Synthesis and in vivo evaluation of [18F]N-(2- benzofuranylmethyl)-N′-[4-(2-fluoroethoxy)benzyl]piperazine, a novel σ1 receptor PET imaging agent

Article abstract of DOI:10.1016/j.bmcl.2011.09.028

N-(2-Benzofuranylmethyl)-N′-[4-(2-fluoroethoxy)benzyl]piperazine (6, σ₁ K_i = 2.6 nM) was radiolabeled with fluorine-18 to provide a potential σ₁ receptor radioligand for use in positron emission tomography (PET). Radiofluo

Full text of DOI:10.1016/j.bmcl.2011.09.028

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6820–6823
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vivo evaluation of [18F]N-(2-benzofuranylmethyl)-
N0-[4-(2-fluoroethoxy)benzyl]piperazine, a novel r1 receptor PET imaging agent
Iman A. Moussa a, Samuel D. Banister a, Nicolas Giboureau b,c, Steven R. Meikle b,c, Michael Kassiou a,b,c,
⇑
a School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
b Brain and Mind Research Institute, Sydney, NSW 2050, Australia
c Discipline of Medical Radiation Sciences, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
N-(2-Benzofuranylmethyl)-N0-[4-(2-fluoroethoxy)benzyl]piperazine (6, r1 Ki = 2.6 nM) was radiolabeled
with fluorine-18 to provide a potential 1 receptor radioligand for use in positron emission tomography
(PET). Radiofluorination of the appropriate tosylate precursor furnished [18F]6 with a specific activity of
45 GBq/ mol, in an average radiochemical yield of 18% and greater than 98% radiochemical purity. Micro-
PET imaging in Papio hamadryas baboon brain revealed [18F]6 uptake consistent with
receptor distri-
bution, and specificity for
receptors was demonstrated in a haloperidol pre-treated animal. [18F]6
Article history:
Received 28 July 2011
Revised 7 September 2011
Accepted 7 September 2011
Available online 13 September 2011
r
l
r
r
Keywords:
Piperazines
Sigma receptors
Positron emission tomography
Fluorine-18
possesses suitable properties for PET imaging of
tracer is warranted.
r
1 receptors, and further investigation of this
r1 receptor
Ó 2011 Elsevier Ltd. All rights reserved.
The
r
receptors—subdivided into
r
1 and
r
2 subtypes—represent
to non-invasively image cerebral r1 receptors has prompted the
development of numerous r1 radioligands for use in PET.25
One of the earliest fluorinated radioprobes with selectivity for
the r1 receptor was [18F]FPS ([18F]1, Fig. 1), which possessed suit-
able lipophilicity (LogP7.5) for penetration of the blood–brain bar-
rier.26 Evaluation of [18F]1 in PET imaging studies in rat revealed
high specific binding in the brain, and biodistribution in the
unique mammalian proteins that are widely distributed in both the
central and peripheral nervous systems (CNS and PNS, respec-
tively).1–4 While less is known about the
r2 receptor, the r1 recep-
tor has been relatively well characterized biochemically and
pharmacologically,5–7 and its genetic sequence in humans has been
reported.8 It is believed that r1 receptors primarily reside at the
interface between the endoplasmic reticulum (ER) and mitochon-
dria, and modulate Ca2+ flux by acting as molecular chaperones
for type 3 inositol-1,4,5-triphosphate receptors.9,10 However, r1
receptors can translocate to the plasma membrane where they reg-
ulate voltage-dependent Ca2+ channels, K+ channels, and other
membrane-bound proteins, accounting for their diverse pharma-
cology.11–13
peripheral nervous system (PNS) consistent with
r receptor
expression patterns.27 Radiotracer [18F]1 has been evaluated in ba-
boon and human brain.28,29 Despite adequate uptake and clear-
ance, and >90% saturable binding of [18F]1 in baboon brain,28 PET
scanning in healthy participants revealed that the r1 affinity of
[
18F]1 is so high that it does not reach transient equilibrium by
4 h post-administration.30–32
The r1 receptor has been increasingly implicated in a range of
CNS diseases, including affective disorders,14–16 psychosis and
schizophrenia,17 substance abuse,18–21 Parkinson’s disease,22 and
Alzheimer’s diseases.23 Although the r1 receptor has generated
much interest as novel drug target for multiple CNS disorders,
the precise mechanistic details linking r1 receptors to many of
the aforementioned diseases are unknown. The ability to image
r1 receptors in vivo using positron emission tomography (PET)
would enable a better understanding of disease processes in which
Piperidine-derived fluorobenzamide 2, a high affinity, selective
r1 ligand has also been labeled with fluorine-18.33 Anatomical
distribution of [18F]2 in the rat brain showed uptake in r1 rich re-
gions—brain stem, cerebellum, hippocampus—and the radioactivity
levels in these regions were significantly reduced by pretreatment of
rats with the high affinity
r
ligand, haloperidol.33 More recently,
[
18F]fluspidine ([18F]3) was evaluated as a potential r1 receptor
PET radiotracer in mice, where it showed high specific binding in
the brain, and distribution consistent with the known distribution
r
1 receptors are involved, and how
r
1 receptor expression or func-
of r
1 receptors.34
tion changes during the course of disease progression.24 This desire
In addition to the piperidine heterocycle, another common scaf-
fold for many r1 PET tracers is piperazine, exemplified by
18F]FESA5845 ([18F]4). [18F]FESA5845 was evaluated as a
1 recep-
tor radioligand in the monkey brain, demonstrating high specific
[
r
⇑
Corresponding author.
E-mail address: michael.kassiou@sydney.edu.au (M. Kassiou).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.028

