Synthesis of three novel fluorine-18 labeled analogues of l -deprenyl for Positron Emission Tomography (PET) studies of Monoamine Oxidase B (MAO-B)

Article abstract of DOI:10.1021/jm200710b

The aim in this project was to synthesize and to study fluorine-18 labeled analogues of l-deprenyl which bind selectively to the enzyme monoamine oxidase B (MAO-B). Three fluorinated l-deprenyl analogues have been generated in multistep organic syntheses. The most promising fluorine-18 compound N-[(2S)-1-[¹⁸F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography on human brain tissue sections demonstrated specific binding for compound 4c to brain regions known to have a high content of MAO-B. In addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited recombinant human MAO-B with an IC₅₀ of 170.5 ±29 nM but did not inhibit recombinant human MAO-A (IC₅₀ > 2000 nM), demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ±0.04%ID/g after 2 min post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a candidate for further investigation in PET.

Full text of DOI:10.1021/jm200710b

ARTICLE
pubs.acs.org/jmc
Synthesis of Three Novel Fluorine-18 Labeled Analogues of L-Deprenyl
for Positron Emission Tomography (PET) studies of Monoamine
Oxidase B (MAO-B)
Sangram Nag,*,† Lutz Lehmann,‡ Tobias Heinrich,‡ Andrea Thiele,‡ Georg Kettschau,‡ Ryuji Nakao,†
Balꢀazs Gulyꢀas,† and Christer Halldin†
†Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
‡Bayer Healthcare AG, Global Drug Discovery, Berlin, Germany
S Supporting Information
b
ABSTRACT: The aim in this project was to synthesize and to study fluorine-18
labeled analogues of L-deprenyl which bind selectively to the enzyme monoamine
oxidase B (MAO-B). Three fluorinated L-deprenyl analogues have been generated
in multistep organic syntheses. The most promising fluorine-18 compound N-[-
(2S)-1-[18F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was
synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Auto-
radiography on human brain tissue sections demonstrated specific binding for
compound 4c to brain regions known to have a high content of MAO-B. In
addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited
recombinant human MAO-B with an IC50 of 170.5 ( 29 nM but did not inhibit recombinant human MAO-A (IC50 > 2000 nM),
demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ( 0.04%ID/g after 2 min
post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a
candidate for further investigation in PET.
’ INTRODUCTION
activity.8 Cerebral MAO-B levels increase with age and are
further up-regulated in the brains of AD patients, mostly due
to an increase of reactive astrocytes.9 As astrocyte activity and,
consequently, the activity of the MAO-B system, is up-regulated
in neuroinflammatory processes, radiolabeled MAO-B inhibitors
may serve as an imaging biomarker in neuroinflammation and
neurodegeneration, including Alzheimer’s disease.10,11
Positron emission tomography (PET), a high-resolution,
sensitive, and noninvasive imaging technique, has been widely
utilized in visualizing the localization of MAO-B. Imaging brain
MAO-B activity with PET in humans has been useful for studying
neurodegenerative diseases12,13 and epilepsy.14 Carbon-11 labeled
L-deprenyl was demonstrated as useful tracer for assessing MAO-
B.15ꢀ17 The short half-life of carbon-11 (20.4 min) makes these
tracers less suitable for distribution to PET centers not equipped
with an on-site cyclotron. Therefore, there has been much
interest in the development of labeled MAO inhibitors with
longer half-life as biological probes to map MAO activity. A
fluorine-18 labeled MAO-B inhibitor would offer a longer half-
life of 110 min, but so far no successful PET radioligand has been
fully validated. Clorgyline was previously labeled with fluorine-18
at aromatic and aliphatic positions, and the resulting tracers were
shown to have favorable properties for mapping MAO-A activity
Monoamine oxidases (MAO) are important enzymes regulating
the levels of monoaminergic neurotransmitters and of bioactive
monoamines by catalyzing their deamination.1 Biochemical and
pharmacological studies indicate that the MAO enzyme exists in
two isoforms known as “MAO type A” (MAO-A) and “MAO type
B” (MAO-B).2 The isoforms differ in their distribution in body
organs and in their substrate specificity. In general, MAO-A
selectively oxidizes the “neurotransmitter” monoamines such as
epinephrine, norepinephrine, and 5-hydroxytryptamine, while
MAO-B selectively oxidizes the monoamines such as O-tyramine,
phenethylamine and tele-N-methyl histamine and both MAO-A
and MAO-B isoforms generate hydrogen peroxide which can form
highly reactive oxygen species. Both MAO-A and MAO-B oxidize
tyramine, tryptamine, and dopamine.3 The MAO isoforms also
differ according to their inhibition selectivity and thus can be
inhibited depending upon the chemical structure of the inhibitor or
the relative concentrations of the inhibitor and the enzyme. MAO
inhibitors are used for the treatment of psychiatric and neurological
disorders. MAO-A inhibitors are prescribed mainly for depression,4
and MAO-B inhibitors are mostly used to treat Parkinson’s disease
(PD)5 as well as depression.6,7 MAO-A is selectively inhibited by,
e.g. pirlindole and clorgyline, whereas MAO-B is selectively
inhibited by e.g. L-deprenyl and rasagiline (Figure 1).
In the human brain, the presence of MAO-B predominates
over MAO-A and constitutes up to ∼70% of total brain MAO
Received: March 22, 2011
Published: September 19, 2011
r
2011 American Chemical Society
7023
dx.doi.org/10.1021/jm200710b J. Med. Chem. 2011, 54, 7023–7029
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of common MAO inhibitors.
