Radiolabelling of 1,4-disubstituted 3-[18F]fluoropiperidines and its application to new radiotracers for NR2B NMDA receptor visualization

Article abstract of DOI:10.1039/c2ob26378e

In order to develop a novel and useful building block for the development of radiotracers for positron emission tomography (PET), we studied the radiolabelling of 1,4-disubstituted 3-[¹⁸F]fluoropiperidines. Indeed, 3-fluoropiperidine became a useful building block in medicinal chemistry for the pharmacomodulation of piperidine-containing compounds. The radiofluorination was studied on substituted piperidines with electron-donating and electron-withdrawing N-substituents. In the instance of electron-donating N-substituents such as benzyl or butyl, configuration retention and satisfactory fluoride-18 incorporation yields up to 80% were observed. In the case of electron-withdrawing N-substituents leading to carbamate or amide functions, the incorporation yields depend on the 4-susbtitutent (2 to 63%). The radiolabelling of this building block was applied to the automated radiosynthesis of NR2B NMDA receptor antagonists and effected by a commercially available radiochemistry module. The in vivo evaluation of three radiotracers demonstrated minimal brain uptakes incompatible with the imaging of NR2B NMDA receptors in the living brain. Nevertheless, moderate radiometabolism was observed and, in particular, no radiodefluorination was observed which demonstrates the stability of the 3-position of the fluorine-18 atom. In conclusion, the 1,4-disubstituted 3-[¹⁸F]fluoropiperidine moiety could be of value in the development of other radiotracers for PET even if the evaluation of the NR2B NMDA receptor antagonists failed to demonstrate satisfactory properties for PET imaging of this receptor.

Full text of DOI:10.1039/c2ob26378e

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PAPER
Radiolabelling of 1,4-disubstituted 3-[18F]fluoropiperidines and its application
to new radiotracers for NR2B NMDA receptor visualization†
Radouane Koudih,‡a,b,c Gwénaëlle Gilbert,‡a,b,c Martine Dhilly,a,b,c Ahmed Abbas,d Louisa Barré,a,b,c
Danièle Debruynea,b,c and Franck Sobrio*a,b,c
Received 17th July 2012, Accepted 5th September 2012
DOI: 10.1039/c2ob26378e
In order to develop a novel and useful building block for the development of radiotracers for positron
emission tomography (PET), we studied the radiolabelling of 1,4-disubstituted 3-[18F]fluoropiperidines.
Indeed, 3-fluoropiperidine became a useful building block in medicinal chemistry for the
pharmacomodulation of piperidine-containing compounds. The radiofluorination was studied on
substituted piperidines with electron-donating and electron-withdrawing N-substituents. In the instance of
electron-donating N-substituents such as benzyl or butyl, configuration retention and satisfactory fluoride-
18 incorporation yields up to 80% were observed. In the case of electron-withdrawing N-substituents
leading to carbamate or amide functions, the incorporation yields depend on the 4-susbtitutent (2 to
63%). The radiolabelling of this building block was applied to the automated radiosynthesis of NR2B
NMDA receptor antagonists and effected by a commercially available radiochemistry module. The in vivo
evaluation of three radiotracers demonstrated minimal brain uptakes incompatible with the imaging of
NR2B NMDA receptors in the living brain. Nevertheless, moderate radiometabolism was observed and,
in particular, no radiodefluorination was observed which demonstrates the stability of the 3-position of the
fluorine-18 atom. In conclusion, the 1,4-disubstituted 3-[18F]fluoropiperidine moiety could be of value in
the development of other radiotracers for PET even if the evaluation of the NR2B NMDA receptor
antagonists failed to demonstrate satisfactory properties for PET imaging of this receptor.
cases, to improved pharmacological properties4 with, as
Introduction
examples, serotoninergic 5-HT1D receptor ligands,5 T-type
calcium channel inhibitor 1,6–9 PAR1 protease activated receptor
Piperidines are widely present in natural bioactive substances
and are also used in medicinal chemistry which has led to
numerous original drugs. In structure–activity relationship
antagonist 2,10 kinesin spindle protein KSP inhibitors8 and
NR2B NMDA antagonist 311 (Chart 1).
