NZ714831B2 - Diazacarbazole derivatives as tau-pet-ligands - Google Patents

Abstract

The present invention relates to a compound of general formula (I) wherein R is hydrogen or tritium; F is fluoro or 18fluoro; or to a pharmaceutically acceptable acid addition salt. Compounds of formula I include 2-(6-fluoro-pyridin-3-yl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole, 3H-2-(6-fluoro-pyridin-3-yl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole and [18F]-2-(6-fluoro-pyridin-3-yl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole. The compounds may be used for binding and imaging tau aggregates and related beta-sheet aggregates including besides others beta-amyloid aggregates or alpha-synuclein aggregates. l)-9H-dipyrido[2,3-b;3',4'-d]pyrrole and [18F]-2-(6-fluoro-pyridin-3-yl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole. The compounds may be used for binding and imaging tau aggregates and related beta-sheet aggregates including besides others beta-amyloid aggregates or alpha-synuclein aggregates.

Description

ARBAZOLE DERIVATIVES AS TAU-PET-LIGANDS The t invention generally relates to a compound of general a N N N R H I wherein R is hydrogen or tritium; and F is fluoro or 18fluoro; or to a ceutically acceptable acid addition salt.

Compounds of formula I include luoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole, 3H(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole and [18F](6-fluoro-pyridin yl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole.

Compounds with similar generic basic structure are described in WO2009/102498 for in vivo imaging of amyloid deposits for diagnosing Alzheimer’s disease. No tricyclic compounds with 3 N atoms are described specifically.

It has been shown that the present compounds may be used for binding and imaging tau aggregates and related beta-sheet aggregates including besides others beta-amyloid aggregates or alpha-synuclein aggregates, especially for use in binding and imaging tau aggregates in Alzheimer ts.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder terized by cognitive e, irreversible memory loss, entation and language impairment (Arch. . 1985, 42(11), 1097-1105). Postmortem examination of AD brain sections reveals abundant senile plaques (SPs), composed of beta amyloid (Aβ) peptides, and numerous neurofibrillary tangles (NFTs) formed by filaments of hyperphosphorylated tau protein.

Tau belongs to the family of microtubule-associated proteins and is mainly expressed in s where it plays an important role in the assembly of tubulin monomers into microtubules to constitute the neuronal microtubule network as tracks for axonal transport (Brain Res. Rev. 2000, 33(1), 95-130). Tau is translated from a single gene located on chromosome 17 and the expression is developmentally regulated by an alternative splicing mechanism generating six Pop/28.05.2014 ent isoforms in the human adult brain that can be guished by their number of binding domains. The underlying mechanisms leading to tau hyperphosphorylation, ding and aggregation are not well understood, but the deposition of tau aggregates follows a stereotyped spatiotemporal pathway both at the intracellular levels as well as on the level of brain aphy.

The recent discovery of tau gene mutations leading to frontotemporal dementia (FTD) with parkinsonism linked to chromosome 17 has reinforced the predominant role attributed to tau in the pathogenesis of neurodegenerative disorders and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to ent pathologies (Biochim. Biophys. Acta 2005, 1739(2) 240-250). egenerative diseases terized by pathological tau accumulation are termed ‘tauopathies’ (Ann. Rev. Neurosci. 2001, 24, 1121- 1159). s AD and FTD, other tauopathies include progressive supranuclear palsy (PSP), -predominant dementia, Pick's e, frontotemporal lobar degeneration (FTLD), Down’s syndrome and .

A direct correlation has been established between the progressive involvement of neocortical areas and the increasing severity of ia, suggesting that pathological tau aggregates such as NFTs are a reliable marker of the neurodegenerative process. The degree of NFT involvement in AD is defined by Braak stages (Acta Neuropathol. 1991, 82, 239-259).

Braak stages I and II are defined when NFT involvement is confined mainly to the transentorhinal region of the brain, stages III and IV are diagnosed when limbic regions such as the hippocampus are involved, and stages V and VI when extensive neocortical involvement is found.

