Radiosynthesis and pharmacological evaluation of [11C]EMD-95885: A high affinity ligand for NR2B-containing NMDA receptors

Article abstract of DOI:10.1016/j.bmc.2004.03.065

EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2- one (1) has been described as a selective antagonist for the NMDA receptors containing NR2B subunits, displaying an IC₅₀ of 3.9nM for this subtype. EMD-95885 (1) has been

Full text of DOI:10.1016/j.bmc.2004.03.065

Bioorganic & Medicinal Chemistry 12 (2004) 3229–3237
Radiosynthesis and pharmacological evaluation of [11C]EMD-
95885: a high affinity ligand for NR2B-containing NMDA receptors
a
a
b
c
ꢀ a
ꢀ a
G. Roger, F. Dolle, B. de Bruin, X. Liu, L. Besret, Y. Bramoulle,
C. Coulon,a M. Ottaviani,a M. Bottlaender,a H. Valettea and M. Kassioub,d,
*
aService Hospitalier Frꢀedꢀeric Joliot, Dꢀepartement de Recherche Mꢀedicale, CEA/DSV, 4 Place du Gꢀenꢀeral Leclerc,
F-91401 Orsay, France
bDepartment of Pharmacology, University of Sydney, Sydney NSW 2006, Australia
cURA CEA/CNRS 2210, Service Hospitalier Frꢀedꢀeric Joliot, Dꢀepartement de Recherche Mꢀedicale,
CEA/DSV, 4 Place du Gꢀenꢀeral Leclerc, F-91401 Orsay, France
dDepartment of PET and Nuclear Medicine, Royal Prince Alfred Hospital, Missenden Road,
Camperdown, Sydney NSW 2050, Australia
Received 5 February 2004; revised 29 March 2004; accepted 30 March 2004
Available online 10 May 2004
Abstract—EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one (1) has been described as a selective
antagonist for the NMDA receptors containing NR2B subunits, displaying an IC50 of 3.9 nM for this subtype. EMD-95885 (1) has
been synthesized in good overall yield and labelled with carbon-11 (T1=2: 20.4 min) at its benzoxazolinone moiety using
[11C]phosgene. The pharmacological profile of [11C]EMD-95885 ([11C]-1) was evaluated in vivo in rats with biodistribution studies
and brain radioactivity monitored with intracerebral radiosensitive b-microprobes. The brain uptake of [11C]-1 was homogeneous
(0.4–0.6% ID/mL) across the different brain structures studied. This in vivo brain regional distribution of [11C]-1 was not consistent
with the known distribution of NR2B subunits. Also as a measure of specificity the hippocampus/cerebellum ratio reached 0.8
throughout the time course of the experiment supporting the lack of specificity. Competition studies with the NR2B prototypic
ligand ifenprodil and EMD-95885 (1), 30 min before the radioligand injection, displayed homogeneous reduction of [11C]-1 uptake
of 40–60%. Pre-treatment of rats with DTG (r ligand), MDL105519 (glycine site antagonist) and MK801 (ion channel blocker) had
no inhibitory effect on [11C]-1 uptake. Use of haloperidol as a blocking drug also resulted in a homogeneous inhibition of [11C]-1
uptake by 66–60%, which does not reflect binding to dopamine or r receptors. Due to the homogeneous radioligand uptake and
inhibition and no measure of cerebral blood flow effects during these blocking studies it is uncertain whether any specific binding is
observed. In view of these results, [11C]EMD-95885 ([11C]-1) does not have the required properties for imaging NR2B containing
NMDA receptors using positron emission tomography.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
heteromers composed of both members of the two
subunit families, namely NR1 (eight different splice
variants) and NR2 (A–D) subunits.3 The NR2 subunits
contain glutamate binding sites4 whereas the NR1 sub-
units contain the glycine binding site.5 The subunit
composition of the NMDA receptor confers diverse
functional properties along with significant effect on the
sensitivity of the allosteric modulators.
The N-methyl-
D
-aspartate (NMDA) receptor is a ligand
gated ion channel with the channel opening triggered by
synaptically released glutamate, the principal excitatory
neurotransmitter in the CNS.1 However, binding of
glycine, a co-agonist, is required for receptor activa-
tion.2 Allosteric modulators, which include protons,
polyamines, Zn2þ, Mg2þ and redox reagents also affect
receptor activity.1 Functional NMDA receptors are
NMDA receptors mediate a number of physiological
and pathophysiological processes in the mammalian
CNS. Abnormal expression, function or regulation of
NMDA receptors is involved in a number of neurode-
generative and psychiatric disorders.1;6 Therefore,
ligands that block or reduce the activity of NMDA
receptors should be useful as neuroprotective agents,
Keywords: EMD-95885; NMDA receptor; NR2B subunit; Carbon-11;
Positron emission tomography (PET).
* Corresponding author. Tel.: +61-2-9515-5150; fax: +61-2-9515-6381;
e-mail: mkassiou@med.usyd.edu.au
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.065

3230
G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
O
anticonvulsants, analgesics and possibly for the therapy
of motor disorders such as Parkinson’s disease.7 The
hypothesis that excessive activation of NMDA receptors
is involved with a number of neurodegenerative and
psychiatric disorders has generated extensive interest in
the NMDA receptor as a target for new pharmacother-
apies. However, the contribution of NMDA receptor
subtypes in these disorders is not well understood due to
the lack of subunit selective drugs. This deficiency is
currently been addressed since the discovery of the pro-
totypic NR2B subunit selective antagonist ifenprodil.8;9
O
O
N
O
N
Cl
Cl
Cl
O
O
AlCl3, DMF
75°C, 2 hrs
H
H
3
4
F
O
NH
F
O
N
N
O
NEt3, EtOH
RT, 2 hrs
H
1
Scheme 1. Synthesis of 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-
3H-benzoxazol-2-one (EMD-95885) (1).