Scheme 1. Synthesis of Precursor and Reference for 4a, 4b,
and 4ca
Figure 2. Structures of two [18F]labeled MAO-B inhibitors.
in the brain.18 Two fluorine-18 labeled MAO-B PET radioligands
(Figure 2), 6-[18F]fluoro-N-methyl-N-(prop-2-yn-1-yl)hexan-1-
amine19 and DL-4-[18F]fluorodeprenyl20 were synthesized for
mapping MAO-B activity. The 6-[18F]fluoro-N-methyl-N-(prop-
2-yn-1-yl)hexan-1-amine has not been fully validated for clinical
study, and the multistep radiosynthesis of DL-4-[18F]fluorode-
prenyl precludes its routine synthesis.
In this project, our aims were (i) to develop a fast and efficient
synthetic method for labeling novel L-deprenyl analogues with
fluorine-18 at the aliphatic chain and (ii) to evaluate their binding
to MAO-B in various brain structures in post mortem human
brain slices using an autoradiography technique in order to select
a suitable fluorine-18 labeled radioligand for in vivo molecular
imaging studies using PET.
’ RESULTS AND DISCUSSION
a Conditions: (a) NaOH/1-fluoro 2-bromo ethane; (b) NaOH/1-bro-
moethanol; (c) TEA/mesyl chloride; (d) LAH; (e) K2CO3/propargyl
bromide; (f) DAST; (g) TEA/mesyl chloride; (h) LAH; (i) K2CO3/
propargyl bromide; (j) DAST; (k) TEA/mesyl chloride.
Chemistry. Three novel fluorinated analogues of L-deprenyl 2,
8, and 13 (Scheme 1) were prepared. To label compounds 2, 8,
and 13 with fluorine-18 to obtain 4a, 4b, and 4c, respectively,
three appropriate chloro-precursors 4, 9, and 15, needed to be
synthesized. The chloro precursor 4 for labeling with fluorine-18
was prepared according to Scheme 1. From commercially avail-
able 1 (desmethyl deprenyl hydrochloride salt), fluoride 2 and
alcohol 3 were synthesized by alkylation of secondary amine
using sodium hydroxide as strong base. In the next step, alcohol 3
was treated with mesyl chloride and instead of N-(2-mesylethyl)-
N-(1-phenylpropan-2-yl)prop-2-yn-1-amine, N-(2-chloroethyl)-
N-(1-phenylpropan-2-yl)prop-2-yn-1-amine (4) was produced.
Initially the reaction was performed at ꢀ7 °C; however, the
reaction did not proceed at that low temperature. Subsequently,
the reaction temperature was allowed to increase slowly, and
progress of the reaction was monitored by TLC. At 10 °C, the
reaction commenced but went directly through to give chloride 4
by immediate substitution of the intermediate mesylate group by
SN2 substitution reaction with concomitant chloride formed in
the initial mesylation reaction.
chloride. A mixture of two chlorinated isomers 14 and 15 was
formed from 12. Fluorides 8 and 13 were synthesized from amino
alcohols 7 and 12 by fluorination with diethylamino sulfurtri-
fluoride (DAST). The formation of chlorides 14 and 15 can be
explained by an intermediate aziridinium ion 17 resulting from an
intramolecular nucleophilic attack (SNi) of the free electron pair of
the nitrogen.23
Chlorides 14 and 15 were then formed by subsequent nucleo-
philic attack (SN2) of Clꢀ atthe corresponding reactive position of
aziridinium ion 17 (Scheme 2). Compounds 14 and 15 were
formed in a molar ratio of 2:1 in the isomeric mixture as indicated
by the N-Me 1H NMR signals.
Radiochemistry. The radiolabeling was achieved by one-
step nucleophilic substitution reactions of the chloride pre-
cursors (4, 9, and mixture of 14 and 15) by [18F]fluoride in
presence of K2.2.2 and K2CO3 as shown in Scheme 3. Azeo-
tropic drying was performed before the dried K[18F]FꢀK2.2.2
complex was treated with the adequate chloride precursor in
anhydrous DMSO. The reaction mixtures were heated at
120 °C for 20 min to form the respective fluorine-18 labeled
Amines 6 and 11 were prepared from commercially available
carbamate 5 and amino acid 10 by reduction with LiAlH4
following a previously described procedure.21 Amino alcohols 6
and 11 were alkylated with propargyl bromide,22 and the obtained
alcohols 7 and 12 were chlorinated upon treatment with mesyl
7024
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029

Journal of Medicinal Chemistry
ARTICLE
product. Compound 4c was purified from the isomeric
mixture of 4c and 4d by HPLC. The overall radiosynthesis,
including fluorination, HPLC purification, and evaporation
of the solvent and radiotracer formulation, was completed in
a time range of 70ꢀ80 min.
and rasagiline both abolished the binding, whereas pirlindole
did not block the binding, indicating that 4b displays a weak but
specific binding to MAO-B rich cortical structures (data not
shown).
Compound 4c demonstrated total binding patterns, i.e. bind-
ing comprising specific and nonspecific binding within brain
slices of AD brains and age matched controls. The baseline
condition (total binding) is shown in panel A (Figure 3). For
blocking, the MAO-B ligands L-deprenyl (panel B) and rasagiline
(panel C), the MAO-A ligand pirlindole (panel D) have been
used in 10 μM concentrations (Figure 3). Using the MAO-B
ligands rasagiline and L-deprenyl, the binding of 4c was com-
pletely blocked, whereas the MAO-A ligand pirlindole did not
block the total binding. This indicates that 4c is a selective
radioligand of the MAO-B isoform, whereas it displays no
binding to MAO-A. Compared with 4a and 4b, the binding of
4c in all brain structures was higher. The signal intensities were
highest in the hippocampus, the perihippocampal gyrus, the
temporal lobe, and to some extent, the parietal lobe. There was
also increased signal intensity in the white matter adjacent to
layer 6 of the cortex.
