WO2008132454A1

WO2008132454A1 - Carbolines and their use as imaging agents

Info

Publication number: WO2008132454A1
Application number: PCT/GB2008/001455
Authority: WO
Inventors: Denis Raymond Christophe Bouvet; Ian Martin Newington
Original assignee: GE Healthcare Ltd
Current assignee: GE Healthcare Ltd
Priority date: 2007-04-26
Filing date: 2008-04-24
Publication date: 2008-11-06
Anticipated expiration: 2009-10-26

Abstract

The invention relates to carbolines of formula (I) which are selective ligands for the GABAA receptor and which carry a radiolabel suitable for imaging with PET or SPECT or a radiolabel suitable for localisation of GABAA receptors in vitro. The compounds of the present invention are thus useful for in vitro diagnostics and in vivo imaging of the GABAA receptor.

Description

CARBOLINES AND THEIR USE AS IMAGING AGENTS

The present invention relates to the field of medical diagnostics and imaging, in one 5 particular aspect to positron emission tomography (PET) and or single photon emission computed tomography (SPECT) and provides compounds and methods for visualising central nervous system (CNS) receptors. In particular, this invention relates to carbolines which are selective ligands for the GABAA receptor and which carry a radiolabel suitable for imaging with PET or SPECT or a radiolabel suitable for o localisation of GABAA receptors in vitro. The compounds of the present invention are thus useful for in vitro diagnostics and in vivo imaging of the GABAA receptor.

GABA (γ -aminobutyric acid) is the most important inhibitory neurotransmitter in the human brain. Dysfunctions of GABAergic neurotransmission are implicated in many5 neurological and psychiatric disorders - for example, epilepsy and anxiety disorders, Parkinson's disease and chronic pain. Current evidence indicates that changes in the receptors which mediate GABAergic transmission are associated with particular CNS (central nervous system) disorders. GABA receptors fall into two main types - GABAA and GABAB receptors. The development of new radioligands selective for GABA o receptor subtypes will lead to major breakthroughs in terms of brain imaging studies in living human patients. At this stage the GABAA receptors are worthy of particular attention owing to the discovery of many GABAA receptor subtypes and development of novel chemical structures which are selective for these subtypes. The field of GABA receptor imaging therefore appears to be poised at an exciting stage where5 development of radioligands for quantifying GABA receptor changes offer the potential to provide useful insight into numerous CNS disorders, such as those associated with epilepsy, anxiety and cognitive (neurodegenerative) disorders.

EP0161575, EP0030254, and EP0054507 describe various β-carbolines and their 0 pharmacalogical properties.

Karimi et a/, Eur. J. Org. Chem. 2003, 2132-2137 describes methods for synthesis of various nc-labelled amides, including N-methyl-9H-β-carboline-3-[carbonyl- ⁿC]carboxamide.

Braestrup et a/, Journal of Neurochemistry, 37(2), 333-41 describes [³H]propyl- β- carboline-3-carboxylate as a ligand for the BZi benzodiazepine receptor subclass.

Bye et al, Trends in Neurosciences 4)8), 1981, describes β-[6-³H]carbo!ine-3-carboxylic acid ethyl ester for benzodiazepine receptor studies.

A number of compounds have been investigated as potential radioligands for studying the GABAA receptor in vivo using PET including [ⁿC]flumazenil ([¹¹C]FMZ), [¹⁸F]fluoroflumazenil ([^1SF]FFMZ), [¹⁸F]fluoroethylflumazenil ([¹⁸F]FEFMZ), and [¹²³l]iomazenil ([¹²³I]IMZ). However, the majority of these compounds have suffered one or more disadvantages including short half-life, metabolic instability, and being difficult to prepare by radiochemical methods. The present invention seeks to provide radioligands suitable for studying the GABAA receptor in vitro and in vivo having improved properties over those in the prior art.

According to the invention, there is provided a compound of formula (I):

or a salt or solvate thereof, wherein:

X is oxygen, sulphur, or NR¹⁰ wherein R¹⁰ is hydrogen or Ci-βalkyl; R¹ is either: (a) Ci-6alkoxy, aryloxy, or arylCi-6alkoxy each optionally substituted by 1 to 3 groups selected from halo, hydroxy, Ci-βalkyl , Ci-βhaloalkyl, Ci-εalkoxy, Ci-εhaloalkoxy, -NR¹¹R¹², and -C(O)OR¹¹ wherein R¹¹ and R¹² are each independently selected from hydrogen, Ci-6haloalkyl and Ci-εalkyl; or (b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, hydroxy, Ci- ealkyl, aryl, arylCi-ealkyl and are each optionally substituted by a group selected from halo, hydroxy, Cualkyl, -C(O)NR¹⁴R^1S , -C(O)OR¹⁴ wherein R¹⁴ and R¹⁵ are each independently selected from hydrogen, Ci-βalkyl and Ci-e haloalkyl or wherein R¹³ and R¹⁴ together with the nitrogen to which they are bonded form a 5-, 6-, or 7-membered nitrogen containing ring optionally substituted by one or two groups selected from those described as optional substituents for R¹³ and R¹⁴;

