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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0468—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K51/047—Benzodiazepines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics

Definitions

• the present invention relates to in vivo imaging and in particular to in vivo imaging of gamma-aminobutyric acid (GABA) receptors of the central nervous system (CNS).
• GABA gamma-aminobutyric acid
• the invention provides novel radiofluorinated compounds based on the benzodiazepine antagonist flumazenil.
• GABA Gamma-aminobutyric acid
• GABA receptors are transmembrane receptors and fall into two main types, GABA A receptors and GABA B receptors.
• GABA A receptors have been the major focus of pharmalogical development to date.
• Many GABA A receptor subtypes have been discovered and novel chemical structures have been developed which are selective for these subtypes.
• Normal activation of the GABA A receptor results in chloride ion being selectively conducted through its pore. This chloride channel gating is generally inhibitory on a neuron by virtue of stabilising the membrane potential near to resting level.
• Flumazenil (also known as flumazepil, code name Ro 15-1788, trade names Anexate, Lanexat, Mazicon, Romazicon) is an imidazo[l,5-a][l,4]benzodiazepine that is a neutralising allosteric modulator of GABA A receptors in the CNS (Johnston 1996 Pharmacol Ther; 69(3): 173-198).
• the chemical structure of flumazenil is as follows:
• flumazenil has been as an antidote to benzodiazepine overdose as it reverses the effects of benzodiazepines by competitive inhibition at the benzodiazepine binding site of the GABA A receptor.
• radio labelled versions thereof have been developed as positron emission tomography (PET) radiotracers.
• Radiofluorinated derivatives of flumazenil known in the art are: [ 18 F] flumazenil
• [ 18 F]FMZ binds to the GABA A receptor with high affinity (K ; around 0.5nM) and selectivity.
• Ryzhikov et al (2005 Nuc Med Biol; 32: 109-116) describe the preparation of [ 18 F]FMZ from a nitro precursor compound. This synthesis, however, has been found by the present inventors to have a less than optimal end of synthesis (EOS) yield of 2.7- 7.7% (described herein as a comparative example). Furthermore, the synthesis as described by Ryzhikov et al uses a high reaction temperature which is not amenable to automation on all radiosynthesis platforms. These EOS yields are comparable to those reported by Odano et al (Neuroimage 2009 45(3) 891-902).
• [ 18 F]FFMZ is an 18 F-labelled derivative of flumazenil wherein 18 F is incorporated by fluoroethylation of a carboxylic acid precursor compound (Mitterhauser et al 2004 Nuc Med Biol; 31 : 291-295):
• the present invention seeks to provide alternative radio fluorinated compounds suitable for studying the GABA A receptor in vivo wherein said compounds have improved properties over those known in the prior art.
• the present invention provides novel radiofluorinated compounds useful for in vivo imaging GABA A receptors.
• the synthesis of the radiofluorinated compounds of the invention is high-yielding.
• a method of synthesis for the radiofluorinated compounds of the invention in particular an automated method of synthesis.
• a further aspect of the invention is a cassette suitable for carrying out the automated method of synthesis of the invention.
• the present invention relates to a radiofluorinated compound of Formula I:
• Suitable salts according to the invention include (i) physiologically acceptable acid addition salts such as those derived from mineral acids, for example hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and those derived from organic acids, for example tartaric, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic, methanesulphonic, and para-toluenesulphonic acids; and (ii) physiologically acceptable base salts such as ammonium salts, alkali metal salts (for example those of sodium and potassium), alkaline earth metal salts (for example those of calcium and magnesium), salts with organic bases such as
• alkoxy means an alkyl ether radical wherein the term alkyl is as defined above.
• suitable alkoxy groups include, methoxy, ethoxy, and propoxy.
• [ 18 F]fluoroalkyl” and “[ 18 F]fluoroalkoxy” refer to alkyl and alkoxy groups, respectively, as defined above, substituted with 18 F.
• 18 F replaces one of the hydrogens at the distal terminus of the substituent, i.e. Ci_ 4 [ 18 F]fluoroalkyl is -(CH 2 ) n - 18 F and Ci_ 4 [ 18 F]fluoroalkoxy is -0-(CH 2 ) n - 18 F, wherein n in both cases is 1-4.