Plasma Protein Binding. Plasma protein binding was mea-
sured using an ultrafiltration method. The results were corrected
for the membrane binding as measured with the control samples.
Binding of 4c to monkey and human plasma proteins were
75.9 ( 0.1% and 83.2 ( 0.5%, respectively. Therefore, there
was a measurable free fraction of 4c in plasma: 24.1 ( 0.1% and
16.8 ( 0.5% for monkey and human, respectively.
Biodistribution. The biodistribution of compound 4c was
investigated in NMRI mice at five time points, namely 2, 5, 30, 60,
and 240 min, and showed a high initial brain uptake of radio-
activity leveling at 5.2 ( 0.04%ID/g at 2 min post injection. In
addition, a high initial elimination of radioactivity from the brain
was observed, reaching a level of 1.9 ( 0.33% ID/g at 30 min pi
and decreased further to 1.4 ( 0.26% ID/g at the 4 h time point
pi. The initial brain uptake was higher as the measured blood
value. This is shown in Figure 4. In addition, the biodistribu-
tion result of 4c in NMRI mice differs to the literature data for
[11C]deprenyl brain kinetics in mice.16 For [11C]deprenyl,
the brain over blood ratio increased with time whereas for
compound 4c a decrease of this ratio had been observed. A
probable explanation behind this observation is that compound
4c (fluorodeprenyl) is a different compound as compared to
deprenyl, and for that reason its behavior in biodistribution
experiments in mice could differ from the data observed with
[11C]deprenyl. Another reason for the differences could be the
use of another mouse strain that could lead to different results.
The incorporation yield of the fluorination reactions varied
from 40% to 70%, and the radiochemical purity was greater than
99% for fluorine-18 labeled 4a, 4b, and 4c throughout (Table 1).
The identities of the labeled compounds were confirmed by
coinjection of their corresponding fluorine-19 analogues 2, 8,
and 13 using analytical HPLC. All radioligands were found to
be stable in PBS buffered solution (pH = 7.4) for the duration of
180 min with a half-life of 110 min.
In Vitro Inhibition of MAO-A and MAO-B. IC50 values for
inhibition of MAO-B and MAO-A were determined based
on the rate of kynuramine oxidation for the analogues of
L-deprenyl in order to determine their specificity for binding
to MAO-B. Compounds 2 and 8 inhibited MAO-B with an
IC50 greater than 2 μM and were therefore not further
investigated for their specificity toward MAO-A. Compound
13 featured an IC50 of 170.5 ( 29 nM for its MAO-B
inhibitory activity while it was not inhibiting MAO-A
(IC50 > 2 μM). L-Deprenyl was used as reference compound.
It inhibited MAO-B with an IC50 of 85.2 ( 26.9 nM. Thus,
compound 13 inhibited MAO-B in a range comparable to
that of the therapeutically used compound. Clorgyline in-
hibited MAO-A with an IC50 of 30.2 ( 11.5 nM.
Autoradiography. All three novel MAO-B fluorine-18
fluorodeprenyl analogues, 4a, 4b, and 4c, were tested in
human whole hemisphere autoradiography experiments on
brain tissue, obtained from deceased subjects with no sign of
any brain disorders. First, 4a featured only a moderate
binding to the white matter and no binding to the cortical
gray matter. The white matter binding could not be blocked
with the cold ligand, L-deprenyl itself, or with the specific
MAO-A and MAO-B ligands pirlindole and rasagiline, re-
spectively, indicating that the observed binding is mainly non-
specific. Using 4b, there a low level binding to the cortex was
found, with the highest but still moderate binding affinity to
the hippocampus at baseline condition. Blocking with L-deprenyl
Scheme 2. ProposedMechanism for theFormation of 14and 15
Scheme 3. Radiolabeling of 4a, 4b, and 4c
7025
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029

Journal of Medicinal Chemistry
ARTICLE
Table 1. Result of Radiochemistry
total synthesis time radiochemical
radiochemical
purity (%)
preparative HPLC retention analytical HPLC retention
specific radioactivity TLC-
radioligand
(min)
yield (%)
time (min)
time (min)
(GBq/μmol)
Rf
4a
4b
4c
70ꢀ80
70ꢀ80
70ꢀ80
41 (N = 6)
46 (N = 2)
68 (N = 3)
>99
>99
>99
11ꢀ13
9ꢀ11
14ꢀ16
5.3
5.4
6.7
240
310
280
0.68
0.63
0.60
referenced internally on CDCl3 (δ1H 7.26) and 13C NMR spectra were
referenced internally on CDCl3 (δ13C 77.20). Liquid chromatographic
analysis (LC) was performed with a MerckꢀHitachi gradient pump and a
MerckꢀHitachi L-4000 variable wavelength UV-detector. A μ-Bondapak
C-18 column (300 mm ꢁ 7.8 mm, 10 μm; Waters instruments) was used
with a flow of 2 mL/min. LC-MS was performed using a Waters Quattra-
Tof Premier micro mass spectrometer or Waters SQD 3001 single
quadrupole mass spectrometer, coupled to Waters Acquity UPLC instru-
ments. The ionization mode used was electro spray positive ionization
(ESI+). Analytical TLC was carried out on 0.25 mm silica gel plates.
All solvents and chemicals were obtained from commercial sources
and used without further purification. Purity of all compounds is g95%
and was determined by HPLC and UPLC and LC-MS.