R² is selected from hydrogen, Ci-εalkyl, aryl, arylCi-δalkyl and is optionally substituted by 1 to 3 groups selected from halo, hydroxy, Ci-βalkyl , Ci-εhaloalkyl, Ci-6alkoxy, -NR¹⁶R¹⁷, and -C(O)OR¹⁶ wherein R¹⁶ and R¹⁷ are each independently selected from hydrogen Ci-ealkyl and Ci-ehaloalkyl;

R³, R⁴, R⁵, R⁶ , and R⁸ are each independently selected from hydrogen, halo, nitro, Ci-6alkyl, Ci-6haloalkyl, hydroxy, Ci-6alkoxy, Ci-βhaloalkoxy, aryl, arylCi-εalkyl, aryloxy, arylCi-ealkoxy, cyano. thiocyanate, -NR¹⁸R¹⁹, -NR¹⁸C(O)R¹⁹, -SR^1S -SO₂NR¹⁸R¹⁹, -C(O)R¹⁸ and -C(O)OR¹⁸ wherein R¹⁸ and R¹⁹ are each independently selected from hydrogen, Ci-δhaloalkyl, and Ci-εalkyl;

R⁷ is selected from hydrogen, Ci-βalkyl, Ci-6 haloalkyl, -CO2H, Ci-6 haloalkoxycarbonyl and Ci-βalkoxycarbonyl;

and wherein at least one Of -C(X)R¹ or R² to R⁸ comprises a detectable label; provided that the compound of formula (I) is not N'-methyl-9H-β-carboline-3-[carbonyl- ⁿC]carboxamide or β-[6-³H]carboline-3-carboxylic acid ethyl ester, or [³H]propyl- β- carboline-3-carboxylate.

In a compound of formula (I), and in following aspects of the invention:

X is preferably oxygen;

R¹ is preferably either: (α) Ci-eαlkoxy or Ci-e hαloαlkoxy; or

(b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, Ci-ealkyl, aryl, and arylCi-δalkyl and are each optionally substituted by a group selected from halo, hydroxy, and Ci--₊alkyl;

R² is preferably selected from hydrogen, Ci-βalkyl, Ci-βhaloalkyl, Ci-6 hydroxyalkyl, Ci-ehalohydroxyalkyl, Ci-ealkoxyalkyl, and Ci-ehaloalkoxyalkyl;

R³, R⁴, R⁵, R⁶, and R⁸ are preferably each independently selected from hydrogen, halo, nitro, Ci-ealkyl, Ci-βhaloalkyl, hydroxy, Ci-εalkoxy, and Ci-βhaloalkoxy ; suitably R³, R⁵, R⁶, and R⁸ are each hydrogen, and R⁴ is selected from halo, nitro, Ci-βalkyl, Ci-ehaloalkyl, hydroxy, Ci-ealkoxy, and Ci-6haloalkoxy; and

R⁷ is preferably selected from hydrogen, Ci-ealkyl, and Ci-6 haloalkyl.

Suitable detectable labels in a compound of formula (I) and in more specific aspects of the invention below are a radiolabel suitable for imaging with PET or SPECT such as ¹³¹- 123.124^,122I₁ 75βr, 76Br,⁷⁷Br, ¹³N, ¹¹C, or ¹⁸F, or a radiolabel suitable for localisation of GABAA receptors in vitro or in vivo such as ³H₁ ¹⁴C, ³⁵S, or ¹²⁵I. In one aspect of the invention, the detectable label is a radiolabel suitable for imaging with PET or SPECT, suitably selected from "l.123^, 124^, 122I₁ 75_Br, 76_Brι 77_Br> i3N, 1¹C, and ¹⁸F, and is preferably ¹⁸F. In an alternative aspect of the invention, the detectable label is a radiolabel suitable for localisation of GABAA receptors in vitro, suitably selected from such as ³H, ¹⁴C, ³⁵S₁ or ¹²⁵I.

The detectable label is incorporated as part of one or more of groups -C(X)R¹ or R² to R⁸, for example where the detectable label is "l.123^, 124^, 125^, 122I₁ 75B_Γ, 76B_n 77Br₁ or ¹⁸F it may be incorporated where one or more of groups -C(X)R¹ or R² to R⁸ comprises halo and where the detectable label is ¹¹C, it may be incorporated where one or more of groups -C(X)R¹ or R² to R⁸ comprises carbon. In one aspect of the invention, the detectable label is incorporated as part of group -C(X)R¹ .

