WO2017103257A1

WO2017103257A1 - Monoamine oxidase b binders for use in the treatment and the diagnostic of alzheimer disease

Info

Publication number: WO2017103257A1
Application number: PCT/EP2016/081704
Authority: WO
Inventors: Shozo Furumoto; Yukitsuka Kudo; Nobuyuki Okamura; Ryuichi HARADA
Original assignee: GE Healthcare Ltd; CLINO Ltd
Current assignee: GE Healthcare Ltd; CLINO Ltd
Priority date: 2015-12-18
Filing date: 2016-12-19
Publication date: 2017-06-22
Anticipated expiration: 2018-06-18
Also published as: GB201522414D0

Abstract

The present invention provides methods useful in the management neuropathological conditions where expression of monoamine oxidase B (MAO-B) deviates from that seen in healthy subjects. The invention provides for the use of compounds in therapeutic and diagnostic applications.

Description

MONOAMINE OXIDASE B BINDERS FOR USE IN THE TREATMENT AND THE

DIAGNOSTIC OF ALZHEIMER DISEASE

Technical Field of the Invention

The present invention relates to the field of neurology. Specifically, the present invention provides methods useful in the management of certain neuropathological conditions and in particular those where expression of monoamine oxidase B (MAO-B) deviates from that seen in healthy subjects.

Description of Related Art

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). A modest MAO-B increase with age has been observed in the neocortex (Saura et al. 1997 Neurobiol Aging; 18(5): 497- 507).

In contrast to MAO-B, which mainly metabolizes dopamine (DA), monoamine oxidase A (MAO-A) generally metabolizes tyramine, norepinephrine (NE), serotonin (5-HT), and dopamine (DA). Inhibitors not specific to MAO-B can pose problems when taken concomitantly with tyramine-containing foods such as cheese, because inhibition of MAO-A causes a dangerous elevation of serum tyramine levels, which can lead to hypertensive symptoms. Selective MAO-B inhibitors bypass this problem by preferentially inhibiting MAO-B. A variety of classes of compounds with MAO-B selectivity have been evaluated. Focally elevated expression of MAO-B in the cerebral neocortex has been observed with early Alzheimer's disease (AD) neurodegenerative changes. Conversely, in late stage AD neurodegeneration the neocortical expression of MAO-B decreases concurrent with increased neocortical expression with Tau (Gulyas et al., 2011

Neurochem Int; 58(1): 60). 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.

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

Chromone 6 below has been described as a scaffold for MAO inhibitors with some analogues being particularly selective for MAO-B (Gaspar et al 201 1 J Med Chem; 54: 5165; Legoabe et al 2012 Eur J Med Chem; 49: 343-5). This scaffold is similar to the flavones 7 below:

In particular, compounds having structures 8 and 9 below have been shown to have good selectivity for MAO-B over MAO-A:

8 9

Where R is typically substituted aryl and R¹ is a substituted aralkyl. Chimenti et al (Bioorg Med Chem 2010; 18: 1273-1279) describes a series of flavone compounds of structure 10 below and chromone compounds of structure 11 below (in both cases where R and R¹ are selected from H, F, CF or OCH3):

The radiolabeled compound [ⁿC]SL25.1 188 has been characterized in vivo by reversible binding, high brain uptake and very slow plasma metabolism, strongly suggesting that this radioligand is a potent tool for the in vivo study of brain MAO-B (Saba et al Synapse 2010; 64(l):61-69).

A number of publications have described of [¹⁸F]fluororasagiline (12 below) and of [¹⁸F]Fluorodeprenyl (13 below) as a novel PET radioligand for MAO-B (Nag et al J Label Compd Radiopharm 201 1 ; 54: S269; Nag et al Bioorg Med Chem 2012; 20: 3065-3071 ; 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 14 and 15 (illustrated below) have been shown to have good affinity for MAO-B, ICso = 13 nM, and selectivity over MAO-A.

Evaluation of MAO-B in conjunction with other biological markers may facilitate differentiation between age related changes and early and late stage AD related neurodegenerative changes.

Summary of the Invention

In one aspect the present invention provides a compound of Formula I:

wherein:

A¹ is NH or O;

A²-A³ is CH-CH, CH-N or N-CH;

R¹ is C 1-3 alkylene or C 1-3 hydroxyalkylene;

X is hydrogen, halogen, OH, O-C1-3 alkyl, or NR2R3 where R² and R³ are independently hydrogen or C 1-3 alkyl; for use in a method of targeting monoamine oxidase B (MAO-B) expression in a subject. In another aspect the present invention provides a method of targeting MAO-B expression in a subject wherein said method comprises administration of a compound of Formula I.