Introduction of noninvasive imaging techniques such as
positron emission tomography (PET) has made possible
the study of neuroreceptors in the living human brain.
Such studies have proven useful in the localization and
quantification of neuroreceptors and offer insight into
the relationship of these receptors in normal and path-
ological states.10 Attempts to image NMDA receptors
using PET have mainly focused on development of
carbon-11-labelled radioligands for the glycine binding
site11–14 and the ion channel,15–19 which have proved
unsuccessful. Recently, ifenprodil analogues selective for
the NR2B subunit have also been radiolabelled and
evaluated which include (ꢀ)threo-1-(4-hydroxyphenyl)-
2[4-hydroxy-4-(p-[11C]methoxyphenyl)piperidino]-1-pro-
panol ([11C]CP-101,606) (IC50 ¼ 10 nM for protection
against glutamate toxicity)20 and 5-[3-(4-benzylpi-
peridin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-
[11C]one (IC50 ¼ 5.3 nM for inhibition of NMDA
responses).21 Even though both radioligands display in
vitro selectivity for the NR2B subunit, this selectivity
was not observed in vivo.
ical steps, respectively (Schemes 1 and 2). Both
compounds were synthesized according to literature
procedures.23–25
Commercially available 2-benzoxazolinone 3, was added
to a mixture of aluminium chloride and DMF followed
by 3-chloropropionyl chloride resulting in the formation
of 6-(3-chloropropionyl)-3H-benzoxazol-2-one 4 in 29%
yield.25 Compound 4 was then treated with 4-(4-fluo-
robenzyl)piperidine in ethanol containing triethylamine
to yield the title compound 1 in 65% yield.23
The hydrolysis of the benzoxazolinone moiety of 1 in
alkaline conditions,26;27 10% KOH in ethanol stirring at
room temperature for 3 h, failed to yield the radiol-
abelling precursor 2. A complex mixture of products
resulted in which 2 could not be identified or isolated. A
two step approach was found to be more successful with
the initial step involving formation of the carbamate 5
from the reaction of 1 with di-tert-butyl dicarbonate in
THF in the presence of nucleophilic addition catalyst
dimethylaminopyridine (DMAP) at room temperature
for 1.5 h.24 The t-Boc group was removed by treating 5
with trifluoroacetic acid resulting in the radiolabelling
precursor 2 in 80% yield.
Further optimization of ifenprodil for better selectivity
and potency has resulted in the discovery of 6-[3-[4-(4-
fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one
(EMD-95885, 1).22 This compound was reported to have
high affinity for the NR2B subunit (IC50 ¼ 3.9 nM) and
does not interact with other sites on the NMDA
receptor. Its benzoxazolinone structure permits its
radiolabelling with carbon-11 in the form of [11C]phos-
gene and therefore was considered an appropriate can-
didate for potential imaging of NMDA receptors
containing NR2B subunits.
2.2. Radiochemistry
The benzoxazolinone 1 was labelled with carbon-11
(T1=2: 20.4 min) using no-carrier-added [11C]phosgene
and the aminohydroxyphenyl precursor 2 (Schemes 3
and 4).
The present work represents (a) a concise synthesis of
EMD-95885 (1), as the nonradioactive reference and its
precursor 2 for radiolabelling; (b) the labelling of 1 with
[11C]phosgene at the benzoxazolinone moiety; (c) the in
vivo evaluation of this radioligand in rodents in bio-
distribution studies and brain radioactivity monitoring
with intracerebral radiosensitive b-microprobes.
[11C]Phosgene ([11C]COCl2) was synthesized from
cyclotron-produced [11C]methane ([11C]CH4) via
[11C]carbon tetrachloride ([11C]CCl4) using minor mod-
ifications of published processes (Scheme 3).28–31 Briefly,
[11C]CH4 was separated from the target contents, trap-
ped and concentrated on Porapak-Q. [11C]CH4 was then
carried off by a flow of helium gas, mixed with 3 mL of
chlorine and the mixture passed through an empty linear
horizontal glass tube at a temperature of 510 °C.30 The
on-line synthesized [11C]CCl4 was continuously swept
away by the same helium vector gas and finally passed
through a glass-U-tube containing iron filings at a
temperature of 290–310 °C,31 without intentional addi-
2. Results and discussion
2.1. Chemistry
6-[3-[4-(4-Fluorobenzyl)piperidino]propionyl]-3H-benz-
oxazol-2-one (EMD-95885, 1, as reference) and 1-(4-
fluorobenzyl)piperidin-1-yl]propan-1-one (2, radiolabel-
ling precursor) were synthesized in two and four chem-

G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
3231
1.
2.
Boc2O, DMAP
NEt3, THF
RT, 2 hrs
O
O
F
OH
F
O
N
N
N
O
K2CO3, MeOH
RT, 1.5 hrs
NHBoc
H
1
5
O
F
OH
TFA, CH2Cl2
RT, 4 hrs
N
NH2
2
Scheme 2. Synthesis of the radiolabelling precursor 1-(4-amino-3-hydroxy-phenyl)-3-[4-(4-fluorobenzyl)piperidin-1-yl]propan-1-one (2).
Cl2 (3 mL)
510ºC
Fe filings
[
11C]CH4
[
11C]CCl4
[
11C]COCl2
290-315ºC
vector gas : helium
Scheme 3. Preparation of [11C]phosgene from [11C]methane.
O
O
11C]COCl2
CH2Cl2
25ºC
30-60 sec
F
F
OH
O
[
N
N
O
*
N
NH2
[
11C] = *
H
2
[
11C]-1
[
11C]EMD-95885
Scheme 4. Radiosynthesis of 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-[11C]one ([11C]EMD-95885, [11C]-1).
tion of oxygen to the system, giving [11C]COCl2 in 12–
13 min and an average of 30–50% decay-corrected
radiochemical yield, based on starting [11C]CH4.
chemically and radiochemically stable for at least
60 min. Administration to animals was done within
15 min following end of synthesis.
