FE@SNAP - A new PET tracer for the melanin concentrating hormone receptor 1 (MCHR1): Microfluidic and vessel-based approaches

Article abstract of DOI:10.1016/j.bmc.2012.07.051

Changes in the expression of the melanin concentrating hormone receptor 1 (MCHR1) are involved in a variety of pathologies, especially obesity and anxiety disorders. To monitor these pathologies in-vivo positron emission tomography (PET) is a suitable met

Full text of DOI:10.1016/j.bmc.2012.07.051

Bioorganic & Medicinal Chemistry 20 (2012) 5936–5940
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
[18F]FE@SNAP—A new PET tracer for the melanin concentrating hormone
receptor 1 (MCHR1): Microfluidic and vessel-based approaches
Cécile Philippe a,b,1, Johanna Ungersboeck a,c,1, Eva Schirmer d, Milica Zdravkovic b, Lukas Nics a,e
Markus Zeilinger a, Karem Shanab d, Rupert Lanzenberger f, Georgios Karanikas a, Helmut Spreitzer d,
,
Helmut Viernstein b, Markus Mitterhauser a,b,g, Wolfgang Wadsak a,c,
⇑
a Radiochemistry and Biomarker Development Unit, Department of Nuclear Medicine, Medical University of Vienna, Waehringer Guertel 18-20, Vienna 1090, Austria
b Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Vienna 1090, Austria
c Department of Inorganic Chemistry, University of Vienna, 1090 Vienna, Austria
d Department of Drug and Natural Product Synthesis, University of Vienna, Vienna 1090, Austria
e Department of Nutritional Sciences, University of Vienna, Vienna 1090, Austria
f Department of Psychiatry and Psychotherapy, Medical University of Vienna, 1090 Vienna, Austria
g Hospital Pharmacy of the General Hospital of Vienna, 1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Changes in the expression of the melanin concentrating hormone receptor 1 (MCHR1) are involved in a
variety of pathologies, especially obesity and anxiety disorders. To monitor these pathologies in-vivo pos-
itron emission tomography (PET) is a suitable method. After the successful radiosynthesis of [11C]SNAP-
7941—the first PET-Tracer for the MCHR1, we aimed to synthesize its [18F]fluoroethylated analogue:
Received 31 May 2012
Revised 23 July 2012
Accepted 23 July 2012
Available online 7 August 2012
[
18F]FE@SNAP. Therefore, microfluidic and vessel-based approaches were tested. [18F]fluoroethylation
was conducted via various [18F]fluoroalkylated synthons and direct [18F]fluorination. Only the direct
Keywords:
MCHR1
Fluorine-18
PET
[
[
18F]fluorination of a tosylated precursor using a flow-through microreactor was successful, affording
18F]FE@SNAP in 44.3 2.6%.
Ó 2012 Elsevier Ltd. All rights reserved.
Microfluidic
SNAP-7941
Radioligand
1. Introduction
Several MCHR1 antagonists were presented in the last decade;
some of them have entered clinical trials for the treatment of obes-
Melanin concentrating hormone (MCH) is a cyclic neuropeptide
originally isolated from the salmon pituitary gland as a hormone
responsible for skin pigmentation in teleost fish.1 In mammals,
MCH is predominantly expressed in the lateral hypothalamus
and zona incerta,2,3 but is also found in peripheral organs and tis-
sues, such as the pancreas,4 colonic epithelial cells5 or adipo-
cytes.6,7 It plays a key role in energy homeostasis, e.g. the control
of food intake, body weight and metabolism.8,9 Furthermore, it is
involved in anxiety,10–14 diabetes,4 gut inflammation5 and adipos-
ity.6,7 The biological function of MCH is mediated by two G-protein
coupled receptors, MCH receptor 1 and 2 (MCHR115–18 and
MCHR2.19–22) The widespread distribution of MCH and its recep-
tors and the involvement in a variety of pathologies makes the
MCH system interesting as a target to treat human disorders.
