(R)-N-Methyl-3-(3′-[18F]fluoropropyl)phenoxy-3- phenylpropanamine (18F-MFP3) as a potential PET imaging agent for norepinephrine transporter

Article abstract of DOI:10.1002/jlcr.1744

A decline of norepinephrine transporter (NET) level is associated with several psychiatric and neurological disorders. Therefore positron emission tomography (PET) imaging agents are greatly desired to study the NET pathway. We have developed a C-fluoropr

Full text of DOI:10.1002/jlcr.1744

Research Article
Received 20 October 2009,
Revised 13 December 2009,
Accepted 30 December 2009
Published online 9 February 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1744
(R)-N-Methyl-3-(30-[18F]fluoropropyl)phenoxy)-
3-phenylpropanamine (18F-MFP3) as
a potential PET imaging agent for
norepinephrine transporter
Vivien L. Nguyen,a Rama Pichika,b Paayal H. Bhakta,a Ritu Kant,a
Ã
and Jogeshwar Mukherjeea
A decline of norepinephrine transporter (NET) level is associated with several psychiatric and neurological disorders.
Therefore positron emission tomography (PET) imaging agents are greatly desired to study the NET pathway. We have
developed a C-fluoropropyl analog of nisoxetine: (R)-N-methyl-3-(30-[18F]fluoropropyl)phenoxy)-3-phenylpropanamine
(
18F-MFP3) as a new potential PET radiotracer for NET with the advantage of the longer half-life of fluorine-18 (110 min
compared with carbon-11 (20 min). Synthesis of (R)-N-methyl-3-(30-fluoropropyl)phenoxy)-3-phenylpropanamine (MFP3)
was achieved in five steps starting from (S)-N-methyl-3-ol-3-phenylpropanamine in approx. 3–5% overall yields. In vitro
binding affinity of nisoxetine and MFP3 in rat brain homogenates labeled with 3H-nisoxetine gave Ki values of 8.02 nM and
23 nM, respectively. For radiosynthesis of 18F-MFP3, fluorine-18 was incorporated into a tosylate precursor, followed by the
deprotection of the N-BOC-protected amine group with a 15% decay corrected yield in 2.5 h. Reverse-phase
chromatographic purification provided 18F-MFP3 in specific activities of 42000 Ci/mmol. Fluorine-18 labeled 18F-MFP3
has been produced in modest radiochemical yields and in high specific activities. Evaluation of 18F-MFP3 in animal imaging
studies is in progress in order to validate this new fluorine-18 radiotracer for PET imaging of NET.
Keywords: 18F-MFP3; PET; norepinephrine transporter; nisoxetine; autoradiography
major depression and has been identified as a noradrenergic
and serotonergic antidepressant.14,15
Introduction
The norepinephrine transporter (NET) present in presynaptic
adrenergic neurons is responsible for the reuptake of the
neurotransmitter norepinephrine (NE), regulating the synaptic
NE concentration.1 High densities of NET can be found in the
hippocampus, thalamus, and especially the locus coeruleus (LC),
from which the NET pathway is projected.2,3 Low densities are
found in the cerebellum and the striatum, which receive sparse
projections from the LC.2,4 Decreased NET concentration is
associated with numerous psychiatric and neurological syn-
dromes such as major depressive disorder and Alzheimer’s
disease.5–7
Currently, there are several clinically used antidepressants that
affect NE levels in the brain. Antidepressants, such as venlafaxine
(or effexor) and duloxetine (or cymbalta), are serotonin–
norepinephrine reuptake inhibitor (SNRI), and block presynaptic
reuptake of both NE and serotonin (via serotonin reuptake
transporter or SERT), thereby increasing the availability of both
neurotransmitters in the synapse.8 These compounds increased
the levels of NE and serotonin in rat prefrontal cortex.9,10 Clinical
Many positron emission tomography (PET) and single photon
emission computed tomography (SPECT) tracers radiolabeled with
carbon-11 (20.4 min half-life), fluorine-18 (109.8 min half-life), and
iodine-123 (13 h half-life) have been reported (Figure 1, 1–9).
