LABELLED COUMARIN 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 coumarin derivative. A labelled coumarin 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 [ ⁿ C] SL25.1188 and [ ¹⁸ F]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 nM 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).

Coumarins are a large family of compounds, of natural and synthetic origin, that present different pharmacological activities. Some coumarins are monoamine oxidase (MAO) inhibitors (MAOI). Recent findings have shown that MAO-A and MAO-B affinity and selectivity can be efficiently modulated by appropriate substitutions in the coumarin moiety (Matos et al Bioorganic & Med Chem Letts 2009; 19: 3268-3270). Matos et al have synthesized analogues of 3-phenylcoumarins that have good selectivity towards MAO-B compared to MAO-A (compounds 1 and 2 illustrated below). The 3-phenylcoumarins have been demonstrated to be better than the reported standard compounds in terms of potency and selectivity (Matos et al Bioorg & Med Chem Letts 2009; 19: 3268-3270; Matos et al 2009 Bioorg Med Chem Letts 2009; 19: 5053-5055).

CLogP: 4.03 CLogP: 4.03

MAO-B IC50 : 0.80 nM

MAO-B IC50 : 13.05 nM

MAO-A: MAO-B : 124595

MAO-A: MAO-B = 7663

2

1

Another publication from the same group related to halogenated 3-phenylcoumarins having good affinity and selectivity for MAO-B over MAO-A (Matos et al Bioorganic & Medicinal Chemistry Letters 20 (2010) 5157-5160). These compounds are illustrated below as compounds 3-5.

CLogP: 4.872 CLogP: 4.9015 CLogP: 4.86492

MAO-B IC50 : 1 1 .05 nM MAO-B IC50 : 3.23 nM MAO-B IC50 : 7.12 nM MAO-A: MAO-B = 9050 MAO-A: MAO-B = 30960 MAO-A: MAO-B = 14045

3 4 5 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 can find use for in vivo imaging applications. 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:

R ¹ is C i_3 alkyl, Ci_ ₃ alkoxy or -0-(CH ₂ ) _n -F wherein n is an integer from 1 to 3;

R ² is -Ar'-R ⁴ wherein Ar ¹ is an aryl and R ⁴ is selected from fluoro, Ci_ ₃ alkoxy, Ci_ ₃ fluoroalkyl and Ci_ ₃ fluoroalkoxy; and,

R ³ is hydrogen, halo or Ci_ ₃ alkyl with the proviso that at least one of R ¹ and R ⁴ comprises fluoro.

Unless otherwise specified, the term "alkyl" alone or in combination, means a straight- chain alkyl radical containing preferably from 1 to 3 carbon atoms. The term "alkoxy" means an alkyl radical comprising an ether linkage, and the term "ether linkage" refers to the group -C-0-C-.

"Aryl" is defined herein as any mono-cyclic C ₂ - ₆ molecular fragment, preferably having 5 to 6 ring members the ring. The term "aryl" is intended to encompass also heteroaryl groups, defined as an aryl including at least one N, O or S atom ring member. In one embodiment said aryl is phenyl. In one embodiment said aryl is pyridyl. In one embodiment said aryl is a triazole.

The term "halogen" or "halo" means a substituent selected from fluorine, chlorine, bromine or iodine, including radioactive isotopes thereof suitable for in vivo imaging as

18 18 123 127 131 defined hereinbelow, e.g. fluorine can be F or F, iodine can be I, I or I.

"Fluoroalkyl" and "fluoroalkoxy" are alkyl and alkoxy groups, respectively, as defined above substituted with one or more fluorine atoms in place of hydrogen atoms. Suitably in the case of fluoroalkyl and flujoroalkoxy substituents, the fluorine replaces a hydrogen at the terminal end of the radical, i.e. -alkylene-fluoro or -alkoxylene-fluoro. The term "alkylene" refers to the bivalent group -(CH ₂ ) _Z - wherein z is 1-3, and the term "alkoxy lene" refers to an alkylene group comprising an ether linkage, wherein an ether linkage is as defined above.