Thus, in a preferred aspect of the invention, there is provided a compound of formula (Ia):

or α salt or solvate thereof, wherein:

R¹ is either:

(a) Ci-6alkoxy or Ci-6 haloalkoxy; or

(b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, Ci-ealkyl, aryl, and arylCi-6alkyl and are each optionally substituted by a group selected from halo, hydroxy, and

R² is selected from hydrogen, Ci-βalkyl, Ci-βhaloalkyl, Ci-β hydroxyalkyl, Ci-6halohydroxyalkyl, Ci-6alkoxyalkyl, and Ci-ehaloalkoxyalkyl;

R³, R⁴, R⁵, R⁶, and R⁸ are each independently selected from hydrogen, halo, nitro, Ci-εalkyl, Ci-6haloalkyl, hydroxy, Ci-εalkoxy, and Ci-εhaloalkoxy ;

R⁷ is selected from hydrogen, Ci-βalkyl, and Ci-e haloalkyl;

and wherein at least one of -C(X)R¹ or R² to R⁸ comprises a detectable label selected from "¹.¹²³.^{124, i22}|_ 75_Br, ⁷⁶Br,77Br, ¹³N₁ ¹¹C, and ¹⁸F; most suitably ¹⁸F.

In a further preferred aspect of the invention, there is provided a compound of formula (Ib):

or α salt or solvate thereof, wherein:

R¹ is either:

(a) Ci-ealkoxy or Ci-6 haloalkoxy; or

(b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, Ci-βalkyl, aryl, and arylCi-εalkyl and are each optionally substituted by a group selected from halo, hydroxy, and

R² is selected from hydrogen, Ci-βalkyl, Ci-βhaloalkyl, Ci-e hydroxyalkyl, Ci-εhalohydroxyalkyl, Ci-βalkoxyalkyl, and Ci-βhaloalkoxyalkyl;

R³, R⁵, R⁶, and R⁸ are each hydrogen and R⁴ is selected from halo, Ci-βalkyl, Ci-ehaloalkyl, hydroxy, Ci-6alkoxy, and Ci-ehaloalkoxy;

R⁷ is selected from hydrogen, Ci-εalkyl, and Ci-e haloalkyl and is suitably hydrogen;

and wherein at least one of -C(X)R¹ or R² to R⁸ comprises a detectable label.

In a compound of formula (Ib), R⁴ is suitably Ci-βhaloalkoxy, more suitably [¹⁸F]Ci-6fluoroalkoxy such as [¹⁸F]Ci-6fluoroethoxy.

Particular compounds of formula (I) include (2-[¹⁸F]Fluoroethyl)-β-carboline-3-carboxylate 3-[¹⁸F]Fluoropropyl)- β-carboline-3-carboxylate (2-[¹⁸F]Fluoroethyl)- β-carboline-3-carboxamide (3-[¹⁸F]Fluoropropyl)- β-carboline-3-carboxamide (2-[¹⁸F]Fluoroethyl)-6-methoxy-4-methoxymethyl- β-cαrboline -3-cαrboxαmide (3-[¹⁸F] Fluoropropyll-β-methoxy^-methoxymethyl-β-cαrboline-S-cαrboxαmide (2-[ⁱ⁸F]Fluoroethyl)-6-fluoro-A-methoxymethyl-β-cαrboline-3-cαrboxαmide (3-[¹⁸F]Fluoropropyl)-6-fluoro-4-methoxymethyl-β-cαrboline-3-cαrboxαmide or α salt or solvate of any thereof .

Further particular compounds of formula (I) include : 6-(2-[¹⁸F] Fluoroethoxy)-9H- β-carboline-3-carboxylic acid ethyl ester; 6-(2-[¹⁸F]Fluoroethoxy)-9H- β-carboline-3-carboxylic acid ethylamide; 6-(2-[¹⁸F]Fluoroethoxy)-4-methoxymethyl-9H- β-carboline-3-carboxylicacid ethyl ester; 6-(2-[¹⁸F]Fluoroethoxy)-4-methoxymethyl-9H- β-carboline-3-carboxylic acid ethylamide; or a salt or solvate of any thereof .

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 triethanolamine, N-methyl-D-glucamine, piperidine, pyridine, piperazine, and morpholine, and salts with amino acids such as arginine and lysine.

Suitable solvates according to the invention include those formed with ethanol, water, saline, physiological buffer and glycol.

As used herein the term "halo" either alone or as part of another term means iodo, bromo, chloro, or fluoro.

As used herein the term "alkyl" either alone or as part of another term means a straight, branched or cyclic alkyl group.

As used herein the term "aryl" either alone or as part of another term means a cαrbocyclic aromatic system, suitable examples being phenyl or naphthyl, more suitably phenyl.

As demonstrated below, the compounds of formula (I) , (Ia), and (Ib) have use as radioligands for the GABAA receptor. Therefore, according to a further aspect of the invention, there is provided a compound of formula (I) , (Ia), or (Ib) as defined above, or a salt or solvate thereof, for use in an in vivo diagnostic or imaging method such as PET or SPECT. Suitably, a compound of formula (I), (Ia), or (Ib) as defined above, or a salt or solvate thereof may also be used to image the GABAA receptor in healthy human volunteers for example for clinical research purposes.