Synthesis of N-(2-Chloroethyl)-N-[(2R)-1-phenylpropan-2-yl]prop-
2-yn-1-amine (4). A mixture of 3 (150 mg, 0.69 mmol) and triethyl
amine (1.5 mmol) in THF (3 mL) was stirred at room temperature for
30 min. To the stirred mixture mesyl chloride (1.4 mmol) was added
dropwise at ꢀ7 °C, and the reaction mixture was stirred at 10 °C for
additional 30 min. Saturated Na2CO3 solution (2 mL) was added and
stirred for 30 more min. The organic layer was partitioned between
CH2Cl2 (20 mL) and water (10 mL). The organic phase was separated
and washed with saturated NaHCO3 solution (10 mL) and brine
(10 mL) and dried over MgSO4 and filtered. The solvent was removed
under reduced pressure to obtain the crude product as light-yellow oil.
The crude product was purified by silica-gel column chromatography
(hexane/ether 3:1), and the final product resulted as colorless oil
(85 mg, 0.63 mmol, 52%). The product was analyzed by NMR and LC-MS.
1H NMR (1H, 400 MHz, CDCl3) δH: 1.0 (3H, d), 2.2 (1H, s), 2.4
(1H, m), 2.9ꢀ3.1 (4H, m), 3.4ꢀ3.6 (4H, m), 7.1ꢀ7.4 (5H, m).
13C NMR (13C, 100 MHz, CDCl3) δC: 16.1, 40.1, 40.6, 42.6, 51.6,
59.9, 71.1, 126.0, 127.6, 129.2, 140.0.
Figure 3. Coronal slices of human brain autoradiograms labeled with
4c. (A) Baseline conditions. (B) Incubation with L-deprenyl (10 μM).
(C) Incubation with rasagiline (10 μM). (D) Incubation with pirlindole
(10 μM). The brain slices were obtained from a 58 y old female control
subject; the post mortem time was 11 h.
LC-MS (ESI): m/z = 236 (M + 1).
Synthesis of N-((1R,2S)-1-Fluoro-1-phenylpropan-2-yl)-N-methyl-
prop-2-yn-1-amine(8). Tothestirred solutionof 7 (150mg, 0.74 mmol)
in dichloromethane (3 mL), diethylamino sulfurtrifluoride (DAST) (1.0
mmol) was addeddropwise at ꢀ5 °C and thereaction mixturewas stirred
for additional 20 min at the same temperature. Saturated aqueous sodium
carbonate (2.0 mL) was added to quench unreacted DAST. The organic
layer was partitioned between CH2Cl2 (15 mL) and water (10 mL). The
organic phase was separated and washed with brine (10 mL) and dried
over MgSO4 and filtered. The solvent was removed under reduced
pressure to obtain the crude product as light-yellow oil. The crude
product was purified by silica-gel column chromatography (hexane/ether
4:1). Final product was obtained as light-yellow oil (75 mg, 0.37 mmol,
49%) and was analyzed by NMR, HPLC and LC-MS.
Figure 4. Time course distribution of compound 4c in brain and blood
of male NMRI mice.
’ CONCLUSION
Radiolabeling of three new fluorine-18 analogues of L-deprenyl
was successfully accomplished with adequate radiochemical yields,
high specific radioactivity, and radiochemical purity higher than
99%. Biodistribution in mice showed that compound 4c ade-
quately passed the bloodꢀbrain barrier. The fluorine-18 labeled
compound 4c binds specifically to MAO B in post mortem human
brain sections. Consequently, radioligand 4c appears to be a
potential molecular imaging biomarker candidate for PET studies
in neuroinflammation and neurodegeneration, accompanied with
astrocyte activation.
1H NMR (1H, 400 MHz, CDCl3) δH: 1.1 (3H, d), 2.3 (1H, s), 2.4
(3H, s), 3.1ꢀ3.2 (1H, m), 3.5 (2H, s), 5.7 (1H, d) and 7.3ꢀ7.5 (5H, m).
13C NMR (13C, 100 MHz, CDCl3) δC: 8.4, 39.6, 43.8, 62.5, 73.5,
93.5, 95.2, 125.5, 128.1, 129.9 and 141.5.
LC-MS (ESI): m/z = 206 (M + 1).
’ EXPERIMENTAL SECTION
Synthesis of N-((1R,2S)-1-Chloro-1-phenylpropan-2-yl)-N-methyl-
prop-2-yn-1-amine (9). A mixture of 7 (120 mg, 0.54 mmol) and
triethyl amine (88 μL, 1.0 mmol) in THF (2 mL) was stirred at room
temperature for 30 min. To the stirred mixture mesyl chloride (46.44 μL,
Chemistry. NMR spectra were recorded on Varian Unity-400 and
Bruker Avance 400 (1H, 400 MHz; 13C, 100 MHz) and Bruker Avance
600 III (1H, 600 MHz) NMR instruments. 1H NMR spectra were
7026
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029

Journal of Medicinal Chemistry
ARTICLE
0.60 mmol) was added dropwise at ꢀ7 °C, and the reaction mixture was
stirred at room temperature for additional 30 min. Saturated Na2CO3
solution (1 mL) was added and stirred for additional 30 min. The
organic layer was partitioned between CH2Cl2 (15 mL) and water
(10 mL). The organic phase was separated and washed with saturated
NaHCO3 solution (10 mL) and brine (10 mL) and dried over MgSO4
and filtered. The solvent was removed under reduced pressure to obtain
the crude product as light-yellow oil. The crude product was purified by
silica-gel column chromatography (hexane/ether 3:1). Final product
was obtained as a light-yellow oil (91 mg, 0.41 mmol, 69%) and was
analyzed by NMR, HPLC, and LC-MS.
Fluorine-18 fluoride was produced by the 18O(p, n)18F nuclear reaction
using a GEMS PETtrace cyclotron. A solution of [18F]fluoride in 18
O
enriched water was flashed through a Sep-Pak QMA light cartridge
(preconditioned with K2CO3 (0.5 M, 10 mL), 18 MΩ H2O, 15 mL) in
order to isolate [18F]fluoride. [18F]Fluoride was then eluted from the
cartridge with a solution of K2CO3 (1.8 mg, 13 μmol), Kryptofix 2.2.2
(9.8 mg, 26 μmol) in water (18 MΩ, 43 μL), and acetonitrile (2 mL).