Radiolabeled 11C-nisoxetine (Figure 1, 1) demonstrated modest
specific NET binding, with findings of 60–75% nonspecific binding.16
The compound 11C-MeNER (Figure 1, 4, IC50 =2.5nM) is an O-methyl
analog of reboxetine (IC50 = 8.2 nM) and showed a hypothalamus to
striatum ratio of 2.5.17,18 Another methyl analog of reboxetine,
11C-(S,S)-MRB, showed a ratio of approximately 1.5–1.6 between
thalamus and striatum.19 The fluorine-18 radiolabeled analog
of reboxetine 18F-FRB (Figure 1, 6) displayed a thalamus to reference
region ratio of 1.3–1.6.20 Talopram (1,3-dihydro-N-3,3-trimethyl-
1-phenylbezo[c]furan-1-propanamine, 8) and talsupram (1,3-dihy-
dro-N-3,3-trimethyl-1-phenylbezo[c]thiophene-1-propanamine,
aDepartment of Psychiatry and Human Behavior, University of California, Irvine,
CA, USA
studies have shown these drugs to be effective antidepres- bDepartment of Radiology, University of California, San Diego, CA, USA
sants.11,12 More selective NET inhibitors (norepinephrine reup-
*Correspondence to: Jogeshwar Mukherjee, Preclinical Imaging Facility, Department
take inhibitor (NRI)) such as reboxetine have been reported with
of Psychiatry and Human Behavior, University of California – Irvine, 162 Irvine Hall,
Irvine, CA 92696-3960, USA.
relatively few side effects.13 Other compounds such as
mirtazapine is an a-2 adrenoreceptor antagonist effective in E-mail: j.mukherjee@uci.edu
J. Label Compd. Radiopharm 2010, 53 172–177
Copyright r 2010 John Wiley & Sons, Ltd.

V. L. Nguyen et al.
X
X
X
NHCH
O
3
O
H
O
NH
NH
CH3
(a)
(b)
(c)
1. X = OCH3 (Nisoxetine; 11C) 4. X = OCH3 (MeNER; 11C)
8. X = O (Talopram; 11C)
9. X = S (Talsupram; 11C)
2. X = I (MIPP; 123I and 125I)
5. X = OCH2CH3 (Reboxetine)
6. X = OCH2CH2F (18F)
3. X = CH3 (Tomoxetine; 11C)
7. X = I (IPBM; 125I or INER; 123I)
Figure 1. Radiolabeled imaging agents for NET. (a) Nisoxetine analogs; (b) reboxetine analogs; (c): other backbones.
9) exhibited high affinity and selectivity for the human NET in vitro.
However, neither compound was able to enter the central nervous
system in adequate amounts to be used in PET studies.21
19F/18
F
Radioiodinated MIPP has high affinity to NET (Ki = 1.33 nM)
compared with nisoxetine (Ki = 7.35 nM) but also has some
O
affinity for SERT.22–24 The compound 125I-(S,S)-IPBM is
a
NH
derivative of reboxetine that is iodinated at position 2 of the
phenoxy ring with Ki value of 4.2 nM. Ex vivo experiments of
125I-IPBM showed high levels of radioactivity in the LC and the
anteroventricular thalamic nucleus and regional distribution
correlated well with reported 3H-nisoxetine binding.25 The same
chemical structure 7, radiolabeled with iodine-123 referred as
123I-INER (Ki = 0.84 nM) was found to have comparable properties
reported for MIPP.26 This group has recently reported additional
carbon-11 and fluorine-18 derivatives of reboxetine, with
promising properties for carbon-11 labeled analogs.27
CH3
10a. MFP3; 10b. 18F-MFP3
Figure 2. Chemical structure of (R)-N-Methyl-3-(30-fluoropropyl)phenoxy)-3-phe-
nyl-propanamine (MFP3, 10a) and (R)-N-Methyl-3-(18F-30-fluoropropyl)phenoxy)-3-
phenyl-propanamine (18F-MFP3 10b).