In a further aspect the present invention provides a method for detection of early stage Alzheimer's disease (AD) comprising:

(a) administering an in vivo imaging agent to a subject wherein said in vivo imaging agent comprises a compound of Formula I and wherein said subject has suspected early stage Braak stage II/III AD;

(b) carrying out in vivo imaging on said subject following said administering step wherein signals emitted by said in vivo imaging agent are detected from the subject or parts of the subject into which the in vivo imaging agent has distributed;

(c) generating images from the detected signals.

The compounds defined herein are known to be binders of tau (EP 2634177 Al) and the present inventors have shown that they also have affinity for MAO-B. This combined affinity is proposed to facilitate differentiation between age related changes and early and late stage AD.

Furthermore it is expected that binding of compounds of Formula I described herein to MAO-B may contribute to the observed uptake in the mesial temporal region in early stages of Alzheimer's Disease (AD) and boost the signal from binding to tau thereby enabling early detection and potentially staging of early disease e.g. Braak & Braak stages II/III.

Brief Description of the Figures

Figure 1 shows the results of in vitro autoradiography of ³H-THK5351 binding to putamen tissue slices fixed with 70% ethanol. Inhibition of ³H-THK5351 binding to the slices was assessed by incubating with a variety of agents.

Figure 2 graphs the % signal observed from each of the autoradiography slices from Figure 1.

Figure 3 shows the results of in vitro autoradiography of ³H-THK5351 binding to putamen tissue slices without fixation. Inhibition of ³H-THK5351 binding to the slices was assessed by incubating with a variety of agents.

Figure 4 graphs the % signal observed from each of the autoradiography slices from Figure 3.

Detailed Description of the Preferred Embodiments

The invention relates to a compound of Formula I for use in a method of targeting MAO-B expression in a subject. In the alternative, the present invention can be understood to relate to a method of targeting MAO-B expression in a subject wherein said method comprises administration of a compound of Formula I as defined herein. The definitions and embodiments defined hereunder apply equally to each aspect of the invention.

To more clearly and concisely describe and point out the subject matter of the claimed invention, definitions are provided herein below for specific terms used throughout the present specification and claims. Any exemplification of specific terms herein should be considered as a non-limiting example. 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.

The term "alkylene" refers to the bivalent group -(CH₂)_n- wherein n is preferably an integer from 1-4.

The term ' 'hy droxyalky lene" refers to an alkylene group as defined herein comprising a hydroxyl substituent wherein the term "hydroxy 1" refers to the group -OH. The term "alkyl", alone or in combination, means a straight-chain or branched-chain alkyl radical having the general formula C_nH_2n+i . Examples of such radicals include methyl, ethyl, and isopropyl.

The term "targeting" as used herein is intended to encompass any diagnostic or therapeutic method wherein a compound of Formula I interacts with MAO-B in a subject or a tissue sample taken from said subject. The "subject" of the invention can be any human or animal subject. In one embodiment the subject of the invention is a mammal. In one embodiment said subject is an intact mammalian body in vivo. In another embodiment, the subject of the invention is a human. In one embodiment A¹ of Formula I is NH.

In one embodiment A¹ of Formula I is O.

In one embodiment A²-A³ of Formula I is CH-CH.

In one embodiment A²-A³ of Formula I is CH-N.

In one embodiment A²-A³ of Formula I is N-CH. In one embodiment R¹ of Formula I is C_1-3 alkylene.

In one embodiment R¹ of Formula I is C_1-3 hydroxy alkylene.

In one embodiment R² of Formula I is hydrogen.

In one embodiment R² of Formula I is methyl.

In one embodiment R² of Formula I is ethyl. In one embodiment R³ of Formula I is hydrogen.

In one embodiment R² of Formula I is methyl.

In one embodiment R² of Formula I is ethyl.

The compound of Formula I may be a racemic mixture or an enantiomerically-enriched mixture, or the racemic mixture may be separated using well-known techniques to obtain an individual enantiomer.