ity,23 some are in discussion of becoming anti-diabetic drugs.24
However, to enable confidence in preclinical to clinical translation
of central MCHR1 pharmacology, a suitable positron emission
tomography (PET) tracer needs to be developed. Borowsky et al.25
presented the evaluation of the very potent MCHR1 antagonist
SNAP-7941 ((+)-methyl (4S)-3-{[(3-{4-[3-(acetylamino)phenyl]-
1-piperidinyl}propyl)amino]carbonyl}-4-(3,4-difluorophenyl)-6-
(methoxymethyl)-2-oxo-1,2,3,4-tetrahydro-5-pyrimidinecarboxylate
hydrochloride, 1, Figure 1) (Kd = 0.18 nM, evaluated on Cos-7 cells
expressing the human MCHR1 (hMCHR1)).25 We previously re-
ported the successful radiosynthesis of its radiolabelled analogue,
[
11C]SNAP-7941—the first PET tracer for the MCHR1.26 Due to the
practical advantages of the longer-lived radioisotope 18F (t1/
2 = 110 min) instead of 11C (t1/2 = 20 min) and the comparable sta-
bility of methyl- and fluoroethylesters,27 we aimed to synthesize
a [18F]fluoroethylated analogue: [18F]FE@SNAP (2, Fig. 1). We per-
formed preliminary stability tests by incubation in human plasma
at 37 °C which showed almost no degradation (<5%) within
120 min; and incubation with carboxyl esterase in a standard assay
⇑
Corresponding author. Tel.: +43 1 40400 5255; fax: +43 1 40400 1559.
E-mail address: wolfgang.wadsak@meduniwien.ac.at (W. Wadsak).
1
These authors contributed equally to this manuscript.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.051

C. Philippe et al. / Bioorg. Med. Chem. 20 (2012) 5936–5940
5937
F
presented in Figure 2. HPLC retention times were 2.3–2.6 min
(k0 = 0.5–0.7) for Tos@SNAP and 5.1–5.9 min (k0 = 2.4–2.9) for
F
[
18F]FE@SNAP. All values were verified by inactive reference sub-
O
O
O
stances. The TLC Rf-values of the two additional, unknown radio-la-
belled side products were 0.76–0.82 and 0.87–0.93, ‘respectively’;
HPLC retention times were 3.6–4.3 min (k0 = 1.4–1.7) and 6.9–
7.7 min (k0 = 3.6–4.1).
1: R=CH3
2
3
: R=(CH2)2 [18F]
: R=H
R
N
O
N
N
H
H
N
N
H
O
4: R=(CH2)2OTos
O
2.2. Vessel-based preparation
Figure 1. SNAP-7941 derivatives 1–4 (1: SNAP-7941; 2: [18F]FE@SNAP; 3: SNAP-
acid; 4: Tos@SNAP).
The production of all [18F]fluoroalkylation agents ([18F]BFE,
18F]FEtTos, 18F]FEtTf) was successful: 2.9 0.9 GBq 18F]BFE
[ [
revealed only 20% cleavage within 6 h. Additionally, in preliminary
binding experiments on CHO-hMCHR1 cell membranes, FE@SNAP
evinced similar binding affinity as the parent compound SNAP-
7941 (Kd = 0.18 nM).
[
(23.1 10.4% EOB), 0.3 0.1 GBq [18F]FEtTos (24.0 0.7% EOB)
and 0.3 0.2 GBq [18F]FEtTf (19.2 9.6% EOB) were achieved. How-
ever, the [18F]fluoroethylation of SNAP-acid via these synthons did
not succeed. No radiochemical incorporation was observed regard-
less of the conditions applied (reaction temperature, reaction time,
solvent, synthon). Furthermore, the direct fluorination of Tos@
SNAP in the conventional approach was not successful, either.
Again, no radiochemical incorporation was observed.
In general, 18F-fluoroalkyl radiolabels can be introduced using
either a direct [18F]fluorination of a suitable precursor-compound
(already containing a leaving group at the alkyl side chain) or via
two step labelling reaction using an 18F-fluoroalkylating synthon.
Even for flow-through microreactor procedures both synthesis
strategies have already been reported for various radiotracer syn-
theses: For example, direct radio-fluorinations were presented
2.3. Microfluidic preparation
for 2-[18F]FDG,28–31
[
[
18F]fallypride,32
[
18F]annexin,29,31 and N-
18F]fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline;33
Radiolabelling of the [18F]fluoroalkylation agent [18F]FEtTos was
in accordance to the literature.35,36 However, the second step—the
furthermore, the
[
18F]fluoroethylation of carboxylic acids,34
18F]fluoroethylation of SNAP-acid—was not successful using this
[
18F]fluoroalkylation of diverse [18F]fluorocholine derivatives using
[
either [18F]fluoroethyl tosylate or [18F]fluoropropyl tosylate35,36
and the syntheses of [18F]fluoroarenes from diaryliodonium salts37
have been presented.
approach.