Our goal was to develop a fluorine-18 labeled analog of
nisoxetine for the following reasons: (1) Nisoxetine has a high
affinity for NET and has been shown to bind to the transporter
sites selectively4; (2) As the clearance of the reported agents,
including 11C-nisoxetine, from nonspecific binding regions is
slow, longer imaging times with fluorine-18 will be needed; (3).
Concentration of NET in the brain is low, thus high specific
activity of the radiotracer would be desired28; (4) Pharmacolo-
gical challenge experiments may be carried out with greater
ease with the longer half-life; (5) Fluorine-18 is suitable for high-
resolution PET studies; (6) Fluorine-18 is clinically more
amenable because centers without a cyclotron may be able to
use these agents; and (7) As defluorination has been an issue
with O-fluoroalkyl derivatives of reboxetine,20 a C-fluoroalkyl
derivative may be more stable to defluorination. We report here
synthesis of a fluorine-18 radiolabeled derivative (methoxy
group in nisoxetine was replaced with a fluoropropyl group) to
provide (R)-N-methyl-3-(30-fluoropropyl)phenoxy)-3-phenylpro-
panamine (Figure 2, 10a; MFP3) as a potential NET imaging
agent for PET when radiolabeled to give (R)-N-methyl-3-(30-
[18F]fluoropropyl)phenoxy)-3-phenylpropanamine (Figure 2,
10b; 18F-MFP3).
OH
OH
a
NCH3
BOC
NHCH3
12
11
b
13
OH
OH
c
O
O
NCH3
BOC
NCH3
BOC
15
14
d
OTs
F
e
O
O
NCH3
BOC
NHCH3
10a, MFP3
Results and discussion
16
Our goal has been to develop a fluorine-18 labeled derivative for
imaging NET in the living brain. Previous efforts have focused
on carbon-11 and fluorine-18 derivatives of nisoxetine and
reboxetine. These derivatives were radiolabeled by either
Figure 3. Chemical synthesis of MFP3: (a) Di-tert-butyl dicarbonate, THF, 0–51C,
90 min; (b) 13, Ph3P, DIAD, THF, 0–51C-221C, 24 h; (c) (i) BH3-THF, 0–51C-221C,
24 h, (ii) H2O2, NaOH, 221C, 12 h; (d) p-TsCl, Et3N, CH2Cl2, 221C, 24 h; (e) TBAF, THF,
681C, 1.5 h.
J. Label Compd. Radiopharm 2010, 53 172–177
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

V. L. Nguyen et al.
N-methylation or O-methylation using 11C-methyl iodide or by
O-fluoroalkylation using 18F-fluoroalkyl groups. Our approach in
this work was to develop a C-fluoropropyl derivative, since our
previous work on dopamine and nicotine receptor imaging
agents has demonstrated their in vivo stability, particularly
towards defluorination.29,30 For this purpose, we undertook the
synthesis of MFP3, Figure 2, 10a, which is an analog of
nisoxetine (where the methoxy group in nisoxetine, Figure 1, 1
is replaced by the fluoropropyl group) or MIPP (where the iodine
in MIPP, Figure 1, 2 is replaced by the fluoropropyl group). As
nisoxetine appears to be tolerant to the replacement of the
methoxy group (as in tomoxetine, Figure 1, 3) and reboxetine
appears to retain affinity upon incorporation of two carbon
chain at the position (Figure 1, 5, 6), we envisaged that MFP3
would maintain binding characteristics to NET sites.
100
80
60
40
20
0
Nisoxetine
MFP-3
10-12
10-11
10-10
10-9
10-8
10-7
10-6
10-5
10-4
The synthesis of MFP3 and the tosylate 16 were carried out
using general procedures for this class of compounds (Figure 3).