Presently, detection of tau aggregates is only possible by histological analysis of biopsy or autopsy materials. In vivo imaging of tau pathology would provide novel insights into deposition of tau aggregates in the human brain and allow to non-invasively examine the degree of tau pathology, quantify changes in tau deposition over time, assess its correlation with cognition and analyze the efficacy of an anti-tau therapy. Potential s for detecting tau aggregates in the living brain must cross the blood-brain barrier and possess high ty and specificity for tau aggregates. To this end, successful neuroimaging radiotracers must have appropriate lipophilicity (logD 1-3) and low molecular weight (<450), show rapid clearance from blood and low non-specific binding.

The object of the present application is to find an imaging tool which will improve sis by identifying potential patients with excess of tau aggregates in the brain, which may be likely to develop Alzheimer’s disease, and/or to at least provide the public with a useful . It will also be useful to monitor the progression of the disease. When an au aggregate drug becomes available, imaging tau tangles in the brain may e an essential tool for monitoring treatment.

Described herein is a method of imaging tau-aggregate deposits, comprising - introducing into a mammal a detectable quantity of a composition - ng sufficient time for the compound of formula I to be associated with tauaggregate deposits, and - detecting the compound associated with one or more tau-aggregate deposits. bed herein is a ceutical ition, containing compounds of formula I and pharmaceutically able carriers, which may be used for identifying potential patients.

The following definitions of the general terms used in the present description apply irrespective of whether the terms in question appear alone or in combination.

As used , the term "lower alkyl" denotes a saturated, i.e. aliphatic hydrocarbon group including a straight or branched carbon chain with 1 – 7 carbon atoms. Examples for “alkyl” are methyl, ethyl, n-propyl, and isopropyl. 3H denotes a tritium atom.

F denotes a fluoro atom or a 18fluoro atom.

The term “leaving group” denotes halogen or sulfonate. Examples of sulfonate are tosylate, mesylate, triflate, nosylate or ate.

The term "pharmaceutically acceptable salt" or “pharmaceutically acceptable acid addition salt” embraces salts with inorganic and organic acids, such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, fumaric acid, maleic acid, acetic acid, succinic acid, tartaric acid, methane-sulfonic acid, p-toluenesulfonic acid and the like.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this ication and claims which include the term “comprising”, other features besides the features prefaced by this term in each statement can also be present. Related terms such as “comprise” and “comprises” are to be interpreted in similar manner.

It has been found that the compounds of formula I may be used for binding and imaging tau aggregates and d beta-sheet aggregates including besides others beta-amyloid aggregates or alpha-synuclein aggregates.

In a first aspect, the invention provides a compound selected from: luoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole; 3H(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole;and [18F](6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole, or a pharmaceutically acceptable acid addition salt thereof.

In a second aspect, the invention provides use of a nd according to the first aspect in the manufacture of a composition for use in binding and imaging tau aggregates, myloid aggregates or alpha-synuclein aggregates.

In a third aspect, the invention provides use of a compound according to the first aspect in the manufacture of a composition for use in binding and imaging tau ates in Alzheimer patients.

In a fourth aspect, the ion provides use of a compound according to the first aspect in the manufacture of a composition for use in a tau-binding study.

In a fifth aspect, the ion provides use of a compound according to the first aspect in the manufacture of a composition for use in diagnostic imaging of tau-aggregates in the brain of a mammal.

In a sixth aspect, the invention provides a pharmaceutical preparation comprising a compound according to the first aspect and a pharmaceutically acceptable carrier.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of ing a t for discussing the es of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter which is not within the scope of the appended claims. That subject matter should be y identifiable by a person d in the art and may assist in putting into practice the invention as defined in the appended claims.

One embodiment of the present invention are compounds of formula I which compounds are luoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole, 3H(6-fluoro-pyridinyl)-9H- dipyrido[2,3-b;3',4'-d]pyrrole and [18F](6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'- d]pyrrole One embodiment of the present invention are further compounds of formula I wherein R is en, which compound is 2-(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole.