• heterocycle refers herein to an aliphatic or aromatic cyclic radical wherein the cycle comprises one or more heteroatoms selected from nitrogen, oxygen or sulfur.
• one ofR andR 2 is C [ 18 F]fluoroalkyl, most preferably R 1 .
• Preferred Ci_ 4 [ 18 F]fluoroalkyl groups are [ 18 F]fluoromethyl and [ 18 F]2-fluoroethyl.
• R la and R 2a are a precursor group, and the other is H, wherein when R la is a precursor group it is selected from C alkyl-LG, Ci_ 4 alkoxyl-LG and hydroxyl, and wherein when R 2a is a precursor group it is selected from Ci_ 4 alkyl-LG and C1-4 alkoxyl- LG, wherein LG is a leaving group selected from bromide, mesylate or tosylate; and, R 3a is as defined for R 3 of Formula I.
• a “suitable source of 18 F” means 18 F in a chemical form that is reactive with a precursor group in the precursor compound such that the 18 F becomes covalently attached, resulting in the radiofluorinated compound of Formula I.
• the choice of suitable source of 18 F depends on the precursor group with which it is intended to react. Further discussion is provided below.
• a “precursor compound” of the present invention comprises a non-radioactive derivative of the radiofluorinated compound of Formula I, comprising a precursor group at the desired location of the 18 F label so that chemical reaction with a convenient chemical form of 18 F occurs site-specifically.
• the precursor compound is designed so that radiofluorination can be conducted in the minimum number of steps (ideally a single step) and without the need for significant purification (ideally no further purification) to give the desired radiofluorinated compound of Formula I.
• Such precursor compounds are synthetic and can conveniently be obtained in good chemical purity.
• the precursor compound may be provided in solution in a kit, or in a cassette suitable for use with an automated synthesis apparatus. The kit and cassette form additional aspects of the invention and will be discussed in more detail below.
• a "precursor group” is a substituent of the precursor compound as defined above which reacts with the source of 18 F such that 18 F is incorporated site-specifically to result in the desired radiofluorinated compound of Formula I.
• a “leaving group” is an atom or group of atoms that is displaced as a stable species taking with it the bonding electrons.
• Suitable leaving groups in the context of the present invention include bromide, mesylate and tosylate.
• R 2 is a precursor group
• the precursor compound can be obtained using the chemistry described in Scheme 2 below, where the appropriate isocyanate acetate is prepared using standard alkylation conditions from commercially available materials.
• Compound 8 can be appropriately modified using standard chemical transformations to generate the desired precursor.
• Precursor compounds of Formula la wherein R 3a comprises a heterocycle can be obtained by methods described by Watjen et al (J Med Chem 1989; 32(10): 2282-2291).
• 18 F may be achieved via direct labelling comprising reaction of a precursor compound comprising a leaving group (LG), i.e. bromide, mesylate or tosylate, preferably tosylate, with 18 F-fluoride as the suitable source of 18 F.
• LG leaving group
• [ 18 F]fiuoride ( 18 F ⁇ ) for radio fluorination reactions is normally obtained as an aqueous solution from the nuclear reaction 18 0(p,n) 18 F and is made reactive by the addition of a cationic counterion and the subsequent removal of water.
• Suitable cationic counterions should possess sufficient solubility within the anhydrous reaction solvent to maintain the solubility of 18 F " .
• counterions that have been used include large but soft metal ions such as rubidium or caesium, potassium complexed with a cryptand such as KryptofixTM, or tetraalkylammonium salts.
• a preferred counterion is potassium complexed with a cryptand such as Kryptofix because of its good solubility in anhydrous solvents and enhanced 18 F " reactivity.
• the alkyl or alkoxy in the Ci_ 4 alkyl-LG or Ci_ 4 alkoxy-LG correspond to the alkyl or the alkoxy in the Ci_ 4 [ 18 F] -fluoroalkyl or Ci_ 4 [ 18 F]-fluoroalkoxy, respectively, wherein Ci_ 4 [ 18 F] -fluoroalkyl or Ci_ 4 [ 18 F]-fluoroalkoxy are as suitably and preferably defined above for Formula I.
