shoghi-Jadid et. al, Am J Geriatr Psychiatry 2002; 10:24-35), [C-1 1]PIB (Klunk e. al, Ann Neurol. 2004 55:306-319), [C-1 1]SB-13 (Verhoeff et. al, Am J Geriatr Psychiatry 2004; 12:584-595), [F-18]Bay 94-9172 (Rowe et al. Lancet Neurol 2008, 7:129-135), [C- 1 1 ]BF227 (Kudo et. al, J Nucl. Med 2007; 49:554-561 ), and [F-18]PIB (Farrar et. al Turku PET Symposium 2007, Abstract 49). For recent reviews see Lockhardt, Drug Discov Today, 2006 1 1 :1093-1099, Henriksen et al., Eur J Nucl Med Mol Imaging. 2008 Mar;35 Suppl 1 :S75-81 , Cohen, Mol. Imaging Biol. 2007 9:204-216, Nordberg, Curr. Opin Biol. 2007, 20:398-402, Small et al., Neurology 2008 7:161-172, Nordberg, Eur. J. Nucl. Med. Mol. Imaging 2008, 35, S46-S50.

Besides their specific binding to amyloid deposits in the brain, the currently most promising PET tracers show a disadvantageous non-specific accumulation, especially in white matter brain regions in AD patients as well as in healthy controls. Generally, non-specific background binding interferes with the image quality and could e.g. impair the quantification of amyloid and the diagnosis of very early stages of the disease.

The current invention relates to the compound of formula

which has been found to be a suitable PET tracer for the detection of amyloid deposits in patients with amyloid-related diseases with high specificity at an early stage of the disease. The invention also relates to suitable precursors for the preparation of that compound as well as to the process of preparation of the novel PET tracer. Furthermore, the invention relates to a commercially useful process for producing the tracer with high yield and for a precursor useful in such method. This problem has been solved by the provision of the respective precursor using Iodine as a leaving group:

Description of the invention

The invention relates to a compound of formula

, wherein X is selected from the group consisting of

F, CI, Br, I, ¹⁸F, F, nitro, trimethyl ammonium, and further leaving groups or a pharmaceutically acceptable salt thereof.

The invention relates to a compound of formula

, wherein X is selected from the group consisting of

F, CI, Br, I, ¹⁸F, F, nitro, and trimethyl ammonium,

or a pharmaceutically acceptable salt thereof. One further embodiment of the invention is the compound of the formula

or of formula

or of formula

or of formula

or a pharmaceutically acceptable salt thereof.

In the context of the present invention, pharmaceutically acceptable salts of the compounds according to the invention are pharmaceutically acceptable salts for PET purposes. These salts also comprise salts which for their part are not suitable for other pharmaceutical appli- cations, but which can be used, for example, for isolating or purifying the compounds according to the invention.

Pharmaceutically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalene disul- phonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Part of the invention is a method for the preparation of a compound of the formula

, wherein X is selected from the group consisting of

Br, I, CI, nitro and trimethyl ammonium, is reacted with a radiofluorination agent.

Also part of the invention is a method for the preparation of a compound of the formula

wherein the com ound of formula

is reacted with a radiofluorination agent.

Furthermore, another embodiment of the invention is a method for the preparation of ound of the formula

wherein the compound of formula

is reacted with a radiofluorination agent.

In a preferred embodiment [¹⁸F]fluoride/TBAOH and a solvent is used as a fluorination agent.

In a preferred embodiment, the fluorination agent is 4,7,13, 16,21 ,24-Hexaoxa-1 , 10 diazabi- cyclo[8.8.8]-hexacosane K¹⁸F (crownether salt Kryptofix K¹⁸F), K¹⁸F, H¹⁸F, KH¹⁸F₂ or tetraal- kylammonium salt of ¹⁸F. More preferably, the fluorination agent is K¹⁸F, H¹⁸F, or KH¹⁸F₂.

The solvents used can be Ν,Ν-Dimethylformamide (DMF), Dimethylsulfoxyde (DMSO), Ace- tonitrile (MeCN), Ν,Ν-Dimethylacetamide (DMA) etc., preferably DMSO, MeCN or DMF. The solvents can also be a mixture of solvents as indicated above.

The invention also relates to a method for the preparation of a compound of formula

, wherein X is selected from the group consisting of Br, I, CI, nitro and trimethyl ammonium. Another embodiment is a method fort he preparation of a compound of formula

One embodiment of the invention is a method for the preparation of a compound of formula

wherein

the com ound of formula

is reacted with 2-fluoropyridine-4-carboxylic acid.