One embodiment of the present ion are further compounds of formula I wherein R is tritium, for e the following compound 3H(6-fluoro-pyridinyl)-9H-dipyrido[2,3- b;3',4'-d]pyrrole.

One ment of the invention are further compounds of formula I, wherein F is ro, for example [18F](6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole The position for R in formula I, if R is tritium, is the most likely ones. But tritium may also be found in small amounts in other positions of the molecule. Normally, only one of R is tritium.

The compounds of formula I may be used in binding and imaging tau aggregates, betaamyloid aggregates, alpha-synuclein aggregates or huntingtin aggregates.

The preferred use of compounds of formula I is the use in binding and imaging tau aggregates in Alzheimer patients.

Furthermore, the compounds of formula I may be used in a tau-binding study.

The nds of formula I are suitable for diagnostic imaging of tau-aggregates in the brain of a .

The invention is also used for diagnostic imaging of tau-aggregate deposits in the brain of a mammal.

The present compounds of formula I N N N R H I and their ceutically acceptable salts can be prepared by processes described below, which process comprises a) coupling a compound of a 2 (X = Cl, Br) N N X H 2 with suitable boronic acids or boronic acid esters of formula 3 OR' R R N F 3, n R’ is hydrogen or lower alkyl, to afford compounds of formula I N N N R H I n R is hydrogen, and, if desired, converting the obtained compound into pharmaceutically acceptable acid addition salts or into compounds of a I, wherein R is tritium,; b) coupling compounds of formula 4 N N R N X 4 (X = Br, Cl, NO2) with suitable fluorinating reagents selected from potassium fluoride or tetrabutylammonium fluoride, to afford compounds of formula I N N N R H I wherein the substituent R is hydrogen and, if d, converting the compound obtained into pharmaceutically acceptable acid addition salts or into compounds of formula I, wherein R is tritium c) reacting a compound of formula I N N N R H I wherein R is hydrogen, with tritium gas in the ce of a catalyst, e.g. iridium, ium, rhodium or palladium containing complexes in a suitable solvent, e.g. dichloromethane, chlorobenzene, DMF, DMSO or mixtures thereof to afford compound of formula I N N N R H I wherein R is tritium, and, if desired, converting the compounds obtained into pharmaceutically acceptable acid addition salts, or d) dissolving a compound of formula N R H 10 in dimethylsulfoxide and sonicate prior to end of dment with aqueous [18]fluoride ion to a compound of formula N R H I and, if desired, converting the compounds obtained into pharmaceutically able acid addition salts.

The following schemes 1-2 describe the processes for the preparation of compounds of formula I in more detail. The starting materials are known compounds or may be prepared according to methods known in the art.

The preparation of compounds of formula I of the present disclosure may be carried out in sequential or convergent synthetic routes. The skills required for carrying out the reactions and purifications of the ing products are known to those d in the art. The substituents and indices used in the following description of the processes have the icance given herein before unless indicated to the contrary.

In more detail, the compounds of formula I can be manufactured by the methods given below, by the methods given in the examples or by analogous methods. Appropriate reaction conditions for the individual reaction steps are known to a person d in the art. The reaction sequence is not limited to the one displayed in schemes 1-2, however, depending on the starting materials and their respective reactivity the sequence of reaction steps can be freely altered.

Starting als are either commercially ble or can be ed by methods analogous to the methods given below, by methods described in references cited in the description or in the examples, or by methods known in the art.

