WO2016097351A1

WO2016097351A1 - 18f labelled thiazolylhydrazone derivatives

Info

Publication number: WO2016097351A1
Application number: PCT/EP2015/080609
Authority: WO
Inventors: Alexander Jackson; Veronique Morisson-Iveson; Duncan George Wynn; Jinto JOSE; Chitralekha Rangaswamy
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 of Formula (I) wherein R¹ and R²are independently selected from ¹⁹F and ¹⁸F 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.

Description

18F LABELLED THIAZOLYLHYDRAZONE 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 thiazolylhydrazone derivative. A labelled thiazolylhydrazone 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 nM and selectivity for MAO-B of -100 (Bioorg Med Chem 2012; 20: 3065-3071; W. Saba et al, Synapse, 2010, 64:61-69). A number of publications have described of [¹⁸F]fluororasagiline (1 below) and of [¹⁸F]Fluorodeprenyl (2 below) as a novel PET radioligand for MAO-B (Nag et al J Label Compd Radiopharm 2011; 54: S269; WO2009/52970 A2; Nag et al Synapse 2012; 66: 323-330). [¹⁸F]fluororasagiline binds specifically to MAO-B in vitro and has a MAO-B specific binding pattern in vivo. It has an IC₅₀ of 70 nM for MAO-B (L- deprenyl = 40- 66 nM) and 950 nM for MAO- A inhibitory activity. For the latter,

[¹⁸F]Fluorodeprenyl has been shown to have relatively slow metabolism with the presence of two radio metabolite peaks with similar retention time as the labeled metabolites of [ⁿC]deprenyl.

Analogues of 3 and 4 (illustrated below) have been shown to have good affinity for MAO-B, IC₅₀ = 13 nM, and selectivity over MAO-A.

Similar compounds are taught in CN103006653A for use in treating diseases related to DHODH (dihydroorotate dehydrogenase) and in WO2014159938 A 1 for the treatment of tuberculosis.

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 provides a compound having selective binding for MAO-B of Formula I:

wherein R¹ and R² are independently selected from ¹⁹F and ¹⁸F.

In one embodiment of the compound of the invention R¹ is ¹⁸F and R² is ¹⁹F.

In one embodiment of the compound of the invention R¹ is ¹⁹F and R² is ¹⁸F.

In a second aspect the present invention provides a precursor compound of Formula II:

wherein one of R^{1 1} and R¹² is selected from Ci_₃ trialkyl ammonium, iodonium, chloro and nitro, and the other is ¹⁹F.

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 ' 'trialky lammonium' ' means the substituent NR wherein R is an alkyl. 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 "iodonium" refers to a salt of a halonium ion R-I⁺ where R is a hydrocarbon.

The term "chloro" refers to a CI substituent. The term "nitro" means the substituent -N0₂.

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 one of R¹¹ and R¹² is selected from chloro and nitro, and the other is ¹⁹F.

In one embodiment of the precursor compound of the invention R¹¹ is ¹⁹F. In one embodiment of the precursor compound of the invention R¹² is ¹⁹F.

Non-radioactive compounds of the invention can be obtained according to the method as described in Example 1. Precursor compounds of the invention are obtained in a straightforward manner by adaption of the method of Example 1, e.g. as described in Example 2. 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.).

Additional details of synthetic routes to ¹⁸F-labelled derivatives are described by Bolton (J Lab Comp Radiopharm 2002; 45: 485-528). In a third aspect the present invention provides a method for the synthesis of the compound of Formula I as defined hereinabove wherein at least one of R¹ and R² is r, wherein said method comprises reaction of the precursor compound of Formula II as defined hereinabove with [¹⁸F]fluoride. In a fourth aspect, the present invention provides a pharmaceutical formulation comprising the compound of Formula I 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 fifth 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 Formula I as defined hereinabove wherein at least one of R¹ and R² is F ;

(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. 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 sixth 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 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

Figure 1 illustrates the NMR data for (E)-4-(4-fluorophenyl)-2-(2-(l-(2-fluoropyridin- 4-yl)ethylidene)hydrazinyl)thiazole.

Brief Description of the Examples

Example 1 describes the synthesis of (E)-4-(4-fluorophenyl)-2-(2-(l-(2-fluoropyridin-4- y l)ethy lidene)hy draziny l)thiazo le .

Example 2 describes the synthesis of a fluorine- 18 radiochemistry precursor for the compound of Example 1.

Example 3 describes the radiosynthesis of (E)-4-(4-fluorophenyl)-2-(2-(l-(2- [¹⁸F]fluoropyridin-4-yl)ethylidene)hydrazinyl)thiazole.

Example 5 describes the assay used to meature in vitro inhibition of MAO- A and MAO-B.