Suitably, the compounds of formula (I) , (Ia), or (Ib) or salt or solvate thereof are useful for in vivo imaging of GABAA receptors and thus have utility in the diagnosis of GABAA- mediated disorders.

The term "GABAA-mediated disorders" means neurological and neuropsychiatric disorders such as stroke, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, sleep disorders, alcoholism , and neuropathic pain. One important GABAA-mediated disorder is epilepsy and in particular post-traumatic epilepsy.

Accordingly, there is further provided use of a compound of formula (I), (Ia), or (Ib) or a salt or solvate thereof in the manufacture of a radiopharmaceutical for the in vivo diagnosis or imaging of a GABAA-mediated disorder. In the alternative, there is provided a compound of formula (I), (Ia), or (Ib) or a salt or solvate thereof for use in the in vivo diagnosis or imaging of a GABAA-mediated disorder.

In a further aspect, there is provided a method for the in vivo diagnosis or imaging of GABAA- mediated disorder in a subject, preferably a human, comprising administration of a compound of formula (I) , (Ia), or (Ib) or a salt or solvate thereof and detecting the uptake of said compound by an in vivo imaging technique such as SPECT or PET. The method is especially preferred for the in vivo diagnosis or imaging of stroke, epilepsy, Alzheimer's disease, Parkinson's disease, and neuropathic pain, especially epilepsy and in particular post-traumatic epilepsy.

The invention further provides a method of monitoring the effect of treatment of a subject, preferably a human with a drug to combat a GABAA- mediated disorder, said method comprising administering to said subject a compound of formula (I) , (Ia), or (Ib) or α salt or solvate thereof and detecting the uptake of said compound by an in vivo imaging technique such as SPECT or PET, said administration and detection optionally but preferably being effected repeatedly, e.g. before, during and after treatment with said drug.

A compound of formula (I) , (Ia), or (Ib) or a salt thereof is preferably administered for in vivo use in a radiopharmaceutical formulation comprising the compound of the invention and a pharmaceutically acceptable excipient A "radiopharmaceutical formulation" is defined in the present invention as a formulation comprising compound of formula (I) , (Ia), or (Ib) or a salt thereof in a form suitable for administration to humans. Administration is preferably carried out by injection of the formulation as an aqueous solution. Such a formulation 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 porσ-aminobenzoic acid).

The effective in vivo dose of a compound of formula (I), (Ia) , (Ib), or a salt thereof will vary depending on the exact compound to be administered, the weight of the patient, and other variables as would be apparent to a physician skilled in the art. Generally, the dose would lie in the range 0.001 μg/kg to 10 μg /kg, preferably 0.01 μg /kg to

1.0 μg/kg.

As mentioned above, the compounds of formula (I), (Ib), and salts thereof may also have use in the field of in vitro diagnostics for localisation of GABAA receptors in vitro. In this aspect of the invention, the detectable label in the compound of formula (I)₁ (Ib), or a salt thereof is suitably ³H, ¹⁴C, ³⁵S₁ or ¹²⁵I. GABAA may be localised in biopsy or postmortem tissue by incubating the tissue with a solution comprising a compound of formula (I)₁ (Ib), or a salt thereof. The solution is suitably aqueous, or an aqueous organic solvent mix such as aqueous ethanol. Upon incubation the compound of formula (I), (Ib), or a salt thereof labels the GABAA receptor in the tissue and may be detected by any standard technique, for example by autoradiography, or gamma counter.

Compounds of formula (I) wherein the group R¹ comprises a detectable label may be prepared from the corresponding compound of formula (II):

or α protected derivative thereof, wherein X and R² to R⁸ are as defined for the compound of formula (I) by reaction with the appropriate alcohol or amine compound of formula (III):

R^!-H (III)

wherein R¹ is as defined for the compound of formula (I) and comprises a detectable label. The reaction of a compound of formula (II) with a compound of formula (III) may be effected by any standard esterification or amidation method. Suitably the compound of formula (I I) is converted to the corresponding activated ester, for exam pie to an acid chloride by reaction with thionyl chloride or to a pentafluorophenyl ester before addition of the compound of formula (III) in the presence of a base, such as a trialkylamine, for example diisopropylethylamine or triethylamine.

Compounds of formula (II) are either commercially available or may be prepared by methods such as those described in EP0030254. Compounds of formula (III) may be prepared by radiohalogenation, radiohaloalkylation, or [ⁿC]alkylation methods, such as those described in Lasne et a/, Topics in Current Chemistry, VoI 222, pp201-256 and MJ Welch and CS Redvanly, Handbook of Radiopharmaceuticals, Radiochemistry and

Applications (Wiley, 2003) using a corresponding precursor amine or alcohol, suitably wherein the amine or hydroxyl group is protected as described below.