The term "at least one of R ¹ and R ⁴ comprises fluoro" is meant that the compound of Formula I comprises at least one fluoro, wherein the term "at least one fluoro" in one embodiment means one fluoro. In some embodiments R ¹ is -0-(CH ₂ ) _n -F and in other embodiments R ⁴ is selected from the group comprising fluoro, Ci_ ₃ fluoroalkyl and Ci_ ₃ fluoroalkoxy, or in an alternative embodiment R ⁴ is fluoro.

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 compound of the invention R ¹ is -0-(CH ₂ ) _n -F wherein n is as defined herein.

In one embodiment of the compound of the invention n is 2.

In one embodiment of the compound of the invention Ar ¹ is phenyl.

In one embodiment of the compound of the invention Ar ¹ is pyridyl.

In one embodiment of the compound of the invention Ar ¹ is triazole. In one embodiment of the compound of the invention R ⁴ is Ci_ ₃ alkoxy. In one embodiment of the compound of the invention R ⁴ is methoxy.

In one embodiment of the compound of the invention R ⁴ is ethoxy.

In one embodiment of the compound of the invention R ⁴ is Ci_ ₃ fluoroalkyl.

In one embodiment of the compound of the invention R ³ is halo. In one embodiment of the compound of the invention R ³ is bromo.

In one embodiment the compound of the invention comprises a radioactive isotope. In one embodiment said radioactive isotope is a radioactive version of an atom as defined for Formula I herein. In one embodiment R ¹ is -0-(Ο¼) _η - ¹⁸ Ρ. In another embodiment

4 18 18 18

R is selected from F, Ci_ ₃ [ F] fluoroalkyl and Ci_ ₃ [ FJfluoroalkoxy. In a further embodiment R ⁴ is ¹⁸ F.

In one embodiment said radioactive isotope is suitable for in vivo imaging, i.e. it can be detected externally in a non- invasive manner following administration in vivo. Examples of suitable such radioactive isotopes include gamma-emitting radioactive halogens and positron-emitting radioactive non-metals, particularly those suitable for imaging using SPECT or PET.

In one embodiment said radioactive isotope is a gamma-emitting radioactive halogen or a positron-emitting radioactive non-metal. In one embodiment said radioactive isotope

123 131 11

is a gamma-emitting radioactive halogen selected from I, I and Br. In one embodiment said gamma-emitting radioactive halogen is selected from ¹²³ I and ¹³¹ I. In one embodiment said gamma-emitting radioactive halogen is ¹²³ I. In one embodiment said radioactive isotope is a positron-emitting radioactive non-metal selected from ⁿ C,

13 18 124

^1J N, ^{i a} F and ^1Ζ Ί. In one embodiment said positron-emitting radioactive non-metal is selected from ⁿ C and ¹⁸ F. In one embodiment said positron-emitting radioactive non- metal is ¹⁸ F. 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.).

Bolton (J Lab Comp Radiopharm 2002; 45 : 485-528) and Kabalaka et al (Nuc Med Biol 2002; 29: 841-843; Nuc Med Biol 2003; 30: 369-373; Nuc Med Biol 2004; 31 : 935-938) provide detail for methods of radio iodination and radiobromination.

Additional details of synthetic routes to ¹⁸ F-labelled derivatives are described by Bolton (J Lab Comp Radiopharm 2002; 45: 485-528).

In one embodiment the compound of the invention is selected from:

In one embodiment of the compound of the invention R ⁴ comprises ¹⁸ F. In one embodiment the compound of the invention is selected from:

In a second aspect the present invention provides a precursor compound useful in the preparation of the compound of the invention as defined hereinabove, said precursor compound being of Formula II:

wherein said precursor compound comprises one precursor group and wherein:

R ^{1 1} is Ci_3 alkyl, Ci_ ₃ alkoxy or -0-(CH ₂ ) _m -R ¹⁵ wherein m is an integer from 1 to 3 and R ¹⁵ is fluoro or said precursor group;

12 2 14 2 14

R is -Ar -R wherein Ar is an aryl and R is selected from fluoro, Ci_ ₃ alkoxy Ci_ ₃ fluoroalkyl and Ci_ ₃ fluoroalkoxy or is said precursor group;

R ¹³ is hydrogen, halo or Ci_ ₃ alkyl; and wherein: when R ¹⁴ is said precursor group it is selected from Ci_ ₃ trialkylammonium or iodonium; and, when R ¹⁵ is said precursor group it is a leaving group; with the proviso that at least one of R ¹⁴ and R ¹⁵ is said precursor group.