The direct [18F]fluorination via the microfluidic system finally
resulted in the production of [18F]FE@SNAP. The RCIY was increas-
ing with higher reaction temperatures and increasing amounts of
precursor. Highest RCIYs were obtained at 170 °C using 4 mg/mL
of precursor in the reaction mixture (43.8 2.2%). Further raise of
precursor amount did not improve synthetic outcome (Fig. 3). Rea-
sonable radiolabelling yields and no formation of any side products
could be observed at 100 °C. At 140 °C radiochemical incorporation
was increased but two unknown radiolabelled side products were
formed additionally. When increasing the reaction temperature up
to 170 °C side product formation was even more pronounced. At
the same time degradation of precursor with increasing tempera-
ture could be observed in the UV channel of the HPLC chromato-
gram. This degradation also increased with slow flow rates.
Therefore, the dependency upon the flow rate was determined
The aim of this work was the preparation of [18F]FE@SNAP in
sufficient amounts for future pre-clinical applications. For this pur-
pose, vessel-based as well as microfluidic approaches were con-
ducted, starting from two different precursors: SNAP-acid (3,
Fig. 1) (for the two step [18F]fluoroalkylation) and Tos@SNAP (4,
Fig. 1) (for direct radiolabelling).
2. Results and discussion
2.1. General
TLC Rf-values were 0.00–0.02 for [18F]fluoride and 0.63–0.69 for
[
18F]FE@SNAP.
A typical radio-TLC sample chromatogram is
Figure 2. Typical radio-TCL chromatogram of the crude reaction mixture (7.5 mg/mL, 170 °C, 150 lL/min) with results after integration.

5938
C. Philippe et al. / Bioorg. Med. Chem. 20 (2012) 5936–5940
Table 1
Optimum reaction conditions and outcome for the preparation of [18F]FE@SNAP
Amount of precursor (mg/mL)
4
Reaction temperature (°C)
170
150
Flow rate (
lL/min)
RCIY (%)
43.8 2.2
to SNAP-acid. Once again, flow-through microreactor technology
was demonstrated to be a valuable addition in the portfolio of
radiolabelling techniques for the preparation of new PET tracers.
Solely, a microfluidic procedure enabled the radiosynthesis of
[
18F]FE@SNAP. Further experiments comprising an up-scaling,
purification and formulation of [18F]FE@SNAP will enable its use
in preclinical investigations.
4. Experimental section
4.1. General
Figure 3. Dependency of the RCIY upon precursor concentration. Reactions were
performed with a flow rate of 60 lL/min at 100 °C, 140 °C and 170 °C. Values are
decay-corrected and given as arithmetic means SD (n P 3). Error bars are depicted
unidirectional for better visualization but represent deviations in both directions.
Chemicals and solvents were purchased from Merck (Darms-
tadt, Germany) and Sigma-Aldrich (Vienna, Austria). All reagents
were at least of analytical grade and used without purification.
both at 140 °C and 170 °C (7.5 mg/mL Tos@SNAP, Fig. 4). At 170 °C
the RCIY increased with increasing flow rates. The inverse trend
was observed at 140 °C. RCIYs decreased with increasing flow
rates. However, best RCIYs were observed using fast flow rates
[
18F]fluoride was produced via the 18O(p,n)18F reaction in a GE PET-
trace cyclotron (16.5-MeV protons; GE Medical Systems, Uppsala,
Sweden). H218O (HYOX18; >98%) was purchased from Rotem Eur-
ope (Leipzig, Germany). Anion-exchange cartridges (PS-HCO3) for
up to 150
lL/min at 170 °C. In this set-up the residence time with-
in the heated microreactor (15.6
lL) was only 6.24 s. This time was
[
18F]fluoride trapping were obtained from Macherey-Nagel (Duer-
sufficient to radiolabel the tosylated precursor and short enough to
en, Germany). 2-bromoethyl triflate39 (BETfO) and ethylene glycol
bis triflate40 were synthesized in cooperation with the Department
of Drug and Natural Product Synthesis of the University of Vienna
(Austria). Alumina cartridges (Sep-PakÒ Light Alum N) were pur-
chased from Waters (WatersÒ Associates Milford, MA, USA). For
conventional small-scale preparations, the reactions were carried
out in Wheaton 1.0 mL v-vials (clear glass) assembled with TFE/sil-
icone septa. Microfluidic reactions were carried out within an Adv-
ion NanoTekÒ unit (Ithaca, NY, USA) comprising a concentrator
unit (CE) and a liquid flow reaction unit (LF) with dedicated control
software (Advion, version 1.4). The set-up is illustrated in Figure 5.