A further embodiment of the invention is a method for the preparation of a compound of for- mula

, wherein

a salt of the com ound of formula

is reacted with 2-fluoropyridine-4-carboxylic acid.

Furthermore, the invention relates to a method for the preparation of a compound of formula

wherein

the h drochloride of the compound of formula

is reacted with 2-fluoropyridine-4-carboxylic acid.

The invention also relates to a com ound according to formula

or a pharmaceutical acceptable salt thereof as a diagnostic compound.

Another aspect of the invention is a com ound according to formula

or a pharmaceutical acceptable salt thereof as a diagnostic compound for PET imaging of Alzheimer's disease. A further embodiment is the use of a com ound according to formula

or a pharmaceutically acceptable salt thereof for the preparation of a diagnostic composition useful for PET imaging of Alzheimer's disease.

The invention also relates to a dia nostic com osition comprising the compound of formula

or a pharmaceutically acceptable salt thereof.

A preferred embodiment is diagnostic composition for PET imaging of Alzheimer's disease com rising the com ound of formula

or a pharmaceutically acceptable salt thereof.

Furthermore, part of the invention is a method for the preparation of a compound having the formula

The invention also relates to a method for the preparation of a compound having the formula

, wherein

the compound of formula

is reacted with 2-iodopyridine-4-carboxylic acid.

wherein

a salt of a compound of formula

is reacted with 2-iodopyridine-4-carboxylic acid.

A preferred embodiment of the invention is a method for the preparation of a compound having the formula

, wherein

the hydrochloride of a compound of formula

is reacted with 2-iodopyridine-4-carboxylic acid.

A further embodiment is a method for the preparation of a compound of formula

Also part of the invention is a method of diagnosing Alzheimer's disease in a patient comprising the steps of administering a compound of formula

or a pharmaceutically acceptable salt thereof to said patient and performing PET analysis. The F-18 labeled compound as described above and herein is, in a preferred embodiment of the invention, bound to an Αβ peptide.

Another aspect of the invention is the use of the F-18 labeled compound as described above and herein for diagnosing Alzheimer's disease and/or amyloidoses in a patient, in particular in a mammal, such as a human.

Preferably, the use of the F-18 labeled compound of the invention in the diagnosis is performed using positron emission tomography (PET).

Another aspect of the invention is directed to a method of imaging amyloid deposits. Such a method comprises a) administering to a mammal the F-18 labeled compound as described above and herein, and b) detecting the signal stemming from the compound that is specifically bound to the amyloid deposits. The specific binding is a result of the high binding affinity of the compounds of the present invention to the amyloid deposits.

In a further aspect, the invention is directed to a method of diagnosing a patient with Alz- heimer's disease or amyloidoses. This method comprises a) administering to a human in need of such diagnosis the F-18 labeled compound of the invention for detecting the compound in the human as described above and herein, and b) measuring the signal from the detectable label arising from the administration of the compound to the human, preferably by using a gamma camera, by positron emission tomography (PET).

A further embodiment of the invention includes a diagnostic method for other neurological disorders as Alzheimer's disease comprising the exclusion of Alzheimer's disease in a patient, that method comprising administering the F-18 labeled compound of the invention to a patient and applying an imaging method of the invention. The diagnostic methods of the invention can also be used as post-mortem diagnostic methods.

Furthermore, the diagnostic methods of the invention by using the F-18 labeled compound of the invention can also be used for monitoring the therapy of Alzheimer's disease, a neurodegenerative disorder or an amyloidoses.

Furthermore, the diagnostic methods of the invention using the F-18 labeled compound of the invention can also be used in diagnosing neurological disorders other than Alzheimer's disease by excluding Alzheimer's disease.

Furthermore, the compounds of the invention can also be used as tools in screening, for example high throughput screening methods and in vitro assays. The invention further relates to a method for diagnosing a disease in a mammal selected from the group consisting of Alzheimer's disease, a neurodegenerative disorder, or an amyloidosis, this method comprising administering to said mammal the F-18 labeled compound of the invention. Furthermore, this method may comprise imaging of said mammal and detecting the imaging signal. Additionally, said imaging may be per- formed using an imaging method selected from the group consisting of PET, MR- spectroscopy, and MR-tomography. Furthermore, this method may be used to monitor the effect of a therapy.