Scheme 1 N OR' N X + NH Cl N X' NH PG PG Cl N X' 6 OR'R N R N F N 3 R N N X' N N H H 2 R N F OR'R N R N F N 3 R N N X' N N PG PG 8 9 R N F According to scheme 1, compounds of formula I, wherein R is hydrogen, can be prepared starting from protected 4-amino-pyridines 5 (X = Cl, Br) and 2,6-di-halogenated pyridine boronic acids 6 (R’ = H, lower alkyl). Transition-metal catalyzed cross-coupling ions using a st system like e.g. Pd(OAc)2 and PPh3 and a base like e.g. triethylamine in a le solvent like DMF results in bipyridines 7. Deprotection and olecular cyclisation can be performed as one-pot procedure using a base like e.g. potassium carbonate and an activator like e.g. 18-crown-6 in a suitable solvent like DMF and affords 1,6-diazacarbazole intermediate 2. Final transformation into compounds of formula I can be accomplished by a direct transition-metal catalyzed coupling on using appropriate pyridine boronic acids 3, a catalyst like e.g. Pd(dppf)Cl2 and a base like e.g. potassium ate in a suitable solvent like DMF. Alternatively, 1,6-diazacarbazole 2 is first converted into protected intermediates 8 via reaction with a suitable reagent, e.g. di-tert-butyldicarbonate, in a suitable solvent like e.g.

DMF, followed by transition-metal catalyzed cross-coupling reaction towards intermediates 9 using appropriate pyridine boronic acids 3, a catalyst like e.g. Pd(dppf)Cl2 and a base like e.g. potassium carbonate in a solvent like DMF. Deprotection is then leading to compounds of formula I.

Scheme 2 N N R R N N N H N R N X R N F 4 I According to scheme 2, a compound of formula I, wherein R is en, can be prepared by treating a nd of formula 4 (X = Br, Cl, nitro); prepared according to the synthesis of compounds of formula I described in scheme 1, with a suitable nating reagent like e.g. potassium de or tetrabutylammonium fluoride in a suitable solvent like e.g. DMF or DMSO.

Compounds of formula I with R being tritium or F being 18F N N R N F I may be ed in conventional manner as described in the specific examples. Compounds of formula I do not contain simultaneously tritium and 18F.

Isolation and purification of the compounds Isolation and purification of the compounds and intermediates described herein can be performed, if desired, by any le separation or purification procedure such as, for example, filtration, extraction, llization, column tography, thin-layer tography, thick-layer chromatography, preparative low or high-pressure liquid chromatography or a combination of these procedures. ic illustrations of suitable separation and isolation procedures can be found by reference to the preparations and examples herein below. However, other equivalent separation or isolation procedures could, of course, also be used. Racemic mixtures of chiral compounds of formula I can be separated using chiral HPLC.

Salts of compounds of formula I The compounds of formula I are basic and may be converted to a corresponding acid addition salt. The conversion is accomplished by treatment with at least a iometric amount of an appropriate acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, nic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Typically, the free base is dissolved in an inert organic t such as diethyl ether, ethyl acetate, form, ethanol or methanol and the like, and the acid added in a similar solvent. The temperature is ined between 0 °C and 50 °C. The resulting salt precipitates spontaneously or may be precipitated by addition of a less polar solvent.

The acid addition salts of the basic nds of formula I may be converted to the corresponding free bases by treatment with at least a stoichiometric lent of a suitable base such as sodium or potassium hydroxide, potassium carbonate, sodium bicarbonate, ammonia, and the like.

The compounds were investigated in accordance with the test given hereinafter.

TAU Radioligand-In-Vitro Displacement assay This in vitro binding assay assesses the affinity of compounds for native tau aggregates. The compounds are co-incubated with the well-established tau specific radioligand [3H]T808 and the compound’s displacement y of 08 binding is determined by in vitro autoradiography using human Alzheimer’s disease (AD) brain sections.

Materials AD human brains are purchased from Banner Sun Health Research Institute (Sun City, AZ, USA). Pathological diagnosis of AD is made according to standard NIA-Reagan Institute criteria based on neuropathological data. The radioligand [3H]T808 was synthesized in-house ([3H][4- (2-Fluoro-ethyl)-piperidinyl]-benzo[4,5]imidazo[1,2-a]pyrimidine, radiochemical purity 99.0 %). As a reference cold T808 is used (2-[4-(2-Fluoro-ethyl)-piperidinyl]- benzo[4,5]imidazo[1,2-a]pyrimidine). For the autoradiography lm Imaging Plates (BAS-IP TR 2025) are exposed to the sections and read with a FujiFilm IP reader (BAS-5000).