As the R-isomer of nisoxetine is more pharmacologically active,
we started out with (S)-N-methyl-3-ol-3-phenylpropanamine 11
reported previously.31 Using previously synthesized (S)-N-methyl-
3-ol-3-phenylpropanamine31 the secondary amine was protected
using the tert-butyloxycarbonyl group in modest yields.32
This N-BOC-protected secondary alcohol was coupled with
2-allylphenol using the Mitsunobu reaction. The yields were in
the range of approximately 40% for this reaction which was carried
out in the presence of diisopropyl azodicarboxylate (DIAD).
Similar yields were observed with other phenol–alcohol coupling
reactions.33 The reaction provided (R)-N-tert-butyloxycarbonyl-
N-methyl-3-(20-allyl)phenoxy)-3-phenylpropanamine 14, since an
inversion of configuration occurs in the Mitsunobu reaction.34
Hydroboration of the allyl group followed by peroxide treatment
under basic conditions provided the propanol derivative 15 in
modest yields. The alcohol was treated with p-toluenesulfonyl
chloride in the presence of triethylamine, using previously des-
cribed methods.30 Yields of this reaction were surprisingly poor. It
is unclear whether steric effects contributed to the low yields of the
tosylate. Nucleophilic substitution of the tosylate with TBAF
followed by deprotection of the N-BOC protecting group with
trifluoroacetic acid (TFA) provided MFP3, 10a in modest yields.
We performed in vitro experiments on rat brain homogenates
in order to measure the binding affinity of MFP3. Competition
Log [drug, mol/L]
Figure 4. Specific Binding of 3H-Nisoxetine and MFP-3. Increase concentration of
the drug, decrease binding affinity. MFP-3: IC50 = 2.30 ꢀ 10ꢁ8 M. 3H-Nisoxetine:
IC50 = 8.02 ꢀ 10ꢁ9 M.
Deprotection of the N-BOC group was accomplished with a
strong acid, TFA, similar to the procedures we have used for the
nicotinic receptor radioligands.33 After the reaction, the neutra-
lized mixture was separated by reverse-phase high-performance
liquid chromatography (HPLC) purification. It must be noted that
at a radioactive peak at approximately 7 min (Figure 4(b)) there
was a major volatile side-product. This may be a result of the
breakdown of the ether linkage under the strong acidic
conditions of deprotection. To minimize this, milder conditions
will have to be explored. The retention time of 18F-MFP3 was
18min (Figure 5(b)). The total synthesis time was 2.5 h with
radiochemical yields of 15–20%, decay corrected. The specific
activities of 18F-MFP3 were 1500–2000 Ci/mmol.
In vitro and in vivo binding studies are currently underway
with 18F-MFP3 in order to evaluate its ability to bind to NET and
if this binding can be displaced by nisoxetine.
Materials and methods
General
studies were performed using different concentrations (between All chemicals and solvents were of analytical or HPLC grade from
1 nM and 50 mM) of the NET-selective agent nisoxetine and the Aldrich Chemical Co. and Fisher Scientific. Nisoxetine was
cold compound MFP3 (Figure 4). The reported Ki value for purchased from Sigma Biochemicals. Electrospray mass spectra
nisoxetine is 1–1.5 nM25,35 and is lower compared with our (MS) were obtained on a Model 7250 mass spectrometer
findings of 8.02 nM for nisoxetine. The Ki of 23 nM for MFP3 (Micromass LCT) and proton NMR spectra were recorded on a
