WO2016097345A1

WO2016097345A1 - Labelled chromone derivatives

Info

Publication number: WO2016097345A1
Application number: PCT/EP2015/080600
Authority: WO
Inventors: Alexander Jackson
Original assignee: Ge Healthcare Limited
Priority date: 2014-12-19
Filing date: 2015-12-18
Publication date: 2016-06-23

Abstract

The present invention relates to compounds having selective binding for MAO-B as compared with MAO-A. The invention also provides radioactive versions of these compounds, and precursor compounds for the synthesis of these radioactive compounds. The radioactive compounds of the invention are particularly useful for in vivo imaging applications.

Description

LABELLED CHROMONE DERIVATIVES

Technical Field of the Invention

The present invention concerns compounds that target monoamine oxidase B (MAO- B). Specifically, the compound of the invention is a chromone derivative. A labelled chromone derivative is also provided having use as an in vivo imaging agent for MAO- B. The present invention also provides a precursor compound useful in the synthesis of this in vivo imaging agent, as well as a method for synthesis of said precursor compound. Other aspects of the invention include a method for the synthesis of the in vivo imaging agent comprising use of the precursor compound, a kit for carrying out said method, and a cassette for carrying out an automated version of said method. In addition, the invention provides a radiopharmaceutical composition comprising the in vivo imaging agent, as well as methods for the use of said in vivo imaging agent.

Description of Related Art

Monoamine oxidase B (MAO-B) is found in the brain primarily in nonneuronal cells such as astrocytes and radial glia (Westlund et al. (1988) Neuroscience 25: 20 439-456; Westlund et al. (1985) Science 230: 181-183; Levitt et al. (1982) Proc. Natl. Acad. Sci., USA, 79: 6385-6389). Its levels are known to increase with age and in association with neurodegenerative disease in both humans and mice (Saura et al. (1994) J Neural Transm Suppl41 : 89-94; Fowler et al. (1980) J Neural Transm 49: 1-20; Riederer et al. (1987) Adv Neurol45: 111-118; Gerlach et al. (1996) Neurology 47: S137-145).

MAO-B activity levels have been found to be doubled in the substantia nigra in

Parkinson's disease, and to correlate with the percentage of dopaminergic substantia nigra cell loss (Damier et al. (1996) Neurology 46: 1262-1269). Activated MAO-B has also been demosntrated in the brain of Alzheimer patients (Gulyas et al. Neurochem Int. 2011; 58(1): 60-8).

A number of radiolabeled compounds have been reported in literature such as [ⁿC] SL25.1188 and [¹⁸F]Fluororasagiline that are used for imaging MAO-B. They are selective for MAO-B over MAO-A. These known compounds have IC50 values in the range of 11-27 iiM and selectivity for MAO-B of -100 (Nag et al Bioorg Med Chem 2012; 20: 3065-3071; Saba et al Synapse 2010; 64:61-69).

Chromone 1 below has been described as a scaffold for MAO inhibitors with some analogues being particularly selective for MAO-B (Gaspar et al 2011 J Med Chem; 54: 5165; Legoabe et al 2012 Eur J Med Chem; 49: 343-5). This scaffold is similar to the flavones 2 below:

In particular, compounds having structures 3 and 4 below have been shown to have good selectivity for MAO-B over MAO-A:

Where R is typically substituted aryl and R is a substituted aralkyl.

Chimenti et al (Bioorg Med Chem 2010; 18: 1273-1279) describes a series of flavone compounds of structure 5 below and chromone compounds of structure 6 below (in both cases where R and R¹ are selected from H, F, CH₃ or OCH₃):

In this paper, Chimenti et al note that, while the inhibitory activity of the chromone derivatives remains selective for MAO-B, there is a relevant decrease of inhibitory activity with the introduction of the double bond.

There is still scope for further agents having selective binding affinity for MAO-B and in particular for agents suitable as in vivo imaging agents.

Summary of the Invention

The present invention relates to compounds having selective binding for MAO-B as compared with MAO-A. The invention also provides radioactive versions of these compounds, and precursor compounds for the synthesis of these radioactive compounds. The radioactive compounds of the invention are particularly useful for vivo imaging applications, such as in positron-emission tomography (PET). The compounds of the invention are novel over those of the prior art and display good properties for binding to MAO-B.