• Suitable and preferred leaving groups LG are as defined above.
• 18 F can also be introduced by O-alkylation of hydroxyl groups in the precursor compound with a synthon comprising 18 F, e.g.
• Example 2(iii) describes the radiofluorination of Precusor Compound 1, which comprises a hydroxyl precursor group, with [ 18 F]-fluoroethyltosylate to obtain [ 18 F] -Compound 1.
• the 3 ⁇ 4 of non-radioactive Compound 1 was found to be 2.4nM (see Example 5).
• Bio distribution of [ 18 F] -Compound 1 in an in vivo model showed good regional differentiation, i.e. between GABA-rich and GABA-poor regions of the brain (see Example 6).
• Example 4(v) describes the radiofluorination of Precusor Compound 2, which also comprises a hydroxyl precursor group, with [ 18 F]-fluoroethyltosylate to obtain [ 18 F]- Compound 2.
• the K ; of non-radioactive Compound 2 was found to be 0.53nM (see Example 5).
• Biodistribution of [ 18 F] -Compound 2in an in vivo model showed good regional differentiation, i.e. between GABA-rich and GABA-poor regions of the brain (see Example 7).
• the method of the invention is automated.
• the radiochemistry is performed on the automated synthesis apparatus by fitting a "cassette" to the apparatus.
• Such a cassette normally includes fluid pathways, a reaction vessel, and ports for receiving reagent vials as well as any solid-phase extraction cartridges used in post- radiosynthetic clean up steps.
• a cassette for carrying out the automated method of the invention comprising:
• a "radiopharmaceutical composition” which comprises the radiofluorinated compound as defined herein together with a biocompatible carrier in a form suitable for mammalian administration.
• the "biocompatible carrier” is a fluid, especially a liquid, in which the radiofluorinated compound is suspended or dissolved, such that the radiopharmaceutical composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort.
• the biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the like).
• injectable carrier liquid such as sterile, pyrogen-free water for injection
• an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic)
• the biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations.
• the biocompatible carrier is pyrogen-free water for injection, isotonic saline or an aqueous ethanol solution.
• the pH of the biocompatible carrier for intravenous injection is suitably in the range 4.0 to 10.5.
• radiofluorinated compound when comprised in the radiopharmaceutical composition of the invention are as already described herein.
• the radiopharmaceutical composition may be administered parenterally, i.e. by injection, and is most preferably an aqueous solution.
• a composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or para- aminobenzoic acid).
• the method for preparation of said radiofluorinated compound may further comprise the steps required to obtain a radiopharmaceutical composition, e.g. removal of organic solvent, addition of a biocompatible buffer and any optional further ingredients.
• steps to ensure that the radiopharmaceutical composition is sterile and apyrogenic also need to be taken.
• the present invention provides in a further aspect the radiofluorinated compound as suitably and preferably defined herein for use in a method of in vivo imaging.
• the radiofluorinated compound for use in a method of in vivo imaging is provided as the radiopharmaceutical composition as suitably and preferably defined herein.
• the present invention provides a positron emission tomography (PET) method for determining the distribution of GABA A receptors in the central nervous system (CNS) of a subject comprising:
• radiofluorinated compound for the PET method of the invention, suitable and preferred aspects of the radiofluorinated compound are as defined earlier in the specification.
• administering the radiofluorinated compound is preferably carried out parenterally, and most preferably intravenously.
• the intravenous route represents the most efficient way to deliver the radiofluorinated compound throughout the body of the subject, and therefore also across the blood-brain barrier (BBB) and into contact with GABA A receptors expressed in the CNS of said subject.
• the radiofluorinated compound of the invention is preferably administered as the radiopharmaceutical composition of the invention, as defined herein.
• the radiofluorinated compound is allowed to bind to GABA A receptors.
• the radiofluorinated compound moves dynamically through the mammal's body, coming into contact with various tissues therein. Once the radiofluorinated compound comes into contact with GABA A receptors, a specific interaction takes place such that clearance of the radiofluorinated compound from tissue with GABA A receptors takes longer than from tissue without, or having less GABA A receptors.