In one embodiment said compound of Formula I is:

In one embodiment the compound of Formula I as defined herein is for use in a method of treatment. In one embodiment the compound of Formula I as defined herein is for use in a method of treatment comprising administration of a therapeutically effective amount of said compound to said subject. The term "therapeutically effective amount" means an amount of a compound of Formula I as defined herein that produces a therapeutic response or desired effect. In one embodiment the therapeutic response or desired effect is produced in a subject having a MAO-B condition. The term "MAO-B condition" refers to any condition is which MAO-B expression is abnormal and is linked to a pathophysiological effect. Non-limiting examples of MAO-B conditions include neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease (AD). In one embodiment of the invention the MAO-B condition is AD.

In one embodiment the compound of Formula I as defined herein is for use in a method of diagnosis. In one embodiment the compound of Formula I as defined herein is for use in a method of diagnosis which is a method of in vitro diagnosis. In one embodiment the compound of Formula I as defined herein is for use in a method of diagnosis which is a method of in vivo diagnosis. The method of diagnosis may be used to study MAO-B expression in subjects known or suspected to have a pathological condition associated with abnormal expression of MAO-B (a MAO-B condition as defined herein).

Diagnostic methods and strategies for using compounds that target biological markers in diagnostic methods are well known to those of skill in the art.

Compounds of Formula I can be used, for example, for detecting and quantifying MAO-B with or without labeling by contacting with sample specimens in vitro. For example, the compounds of Formula I can be used for staining MAO-B in microscopic specimens, for colorimetric determination of MAO-B in samples, or for quantifying MAO-B using a scintillation counter. Preparation of a microscope specimen and staining using the compound of Formula I can be carried out by a conventional method known to a person with ordinary skill in the art.

For use in a therapeutic method or an in vivo diagnostic method the compound of Formula I is provided in one embodiment as a pharmaceutical composition. Forms of such pharmaceutical compositions are not limited in particular, but in one embodiment said composition is a liquid composition, in another embodiment said composition is suitable for injection. A composition for injection can either be infused directly into the brain, or alternatively the pharmaceutical composition can be formulated for intravenous injection or drip and administered, since the compounds of Formula I have high permeability through the blood-brain barrier. Such liquid compositions can be prepared by methods well known in the art. Solutions can be prepared, for example, by dissolving the compound of Formula I in an appropriate carrier, water for injection, physiological saline, Ringer's solution or the like, sterilizing the solution through a filter or the like, and then filling the sterilized solution into appropriate containers, for example, vials or ampules. Solutions also can be lyophilized and when used, reconstituted with an appropriate carrier. Suspensions can be prepared, for example, by sterilizing the compound of Formula I, for example, by exposure to ethylene oxide, and then suspending it in a sterilized liquid carrier.

When such a pharmaceutical composition is used in a liquid formulation, particularly a formulation for injection, an injection can be prepared by adding a solubilizing agent to a compound of Formula I according to the present invention.

In one embodiment the solubilizing agent is selected from nonionic surfactants, cationic surfactants, amphoteric surfactants and the like used in the art. Non-limiting examples of these solubilizing agents include Polysorbate 80, polyethylene glycol, ethanol or propylene glycol is preferable, and Polysorbate 80 is more preferable.

The amount of the compound of Formula I to be administered to a subject in a method of treatment varies depending on the condition, gender, age, weight of the patient and the like, and is generally within a range from 0.1 mg to 1 g, preferably from 1 mg to 100 mg, per day for adult humans weighing 70 kg. It is possible to conduct a treatment with such a dose for a specified period of time, followed by increasing or reducing the dose according to the outcome. The compound of Formula I comprises in one embodiment a radioactive label. In one embodiment the radioactive label is suitable for detection in a positron emission tomography (PET) imaging procedure. Suitable such radioactive labels include ⁿC, ¹³N, ¹⁵0 and ¹⁸F. In one embodiment of the compound of Formula I as defined herein said fluoro substituent is ¹⁸F and said compound is use in a method of in vivo diagnosis is PET imaging.

In a particular aspect, the present invention relates to a method for detection of early stage Alzheimer's disease (AD) wherein the compound of Formula I comprises a radioactive label and is provided as a pharmaceutical composition for administering to a subject. ' 'Administering' ' the compound of Formula I is in one embodiment carried out parenterally, and in another embodiment intravenously. The intravenous route represents an efficient way to deliver the compound of Formula I throughout the body of the subject and therefore into contact with MAO-B expressed in said subject.