studies, the piperidine is mainly employed either as an N-substi-
tuted aliphatic amine or substituted in the 4-position. Among
various modifications of the piperidine pharmacophore, the
3-fluoropiperidine moiety is present in some bioactive
compounds such as Y1 neuropeptide Y receptor antagonists1,2
and M1 muscarinic receptor agonists3 and has led, in some
Positron emission tomography (PET) is an imaging technique
used for diagnosis and prognosis, and to explore the disease
states as well as to validate innovative therapies or drugs. The
development of this non-invasive technique and its extensive
clinical use are due to the availability of commercial radiophar-
maceuticals labelled with fluorine-18. The weak energy of the
emitted positron affords high resolution images. The production
capacities in quantity of fluoride-18 as well as a half-life of
109.7 min are compatible with transportation and in consequence
fluorine-18 became the radioisotope of choice for PE. The devel-
opment of this radiofluorinated family of radiotracers is linked to
the increased number of fluorinated building blocks and radio-
labelling methods.12,13
aCEA, I2BM, LDM-TEP, UMR 6302 ISTCT, GIP Cyceron, BP5229
F-14074 Caen, France. E-mail: sobrio@cyceron.fr;
Fax: +33 2 3147 0225; Tel: +33 2 3147 0264
bUniversité de Caen Basse-Normandie, UMR 6302 ISTCT, LDM-TEP,
GIP Cyceron, F-14074 Caen, France
cCNRS, UMR 6302 ISTCT, LDM-TEP, GIP Cyceron, F-14074 Caen,
France
dINSERM U1077, GIP Cyceron, F-14074 Caen, France
†Electronic supplementary information (ESI) available: Synthetic
procedures for 3–32, in silico pharmacological properties and NMR
spectra for 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 19, 20, 21 and 29. See DOI:
10.1039/c2ob26378e
To enrich the number of radiofluorinated building blocks, we
studied the radiolabelling of different N-substituents containing
4-substituted-3-fluoro-piperidines. To evaluate the usefulness of
this radiofluorinated moiety, we labelled NR2B subunit con-
taining N-methyl-D-aspartate receptor (NMDAR) antagonists.
‡These authors had equally contributed to this work.
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bioactive compounds.5–7 As carbamate and amide functions are
found in several ligands,10,21 the benzoyl and tert-butyl carboxy-
late were selected as electron-withdrawing groups to study the
radiofluorination reaction. The addition of a trityl group could be
selectively performed on the primary alcohol. Then the second-
ary alcohol could react with mesyl chloride to form the mesylate
precursor. The presence of the trityl chromophore group allowed
the ready detection of the compound in HPLC. Thereafter, the
labelling precursors 13–16 with the cis-configuration were pre-
pared (Scheme 1) from the N-benzyl diol 4 by the removal of the
N-benzyl group followed by reaction with the corresponding
alkyl or benzoyl halide. After selective O-alkylation with trityl
chloride of the primary alcohol, the mesylation was achieved
with mesylate chloride in the presence of silver triflate in pyri-
dine24 to give the labelling precursors 13–16. The preparation of
the fluorinated O-trityl compounds (22–25) was performed after
deprotection of the Boc group following the same synthetic
pathway (Scheme 2). Addition of the trityl group created the
fluorinated cis and trans isomers 22–25 needed for the identifi-
cation of the radiolabelled compounds by co-migration in
HPLC. To further explore the scope and applications of radio-
fluorination reaction, the N-Boc-4-(pyrimidin-2-yl)aminomethyl-
3-mesyloxy-piperidine precursor cis-31 was obtained from the
3-hydroxy analogue21 by mesylation (Scheme 3). The prep-
aration of the fluorinated reference compound 32 as well as the
synthesis of NR2B NMDAR antagonists cis- and trans-3 and
their radiolabelling precursors cis-30 and trans-30 have been
described previously (Scheme 3).21 Amide coupling of trans-2-
phenylcyclopropane-1-carboxylic acid and the piperidine
obtained from the removal of the Boc group of trans-32 gave
trans-33. The labelling precursor cis-29 was prepared following
the same methodology (Scheme 3).
Chart 1 Structure of 3-fluoropiperidines containing bioactive com-
pounds.