List of Abbreviations used in the Examples

CRC: concentration response curve

DCM: dichloromethane DMF: dimethylformamide DMSO: dimethyl sulfoxide

MAO A: monoamine oxidase A

MAO B: monoamine oxidase A TLC: thin-layer chromatography Examples

Example 1: Synthesis of (E)-4-(4-fluorophenyl)-2-(2-(l-(2-fluoropyridin-4- yl)ethylidene)hydrazinyl)thiazole

Step 1 :

Pyridine, RT, 16h

Molecular Weight: 184.17

To a stirred solution of 2-fluoroisonicotinic acid (lg 7.087 mmol) in dry dichloromethane (15 ml), was added DMF (catalytic amount). Then oxalyl chloride (0.73ml 8.504mmol was added dropwise at 0° C and stirred for an hour at same temperature. Formation of the acid chloride was monitored by TLC (3:7, ethyl acetate : pet ether). Ν,Ο-dimethylhydroxyl amine hydrochloride (0.760g 7.795mmol) and pyridine (1.25ml 15.591mmol) was added at 0° C. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC (3:7, ethyl acetate : pet ether). The reaction mixture was cooled to 0° C and quenched with saturated sodium bicarbonate solution and then extracted with DCM (2 x 100 ml). The combined organic layer was washed with water (120 ml) followed by brine (80 ml) and then dried over anhydrous Na₂S0₄. The organic layer was concentrated under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 23% ethyl acetate in pet ether as eluent to yield 2-fluoro- V-methoxy- V-methylisonicotinamide (0.72g; 55.17%) as a yellow liquid.

Step 2:

Molecular Weight: 139.13

To a stirred solution of 2-fluoro-N-methoxy-N-methylisonicotinamide (0.72 g 3.913 mmol) in dry THF (15 ml), was added methylmagnesiumbromide [3M in Diethyl ether; (0.68ml 5.869mmol)] dropwise at 0° C. The reaction mixture was stirred at 0° C for 2 hours. The progress of the reaction was monitored by TLC (2:8, Ethyl acetate : pet ether). The reaction mixture was quenched with saturated ammonium chloride solution and then extracted with ethyl acetate (2 x 70 ml). The combined organic layer was washed with water (100 ml) followed by brine (60 ml) and then dried over anhydrous Na₂S0₄. The organic layer was concentrated under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 6% ethyl acetate in pet ether as eluent to yield l-(2-fluoropyridin-4-yl)ethan-l-one (0.35g; 64.3%) as a yellow liquid.

Step 3:

To a stirred solution of l-(2-fluoropyridin-4-yl)ethan-l-one (0.35g, 2.517 mmol) in dry ethanol (15 ml), was added hydrazinecarbothioamide (0.230 g 2.517 mmol) and acetic acid (catalytic amount). The reaction mixture was stirred at room temperature for 20h. The progress of the reaction was monitored by TLC (5:5: ethyl acetate: pet ether). The reaction mixture was filtered and washed with ethanol then dried under high vacuum. The white solid was stirred in water (20ml) for 20minutes then filtered and dried under vacuum to yield (£)-2-(l-(2-fluoropyridin-4-yl)ethylidene)hydrazinecarbothioamide

(0.250g; 46.8%) as a off white solid. Step 4:

To a stirred solution of (E)-2-(l-(2-fluoropyridin-4- yl)ethylidene)hydrazinecarbothioamide (0.25g, 1.179 mmol) in dry Ethanol (15 ml), was added 2-bromo- 1 -(4-fluorophenyl)ethan- 1 -one (0.256g 1.179 mmol). The reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by TLC (4:6, ethyl acetate : pet ether). The reaction mixture was filtered and washed with ethanol then dried under vacuum. The light yellow solid was suspended and stirred in a mixture of water and ethanol in the ratio 70:30 for 40minutes and filtered. The solid was again recrystallized from ethanol to yield (E)-4-(4- fluorophenyl)-2-(2-(l-(2-fluoropyridin-4-yl)ethylidene)hydrazinyl)thiazole (200mg, 51.4% ) as a yellow solid.

Figure 1 illustrates the NMR characterization data for this compound.

Example 2: Synthesis of a fluorine-18 radiochemistry precursor for the compound of Example 1.

Step 1:

To a stirred solution of 2-chloroisonicotinic acid (3g 19.04 mmol) in dry

Dichloromethane (30 ml), was added DMF (catalytic amount). Then oxalyl chloride (2ml 22.85mmol) was added drop wise at 0° C and stirred for an hour at 0° C. The formation of the acid chloride was monitored by TLC (3:7, Ethyl acetate : pet ether). Ν,Ο-hydroxyl amine hydrochloride (2.04g 20.944mmol) and pyridine (1.7ml 20.965 mmol) was added at 0° C. The reaction mixture was slowly warmed to room

temperature and stirred for 16hours. The progress of the reaction was monitored by TLC (3:7, Ethyl acetate : pet ether). The reaction mixture was cooled to 0° C and quenched with half saturated sodium bicarbonate solution and then extracted with DCM (2 x 100 ml). The combined organic layers were washed with water (120 ml) followed by brine (80 ml) and then dried over anhydrous Na₂S0₄. The organic layer was concentrated under reduced pressure. The crude was purified by column

chromatography (60-120 mesh silica gel) using 20% Ethyl acetate in pet ether as eluent to yield 2-chloro-N-methoxy-N-methylisonicotinamide (1.50g; 39.47%) as a yellow liquid.