[11C]COCl2 was trapped (bubbling through) at room
temperature in CH2Cl2 (500 lL) containing the labelling
precursor 2 (0.5–1.0 mg) (Scheme 4). The cyclization
reaction of [11C]phosgene was fast (almost instanta-
neously), but gave [11C]-1 in low radiochemical yields.
Typically, starting from a 1.2 Ci (44.4 GBq) [11C]CH4
production batch, 10–20 mCi (0.37–0.74 GBq) of [11C]-1
with a radiochemical- and chemical purity of more than
99% was routinely obtained within 25 min of radiosyn-
thesis, including HPLC purification. The total decay-
corrected radiochemical yield of [11C]-1, based on
starting [11C]CH4, was only 6.5–7.8% (n ¼ 5). Addition
of 2 equiv of triethylamine did not improve the cycliza-
tion reaction yields. No further efforts were made in
order to increase the observed low yields. The specific
radioactivity measured at the end of the radiosynthesis
was 1–2 Ci/lmol (37-74 GBq/lmol).
2.3. Pharmacology
2.3.1. Radiosensitive b-microprobe acquisition. The brain
uptake of [11C]-1 was investigated in rats using intrace-
rebral radiosensitive b-microprobes32;33 (Fig. 1A and B).
Following i.v. injection of [11C]-1 (0.5–0.8 mCi with
specific radioactivity greater than 1 Ci/lmol), the uptake
in the cerebellum during the 45 min acquisition was
higher than that in the left striatum, 0.50% and
0.35% ID/mL, respectively. In both structures, the
radioactive concentration was maximal 2 min post-
injection. The washout phase was very slow and similar
in both structures. Pre-treatment of rats with EMD-
95885 (1, 3 mg/kg, i.p., 30 min before injection of the
radioligand) resulted in a similar uptake of [11C]-1 in the
cerebellum as in the control experiment at earlier time
points, but displayed a faster washout, which was evi-
dent as early as 20 min. At 45 min the uptake in the
cerebellum was only 0.3% ID/mL compared to
0.5% ID/mL for the control experiment. The uptake of
[11C]-1 in the cerebellum (chosen for its highest accu-
mulation level) was also inhibited by pre-administration
of the NR2B prototypic ligand ifenprodil (20 mg/kg, i.p.,
30 min before injection of the radioligand) (Fig. 1B). In
this case reduction of radioligand uptake was evident as
early as 2 min (0.4% ID/mL) followed by washout,
which reached levels of 0.3% ID/mL at 45 min.
Formulation of labelled product for i.v. injection was
effected as follows: (1) HPLC solvent removal by evap-
oration; (2) reconstitute the residue in 5 mL of physio-
logical saline containing 10% ethanol; (3) sterile
filtration through a 0.22 lm filter. The solution for
injection was a clear and colourless solution and its pH
was between 5 and 7. The preparation was found to be
>99% chemically and radiochemically pure, as demon-
strated by HPLC analysis. The preparation was free
from starting labelling precursor and was shown to be

3232
G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
cerebellum
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
control experiment
presaturation Ifenprodil
presaturation EMD-95885
cerebellum
striatum
plasma
0
5
10 15 20 25 30 35 40 45
0
5
10 15 20 25 30 35 40 45
(B)
(A)
Time (min)
Time (min)
Figure 1. Radiosensitive b-microprobe acquisition: (A) time course of [11C]EMD-95885 ([11C]-1) in rat striatum and cerebellum; (B) effect of various
drugs on [11C]EMD-95885 ([11C]-1) binding in rat cerebellum.
pons
liver
kidney
heart
lung
muscle
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
30.0
25.0
20.0
15.0
10.0
5.0
cerebellum
hippocampus
frontal cortex
blood
thalamus
striatum
posterial cortex
plasma
0.0
0
10 20 30 40 50 60
0
10 20 30 40 50 60
(A)
(B)
Time (min)
Time (min)
Figure 2. Biodistribution studies in rats: (A) time course of [11C]EMD-95885 ([11C]-1) in rat brain; (B) time course of [11C]EMD-95885 ([11C]-1) in rat
peripheral organs.
2.3.2. Biodistribution studies. Uptake of [11C]-1 in the rat
brain was determined in biodistribution experiments
(Fig. 2A). Following i.v. injection of [11C]-1 (22–26 lCi
with specific radioactivity greater than 1 Ci/lmol), the
distribution was rather homogeneous in all brain
structures studied. The uptake in the cerebellum and
striatum at 5 min was 0.6% and 0.4% ID/mL, respec-
tively, which was consistent with that observed using the
radiosensitive b-microprobes. NR2B subunits are pri-
marily localized in forebrain regions, hippocam-
pus > cortex > striatum > thalamus > cerebellum.34
receptor subunits including NR2A and NR2C which are
expressed in high numbers in the cerebellum.3;35 In the
peripheral organs, the lung showed the highest uptake
(28% ID/mL at 5 min) which may reflect a high lipo-
philicity of 1 (cLogP ¼ 4:0) (Fig. 2B).
In vivo specificity and selectivity of [11C]-1 binding was
examined by studying the effects of ifenprodil (NR2B/r
antagonist), MK801 (NMDA channel blocker),
MDL105519 (glycine site antagonist), haloperidol (D2/
r antagonist), and DTG (r ligand), and the unlabelled
drug itself, EMD-95885 (1), which were administered at
doses of 20, 0.5, 16, 1.5, 2 and 3 mg/kg i.p., respectively,
30 min prior to [11C]-1 (Fig. 3). Pre-treatment of rats
with 1 resulted in a 40–50% global reduction [11C]-1
uptake demonstrating some specific binding in the brain.