NMDARs are heteromeric complexes of NR1 and NR2 subunits
that mediate rapid excitatory neurotransmission throughout the
central nervous system (CNS). NR2 subunits result from four
distinct genes that construct NR2A-D subunits that have diverse
pharmacological properties and substantially different expression
patterns in the brain.14 The NR2B subunit containing NMDARs
is the focus of increasing interest as a therapeutic target15 in
many of CNS pathologies, including stroke, acute and chronic
pain, drug-induced dyskinesia and dementia.16,17 The radio-
tracers to study the NMDARs are the subject of intensive studies
but none so far have been suitable for clinical or even preclinical
imaging.18–20 Recently, we reported the radiosynthesis of two
radiotracers, [18F]MK-0567 ([18F]cis-3) and its trans-isomer,
[18F]trans-3.21 Their in vitro evaluation based on the autoradio-
graphy of the rat brain demonstrated high specific binding for
NR2B subunit containing NMDARs located in rich NR2B
regions. The Kd was 9.1 2.1 nM and 7.1 1.7 nM in the hip-
pocampus for [18F]MK-0567 and its trans-[18F]isomer, respect-
ively. The Kd/Bmax ratios for both radiotracers varied from 17 to
37 as a function of the anatomical localization as well as the log
D7.4 (2.66 and 2.80 for [18F]trans-3 and [18F]cis-3, respectively)
indicates favourable properties for the CNS imaging of the
NR2B NMDARs.
Radiolabelling
The radiolabelling was performed under classical nucleophilic
radiofluorination conditions based on the complex [K18F/K2.2.2
]
as the source of reactive “naked” fluoride (Scheme 4). In brief,
[18F]fluoride, produced by the 18O(p,n)18F nuclear reaction, was
trapped in a quaternary ammonium resin solid phase extraction
(SPE) cartridge. The [18F]fluoride was eluted by a potassium car-
bonate solution and dried in the presence of Kryptofix 2.2.2
(K2.2.2) by successive azeotropic evaporation with acetonitrile.
The amount of the complex and the solvent of reaction as well
as the reaction temperature were varied (Table 1). The incorpor-
ation yield of fluorine-18 into compounds [18F]22–25, [18F]-
32–33 and [18F]3 was established by radioTLC and the identity
of the radiolabelled product was established by co-migration on
HPLC with the non-radioactive reference compound. For each
compound, the two diastereoisomers were prepared and separ-
ated by HPLC (Table 2). The kinetics of the reaction were
studied over 20 min which was the reaction time leading to a
high incorporation yield with low variability. The radiofluorina-
tion of the cis-mesyloxy N-butyl and N-benzyl precursors (15
and 16) was performed with moderate to excellent yields (entries
1–4) of the cis products, [18F]cis-24 and [18F]cis-22 respectively.
The conservation of the configuration resulted from a double
SN2 substitution also called neighbouring-group participation,
Herein we report the radiolabelling of 4-trityloxymethyl-3-
[18F]fluoropiperidine model compounds that contain different N-
substituents, their application to the radiosynthesis of radiofluori-
nated NR2B NMDAR antagonists and their in vivo evaluation in
μPET imaging studies.
Results and discussion
Chemistry
The radiofluorination of 3-[18F]fluoropiperidines was envisaged
based on the mesylate leaving group which could be obtained
from the corresponding alcohol. The 4-hydroxymethylpiperidi-
nyl moiety was chosen because piperidin-3-ol (4) was previously
described22,23 and the corresponding 3-fluoropiperidine (17) was
synthesized by DiFabio and coll.1 Butyl and benzyl were chosen
as electron-donating groups given their presence in many
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Scheme 1 Synthesis of labelling precursors 13–16.
Scheme 2 Synthesis of reference compounds 22–25.
Scheme 3 Synthesis of NR2B NMDAR antagonists 3 and 33 and their radiolabelling precursors.
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was less (40% vs. 62%) but identical for the both diastereo-
isomers (entries 17–18). The radiolabelling of the N-carbonyl
compound, [18F]trans-33, was obtained with an incorporation
yield of fluorine-18 around 26% (entry19). When the standard
quantity of the [K18F/K2.2.2] complex was employed (entries 2,
4, 6, 10, 14, 16 and 20), the radiochemical yields were signifi-
cantly lower than when a reduced amount of complex was used.
These poor yields could be due to the competing elimination
reaction which was shown by the characterization of the alkene
resulting from the elimination reaction with 31. In this case, the
percentage of the elimination product was about 60% of the pre-
cursor versus 23% under the most optimal conditions (entry 13).