Step 2: To a stirred solution of 2-chloro-N-methoxy-N-methylisonicotinamide (1.50 g 7.476 mmol) in dry THF (25 ml), was added methylmagnesiumbromide [3M in Diethyl ether; (3.8ml 12.4mmol)] drop wise at 0° C. The reaction mixture was stirred at 0° C for 2hours. The progress of the reaction was monitored by TLC (2:8, Ethyl acetate: pet ether). The reaction mixture was quenched with saturated ammonium chloride solution and then extracted with ethyl acetate (2 x 75 ml). The combined organic layer was washed with water (100 ml) followed by brine (50 ml) and then dried over anhydrous Na₂S0₄. The organic layer was concentrated under reduced pressure. The crude was purified by column chromatography (60-120 mesh silica gel) using 5% Ethyl acetate in pet ether as eluent to yield l-(2-chloropyridin-4-yl)ethan-l-one (850mg; 61.4%) as a yellow liquid.

Step 3:

To a stirred solution of l-(2-chloropyridin-4-yl)ethan-l-one (0.85g, 3.716 mmol) in dry Ethanol (35 ml), was added hydrazinecarbothioamide (0.339 g 3.716 mmol) and acetic acid (catalytic amount). The reaction mixture was stirred at room temperature for 20h. The progress of the reaction was monitored by TLC (5:5, Ethyl acetate: pet ether). The reaction mixture was filtered and washed with ethanol then dried under high vacuum. The white solid was stirred in water (20 ml) for 20minutes then filtered and dried under vacuum to yield (E)-2-(l-(2-chloropyridin-4-yl)ethylidene)hydrazine-l- carbothioamide (0.570g; 45.63%) as a white solid.

Step 4:

To a stirred solution of (E)-2-(l-(2-chloropyridin-4-yl)ethylidene)hydrazine-l- carbothioamide (0.570 g, 1.643 mmol) in dry Ethanol (25ml), was added 2-bromo-l-(4- fluorophenyl)ethan-l-one (0.357 g 1.643 mmol). The reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC (4:6, Ethyl acetate: pet ether). The reaction mixture was filtered and washed with ethanol then dried under vacuum. The light yellow solid was stirred in a mixture of water and ethanol in the ratio 70:30 for 40minutes and filtered. The solid was again recrystallized from ethanol to yield (£)-2-(2-(l-(2-chloropyridin-4-yl)ethylidene)hydrazinyl)-4-(4- fluorophenyl)thiazole (215mg; 24.88% ) as a yellow solid.

LCMS gave m.z = 347 [M+H]⁺

Example 3: Radiosynthesis of (E)-4-(4-fluorophenyl)-2-(2-(l-(2-f^lsF]fluoropyridin-4- yl)ethylidene)hydrazinyl)thiazole Suitable methods are described in Jackson et al (Bioorganic & Medicinal Chemistry

Letters 23 (2013) 821-826). Fluorine- 18 radio labelling is achieved using direct labelling of the compound of Example 2 with [¹⁸F]fluoride.

Example 5: In vitro inhibition ofMAO-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 folio ws-

□ 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. Compound MAO-A IC50 MAO-B ICso Selectivity index (umol) (umol)

>10 0.021 >484

CLogP: 3.56914

C lor gy line 0.001

Deprenyl 0.035

Claims

A compound having selective binding for MAO-B of Formula I

wherein R¹ and R² are independently selected from ¹⁹F and ¹⁸F.

(2) The compound as defined in Claim 1 wherein R¹ is ¹⁵F and R² is ¹⁹F. (3) The compound as defined in Claim 1 wherein R¹ is ¹⁹F and R² is ¹⁸F. (4) A precursor compound of Formula II:

wherein one of R¹¹ and R¹² is selected from Ci_₃ trialkylammonium, iodonium, chloro and nitro, and the other is ¹⁹F.

(5) The precursor compound as defined in Claim 4 wherein one of R¹¹ and R¹² is selected from chloro and nitro, and the other is ¹⁹F.

(6) The precursor compound as defined in Claim 5 wherein R¹¹ is ¹⁹F. (?) The precursor compound as defined in Claim 5 wherein R¹² is ¹⁹F. (8) A method for the synthesis of the compound as defined in Claim 1 wherein at least one of R¹ and R² is ¹⁸F wherein said method comprises reaction of the precursor compound of Formula II as defined in any one of Claims 4-7 with [¹⁸F]fluoride. (9) A pharmaceutical formulation comprising the compound as defined in any one of Claims 1-3 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 1 wherein at least one of R¹ and R² is ¹⁸F;

(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.