The solvent was evaporated at 160 °C under continuous nitrogen flow.
The residue was then cooled to 25 °C, followed by addition of the
precursor 4, 9, or 15 (2.0 mg, ∼0.01 mmol, in DMSO (600 μL)) was
added. The closed reaction vessel was then heated at 120 °C for 20 min.
The reaction vessel was cooled to room temperature, and 18 MΩ H2O
(1 mL) was added before injecting to the HPLC.
All three fluorine-18 labeled radioligands 4a, 4b, and 4c were purified
by reverse phase HPLC on a μ-Bondapak C-18 column (300 mm ꢁ
7.8 mm, 10 μm; waters instruments) and MeCNꢀH3PO4 (0.01 M)
(15:85 v/v) was used as the eluting solvent at a flow rate of 4 mL/min.
Elute was monitored by a UV absorbance detector (λ = 214 nm) in series
with a GM tube radioactivity detector. The isomeric mixture of 4c and
4d was separated by HPLC (tR(4c) = 14ꢀ16 min, tR(4d) = 18ꢀ19 min).
The fraction of the desired compounds was collected and evaporated to
dryness. The residue was dissolved in sterile disodiumphosphate phos-
phate buffered saline (PBS; pH = 7.4; 10 mL) and filtered through a
sterile filter (0.22 μm; Millipore, Bedford, MA), yielding a sterile and
pyrogen-free (<1.25 EU) solution of the radioligand.
The radiochemical purity of each radioligand (4a, 4b, and 4c) was
analyzed by a reverse phase HPLC on a μ-Bondapak C-18 column
(300 mm ꢁ 3.9 mm, 10 μm; waters instruments) and MeCNꢀH3PO4
(0.01 M) (15:85 v/v) wasusedastheeluting solvent at aflowrateof 2mL/
min. Elute was monitored by a UV absorbance detector (λ = 214 nm) in
series with a radioactivity detector (β-flow; Beckman, Fullerton, CA). The
radiochemical purity was >99% for all three compounds.
The stability and radiochemical yield was tested with HPLC and TLC
on silica gel (100% CH3COOC2H5 was used as the eluting solvent).
TLC plate was scanned with an AR-2000 imaging scanner and analyzed
with Winscan 2.2 software.
Determination of MAO Inhibition. Human recombinant MAO-
B and MAO-A enzymes (Sigma) prepared from insect cells were
purchased from Sigma. The assays were designed to determine the
inhibition of kynuramine oxidation in the presence of the compounds of
interest according to Weissbach et al.24 A calibration curve of kynur-
amine hydrobromide (Sigma) was determined at 360 nm and used for
calculation of the enzyme activity (pmol/min) at the respective com-
pound concentration. This relation was plotted and the IC50 determined
using the software GraFit 5 (Version 5.0.6). The assays were performed
as follows. The compounds were diluted 1:2 in each step in 50 mM
phosphate buffer (pH 7.4) so that a concentration curve between 0.49
and 1000 nM was generated to determine the IC50 for MAO-B and
between 0.98 and 2000 nM for determination of inhibition of MAO-A,
respectively. Kynuramine hydrobromide at a concentration of 125 μM
for MAO-B and 100 μM for MAO-A, respectively, and 2.5 U/mL
enzyme were added and the reaction followed measuring the absorption
at 360 nm in a 5 min interval over 30 min at 37 °C. The 30 min time
point was used to determine IC50 values. As internal standards for MAO-
B, pargyline and L-deprenyl and for MAO-A clorgyline were used.
In Vitro Autoradiography. Human brains without pathology
were obtained from the Department of Forensic and Insurance Medi-
cine, Semmelweis University, Budapest. The brains had been removed
during forensic autopsy (control brains) and were handled in a manner
similar to that described previously.25ꢀ27 Ethical permissions were
obtained from the relevant Research Ethics Committee of the respective
institutions. The sectioning of the brains and the autoradiography
experiments were performed at the Department of Neuroscience,
Karolinska Institutet. The sectioning took place on a Leica cryomacrocut
1H NMR. (1H, 400 MHz, CDCl3) δH: 1.3 (3H, d), 2.3 (1H, s), 2.4
(3H, s), 3.2ꢀ3.3 (1H, m), 3.5 (2H, s), 4.5 (1H, d) and 7.2ꢀ7.4 (5H, m).
13C NMR (13C, 100 MHz, CDCl3) δC: 22.9, 31.6, 39.7, 43.9, 57.1,
71.4, 73.8, 127.5, 128.1, 129.9 and 136.1.
LC-MS (ESI): m/z = 222 (M + 1).
Synthesis of (S)-N-(1-Fluoro-3-phenylpropan-2-yl)-N-methylprop-
2-yn-1-amine (13). To the stirred solution of 12 (300 mg, 1.48 mmol)
in dichloromethane (5 mL), diethylamino sulfurtrifluoride (264 μL,
2.0 mmol) was added dropwise at ꢀ5 °C and the reaction mixture was
stirred for additional 20 min at the same temperature. Saturated sodium
carbonate (4.0 mL) was added to quench unreacted DAST. The organic
layer was partitioned between CH2Cl2 (25 mL) and water (15 mL). The
organic phase was separated and washed with brine (10 mL) and dried
over MgSO4 and filtered. The solvent was removed under reduced
pressure to obtain the crude product as a light-yellow oil. The crude
product was purified by silica-gel column chromatography (hexane/
ether 3:1) and gave the final product (65 mg, 0.32 mmol, 21%). The
product was analyzed by NMR, HPLC, and LC-MS.