Furthermore, the invention relates to a method for diagnosing or therapy monitoring of a dis- ease selected from the group consisting of Alzheimer's disease, a neurodegenerative disorder, or an amyloidosis in a mammal, said method comprising analyzing in vitro a sample of said mammal, wherein said mammal or sample has been treated with the F-18 labeled compound of the invention. This sample can be cerebrospinal fluid. The diagnostic methods and the novel PET tracer of the invention may also be used for the stratification of AD patients.

Part of the invention is also a kit, comprising a compound having the formula

, wherein X is selected from the group consisting of:

F, CI, Br, I, ¹⁸F, leaving groups such as nitro and trimethyl ammonium, or a pharmaceutically acceptable salt thereof, in sealed container.

The kit also may comprise a compound having the formula

wherein X is selected from the group consisting of:

Br, I, CI, nitro and trimethyl ammonium, or a pharmaceutically acceptable salt thereof, in a sealed conainer.

The invention furthermore relates to a kit, comprising a compound selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

The kit may contain one or more sealed vials comprising the compounds.

Furthermore, the kit may comprise buffers and/or reactants for radiofluorination. F-18 radiolabeling procedures

[F-18] radiolabeling procedures are well known to the person skilled in the art. For example, radiolabeling can be preformed as described in the following.

[F-18]Fluoride can be produced by proton bombardment in a cyclotron using a silver target (1 ml.) filled with [0-18] water for the ¹⁸0 (p,n)¹⁸F reaction. The aqueous [F-18]fluoride can be passed through a cartridge (e.g. QMA-resin cartridge Waters, Sep Pak Light QMA Part.No.: WAT023525 ). The trapped [F-18]fluoride can then be eluted from the cartridge by adding e .g . a Kryptofix K2.2.2/ K₂C0₃ solution (Kryptofix is 4,7, 13, 16,21 ,24-Hexaoxa-1 , 10- diazabicyclo[8.8.8]-hexacosane). The nucleophilic substitution of the precursor works preferably in the presence of a base such as Tetrabutylammonium hydroxide (NBu₄OH), Tetrabutylammonium carbonate ((NBu₄)₂C03), Tetrabutylammonium hydrogencarbonate (NBu₄HC0₃), K₂C0₃ etc. and at elevated temperatures. The addition of crown ethers such as Kryptofix (K2.2.2) can influence the reaction positively, especially in the presence of K₂C0₃ as the base.

The potassium fluoride Kryptofix complex is preferably dried by repeated azeotropic distillation with sequential addition of acetonitrile. Solvents such as acetonitrile, DMF, DMSO etc. can be used as a reaction solvent. The labeling product can be purified by solid phase extraction using cartridges. Preferred cartridges are Sep-Pak Plus C18 cartridge (Waters, WAT020515). The cartridge can be rinsed with water and the compound can be eluted with acetonitrile. The eluted compound can be diluted with water and can then be subjected to preparative HPLC purification. Preferred HPLC columns are reversed phase columns such as Gemini 5 μ C 18 1 10 A, 250 * 10 mm (Phenomenex, 00G-4435-N0). Mixtures of buffer solution, acids, water etc. with organic solvents such as acetonitrile, methanol, ethanol etc. can be used as mobile.

The solution can then be diluted with e.g. water to be passed through a cartridge for concentration and solvent change. [1] Dolle F.; [¹⁸F]Fluoropyridines: From conventional radiotracers to the labeling of macro- molecules such as proteins and oligonucleotides. Ernst Schering Research Foundation workshop(62): 113-57, 2007, ISBN 978-3-540-32623-6.

[2] Zhang Y.; Synthesis of 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-[¹⁸F]fluoropyridin-4- yl)pyridine ([¹⁸F]NIDA522131 ), a novel potential radioligand for studying extrathalamic nico- tinic acetylcholine receptors by PET. J Label Compd Radiopharm 2004; 47: 947-952.

Brief description of the figures Figure 1 : Preparative HPLC for the compound of Example 3.

Figure 2: Analytical HPLC chromatogram for the compound of Example 3 (gamma- detection). Figure 3: Analytical HPLC chromatogram for the compound of Example 3 (UV detection). Figure 4: Autoradiographical analysis of binding of compound Ex. 3 to brain sections from cortex of Alzheimer^'s disease patients (AD) with amyloid beta plaques. Control sections without amyloid beta pathology stem from healthy volunteers (HC) and fro n to-temporal dementia (FTD) patients. Blocking of specific signals was performed with an excess of cold compound. Arrows point to plaque-specific signals.

