LABELLED COUMARIN DERIVATIVES
Technical Field of the Invention
The present invention concerns compounds that target monoamine oxidase B (MAOB).
Specifically, the compound of the invention is a coumarin derivative. A labelled
5 coumarin derivative is also provided having use as an in vivo imaging agent for MAOB.
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
10 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
15 such as astrocytes and radial glia (Westlund et al. (1988) Neiiroscience 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.
20 (1987)AdvNeurol45: 111-118; Gerlachetal. (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 ct al. (1996) Neurology 46: 1262-1269). Activated MAO-B has
also been demosntrated in the brain of Alzheimer patients (Gulyas ct al. Neurochem Int.
25 2011; 58(1): 60-8).
A number of radiolabeled compounds have been reported in literature such as [ C]
i a
SL25.1188 and [ FjFIuororasagiline 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 nMand selectivity for MAO-B of-100 (Nag et al Bioorg Med Chem
30 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
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PZ1477 IN Priority (spec DRAFT)
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).
5 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:
10 5053-5055).
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
15 illustrated below as compounds 3-5.
O^
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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
5 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.
10 Detailed Description of the Preferred Embodiments
In a first aspect the present invention relates to a compound of Formula I:
wherein:
R1 is C1-3 alkoxy or -0-(CH2)n-F wherein n is an integer from 1 to 3;
15 R2 is -Ar'-R4 wherein Ar1 is an aryl and R4 is selected from fluoro, C1-3 alkoxy,
C1-3 fluoroalkyl and C1-3 fluoroalkoxy, with the proviso that when Ar1 is phenyl
R4 is not C1-3 alkoxy;
R3 ishaloor C1-3 alkyl.
Unless otherwise specified, the term "alkyl" alone or in combination, means a straight-
20 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 C2-6 molecular fragment, preferably having
5 to 6 ring members the ring. The term "aryl" is intended to encompass also heteroaryl
25 groups, defined as an aryl including at least one N, 0 or S atom ring member.
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The term "halogen" or "halo" means a substituent selected from fluorine, chlorine,
bromine or iodine.
"Fluoroaikyi" and "fluoroalkoxy" are alkyl and alkoxy groups, respectively, as defined
above substituted with one or more fluorine atoms in place of hydrogen atoms.
5 Suitably in the case of fluoroaikyi 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 -(CH2)Z- wherein z is 1-3, and the term
"alkoxylene" refers to an alkylene group comprising an ether linkage, wherein an ether
linkage is as defined above.
10 In one embodiment of the compound of the invention R is -0-(CH2)n-F wherein n is as
defined in Claim 1.
In one embodiment of the compound of the invention n is 2.
In one embodiment of the compound of the invention Ar1 is phenyl.
In one embodiment of the compound of the invention Ar is pyridyl.
15 In one embodiment of the compound of the invention Ar1 is triazole.
In one embodiment ofthe compound of the invention R4 is C1-3 alkoxy.
In one embodiment ofthe compound ofthe invention R4 is methoxy.
In one embodiment ofthe compound ofthe invention R4 is ethoxy.
In one embodiment ofthe compound ofthe invention R is C 1.3 fluoroaikyi.
20 In one embodiment ofthe compound ofthe invention R3 is halo.
In one embodiment ofthe compound ofthe invention R is bromo.
In one embodiment the compound ofthe invention comprises a radioactive isotope.
In one embodiment said radioactive isotope can be detected externally in a non-invasive
manner following administration in vivo. Examples of suitable such radioactive
25 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
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1-11 111 *7"7
is a gamma-emitting radioactive halogen selected from '"I, '""I and "Br. In one
embodiment said radioactive isotope is a positron-emitting radioactive non-metal
selected from nC, ,3N, ,8F and 124I.
Precursor compounds and methods of introducing radioactive isotopes into organic
5 molecules arc 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:
10 935-938) provide detail for methods of radioiodination and radiobromination.
Additional details of synthetic routes to 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:
18p
15
In one embodiment of the compound of the invention R4 comprises l8F.
In one embodiment the compound of the invention is selected from:
-5-
In a second aspect the present invention provides a precursor compound useful in the
5 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:
R11 is C1-3 alkoxy or -0-(CH2)m-R15 wherein m is an integer from 1 to 3 and R15
10 is fluoro or said precursor group;
R12 is -Ar2-R14 wherein Ar2 is an aryl and R14 is selected from fluoro, Cj.3
alkoxy C1-3 fluoroalkyl and C\.3 fluoroalkoxy or is said precursor group, with
the proviso that when Ar2 is phenyl R14 is not C1-3 alkoxy;
R13 is halo or C1-3 alkyl, or is said precursor group;
15 and wherein:
when either R13 or R14 is said precursor group it is selected from CM
trialkylammonium or iodonium; and,
when R15 is said precursor group it is a leaving group.