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 "leaving group" refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. A suitable leaving group can be a halo, e.g. selected from chloro, iodo, or bromo, or an aryl or alkyl sulphonate. Non-limiting examples include CI, Br, I, tosylate (OTs), mesylate (OMs) and triflate (OTf).

In one embodiment of the precursor compound of the invention R ¹⁴ is said precursor group.

In one embodiment of the precursor compound of the invention said precursor group is Ci_3 trialkyltin.

In one embodiment of the precursor compound of the invention R ¹⁵ is said precursor group.

In one embodiment of the precursor compound of the invention R ¹⁵ is selected from tosylate (OTs), mesylate (OMs), and trifiate (OTf).

Compounds and precursor compounds of the invention can be synthesized according to Scheme 1 below, which based on the disclosures of Matos et al (Bioorg Med Chem Letts 2009; 19: 3268-3270; Bioorg Med Chem Letts 2009; 19: 5053-5055; Bioorg Med Chem Letts 2010; 20: 5157-5160):

X = C, N

Y = H, OCH3, CH2CH2F, F, N02, Me, Et, NHCH2F, NHCH2CH2F

Reagents and Conditions: a. DCC, DMSO, 1 10 C, 24 h

b. NBS, AIBN, CCL4, reflux, 18h

Other compounds of the invention can be obtained via click chemistry according to Scheme 2 below:

In a third aspect the present invention provides a method for synthesis of a compound of the invention comprising a radioactive isotope as defined hereinabove wherein said method comprises reaction of the precursor compound of the invention as defined hereinabove with a suitable source of the radioactive isotope as defined hereinabove.

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

wherein:

21 22 21 22 either R is hydroxyl or R is ethynyl, otherwise R and R are as respectively defined for R ¹ and R ² hereinabove; with a compound of Formula IVa when R ²¹ is hydroxyl: wherein LG is a leaving group and x is an integer of 1-3; or with a compound of Formula IVb when R ²² is ethynyl:

^{1 8} F. >N ₃

y IVb wherein y is an integer of 1-3.

In a fifth aspect the present invention provides a pharmaceutical formulation comprising the compound of the invention 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 of the invention comprising a radioactive isotope as defined hereinabove;

(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 "m vivo imaging" as used herein refers to those techniques that noninvasively produce images of all or part of the internal aspect of a subject. Examples of suitable in vivo imaging procedures for use with this aspect of the invention are single-photon emission tomography (SPECT) and 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.

In one embodiment of the in vivo imaging method of the invention 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.

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. Preferably for this embodiment, the in vivo imaging procedure is PET. PET 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 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 of the invention 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 a 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 1-4 present NMR spectra for various compounds that have been synthesised as described in the examples.

Figure 5 shows the results of autoradiography.

Brief Description of the Examples

Example 1 describes the synthesis of 8-bromo-3-(4-methoxyphenyl)-6-(fluoroethoxy)- 2H-chromen-2-one. Example 2 describes the synthesis of 8-bromo-3-(4-ethoxyphenyl)-6-(fluoroethoxy)- 2H-chromen-2-one.

Example 3 describes the synthesis of precursor compounds for radio labelling with fluorine- 18.

Example 4 describes the synthesis of [ ¹⁸ F]-8-bromo-3-(4-ethoxyphenyl)-6- (fluoroethoxy)-2H-chromen-2-one.