Examples

A method for synthesizing and labeling is exemplified in the following Examples. These Examples illustrate certain aspects of the above-described method and advantageous results and are shown by way of illustration and not by way of limitation.

Example 1

N-{2-[4-(5-Benzyloxy-pyrimidin-2-yl)-piperazin-1 -yl]-2-oxo-ethyl}-2-iodopyridine-4- carboxamide -(5-Benzyloxy-pyridimin-2-yl)-piperazine

76.2 mg (0.4 mmol) of copper(l) iodide and 1.955 g (6 mmol) of cesium carbonate were dried overnight at 120°C in vacuo and suspended in 4.12 mL (40 mmol) of benzyl alcohol and 5 mL of toluene. 972.45 mg (4 mmol) of 5-bromo-2-(piperazin-1-yl)pyrimidine (Scientific Frontier) and 144 mg (0.8 mmol) of 1 ,10-phenanthroline were added and the mixture was stirred under nitrogen at 1 10°C for 24h. After the insoluble salts were filtered off, the solution was evaporated and the residue taken up in 20 mL ethyl acetate. The precipitate was collected and dried in vacuo.

Yield: 460 mg (42.5 %).

MS (ESIpos): m/z = 271 razin-1 -yl}-2-oxoethyl)carbamate

To a solution of 219 mg (1.25 mmol) i-Butoxycarbonyl-glycine (Aldrich) in 15 mL THF and 0.7 mL (5.0 mmol) triethyl amine at -15°C, 0.18 mL (1 .36 mmol) isobutyl chloroformate were added dropwise and the solution was maintained at this temperature for another 15 min. Then, 340 mg (1.26 mmol) of 1-(5-Benzyloxy-pyridimin-2-yl)-piperazine (1 a) and 0.17 mL triethyl amine (1 .24 mmol) in 20 mL THF were added slowly to this cold solution, the temperature was kept below -10°C for another 15 min and was then allowed to reach room temperature. After stirring overnight the solvent was evaporated and the residue was taken up in ethyl acetate. This solution was washed successively with aqueous sodium carbonate, water, 1 M aqueous hydrochloric acid (HCI) solution, saturated aqueous sodium chloride solution, finally dried over magnesium sulfate and then evaporated. This residue was chromatogra- phed on silica gel using a hexane/ethyl acetate gradient.

Yield: 156 mg (29.0 %). MS (ESIpos): m/z = 428 [M+H]⁺

c) N-{2-[4-(5-Benzyloxy-pyrimidin-2-yl)-piperazin-1-yl]-2-oxo-ethyl}-2-iodopyridine-4-

120 mg (0.28 mmol) of ierf-Butyl (2-{4-[5-(benzyloxy)pyrimidin-2-yl]piperazin-1-yl}-2-oxo- ethyl)carbamate (1 b) were suspended in 10 mL 2N HCI in diethyl ether and stirred overnight at room temperature. The precipitate was filtered off and washed with ether and dried at 40°C in vacuo.

Yield: 57 mg (55.8%). The product was used in the next step without further purification. MS (ESIpos): m/z = 328 [M+H]⁺

To a solution of 41.1 mg (0.157 mmol) of 2-iodopyridine-4-carboxylic acid (Alfa Aesar) and 57 mg (0.157 mmol) of hydrochloride prepared above in 3 mL DMF were added 81 .6 mg (0.157 mmol) PyBOP and 104 microL (0.597 mmol) N-ethyl-N,N-diisopropylamine and the reaction mixture was stirred overnight at room temperature. After evaporation of the solvent the residue was taken up in ethyl acetate. This solution was washed with water and saturated aqueous sodium chloride solution, dried over sodium sulfate and then evaporated. This residue was chromatographed on silica gel using an dichloromethane/methanol gradient and the appropriate fractions were combined and concentrated.

Yield: 88 mg (quantitative).