A "precursor compound" comprises a non-radioactive derivative of a radiolabelled
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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
5 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).
10 In one embodiment of the precursor compound of the invention R is said precursor
group.
In one embodiment of the precursor compound of the invention R is said precursor
group.
In one embodiment of the precursor compound of the invention said precursor group is
15 Ci.3trialkyltin.
In one embodiment of the precursor compound of the invention R15 is said precursor
group.
In one embodiment of the precursor compound of the invention R15 is selected from
tosylate (OTs), mesylate (OMs), and triflate (OTf).
20 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):
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X = C, N
Y = H, OCH3, CH2CH2F, F, N02, Me, Et, NHCH2F, NHCH2CH2F
Reagents and Conditions: a. DCC, DMSO, 110 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:
Scheme 2
In a third aspect the present invention provides a method for synthesis of a compound
of the invention as defined hereinabove wherein said method comprises reaction of the
precursor compound of the invention as defined hereinabove with a suitable source of
10 the radioactive isotope as defined hereinabove.
In a fourth aspect the present invention provides a method for synthesis of a compound
of the invention as defined hereinabove wherein said method comprises reaction of a
PZ1477 IN Priority (spec DRAFT)
compound of Formula III:
wherein:
either R is hydroxyl or R is ethynyl, otherwise R and R are as
respectively defined for R and R hereinabove;
with a compound of Formula IVa when R is hydroxyl:
18c LG K IVa
wherein LG is a leaving group and x is an integer of 1-3;
or with a compound of Formula IVb when R is ethynyl:
10 ^V 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.
15 A "pharmaceutical formulation" is a composition comprising the compound of the
invention, together with a biocompatible carrier in a form suitable for mammalian
administration. Jn cevta'm 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 lo the mammalian
20 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
25 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. polyethyleneglycols,
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PZH77 IN Priority (spec DRAFT)
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
5 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:
10 (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.
15 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
20 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
25 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, nonlimiting
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,
30 during and after treatment with a drug to combat a MAO-B condition. In this way, the
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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.
5 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.
10 In a yet farther 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
15 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
20 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.
25 Brief Description of the Figures
Figures 1-4 present NMR spectra for various compounds that have been synthesised as
described in the examples.
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10
15
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 radiolabelling with
fluorine-18.
Example 4 describes the synthesis of [ F]-8-bromo-3-(4-ethoxyphenyl)-6-
(iiuoroethoxy)-2H-chromen-2-one.
Example 5 describes in vitro testing of affinity of compounds of the invention.
List of Abbreviations used in the Examples
20
AcOH:
DCM:
DMF:
EtOAc:
MAO:
NaOAc:
NMR:
TEA:
TLC:
acetic acid
dichloromethane
dimethylformamide
ethyl acetate
monoamine oxidase
sodium acetate
nuclear magnetic resonance
triethanolamine
thin-layer chromatography
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Examples
Example 1: 8-bromo-3-(4-methoxvphenvl)~6-(fIttoroethoxy)-2H-chromen-2-one
Step 1:
5 To a stirred solution of 2-hydroxy-5-methoxy-benzaldehyde (14g, 92 mmol) in glacial
AcOH (400 ml) was added Br2 (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.
10 The cmde 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
NaC! (200 mL) solution, dried over anhydrous Na2SC>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-
15 benzaldehyde (17 g, 79.97%) as a orange solid.
Figure 1 illustrates the NMR characterization data for this compound.
Step-2:
20 To a stirred suspension of 3-bromo-2-hydroxy-5-methoxy-benzaldehyde (4g, 17.31
mmol) in dry DCM (40 ml) was added BBr3 [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
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P7.1477 IN Priority (spec DRAhT)
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 Na2SO.j, filtered and concentrated. The crude was
purified by column chromatography (60-120 mesh silica gel) using 14% Ethyl acetate
5 in pet ether as eluent to yield pure 3-bromo-2,5-dihydroxy bcnzaldehyde (2.8 g,
69.2%) as a yellow solid.
Step-3:
To a stirred solution of 3-bromo-2,5-dihydroxy benzaldehyde (0.3 g, 1.38 mmol, 1 eq)
10 and 2-(4-methoxyphenyI)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
15 with saturated NaCl (50 mL) solution, dried over anhydrous Na2S04, 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-2/f-chromen-6-yl
acetate (0.19 g, 35.37%) as a pale yellow solid.
