A positron emission tomography radioligand for the in vivo labeling of metabotropic glutamate 1 receptor: (3-Ethyl-2-[11C]methyl-6- quinolinyl)(cis-4-methoxycyclohexyl)methanone

Article abstract of DOI:10.1021/jm050263+

A selective metabotropic glutamate 1 receptor (mGlu1) antagonist was labeled with the positron-emitting radioisotope carbon-11 and evaluated in ex vivo biodistribution studies and micro-positron emission tomography (micro-PET) imaging experiments in rats.

Full text of DOI:10.1021/jm050263+

5096
J. Med. Chem. 2005, 48, 5096-5099
Chart 1. Metabotropic Glutamate 1 (mGlu1)
Antagonists
A Positron Emission Tomography
Radioligand for the in Vivo Labeling of
Metabotropic Glutamate 1 Receptor:
(3-Ethyl-2-[11C]methyl-6-quinolinyl)(cis-
4-methoxycyclohexyl)methanone
Yiyun Huang,*,†,‡ Raj Narendran,† Franc¸ois Bischoff,§
Ningning Guo,† Zhihong Zhu,† Sung-A Bae,†
Anne S. Lesage,# and Marc Laruelle†,‡
Departments of Psychiatry and Radiology, Columbia
University College of Physicians and Surgeons, New York,
New York 10032, and Department of Medicinal Chemistry
and CNS Discovery Research, Johnson & Johnson
Pharmaceutical Research and Development,
[3H]methoxy-PEPy (PEPy ) 5-(pyridin-2-ylethynyl)-
pyridine) should further aid in the elucidation of mGlu5
functions.8-10 On the other hand, a detailed study of the
mGlu1 receptor has heretofore been hindered by the
lack of high affinity, selective ligands, and radioligands
for this receptor subtype. Lavreysen et al. recently
reported the characterization of radioligand 1, [3H]1-
(3,4-dihydro-2H-pyrano[2.3-b]quinolin-7-yl)-2-phenyl-
ethanone ([3H]R214127, Chart 1), as a selective an-
tagonist radioligand for mGlu1 receptor and demon-
strated that it can be used to label the mGlu1 receptor
in vitro and ex vivo,11,12 while Mabire et al. disclosed a
large series of substituted quinolines, including 2 and
3, as selective mGlu1 antagonists.13,14 Still lacking are
radioligands suitable for the in vivo labeling of mGlu1
receptor using the noninvasive imaging technology
positron emission tomopraphy (PET). In this paper we
report on the synthesis and characterization of a 11C-
labeled mGlu1 antagonist, (3-ethyl-2-[11C]methyl-6-
quinolinyl)(cis-4-methoxycyclohexyl)methanone ([11C]2,
or [11C]JNJ-16567083), as the first selective PET radio-
ligand for in vivo labeling of the mGlu1 receptor.
The synthesis of compound 2 and its radiolabeled
form [11C]2 is presented in Scheme 1.14 N-(4-Bromo-
phenyl)butanamide (4) was reacted with DMF and
POCl3 to produce 6-bromo-2-chloro-3-ethylquinoline (5)
in 84% yield. Lithiodebromination of compound 5 with
n-BuLi and subsequent reaction of the resulting aro-
matic anion with the Weinreb amide N,4-dimethoxy-N-
methyl-cis/trans-cyclohexanecarboxamide (6) afforded a
2:1 mixture of cis/trans ketones, which were separated
by column chromatography on silica gel to provide 7 in
29% yield. Stille coupling of 7 with tetramethyltin
afforded 2 in 56% yield. Similarly, reaction of 7 with
hexamethylditin under Pd-catalyzed conditions gave the
11C-labeling precursor 8 in 41% yield. Incorporation of
a [11C]methyl group to form the labeled compound [11C]2
was accomplished by reaction of the trimethyltin pre-
cursor 8 with [11C]methyl iodide under the catalysis of
Pd2(dba)3 and (o-tolyl)3P.15 The crude product was
purified by semipreparative HPLC (Phenomenex Prodigy
ODS-prep C18 column, 10 µm, 10 mm × 250 mm; mobile
phase, 50:50 mixture of MeCN and 0.1 M ammonium
acetate; flow rate of 8 mL/min). Compound [11C]2,
eluting at 12-13 min from the HPLC column, was
obtained with a decay-corrected radiochemical yield of
47 ( 17% (n ) 10, based on [11C]MeI) and specific
activity of 607 ( 228 Ci/mmol at end of synthesis (EOS,
A Division of Janssen Pharmaceutica, N.V., Beerse, Belgium
Received March 22, 2005
Abstract: A selective metabotropic glutamate 1 receptor
(mGlu1) antagonist was labeled with the positron-emitting
radioisotope carbon-11 and evaluated in ex vivo biodistribution
studies and micro-positron emission tomography (micro-PET)
imaging experiments in rats. Results from animal experiments
demonstrate that the radioligand [11C]2 is the first PET tracer
capable of labeling the rat mGlu1 receptor in vivo.
The metabotropic glutamate (mGlu) receptors are
G-protein-coupled receptors in the central nervous
system that regulate cell excitability and synaptic
transmission.1 They have been classified into three
major groups with eight subtypes (group I, mGlu1 and
5; group II, mGlu2 and 3; group III, mGlu4, 6, 7, and 8)
based on their sequence homology, signal trans-
duction mechanism, and pharmacology.2 The group I
metabotropic receptors, which encompass the mGlu1
and 5 subtypes, are mainly postsynaptic receptors and
have been implicated in disorders such as ischemia,
epilepsy, neuropathic pain, anxiety, and schizophre-
nia.3,4 As a result, the group I metabotropic glutamate
receptors have been the targets of intensive drug
development effort.5
Over the past few years understanding of the mGlu5
receptor and investigation of its involvement in neuro-
logical and psychiatric conditions have been greatly
advanced by the discovery of 2-methyl-6-(phenylethynyl)-
pyridine (MPEP) as a selective mGlu5 antagonist.6,7 And
the recent emergence of selective radioligands such