As would be appreciated by a person skilled in the art, protecting groups may be required during synthesis of a compound of formula (I) to prevent unwanted side- reactions. Therefore, protected derivatives of synthetic intermediates such as a compound of formula (II) comprise one or more protecting groups to prevent unwanted reaction of certain reactive groups. Suitable protecting groups may be found in Protecting Groups in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts, published by John Wiley & Sons Inc. which describes methods for incorporating and removing such protecting groups.

Compounds of formula (I) wherein one of the groups R² to R⁶ or R⁸ comprises a detectable label may be prepared by conventional radiolabelling techniques, such as aromatic nucleophilic [¹⁸F]fluorination involving displacement of a nitro, iodonium, or tetraalkylammonium group, or by radioiodide or radiobromide displacement of a trialkyltin precursor, such as a tributylstannyl precursor, in the presence of an oxidising agent such as peracetic acid, N-chlorosuccinimide, and N-chlorotolylsulphonamide (for example chloramine-T or iodogen), at non-extreme temperature and in a suitable solvent such as an aqueous buffer. Radiohalogenation methods are reviewed in detail in Bolton, J Label. Compd Radiopharm 2002, 45, 485-528.

A thorough review of ⁿC-labelling techniques, suitable for incorporation of a ¹¹C detectable label may be found in Antoni et al "Aspects on the the Synthesis of ¹¹C- Labelled Compounds" in Handbook of Radiopharmaceuticals, Ed. MJ. Welch and CS. Redvanly (2003, John Wiley and Sons).

For example, a compound of formula (I) wherein R⁴ comprises an ¹⁸F detectable label may be prepared according to the following scheme 1. Other compounds of formula (I) for example having an ¹⁸F detectable label in a different ring position may be prepared by analogy.

Scheme 1

The starting phenol in Scheme 1 may be prepared by the method of Scheme 2, or by analogy.

Scheme 2

Pb(OAc)₄ AcOH

Compounds of formula (I) wherein R⁷ comprises a detectable label may be prepared by radiohalogenation, radiohaloalkylation, or [ⁿC]alkylation reactions such as those described in Lasne et a/, Topics in Current Chemistry, VoI 222, pp201-256 and MJ Welch and CS Redvanly, Handbook of Radiopharmaceuticals, Radiochemistry and Applications (Wiley, 2003).

According to a further aspect of the invention there is provided a kit for the preparation of a radiopharmaceutical formulation, said kit comprising a compound of formula (II) as defined above. In use of the kit, the compound of formula (II) would be converted to the corresponding compound of formula (I) by reaction with a compound of formula (III) using the process described above.

The invention will now be illustrated by way of the Examples in which the following abbreviations are used:

THF: tetrahydrofuran TLC : thin layer chromatography NMR : nuclear magnetic resonance DMSO : dimethyl sulphoxide MS : mass spectroscopy HPLC : high performance liquid chromatography

UV : ultraviolet

Rt : retention time

HATU : O-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

NMM ; N-methyl morpholine

BpyPOP : (benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate.

DMF : N,N-dimethyl formamide rt : room temperature eq : equivalent(s)

LCMS : liquid chromatography mass spectroscopy

Intermediate 1

Preparation of Intermediate 2

Intermediate 1 (Aldrich, Ig₁ 4.1 10-3 mol) is in suspension in THF (50 mL). Lithium hydroxide (1 eq, 100 mg) is added followed by 10 mL of water. All the material is then getting dissolved. The mixture is stirred at room temperature until completion. A precipitate appears during the reaction. TLC is done to monitor progress. Lithium hydroxide is added to drive the reaction to completion. (TLC conditions: Ethyl acetate/Dichloromethane : 80/20). The precipitate is filtered and dried. Proton NMR shows the correct material.

Preparation of Compounds 1 to 4 Typical procedure :

Intermediate 2 (150 mg, 6.2 10-4 moles) is put in suspension in neat thionyl chloride (10 mL). A few drops of dimethylform amide are then added. The reaction is then refluxed for a minimum of 2 hours. The suspension has disappeared and the acyl chloride moiety is completely dissolved in the thionyl chloride as a solvent. Thionyl chloride is then fully evaporated under vacuum. The remaining residue is dissolved back in dichloromethane. The alcohol or amine (7.1 10-4 moles) is then added drop wise followed immediately by diisopropylethylamine as a base (7.1 10-4 moles in case of the alcohol and 1.4 10-3 moles in case using amine ammonium salts). When ammonium salt of amines are used, those later are first neutralised with the base prior addition to the reaction.

The reaction is then stirred for a few hours at room temperature. The reaction is monitored by TLC (Ethyl acetate/Dichloromethane : 80/20 or methanol/dichloromethane : 10/90). New faster running spot is appearing. Solvent is then evaporated. The crude solid is then chromatographed using the companion equipment and Ethyl acetate/Dichloromethane as a solvent system. For Compound 4 : the purified material was still containing some of intermediate 1. Addition of acetone made intermediate 1 precipitate as the desired product remained in solution. So filtration and evaporation of the filtrate solvent afforded the desired compound 4 product with proper purity.