20 Step-4:
P
To a stirred solution of 8-bromo-3-(4-methoxyphenyl)-2-oxo-2//-chromen-6-yl acetate
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(0.190 g, 0.488 mmol, 1 eq), in DMF (6 mL), was added K2C03 (0.202 g, 1.46 mmol, 3
eq). After stirring at room temperature for 30 minutes, 1 -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
5 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 Na2S04, filtered and concentrated. The crude was purified by column
chromatography (60-120 mesh silica gel) using 30% Ethyl acetate in pet ether as eluent.
10 The product was further re-crystallized from ethyl acetate to yield 8-bromo-3-(4-
methoxyphcnyI)-6-(fluorGethoxy)-2Z/-chromeii-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 of8-bromo-3-(4-ethoxvvhenvl)-6-(fluoroethoxy)-2H-chromen~
15 2-Q/i e
Step-1:
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,
20 2.76 mmol, 1 eq) in acetic anliydride (3 mL), was added TEA (2 mL). The reaction
mixture was heated to at 120° C for 12 h. Reaction progress was monitored b}' 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
25 anhydrous Na2SCj, 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-
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PZI477 IN Priority (spec DRAFT)
ethoxyphenyl)-2-oxo-2//-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-2i/-chromen-6-yl acetate
(0.380 g, 0.942 mmol, 1 eq), in DMF (6 mL), was added K2C03 (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
10 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 (4x30 mL). The
combined organic layer was washed with saturated NaCl (20 mL) solution, dried over
anhydrous Na2S04, filtered and concentrated.. The crude was purified by column
15 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.
P7.1477 IN Priority (spec DRAFT)
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-2#-chromen-6-yl acetate
(0.380 g, 0.942 mmol, 1 eq), in DMF (6 mL), is added K2C03 (0.39 g, 2.82 mmol, 3
eq). After stirring at room temperature for 30 minutes, the mixture is added drop wise to
5 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 Na2SC>4, filtered and
10 concentrated. The cmde is purified by column chromatography (60-120 mesh silica gel)
using ethyl acetate in pet ether as eluent.
to
Example 4: Radiosynthesis off F18-bromo-3-(4-ethoxwhenyl)-6-(fluoroethoxv)-2Hchromen-
2'One
Suitable methods are described by Jackson et a! (Bioorg Med Chem Letts 2013; 23:
15 821 826). Fluorine-18 radiolabelling is achieved using ether method:
1 ft
1) Direct labelling of the compound of Example 3 with [ F] fluoride, or
2) Indirect labelling of the product compound of e.g. Example 2, step 3. In this
1 Q
case [ F]2-fluoroethyltosylate is prepared as an intermediate labelling synthon.
These methods are also suitable for e.g. the compound of Example 1.
20 Example 5: lit 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 Teclinologies.
General parameters for screening of the compounds was as follows:
O Assay Type: Amplex Red Monoamine Oxidase Assay (Invitrogen Cat # A12214)
25 n Assay Format: 96 well plate (Black, Corning)
El Assay Volume: 200uL
• Enzyme Source: MAO A and MAO B enzymes (Sigma-Aldrich Cat # M7316 and
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FZ1477 IN Priority (spec DRAFT)
M7441)
D Substrate : For MAO-A/B : Tyramine, For MAO-B : Benzylamine
fl Reference Compounds: For MAO-A: Clorgyline (Irreversible), Moclobemide
(Reversible), for MAO-B: Pargyline (Irreversible), Deprenyl (Irreversible), Lazabemide
5 hydrochloride (Reversible).
• Screening Format: Concentration Response Curve (CRC) for all compounds
• Replicates: Duplicates (n=2)
U Number of Runs: One (N=l)
G Controls:
10 D Negative control/"MIN": 1% DMSO Buffer without enzyme (substrate alone)
D Positive control/ "Max": 1% DMSO Buffer with enzyme and substrate
D Reading: End point or Kinetic
• Detection: Fluorescence Ex/Em - 560nm/590nm
D Detection Instrument: Envision [Perkin Elmer]
15 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:
PZ1477 IN Priority (spec DRAFT)

Claims
(1) A compound of Formula I:
wherein:
5 R1 is Ci_3 alkoxy or -0-(CH2)n-F wherein n is an integer from 1 to 3;
R2 is —Ar*-R4 wherein Ar1 is an aryl and R is selected from fluoro, C\.j alkoxy,
Ci-3 fluoroalkyl and C1.3 fluoroalkoxy, with the proviso that when Ar1 is phenyl
R is not C1-3 alkoxy;
-j
R is halo or C1-3 alkyl.
10 (2) The compound as defined in Claim 1 wherein R1 is -0-(CH2)n-F wherein n is as
defined in Claim 1.