Radiotracer radiosyntheses
Recently, we described the radiosyntheses of the radiotracers
[18F]cis-3, [18F]trans-3 through the use of a widely available
commercial TRACERlab FX-FN module (GE Healthcare).21
The same conditions were used for the preparation of [18F]trans-
33. After azeotropic drying, the [K18F/K2.2.2] complex was
reacted with the precursor cis-30, trans-30 or 29 (10 μmol
respectively) in acetonitrile (1 mL) at 90 °C for 20 min. After
dilution with water, the prepurification was achieved through a
C18 SPE cartridge. The radioactive product was eluted from the
cartridge by acetonitrile and purified by reversed phase HPLC.
The pure fraction containing the [18F]-product was collected and
submitted to a reformulation by SPE to obtain a radioactive con-
centrated saline solution for i.v. injection containing less than
10% of ethanol. The data concerning the radiosynthesis are
described in Table 3. 400 to 750 MBq of the radiolabelled
product were obtained with acceptable and reliable radiochemi-
cal yields after reformulation. High specific radioactivities were
measured between 170 to 590 GBq μmol−1 and the radiochemi-
cal purities of the radiotracers were established by HPLC and
were greater than 98%.
Scheme 4 Radiofluorination reactions.
which proceeds by an aziridinium intermediate (Scheme 5),25
the formation of which was due to the availability of the elec-
tronic doublet of the nitrogen atom in the piperidine25 enriched
by the electron-donating substituents. The selective opening of
the aziridinium ring by the [18F]fluoride occurred on the more
hindered atom (Scheme 4).26 In the case of an electron-with-
drawing substituent on piperidine like carboxylate or carbonyl,
the inversion of conformation indicated an SN2 mechanism
(entries 5–21). With methoxytrityl in the 4-position and N-Boc
or N-benzoyl substituents (13 and 14), the radiofluorination gave
the [18F]trans-25 and [18F]trans-23 in very low yields (entries
5–12). Further, we studied the scope of the reaction and excluded
a steric effect of the trityl group which could explain the poor
incorporation rate for the compounds 13 and 14, by the reaction
with the 4-(pyrimidin-2-ylamino)methyl substituted piperidine
31.21 The fluorine-18 incorporation was effected in acetonitrile
with a good yield (entry 13). No effect other than a steric one
was proposed for the difference of incorporation yields between
pyrimidin-2-ylamino and trityloxy substituted compounds [18F]-
trans-25 and [18F]trans-32. For the radiolabelling of [18F]trans-
32, a reaction temperature of 80 °C led to a 19–27% incorpor-
ation yield and could be improved to 32–47% at 95 °C (entries
14 and 16). The use of DMSO at 120 °C did not improve the
labelling rate (entries 8, 15, 12 and 21). The radiofluorinations of
the NR2B antagonists, cis-3 and trans-3, were performed under
the same conditions as those of the N-Boc-piperidine 31 since
the N-substituent was also a carbamate. The incorporation yield
In vivo pharmacological evaluation of the radiotracers
Compound cis-3 (MK-0657) was developed by Merck labora-
tories27 and clinical trials were conducted to evaluate its effects
in the treatment of major depression and in the motor function
improvement in Parkinson’s disease patients.28 Our recent in
vitro findings on [18F]cis-3 and [18F]trans-3 warrant the in vivo
evaluation of these radiotracers.21 In the same patent describing
cis- and trans-3,27 several antagonists presenting similar activity
were described among them, an amide (33) caught our attention
due to the in silico predicted properties of this compound§ and
to the presence of the amide function which could validate our
radiolabelling strategy. The radiotracers (16
5 MBq) were
administered to rats and were evaluated by μPET and ex vivo
measurements. The brain uptake kinetics were determined by
reconstruction of PET time activity curves from 0 to 90 min. The
radioactivity peaked by 1 min post-injection; thereafter, all three
radiotracers were rapidly cleared from the brain (Fig. 1). At
90 min, the brain/blood ratios were 1.97 ([18F]trans-3) and 1.88
([18F]cis-3) and the brain showed a poor contrast corresponding
§See ESI for the in silico predicted pharmacological properties.