Method Ten µm thick human AD brain sections are generated with a cryostat (Leica CM3050) at -17 °C chamber ature and -15 °C object temperature. Sections are transferred to Histobond+ microscope slides (Marienfeld Laboratory Glasware). After drying for 3 hours at room temperature the sections are stored at -20 °C. The sections are ted with the radioligand (10 nM) and the respective cold compound (at various concentrations) in 50 mM Tris buffer, pH 7.4 at room temperature for 30 min. After washing 3x 10 min at 4 °C in 50 mM Tris buffer, pH 7.4 and 3 quick dips in H2O dist. at 4 °C the sections are dried at 4 °C for 3 h. The sections are placed in a FujiFilm Cassette (BAS 2025), d with an Imaging Plate for five days and ards scanned with a tion of 25 µM per pixel.

Data is The signal intensity (Dens - PSL/mm2) in the region of st (ROI) of the autoradiogram is quantified with the software MCID analysis (version 7.0, Imaging ch Inc.).The specific binding (SB) of [3H]T808 binding in absence or in presence of a compound is calculated by subtracting the non-specific binding signal in the white matter, thus yielding SB[3H]T808 only and und. The % displacement by the various compounds is calculated as following: % displacement =100-(SBcompund/SB[3H]T808 100.

Validation data In each experiment cold T808 is used as a positive internal control. Co-incubation of equimolar amounts of hot and cold T808 is expected to reduce specific binding by approximately 50 %.

References A.K. Szardenings et al. ‘Imaging agents for detecting neurological disorders’. US Patent Application US20110182812 W. Zhang et al., ‘A highly selective and specific PET tracer for imaging of tau pathologies’.

Journal of Alzheimer’s Disease 31 (2012) 601-612.

Table 1 Structure Name %displacement of Expl. [3H]T808 (10 nM) 2-(6-fluoro-pyridinyl)-9H- 43 1 N N dipyrido[2,3-b;3',4'-d]pyrrole 3H(6-fluoro-pyridinyl)- 2 F 9H-dipyrido[2,3-b;3',4'- N N 3H d]pyrrole N [18F](6-fluoro-pyridin- 3 N 3-yl)-9H-dipyrido[2,3- N '-d]pyrrole Figure 1: diogram of 3H(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole incubated with a human cortical brain n obtained from a Braak V staged AD patient. The radioligand concentration was 3.2 nM. The igand shows a punctate staining of tau aggregates in a layered distribution n.

The compounds of formula I and pharmaceutically acceptable salts thereof can be used in the form of pharmaceutical ations. The pharmaceutical preparations can be administered in form of injection solutions.

The compounds of formula I and pharmaceutically acceptable salts thereof can be processed with pharmaceutically inert, inorganic or organic carriers for the production of pharmaceutical preparations. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, sucrose, invert sugar, glucose and the like. Adjuvants, such as alcohols, polyols, glycerol, vegetable oils and the like, can be used for aqueous ion solutions of water-soluble salts of compounds of formula I, but as a rule are not necessary. Suitable carriers for itories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.

In addition, the pharmaceutical preparations can contain vatives, solubilizers, stabilizers, g agents, emulsifiers, sweeteners, colorants, flavorants, salts for g the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.

The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case.