Detailed Description of the Preferred Embodiments

In a first aspect, the present invention relates to a compound of Formula I:

wherein one of A¹ and A² is N and the other is C-F.

In one embodiment of the compound of the invention A¹ is N and A² is C-F.

In one embodiment of the compound of the invention A² is N and A¹ is C-F.

In one embodiment of the compound of the invention C-F is C-¹⁸F.

In a second aspect, the present invention provides a precursor compound for the synthesis of the compound of the invention comprising ¹⁸F, wherein said precursor is a compound of Formula II:

wherein one of A¹¹ and A¹² is C-R¹ and the other is N, wherein R¹ is selected from nitro, quaternary ammonium salt, diazonium salt, iodonium salt or chloro.

A "precursor compound" comprises a non-radioactive derivative of a radiolabeled compound, designed so that chemical reaction with a convenient chemical form of the detectable label occurs site-specifically; 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 in vivo imaging agent. Such precursor compounds are synthetic and can conveniently be obtained in good chemical purity. The term "nitro" means the substituent -N0₂.

The term "quaternary ammonium salt" refers to a salt of a quaternary ammonium cation with an anion wherein a quaternary ammonium cation is a positively charged polyatomic ions of the structure NP ⁺, R being an alkyl group or an aryl group.

The term "diazonium salt" refers to the functional group R-N₂ ⁺ X^" where R can be any organic residue such alkyl or aryl and X is an inorganic or organic anion such as a halogen

The term "iodonium salt" refers to a salt of a halonium ion R-I⁺ where R is a hydrocarbon.

The terms "comprising" or "comprises" have their conventional meaning throughout this application and imply that the agent or composition must have the essential features or components listed, but that others may be present in addition. The term 'comprising' includes as a preferred subset "consisting essentially of which means that the composition has the components listed without other features or components being present. In one embodiment of the precursor compound of the invention R¹ is chloro or nitro.

Precursor compounds and non-radioactive compounds of the present invention readily obtained by straightforward adaptation methods known in the art according to Scheme 1 below:

AcOH

NBS, Bz₂0₂

CCI₄, 80°C

DMFDMA

90°C

Scheme 1

Cone. HCI

DCM

45°C

In a third aspect, the present invention provides a method for the synthesis of the compound of the invention comprising ¹⁸F wherein said method comprises reaction of the precursor compound of the invention with [¹⁸F]fluoride.

Precursor compounds and methods of introducing radioactive isotopes into organic molecules are well-known in the art. A good overview is provided in the "Handbook of Radiopharmaceuticals: Radiochemistry and Applications" (Wiley 2003; Welch and Redvanley, Eds.). Well-known synthetic routes to ¹⁸F-labelled derivatives are also described by Bolton (J Lab Comp Radiopharm 2002; 45: 485-528).

In a fourth aspect the present invention provides a method for the synthesis of the compound of the invention comprising ¹⁸F wherein said method comprises reaction of a compound of Formula III:

with a compound of Formula IV:

wherein LG is a leaving group. A review by Dolle (2005 Curr Pharm Des; 11 : 3221-3235) describes how a variety of [¹⁸F]fluoropyridyl-containing compounds can be obtained by nucleophilic heteroaromatic substitution at the ortho position with [¹⁸F]fiuoride. These methods can be easily adapted by the person skilled in the art to arrive a method of the fourth aspect of the invention. In a fifth aspect, the present invention provides a pharmaceutical formulation comprising the compound as defined hereinabove together with a biocompatible carrier in a form suitable for mammalian administration.

A "pharmaceutical formulation" is a composition comprising the compound of the invention, together with a biocompatible carrier in a form suitable for mammalian administration. In certain embodiments the "biocompatible carrier" is a fluid, especially a liquid, in which the compound is suspended or dissolved, such that the formulation 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. poly ethylenegly cols, propylene glycols and the like). The biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably 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. In a sixth aspect, the present invention provides an in vivo imaging method for determining the distribution and/ or extent of expression of monoamine oxidase B (MAO-B) in a subject comprising:

(i) administration of the compound as defined herein comprising

18p. (ii) detecting by an in vivo imaging procedure signals emitted by the radioactive isotope comprised in said compound; and,

(iii) generating an image representative of said signals.