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Table 1 Summary of 18F labelling reaction
Reaction entry
Precursor
K222/K2CO3 (mg)
Solvent
Reaction temperaturea
[
18F]-product
18F incorporation yieldb (%)
1
2
3
4
5
6
7
8
cis-15
cis-15
cis-16
cis-16
cis-13
cis-13
cis-13
cis-13
cis-14
cis-14
cis-14
cis-14
cis-31
cis-31
cis-31
cis-31
cis-30
trans-30
cis-29
cis-29
cis-29
6.8/2.1
22/7
6.8/2.1
22/7
6.8/2.1
22/7
6.8/2.1
6.8/2.1
6.8/2.1
22/7
6.8/2.1
6.8/2.1
6.8/2.1
22/7
6.8/2.1
22/7
6.8/2.1
6.8/2.1
6.8/2.1
22/7
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
DMF
90 °C
90 °C
90 °C
90 °C
90 °C
90 °C
100 °C
100 °C
90 °C
90° C
100 °C
100 °C
95 °C
95 °C
120 °C
80 °C
95 °C
95 °C
95 °C
95 °C
120 °C
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
18F]cis-24
78–81%
48–55%
29–37%
10–14%
2–5%
<2%
1–3%
4–6%
2–4%
<2%
2–4%
18F]cis-24
18F]cis-22
18F]cis-22
18F]trans-25
18F]trans-25
18F]trans-25
18F]trans-25
18F]trans-23
18F]trans-23
18F]trans-23
18F]trans-23
18F]trans-32
18F]trans-32
18F]trans-32
18F]trans-32
18F]trans-3
18F]cis-3
DMSO
9
Acetonitrile
Acetonitrile
DMF
10
11
12
13
14
15
16
17
18
19
20
21
DMSO
<2%
Acetonitrile
Acetonitrile
DMSO
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
DMSO
60–65%
32–47%
63–64%
19–27%
33–42%
31–45%
25–27%
None
18F]trans-33
—
6.8/2.1
[
18F]trans-33
23–38%
a Reaction conditions: precursor, 10 μmol; solvent, 1 mL; reaction time, 20 min. b Incorporation yields were determined by radio-TLC. n ≥ 3
experiments.
Table 2 HPLC conditions used for the identification of the
radiolabelled products
Table 3 Summary of the radiotracer syntheses
Specific
Retention time (min)
HPLC conditionsa
H2O/CH3CN/TFA
Radiochemical
radioactivityb
Radiosynthesis
time
[
18F]product
yielda
(GBq μmol−1
)
Precursor
Cis-product
Trans-product
(v : v : v)
[
[
[
18F]cis-3
13 5%
18 6%
170 80
326 45
592 150
100 min
89 min
123 min
18F]trans-3
18F]trans-33
9.5
5.9
(cis-13)
(cis-14)
15.1 (cis-25) 18.7 (trans-25) 80/20b
9
1%
9.7 (cis-23)
8.5 (trans-23) 80/20b
a Relative to
[
18F]-fluoride and decay corrected to the end of
10.8 (cis-15)
12.4 (cis-16)
9.9
10.1 (cis-30)
19.7 (trans-30) 18.6 (cis-3)
8.2 (cis-31)
14.8 (cis-24) 16.9 (trans-24) 50/50/0.1b
10.3 (cis-22) 11.5 (trans-22) 50/50/0.1b
12.5 (trans-33) 55/45b
bombardment. b At the end of synthesis.
(cis-29)
12.5 (cis-3)
14.6 (trans-3)
23.7 (trans-3)
55/45c
60/40c
9.3 (cis-32) 12.1 (trans-32) 40/60c
a Conditions: flow: 1 mL min−1, UV detection: 234 nm. b Column
Nucleodur C18 Isis (Macherey-Nagel), 250 × 4 mm, 5 μm. c Column
Nucleodur C18 Pyramid (Macherey-Nagel), 250 × 4 mm, 5 μm.
Scheme 5 Radiolabelling mechanism for [18F]-22 and [18F]-24.
Fig. 1 μPET radioactivity kinetics in rat brain of the radiotracers (n = 3).
to an uptake index (UI) lower than 1 (Fig. 2). Only a slight
difference of brain uptake was observed with a UI = 0.55 for
[18F]cis-3 related to UI = 0.39 for [18F]trans-3. The brain/blood
ratio for [18F]33 was evaluated to be 0.35 with a very low UI =
0.14 which could be due to a lower stock level in fat. No signifi-
cant differences were observed between the accumulations of
radioactivity within the different brain structures (Fig. 3). The
target to non-target ratio, such as the cortex to cerebellum ratio,
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Fig. 2 Ex vivo organ and tissue distribution in rat at 90 min post-injec-
tion: black bars, [18F]trans-3; white bars, [18F]cis-3; grey bars, [18F]33
(n = 3).