Microreactors were made of fused silica tubing (inner diameter
avoid extensive precursor degradation. RCIY of [18F]FE@SNAP was
increased from 25.1 1.8% (20 lL/min) to 44.3 2.6% (150 lL/
min) applying 7.5 mg/mL precursor at 170 °C.
Due to previous findings, 38 the reaction volume was examined
as additional parameter. No ‘‘bolus volume effect’’ could be ob-
served for this set-up. For reactions using 3 mg/mL Tos@SNAP at
170 °C and a flow rate of 150
scale reactions (20 L) and for enlarged reaction boluses
(300 L): 30.5 8.9%.
All these results led to the optimum reaction conditions for the
lL/min, RCIY was similar for small-
l
l
radiochemical preparation of
Table 1.
[
18F]FE@SNAP summarized in
0.1 lm; length 2.0 m), wound-up and held in a brass ring filled
with a thermoresistant polymer to hold the tubing in its place. Dry-
ing and collecting v-vials (3.0 and 5.0 mL, clear glass) were ob-
tained from Alltech (Deerfield, IL, USA). Radiochemical
incorporation yields (RCIYs) were determined by analysing the
reaction mixtures by radio-analytical thin-layer chromatography
(radio-TLC) and analytical high performance liquid chromatogra-
phy (HPLC). Radio-TLC was performed using silica gel 60 RP-18
3. Conclusion
[
18F]FE@SNAP could not be synthesized in any vessel-based
approach neither using direct radio-fluorination of the tosylated
precursor nor through the coupling of a [18F]fluoroalkylation agent
F254S plates from Merck (Darmstadt, Germany) (mobile phase: ace-
tonitrile/water 95/5 v/v). Analyses of radio-TLC plates were done
using a Canberra-Packard Instant Imager (Perkin Elmer, Watford,
UK). Analytical HPLC was performed using an Agilent system
(Boeblingen, Germany) consisting of an autosampler 1100, a qua-
ternary pump 1200 (flow: 1 mL/min), a diode array detector
1200 (operated at 255 nm) and a lead-shielded NaI-radiodedector
(Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).
HPLC column (ChromolithÒ Performance RP-18e; 100–4.6 mm)
was purchased from Merck (Darmstadt, Germany). HPLC mobile
phase consisted of (water/acetic acid 97.5/2.5 v/v; 2.5 g/L ammo-
nium acetate; pH 3.5)/acetonitrile 70/30 v/v.
4.2. Preparation of precursor compounds and reference
standard
Figure 4. Influence of the overall flow rate on the RCIY. Reactions were conducted
using 7.5 mg/mL Tos@SNAP in reaction mixture. Values are decay-corrected and
given as arithmetic means SD (n P 3).
Syntheses of precursor compounds (SNAP-acid26 and Tos@
SNAP41,42) and reference standard (FE@SNAP43) were performed

C. Philippe et al. / Bioorg. Med. Chem. 20 (2012) 5936–5940
5939
Figure 5. Schematic illustration of the set-up of the Advion NanoTekÒ system for two-step reactions.
in cooperation with the Department of Drug and Natural Product
Synthesis of the University of Vienna (Austria). 1H- and 13C-NMR
data and spectra as well as 19F-NMR, MS, HRMS and IR data, are
available as Supplementary data.
85 °C for 15 min. The resulted [18F]FEtTos was added to SNAP-acid
(activated with TBAH, 4 mg/mL in acetonitrile) (1:1) and the reac-
tion mixture was heated at 75 °C for 10–30 min.
4.4.3. Fluoroethylation via [18F]fluoroethyl triflate ([18F]FEtTf)
4.3. Azeotropic drying
[
18F]FEtTf was produced according to Kiesewetter et al.40
Briefly, a solution of ethylene glycol bis triflate (1.0 mg in 100 lL
Azeotropic drying of cyclotron produced [18F]fluoride was
always performed within the concentrator unit of the Advion
NanoTekÒ synthesizer using a programmed procedure, in order
to avoid differences between manual and automatic azeotropic
drying. N.C.A [18F]fluoride (1.5–31.7 GBq) was trapped on to an
anion exchange cartridge (PS-HCO3) and released with a solution
containing Kryptofix 2.2.2 (4,7,13,16,21,24-hexaoxa-1,10-diaza-
acetonitrile) was added to the dried [18F]fluoride-aminopolyether.