Pre-injection of the NR2B ligand ifenprodil reduced the
uptake of [11C]-1 in all brain regions by 46–58%. Al-
though ifenprodil binds to NR2B subunits, the inhibi-
tion pattern was inconsistent for this site. Binding to
When specific binding was defined as the ratio of the
radioactivity concentration in hippocampus and cere-
bellum, a value of 0.8 was obtained throughout the time
course of the experiment. Therefore, the in vivo regional
brain distribution of [11C]-1 did not correlate with the
known distribution of NR2B subunits. This uptake does
not demonstrate exclusively binding to NR2B subunits
but might also reflect in vivo binding to other NMDA

G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
3233
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
EMD-95885
MK801
Haloperidol
Ifenprodil
MDL105519
DTG
Figure 3. Effect of various drugs on [11C]EMD-95885 ([11C]-1) binding in rat brain. Data are means ꢀ SD (n ¼ 3).
sigma (r) receptors was also examined as ifenprodil also
shows high affinity for this site (IC50 ¼ 3.9 nM).36 The r
ligand DTG had no inhibitory effect on [11C]-1 binding
in any brain region. Pre-treatment with haloperidol re-
sulted in inhibition of [11C]-1 binding in all brain regions
by 60–66%. This does not reflect dopaminergic binding
of the radioligand since highest inhibition was observed
in the cerebellum (66%), and unlikely to reflect r
receptor binding as the r ligand DTG had no inhibitory
effect on [11C]-1 uptake in any brain region. However,
haloperidol has been reported to block NMDA recep-
tors with efficacy in the low lmol range.37 This inter-
action was assumed to be related to interactions with the
glycine site38 with subsequent electrophysiological
studies showing that haloperidol specifically inhibited
NR2B-containing-NMDA receptors.39 Nevertheless, the
homogeneous pattern of radioligand inhibition dis-
played by haloperidol is not consistent with selective
binding to NR2B subunits. As there are no independent
measures on the effect of cerebral blood flow during
blocking studies, it is uncertain whether any inhibition
observed is a result of specific binding to possibly a
combination of subunits, including NR2A and NR2C,
or binding at a different site. Pre-treatment of rats with
the glycine site antagonist, MDL105519 and the ion
channel blocker, MK801, displayed no inhibition of
[11C]-1 uptake indicating that no radioligand binding
was evident at these sites.
4. Experimental
4.1. General
4.1.1. Chemicals, TLCs and HPLCs. Chemicals were
purchased from Aldrich and used with no further puri-
fication. 4-(4-Fluorobenzyl)piperidineÆHCl was a gener-
ous gift from Pfizer (Dr. Yuen, Ann Arbor MI). TLCs
were run on pre-coated plates of silica gel 60F254
(Merck) and compounds were visualized at 254 nm using
a UV lamp. HPLCs: [HPLC A]: equipment: waters 600
Pump and Waters 600 Controller, a Shimadzu SPD10-
AVP UV-multi-wavelength detector; column: Hewlett
Packard Zorbaxâ RX-Sil (250 ꢁ 9.4 mm); porosity:
5 lm; eluent CH2Cl2/MeOH/aq 28% NH4OH: 93:7:0.5
(v:v:v); flow rate: 5 mL/min; temperature: rt; absorbance
detection at k ¼ 254 nm. [HPLC B]: equipment: Waters
Alliance 2690 equipped with a UV spectrophotometer
(photodiode array detector, Waters 996) and a Berthold
LB509 radioactivity detector; column: analytical Sym-
metry-Mâ C-18, Waters (4.6 ꢁ 50 mm); porosity: 3.5 lm;
conditions: isocratic elution with solvent A/solvent B:
39:61 (v:v) [solvent A: H2O containing low-UV PICâ B7
reagent (waters, % by weight: methanol (18–22%), hep-
tane sulfonic acid–sodium salts (4–6%), phosphate buf-
fer solution (3–7%), water (65–75%), pH 3), 20 mL for
1000 mL; solvent B: H2O/CH3CN: 50:50 (v:v) contain-
ing low-UV PICâ B7 reagent, 20 mL for 1000 mL]; flow
rate: 2.0 mL/min; temperature: 30 °C; absorbance
detection at k ¼ 254 nm.
3. Conclusion
The NR2B subtype-selective antagonist, 6-[3-[4-(4-fluo-
robenzyl)piperidino]propionyl]-3H-benzoxazol-2-one
(EMD-95885, 1), has been synthesized in good yield and
labelled with carbon-11 (T1=2: 20.4 min) at its benzox-
azolinone moiety using [11C]phosgene. The evaluation of
its in vivo pharmacological profile (biodistribution
studies and brain radioactivity monitoring using intra-
cerebral radiosensitive b-microprobes) clearly indicates
that it does not have the required properties for imaging
NR2B containing NMDA receptors using positron
emission tomography.
4.1.2. Spectroscopies. NMR spectra were recorded on a
Varian Gemini (300 MHz) instrument using the hydro-
genated residue of the deuteriated solvents (CDCl3,
d ¼ 7:26 ppm; CD3OD, d ¼ 3:34 and 4:11 ppm) and/or
1
TMS as internal standard for H NMR. The chemical
shifts are reported in ppm downfield from TMS (br m,
d, dd, dt, m, t; broad multiplet, doublet, doublet of
doublet, doublet of triplet, multiplet and triplet,
respectively). The mass spectra MS were recorded on a
Finnigan/MAT TSQ46 using electron impact.

3234
G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
4.1.3. Radioisotope production. No-carrier-added
[11C]CH4 was produced on a CGR-MeV 520 cyclotron
by irradiation of a target consisting of an ultrapure Air
Liquide 95/5 mixture of N2/H2 (target holder: 668 mL
operated at 8 bars) using a 20 MeV proton beam by the
14N[p, a]11C nuclear reaction. Typical production:
1.20 Ci (44.40 GBq) of [11C]CH4 at the end of bom-
bardment (EOB) for a 30 lA, 30 min (54,000 lC) irra-
diation.