Example 5 describes in vitro testing of affinity of compounds of the invention. List of Abbreviations used in the Examples

AcOH: acetic acid

DCM: dichloromethane

DMF: dimethylformamide

EtOAc: ethyl acetate

MAO: monoamine oxidase

NaOAc: sodium acetate

NMR: nuclear magnetic resonance

TEA: triethanolamine

TLC: thin-layer chromatography

Examples

Example 1: 8-bromo-3-(4-methoxyphenyl)-6-(fluoroethoxy)-2H-chromen-2-on e

Step 1 :

To a stirred solution of 2-hydroxy-5-methoxy-benzaldehyde (14g, 92 mmol) in glacial AcOH (400 ml) was added Br ₂ (6.1ml, 117.8 mmol) followed by NaOAc (12g, 146 mmol) at 0°C. Then reaction mixture was stirred at ambient temperature for further lh. The progress of the reaction was monitored by TLC (2:8, Ethyl acetate : pet. ether). After completion of reaction, solvent was removed completely under reduced pressure. The crude was diluted with cold water and extracted with DCM (3 x 300 mL). The combined organic layer was washed with water (2 X 200 ml) followed by saturated NaCl (200 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 4-10% Ethyl acetate in pet ether as eluent to yield pure 3-bromo-2-hydroxy-5-methoxy- benzaldehyde (17 g, 79.97%) as a orange solid.

Figure 1 illustrates the NMR characterization data for this compound.

Step-2:

Molecular Weight: 217.02

To a stirred suspension of 3-bromo-2-hydroxy-5-methoxy-benzaldehyde (4g, 17.31 mmol) in dry DCM (40 ml) was added BBr ₃ [1M in DCM; 86.5ml, 86.56 mmol] at 0°C Then reaction mixture was stirred at ambient temperature for further 24h. The progress of the reaction was monitored by TLC (3:7, Ethyl acetate: pet. ether). After completion of reaction, reaction mixture was quenched with cold water and extracted with EtOAc (4 x 200 mL). The combined organic layer was washed with saturated NaCl (100 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 14% Ethyl acetate in pet ether as eluent to yield pure 3-bromo-2,5-dihydroxy benzaldehyde (2.8 g, 69.2%) as a yellow solid.

Step-3:

Molecular Weight: 389.2

To a stirred solution of 3-bromo-2,5-dihydroxy benzaldehyde (0.3 g, 1.38 mmol, 1 eq) and 2-(4-methoxyphenyl)acetic acid (0.229 g, 1.38 mmol, 1 eq) in acetic anhydride (2 mL), was added TEA (1.2 mL). The reaction mixture was heated to at 120°C for 12 h. Reaction progress was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of reaction, reaction mixture was diluted with ethyl acetate (100 ml) and thoroughly washed with water (4 x 100 mL). The combined organic layer was washed with saturated NaCl (50 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 20%- 16% Ethyl acetate in pet ether as eluent and the product was washed with ethyl acetate to yield 8-bromo-3-(4-methoxyphenyl)-2-oxo-2H-chromen-6-yl acetate (0.19 g, 35.37%) as a pale yellow solid.

Step-4:

Molecular Weight: 393.2

To a stirred solution of 8-bromo-3-(4-methoxyphenyl)-2-oxo-2H-chromen-6-yl acetate (0.190 g, 0.488 mmol, 1 eq), in DMF (6 mL), was added K ₂ C0 ₃ (0.202 g, 1.46 mmol, 3 eq). After stirring at room temperature for 30 minutes, l-bromo-2-fluoroethane (0.185 g, 1.46 mmol, 3 eq) was added drop wise. The reaction mixture was heated to at 70°C for 24 h. Reaction progress was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of reaction, reaction mixture was cooled to room temperature, was diluted with ethyl acetate (40 ml) and thoroughly washed with water (4 x 30 mL). The combined organic layer was washed with saturated NaCl (20 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 30% Ethyl acetate in pet ether as eluent. The product was further re-crystallized from ethyl acetate to yield 8-bromo-3-(4- methoxyphenyl)-6-(fluoroethoxy)-2H-chromen-2-one (0.70 g, 36.48%) as an off white solid.