Experimental Section: Example 1 2-(6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole N N Step 1: tert-Butyl N-[3-(2,6-dichloropyridyl)pyridyl]carbamate A pre-heated flask was evacuated and back-filled with argon several times and charged with tert- butyl 3-iodopyridinylcarbamate (4.56 g, 14.2 mmol), 2,6-dichloropyridinylboronic acid (5.46 g, 28.4 mmol), Pd(OAc)2 (320 mg, 1.42 mmol) and triphenylphosphine (371 mg, 1.41 mmol) under argon atmosphere. Triethylamine (4.32 g, 5.94 mL, 42.7 mmol) in DMF (137 mL) was added and the reaction mixture was heated to 100 °C and stirred for 3 h. The solvent was evaporated almost completely. Water was added and the crude product suspension was extracted with ethyl acetate twice. The combined organic layer was washed with water (3 x), dried over Na2SO4, filtered and the solvent was evaporated. ation of the crude product with dichloromethane afforded 1.92 g of the desired product. The romethane phase was evaporated and ed by flash chromatography (using silica gel and an ethyl acetate/heptane gradient) to yield in total 3.39 g (~90 % purity, 63 % yield) of tert-butyl N-[3-(2,6-dichloro pyridyl)pyridyl]carbamate as light yellow solid.

MS: m/z =340.1 (M+H)+.

Step 2: 2-Chloro-9H-dipyrido[2,3-b;3',4'-d]pyrrole A suspension of tert-butyl N-[3-(2,6-dichloropyridyl)pyridyl]carbamate (264 mg, 776 µmol), potassium carbonate (215 mg, 1.55 mmol) and 18-crown-6 (226 mg, 854 µmol) in DMF (15.8 mL) was heated to 100 °C and stirred for 3 h under an here of argon. Water was added and the crude product suspension was extracted with ethyl acetate twice. The combined organic layer was washed with water (twice), brine, dried over Na2SO4, filtered and the solvent was evaporated. Trituration of the crude product with little methanol afforded ro-9H- dipyrido[2,3-b;3',4'-d]pyrrole (105 mg, 63 % yield) as light yellow solid.

MS: m/z = 204.3 (M+H)+.

Step 3: 2-Chloro-dipyrido[2,3-b;3',4'-d]pyrrolecarboxylic acid tert-butyl ester A suspension of sodium hydride (26.5 mg, 607 µmol) in dry DMF (1.5 mL) was cooled to 0 °C and under argon a solution of 2-chloro-9H-dipyrido[2,3-b;3',4'-d]pyrrole (103 mg, 506 µmol) in dry DMF (3.0 mL) was added. Stirring was continued at 0 °C for 10 min and at r.t. for 30 min.

After cooling down to 0 °C and addition of di-tert-butyl dicarbonate (132 mg, 141 µL) in dry DMF (0.75 mL) was added and stirring was continued at r.t. overnight. Water was added and the reaction mixture was ted twice with ethyl e. The combined organic layer was washed with water (twice) and brine, dried over Na2SO4, filtered and evaporated. 2-Chloro-dipyrido[2,3- b;3',4'-d]pyrrolecarboxylic acid utyl ester was obtained after purification by flash tography (using silica gel and a methanol/dichloromethane gradient) as off-white solid (113 mg, 73.5 %).

MS: m/z = 304.1 (M+H)+.

Step 4: 2-(6-Fluoro-pyridinyl)-dipyrido[2,3-b;3',4'-d]pyrrolecarboxylic acid utyl ester A microwave vessel was charged under argon atmosphere with 2-chloro-dipyrido[2,3-b;3',4'- d]pyrrolecarboxylic acid tert-butyl ester (100 mg, 329 µmol), 2-fluoro(4,4,5,5-tetramethyl- 1,3,2-dioxaborolanyl)pyridine (147 mg, 658 µmol), potassium carbonate (137 mg, 988 µmol) and 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (10.8 mg, 13.2 µmol) and the vessel was sealed, evacuated and back-filled with argon. DMF (7 mL) was added and the reaction mixture was heated to 90 °C and stirred for 17 h. The reaction mixture was filtered. Water was added to the filtrate and the reaction mixture was extracted twice with ethyl acetate. The combined organic layer was washed with water (3 x), dried over Na2SO4, filtered and evaporated. Trituration of the crude product e with little methanol afforded 2-(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole (m/z = 265.1 (M+H)+) as light red solid (23 mg with 80 % , 21 %). The liquid was evaporated and purified by flash chromatography (using silica gel and a methanol/dichloromethane gradient) to afford 2-(6- fluoro-pyridinyl)-dipyrido[2,3-b;3',4'-d]pyrrolecarboxylic acid tert-butyl ester as off-white solid (12 mg, 10 %).