(3) The compound as defined in Claim 2 wherein n is 2.
(4) The compound as defmed in any one of Claims 1-3 wherein Ar is phenyl.
(5) The compound as defined in any one of Claims 1-3 wherein Ar1 is pyridyl.
15 (6) The compound as defined in any one of Claims 1-3 wherein Ar is triazole.
(7) The compound as defined in any one of Claims 1-6 wherein R4 is C1-3 alkoxy.
(8) The compound as defined in Claim 7 wherein R4 is mcthoxy.
(9) The compound as defined in Claim 7 wherein R4 is ethoxy.
(10) The compound as defined in any one of Claims 1-6 wherein R'1 is C1-3
20 fluoroalkyl.
(11) The compound as defmed in any one of Claims 1-7 wherein R3 is halo.
(12) The compound as defined in Claim 11 wherein R is bromo.
(13) The compound as defined in any one of Claims 1-12 which comprises a
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PZ1477 IN Priority (spec DRAFT)
radioactive isotope.
(14) The compound as defined in Claim 13 wherein said radioactive isotope is a
gamma-emitting radioactive halogen or a positron-emitting radioactive nonmetal.
5 (15) The compound as defined in Claim 14 wherein said radioactive isotope is a
gamma-emitting radioactive halogen selected from l23I, l31I and 77Br.
(16) The compound as defined in Claim 14 wherein said radioactive isotope is a
positron-emitting radioactive non- metal selected from nC, l3N, ,SF and l24T.
(17) The compound as defined in an}' one of Claims 13-16 wherein R1 is -0-(CH2)„-
10 F wherein n is as defined in any one of Claims 1-3.
(18) The compound as defined in Claim 17 which is selected from:
Br
(19) The compound as defined in any one of Claims 13-16 wherein K4 comprises ,SF.
15 (20) The compound as defined in Claim 19 which is selected from:
N
(21) A precursor compound useful in the preparation of the compound as defined in
any one of Claims 9-14, said precursor compound being of Formula II:
wherein said precursor compound comprises one precursor group and wherein:
RH is Cj_3 alkoxy or -0-(CH2)m-R15 wherein m is an integer from 1 to 3 and R15
is fluoro or said precursor group;
R is —Ar -R wherein Ar is an aryl and R is selected from fluoro, C1-3
alkoxy C1.3 fluoroalkyl and C1.3 fluoroalkoxy or is said precursor group, with
the proviso that when Ar2 is phenyl R1 is not CM alkoxy;
R13 is halo or C1.3 alkyl, or is said precursor group;
and wherein:
when either R13 or R14 is said precursor group it is selected from Ci_3
trialkylammonium or iodonium; and,
when R15 is said precursor group it is a leaving group.
(22) The precursor compound as defined in Claim 21 wherein R13 is said precursor
group.
(24) The precursor compound as defined in Claim 21 wherein R14 is said precursor
group.
(25) The precursor compound as defined in Claim 22 or Claim 23 wherein said
precursor group is C1-3 trialkyltin.
(26) The precursor compound as defined in Claim 15 wherein R15 is said precursor
-22-
group.
(27) The precursor compound as defined in Claim 26 wherein R15 is selected from
tosylate (OTs), mesylate (OMs), and triflate (OTf).
(28) A method for synthesis of a compound as defined in any one of Claims 13-20
wherein said method comprises reaction of the precursor compound as defined
in any one of Claims 21-26 with a suitable source of the radioactive isotope as
defined in any one of Claims 13-16.
(29) A method for synthesis of a compound as defined in any one of Claims 13-20
wherein said method comprises reaction of a compound of Formula III:
wherein:
11 11 II 11
either R is hydroxyl or R is ethynyl, otherwise R and R are as
1 1
respectively defined for R and R in Claim 1;
11
with a compound of Formula IVa when R is hydroxyl:
1 8 R < v LG
^ x IVa
wherein LG is a leaving group and x is an integer of 1-3;
or with a compound of Formula IVb when R is ethynyl:
^ y ivb
wherein y is an integer of 1-3.
(30) A pharmaceutical formulation comprising the compound as defined in any one
of Claims 1-20 together with a biocompatible carrier in a form suitable for
mammalian administration.
(31) 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 any one of Claims
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PZ1477 IN Priority (spec DRAFT)
13-20;
(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.
5 (32) The in vivo imaging method as defined in Claim 31 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.
(33) A method of diagnosis of a condition in which MAO-B expression is abnormal
10 wherein said method of diagnosis comprises the in vivo imaging method as
defined in Claim 31 as well as the further step (iv) of attributing the distribution
and extent of MAO-B expression to a particular clinical picture.