Figure 2 illustrates the NMR characterization data for this compound. Example 2: Synthesis of 8-bromo-3-(4-ethoxyphenyl)-6-(fluoroethoxy)-2H-chromen-

2-one

Step-1 :

Molecular Weight: 403.22 To a stirred solution of 3-bromo-2,5-dihydroxy benzaldehyde (0.6 g, 2.76 mmol, 1 eq; obtained as per Example 1 Steps 1 and 2) and 2-(4-ethoxyphenyl)acetic acid (0.498 g, 2.76 mmol, 1 eq) in acetic anhydride (3 mL), was added TEA (2 mL). The reaction mixture was heated to at 120° C for 12 h. Reaction progress was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of reaction, reaction mixture was diluted with ethyl acetate (100 ml) and thoroughly washed with water (4 x 100 mL). The combined organic layer was washed with saturated NaCl (50 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The crude was purified by column chromatography (60-120 mesh silica gel) using 22% Ethyl acetate in pet ether as eluent. The product was further washed with ethyl acetate to yield 8-bromo-3-(4- ethoxyphenyl)-2-oxo-2H-chromen-6-yl acetate (0.380 g, 34.13%) as a pale yellow solid.

Figure 3 illustrates the NMR characterization data for this compound.

Step-2:

To a stirred solution of 8-bromo-3-(4-ethoxyphenyl)-2-oxo-2H-chromen-6-yl acetate

(0.380 g, 0.942 mmol, 1 eq), in DMF (6 mL), was added K ₂ C0 ₃ (0.39 g, 2.82 mmol, 3 eq). After stirring at room temperature for 30 minutes, l-bromo-2-fluoroethane (0.358 g, 2.82 mmol, 3 eq) was added drop wise. The reaction mixture was heated to at 70° C for 30 h. Reaction progress was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of reaction, reaction mixture was cooled to room temperature, was diluted with ethyl acetate (40 ml) and thoroughly washed with water (4 x 30 mL). The combined organic layer was washed with saturated NaCl (20 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated.. The crude was purified by column chromatography (60-120 mesh silica gel) using 38% Ethyl acetate in pet ether as eluent. The product was further re-crystallized from ethyl acetate to yield 8-bromo-3-(4- ethoxyphenyl)-6-(fluoroethoxy)-2H-chromen-2-one (0.180 g, 46.92%) as an off white solid. Figure 4 illustrates the NMR characterization data for this compound. Example 3: Synthesis of precursors for radio labelling with fluorine-18.

The following example in principle also applies to the compound of Example 1.

To a stirred solution of 8-bromo-3-(4-ethoxyphenyl)-2-oxo-2H-chromen-6-yl acetate (0.380 g, 0.942 mmol, 1 eq), in DMF (6 mL), is added K ₂ C0 ₃ (0.39 g, 2.82 mmol, 3 eq). After stirring at room temperature for 30 minutes, the mixture is added drop wise to ethylene ditosylate (2.82 mmol, 3 eq). The reaction mixture is heated to at 70° C for 30 h. Reaction progress is monitored by TLC. After completion of reaction, reaction mixture is cooled to room temperature, diluted with ethyl acetate (40 ml) and thoroughly washed with water (4 x 30 mL). The combined organic layer is washed with saturated NaCl (20 mL) solution, dried over anhydrous Na ₂ S0 ₄ , filtered and

concentrated. The crude is purified by column chromatography (60-120 mesh silica gel) using ethyl acetate in pet ether as eluent.

Example 4: Radiosynthesis of f ¹⁸ F18-bromo-3-(4-ethoxyphenyl)-6-(fluoroethoxy)-2H- chromen-2-one Suitable methods are described by Jackson et al (Bioorg Med Chem Letts 2013; 23: 821-826). Fluorine- 18 radio labelling is achieved using ether method:

1) Direct labelling of the compound of Example 3 with [ ¹⁸ F] fluoride, or

2) Indirect labelling of the product compound of e.g. Example 2, step 3. In this case [ ¹⁸ F]2-fluoroethyltosylate is prepared as an intermediate labelling synthon.