The term "in vivo imaging" as used herein refers to those techniques that noninvasively produce images of all or part of the internal aspect of a subject. One suitable in vivo imaging procedures for use with this aspect of the invention is positron-emission tomography (PET).

The compound of the invention may be administered as the pharmaceutical formulation of the invention, e.g. parenterally, i.e. by injection. For parenteral administration, steps to ensure that the radiopharmaceutical composition is sterile and apyrogenic also need to be taken.

The term "MAO-B condition" refers to any condition is which MAO-B activity is abnormal. In particular, MAO-B conditions include neurodegenerative diseases, non- limiting examples of which include Parkinson's disease and Alzheimer's disease.

For example, the in vivo imaging method of the invention can be carried out before, during and after treatment with a drug to combat a MAO-B condition. In this way, the effect of said treatment can be monitored over time. PET is particularly suitable in this aspect as it has excellent sensitivity and resolution, so that even relatively small changes in a lesion can be observed over time, which is particularly advantageous for treatment monitoring. In one embodiment, the in vivo imaging method of the invention is carried out repeatedly during the course of a treatment regimen for said subject, said regimen comprising administration of a drug to combat a MAO-B condition.

In an alternative embodiment, said in vivo imaging method of the sixth aspect of the invention can be understood to be carried out from step (ii) wherein the subject is already administered with said compound of the invention.

In a further alternative embodiment, the sixth aspect of the invention can be understood to be the compound of the invention for use in said in vivo imaging method.

In a yet further alternative embodiment, said in vivo imaging method can be understood to be the use of said compound of the invention in the manufacture of the pharmaceutical composition of the invention for the in vivo imaging of a MAO-B condition.

In a seventh aspect the present invention provides a method of diagnosis of a condition in which MAO-B expression is abnormal wherein said method of diagnosis comprises the in vivo imaging method as defined hereinabove as well as the further step (iv) of attributing the distribution and extent of MAO-B expression to a particular clinical picture.

In an alternative embodiment, the seventh aspect of the invention can be understood to be the compound of the invention for use in said method of diagnosis.

In yet further alternative embodiment of the seventh aspect of the invention, said method of diagnosis can be understood to be the use of said compound of the invention in the manufacture of the pharmaceutical composition of the invention for the diagnosis of a MAO-B condition.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. All patents and patent applications mentioned in the text are hereby incorporated by reference in their entireties, as if they were individually incorporated.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Brief Description of the Figures

Figures 1 and 2 present NMR spectra for certain compounds synthesised as described in the examples.

Figures 3 and 4 show the results of an autoradiography study of certain compounds of the invention.

Brief Description of the Examples

Example 1 describes the synthesis of 6-((6-fluoropyridin-3-yl)methoxy)-4H-chromen-4- one.

Example 2 describes the synthesis of 6-((2-fluoropyridin-4-yl)methoxy)-4H-chromen-4- one.

Example 3 describes the synthesis of a fluorine- 18 radiochemistry precursor for the compound of Example 2.

Example 4 describes the radiosynthesis of [¹⁸F]6-((2-fluoropyridin-4-yl)methoxy)-4H- chromen-4-one. Example 5 describes an assay to measure the in vitro inhibition of MAO- A and MAO- B.

List of Abbreviations used in the Examples

CRC: concentration response curve

DCM: dichloromethane DMFDMA: dimethylformamide dimethylacetal

DMSO: dimethyl sulfoxide

MAO A: monoamine oxidase A

MAO B: monoamine oxidase B Max: maximum

MIN: minimum

TLC: thin-layer chromatography

Examples

Example 1: 6-((6-fluoropyridin-3-yl)methoxy)-4H-chromen-4-one

Step 1 :

AcOH

Molecular Weight: 190.01

To a stirred solution of 2-fluoro-5-methylpyridine (1.44 g, 12.95 mmol) in dry carbon tetrachloride (13 ml), N-bromo succinimide (2.32 g, 12.95 mmol) and Benzoyl peroxide (157 mg, 0.64 mmol) was added. The reaction mixture was stirred at 80° C for 2h. The progress of the reaction was monitored by TLC (1 :9, Ethyl acetate : pet. ether). The reaction mixture was cooled to ambient temperature and filtered under suction. The solid was washed with Carbon tetrachloride. The solvent was removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 3% Ethyl acetate in pet ether as eluent to yield pure 5-(bromomethyl)-2- fluoropyridine (1.2 g, 48.7%) as a pale yellow oil.