6H); 7.0 (t, J ¼ 8:6 Hz, 2H); 7.2 (m, 3H); 7.9 (d,
J ¼ 1:8 Hz, 1H); 7.9 (dd, J ¼ 1:8, 8.6 Hz, 1H). MS:
C22H23F1N2O3: 383 [Mþ1].
4.2.3. (4-[3-[4-(4-Fluorobenzyl)piperidino]propionyl]-2-hy-
droxy-phenyl)-carbamic acid tert-butyl ester (5). 6-[3-[4-
(4-Fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-
2-one (1, 0.3 g, 0.7 mmol), NEt3 (0.1 mL, 0.9 mmol) and
DMAP (19 mg, 0.2 mmol) were dissolved in THF
(7 mL). To the mixture was then added di-tert-butyl
dicarbonate (0.2 g, 1.0 mmol) in three portions over
10 min and the mixture was stirred at room temperature
for 2 h. The reaction mixture was concentrated in vacuo
to give a light-yellow oil, to which was added MeOH
(6 mL) followed by stirring for an additional 40 min at
room temperature. A light-yellow precipitate was
formed to which was added K2CO3 (107 mg, 0.8 mmol)
and stirring was continued for 1.5 h. The mixture was
concentrated by evaporation to reduce the volume of
MeOH and water was added resulting in an suspension,
which was partitioned with EtOAc and water. The or-
ganic phase was filtered through silica gel (1.9 g) to give
a light-yellow clear solution, which was concentrated in
vacuo to give the title compound (5) as a light-yellow oil,
which solidified on cooling (0.3 g, 89%). 1H NMR
(CDCl3): 1.2–1.4 (m, 2H); 1.5 (m, 10H); 1.6–1.8 (d,
J ¼ 12:6 Hz, 2H); 2.0–2.2 (t, J ¼ 11:4 Hz, 2H); 2.5 (d,
J ¼ 6:9 Hz, 2H); 2.9 (t, J ¼ 7:4 Hz, 2H); 3.1 (d,
J ¼ 11:4 Hz, 2H); 3.2 (t, J ¼ 7:4 Hz, 2H); 6.95 (m, 2H);
7.1 (m, 2H); 7.4 (m, 2H); 7.9 (d, J ¼ 10:5 Hz, 1H).
4.1.4. Miscellaneous. Radiosyntheses using carbon-11,
including the HPLC purifications, were performed in a
5 cm-lead shielded cell. Specific radioactivity was deter-
mined as follows: the area of the absorbance peak cor-
responding to the radiolabelled product was measured
on the HPLC chromatogram and compared to a stan-
dard curve relating mass to absorbance.
4.2. Chemistry
4.2.1. 6-(3-Chloropropionyl)-3H-benzoxazol-2-one (4).
Dimethylformamide (6.0 mL, 80 mmol) was slowly
added to aluminium chloride (37.3 g, 280 mmol) while
stirring and maintaining a temperature of 45 °C. To the
mixture was then added 2-benzoxazolinone (3, 5.4 g,
40 mmol) and 3-chloropropionyl chloride (6.2 g,
60 mmol) followed by warming at 75 °C for 2 h. The
reaction mixture was then poured on ice and the crude
product (4.6 g) was collected by filtration, air-dried and
recrystallized from dioxane. This yielded the title com-
pound (4) as a white solid (2.6 g, 29%). 1H NMR
(CDCl3) d: 3.45 (m, 2H); 3.95 (m, 2H); 7.2 (m, 2H); 7.9
(m, 2H); 8.6 (br m, 1H).
4.2.4. 1-(4-Amino-3-hydroxy-phenyl)-3-[4-(4-fluorobenz-
yl)piperidin-1-yl]propan-1-one (2).
A suspension of
(4-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-2-hydroxy-
phenyl)-carbamic acid tert-butyl ester (5, 0.3 g,
0.7 mmol) in CH2Cl2 (6.9 mL) was cooled to 0 °C. Tri-
fluoroacetic acid (0.7 mL) was added and the resulting
light-brown solution was stirred for 0.5 h at 0 °C and
then for 4 h at room temperature. The reaction mixture
was evaporated to dryness and basified with a concen-
trated Na2CO3 solution, then extracted with CH2Cl2.
The organic layer was dried with Na2SO4 and evapo-
rated in vacuo and the residue was purified by column
chromatography to yield the title compound as a brown
4.2.2. 6-[3-[4-(4-Fluorobenzyl)piperidino]propionyl]-3H-
benzoxazol-2-one (1). 4-(4-Fluorobenzyl)piperidineÆHCl
(1.0 g, 4.4 mmol) and NEt3 (1.3 mL, 9.3 mmol) were
added to a suspension of 6-(3-chloropropionyl)-3H-
benzoxazol-2-one (4, 1.0 g, 4.4 mmol) in EtOH (10 mL).
The mixture was stirred at room temperature for 2 h and
then basified using excess NEt3. The reaction mixture
was concentrated by rotary evaporation and the residue
suspended in isopropanol. The resulting solid was fil-
tered and washed with water and isopropanol and air-
dried to yield the title compound (1) as a white powder
1
solid (0.2 g, 80%). H NMR (CDCl3) d: 1.4 (dt, J ¼ 3:6,
12.6 Hz, 2H); 1.5 (m, 1H); 1.65 (d, J ¼ 12:6 Hz, 2H);
2.0–2.2 (t, J ¼ 11:4 Hz, 2H); 2.5 (d, J ¼ 6:9 Hz, 2H);
2.85 (t, J ¼ 7:4 Hz, 2H); 3.05 (d, J ¼ 11:4 Hz, 2H); 3.15
(t, J ¼ 7:4 Hz, 2H); 6.65 (d, J ¼ 8:7 Hz, 1H); 6.95 (t,
J ¼ 8:7 Hz, 2H); 7.05 (dd, J ¼ 5:4, 8.7 Hz, 2H); 7.4 (m,
2H). MS: C21H25F1N2O2: 357 [Mþ1].