1H NMR (600 MHz, chloroform-d) δ ppm: 2.3 (t, 1 H), 2.55 (s, 3 H),
2.77 (dd, 1 H), 2.97ꢀ3.03(m, 1H), 3.03ꢀ3.14 (m, 1 H), 3.53 (t, 2 H), 4.38
(ddd, 1 H), 4.51 (ddd, J = 48.05, 10.27, 2.57 Hz, 1 H), 7.20ꢀ7.32 (m, 5H).
13C NMR (151 MHz, CDCl3) δ ppm 39.7, 42.4, 46.3, 58.5, 73.4, 78.2,
92.8, 126.6, 128.4, 129.4 and 136.8.
LC-MS (ESI): m/z = 206 (M + 1).
Synthesis of (S)-N-(1-Chloro-3-phenylpropan-2-yl)-N-methylprop-
2-yn-1-amine (14) and N-(2-Chloro-3-phenylpropyl)-N-methylprop-
2-yn-1-amine (15). A mixture of 12 (100 mg, 0.49 mmol) and triethyl
amine (139 μL, 1.0 mmol) in THF (2 mL) was stirred at room
temperature for 30 min. To the stirred mixture mesyl chloride
(68.7 mg, 46.4 μL, 0.60 mmol) was added dropwise at ꢀ7 °C, and
the reaction mixture was stirred at room temperature for additional
30 min. Saturated aqueousNa2CO3 solution (1 mL) was addedand stirred
for further 30 min. The organic layer was partitioned between CH2Cl2
(15 mL) and water (10 mL). The organic phase was separatedand washed
with saturated NaHCO3 solution (10 mL) and brine (10 mL), dried over
MgSO4, and filtered. The solvent was removed under reduced pressure to
obtain the crude product as a light-yellow oil. The crude product was
purified by silica-gel column chromatography (hexane/ether 3:1) and final
product (90 mg, 82%, 0.41 mmol) was obtained as a light-yellow oil. The
product was analyzed by NMR, HPLC, and LC-MS. The final product was
a mixture of 14 (major) and 15 (minor).
14 (major): 1H NMR (1H, 400 MHz, CDCl3) δH 2.28 (t, 1 H), 2.51
(s, 3 H), 2.78 (d, 2 H), 3.06 (dd, 1 H), 3.50 (dd, 2 H), 3.55ꢀ3.65 (m, 2
H), 7.30ꢀ7.41 (m, 5 H).
LC-MS (ESI): m/z = 222 (M + 1).
15 (minor): 1H NMR (1H, 400 MHz, CDCl3) δH 2.21 (t, 1 H), 2.38
(s, 3 H), 2.74 (d, 2 H), 2.94 (dd, J = 14.22, 1 H), 3.23 (dd, 1 H), 3.43 (t, 2
H), 4.08ꢀ4.20 (m, 1 H), 7.22ꢀ7.26 (m, 2 H), 7.26ꢀ7.36 (m, 3 H).
LC-MS (ESI): m/z = 222 (M + 1).
Radiochemistry. Synthesis of N-[2-[18F]Fluoroethyl]-N-[(2R)-1-
phenylpropan-2-yl]prop-2-yn-1-amine (4a), N-[(1S,2R)-1-[18F]Fluoro-
1-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4b), and N-[(2S)-
1-[18F]Fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c).
7027
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029

Journal of Medicinal Chemistry
ARTICLE
system. The resulting slices were horizontal brain slices of 100 μm
thickness. The brain slices were obtained from a 58-year-old female
control subject; the post mortem time was 11 h, until which the cadaver
was stored at (0 °C. After the removal of the brain, it was kept
at ꢀ85 °C until sectioning. The whole hemisphere brain slices were
kept at ꢀ25 °C until the autoradiographic procedures.
’ ACKNOWLEDGMENT
We thank all the members of the PET group at Karolinska
Institutet, including special thanks to Siv Eriksson for excellent
technical assistance with autoradiography. We also thank Bayer
HealthCare AG for their support.
The autoradiographic procedures were basically identical with those
used by us in former studies.28 Briefly, 100 μm thick whole hemisphere
sections were incubated for 90 min at room temperature with 4 MBq/
200 mL of the corresponding radiotracer in 50 mM TRIS buffer pH 7.4
containing sodium chloride (120 mM), potassium chloride (5 mM),
calcium chloride (2 mM), and albumin (0.1% w/v). After the incubation,
the sections were washed in the same buffer three times for 5 min each
time at room temperature, briefly dipped in ice-cold distilled water,
dried, and exposed to phosphorimaging plates. The readings were made
in a Fujifilm BAS-500 phosphorimager and digitized using a Fujifilm IP
Eraser 3. Standards were prepared by serial dilution of the radioligand
stock solution in assay buffer.
’ ABBREVIATIONS USED
PET, positron emission tomography; MAO, monoamino oxidase;
PD, Parkinson’s disease; AD, Alzheimer’s disease; DAST, diethy-
lamino sulfurtrifluoride; NMR, nuclear magnetic resonance;
UPLC, ultra-performance liquid chromatograpy; DMSO, di-
methylsuphoxide; DMF, dimethylformamide; PBS, phosphate
buffer solution; IC, inhibitory concentration
’ REFERENCES
(1) Fuller, R. W.; Roush, B. W. Substrate-selective and tissue-
selective inhibition of monoamine oxidase. Arch. Int. Pharmacodyn. Ther.
1972, 198, 270–276.
(2) Bach, A. W. J.; Lan, N. C.; Bruke, D. J.; Abell, C. W.; Bembenek,
M. E.; Kwan, S. W.; Seeburg, P. H.; Shih, J. C. Molecular-cloning of
human monoamine oxidase-A and oxidase-B (MAO-A and MAO-B).