suggests that MFP3 is about three times weaker in binding Bruker OMEGA 500 MHz spectrometer. Analytical thin layer
compared with nisoxetine. Incorporation of the 3-carbon long chromatography (TLC) was carried out on silica-coated plates
propyl group could potentially result in some steric effects in the (Baker-Flex, Phillipsburg, NJ). Chromatographic separations were
binding site resulting in a lower affinity. Similar lowering of carried out on preparative TLC (silica gel GF 20 ꢀ 20 cm 2000 mm
affinity was reported in fluoroalkyl derivatives of reboxetine.27
thick; Alltech Assoc. Inc., Deerfield, IL) or silica gel flash column
Radiosynthesis of 18F-MFP3 involved two steps as described or semi-preparative reverse-phase columns using the Gilson
in Figure 5(a). The first step involves incorporation of fluorine-18 HPLC systems. High specific activity aqueous 18F-fluoride was
into (R)-N-tert-butyloxycarbonyl-N-methyl-3-(30-tosyloxypropyl)- produced in the MC-17 cyclotron or the CTI RDS-112 cyclotron
phenoxy)-3-phenylpropanamine 16, and the second step using the 18O (p,n)18F reaction. The high specific activity
involved deprotection of the N-BOC-protected amine group. 18F-fluoride was used in subsequent reactions which were
The radiolabeling step by 18F-fluorine was carried out using the carried out in automated radiosynthesis units (either a chemistry
nucleophilic substitution reaction in the presence of Kyptofix processing control unit (CPCU) or a nuclear interface fluorine-18
and K2CO3 in acetonitrile at 951C for 30min. This reaction module). Fluorine-18 radioactivity was counted in a Capintec
provided high, reproducible radiochemical yields as previously dose calibrator and radioactive thin layer chromatographs were
observed in the case of propyl tosylates.29,30 After radiolabeling, obtained by scanning in a Bioscan system 200 Imaging scanner
product was taken directly to the second step of deprotection. (Bioscan, Inc., Washington, DC). Tritium was assayed by using a
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 172–177

V. L. Nguyen et al.
18F
OTs
1. 18F, K2CO3,
Kryptofix, CH3CN
O
O
2. CF3CO2H
NHCH3
10b, 18F-MFP3
NCH3
BOC
16
(a)
(b)
Figure 5. (a) Radiosynthesis of 18F-MFP3 (b) HPLC chromatogram of 18F-MFP3 purification (Reverse phase C18, CH3CN:H2O 0.25%Et3N, 68%:32%; flow rate = 2.5 mL/min).
The retention time of 18F-MFP3 was 18 min.
Packard Tri-Carb Liquid scintillation counter with 65% efficiency. triphenylphosphine (0.27 g, 1 mmol) in THF (10 mL), stirred
All animal studies were approved by the Institutional Animal under nitrogen and cooled at 01C, DIAD (0.22 mL, 1.1 mmol) in
Care and Use Committee of University of California-Irvine.
THF (2 mL) was added. The reaction mixture was stirred at room
temperature for 24 h and evaporated until dried. The residue
was purified by preparative TLC (30% EtOAc in hexane) to give
pure product (R)-N-BOC-N-methyl-3-(20-allyl)phenoxy)-3-phenyl-
propanamine 14 0.15 g (42% yield). MS, m/z, 382 (9%, [M1H]1),
404 (100%, [M1Na]1). NMR (500 MHz; CDCl3; d ppm): 7.65–7.69
(m, 3H, Ar); 7.56–7.53 (m, 1H, Ar); 7.48–7.45 (m, 3H, Ar); 7.12–7.07
(m, 1H); 6.86–6.83 (m, 1H); 5.12 (m, 1H), 4.98 (m, 2H), 4.12 (m, 1H,
CH); 2.83 (s, 3H, NCH3); 2.81 (m, 2H, CH2N); 1.74 (m, 2H, CH2 b to
N); 1.26 (s, 9H, t-butyl).