MS (ESIpos): m/z = 558 [M+H]⁺

¹H-NMR (400MHz, DMSO-d₆): δ [ppm]= 3.50-3.75 (m, 8H), 4.20 (d, 2H), 5.1 1 (s, 2H), 7.29- 7.48 (m, 5H), 7.79 (dd, 1 H), 8.21 (s, 1 H), 8.28 (s, 2H), 8.52 (d, 1 H), 9.00 (t, 1 H). Example 2 N-{2-[4-(5-Benzyloxy-pyrimidin-2-yl)-piperazi

carboxamide

To a solution of 15 mg (0.1 1 mmol) of 2-fluoropyridine-4-carboxylic acid (Aldrich) and 40 mg (0.1 1 mmol) of hydrochloride prepared above in 2.5 mL DMF were added 57.2 mg (0.1 1 mmol) Benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 38 microL N-ethyl-N,N-diisopropylamine and the reaction mixture was stirred overnight at room temperature. After evaporation of the solvent the residue was chromatographed on silica gel using an ethyl acetate/ethanol gradient.

Yield: 15 mg (30.3 %).

MS (ESIpos): m/z = 451 [M+H]⁺

¹H-NMR (400MHz, DMSO-d₆): δ [ppm]= 3.38-3.70 (m, 8H), 4.16 (d, 2H), 4.91 (s, 2H), 7.13- 7.27 (m, 6H), 7.39-7.43 (m, 2H), 8.01 (d, 2H), 8.22 (m, 1 H).

Example 3 [¹⁸F]-N-{2-[4-(5-Benzyloxy-pyrimidin-2-yl)-piperazin-1-yl]-2-oxo-ethyl}-2-fluoropyridine-4- carboxamide

Aqueous [¹⁸F]Fluoride (2 GBq) was trapped on a QMA cartridge (Waters) and eluted with 2 ml of a Kryptofix/cesiumcarbonate (Cs₂C0₃)-solution (5 mg Kryptofix in 1 .5 mL ace- tonitrile (MeCN), 2.3 mg CS2CO3 in 0.5 mL water) into the reactor. The solvent was removed by heating at 120 °C for 10 min under a stream of nitrogen. Anhydrous MeCN (3x1 mL) was added and evaporated as before. A solution of precursor 1c (5 mg) in 500 μΙ anhydrous dimethylsulfoxide (DMSO) was added. After heating at 180 °C for 20 min the crude reaction mixture was diluted with 4 mL water/MeCN (3:1 ; v/v) and purified by preparative HPLC: Synergi 4 μ Hydro-RP 80A; 250 x 10.00 mm 4 micron; mobile phase: A: 0.1 M aqueous ammonium formate B: acetonitrile.

For the preparative purification a linear gradient was run 40% B - 50% B in 20 min and the product fraction was collected at t_R=19 min (29 MBq)(Figure 1 ).

The collected HPLC fraction was diluted with 20mL water and immobilized on a Sep-Pak C18 light cartridge (Waters), which was washed with 5 mL water and eluted with 1 mL etha- nol into the product vial to deliver the F-18 labeled product 3 (27 MBq) In a overall synthesis time of -120 min and in a radiochemical yield of -3% corrected for decay (radiochemical purity >99% (HPLC).

The desired F-18 labeled product 3 (t_R=5.8 min, Fig. 2) was analyzed using analytical HPLC: SepServ UltraSep ES AMID RP 18A 5 μιτι; 150 x 3 mm; solvent gradient: start 5 % acetonitrile - 95 % acetonitrile in 0.1 % trifluoroacetic acid in 7 min., flow: 1 mL/min and confirmed by co-injection with the corresponding non-radioactive F-19 fluoro-standard 2 on the analytical HPLC (t_R=5.6 min, Fig. 3).

Example 4

Biological methods and results

Binding studies using human brain homoqenate

A competition assay with a tritiated amyloid ligand was performed in 96-well plates (Greiner bio-one; Cat. 651201 ; Lot. 06260130) using brain homogenate from AD patients.

Homogenates were prepared by homogenizing (Ultra-Turrax, setting 2, 30 s, 24000 rpm) dissected frontal cortex containing grey matter and white matter from AD patients in phos- phate buffered saline (PBS, pH 7.4). The homogenate with a concentration of 100 mg wet tissue/ml was divided into aliquots of 300 μΙ and stored at -80°C.

Varying concentrations of the unlabeled test substances were incubated with 100 μg/ml homogenate and 10 nM of the tritiated ligand in PBS, 0.1 % BSA (final volume 200 μΙ) for 3 h at room temperature. Subsequently the binding mixture was filtered through Whatman GF/B filters (wetted with PBS, 0.1 % BSA) using a Filtermate 196 harvester (Packard). Filters were then washed twice with PBS, 0.1 %BSA and 40 μΙ scintillator was added to each well before the bound radioactivity was measured in a TopCount devise (Perkin Elmer). Non-specific binding was assessed by adding an excess of 1000x of the tritiated ligand to the reaction mixture. Finally IC50 values were calculated with the help of appropriate analysis software.