FASEB J. 1988, 2, A1733–A1733.
(3) Tipton, K. F.; Boyce, S.; O’Sullivan, J.; Davey, G. P.; Healy, J.
Monoamine oxidases: certainties and uncertainties. Curr. Med. Chem.
2004, 11, 1965–1982.
Blocking experiments were performed with 10 μM L-deprenyl,
rasagiline, and pirlindol for fluorine-18 labeled deprenyl analogues 4a
and 4b. For compound 4c, blocking experiments were done with
L-deprenyl, rasagiline, and pirlindol.
Biodistribution. Biodistribution was investigated in male NMRI
mice at five time points, namely 2, 5, 30, 60, and 240 min. The mice used
in the experiments weighted between 25.0 and 31.5 g. For each time
point, three mice were examined. The mice were injected each with
0.178 MBq radioactively labeled deprenyl analogues dissolved in 0.9%
NaCl solution. At the respective post-injection time points, the mice
were euthanized with an overdose of isofluorane, sacrificed, the organs
excised, and blood taken, respectively, before measuring the organ and
blood radioactivity levels in a γ counter. In addition, faeces and urine
were collected and measured in a γ counter, as well. The results were
decay corrected and normalized to 1 g of tissue and to whole blood
volume, respectively, and expressed as percent injected dose per gram
tissue and blood (%ID/g), respectively.
Plasma Protein Binding. Plasma protein binding of 4c was
measured in duplicate in baseline conditions. Monkey plasma (500 μL),
human plasma (500 μL), and phosphate buffered saline solution (pH 7.4,
KCl 0.2 mg, KH2PO4 0.2 mg, Na2HPO4 1.42 mg, NaCl 8 mg in 1 mL)
serving as control, respectively, were mixed with 4c radiopharmaceutical
formulation (50 μL, approximately 20 MBq in PBS) and incubated at
room temperature for 10 min. Samples (20 μL) from each incubation
mixture were measured in a well-counter. After incubation, 200 μL portions
of the incubation mixtures were pipetted into ultrafiltration tubes (Millipore
Centrifree YM-30) and centrifuged for 15 min in 3000 rpm. Samples
(20 μL) from each filtrate were counted in a well-counter. The fraction of
protein-bound ligand was calculated using the following formula;
(4) Andrews, J. M.; Nemeroff, C. B. Contemporary management of
depression. Am. J. Med. 1994, 97, S24–S32.
(5) Drukarch, B.; van Muiswinkel, F. L. Drug treatment of Parkinson’s
disease—time for phase II. Biochem. Pharmacol. 2000, 59, 1023–1031.
(6) Youdim, M. B. H.; Bakhle, Y. S. Monoamine oxidase: isoforms and
inhibitors in Parkinson’s disease and depressive illness. Br. J. Pharmacol.
2006, 147, S287–S296.
(7) Holtzheimer, P. E.; Nemeroff, C. B. Emerging treatments for
depression. Expert Opin. Pharmacother. 2006, 7, 2323–2339.
(8) Cesura, A. M.; Pletscher, A. The new generation of monoamine
oxidase inhibitors. Prog. Drug Res. 1992, 38, 171–297.
(9) Saura, J.; Luque, J. M.; Cesura, A. M.; Da Prada, M.; Chan-Palay,
V.; Huber, G.; Loffler, J.; Richards, J. G. Increased monoamine oxidase B
activity in plaque-associated astrocytes of Alzheimer brains revealed by
quantitative enzyme radioautography. Neuroscience 1994, 62, 15–30.
(10) Gulyas, B.; Pavlova, E.; Kasa, P.; Gulya, K.; Bakota, L.; Varszegi,
S.; Keller, E.; Horvath, M. C.; Nag, S.; Hermecz, I.; Magyar, K.; Halldin,
C. Activated MAO-B in the brain of Alzheimer patients, demon-
strated by [(11)C]-l-deprenyl using whole hemisphere autoradio-
graphy. Neurochem. Int. 2011, 58, 60–68.
(11) Bench, C. J.; Price, G. W.; Lammertsma, A. A.; Cremer, J. C.;
Luthra, S. K.; Turton, D.; Dolan, R. J.; Kettler, R.; Dingemanse, J.; Da
Prada, M.; et al. Measurement of human cerebral monoamine oxidase
type B (MAO-B) activity with positron emission tomography (PET): a
dose ranging study with the reversible inhibitor Ro 19ꢀ6327. Eur. J. Clin.
Pharmacol. 1991, 40, 169–173.
CplaðfiltrateÞ
CplaðtotalÞ
C
÷
PBSðfiltrateÞ ꢁ 100ð%Þ
CPBSðtotalÞ
where Cpla(total) and CPBS(total) represent the radioactivity concentration of
incubation mixture in plasma and PBS (control), respectively. Cpla(filtrate)
and CPBS(filtrate) are the radioactivity concentrations from filtrate samples.
(12) Fowler, J. S.; Logan, J.; Volkow, N. D.; Wang, G. J.; MacGregor,
R. R.; Ding, Y. S. Monoamine oxidase: radiotracer development and
human studies. Methods 2002, 27, 263–277.
(13) Fowler, J. S.; Volkow, N. D.; Wang, G. J.; Pappas, N.; Shea, C.;
MacGregor, R. R.; Logan, J. Visualizationofmonoamineoxidaseinhuman
brain. Adv. Pharmacol. 1998, 42, 304–307.
(14) Bergstrom, M.; Kumlien, E.; Lilja, A.; Tyrefors, N.; Westerberg,
G.; Langstrom, B. Temporal lobe epilepsy visualized with PET with
C-11-L-deuterium-deprenyl—analysis of kinetic data. Acta Neurol.