Chemistry
(S)-N-tert-butyloxycarbonyl-N-methyl-3-ol-3-phenylpropanamine (12)
The synthesis of (S)-N-methyl-3-ol-3-phenylpropanamine (11) was
previously described.31 The secondary amine 11 (0.62 g;
3.74mmol) was dissolved in tetrahydrofuran (8 mL) and cooled
to 0–51C. Dissolved di-tert-butyl dicarbonate (1.33 g; 6.1 mmol) in
tetrahydrofuran (8mL) was further added to this cold (0–51C)
mixture and allowed to react for 90min. The reaction was then
(R)-N-tert-butyloxycarbonyl-N-methyl-3-(30-propanol)phenoxy)-3-
permitted to reach ambient temperature and allowed to react phenylpropanamine (15)
overnight. Tetrahydrofuran was removed and 20mL of dichlor-
The olefin, (R)-N-BOC-N-methyl-3-(20-allyl)phenoxy)-3-phenylpro-
omethane was added and washed with saturated NaHCO3
(2ꢀ 20mL) and water (1ꢀ 20mL). The organic layer was dried
(magnesium sulfate) and filtered while the crude product was
purified on a silica column (30% ethyl acetate in hexane) to
provide 0.56g of pure (S)-N-BOC-N-methyl-3-ol-3-phenylpropana-
mine, 12 (57% yield) comparable to previous findings.32 MS, m/z,
266 (9%, [M1H]1), 288 (100%, [M1Na]1). NMR (500MHz; CDCl3;
d ppm): 7.30–7.36 (m, 5H, Ar); 4.59 (br, 1H, CH); 2.87 (s, 3H, NCH3);
2.81 (m, 2H, CH2N); 1.68 (m, 2H, CH2 b to N); 1.42 (s, 9H, t-butyl).
panamine 14 (0.11 g, 0.28 mmol) in THF (5 mL) was treated with
borane-tetrahydrofuran complex (0.58 mL, 0.3 mmol) drop wise
at 01C stirred under nitrogen atmosphere. The reaction mixture
was stirred at room temperature over night and then was
treated with NaOH (3N, 5 mL) followed by the addition of 30%
H2O2 (200 mL) at 01C. This mixture was stirred at room
temperature for 12 h and dried. The residue was poured into
saturated ammonium chloride solution and extracted with ethyl
acetate. The organic layer was dried over MgSO4 and the solvent
evaporated. The residue was purified by silica column chroma-
tography (30% ethyl acetate in hexane) to furnish alcohol,
(R)-N-BOC-N-methyl-3-(30-propanol)phenoxy)-3-phenylpropan-
amine 15 (55 mg, 49% yield). MS, m/z 400 (8%, [M1H]1), 422
(100%, [M1Na]1). NMR (500 MHz; CDCl3; d ppm) 7.71–6.75 (m,
(R)-N-tert-butyloxycarbonyl-N-methyl-3-(20-allyl)phenoxy)-3-phe-
nylpropanamine(14)
To a solution of (S)-N-BOC-N-methyl-3-ol-3-phenylpropanamine
12 (0.25 g, 0.94 mmol), 2-allylphenol 13 (0.14 g, 1 mmol) and
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V. L. Nguyen et al.
9H, Ar); 3.40 (m, 2H, CH2–OH); 2.83 (s, 3H, NCH3); 2.35 (m, 2H,
CH2N); 1.66 (m, 2H, CH2 b to N); 1.30 (s, 9H, t-butyl).
Using 3H-nisoxetine (nisoxetine hydrochloride N-methyl 3H,
85 Ci/mmol, Perkin-Elmer, Boston, MA) competition assays were
performed using 100 mL of tissue, 50 mL of 3H-nisoxetine (1.0 nM)
and 25 mL of various drug concentrations in buffer B, with the
final volume made up to 500 mL per tube using buffer B.
Incubation was carried out at 41C for 4 h. The incubation was
terminated by rapid vacuum filtration through Whatman GF/C
filter paper (presoaked in 0.1% polyethyleneimine for 60 min)
using Brandel tissue harvester. The filters were rapidly washed
three times (4 mL each) with cold buffer B and the filters were
suspended in 10 mL of Bio-Safe II scintillation fluid and counted
for 10 min in the scintillation counter. The experiments were
done in duplicates. Data was analyzed using following
procedure: (1) the nonspecific binding of 3H-nisoxetine (mea-
sured using 10 mM nisoxetine) was subtracted for all samples;
(2) the specific binding was normalized to 100% (no competitive
ligand); and (3) the binding isotherms were fit to the Hill
equation (KELL BioSoft software (v 6), Cambridge, UK) to provide
inhibitor concentration (IC50), which is the inflection point of
the isotherm where 50% of the 3H-nisoxetine binding (Figure 4).