Furthermore, intravenous administration does not represent a substantial physical intervention or a substantial health risk. The compound of Formula I of the invention is in one embodiment administered as the pharmaceutical composition, as defined herein. The method for detection of the invention can also be understood as comprising steps (b)-(c) carried out on a subject to whom an in vivo imaging agent has been pre- administered. Following the administering step and preceding the detecting step, the compound of Formula I binds to MAO-B. When the subject is an intact mammal, the compound of Formula I will dynamically move through the mammal's body, coming into contact with various tissues therein. A point in time is reached when detection of compound of Formula I specifically bound to MAO-B is enabled as a result of the ratio between compound of Formula I bound to tissue with MAO-B versus that bound in tissue without, or with significantly less MAO-B.

The "detecting" step involves detection of signals emitted by the compound of Formula I when it includes a radioactive label by means of a detector sensitive to said signals. This detection step can also be understood as the acquisition of signal data. The "generating" step is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate images showing the location and/or amount of signals emitted by said radioactive label. The signals emitted directly correlate with the expression of MAO-B such that evaluation of the image generated enables diagnostic and/or prognostic decisions to be taken. EP 2634177 Al describes 2,6-substituted quinolone derivatives highly specific to tau. The compounds are reported to have high brain transition, low or undetected boneseeking properties and low or undetected toxicity. EP 2634177 Al reports that the compounds can be used in the diagnosis, the treatment and/or prevention of a tauopathy, particularly Alzheimer's disease. Compounds of Formula I as defined herein may be obtained by methods described in EP 2634177 A 1. Briefly, said methods may comprise reacting a compound of Formula (la):

in which A¹ is as defined herein for Formula I, R⁴ represents R^!-F where R¹ is as defined for Formula I or R⁴ represents hydrogen wherein when A^J-R⁴ is hydroxyl it may optionally be protected, and R⁵ represents NH₂ or N0₂, with a compound of the Formula (lb):

in which A², A³, R² and R³ are as defined for Formula I; and, optionally converting the product of Formulas la and lb to obtain the compound of Formula I.

The term "protected" as used herein is takes its ordinary meaning in the art, which is to say that a protecting group is introduced by chemical modification of a functional group to direct chemoselectivity in a subsequent chemical reaction. The use of protecting groups and examples of protecting groups suitable for protecting hydroxyl are described in 'Protective Groups in Organic Synthesis', Theorodora W. Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley & Sons, 2007).

The present inventors observed high uptake of the compounds [¹⁸F]THK-5117 and [¹⁸F]THK-5351 in the basal ganglia in healthy controls, mild cognitive impairment

(MCI) and AD. Tau deposits are not expected in the basal ganglia except in very severe AD and this observation indicated off-target binding which was confirmed by the present inventors to be affinity for MAO-B.

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.

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.

Brief Description of the Examples

Example 1 shows in vitro competitive autoradiography of ³H-THK 5351 to confirm targets in normal human putamen brain slices. Example 2 shows the binding affinity of THK 5351 to MAO enzymes and human putamen homogenate. Example 3 shows MAO enzymes inhibition activity of compounds of Formula I. Example 4 describes a method to assess the binding affinity of compounds of Formula I to MAO-B enzyme.

List of Abbreviations used in the Examples

BSA bovine serum albumin

HTS high-throughput screening MAO monoamine oxidase

MAO-A monoamine oxidase A

MAO-B monoamine oxidase B

PBS phosphate-buffered saline

Examples Example 1

Confirmation of Compounds of Formula I Binding to MAO-B in Human Putamen Brain Slices by In Vitro Competitive Autoradiography

Method

Normal human brain tissue (83/M) was obtained from Tohoku University Brain Bank. Twenty μιη thick brain slices were generated with a cryostat (Microm HM560; Thermo Scientific, Waltham, MA) at -20°C chamber temperature and -15°C object temperature. Sections were transferred to MAS coated glass slides (Matsunami glass ind.,ltd, Osaka, Japan). After drying, the sections were stored at -80°C. A part of the sections was fixed with ice-cold 70% ethanol, rinsed with PBS, and then used for the following process. Both fixed and non- fixed sections were pre-incubated in PBS, 1%BSA for 30 min. The sections were incubated with ³H-THK 5351 (3 nM) as a radioligand and the respective cold compounds (10 μΜ) in PBS, 1%BSA at room temperature for 30 min. Then, it was immersed in PBS, 1%BSA for 5 min and further in PBS for 5 min twice. After drying the sections at room temperature, the sections were exposed to phosphorimaging plates (BAS-TR2040; GE Healthcare, Little Chalfont, UK) for 3 days. The images were obtained using a FLA-7000 phosphorimaging instrument (GE Healthcare) with a spatial resolution of 25x25 μιη. The % signal in the region of interest (ROI) was quantified with the ImageQuant TL software (GE Healthcare).