Fig. 4 Radioactive plasmatic kinetics of the radiotracers in rat: closed
circle and plain line, [18F]-parent compound; open circle and dashed
line, polar [18F]-radiometabolites (n = 3).
Fig. 3 Ex vivo cerebral distribution in rat brain at 90 min post-injec-
tion: black bars, [18F]trans-3; white bars, [18F]cis-3; grey bars, [18F]33
(n = 3).
of [18F]33 differed with a lower uptake in brown fat and pancreas
(Fig. 2). The accumulation of activity in the bone was low with
bone/blood radioactivity concentration ratios of 1.05, 0.62 and
0.52 for [18F]trans-3, [18F]cis-3 and [18F]33 respectively, at
90 min post-injection (Fig. 2). The absence of accumulation in
bones and joints demonstrated the stability of the [18F]-3-fluoro-
piperidine moiety versus radiodefluorination. The high uptake in
fatty tissues at 90 min points towards a lipophilicity such that the
capacity of both [18F]3 radiotracers to image the brain is ques-
tionable in spite of a log D7.4 less than 2.8 (2.80 0.02 and 2.66
0.09 for [18F]cis-3 and [18F]trans-3 respectively). The lower
fat uptake for [18F]33 could be correlated to its lower log D7.4
(2.36 0.09).
was essentially unit and thus in disagreement with the heterogen-
eity observed in vitro.21 In addition, the simultaneous adminis-
tration of a non-radioactive compound did not significantly affect
the regional cerebral uptakes which would imply a high non-
specific binding of these radioligands in vivo. The in vivo meta-
bolism of [18F]3 and [18F]33 was investigated over time by μPET
scanning in the anesthetized rat. Radiometabolite analysis indi-
cated that parent tracers were still prevalent at 45 min (Fig. 4).
The terminal half-life of the parent compound, [18F]trans-3,
[18F]cis-3 and [18F]33, was 58 min, 53 min and 22 min respect-
ively. In the rat brain, about 79% ([18F]trans-3), 71% ([18F]cis-3)
and only 51% ([18F]33) of the initial compound remained
unchanged at 90 min. In tissue dissection studies, the highest
radioactivity concentrations at 90 min post-injection were
measured in the liver and brown fat, followed by the pancreas
and kidney while the lowest levels were in the blood and bones
for [18F]trans-3 and [18F]cis-3. The peripheral tissue distribution
Experimental
Chemistry
The material and reagents part as well as the synthetic pro-
cedures are given in the ESI.†
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Radiochemistry
The radiochemical purity of the radiotracers was determined
by HPLC following the conditions described in Table 2 and was
more than 98%. The specific radioactivity was measured by the
radioactivity concentration/molar concentration obtained by UV
absorption in HPLC and calibration curves established from the
reference compounds ratio.
Radioisotope production. No-carrier-added aqueous [18F]
fluoride was produced by the 18O[p,n]18F nuclear reaction of a
target consisting of 18O-enriched water (97%, Eurisotop) ir-
radiated with a 18 MeV proton beam (IBA Cyclone 18/9
cyclotron).
Preparation of nucleophilic [18F]fluoride ion. [18F]F− was
separated from 18O-enriched water using an ion exchange resin
(QMA light, Waters, ABX) eluted with aqueous potassium car-
bonate (5 mg mL−1, 300 μL). The [18F]fluoride solution was col-
lected into a conical reactivial® containing Kryptofix 2.2.2 (22
or 6.8 mg) and K2CO3 (adjusted to obtain 7 or 2.1 mg) dissolved
in a water–acetonitrile mixture (600 μL, 1 : 1, v : v). The water
was removed azeotropically with acetonitrile (2 × 600 μL) under
a stream of nitrogen at 110 °C, affording a dry residue of
Pharmacological evaluation
μPET imaging. The animal experiments were conducted
according to the appropriate European Directives (86/609/EEC)
and French National Legislation and in conformity with the
Guiding Principles in the Care and Use of Animals as approved
by the University of Caen. Male Sprague–Dawley rats
(250–310 g, C.E.R.J., France) were induced with isoflurane 5%
and maintained under anesthesia with isoflurane 1.5–2.5% in
oxygen and nitrous oxide (30/70) for the duration of the exper-
iment. Rats were positioned in an Inveon® μPET (Siemens,
Saint Denis, France) with brain centred in the field of view. A
transmission scan of 10 min using a cobalt-57 source was per-
formed before tracer injection to provide attenuation correction.
