LABELLED THIAZOLYLHYDRAZONE 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 thiazolylhydrazone derivative. A
5 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
10 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
15 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
20 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
25 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 [ FjFluororasagiline that are used for imaging MAO-B. They are
selective for MAO-B over MAO-A. These known compounds have IC50 values in the
30 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).
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1
A number of publications have described of [ FJfluororasagiline (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 eta! Synapse
2012; 66: 323-330). [18F]fluororasagiIine 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 (Ldeprenyl
= 40- 66 nM) and 950 nM for MAO-A inhibitory activity. Tor the latter,
[18F]Fluorodeprenyl has been shown to have relatively slow metabolism with the
presence of two radiometabolite peaks with similar retention time as the labeled
metabolites of [uC]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.
There is still scope for further agents having selective binding affinity for MAO-B and
15 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
20 compounds. The radioactive compoxinds 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.
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Detailed Description of the Preferred Embodiments
In a first aspect the present invention provides a compound of Formula I:
——-S
rr wherein R1 and Rz are independently selected from iyF and ISF.
1 IS 0 TO
5 In one embodiment of the compound of the invention R is F and R is F.
In one embodiment of the compound of the invention R1 is I9F and R2 is 18F.
In a second aspect the present invention provides a precursor compound of Formula II:
wherein one ofR" and R12 is selected 1 Vom Ci-3 trialkylammonium, iodonium, chloro
10 and nitro, and the other is l9F.
A "precursor compound" comprises a non-radioactive derivative of a radiolabelled
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
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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 "tri alkyl ammonium" means the substituent NR wherein R is an alkyl. Unless
otherwise specified, the term "alkyl" alone or in combination, means a straight-chain
5 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 -NO2.
In one embodiment of the precursor compound of the invention one ofRMandR, z is
10 selected from chloro and nitro, and the other is i9F.
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 l9F.
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
15 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.).
1 it
20 Additional details of synthetic routes to F-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 R and R is F,
wherein said method comprises reaction of the precursor compound of Formula II as
1 o
25 defined hereinabove with [ F] 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
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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
5 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-adjlisting substances (e.g. salts of plasma cations with biocompatible
10 counlerions), 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,
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
15 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
20 (MAO-B) in a subject comprising:
(i) administration of the compound of Formula I as defined hereinabove
wherein at least one of R audR is F;
(ii) detecting by an in vivo imaging procedure signals emitted by the
radioactive isotope comprised in said compound; and,
25 (iii) generating an image representative of said signals.
The term "»? 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).
30 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
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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
5 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,
10 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.
15 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.
20 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
25 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.
30 In a further alternative embodiment of the seventh aspect of the invention, said method
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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.
Brief Description of the Figures
5 Figure 1 illustrates the NMR data for (£)-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-
yi)ethylidene)hydrazinyl)thiazole.
10 Example 2 describes the synthesis of a fluorine-18 radio chemistry precursor for the
compound of Example 1.
Example 3 describes the radiosynthesis of (E)-4-(4-fluorophenyl)-2-(2-(l-(2-
[lsF]fluoropyridui-4-yl)ethylidene)hydrazinyl)thiazole.
Example 5 describes the assay used to meature in vitro inhibition of MAO-A and
15 MAO-B.
List of Abbreviations used in the Examples
CRC: concentration response curve
DCM: dichloromethane
DMF: dimethylformamide
20 DMSO: dimethyl sulfoxide
MAO A: monoamine oxidase A
MAO B: monoamine oxidase A
TLC: thin-layer chromatography
Examples
25 Example 1: Synthesis of(E)-4-(4-fliiorovlienyl)~2-(2-(l-(2-fluovovyiidin-4-
yl) etfi y I id en e)h ydvazin yl) lit iazole
Step 1:
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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). N,0-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-ALmethoxy-A'-methylisonicotinamide (0.72g; 55.17%) as a yellow
liquid.
Step 2:
o O
To a stirred solution of 2-fluoro-ALmethoxywV-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
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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)cthan-l-one (0.35g; 64.3%) as a
yellow liquid.
Step 3:
To a stirred solution of 1 -(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-fIuoropyridin-4-yl)ethyIidene)hydrazinccarbothioamide
(0.250g; 46.8%) as a off white solid.
Step 4:
To a stirred solution of (£)-2-(l-(2-fluoropyridin-4-
yl)ethylidene)hydrazinecarbothioamide (0.25g, 1.179 mmol) in dry Ethanol (15 ml),
was added 2-bromo-l-(4-fluorophenyl)ethan-I-one (0.256g 1.179 mmol). The reaction
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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
5 filtered. The solid was again recrystallized from ethanol to yield (£)-4-(4-
fluorophenyI)-2-(2-(l-(2-fluoropyridiii-4-yl)ethyIidene)hydrazinyl)thiazole (200mg,
51.4% ) as a yellow solid.
Figure 1 illustrates the NMR characterization data for this compound.
Example 2: Synthesis of a fttiovine-18 radiocliemistry precursor for the compound of
10 Example 1.
The experimental procedure as described for Example 1 is carried out, but with the
exception that the starting material in Step 1 is 2-fluoroisonicotinic acid. The
radiochemistry precursor is therefore (£)-4-(4-fluorophenyl)-2-(2-(l-(2-
chloropyridin-4-yl)ethylidcne)hydrazinyl)thiazoIe.
15
to
Example 3: Radiosynthesis of(E)-4-(4-fluorophenyl)-2-(2~(l~(2~[ E]fluoropyridin-4-
yl)ethylidene)hydrazinyl)thiazole
Suitable methods are described in Jackson et al (Bioorganic & Medicinal Chemistry
Letters 23 (2013) 821-826). Fluorine-18 radiolabelling is achieved using direct
20 labelling of the compound of Example 2 with [ Fjfiuoride.
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.
25 General parameters for screening of the compounds was as followsn
Assay Type: Amplex Red Monoamine Oxidase Assay (Invitrogen Cat # A12214)
• Assay Format: 96 well plate (Black, Corning)
• Assay Volume: 200uL
• Enzyme Source: MAO A and MAO B enzymes (Sigma-Aldrich Cat # M7316 and
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M7441)
Q Substrate : For MAO-A/B : Tyramine, For MAO-B : Benzylamine
G 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
G Replicates: Duplicates (n=2)
• Number of Runs: One (N=l)
D Controls:
10 • Negative control/"MIN": 1% DMSO Buffer without enzyme (substrate alone)
• Positive control/ "Max": 1 % DMSO Buffer with enzyme and substrate
D Reading: End point or Kinetic
G Detection: Fluorescence Ex/Em - 560nm/590nm
G 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 lolerability study.
p / l J79 IN Priority (spec DRAFT)

Claims
(1) A compound of Formula I:
wherein R1 and R2 are independently selected from F and F.
5 (2) The compound as defined in Claim 1 wherein R is F and R is F.
(3) The compound as defined in Claim 1 wherein R is F and R is F.
(4) A precursor compound of Formula II:
wherein one of R" and R'* is selected from CM tnaikylammonium, lodonium,
10 chloro and nitro, and the other is l9F.
(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 F.
(6) The precursor compound as defined in Claim 5 wherein R is F.
(7) The precursor compound as defined in Claim 5 wherein R is F.
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(8) A method for the synthesis of the compound as defined in Claim 1 wherein at
least one of R and R is F wherein said method comprises reaction of the
precursor compound of Formula If as defined in any one of Claims 4-7 with
[18F]fluoride.
5 (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:
10 (i) administration of the compound as defined in Claim 1 wherein at
least one ofR1 and R2 is 1SF;
(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 (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
20 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.