Results

Figure 1 and 3 show autoradiograms of the brain slices with or without ethanol- fixation, respectively. The slices were incubated with ³H-THK 5351 in the presence of various unlabeled compounds. ³H-THK 5351 bound to human putamen brain slice and excess unlabeled THK 5351 blocked the binding. Figure 2 and 4 graph the % signal observed from each of the autoradiography slices from Figure 1 and 3, respectively. These graphs clearly indicate that the binding to human putamen brain slice was also displaced in the presence of MAO-B inhibitors comparable to unlabeled THK 5351 , demonstrating that THK 5351 binds to MAO-B in human putamen.

Example 2 Confirmation of Binding Affinity of Compounds Formula I to MAO Enzymes

Method

MAO enzymes (MAO- A: M7316, MAO-B: M7441) were obtained from Sigma- Aldrich (St. Louis, MO). MAO-A, MAO-B membrane fraction (0.5 μg), or human putamen homogenate from normal brain (100 μg) were incubated with various concentrations of ³H-THK 5351 (0.05-50 nM). To account for nonspecific binding of the ³H-THK 5351 , the above-mentioned reactions were performed in the presence of unlabeled THK 5351. The binding reactions were incubated for 2 hours at room temperature in 200 μ L of assay buffer (Dulbecco's PBS, 0.1% BSA). Separation of bound from free radioactivity was achieved by filtration under reduced pressure (MultiScreen HTS Vacuum Manifold, MultiScreen HTS 96-well 0.65 μιη filtration plate; Millipore, Billerica, MA). The filers were washed three times with 200 assay buffer, and the filters were incubated in 2 mL of scintillation fluid (Emulsifier-Safe; Perkin Elemer, Boston, MA), and the radioactivity was counted using a beta counter (LS6500 liquid scintillation counter; Beckman Coulter, Brea, CA). Kd values were calculated using GraphPad Prism Version 5.0 (GraphPad Software, SanDiego, CA).

Results

The results are shown in TABLE 1. THK 5351 bound to MAO-B with high affinity comparable to human putamen homogenate from normal brain, while it showed lower binding affinity for MAO-A. These results suggest that THK 5351 selectively binds to MAO-B over MAO-A.

TABLE 1

Example 3

Confirmation of MAO Enzymes Inhibition Activity of Compounds Formula I

Method The inhibition assay used to determine IC50 values for the compounds against human MAO-A and MAO-B enzyme activity was based on the standard protocol provided in the product insert for Amplex Red Monoamine Oxidase Assay Kit from Life

Technologies (Invitrogen Cat # A12214). MAO-A and MAO-B enzymes were sourced from Sigma- Aldrich (Cat # M7316 and M7441, respectively). Substrates included in the assay kit were Tyramine for MAO-A/B and Benzylamine for MAO-B. Reference compounds included in the assay were Deprenyl (Irreversible binder) and Lazabemide hydrochloride (Reversible binder) for MAO-B and Clorgyline for MAO-A.

The assay was performed using a 96 well plate. Using an assay volume of 200 μΐ,, Amplex red concentration of 200 μΜ and a MAO-A enzyme concentration of 2 μg/mL or a MAO-B enzyme concentration of 5 μg/mL, recombinant enzyme was incubated with substrates/reference or test compounds for 60 min. A 10 point concentration response curve was carried out in duplicate using a test concentration range of 0.0005 - 10 μΜ. Fluorescence was measured (Ex/Em 560nm/590nm) using an Envision (Perkin Elmer) reader. Data analysis was carried out using XLFit or GraphPad Prism software. The concentrations required for 50% inhibition of MAO-A & MAO-B (IC50) was determined by fitting data to a sigmoidal dose-response curve using nonlinear regression analysis (GraphPad Software Inc.).

Results

The results are shown in TABLE 2. In a MAO-B enzyme inhibition assay the above compounds of Formula I showed IC50 values comparable to MAO-B binding reference compounds Deprenyl and Lazabemide and demonstrated selectivity over MAO-A.