These methods are also suitable for e.g. the compound of Example 1.

Example 5: In vitro testing of affinity of compounds of the invention

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.

The affinity of certain compounds of the invention for MAO- A and MAO-B was tested in the in vitro assay with the following results:

Example 6: Synthesis of 3-(2-fluoropyridin-4-yl)methxyl-2H-chromen-2-one

Step 1:

To a stirred solution of DIP A (2.4 g, 23.77 mmol, 2.2 eq) in dry THF (20 mL) was added n-BuLi [1.6M in hexane; (13.5 mL, 21.61 mmol, 2 eq) dropwise at -78°C under nitrogen. The mixture was stirred at same temperature for 30 minutes. A solution of 2- fluoro-4-methyl pyridine (1.2 g, 10.80 mmol, 1 eq) in dry THF (12 mL) was added to the previous solution at -78°C dropwise. The reaction mixture was stirred at -78°C for 2h. Diethyl carbonate (2.55 g, 21.61 mmol, 2 eq) was added dropwise at -78°C and RM was allowed stirred at ambient temperature for further 12 h. The completion of the reaction was monitored by TLC (2:8, ethyl acetate: pet ether). After completion of the reaction the RM was quenched with ice cold saturated NH ₄ C1 solution and crude was extracted with EtOAc (3 X 70 ml). The combined organic layer was dried over anhydrous sodium sulfate. The crude product obtained on concentration was purified by column chromatography (60-120 mesh silica gel, 6 % ethyl acetate in pet ether) to afford ethyl 2-(2-fluororopyridin-4-yl)acetate as colorless oil (0.68 g, 34.3%).

Step 2:

To the stirring solution of compound 2 (0.48 g, 2.62 mmol, 1 eq) in THF: Water [3:2: (7.5 mL)] was added lithium hydroxide monohydrate (0.22 g, 5.24 mmol, 2 eq) at 0°C and stirred for 16 h at ambient temperature. The completion of the reaction was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of the reaction the

Reaction mixture was concentrated under vacuum. The crude was acidified to pH ~ 2 with 1.5 N HC1 and extracted with EtOAc (3 X 100 ml). The combined organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure to afford compound 3 (0.265 g, 65.2%) as off white solid. Step 3:

To a stirred solution of 2-hydroxy-5-methyl-benzaldehyde (0.232 g, 1.70 mmol, 1 eq), was added 2-fluoropyridinyl acetic acid (0.265 g, 1.70 mmol, 1 eq), T ₃ P [50% solution in EtOAc; (2.17 g, 3.41 mmol, 2 eq) in n-BuOAc (3 mL). The reaction mixture was heated to 120°C for 16 h. Progress of the reaction was monitored by TLC (4:6, ethyl acetate: pet ether). After completion of reaction, reaction mixture was cool to room temperature, cold water (15 mL) was added and stirred at room temperature for 20 minutes. The solid appeared was filtered under suction. The solid was washed with 5% aqueous NaHC0 ₃ solution (6 x 10 mL) and then with water (6 x 10 mL) thoroughly. The solid was dried completely under suction. The yellow solid was suspended in 2 mL of EtOAc, sonicated (for 2 minutes) and filtered to afford 3-(2-fluoropyridin-4-yl)-6- methxyl-2H-chromen-2-one as pale yellow solid (0.107 g, 24.65%).