Step 2:

Molecular Weight: 261.25

To a stirred solution of 2,5-dihydroxyacetophenone (0.4 g, 2.62 mmol) in dry acetone (10 ml) was added K₂CO₃ (0.36 g, 2.6 mmol). After stirring at ambient temperature for 30 minutes, 5-(bromomethyl)-2-fluoropyridine (0.494 g, 2.6 mmol) was added drop wise. The reaction mixture was heated to 60° C for 14h. The progress of the reaction was monitored by TLC (2:8, Ethyl acetate : pet. ether). After completion of the reaction the reaction mixture was cooled o room temperature and quenched with aqueous 10% NaHC0₃ solution (30 ml) and extracted with ethyl acetate (4 x 70 ml). The combined organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 9% Ethyl acetate in pet ether as eluent to yield pure l-(5-((6- fluopyridin-3-yl)methoxy)-2-hydroxyphenyl)ethanone (0.345 g, 50.4%) as a pale yellow solid.

Step 3:

Molecular Weight: 316.33 A mixture of l-(5-((6-fluoropyridin-3-yl)methoxy)-2-hydroxyphenyl)ethanone (0.345 g, 1.32 mmol, 1 eq) and DMFDMA (0.32 g, 2.64 mmol, 2 eq) was heated to 90° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate : pet ether). After completion of reaction, reaction mixture was diluted with ethyl acetate (40 ml) and thoroughly washed with water (4 x 40 mL). The combined organic layer was washed with saturated NaCl (20 mL) solution, dried over anhydrous Na₂S0₄, filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 28-37% Ethyl acetate in pet ether as eluent to yield (£)-3-(dimethylamino)- l-(5-((6-fluoropyridin-3-yl)methoxy)-2-hydroxyphenyl)prop-2-en-l-one (0.21 g, mixture of desired and the final) as a pale yellow solid. Step 4:

Molecular Weight: 271.24

To a stirred solution of (E)-3-(dimethylamino)-l-(5-((6-fluoropyridin-3-yl)methoxy)-2- hydroxyphenyl)prop-2-en-l-one (0.21 g, 0.663 mmol, 1 eq), in dichloromethane (6 mL), was added cone. HC1 (0.6 mL) drop wise at room temperature. The reaction mixture was heated to reflux at 45° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate: pet ether). After completion, the reaction mixture was cooled to room temperature and extracted with dichloromethane (2 x 30 mL). The combined organic layers were washed successively with saturated NaHC0₃ solution (20 mL) and saturated NaCl solution (20 mL). After drying over anhydrous Na₂S0₄, it was concentrated and the crude product was purified by column chromatography over 60- 120 mesh silica gel (24% Ethyl acetate in pet ether) followed by crystallization from ethyl acetate afforded 6-((6-fluoropyridin-3-yl)methoxy)-4H-chromen-4-one as off pale yellow solid (0.120 g, 66.72%). Figure 1 shows the NMR characterization data for this compound.

Example 2: Synthesis of 6-((2-fluoroOyridin-4-yl)methoxy)-4H-chromen-4-one

Step-1 :

Molecular Weight: 190.01 To a stirred solution of 2-fluoro-4-methylpyridine (5g, 44.99 mmol) in dry carbon tetrachloride (114 ml) was added glacial Acetic acid (3.3ml) at 25° C. Then reaction mixture was heated to 50° C and N-bromo succinimide (8 g, 44.9 mmol) followed by Benzoyl peroxide (1.1 g, 4.49 mmol) was added. After complete addition reaction mixture was stirred at 80° C for 20h. The progress of the reaction was monitored by TLC (1 :9, Ethyl acetate : pet. ether). After completion of reaction, reaction mixture was cooled to room temperature quenched with cold water and extracted with DCM (4 x 200 mL). The combined organic layer was washed with saturated NaCl (100 mL) solution, dried over anhydrous Na₂S0₄, filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 2% Ethyl acetate in pet ether as eluent to yield pure 4-(bromomethyl)-2-fluoropyridine (2.8 g, 23.4%>) as a pale yellow oil.