I. A. Moussa et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6820–6823
6821
N
H
N
O
O
N
F
F
N
1
2
σ1 Ki = 4.3 nM
σ2 Ki = 144 nM
σ1 K = 3.39 nM
i
σ2 Ki = 406 nM
O
O
F
F
F
N
N
O
N
3
4
σ1 Ki = 0.59 nM
σ2 Ki = 785 nM
σ1 IC50 = 3.1 nM
σ2 IC50 = 6.8 nM
Figure 1. Previously reported fluorine-18-labelled r1 receptor PET tracers.
binding and distribution consistent with
Although 4 is a subtype non-selective ligand, many reports have
described [18F]4 as a potential tumor imaging agent37,38 since both
r
1-rich regions.35,36
for this chemotype. Fluorine-18 also possesses relatively low pos-
itron energy (0.64 MeV) with respect to carbon-11 (0.96 MeV),
which allows PET imaging to be performed with the highest
resolution.42
r
r
receptor subtypes are considered to be cancer-associated
biomarkers.39,40
Cold standard 6 was synthesized by O-alkylation of N-(2-ben-
In our efforts to design and develop increasingly selective r1
radiotracers based on the differential arylalkyl disubstitution of
1,4-piperazine, we recently reported a series of N-(2-benzofura-
nylmethyl)-N0-(alkoxybenzyl)piperazines as selective r1 receptor
ligands.41 Most analogs within this series showed a distinct prefer-
zofuranylmethyl)-N0-(4-hydroxybenzyl)piperazine (7) with 2-flu-
oroethyl tosylate (Scheme 1).41 The synthesis of
[
18F]6 was
achieved in two steps from 7. Mono-alkylation of 7 with 1,2-ethyl-
ene glycol bistosylate furnished tosylated precursor 8. Radiofluori-
nation of
Nucleophilic aliphatic substitution of 8 with cyclotron-produced
[
18F]fluoride as a [18F]KF-Kryptofix-222 complex in acetonitrile at
8 was achieved using a Tracerlab FXF–N module.
ence for the r1 receptor and the most potent compounds were the
4-methoxy (5, Fig. 2) and 4-(2-fluoroethoxy) derivatives (6) deriv-
atives, with Ki values of 2.7 and 2.6 nM, respectively. Methyl ether
5 was identified as a suitable candidate for carbon-11 radiolabel-
ing, in order to assess the in vivo pharmacokinetic profile of this
class of compounds in a healthy baboon. Carbon-11-labeled 5
showed good permeation and distribution in normal baboon
brain,41 consistent with the expression pattern of r1 receptors.3
120 °C for 10 min, followed by reverse-phase semipreparative
HPLC purification, gave [18F]6 in an overall average synthesis time
of 45 min and 18% non-decay corrected radiochemical yield. Co-
injection of nonradioactive 6 was performed using analytical HPLC
to confirm the identity of the product. In the final product, both
radiochemical and chemical purities were greater than 98%, with
Although [11C]5 showed good tissue uptake and selectivity for
r
a specific activity of 45 GBq/lmol. No attempts were made to opti-
receptors in vivo, the relatively short half-life of carbon-11 (t1/
2 = 20 min) limits the use of this radioisotope for pharmacological
evaluation of this class of compounds in living tissue. To further
mize these conditions, as sufficient quantities of the radioligand
were produced to enable preliminary pharmacological evaluation.
Formulation of radiolabeled product was achieved by dilution
of the radioactive fraction of the HPLC mobile phase with water
for injection (WFI, 50 mL). The aqueous phase was passed through
a tC-18 Sep-Pak cartridge, and the trapped the radiolabeled prod-
uct was rinsed with WFI (5 mL). The radiolabeled product was
eluted from the tC-18 Sep-Pak cartridge with EtOH (1 mL) and
WFI (2 mL), the resulting solution was filtered through a sterile
investigate the in vivo pharmacokinetics of this class of
r ligands,
we sought to incorporate fluorine-18 (t1/2 = 109.8 min) into 6,
which possessed higher r1 affinity and selectivity (r1 Ki = 2.6 nM,
r2/r1 = 187), and reduced off-target CNS affinity at 5-HT2B and
D2 dopamine receptors, compared to 5.41 Fluorine-18 is often
considered a superior positron emitter to carbon-11. The longer
half-life of fluorine-18 allows extended imaging studies, ultimately
providing more accurate assessment of biodistribution and kinetics
0.22 lm filter, and the residue was reconstituted in saline solution
(10 mL). Radiochemical stability was followed for 5 h at room tem-
N
N
N
N
F
O
O
O
O
5
6
σ1 Ki = 2.7 0.4 nM
σ2 Ki = 103 18 nM
5-HT2B Ki = 15 2 nM
σ1 Ki = 2.6 0.6 nM
σ2 Ki = 486 151 nM
5-HT2B Ki = 96 13 nM
D2 Ki >10000 nM
D2 K = 2700 295 nM
i
Figure 2. r1 Selective N-(2-benzofuranylmethyl)-N0-(4-(alkoxybenzyl)piperazines 5 and 6.