as [3H]M-MPEP, [3H]methoxymethyl-MTEP (MTEP
) 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine), and
* To whom correspondence should be addressed. Address: Depart-
ment of Psychiatry, Columbia University College of Physicians and
Surgeons, 1051 Riverside Drive, Box #31, New York, NY 10032.
Telephone: 212-543-6629. Fax: 212-568-6171. E-mail: hh285@
columbia.edu.
† Department of Psychiatry, Columbia University.
‡ Department of Radiology, Columbia University.
§ Department of Medicinal Chemistry, Johnson & Johnson Phar-
maceutical Research and Development, A Division of Janssen Phar-
maceutica, N.V.
# CNS Discovery Research, Johnson & Johnson Pharmaceutical
Research and Development, A Division of Janssen Pharmaceutica, N.V.
10.1021/jm050263+ CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/12/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5097
Scheme 1
Table 1. Uptake of the Radioligand [11C]2 in Male Sprague-Dawley Ratsa
time (min)
blood
medulla
frontal cortex
cerebellum
striatum
hippocampus
10
30
60
0.42 ( 0.02
0.26 ( 0.08
0.20 ( 0.06
0.47 ( 0.00
0.13 ( 0.03
0.07 ( 0.01
0.52 ( 0.06
0.13 ( 0.00
0.07 ( 0.00
1.24 ( 0.06
0.61 ( 0.03
0.27 ( 0.06
0.73 ( 0.04
0.17 ( 0.01
0.10 ( 0.01
0.58 ( 0.05
0.17 ( 0.02
0.10 ( 0.01
a Values are expressed as %ID/g. Data are the mean ( SD (n ) 3 animals in each group).
n ) 10). Total synthesis time was about 40 min.
Radiochemical purity of the final product was >99%.
Identity of the labeled compound was confirmed by co-
injection of the product [11C]2 with the cold compound
2 and observation of a single UV peak by an analytical
HPLC method (Phenomenex Prodigy ODS-3 C18 col-
umn, 5 µm, 5 mm × 250 mm; mobile phase, 60:40
mixture of MeCN and 0.1 M ammonium acetate;
flow rate of 2 mL/min). The retention time for 2 was
7.8 min.
The affinities of 2 for the mGlu1 and mGlu5 receptors
were determined in radioligand binding experiments in
vitro using cloned rat mGlu1 and mGlu5 receptors
stably expressed on Chinese hamster ovary cells, ac-
cording to the published procedures.11 Compound 2 was
found to possess high affinity for the rat mGlu1 receptor
(Ki ) 0.87 ( 0.43) and low affinity for the mGlu5
receptor (Ki ) 2366 ( 628 nM).
is some indication of specific binding in other brain
regions as well (ratio greater than 1), including the
striatum and hippocampus. In a second set of experi-
ment, the binding specificity and selectivity of the
radioligand were evaluated by pretreating the animals
with different pharmacological agents. When the rats
were treated with the cold compound 2 (2 mg/kg, iv) 10
min before the injection of radioligand [11C]2 and
sacrificed 30 min after, specific binding of the radio-
ligand in the cerebellum was reduced by 81% when
compared with the control group (Figure 1, bottom).
Similarly, when the rats were treated with compound
3, another selective mGlu1 antagonist,14 at a dose of 2
mg/kg, binding of the radioligand [11C]2 in the cerebel-
lum was also reduced by 81%. On the other hand, when
the animals were treated with the selective mGlu5
antagonist MPEP, there was no significant change in
the specific binding of [11C]2 (Figure 1, bottom). Taken
together, results from ex vivo studies in rats indicate
that the radioligand [11C]2 has excellent brain uptake
and its binding in the brain is saturable, specific, and
selective to the mGlu1 receptor.
To further evaluate the capability of the radioligand’s
binding to the mGlu1 receptor in vivo, we conducted
micro-PET imaging experiments in a living rat. The rat
was first injected with high specific activity [11C]2,
scanned for 90 min, then injected with a low specific
activity dose of [11C]2, and scanned again in a second
90 min session. In the high specific activity scan
(injected dose of 118 µCi; injected mass of 0.15 µg),
radioactivity entered the brain rapidly and over time
was localized in brain regions with high densities of
mGlu1 receptor, such as the cerebellum and striatum
(Figure 2, top). Activity in the cerebellum peaked at ∼10
min after the injection of [11C]2 and decreased there-
after (Figure 2, top). Radioactivity uptake was the
highest in the cerebellum, followed by striatum and
hippocampus, with the lowest uptake in the cortex
Ex vivo binding of the radioligand [11C]2 was evalu-
ated in animal experiments. Male Sprague-Dawley rats
were injected with the radioligand and sacrificed at
different time points. The brain parts were dissected,
and radioactivity was counted to determine the uptake
of the radioligand in each of the dissected brain regions.
The results from this ex vivo biodistribution study are
listed in Table 1. Initial uptake of [11C]2 in the brain
was high, with the percent injected dose per gram of
brain tissue (%ID/g) in various brain regions ranging
from 0.47% in the medulla to 1.24% in the cerebellum
at 10 min after radioligand injection, thus indicating
an excellent entry of the radioligand into the brain. Over
time, radioactivity was highly concentrated in the
cerebellum, where the level of mGlu1 receptor is high.12,16
The ratio of radioactivity in the cerebellum to that in
the medulla, which can be used as a measure of specific
binding, was 2.63 ( 0.11, 4.72 ( 0.77, and 4.06 ( 0.52
at 10, 30, and 60 min, respectively, after intravenous
injection of the radioligand [11C]2 (Figure 1, top). There