1
(1.2 g, 71%). H NMR (CDCl3) d: 1.3 (m, 2H); 1.5 (m,
1H); 1.65 (d, J ¼ 12:6 Hz, 2H); 2.0 (m, 2H); 2.5 (d,
J ¼ 6:9 Hz, 2H); 2.8 (t, J ¼ 7:4 Hz, 2H); 2.95 (d,
J ¼ 11:4 Hz, 2H); 3.2 (t, J ¼ 7:4 Hz, 2H); 6.95 (m, 2H);
7.15 (m, 3H); 7.8 (m, 2H).
The free base was converted into its HCl salt by sus-
pending 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-
3H-benzoxazol-2-one (1, 0.3 g, 0.8 mmol) in a mixture of
isopropanol (3 mL) and diethyl ether saturated with HCl
(2 mL). The suspension was filtered and the solid washed
with small amounts of anhydrous diethyl ether to give
the HCl salt of (1) as a white powder (0.2 g, 67%). H
NMR (CD3OD) d: 1.5 (m, 2H); 1.9 (m, 3H); 2.6 (d,
J ¼ 6:6 Hz, 2H); 3.0 (t, J ¼ 11:7 Hz, 2H); 3.5–3.7 (m,
4.3. Radiochemistry
4.3.1. Preparation of [11C]CCl4 . At the end of the bom-
bardment, the target contents were transferred by
expansion to the 5 cm-lead shielded hot cell dedicated to
the radiosynthesis of the tracer and passed firstly
through an empty tube (stainless steel coil, 500 mm
length, 4 mm internal diameter, cooled at ꢂ186 °C using
1

G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
3235
liquid argon) in order to remove traces of 13N-ammonia
(produced during the irradiation) and secondly through
a guard of P2O5 (glass tube, 70 mm length, 3 mm inter-
nal diameter) in order to remove moisture. [11C]CH4 was
then separated from the target gas by trapping in a
copper-U-tube (150 mm length, 4 mm internal diameter)
filled with Porapak-Q (80–100 mesh, Waters) and cooled
at ꢂ186 °C (liquid argon). [11C]CH4 was released from
the trap by warming the copper-U-tube to room tem-
perature (hot air) and swept away by a flow of helium
gas (40 mL/min). [11C]CH4 was then passed through a
guard of P2O5 (glass tube, 70 mm length, 10 mm internal
diameter) and concentrated in a second smaller copper-
U-tube (150 mm length, 2 mm internal diameter) filled
with Porapak-Q (80–100 mesh, Waters) and cooled at
ꢂ186 °C (liquid argon). On average, about 1.20 Ci or
44.40 GBq (at EOB) of [11C]CH4 is routinely produced
for a 30 lA, 30 min (54,000 lC) irradiation and then
transferred and concentrated in 4–5 min using the pro-
cess described above. [11C]CH4 was released from the
trap by warming the latter to room temperature and
swept (15 mL/min) in a volume of 1–2 mL of helium into
a gas mixing chamber containing 3 mL of chlorine
(99.99%, Air Liquide). Using the same helium as vector
gas (15 mL/min), the [11C]CH4-chlorine mixture was
passed through an empty horizontal glass tube (215 mm
length, 7 mm internal diameter) at a temperature of
510 °C converting [11C]CH4 into [11C]CCl4.
stream, oil bath temperature: 70–80 °C). The residue was
taken up in 2–5 mL of physiological saline containing
10% of ethanol and filtered through a sterile 0.22 lm
filter into a sterile, pyrogen free evacuated vial.
4.3.5. Quality control of 6-[3-[4-(4-fluorobenzyl)piper-
idino]propionyl]-3H-benzoxazol-2-[11C]one ([11C]-1). The
radioligand preparation is a clear and colourless solu-
tion and its pH is between 5 and 7. As demonstrated by
HPLC analysis (HPLC B), the radiolabelled product
was found to be >98% radiochemically pure (HPLC B;
retention time: 3.12 min). The preparation was shown to
be free of nonradioactive precursor and radiochemically
stable for at least 60 min. Specific radioactivity was
calculated from three consecutive HPLC analyses
(average) and determined as follows: The area of the UV
absorbance peak corresponding to the radiolabelled
product was measured (integrated) on the HPLC chro-
matogram and compared to a standard curve relating
mass to UV absorbance. The first injection in animal
experiments was done within 15 min after the end of
synthesis.
4.4. Kinetics, regional distribution and radiopharmaco-
logical characterization in rodents
4.4.1. Animals. Sprague–Dawley male rats weighing
250–350 g were used in all experiments. Animals use
procedures were in accordance with the recommenda-
tions of the EEC (86/609/CEE) and the French National
Committee (decret 87/848) for the care and use of lab-
oratory animals.
4.3.2. Preparation of [11C]COCl2. The synthesized
[11C]CCl4 was then on-line passed through a glass-U-
tube (200 mm length, 4 mm internal diameter) contain-
ing 1.5 g of iron filings (Telar 57, Weber) at a tempera-
ture of 290–310 °C (using the [11C]CCl4 vector gas)
converting it into [11C]COCl2, which was then passed
through an antimony-guard (glass tube, 70 mm length,
3 mm internal diameter, containing a 2/1 ratio [v/v] of
antimony powder (400 mg) and glass beads (1 mm
diameter) in order to remove the excess of chlorine.