Step-2:

Molecular Weight: 261 .25

To a stirred solution of 2,5-dihydroxy acetophenone (0.6 g, 3.94 mmol, 1 eq), in acetone (15 mL), was added K₂C0₃ (0.54 g, 3.9 mmol, 0.99 eq). After stirring at room temperature for 30 minutes, 4-(bromomethyl)-2-fluoropyridine (0.74 g, 3.90 mmol, 0.99 eq) was added drop wise. The reaction mixture was heated to reflux at 60° C for 16 h. Reaction progress was monitored by TLC (2:8, ethyl acetate: pet ether). After completion of reaction, the reaction mixture was cooled to room temperature mixed with aq. NaHC0₃ solution (30 mL) and extracted with ethyl acetate (4 x 50 mL). The combined organic layer was washed with saturated NaCl (100 mL) solution, dried over anhydrous Na₂S0₄, filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 13% Ethyl acetate in pet ether as eluent to yield l-(5-((2-fluopyridin-4-yl)methoxy)-2-hydroxyphenyl)ethanone (0.5 g, 48.2%) as a pale yellow solid.

Step-3:

A mixture of l-(2-hydroxy-5-((4-methylbenzyl)oxy)phenyl)-ethan-l-one (0.5 g, 1.91 mmol, 1 eq) and DMFDMA (0.456 g, 3.82 mmol, 2 eq) was heated to 90° C for 2 h.

Reaction progress was monitored by TLC (5:5, ethyl acetate : pet ether). After completion of reaction, reaction mixture was diluted with ethyl acetate (40 ml) and thoroughly washed with water (4 x 40 mL). The combined organic layer was washed with saturated NaCl (20 mL) solution, dried over anhydrous Na₂S0₄, filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 32-65% Ethyl acetate in pet ether as eluent to yield (£)-3-(dimethylamino)- l-(5-((2-fluopyridin-4-yl)methoxy)-2-hydroxyphenyl)prop-2-en-l-one (0.25 g, mixture of desired and the final) as a pale yellow solid. Step-4:

Molecular Weight: 271.24

To a stirred solution of (E)-3-(dimethylamino)-l-(5-((2-fluopyridin-4-yl)methoxy)-2- hydroxyphenyl)prop-2-en-l-one (0.25 g, 0.79 mmol, 1 eq), in dichloromethane (10 mL), was added cone. HCI (1 mL) drop wise at room temperature. The reaction mixture was heated to reflux at 45° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate: pet ether). After completion, the reaction mixture was cooled to room temperature and extracted with dichloromethane (2 x 30 mL). Combined organic layer was washed successively with saturated NaHC0₃ solution (20 mL) and saturated NaCl solution (20 mL). After drying over anhydrous Na₂S0₄, it was concentrated and the crude was purified by column chromatography over 60-120 mesh silica gel using 32% Ethyl acetate in pet ether as eluent. The product was crystallized from ethyl acetate afforded 6-((2-fluoropyridin-4-yl)methoxy)-4H-chromen-4-one as pale yellow solid (0.092 g, 42.29%). Figure 2 shows the NMR characterization data for this compound.

Example 3: Synthesis of a fluorine-18 radiochemistry precursor for the compound of Example 1.

To a stirred solution of (6-chloropyridin-3-yl)methanol (2 g, 13.93 mmol) in dry DCM (40 ml), PBr₃ (1.6 ml, 16.71 mmol) was added dropwise at 0° C. The reaction mixture was stirred at ambient temperature for 3h. The progress of the reaction was monitored by TLC (3 :7, Ethyl acetate: pet. ether). After completion of the reaction the reaction mixture was cooled o room temperature and quenched with aqueous 10% NaHC0₃ solution (30 ml) and extracted with DCM (3 X 50 ml). The combined organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 8% Ethyl acetate in pet ether as eluent to yield pure 5-(bromomethyl)-2-chloropyridine (2.2 g, 76.65%) as a yellow oil.