Table 1 : Binding affinity of example 2 towards human AD brain homogenate (ADH)

Autoradioqraphical analysis

Fresh frozen as well as paraffin embedded sections of the frontal lobe from Alzheimer's dementia patients, frontotemporal dementia patients and age matched controls were used for the study.

Frozen sections, sliced at 18 μιτι thickness on a cryostate (Leica, Germany) and paraffin sections, sliced on a sliding microtom (Leica) at a thickness of 6 μιτι, were mounted onto glass slides (Superfrost Plus, Fa.Menzel, Braunschweig Germany). Frozen sections were allowed to adhere to the slides for several nights at -20°C. The paraffin sections were deparaffinized using routine histological methods. For binding studies sections were incubated with the [F- 18] labeled test compound at 10 Bq/μΙ diluted in 25mM Hepes buffer, pH 7.4, 0,1 % BSA (200-300 μΙ/slide) for 1 ,5 hour at room temperature in a humidified chamber. For blocking experiments an excess of the unlabeled test substance was added to the incubation mixture. After hybridization, sections were washed four times with Hepes buffer, 0.1 % BSA (or alternatively two times with 40% ethanol) and finally dipped two times into water for 10 sec. The air-dried sections were exposed to imaging plates and signals were detected by a phosphoi- mager device (Fuji BAS5000).

Fig. 4: Autoradiographical analysis of binding of compound Ex. 3 to brain sections from cortex of Alzheimer^'s disease patients (AD) with amyloid beta plaques. Control sections without amyloid beta pathology stem from healthy volunteers (HC) and fronto-temporal dementia (FTD) patients. Blocking of specific signals was performed with an excess of cold compound. Arrows point to plaque-specific signals. The studies described above indicate that the F-18 labeled compound of the invention is useful as imaging agents for amyloid plaques. It bind specifically to amyloid beta deposits with low background binding.

Claims

1. A compound of formula

wherein X is selected from the group consisting of:

F, CI, Br, I, ¹⁸F, F, leaving groups, such as nitro and trimethyl ammonium, or a pharmaceutically acceptable salt thereof.

2. A compound accordin to claim 1 , selected from the roup of compounds consisting of

and

or a pharmaceutically acceptable salt thereof.

3. A method for the preparation of a compound of the formula

wherein the compound of formula

, wherein X is selected from the group consisting of

Br, I, CI, nitro and trimethyl ammonium,

is reacted with a radiofluorination agent.

4. A method for the preparation of a compound of the formula

wherein the com ound of formula

is reacted with a radiofluorination agent.

5. A method according to claim 4, wherein the compound of the formula

is reacted with a radiofluorination agent.

6. A compound according to formula

or a pharmaceutical acceptable salt thereof as a diagnostic compound. com ound according to formula

or a pharmaceutical acceptable salt thereof as a diagnostic compound for PET imaging of Alzheimer's disease.

8. Use of a com ound according to formula

9. A dia nostic composition comprising the compound of formula

or a pharmaceutically acceptable salt thereof.

10. A diagnostic composition for PET imaging of Alzheimer's disease comprising the comound of formula

or a pharmaceutically acceptable salt thereof.

1 1 . A method for the preparation of a compound having the formula

12. A method for the preparation of a compound having the formula

, wherein

a compound of formula

is reacted with 2-iodopyridine-4-carboxylic acid.

13. A method for the preparation of a compound of formula

14. A method for the preparation of a compound of formula

, wherein the com ound of formula

is reacted with 2-fluoropyridine-4-carboxylic acid.

15. A method for the preparation of a compound of formula

Br

16. Method of diagnosing Alzheimer's disease in a patient comprising the steps of administerin a compound of formula

or a pharmaceutically acceptable salt thereof to said patient and performing PET analysis.

17. A kit comprising a compound according to claim 1 in a sealed container.

18. A kit according to claim 17, wherein said compound has the formula

, wherein X is selected from the group consisting of

Br, I, CI, nitro and trimethyl ammonium.

19. A kit comprising the compound of formula

pharmaceutically suitable salt thereof in a sealed container.