TABLE 2

Example 4

Confirmation of Compounds of Formula I Binding Affinity I to MAO-B Enzyme

Method A binding inhibition assay was used to determine Ki values for the compounds against human MAO-B enzyme. MAO-B membrane fraction (0.5 was incubated with various concentrations of cold test compounds (0.1 nM - 10,000 nM) and ³H-THK 5351 (1 nM). The binding reactions were incubated for 2 hours at room temperature in 200 μΐ, of assay buffer (Dulbecco's PBS, 0.1% BSA). Separation of bound from free radioactivity was achieved by filtration under reduced pressure. The filters were washed three times with 200

assay buffer, and the filters were incubated in 2 mL of scintillation fluid, and the radioactivity was counted using a beta counter. Ki values were calculated using GraphPad Prism Version 5.0 (GraphPad Software, SanDiego, CA). Results The results are shown in TABLE 3. Competitive binding assay showed Ki values comparable to MAO-B binding reference compounds Lazabemide and Rasagyline.

TABLE 3.

Rasagyline 3.4 (MAO-B Reference)

Claims

( 1 ) A compound of Formula I

wherein:

A¹ is NH or O;

A²-A³ is CH-CH, CH-N or N-CH;

R¹ is C 1-3 alkylene or C 1-3 hydroxyalkylene;

X is hydrogen, halogen, OH, O-C1-3 alkyl, or NR2R3 where R² and R³ are independently hydrogen or C 1-3 alkyl; for use in a method of targeting monoamine oxidase B (MAO-B) expression in a subject.

(2) The compound as defined in Claim 1 wherein A¹ is NH.

(3) The compound as defined in Claim 1 wherein A¹ is O.

(4) The compound as defined in any one of Claims 1-3 wherein A²- A³ is CH-N.

(5) The compound as defined in any one of Claims 1-3 wherein A²- A³ is N-CH.

(6) The compound as defined in any one of Claims 1-5 wherein R¹ is C_1-3 alkylene.

(7) The compound as defined in any one of Claims 1-5 wherein R¹ is Ci_ hy droxy alky lene .

(8) The compound as defined in any one of Claims 1 therein R² is hydrogen.

(9) The compound as defined in any one of Claims 1

(10) The compound as defined in any one of Claims 1

(11) The compound as defined in any one of Claims 1

(12) The compound as defined in any one of Claims 1

(13) The compound as defined in any one of Claims 1 The compound as defined in Claim 1 wherein said compound of Formula I

-24-

(15) The compound as defined in any one of Claims 1-15 wherein said method of targeting is a method of treatment. (16) The compound as defined in Claim 16 wherein said method of treatment comprises administration of a therapeutically effective amount of said compound to said subject.

(17) The compound as defined in any one of Claims 1-15 wherein said method of targeting is a method of diagnosis. (18) The compound as defined in Claim 18 wherein said method of diagnosis is a method of in vitro diagnosis.

(19) The compound as defined in Claim 18 wherein said method of diagnosis is a method of in vivo diagnosis.

(20) The compound as defined in Claim 20 wherein said fluoro substituent of Formula I is ¹⁸F and said method of in vivo diagnosis is positron emission tomography (PET).

(21) A method of targeting MAO-B expression in a subject wherein said method comprises administration of a compound of Formula I as defined in any one of Claims 1- 15.

(22) The method as defined in Claim 22 wherein said method of targeting is a method of treatment.

(23) The method as defined in Claim 23 wherein said method of treatment comprises administration of a therapeutically effective amount of said compound to said subject generally within a range from 0.1 mg to 1 g, preferably from 1 mg to 100 mg, per day for adult humans weighing 70 kg. (24) The method as defined in Claim 22 wherein said method of targeting is a method of diagnosis.

(25) The method as defined in Claim 25 wherein said method of diagnosis is a method of in vitro diagnosis. (26) The method as defined in Claim 25 wherein said method of diagnosis is a method of in vivo diagnosis.

(27) The method as defined in Claim 27 wherein said compound of Formula I is as defined in Claim 21 and said method of in vivo diagnosis is positron emission tomography (PET). (28) A method for detection of early stage Alzheimer's disease (AD) comprising:

(a) administering an in vivo imaging agent to a subject wherein said in vivo imaging agent comprises a compound of Formula I as defined in Claim 21 and wherein said subject has suspected early stage Braak stage II/III AD;

(c) generating images from the detected signals.