Step 2:

To a stirred solution of 2,5-dihydroxyacetophenone (1.48 g, 9.78 mmol) in dry Acetone (30 mL) was added K₂C0₃ (0.36 g, 1.33 mmol). After stirring at ambient temperature for 30 minutes, a solution of 5-(bromomethyl)-2-chloropyridine (2 g, 9.68 mmol) in acetone (15 mL) was added dropwise. The reaction mixture was heated to 60° C for

14h. The progress of the reaction was monitored by TLC (3 :7, Ethyl acetate: pet. ether). After completion of the reaction the reaction mixture was cooled to room temperature and quenched with aqueous 10%> NaHC0₃ solution (50 ml) and stirred for 15 minutes. The yellow solid appeared was filtered under suction. The solid obtained was suspended in EtOAc (70 ml) and inorganic was filtered under suction. The EtOAc layer was dried over anhydrous sodium sulfate and removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 14%> Ethyl acetate in pet ether as eluent to yield pure l-(5-((6-chloropyridin-3-yl)methoxy)- 2-hydroxyphenyl)ethan-l-one (1.15 g, 42.9%) as a pale yellow solid. Step 3:

A mixture of l-(5-((6-chloropyridin-3-yl)methoxy)-2-hydroxyphenyl)ethan-l-one (1.1 g, 3.96 mmol, 1 eq) and DMFDMA (2.63 g, 19.80 mmol, 5 eq) was heated to 90° C for 2 h. Reaction progress was monitored by TLC (5 :5, ethyl acetate : pet ether). After completion of reaction, reaction mixture was diluted with water (30 ml), the reaction mass gets precipitating out then the reaction mixture was filtered and thoroughly washed with water (2 x 30 mL), followed by pet ether (30 mL). The solid was dried to yield (E)-l-(5-((6-chloropyridin-3-yl)methoxy)-2-hydroxyphenyl)-3- (dimethylamino)prop-2-en-l-one (0.68 g, 42.9%) as a pale yellow solid. Step 4:

To a stirred solution of (E)-l-(5-((6-chloropyridin-3-yl)methoxy)-2-hydroxyphenyl)-3- (dimethylamino)prop-2-en-l-one (0.40 g, 1.20 mmol, 1 eq), in dichloromethane (20 mL), was added cone. HC1 (1.8 mL) drop wise at room temperature. The reaction mixture was heated to reflux at 45° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate: pet ether). After completion, the reaction mixture was cooled to room temperature and extracted with dichloromethane (2 x 40 mL). The combined organic layers were washed successively with saturated NaHC0₃ solution (60 mL) and saturated NaCl solution (70 mL). After drying over anhydrous Na₂S0₄, it was concentrated and the crude product was purified by column chromatography over 60- 120 mesh silica gel (24% Ethyl acetate in pet ether) followed by crystallization from ethyl acetate afforded 6-((6-chloropyridin-3-yl)methoxy)-4H-chromen-4-one (GE- MAO-B-03_4) as off pale yellow solid (0.245 g, 71.01%). LCMS gave m.z = 288 [M+H]⁺

Example 4: Synthesis of a fluorine-18 radiochemistry precursor for the compound of

Cone. HCI DCM

45°C

Step-1:

To a stirred solution of (2-chloropyridin-4-yl)methanol (2g, 13.93 mmol) in dry in dry

DCM (40 ml), PBr₃ (1.6 ml, 16.71 mmol) was added dropwise at 0° C. The reaction mixture was stirred at ambient temperature for 3h. The progress of the reaction was monitored by TLC (3:7, Ethyl acetate: pet. ether). After completion of the reaction the reaction mixture was cooled o room temperature and quenched with aqueous 10% NaHC0₃ solution (30 ml) and extracted with DCM (3 X 50 ml). The combined organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 8% Ethyl acetate in pet ether as eluent to yield pure 4-(bromomethyl)-2- chloropyridine (lg, 34.77%o) as a pale yellow oil.