• a certain point in time is reached when detection of radiofluorinated compound specifically bound to GABA A receptors is enabled as a result of the ratio between radiofluorinated compound bound to tissue with GABA A receptors versus that bound in tissue without, or having less GABA A receptors. Ideally, this ratio is 2: 1 or greater.
• the "detecting" step of the method of the invention involves detection of signals derived from the positron emission decay of 18 F by means of a detector sensitive to said signals, a scintillator present in the PET scanner.
• positron-emission decay which is also known as positive beta decay
• a positron is emitted, and then travels up to a few millimetres until it encounters an electron.
• the encounter of the positron and the electron results in the production of a pair of annihilation (gamma) photons that are emitted at around 180 degrees to each other. It is these annihilation photons that are the "signals derived from the positron emission decay".
• the "generating" step of the method of the invention is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate an image showing the location and/or amount of signals emitted by 18 F.
• the "subject" of the invention can be any human or animal subject.
• the subject of the invention is a mammal.
• said subject is an intact mammalian body in vivo.
• the subject of the invention is a human.
• the PET method of the invention may be carried out repeatedly during the course of a treatment regimen for said subject, said treatment regimen comprising administration of a drug to combat a GABA A condition.
• the PET method as suitably and preferably defined herein can be carried out before, during and after treatment with a drug to combat a GABA A condition.
• PET has excellent sensitivity and resolution, so that even relatively small changes in a lesion can be observed over time, which is advantageous for treatment monitoring.
• PET scanners routinely measure radioactivity concentrations in the picomolar range. Micro-PET scanners now approach a spatial resolution of about 1mm, and clinical scanners about 4-5mm.
• the present invention provides the radiofluorinated compound as suitably and preferably defined herein for use in the method of diagnosis as defined herein.
• the present invention provides the in vivo imaging agent as defined herein for use in the manufacture of a radiopharmaceutical composition as defined herein for use in the method of diagnosis as defined herein.
• Example 1 describes a method for the synthesis of non-radioactive Compound 1.
• Example 2 describes a method for the synthesis of radiofluorinated Compound 1 from Precursor Compound 1.
• Example 3 describes a method for the synthesis of non-radioactive Compound 2.
• Example 4 describes a method for the synthesis of radiofluorinated Compound 2 from Precursor Compound 2.
• Example 5 describes an in vitro assay that was used to evaluate the affinity of nonradioactive Compound 1 and Compound 2 for GABA A receptors.
• Examples 6 and 7 describe the in vivo biodistribution of [ 18 F] -Compound 1 and [ 18 F]- Compound 2, respectively.
• Comparative example 8 describes a known method to obtain [ 18 F]-flumazenil.
• the cut peak was diluted with water (10 mL) and was trapped onto a pre-conditioned sep pak t-C18 light using a vacuum pump.
• the trapped material was washed with water (2 mL) and eluted with ethanol (0.7 mL) and phosphate buffered saline (6.3 mL).
• MeCN/water (MeCN 1400 ⁇ , water 100 ⁇ , TBA.HC0 3 27 mg) from vial 1.
• the solution was dried at 100°C for 10 minutes then 120°C for 20 minutes using nitrogen plus vacuum flow and then cooled to 50°C.
• nitromazenil (18.8 mg) in DMF (1 mL) from vial 3.
• the reaction mixture was heated at 160°C for 30 min then it was cooled to 50°C.
• the reaction mixture was diluted with 10 mM phosphoric acid (2.5 mL) from vial 5 and was transferred to the crude product tube.
• the crude product was then transferred onto the preparative HPLC loop manually.
• Preparative HPLC gave a peak with retention time 17.5 minutes which was cut using into the TRACERlab round bottomed flask containing water (12 mL).
• the prepartative HPLC system was fitted with a liquid flow scintillation counter.
• the mixture in the round bottom flask was trapped on a tC18 plus lite SPE cartridge (pre conditioned with 1 mL ethanol then 2 mL water).
• the SPE cartridge was washed with water (3 mL) and the crude product eluted into a P6 vial using EtOH (0.5 mL) and water (4.5 mL).

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