4.4.2. Intracerebral radiosensitive b-microprobes acquisi-
tion. Anaesthesia of animals was induced with 5% iso-
flurane in a gas mixture of O2/N2O (30/70%) and
maintained with 1.5–2.5% isoflurane during the entire
surgical procedure. Body temperature was monitored
rectally and maintained by means of a thermoregulated
blanket. Catheters were placed in both femoral vein and
artery. Animals were then mounted in a stereotaxic
frame. Craniotomies were performed in order to implant
the radiosensitive b-microprobes (Biospace Mesures,
Paris). Two probes were implanted, the first one in the
left striatum and the second one in the cerebellum using
the coordinates of implantation according to the atlas of
Paxinos and Watson.40 Data were acquired from 30 min
after probes implantation. Local radioactivity count
rates were acquired for 30 min prior to the radioligand
injection to evaluate environmental background
(approximately 5 cps) with a temporal resolution of 1 s.
In control experiments, the radioligand was injected in
the femoral vein ([11C]-1, 0.5–0.8 mCi) in a volume of
1 mL over 1 min. Data were acquired for another 45 min
with a temporal resolution of 1 s. Finally, mean back-
ground noise was subtracted from the raw data and
radioactive decay correction for carbon-11 was applied
to obtain quantitative time activity curves. Samples of
plasma were also obtained for each animal. Results were
expressed as %injected dose per mL (% ID/mL). In vivo
4.3.3. Preparation of 6-[3-[4-(4-fluorobenzyl)piperidino]-
propionyl]-3H-benzoxazol-2-[11C]one ([11C]-1). The on-
line synthesized [11C]COCl2 was trapped (bubbling
through) at room temperature in a reaction vessel con-
taining 0.5–1.0 mg of the labelling precursor 1-(4-amino-
3-hydroxy-phenyl)-3-[4-(4-fluoro-benzyl)-piperidin-1-yl]-
propan-1-one (2, 1.4–2.8 lmol) dissolved in 500 lL of
CH2Cl2. Trapping of [11C]COCl2 was monitored using
an ionization-chamber probe. When the reading had
reached its maximum (2–3 min usually), the reaction
mixture was diluted with 1.0 mL of HPLC solvent and
injected onto the column. (HPLC A; tR: [11C]-1: 7.0–
7.5 min).
4.3.4. Formulation of 6-[3-[4-(4-fluorobenzyl)piperidino]-
propionyl]-3H-benzoxazol-2-[11C]one ([11C]-1). Formula-
tion of labelled product for i.v. injection was effected as
follows: The HPLC-collected fraction containing [11C]-1
was concentrated to dryness (using a rotavapor, water
bath temperature: 40–60 °C or using a helium gas

3236
G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
12. Haradahira, T.; Okauchi, T.; Maeda, J.; Zhang, M.-R.;
Kida, T.; Kawabe, K.; Mishina, M.; Watanabe, Y.;
Suzuki, K.; Suhara, T. Synapse 2002, 43, 131.
13. Waterhouse, R.; Sultana, A.; Laruelle, M. Nucl. Med.
Biol. 2002, 29, 791.
receptor blocking studies were performed by pre-
administration, 30 min before injection of the radio-
ligand, of EMD-95885 (1, 3 mg/kg, i.p.) or ifenprodil
(20 mg/kg, i.p.).
14. Haradahira, T.; Zhang, M.-R.; Maeda, J.; Okauchi, T.;
Kida, T.; Kawabe, K.; Sasaki, S.; Suhara, T.; Suzuki, K.
Chem. Pharm. Bull. Tokyo 2001, 49, 147.
15. Kokic, M.; Honer, M.; Kessler, L.; Grauert, M.; Schubi-
ger, P.; Ametamey, S. J. Recept. Signal Transduct 2002, 22,
123.
16. Ishibashi, N.; Kuwamura, T.; Sano, H.; Yamamoto, F.;
Haradahira, T.; Suzuki, K.; Suhara, T.; Sasaki, S.; Maeda,
J. J. Lab.Compd. Radiopharm. 2000, 43, 375.
17. Haradahira, T.; Sasaki, S.; Maeda, J.; Kobayashi, K.;
Inoue, O.; Tomita, U.; Nishikawa, T.; Suzuki, K. J.
Lab.Compd. Radiopharm. 1998, 41, 843.
4.4.3. Biodistribution studies. Each animal received 15–
30 lCi of the radioligand ([11C]-1), dissolved in 0.1 mL
saline, by injection in a tail vein. At designated times (5,
15, 30, 60 min) after injection of the radioligand, animals
(n ¼ 3 per time point) were killed by decapitation, the
brains as well as peripheral organs were quickly re-
moved, dissected, weighed and assayed for radioactivity
in a c-counter (Cobra Quantum, Packard). Samples of
liver, kidney, heart, lung, muscle, blood, plasma and for
the brain, pons, cerebellum, thalamus, hippocampus,
striatum, frontal cortex, posterial cortex were obtained
for each animal. Results were expressed as %injected
dose per mL (% ID/mL) after correction for the physical
decay of the radioisotope. In vivo receptor blocking
studies were also performed on three animals by pre-
administration, 30 min before injection of the radio-
ligand, of EMD-95885 (1, 3 mg/kg, i.p.), ifenprodil
(20 mg/kg, i.p.), MK-801 (0.5 mg/kg, i.p.), MDL-105519
(16 mg/kg, i.p.), haloperidol (1.5 mg/kg, i.p.) or 1,3-di-
ortho-tolylguanidine (DTG, 2 mg/kg, i.v.). Animals were
then i.v.-injected with the radioligand ([11C]-1, 15–20 lCi
in 0.1 mL saline) and sacrificed 30 min later. The brain
was dissected as mentioned above. Results were
expressed as % of variation compared to the controls
after correction for the physical decay of the radioiso-
tope and correction of plasma variation.
18. Sihver, S.; Sihver, W.; Andersson, Y.; Murata, T.;
Bergstrom, M.; Onoe, H.; Matsumura, K.; Tsukada, H.;
ꢁ
€
Oreland, L.; Langstrom, B.; Watanabe, Y. J. Neural.