MS: m/z = 365.2 (M+H)+.

Step 5: 2-(6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole A light yellow solution of 2-(6-fluoro-pyridinyl)-dipyrido[2,3-b;3',4'-d]pyrrolecarboxylic acid tert-butyl ester (22 mg, 60.4 µmol) and trifluoroacetic acid (33.3 µL, 432 µmol) in romethane (0.5 mL) was d at r.t. overnight. After cooling to 0 °C triethylamine (70 µL, 503 µmol) was added and all volatiles were removed. The crude material was purified by preparative HPLC (using a Gemini C18 column and a water with 0.1% triethylamine / acetonitrile gradient) to afford 2-(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole as offwhite solid (14 mg, 88 %).

MS: m/z = 265.1 (M+H)+.

Example 2 3H(6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole N N N H In a 2 ml tritiation flask, 2-(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole (2.0 mg, 7.6 µmol; example 1) and Crabtree’s st (9.14 mg, 11.4 µmol) were dissolved in dichloromethane (0.8 mL) and DMF (0.2 mL). The flask was ed to the tritium manifold (RC-TRITEC) and ed by freeze-pump-thaw. Tritium gas was uced, and the light orange solution was vigorously stirred for 4 hours in an atmosphere of tritium at 450 mbar. The solution was cooled by liquid nitrogen and the excess tritium gas in the reaction vessel was reabsorbed on a uranium-trap for waste-tritium. The solvent was lized off and labile tritium was removed by lyophilization with a 9:1-mixture of ethanol and water (3 x 1 mL) and toluene (2 x 1 mL). The remaining brownish oil was dissolved in ethanol (1.5 mL) and transferred on a SCX-2 cation exchanger. Remaining catalyst was eluted with MeOH (10 mL) and discarded, and the product was eluted with NH3 in MeOH (3.5 N, 10 mL), collected separately and concentrated under reduced pressure. The crude product was purified by preparative HPLC (XBridge Prep, 5 µm, 10 x 250 mm) using acetonitrile, water, and pH 7 buffer as eluent. 37 MBq (1 mCi) of the title compound were obtained with a radiochemical purity of 99% and a specific activity of 936 GBq/mmol (25.3 Ci/mmol), determined by MS spectrometry.

The compound was stored as a pH 7 buffer/DMSO solution.

MS: m/z = 265.1 , 267.1 [M(3H1)+H]+, 269.1 [M(3H2)+H]+ Example 3 [18F](6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole a) 2-(6-Nitro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole In a 50 mL flask (evaporated and purged with Ar), 2-chloro-dipyrido[2,3-b;3',4'-d]pyrrole carboxylic acid utyl ester (285 mg, 938 µmol), 2-nitropyridineboronic acid pinacol ester (469 mg, 1.88 mmol), 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (34.5 mg, 42.2 µmol) and K2CO3 (389 mg, 2.81 mmol) were ed. DMF (24 mL) was added and the tube was sealed, evaporated and purged with Ar.