After heating at 125 °C for 1 min, the solution was cooled in an ice
bath and triflic anhydride (5
loaded onto an alumina cartridge and [18F]FEtTf was eluted with
acetonitrile (500
L). The solution of [18F]FEtTf was added to
lL) was added. The solution was
l
SNAP-acid (activated with TBAH, 4 mg/mL in acetonitrile) (1:1)
and heated at 75 °C for 10–30 min.
bi-cyclo[8.8.8]hexacosane; 10 mg, 26.6
carbonate (2.25 mg, 16.6 mol) in acetontrile/water (70/30 v/v;
V = 0.5 mL). Iterative azeotropic drying was performed at 110 °C
by addition of 3 times 300 L of dry acetonitrile.
lmol) and potassium
l
4.4.4. Direct [18F]fluorination
The tosylated precursor Tos@SNAP (2 mg/mL) was dissolved in
acetonitrile and added to the dried [18F]fluoride-aminopolyether.
The reaction mixture was heated at 75 °C for 10–90 min.
l
4.4. Vessel-based preparation
4.5. Microfluidic preparation
4.4.1. Fluoroethylation via 1-bromo-2 [18F]fluoroethane
([18F]BFE)
4.5.1. Fluoroethylation via [18F]fluoroethyl tosylate
([18F]FEtTos)
[
18F]BFE was produced according to Zuhayra et al.44 Briefly, to
the dried [18F]fluoride-aminopolyether a mixture of 30
and 500 L 1,2-dichlorobenzene (o-DCB) was added. After heating
lL BETfO
Reaction educts were dissolved in acetonitrile and drawn into
l
the respective loops (loop 1: ethylene glycol di-
mL); loop 2: SNAP-acid (activated with TBAH, 20 mg/mL); loop 3:
dried [18F]fluoride-aminopolyether in 500
L acetonitrile). Before
P-tosylate (10 mg/
at 100 °C for 10 min, [18F]BFE was distilled at 130 °C under helium
flow into the respective solvent (DMF, DMSO and 2-butanone). The
precursor SNAP-acid (4 mg/mL) was activated by adding an equi-
molar amount of tetrabutylammonium hydroxide (TBAH) and dis-
solved in the respective solvent. The [18F]BFE solution was added
to the precursor solution (1:1) and the reaction mixture was
heated at 75–140 °C for 10–20 min.
l
starting the experiments, transfer-lines were filled with the solu-
tions from the storage loops and the reactors were finally swept
with the reaction solvent via pump 3 (Fig. 2). The synthesis was
carried out within the Advion NanoTekÒ using the ‘discovery
mode’ for two step reactions. The conversion to [18F]FEtTos (first
reaction) was performed by pushing the previously filled solutions
simultaneously from loop 1 and 3 through the first temperature
controlled (180 °C) flow-through microreactor (flow rate: 30–
4.4.2. Fluoroethylation via [18F]fluoroethyl tosylate
([18F]FEtTos)
Ethylene glycol di-
P
-tosylate (9.25 mg/mL in acetonitrile) was
50
l
L/min). In a second phase the [18F]FEtTos-mixture was swept
added to the dried [18F]fluoride-aminopolyether and heated at
out of the reactor through the storage line towards the other

5940
C. Philippe et al. / Bioorg. Med. Chem. 20 (2012) 5936–5940
reactor. The second reaction starts when the [18F]FEtTos-mixture
and the precursor solution (loop 2) are pushed through the second
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followed by a final sweeping where the reaction mixture is finally
flushed out of the system into a collecting vial filled with 200 L of
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l
l
lL
l
4.5.2. Direct [18F]fluorination
Dried [18F]fluoride-aminopolyether dissolved in 500
l
L acetoni-
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and 3 of the LF unit. Before starting the experiments, transfer lines
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200
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with defined overall flow rates (60–150 L/min). Subsequently,
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l
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4.6. Statistical analyses
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Quantitative data (both in text and figures) are expressed as
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accepted.
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