[18F]trans-3 or [18F]cis-3 were injected (16.6 5.5 MBq) via a
catheter in the tail vein to animals (n = 3). For co-administration
studies, non-radioactive reference compounds (trans-3 or cis-3,
30 μg, 84 μmol i.e. about 1000-fold the injected radiotracer
amount) were injected simultaneously with the radiotracer. PET
dynamic images were reconstructed from 0 to 30 or 90 min
emission scans with an OSEM 2D algorithm using the Inveon
Acquisition Workplace Software (Siemens Medical solution,
Knoxville, USA). An analytical scatter correction was
implemented for the image reconstruction. Decay corrected
time–activity curves (TAC) were obtained for the regions of
interest (ROI) selected in the upper part of the rat body: brain,
wrist articulation, liver and cervical brown adipose tissue. Radio-
activity data were injected dose- and bodyweight-normalized by
expression of SUV (Standardized Uptake Value).
[K/K222
]
+18F−. Between 1 mCi (37 MBq) and 5 mCi (185
+18F− were obtained in 20 min.
MBq) of dried [K/K222
]
Radiofluorination reaction. A solution of the labelling precur-
sor (10 μmol) dissolved in either DMSO, MeCN or DMF
(1 mL) was added to the dried [K/K222]
+18F− complex and the
sealed reaction vial was heated (45–120 °C) for 20 min. Aliquots
(25 μL) were taken at 5, 10 and 20 min and were subjected to
radioTLC and/or radioHPLC analysis after dilution with an
HPLC mobile phase. The radiochemical yield was determined
from radioTLC representing the percentage of radioactivity area
of the labelled product related to the total radioactivity area. TLC
elution conditions for all compounds were CH2Cl2–MeOH;
95 : 5.
Radiosynthesis of radiotracers [18F]-3 and [18F]-33 with GE
TRACERLab FXFN. The fluorine-18 produced by the cyclotron
was trapped on an ion exchange resin (QMA light, Waters,
ABX), separated from 18O-enriched water then eluted with a sol-
ution of potassium carbonate and Kryptofix 2.2.2 in acetonitrile
(0.3 mL) and water (0.3 mL) (vial 1). The mixture was heated to
95 °C under reduced pressure under a flow of helium. 0.6 mL of
acetonitrile (vial 2) was added and the mixture was further
heated at 95 °C under reduced pressure. About 5 mg (10 μmol)
of the labelling precursor 30 or 29 in 1 mL of acetonitrile (vial
3) was added to the reactor and the reaction mixture was heated
at 95 °C for 20 min, then diluted with 2 mL of water (vial 4).
The reaction mixture was passed through an SPE cartridge
(Shorty C18 ec, 20 mg, Macherey-Nagel) to separate the radio-
labeled product from the reaction mixture. The cartridge was
eluted with 0.5 mL of acetonitrile (vial 5) then washed with
0.5 mL of water (vial 6). 200 μL of this elution solution was
injected into an HPLC column to facilitate the separation
(column: Nucleosil 100-5 C-18 Nautilus, 250 × 10 mm,
Macherey-Nagel; eluent: acetonitrile–H2O, 35 : 65 for tR(cis-3) =
36 min and 37 : 63 for tR(trans-3) = 38 min; eluent: acetonitrile–
H2O, 27 : 73 for tR(trans-33) = 57 min flow rate: 6 mL min−1).
The collected fraction containing the pure radiolabeled product
was diluted with 25 to 40 mL of water and then passed through
an SPE cartridge (Shorty C18 ec, Macherey-Nagel). The car-
tridge was then washed with 2 mL of water (vial 9) and eluted
with 0.2 mL of ethanol (vial 8) and 1.8 mL of saline (vial 7).