Step-2:

To a stirred solution of 2,5-dihydroxyacetophenone (0.740 g, 4.89 mmol) in dry Acetone (20 mL) was added K₂CO₃ (0.668 g, 4.84 mmol). After stirring at ambient temperature for 30 minutes, a solution of 5-(bromomethyl)-2-chloropyridine (1 g, 4.84 mmol) in acetone (15 mL) was added dropwise. The reaction mixture was heated to 60° C for 14h. The progress of the reaction was monitored by TLC (3:7, Ethyl acetate: pet. ether). After completion of the reaction the reaction mixture was cooled to room temperature and quenched with aqueous 10%> NaHC0₃ solution (50 ml) and stirred for 15 minutes. The yellow solid appeared was filtered under suction. The solid obtained was suspended in EtOAc (70 ml) and inorganic was filtered under suction. The EtOAc layer was dried over anhydrous sodium sulfate and removed under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 14% Ethyl acetate in pet ether as eluent to yield pure l-(5-((2-chloropyridin-4-yl)methoxy)- 2-hydroxyphenyl)ethan-l-one (0.7 g, 52.23%) as a pale yellow solid.

Step-3:

A mixture of l-(5-((2-chloropyridin-4-yl)methoxy)-2-hydroxyphenyl)ethan-l-one (0.7 g, 2.52 mmol, 1 eq) and DMFDMA (1.50 g, 12.60 mmol, 5 eq) was heated to 90° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate : pet ether). After completion of reaction, reaction mixture was diluted with water (25 ml), the reaction mass gets precipitating out then the reaction mixture was filtered and thoroughly washed with water (2 x 25 mL), followed by pet ether (30 mL). The solid was dried to yield (E)-l-(5-((2-chloropyridin-4-yl)methoxy)-2-hydroxyphenyl)-3- (dimethylamino)prop-2-en-l-one (0.43 g, 51.2% ) as a yellow solid. Step-4:

To a stirred solution of (E)-l-(5-((2-chloropyridin-4-yl)methoxy)-2-hydroxyphenyl)-3- (dimethylamino)prop-2-en-l-one (0.43 g, 1.29 mmol, 1 eq), in dichloromethane (25 mL), was added cone. HC1 (1.9 mL) drop wise at room temperature. The reaction mixture was heated to reflux at 45° C for 2 h. Reaction progress was monitored by TLC (5:5, ethyl acetate: pet ether). After completion, the reaction mixture was cooled to room temperature and extracted with dichloromethane (2 x 30 mL). Combined organic layer was washed successively with saturated NaHC0₃ solution (20 mL) and saturated NaCl solution (20 mL). After drying over anhydrous Na₂S0₄, it was concentrated and the crude was purified by column chromatography over 60-120 mesh silica gel using 32% Ethyl acetate in pet ether as eluent. The product was crystallized from ethyl acetate afforded 6-((2-chloropyridin-4-yl)methoxy)-4H-chromen-4-one (GE-MAO-B-03 5) as off white solid (0.22 g, 59.45%).

LCMS gave m.z = 288 [M+H]⁺

Example 5: Radiosynthesis of f^lsF 6-((6-fluoropyridin-3-yl)methoxy)-4H-chromen-4- one

The radiosynthesis was carried out on an automated radiochemistry module. Briefly, no-carrier- added [¹⁸F]fluoride was produced via ¹⁸0(p,n)¹⁸F reaction by proton irradiation of an ¹⁸0-enriched water target of the cyclotron. The [¹⁸F]fluoride was trapped on an ion exchange cartridge (QMA) then eluted using a mixture of potassium carbonate, Kryptofix [2.2.2], water and acetonitrile. The solution was dried ith heating with nitrogen plus gas flow. A solution of precursor (5-10 mg) in was added and the mixture heated at 125 C for 10 minutes. The crude product was diluted with water and semi-preparative HPLC was performed (column was ACE 5 C18-HL(250xl0mm); eluent was acetonitrile:ammonium formate (0.1 M), 30:70); flow 5 mL/min; retention time: 25 minutes). The product peak was collected, diluted with water, trapped on a reversed-phase SPE cartridge and formulated.

Analytical HPLC (254 nm; ACE5 C18-HL 250x4.6mm column; flow = 2 mL/min; eluent is 0.1% TFA: acetonitrile 45%-90% gradient over 10 minutes: retention time = 4 minutes. Example 6: In vitro autoradiosraphy of f¹⁸F 6-((6-fluoropyridin-3-yl)methoxy)-4H- chromen-4-one

[¹⁸F 6-((6-fluoropyridin-3-yl)methoxy)-4H-chromen-4-one was applied to whole brain sections from healthy Rhesus monkey (20 μιη thickness) or human (100 μιη thickness). Blocking was performed with either deprenyl (MAO-B) or pirlindole (MAO-A).