5098 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Letters
Figure 2. Regional time-activity curves of the radioligand
[11C]2 in a rat brain. The top panel represents time-activity
curves of the radioligand [11C]2 in the high specific activity
(HSA) scan. The bottom panel represents time-activity curves
in the low specific activity (LSA) scan. Points are activities
measured in the cerebellum (open circles), striatum (open
squares), hippocampus (open triangles), and cortex (closed
circles). Activity data were normalized to the injected doses
to allow for comparison on the same scale. Analysis by
simplified reference tissue model (SRTM) was then performed
on the normalized data. Lines represented the data analyzed
by SRTM in both scans. In the HSA scan differential uptake
among brain regions is detected, with the highest uptake in
the cerebellum. In the LSA scan activity distribution is
homogeneous across brain regions.
Figure 1. Regional specific binding of the radioligand [11C]2
in the rat brain in the time-course experiment (top) and in
the pretreatment experiment (bottom). In the time-course
experiment animals were sacrificed at 10, 30, and 60 min after
the adminstration of [11C]2. In the pretreatment experiment
animals were treated with compound 2, the mGlu1 antagonist
3, or the mGlu5 antagonist MPEP (2 mg/kg each, iv) 10 min
before the administration of [11C]2 and sacrificed 30 min after.
Brain regions examined are cerebellum (CER), prefrontal
cortex (PFC), striatum (STR), and hippocampus (HIP).
Supporting Information Available: Reaction procedures
and elemental analysis results. This material is available free
of charge via the Internet at http://pubs.acs.org.
(Figure 2, top). In the low specific activity scan (injected
dose of 106 µCi; injected mass of 110 µg), distribution
of radioactivity in the rat brain was homogeneous
(Figure 2, bottom), indicating the displacement of
specific uptake in mGlu1 receptor-rich regions by the
cold compound 2, thus demonstrating the binding
specificity of the radioligand [11C]2 to the mGlu1 recep-
tor in vivo.
In conclusion, an mGlu1 antagonist radioligand,
[11C]2, was successfully prepared. Evaluation of this
radioligand for its potential to label mGlu1 receptor was
carried out in ex vivo biodistribution studies and in vivo
micro-PET imaging experiments in rats. Ligand [11C]2
displays highly selective and specific binding to the
mGlu1 receptor in the cerebellum and, to a lesser
degree, in the striatum and hippocampus. Micro-PET
imaging experiments confirm the specific binding of
[11C]2 in vivo in a living rat. Taken together, these
results demonstrate that the radioligand [11C]2 repre-
sents the first selective PET tracer capable of labeling
the mGlu1 receptor in vivo in rat. The emergence of such
an in vivo imaging agent should open new avenues for
the investigation of the mGlu1 receptor.
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