Ex vivo biodistribution. During the μPET examination, suc-
cessive blood samples were taken off from the femoral artery
from 0 to 90 min and were immediately centrifuged. Plasma
samples (5 μl) were placed in the γ-counter Packard Cobra for
measuring the radioactivity. Rats were killed under anesthesia at
the end of the μPET exam. Organs and tissues (heart, liver, lung,
kidney, spleen, skeletal muscle, bone, joint, brown fat and pan-
creas) were collected, blotted, weighed and counted. The brain
was carefully removed, blotted and cut into two parts according
to the sagittal way. Half of the brain was weighed and counted
and the other half was dissected into cerebellum, hypothalamus,
colliculi, cortex, striatum, hippocampus and thalamus. Radio-
activity in each tissue or plasma sample was measured with a
γ-counter Packard Cobra (Perkin Elmer, Courtaboeuf, France)
and corrected for decay. Radioactivity data were injected dose-
and bodyweight-normalized by expression of UI (Uptake Index).
In vivo radiometabolism study. For radiometabolite analyses
of plasma, acetonitrile was added to plasma (1/1: v/v), vortexed
and centrifuged. The supernatant was analysed by radioTLC
using a silica-gel TLC (Merck) and CH2Cl2–MeOH (95 : 5) as a
mobile phase. To assess the radiometabolism of [18F]trans-3 or
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8493–8500 | 8499

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[18F]cis-3 in the rat brain, rats were killed at 30 min after radio-
tracer injection. The whole brains were rapidly removed, homo-
genized in acetonitrile and centrifuged and the supernatant was
analysed by HPLC (eluent: H2O–MeCN; 60 : 40). The percen-
tage of unchanged [18F]trans-3 or [18F]cis-3 with respect to total
plasma radioactivity was obtained by measuring counts related
to parent radiotracer and radiometabolites from each time point.
The identification of the parent compound was achieved on the
sample at the latest time after administration by radioHPLC
(eluent: H2O–MeCN; 60 : 40) and comigration with the non-
radioactive reference.
4 M. Morgenthaler, E. Schweizer, A. Hoffmann-Roder, F. Benini,
R. E. Martin, G. Jaeschke, B. Wagner, H. Fischer, S. Bendels,
D. Zimmerli, J. Schneider, F. Diederich, M. Kansy and K. Muller, Chem-
MedChem, 2007, 2, 1100–1115.
5 Z. Q. Yang, J. C. Barrow, W. D. Shipe, K. A. Schlegel, Y. Shu,
F. V. Yang, C. W. Lindsley, K. E. Rittle, M. G. Bock, G. D. Hartman,
V. N. Uebele, C. E. Nuss, S. V. Fox, R. L. Kraus, S. M. Doran,
T. M. Connolly, C. Tang, J. E. Ballard, Y. Kuo, E. D. Adarayan,
T. Prueksaritanont, M. M. Zrada, M. J. Marino, V. K. Graufelds,
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6 C. D. Cox, P. J. Coleman, M. J. Breslin, D. B. Whitman,
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4239–4252.
Conclusions
We have demonstrated that the 18F-labelled 3-fluoro-4,1-substi-
tuted-piperidine could be obtained with a good yield with vari-
ation of both the 4-position substituent and the N-substituent. In
the case of an alkyl or benzyl substituent on the piperidine (i.e.
electron-donating groups), the incorporation yield was excellent
via a double SN2 mechanism which conserves the configuration.
When the N-substituents were carbonyl or carboxylate (i.e. elec-
tron-withdrawing substituents), the incorporation of fluorine-18
depended on the 4-substitutent and each time inverted the
configuration. We applied the labelling of this moiety, with
success, to the radiosynthesis of three antagonists of the NR2B
subunit-containing NMDA receptor. The radiotracers labelled
with fluorine-18 by a one-step nucleophilic substitution reaction
were efficiently and reliably produced with high specific radio-
activity. Despite promising in vitro properties for [18F]trans-3
and [18F]cis-3, the in vivo μPET studies and ex vivo data of those
radiotracers showed a limited brain uptake and a homogeneous
distribution. These preliminary findings speak against the further
development of, at least, this class of NR2B NMDAR antagon-
ists as radiotracers for PET imaging. Nevertheless, the in vivo
studies demonstrated that the radiolabelling position is stable as
no defluorination was observed. The 3-[18F]fluoropiperidines
could be an advantageous building block to facilitate the devel-
opment of future radiopharmaceuticals containing a piperidine
ring in their structure.
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This research was supported by the French “Ministère de l’en-
seignement supérieur et de la recherche” (RK, GG). The authors
thank Olivier Tirel and Jérôme Delamare for the cyclotron pro-
duction of fluorine-18. The authors are grateful to Dr E.T. Mac-
Kenzie for the proof reading of this article.
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This journal is © The Royal Society of Chemistry 2012