LCMS gave m.z = 256 [M+H] ⁺

Example 7: Synthesis of radio labelling precursor 3-(2-chloropyridin-4-yl)methxyl- 2H-chromen-2-one

Step 1:

To a stirred solution of DIPA (12.21 g, 120.71 mmol, 2.2 eq) in dry THF (100 mL) was added n-BuLi [1.6M in hexane; (67.5 mL, 109 mmol, 2 eq) dropwise at -78°C under nitrogen. The mixture was stirred at same temperature for 30 minutes. A solution of 2- chloro-4-methyl pyridine (7 g, 54.87 mmol, 1 eq) in dry THF (60 mL) was added to the previous solution at -78°C dropwise. The reaction mixture was stirred at -78°C for 2h. Diethyl carbonate (12.96 g, 109 mmol, 2 eq) was added dropwise at -78°C and RM was allowed stirred at ambient temperature for further 12 h. The completion of the reaction was monitored by TLC (2:8, ethyl acetate: pet ether). After completion of the reaction the RM was quenched with ice cold saturated NH ₄ C1 solution and crude was extracted with EtOAc (3 X 200 ml). The combined organic layer was dried over anhydrous sodium sulfate. The crude product obtained on concentration was purified by column chromatography (60-120 mesh silica gel, 8 % ethyl acetate in pet ether) to afford ethyl 2-(2-chloropyridin-4-yl)acetate as colorless oil (3.6 g, 32.9%).

Step 2:

To the stirring solution of compound 2 (2.5 g, 12.52 mmol, 1 eq) in THF: Water [3:2: ( 25 mL) was added lithium hydroxide monohydrate (0.6 g, 25.04 mmol, 2 eq) at 0°C and stirred for 16 h at ambient temperature. The completion of the reaction was monitored by TLC (3:7, ethyl acetate: pet ether). After completion of the reaction the Reaction mixture was concentrated under vacuum. The crude was acidified to pH ~ 2 with 1.5 N HC1 and extracted with EtOAc (3 X 100 ml). The combined organic layer was dried over anhydrous sodium sulfate and removed under reduced pressure to afford compound 3 (1.7 g, 79.1%) as pale yellow solid.

Step 3:

To a stirred solution of 2-hydroxy-5-methyl-benzaldehyde (0.476 g, 3.49 mmol, 1 eq), was added 2-fluoropyridinyl acetic acid (0.60 g, 3.49 mmol, 1 eq), T ₃ P [50%> solution in EtOAc; (2.22 g, 4.60 mmol, 2 eq) in n-BuOAc (5 mL). The reaction mixture was heated to 120°C for 16 h. Progress of the reaction was monitored by TLC (4:6, ethyl acetate: pet ether). After completion of reaction, reaction mixture was cool to room temperature, cold water (15 mL) was added and stirred at room temperature for 20 minutes. The solid appeared was filtered under suction. The solid was washed with 5% aqueous

NaHCC"3 solution (6 x 10 mL) and then with water (6 x 10 mL) thoroughly. The solid was dried completely under suction. The yellow solid was suspended in 2 mL of EtOAc, sonicated (for 2 minutes) and filtered to afford 3-(2-chloropyridin-4- yl)methxyl-2H-chromen-2-one_as pale yellow solid (0.255 g, 26.89%>). LCMS gave m.z = 272 [M+H] ⁺

Example 8: Radiosynthesis of f ¹⁸ F13-(2-fluoropyridin-4-yl)methxyl-2H-chromen-2- one

The radiosynthesis was carried out on an automated radiochemistry module. Briefly, no-carrier- added [ ¹⁸ F]fluoride was produced via ¹⁸ 0(p,n) ¹⁸ F reaction by proton irradiation of an ¹⁸ 0-enriched water target of the cyclotron. The [ ¹⁸ F]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:0.1% TFA in water (45:55); flow 6 mL/min; retention time: 19 minutes). The product peak was collected, diluted with water, trapped on a reversed- phase SPE cartridge and formulated. Analytical HPLC (254 nm; ACE5 C 18-HL 250x4.6mm column; flow = 2 mL/min; eluent is ammonium formate: acetonitrile 20%-90% gradient over 10 minutes: retention time = 6 minutes.

Example 9: In vitro autoradiosraphv of f ¹⁸ F13-(2-fluoropyridin-4-yl)methxyl-2H- chromen-2-one

[ ¹⁸ F]3-(2-f uoropyridin-4-yl)methxyl-2H-chromen-2-one was applied to whole brain sections (20 μιη thickness) from healthy Rhesus monkey. 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