Yields are typically between 15 and 30 %

Compound 2 :

IH NMR (DMSO d6) : 2.2 (2H₁ CH2, m); 4.46 (m. CH2O, 2H, JHH 6.0 Hz); 4.65 (CH2F, 2H₁ dt,

JFH 48 Hz; JHH 6.0 Hz ); 7.36 (m, IH); 7.70 (m, 2H); 8.46 (m. IH); 9.02 Im₁ 2H). MS : ES+: 273.5 M+l

HPLC : column Phenomenex Gemini 3 micron C18 water : MeCN UV 254/214 nm : 30-90% in 30 min Rt : 9,27 min 100%

Compound 1 :

IH NMR (DMSO d6): 4.62 (dt, 2H₁ JHF 27.04 Hz, JHH 6.0 Hz); 4.81 (dt, 2H, JHF 48 Hz₁ JHH

6Hz); 7.37 (m, IH); 7.68 Im₁ 2H); 8.47 (m, IH); 9.04 (m. 2H). MS ES+ : 257.3 M+l

HPLC : column Phenomenex Gemini 3 micron C18 water : MeCN

UV 254/214 nm : 30-90% in 30 min

Rt : 8,41 min 91.89%

Compound 3: IH NMR (DMSO d6): 3.71 (dm, 2H, JHF 27.04 Hz); 4.60 (dt, 2H, JHF 48 Hz, JHH 6HZ); 7.37 (m,

IH); 7.63 (m. 2H); 8.42 (m, IH); 8.85 (IH₁ m. NH); 8.90 (m, 2H).

MS ES+ : 258.3 M+l

HPLC : column Phenomenex Gemini 3 micron C18 water : MeCN

UV 254/214 nm : 30-90% in 30 min Rt : 7.42 min 97.66%

Compound 4 :

IH NMR (DMSO d6) : 2.0 (m, 2H); 3.5 (CH2N, 2H, m); 4.55 (CH2F, 2H, dt, JFH 48 Hz; JHH 6.0

Hz ); 7.4 (m, IH); 7.74 (m, 2H); 8.4 (m, IH); 9.0 (m. 2H), 9.1 (bm, IH, NH)

MS (ES+) : (M+l) 272.5

Compounds 1 to 4 may be prepared with a detectable label, by using the appropriately labelled alcohol or amine in an analogous synthesis.

Thus, Compound 4 was prepared with an ¹⁸F label according to the scheme:

ester

Compound 4 Step (i)

After standard Kryptofix 2.2.2. (5mg) & K₂CO₃(Qq) (0.1M, 50μL) drying, typical labelling used protected tosyl amine (10 - 2mg) in acetonitrile (Vim L). Radiochemical purity 5 (RCP) was typically >90% (n=6), worst RCP 75%

The labelled/protected fluoropropylamine was purified on preparative HPLC followed by solide phase extraction (SPE) removal of mobile phase.

Compound 4 Step (ii) l o Deprotection of the purified product was effected in >95% yield, using an aqueous acid (HCl) in excess (5M excess over starting material)

Compound 4 Step (iii)

The reaction of the 3-fluoropropylamine (pH increased to 10 with triethylamine) with 15 the carboline pentafluorophenyl ester (5mg) in DMF (VέmL) was performed with approximately 30% of the propylamine converted. The product was confirmed by analytical HPLC (UV @254nm)

Methoxymethyl-β-carbolines synthesis. Described in Heterocycles, 1983, VoI 20(7)

glacial acetic acid toluene, HO⁰C

R= F, -OCH₃

pH 4 xylene, reflux

Typical procedure for preparing compounds 5 to 8.

HATU, NMM, BpyPOP, DMF, rt fluoroethylamine/fluoroproprylamine

Intermediate 4a R=F Intermediate 4b R=OCH₃ R1 = Ethylfluoro, propylfluoro

Intermediate 4a or 4b (0.279mmoles, leq) was dissolved in DMF (10ml). Fluoroalkyl amine hydrochloride (0.334mmoles, 1.2eq) was dissolved in DMF (2ml) and N-methyl morpholine (0.837mmoles, 3eq) and added to the reaction mixture. HATU (0.41SmITIoIeS, 1.5eq) and BpyPOP (OAlδmmoles, 1.5eq) were added. The reaction was left stirring at room temperature overnight. (TLC: ethyl acetate/petrol, 8:2). DMF was removed by rotary evaporation and the reaction crude was then submitted to purification by flash chromatography (ethyl acetate 50% to 100% in petrol). Isolated 5 compound was characterised by NMR and LCMS. Typical yield 35-45%.