Scand. 1998, 98, 224–231.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental and spectro-
b
scopic details on compounds 2, 3, 6, 7, 11, and 12. This material
is available free of charge via the Internet at http://pubs.acs.org.
(15) Fowler, J. S.; MacGregor, R. R.; Wolf, A. P.; Arnett, C. D.;
Dewey, S. L.; Schlyer, D.; Christman, D.; Logan, J.; Smith, M.; Sachs, H.;
Aquilonius, S. M.; Bjurling, P.; Halldin, C.; Hartvig, P.; Leenders, K. L.;
Lundqvist, H.; Oreland, L.; Stalnacke, C. G.; Langstrom, B. Mapping
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +46-8-51775018. E-mail: sangram.nag@ki.se.
7028
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029

Journal of Medicinal Chemistry
ARTICLE
human-brain monoamine oxidase-A and oxidase-B with C-11 labeled
suicide inactivators and PET. Science 1987, 235, 481–485.
(16) MacGregor, R. R.; Halldin, C.; Fowler, J. S.; Wolf, A. P.; Arnett,
C. D.; Langstrom, B.; Alexoff, D. Selective, irreversible in vivo binding of
[11C]clorgyline and [11C]-L-deprenyl in mice: potential for measure-
ment of functional monoamine oxidase activity in brain using positron
emission tomography. Biochem. Pharmacol. 1985, 34, 3207–3210.
(17) Fowler, J. S.; Logan, J.; Wang, G. J.; Volkow, N. D.; Telang, F.;
Ding, Y. S.; Shea, C.; Garza, V.; Xu, Y. W.; Li, Z. H.; Alexoff, D.; Vaska, P.;
Ferrieri, R.; Schlyer, D.; Zhu, W.; Gatley, S. J. Comparison of the binding
of the irreversible monoamine oxidase tracers, [C-11]clorgyline and
[C-11]L-deprenyl in brain and peripheral organs in humans. Nucl. Med.
Biol. 2004, 31, 313–319.
0
(18) Mukherjee, J.; Yang, Z. Y. Development of N-[3-(2 ,40-dich-
lorophenoxy)-2-F-18-fluoropropyl]-N-methylpropargylamine (F-18-
fluoroclorgyline) as a potential PET radiotracer for monoamine oxidase-
A. Nucl. Med. Biol. 1999, 26, 619–625.
(19) Mukherjee, J.; Yang, Z. Y.; Lew, R. N-(6ꢀ18F-fluorohexyl)-N-
methylpropargylamine: a fluorine-18-labeled monoamine oxidase B
inhibitor for potential use in PET studies. Nucl. Med. Biol. 1999, 26,
111–116.
(20) Plenevaux, A.; Fowler, J. S.; Dewey, S. L.; Wolf, A. P.;
Guillaume, M. The synthesis of no-carrier-added DL-4-[18F]fluorode-
prenyl via the nucleophilic aromatic substitution reaction. Int. J. Rad.
Appl. Instrum., A 1991, 42, 121–127.
(21) Bowen, R. D.; Harrison, A. G.; Reiner, E. J. Ionꢀdipole
complexes in the unimolecular reactions of isolated organic ions-effect
of N-methylation on olefin and amine loss from protected aliphatic-
amines. J. Chem. Soc., Perkin Trans. 2 1988, 1009–1013.
(22) Couty, F.; Durrat, F.; Prim, D. Expeditive synthesis of homochiral
fusedtri-and tetrazoles-piperazines from beta-amino alcohols. Tetrahedron
Lett. 2004, 45, 3725–3728.
(23) Metro, T. X.; Appenzeller, J.; Pardo, D. G.; Cossy, J. Highly
enantioselective synthesis of beta-amino alcohols. Org. Lett. 2006, 8,
3509–3512.
(24) Weissbach, H.;Smith, T. E.;Daly, J. W.;Witkop, B.; Udenfriend,
S. A rapid spectrophotometric assay of mono-amine oxidase based on the
rate of disappearance of kynuramine. J. Biol. Chem. 1960, 235, 1160–
1163.
(25) Hall, H.; Halldin, C.; Farde, L.; Sedvall, G. Whole hemisphere
autoradiography of the postmortem human brain. Nucl. Med. Biol. 1998,
25, 715–719.
(26) Schou, M.; Halldin, C.; Pike, V. W.; Mozley, P. D.; Dobson, D.;
Innis, R. B.; Farde, L.; Hall, H. Post-mortem human brain autoradio-
graphy of the norepinephrine transporter using (S,S)-[18F]FMeNER-
D2. Eur. Neuropsychopharmacol. 2005, 15, 517–520.
(27) Gillberg, P. G.; Jossan, S. S.; Askmark, H.; Aquilonius, S. M.
Large section cryomicrotomy for invitro receptor autography. J. Phar-
macol. Methods 1986, 15, 169–180.
(28) Gulyas, B.; Makkai, B.; Kasa, P.; Gulya, K.; Bakota, L.; Varszegi,
S.; Beliczai, Z.; Andersson, J.; Csiba, L.; Thiele, A.; Dyrks, T.; Suhara, T.;
Suzuki, K.; Higuchi, M.; Halldin, C. A comparative autoradiography
study in post mortem whole hemisphere human brain slices taken from
Alzheimer patients and age-matched controls using two radiolabelled
DAA1106 analogues with high affinity to the peripheral benzodiazepine
receptor (PBR) system. Neurochem. Int. 2009, 54, 28–36.
’ NOTE ADDED AFTER ASAP PUBLICATION
This paper was published on the Web on September 19, 2011
without the required corrections. The corrected version was
reposted on October 20, 2011.
7029
dx.doi.org/10.1021/jm200710b |J. Med. Chem. 2011, 54, 7023–7029