6822
I. A. Moussa et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6820–6823
N
a
N
N
N
F
O
O
OH
O
7
6
b
c
N
N
N
OTs
N
18 F
O
O
O
O
8
[
18F]6
Scheme 1. Reagents and conditions: (a) TsOCH2CH2F, K2CO3, DMF, reflux, 16 h, 60%; (b) TsOCH2CH2OTs, K2CO3, DMF, reflux, 10 h, 61%; (c) [18F]KF, Kryptofix-222, CH3CN,
120 °C, 10 min, followed by HPLC purification (LichroCART).
perature and was greater than 98% after 5 h. The final injectable
solution was clear and colorless, with a pH of 7.0. The preparation
was free from labeling precursor 8. Administration to the animal
was performed within 30 minutes following the end of synthesis.
Prior to attempting the radiosynthesis of [18F]6 for microPET
imaging, it was necessary to examine the lipophilicity of 6. To en-
sure high uptake in the brain and to minimize non-specific binding,
it is believed that the optimum logD value for therapeutic CNS-ac-
tive compounds is between 2 and 3.5.43 To address this issue, the
logD value of piperazine 6 was evaluated using previously de-
scribed HPLC methods.41 In short, a calibration curve was obtained
by correlating known logD values with the retention times of sev-
eral reference compounds, and the average retention time of 6 over
three runs, gave a logD value of 3.35.
[
18F]6 was observed in the cortex, striatum, thalamus and cerebel-
lum, which are known to contain
receptors.3 This is in agreement
r
with previous baseline studies performed using the fluorine-18
radiotracers depicted in Figure 1.
The in vivo specificity of [18F]6 uptake was further evaluated in
a single blocking study, conducted three weeks after the initial
microPET evaluation in the same baboon. Pretreatment with halo-
peridol (1 mg/kg, i.v.) 5 min prior to [18F]6 administration resulted
in increased radioligand uptake within three minutes p.i., followed
by a rapid decline to washout levels within 5 min p.i. (Fig. 4b). The
net result was an 80% reduction in radioligand uptake in all regions
of the brain at the conclusion of the imaging experiment when
compared to [18F]6 administration alone. Complete washout of
the radioligand in the blocking study demonstrated the in vivo
MicroPET studies were conducted with an anaesthetized Papio
hamadryas baboon to evaluate the in vivo regional distribution
kinetics of [18F]6. Dynamic microPET brain imaging commenced
just prior to intravenous administration of [18F]6 (100 MBq in
3 mL of saline) and was terminated 60 min post-injection (p.i.).
The microPET images confirmed the ability of [18F]6 to penetrate
the blood–brain barrier with considerable accumulation of radio-
activity in the baboon brain (Fig. 3).
specificity of [18F]6 for
r receptors. Similar in vivo pharmacoki-
netic profiles have been reported in our [11C]5 microPET studies,41
as well as for other haloperidol blocking studies,44–46 performed
with the aforementioned fluorine-18 radiotracers shown in Figure
1.
In conclusion, 6 is a highly selective r
1 receptor ligand,41 of suit-
able lipophilicity for use as a potential r1 receptor PET imaging
agent. The corresponding fluorine-18 PET radioligand, [18F]6, has
been reliably prepared and showed good uptake and specific bind-
ing to r1 receptors in the P. hamadryas brain. These data demon-
strate that [18F]6 exhibits promising characteristics for in vivo
use and may provide a superior brain imaging radiotracer.
Time activity curves representing the uptake of [18F]6 in the ba-
boon brain are shown in Figure 4. In a single baseline study, [18F]6
reached maximal levels within 5 min p.i., and remained at a pla-
teau to the end of the scan ( Fig. 4a). Homogenous uptake of
Figure 3. P. hamadryas baboon microPET summation images of selected transaxial brain slices over 60 min.

I. A. Moussa et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6820–6823
6823
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Acknowledgment
The authors would like to thank Ms. Sheruna Naidoo-Variawa
and Mr. Wesley Ng Ping Man for their assistance with the microPET
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.028.
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