Compound 5

0 ¹H-NMR (CD₃OD) δ (ppm): 3.51 (3H, s, -OCHj)₁ 3.56 (2H, t, J= 7.0 Hz), 3.69 (IH, t, J= 4.6 Hz), 3.78 (IH, t, J= 4.6 Hz), 3.92 (3H, s), 4.53 (IH, t, J= 4.9 Hz), 4.69 (IH, t, J= 5.5 Hz), 5.46 (2H, s), 7.23-7.27 (lH.m), 7.51 (IH, d, J= 8.8 Hz)₁ 7.75 (IH, d, J= 2.4 Hz), 8.76 (IH, s). 5 Compound 6.

iH-NMR (CD₃OD) δ (ppm): 1.96-2.13 (2H, m), 3.51 (3H, s, -OCH₃), 3.56 (2H, t, J= 7.0 Hz), 3.91 (3H, s), 4.50 (IH₁ 1, J= 5.9 Hz), 4.66 (IH₁ 1, J= 5.8 Hz)₁ 5.43 (2H₁ s), 7.25 (IH, dd, J= O 2.4, 8.9 Hz), 7.51 (IH₁ d, J= 8.9 Hz)₁ 7.75 (IH₁ d, J= 2.4 Hz)₁ 8.76 (IH₁ s).

Compound 7

¹H-NMR (CD₃OD) δ (ppm): 3.46 (3H, s, -OCH₃), 3.69 (IH, t, J= 5.2 Hz), 3.78 (IH, t, J= 5.2 Hz), 4.53 (IH, t, J= 4.9 Hz), 4.69 (IH₁ 1, J= 5.2 Hz), 5.4 (2H, s), 7.32-7.39 (IH, m), 7.53- 7.58 ((1H, m), 7.89-7.94 (IH, m), 8.77 (IH, s).

Compound 8

¹H-NMR (CD₃OD) δ(ppm): 1.96-2.13 (2H, m), 3.45 (3H, s, -OCH₃), 3.56 (2H, t, J= 7.1 Hz), 4.49 (IH, t, J= 5.8 Hz), 4.65 (IH, t, J= 5.8 Hz), 5.37 (2H, s), 7.31-7.38 (IH, m), 7.52-7.55 ((1H, m), 7.88-7.92 (IH, m), 8.75 (IH, s).

Compounds 5 to 8 may be prepared with a detectable label, by using the appropriately labelled amine in an analogous synthesis.

Biological Examples

To determine in vitro affinities of the compounds, a standard radioligand binding assay for rat GABAA receptor was used. Membranes (ex vivo rat cerebellar membranes, 500ng/ml) were incubated with [³H] flumazenil (1.8nM) (PerkinElmer) in the presence or absence of a range of test compound concentrations. Non-specific binding was performed in presence of excess diazepam (Sigma) (lOμM). Final assay volume was lOOμl). Assay wells were incubated at room temperature for 1 hour and the assay terminated by filtration. Bound radioactivity was detected using a scintillation counter.

Each of Compounds 1 to 3 when tested in this screen gave an ICsoof below 2OnM.

Claims

1. A compound of formula (I):

or a salt or solvate thereof, wherein:

X is oxygen, sulphur, or NR¹⁰ wherein R¹⁰ is hydrogen or Ci-ealkyl; R¹ is either: o (a) Ci-6alkoxy, aryloxy, or arylCi-6θlkoxy each optionally substituted by 1 to 3 groups selected from halo, hydroxy, Ci-βalkyl , Ci-εhaloalkyl, Ci-εalkoxy, Ci-6haloalkoxy, -NR¹¹R¹², and -C(O)OR¹¹ wherein R¹¹ and R¹² are each independently selected from hydrogen, Ci-βhaloalkyl and Ci-βalkyl; or (b) N R¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, hydroxy, Ci-5 ealkyl, aryl, arylCi-ealkyl and are each optionally substituted by a group selected from halo, hydroxy,

-C(O)NR¹⁴R¹⁵ , -C(O)OR¹⁴ wherein R¹⁴ and R¹⁵ are each independently selected from hydrogen, Ci-βalkyl and Ci-e haloalkyl or wherein R¹³ and R¹⁴ together with the nitrogen to which they are bonded form a 5-, 6-, or 7-membered nitrogen containing ring optionally substituted by one or two groups selected from o those described as optional substituents for R¹³ and R¹⁴;

R² is selected from hydrogen, Ci-ealkyl, aryl, arylCi-ealkyl and is optionally substituted by 1 to 3 groups selected from halo, hydroxy, Ci-βalkyl , Ci-βhaloalkyl, Ci-βalkoxy, -NR¹⁶R¹⁷, and -C(O)OR¹⁶ wherein R¹⁶ and R¹⁷ are each independently selected from5 hydrogen Ci-εalkyl and Ci-βhaloalkyl;