The reaction mixture was heated to 90 °C and stirred for 18 h. Filtration through Celite and subsequently through small silica gel pad (neutral, 60A, mesh 32-63) was followed by rinsing with ient DMF and evaporation to dryness. The brown solid is dissolved in DMF (20 mL) and DMSO was added until an almost clear on resulted. After filtration, the solvents are evaporated to almost dryness. Purification by prep-HPLC provided the title compound (37 mg, 13 %) as a yellow solid. MS m/z: 292.2 [M+H] + b) [18F](6-Fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole The precursor, (0.7 ± 0.3 mg) was dissolved in 400 µL dimethylsulfoxide and sonicated prior to end of bombardment (EOB). At EOB, the aqueous [18F]fluoride ion, produced by proton bombardment of [18O]-enriched water, was trapped on an ion exchange column. The ion exchange column was eluted with 150 µL of a stock solution of Krytpofix 2.2.2/potassium carbonate (48 mg of fix 2.2.2 and 10 mg potassium carbonate dissolved in 600 µL of 1:1 acetonitrile:water) into the reaction vial followed by 250 µL acetonitrile. The fluoride solution was ated to dryness at 110 ˚C via nitrogen flow and further dried azeotropically by two additions of itrile (250 µL each). The reaction vial was ly transferred to the microwave cavity (Resonance Instruments) and cooled with compressed air for 2 minutes. The precursor was added and then microwaved at 50 watts for 240 seconds after which the solution was ed with 1 mL of water.

The reaction solution was diluted 3 mL triethylamine (TEA) buffer (pH 7.2) then injected onto the semi-preparative HPLC column (XBridge Cl8, 10 µm, 10x150 mm) eluted with 15:85 methanol:TEA buffer (pH 7.2) at 15 mL/min.

The HPLC effluent was monitored at 254 nm and an in-line radioactivity detector. The semipreparative chromatogram was ed and the [18F]- product peak was collected in 50 mL of water and reformulated using an automated SPE module. The t solution was eluted h a Waters C-18 SepPak Plus, washed with 10 mL of Milli-Q water, then eluted with 1 mL of absolute l followed by 10 mL of normal saline into the final product vial via a 0.22 µm Millipore FG sterilizing filter.

Aliquots were removed from the final bottle for quality control analysis. Analytical HPLC (XBridge C18, 3.5 µm, 4.6x100 mm) elute with 40:60 methanol:TEA buffer (pH 7.2) at 2 mL/min monitored at 350 nm was performed to determine radiochemical and chemical purity, ic activity and chemical identity.

The 57-minute radio-synthesis of [18F](6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'- d]pyrrole produced an average final product of 330.5 mCi, 26.1 % (n=2) non-decay corrected yield. The final product had an average specific radioactivity of 24,684 mCi/µmole and radiochemical purity of 99 %.

Claims (3)

Claims

1. A compound selected from:

2-(6-fluoro-pyridinyl)-9H-dipyrido[2,

3-b;3',4'-d]pyrrole; 5 3H(6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole;and [18F](6-fluoro-pyridinyl)-9H-dipyrido[2,3-b;3',4'-d]pyrrole, or a pharmaceutically acceptable acid addition salt thereof. 2. A compound according to claim 1 for use in binding and imaging tau aggregates, beta-amyloid 10 aggregates or alpha-synuclein aggregates. 3. A compound according to claim 1 for use in binding and imaging tau aggregates in Alzheimer patients. 15 4. A compound according to claim 1 for use in a tau-binding study. 5. A compound according to claim 1 for use in diagnostic imaging of tau-aggregates in the brain of a mammal. 20 6. Use of a compound according to claim 1 in the cture of a ition for use in g and g tau aggregates, beta-amyloid aggregates or alpha-synuclein aggregates. 7. Use of a compound according to claim 1 in the manufacture of a ition for use in binding and imaging tau aggregates in Alzheimer patients. 8. Use of a compound according to claim 1 in the manufacture of a composition for use in a taubinding study. 9. Use of a compound according to claim 1 in the manufacture of a composition for use in 30 diagnostic imaging of tau-aggregates in the brain of a . 10. A pharmaceutical preparation sing a compound according to claim 1 and a pharmaceutically acceptable carrier. 11. A compound ing to any one of claims 1 to 5, ntially as herein described with reference to any example thereof. 12. Use according to any one of claims 6 to 9, substantially as herein described with reference to 5 any example thereof. 13. A pharmaceutical preparation according to claim 10, substantially as herein described with reference to any example thereof. -