Blocking agents (deprenyl and Pirlindole) are available commercially from Sigma- Aldrich.

Conditions:

• Radioligand concentration 0.02 MBq/ml

· Blocker concentrations 10 μΜ

• Incubation 90 min, RT

• Wash 3 x 5 min, 4°C

Buffer:

• Tris HC1 50 mM, pH 7.4 incl

· 120 mM NaCl

• 5 mM KC1

• 2 mM CaC12

• 1 mM MgC12

• 0.1 % BSA Figure 3 shows the results obtained for human tissue and Figure 4 shows the results obtained for Rhesus monkey.

Example 7: In vitro inhibition of MAO-A and MAO-B

Assay development was carried out based on the standard protocol provided in product insert for Amplex Red Monoamine Oxidase Assay Kit from Life Technologies. General parameters for screening of the compounds was as follows:

□ Assay Type: Amplex Red Monoamine Oxidase Assay (Invitrogen Cat # A12214)

□ Assay Format: 96 well plate (Black, Corning)

□ Assay Volume: 200μΙ,

□ Enzyme Source: MAO A and MAO B enzymes (Sigma- Aldrich Cat # M7316 and M7441)

□ Substrate : For MAO-A/B : Tyramine, For MAO-B : Benzylamine

□ Reference Compounds: For MAO-A: Clorgyline (Irreversible), Moclobemide (Reversible), for MAO-B: Pargyline (Irreversible), Deprenyl (Irreversible), Lazabemide hydrochloride (Reversible).

□ Screening Format: Concentration Response Curve (CRC) for all compounds

□ Replicates: Duplicates (n=2)

□ Number of Runs: One (N=l)

□ Controls: □ Negative control/"MIN": 1% DMSO Buffer without enzyme (substrate alone)

□ Positive control/ "Max": 1% DMSO Buffer with enzyme and substrate

□ Reading: End point or Kinetic

□ Detection: Fluorescence Ex/Em - 560nm/590nm

□ Detection Instrument: Envision [Perkin Elmer] Stock solution will be prepared from solid samples and serial dilutions will be made on a semi-log scale while maintaining the final concentration of the DMSO in the assay as determined under tolerability study.

Claims

(1) A compound of Formula I:

wherein one of A¹ and A² is N and the other is C-F.

(2) The compound of Formula I as defined in Claim 1 wherein A is N and A is C- F. (3) The compound of Formula I as defined in Claim 1 wherein A² is N and A¹ is C-

(4) The compound as defined in any one of Claims 1-3 wherein C-F is C- F. (5) A precursor compound for the synthesis of the compound as defined in Claim 4 wherein said precursor compound is a compound of Formula II:

wherein one of A^{1 1} and A¹² is C-R¹ and the other is N, wherein R¹ is selected from nitro, quaternary ammonium salt, diazonium salt, iodonium salt or chloro. (6) The precursor compound as defined in Claim 5 wherein R¹ is chloro or nitro. (?) A method for the synthesis of the compound as defined in Claim 4 comprising reaction of the precursor compound of Formula II as defined in Claim 5 or Claim 6 with [¹⁸F]fluoride.

(8) A method for the synthesis of the compound as defined in Claim 4 comprising reaction of a compound of Formula III:

with a compound of Formula IV:

wherein LG is a leaving group.

(9) A pharmaceutical formulation comprising the compound as defined in any one of Claims 1-4 together with a biocompatible carrier in a form suitable for mammalian administration.

(10) An in vivo imaging method for determining the distribution and/ or extent of expression of monoamine oxidase B (MAO-B) in a subject comprising:

(i) administration of the compound as defined in Claim 4;

(ii) detecting by an in vivo imaging procedure signals emitted by the radioactive isotope comprised in said compound; and,

(iii) generating an image representative of said signals. (11) The in vivo imaging method as defined in Claim 10 wherein said method is carried out repeatedly during the course of a treatment regimen for said subject, said regimen comprising administration of a drug to combat a MAO-B condition.

(12) A method of diagnosis of a condition in which MAO-B expression is abnormal wherein said method of diagnosis comprises the in vivo imaging method as defined in Claim 10 as well as the further step (iv) of attributing the distribution and extent of MAO-B expression to a particular clinical picture.