R³, R⁴, R⁵, R⁶ , and R⁸ are each independently selected from hydrogen, halo, nitro, Ci-ealkyl, Ci-ehaloalkyl, hydroxy, Ci-βalkoxy, Ci-ehaloalkoxy, aryl, arylCi-βalkyl, aryloxy, αrylCi-eαlkoxy, cyαno, thiocyαnαte, -NR¹⁸R¹⁹, -NRI8C(O)R¹⁹ , -SR¹⁸, -SO₂NR¹⁸R¹⁹, -C(O)R¹⁸ and -C(O)OR¹⁸ wherein R¹⁸ and R¹⁹ are each independently selected from hydrogen, Ci-_δhaloalkyl, and Ci-ealkyl;

5 R⁷ is selected from hydrogen, Ci-ealkyl, Ci-e haloalkyl, -CO₂H₁ Ci-6 haloalkoxycarbonyl and Ci-6alkoxycarbonyl;

and wherein at least one of -C(X)R¹ or R² to R⁸ comprises a detectable label; provided that the compound of formula (I) is not N'-methyl-9H-β-carboline-3-[carbonyl-o ^1:LC]carboxamide or β-[6-³H]carboline-3-carboxylic acid ethyl ester, or [³H]propyl- β- carboline-3-carboxylate.

2. A compound of formula (Ia):

or α salt or solvate thereof, wherein:

R¹ is either: o (a) Ci-6alkoxy or Ci-6 haloalkoxy; or

(b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, Ci-βalkyl, aryl, and arylCi-ealkyl and are each optionally substituted by a group selected from halo, hydroxy, and

5 R² is selected from hydrogen, Ci-βalkyl, Ci-βhaloalkyl, Ci-6 hydroxyalkyl, Ci-δhalohydroxyalkyl, Ci-εalkoxyalkyl, and Ci-εhaloalkoxyalkyl;

R³, R⁴, R⁵, R⁶, and R⁸ are each independently selected from hydrogen, halo, nitro, Ci-eαlkyl, Ci-βhαloαlkyl, hydroxy, Ci-eαlkoxy, and Ci-εhaloalkoxy ;

R⁷ is selected from hydrogen, Ci-βalkyl, and Ci-β haloalkyl;

and wherein at least one of -C(X)R¹ or R² to R⁸ comprises a detectable label selected from ¹³L ¹²³> ¹²⁴> ¹²²1, ⁷⁵Br, ⁷⁶Br₁ ⁷⁷Br, ¹³N₁ ¹¹C, and ¹⁸F; most suitably ¹⁸F.

3. A compound of formula (Ib):

or a salt or solvate thereof, wherein:

R¹ is either: (a) Ci-6alkoxy or C1-6 haloalkoxy; or

(b) NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently selected from hydrogen, Ci-ealkyl, aryl, and aryiCi-βalkyl and are each optionally substituted by a group selected from halo, hydroxy, and

R² is selected from hydrogen, Ci-ealkyl, Ci-εhaloalkyl, Ci-6 hydroxyalkyl, Ci-δhalohydroxyalkyl, Ci-ealkoxyalkyl, and Ci-εhaloalkoxyalkyl;

R³, R⁵, R⁵, and R⁸ are each hydrogen and R⁴ is selected from halo, Ci-βalkyl, Ci-δhaloalkyl, hydroxy, Ci-6alkoxy, and Ci-εhaloalkoxy;

R⁷ is selected from hydrogen, Ci-βalkyl, and Ci-ε haloalkyl and is suitably hydrogen;

4. A compound according to claim 1 or 3 wherein the detectable label is selected from 131.123^, 124^, i22|_ ysβp,

¹³N, ¹¹C₁ ^F₁ ³H, we, ³⁵S, and 1²⁵I; and is preferably ¹⁸F.

5. A compound according to any one of claims 1 to 4 selected from:

6-(2-[¹⁸F]Fluoroethoxy)-9H- β-carboline-3-carboxylic acid ethyl ester; 6-(2-[¹⁸F] Fluoroethoxy)-9H- β-carboline-3-carboxylic acid ethylamide; 6-(2-[¹⁸F]Fluoroethoxy)-4-methoxymethyl-9H- β-carboline-3-carboxylic acid ethyl ester; 6-(2-[¹⁸F]Fluoroethoxy)-4-methoxymethyl-9H- β-carboline-3-carboxylic acid ethylamide; or a salt or solvate of any thereof .

6. A compound according to any one of claims 1 to 5 or a salt or solvate thereof for use in an in vivo diagnostic or imaging method such as PET or SPECT.

7. A compound according to any one of claims 1 to 5 or a salt or solvate thereof for use in in vivo diagnosis or imaging of a GABAA-mediated disorder.

8. A radiopharmaceutical formulation comprising the compound as defined in any one of claims 1 to 5 or a salt or solvate thereof and a pharmaceutically acceptable excipient

9. A method for the in vivo diagnosis or imaging of GABAA- mediated disorder in a subject, preferably a human, comprising administration of a compound of formula (I) as defined in any one of claims 1 to 5 or a salt or solvate thereof and detecting the uptake of said compound by an in vivo imaging technique such as SPECT or PET.