The Ki was calculated by the Cheng-Prussof equation and using
(R)-N-tert-butyloxycarbonyl-N-methyl-3-(30-tosyloxypropyl)phe-
noxy)-3-phenylpropanamine (16)
The alcohol, (R)-N-BOC-N-methyl-3-(30-propanol)phenoxy)-3-
phenylpropanamine 15 (50 mg, 0.13 mmol) was reacted with
p-toluenesulfonyl chloride (25 mg, 0.13 mmol) in the presence of
Et3N (60 mL) in CH2Cl2 for 24 h at room temperature. Solvent was
removed by rotary evaporation. Dichloromethane was added
and washed with saturated NaHCO3 and water. The organic
layer was removed, dried with MgSO4, and filtered to give (R)-N-
BOC-N-methyl-3-(30-tosyloxypropyl)phenoxy)-3-phenylpropana-
mine, 16 which was purified by silica column chromatography
using 5–30% ethyl acetate in hexane to provide 14 mg of pure
product (19% yield). MS, m/z, 554 (58%. [M1H]1), 576 (100%. [M1
Na]1). NMR (500 MHz; CDCl3; d ppm) 7.81–7.78 (m, 3H, Ar);
7.75–7.70 (m, 1H, Ar); 7.36–7.31 (dd, 4H, tosyl ArH-2,3,5,6);
7.30–7.28 (m, 3H, Ar); 6.96–6.94 (m, 1H); 6.74–6.71 (m, 1H); 4.10
(m, 2H, CH2–OSO2); 3.95 (m, 1H, CH) 2.81 (s, 3H, NCH3); 2.47 (m, 2H,
CH2N); 2.44 (s, 3H, tosyl CH3); 1.65 (m, 2H, CH2 b to N); 1.32 (s, 9H,
t-butyl).
3
the value of KD as 1 nM for H-nisoxetine.35,36
(R)-N-methyl-3-(30-fluoropropyl)phenoxy)-3-phenylpropanamine (10a)
Radiosynthesis
The radiosynthesis of 18F-MFP3 was carried out using an
automated synthesis procedure using a tosylate precursor
which employs a CPCU (Figure 5(a)). Fluorine-18 in H128O from
a MC-17 cyclotron was passed through a QMA-light sep-pak
(Waters Corp. Milford, MA), preconditioned with 3 mL of K2CO3,
140 mg/mL, followed by 3 mL of anhydrous acetonitrile. The
fluorine-18 trapped in the QMA-light sep-pak was then eluted
(using nitrogen gas) with 1 mL Kryptofix222/K2CO3 (360 mg/
75 mg in 1 mL of water and 24 mL of acetonitrile) and transferred
to the CPCU reaction vessel. The ‘SYNTH1’ program in the CPCU
was used for the synthesis. This involved initial drying of the
18F-fluoride, Kryptofix, and K2CO3 mixture at 1201C for 10 min.
Subsequently, acetonitrile (2 mL) from CPCU reagent vial 2 was
added and evaporated at 1201C for 7 min to ensure dryness
of the 18F-fluoride mixture. Following this, the precursor, (R)-N-
tert-butyloxycarbonyl-N-methyl-3-(30-tosyloxypropyl)phenoxy)-3-
phenylpropanamine, 16 (1-2 mg in 0.5 mL of anhydrous
acetonitrile continued in CPCU reagent vial 3) was added and
the reaction went for 15–30 min at 961C. Subsequent to the
reaction, CH2Cl2 (7 mL contained in CPCU reagent vial 4) was
added to the mixture and the CH2Cl2 contents were passed
through a neutral alumina sep-pak (prewashed with CH2Cl2 in
order to remove any unreacted 18F-fluoride). The collected
CH2Cl2 solution coming out of the CPCU now contained 18F-N-
BOC-MFP3. The solvent was removed and the residue was taken
in dichloromethane (1 mL) and trifluroacetic acid (0.2 mL). The
solution was heated at 981C for 30 min and evaporated until
dried. The residue was neutralized with 10% NaHCO3 solution
until pH of 7.0 was obtained. Semipreparative HPLC purifica-
tion was performed using an Alltech C18 column (10 mm,
250 ꢀ 10 mm) and UV detector (254 nm), mobile phase 60%
acetonitrile: 40% water containing 0.1% triethylamine with a
flow rate of 2.5 mL/min. The retention time of 18F-MFP3 was
found to be 18 min (Figure 5(b), HPLC chromatogram). The
18F-MFP3 fraction was collected into a flask and the solvent was
removed in vacuo using a rotary evaporator. The radiosynthesis
was accomplished in 2.5 h with an overall radiochemical yield of
The tosylate, (R)-N-BOC-N-methyl-3-(30-tosyloxypropyl)phenoxy)-
3-phenylpropanamine 16 (20 mg, 36.1 mmol) in THF (0.5 mL) was
treated with a solution of tetrabutylammonium fluoride TBAF.