Trans. 1998, 105, 117.
19. Andersson, Y.; Tyrefors, N.; Sihver, S.; Onoe, H.;
ꢁ
Watanave, Y.; Tsukada, H.; Langstrom, B. J. Lab.Compd.
€
Radiopharm. 1998, 41, 567.
20. Haradahira, T.; Maeda, J.; Okauchi, T.; Zhang, M.-R.;
Hojo, J.; Kida, T.; Arai, T.; Yamamoto, F.; Sasaki, S.;
Maeda, M.; Suzuki, K.; Suhara, T. Nucl. Med. Biol. 2002,
29, 517.
21. Roger, G.; Lagnel, B.; Besret, L.; Bramoulle, Y.; Coulon,
C.; Ottaviani, M.; Kassiou, M.; Bottlaender, M.; Valette,
H.; Dolle, F. Bioorg. Med. Chem. 2003, 11, 5401.
22. Leibrock, J.; Prucher, H.; Rautenberg, W. Pharmazie
1997, 52, 479.
23. Prucher, H.; Gottschlich, R.; Leibrock, J. WO Patent 98/
18793, 1998.
24. Baine, N.; Clark, W., Jr.; Eldridge, A. WO Patent 00/
12468, 2000.
25. Aichaoui, H.; Lesieur, D.; Henichart, J.-P. J. Heterocycl.
Chem. 1992, 29, 171.
Acknowledgements
26. Moussavi, Z.; Lesieur, D.; Lespagnol, C.; Sauzieres, J.;
Olivier, P. Eur. J. Med. Chem. 1989, 24, 55.
27. Bonte, J.-P.; Lesieur, D.; Lespagnol, C.; Cazin, J.-C.;
Cazin, M. Eur. J. Med. Chem. 1974, 9, 497.
The authors wish to thank the cyclotron operators Mr.
Daniel Gouel, Mr. Christophe Peronne and Mr. Chris-
^
tophe Lechene for performing the irradiations. The
ꢀ
28. Dolle, F.; Valette, H.; Bramoulle, Y.; Guenther, I.;
authors also wish to thank Dr. Dirk Roeda for proof-
reading the manuscript.
Fuseau, C.; Coulon, C.; Lartizien, C.; Jegham, S.; George,
P.; Curet, O.; Pinquier, J.-L.; Bottlaender, M. Bioorg.
Med. Chem. Lett. 2003, 13, 1771.
ꢀ
29. Dolle, F.; Bramoulle, Y.; Hinnen, F.; Demphel, S.;
References and notes
George, P.; Bottlaender, M. J. Lab. Compd. Radiopharm.
2003, 46, 783.
1. Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S.
Pharmacol. Rev. 1999, 51, 7.
30. Link, J.; Krohn, K. J. Lab. Compd. Radiopharm. 1997, 40,
306.
2. Johnson, J.; Ascher, P. Nature 1987, 325, 529.
3. Seeberg, P. Trends Neurosci. 1993, 16, 359.
4. Laube, B.; Hirai, H.; Sturgess, M.; Betz, H.; Kuhse, J.
Neuron 1997, 18, 493.
5. Kuryatov, A.; Laube, B.; Betz, H.; Kuhse, J. Neuron 1994,
12, 1291.
6. Danysz, W.; Parsons, C. Pharmacol. Rev. 1998, 50, 597.
7. Le, D.; Lipton, S. Drugs Aging 2001, 18, 717.
8. Nikam, S.; Meltzer, L. Curr. Pharm. Des. 2002, 8, 845.
9. Chenard, B. L.; Menniti, F. S. Curr. Pharm. Des. 1999, 5, 381.
10. Frost, J., Jr.; Wagner, H. Quantitative Imaging- Neuro-
receptors, Neurotransmitters and Enzymes; Raven: New
York, 1990.
31. Landais, P.; Crouzel, C. Appl. Radiat. Isot. 1987, 38, 297.
32. Pain, F.; Laniece, P.; Mastrippolito, R.; Pinot, L.; Charon,
Y.; Glatiny, A.; Guillemin, M.; Hantraye, P.; Leviel, V.;
Menard, L.; Valentin, L. IEEE Trans. Nucl. Med. 2002,
49, 822.
33. Zimmer, L.; Hassoun, W.; Pain, F.; Bonnefoi, F.; Laniece,
P.; Mastrippolito, R.; Pinot, L.; Pujol, J.; Leviel, V.
J. Nucl. Med. 2002, 43, 227.
34. Mutel, V.; Buchy, D.; Klingelschmidt, A.; Messer, J.;
Bleuel, Z.; Kemp, J. A.; Richards, J. G. J. Neurochem.
1998, 70, 2147.
35. Monyer, H.; Burnashev, N.; Laurie, D.; Sakmann, B.;
Seeberg, P. Neuron 1994, 12, 529.
11. Haradahira, T.; Okauchi, T.; Maeda, J.; Zhang, M.-R.;
Nishikawa, T.; Konno, R.; Suzuki, K.; Suhara, T. Synapse
2003, 50, 130.
36. Chenard, B.; Shalaby, I.; Koe, B.; Ronau, R.; Butler, T.;
Prochniak, M.; Schmidt, A.; Fox, A. J. Med. Chem. 1991,
34, 3085.

G. Roger et al. / Bioorg. Med. Chem. 12 (2004) 3229–3237
3237
37. Monnet, F.; Debonnel, G.; Juien, J.-L.; De Montigny, C.
Eur. J. Pharmacol. 1990, 179, 441.
39. Coughenour, L. L.; Cordon, J. J. Pharmacol. Exp. Ther.
1997, 280, 584.
38. Fletcher, E.; MacDonald, J. Eur. J. Pharmacol. 1993, 235,
291.
40. Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic
Coordinates; Academic: New York, 1986.