THF (1.0 M, 54 mL). The solution was heated at 681C for 1.5 h. The
reaction mixture was cooled to room temperature and dried.
The residue was titrated with ether (3 ꢀ 5 mL) three times until
none of the product showed in the TLC. The ether solution
was concentrated to dryness. The residue was purified by
preparative TLC (1:1 hexane and ethyl acetate) to provide (R)-N-
BOC-N-methyl-3-(30-fluoropropyl)phenoxy)-3-phenylpropanamine
(7.2mg, 49% yield).
Deprotection of (R)-N-BOC-N-methyl-3-(30-fluoropropyl)phe-
noxy)-3-phenylpropanamine was carried out by treatment with
TFA. The N-BOC derivative (7 mg, 17.9 mmol) was dissolved in
dichloromethane (1 mL) and TFA (0.1 mL) was added at ambient
temperatures. The reaction solution was stirred at ambient
temperatures for 24 h and then dried. The resulting residue was
neutralized with saturated NaHCO3 and its product was
extracted into ether. The ether layer was dried (MgSO4), filtered,
and the crude product was purified on preparative TLC to
provide (R)-N-methyl-3-(30-fluoropropyl)phenoxy)-3-phenylpro-
panamine 10a (1.5mg, 28% yield). MS, m/z, 302 (100%. [M1H]1).
NMR (500MHz; CDCl3; d ppm) 7.32–6.65 (m, 9H); 4.52 (dt, 2H,
CH2F); 4.2 (m, 1H, CH); 2.88 (s, 3H, NCH3); 2.6 (br, 2H, CH2N); 2.2
(br, 2H, benzylic CH2); 1.59 (br, 2H, CH2 b to N); 1.35 (br, 2H, CH2).
In vitro binding affinity
Using prior published methods,25 rat cerebral cortex from male
Sprague-Dawley rats was dissected and homogenized using
Tekmar tissumizer (15 s, half maxima speed) in buffer A
(containing 50 mM Tris-HCL, 120 nM NaCl, and 5 nM KCl at a
pH of 7.4) using 1:100 (wt:vol). This homogenized mixture was
then centrifuged at 28,000g for 10 min at 41C. The procedure
was repeated twice with the final pellet suspended in buffer B
(containing 50 mM Tris-HCl, 300 mM NaCl, and 5 mM KCl at a pH
of 7.4).
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 172–177

V. L. Nguyen et al.
approx. 15% decay corrected. Specific activity was measured to
be 42000 Ci/mmol.
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Conclusion
The new NET radioligand, (R)-N-methyl-3-(18F-30-fluoropropyl)-
phenoxy)-3-phenylpropanamine (18F-MFP3) was prepared suc-
cessfully. The compound showed moderate affinity for the NET
pathway in rat brain homogenates. Potential use of 18F-MFP3 in
the study of the NET pathway as well as in evaluating SNRI/NRI
antidepressants remains to be determined.
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Acknowledgements
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H. Nishimura, H. Saji, Nucl. Med. Biol. 2003, 30, 697.
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This work was supported by NIH RO1 EB006110 from the
National Institute of Biomedical Imaging and Bioengineering.
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J. Label Compd. Radiopharm 2010, 53 172–177
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