Radiosynthesis of PET radiotracer as a prodrug for imaging group II metabotropic glutamate receptors in vivo

Article abstract of DOI:10.1016/j.bmcl.2012.01.039

Group II metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological and psychiatric disorders in recent studies. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission to

Full text of DOI:10.1016/j.bmcl.2012.01.039

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1958–1962
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Bioorganic & Medicinal Chemistry Letters
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Radiosynthesis of PET radiotracer as a prodrug for imaging group II
metabotropic glutamate receptors in vivo
⇑
Ji-Quan Wang, Zhaoda Zhang, Darshini Kuruppu, Anna-Liisa Brownell
Athinoula A. Martinos Biomedical Imaging Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 2129, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Group II metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological
and psychiatric disorders in recent studies. As a noninvasive medical imaging technique and a powerful
tool in neurological research, positron emission tomography (PET) offers the possibility to visualize and
study group II mGluRs in vivo under physiologic and pathologic conditions. We synthesized a PET tracer,
(S,S,S)-2-(2-carboxycyclopropyl)-2-(3-[11C]methoxyphenethyl) glycine dimethyl ester ([11C]CMGDE), as a
prodrug for group II mGluRs, and studied its preliminary biological properties in Sprague-Dawley rats to
visualize group II mGluRs. The microPET studies demonstrated that [11C]CMGDE readily penetrated into
the brain and the radiotracer generated from [11C]CMGDE had fast reversible binding in the group II
mGluRs rich regions including striatum, hippocampus and different cortical areas. Blocking studies with
LY341495 showed 20–30% decrease of binding of the radiotracer generated from [11C]CMGDE in all brain
areas with the highest decrease in the striatum 31.5 3.2%. The results show [11C]CMGDE is the first PET
tracer that is brain penetrating and can be used to image group II mGluRs in vivo.
Received 2 January 2012
Accepted 12 January 2012
Available online 21 January 2012
Keywords:
Group II mGluRs
mGluR2
mGluR3
PET
Prodrug
Ó 2012 Elsevier Ltd. All rights reserved.
Glutamate is a major neurotransmitter in the central nervous
system (CNS) and acts on diverse sets of receptors. The mGluRs
are G-protein coupled receptors (GPCRs). Based on sequence simi-
larity, signal transduction mechanism and pharmacological profiles,
mGluRs are classified into three groups. Group II mGluRs (including
mGluR2 and mGluR3 subtypes) modulate glutamate transmission
by second messenger activation to negatively regulate the activity
of adenylyl cyclase. Group II mGluRs have been implicated in a vari-
ety of neurological and psychiatric disorders in recent studies, such
as anxiety disorders,1 depression, schizophrenia,2,3 chronic pain
syndromes,4 seizure disorders,5,6 Parkinson’s disease,7 and sub-
stance abuse.8,9 Thus, compounds that are potent and selective for
mGluR2/3 could provide a valuable tool to investigate the involve-
ment of these receptors in various diseases.
A number of extremely potent group II mGluR selective agonists
and antagonists had been developed as competitive orthosteric li-
gands (examples in Fig. 1).10 LY354740 (EC50 = 11.1 nM for
mGluR2; 38.0 nM for mGluR3),11 LY379268 (EC50 = 2.69 nM for
mGluR2; 4.58 nM for mGluR3),12 and MGS0028 (EC50 = 0.57 nM
for mGluR2; 2.07 nM for mGluR3)13 are the agonists for group II
mGluRs, in which LY354740 (Eglumegad) was developed as a re-
search drug for its potential in the treatment of anxiety and drug
addiction.14 The cyclopropane amino-diacid structures were also
further modified to generate selective antagonists for group II
mGluR, such as LY341495 (IC50 = 21 nM for mGluR2; 14 nM for
mGluR3)15,16 and MGS0039 (IC50 = 20 and Ki = 2.2 nM for mGluR2;
IC50 = 24 nM and Ki = 4.5 nM for mGluR3).17 LY341495 has been
O
H
H
H
O
HO2C
HO2C
HO2C
F
H
H2N
H
H2N
CO2H
H
CO2H
CO2H
H2N
LY354740
LY379268
MGS0028
O
H
HO2C
Cl
H
O
F
Cl
H
H2N
HO2C
CO2H
CO2H
LY341495
Figure 1. Structures of selected agonists and antagonists for group II mGluRs.
H2N
MGS0039
H
R
HO2C
CO2H
H2N
⇑
Corresponding author. Tel.: +1 617 726 3807; fax: +1 617 726 7422.
E-mail address: abrownell@partners.org (A.-L. Brownell).
Figure 2. The S,S,S,-isomer of compounds 1–4.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.039

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1958–1962
1959
Table 1
Binding affinity of the substituted carboxycyclopropylglycines15
H
O11CH3
H
OH
O
O
a
Compound
R (see Fig. 2)
9-Xanthenyl
Stereochemistry
IC50(nM) SEM
10
2.9 0.6
89 36
1
Mixture
1
HO
OH
O
O
H2N
BocHN
1-SSS
2
2-SSS
3
3-SSS
4
(S,S,S)-Isomer
Mixture
(S,S,S)-Isomer
Mixture
(S,S,S)-Isomer
Mixture
O
O
3-Methylbenzyl
2,2-Diphenylethyl
3-Methoxybenzyl
[
11C]CMG
6
12
240 80
35
3
b
5
94 33
O11CH3
H
O11CH3
H
O
O
O
O
c
O
O
BocHN
H2N
O
O
H
O
H
O
O
[
11C]CMGDE
7
HO2C
O
Scheme 1. Reagents and conditions: (a) (1) [11C]CH3I, DMSO, 5 N NaOH, 100 °C; (2)
6 N HCl, 100 °C; (3) 5 N NaOH. (b) [11C]CH3I, aq. K2CO3, DMSO, 120 °C; (c) (1) TFA,
CH2Cl2, rt; (2) aq. K2CO3.
CO2H
O
H2N
H2N
O
4-SSS (CMG)
5-SSS (CMGDE)
Exact Mass: 293.13
tPSA: 109.85
Exact Mass: 321.16
tPSA: 87.85
To better understand group II mGluRs and their role in disease,
we developed an mGluR2/3 selective radiotracer for in vivo study.
As a noninvasive medical imaging technique and a powerful tool in
neurological research, PET offers the possibility to visualize and
study mGluR2/3 under physiologic and pathologic conditions.
Many PET radioligands have been developed for group I mGluRs
(mGluR1 and mostly mGluR5),20 however, no PET tracer is
available for imaging group II mGluRs. Here we report on the
CLogP: -0.5151
CLogP: 1.50902
Figure 3. Structures and calculated properties of CMG and CMGDE.
studied for its antidepressant effect and other neurological effects
in animal models.18,19
Figure 4. HPLC chromatogram of [11C]CMGDE.

1960
J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1958–1962
radiosynthesis of a prodrug, (S,S,S)-2-(2-carboxycyclopropyl)-2-
(3-[11C]methoxyphenethyl) glycine dimethyl ester ([11C]CMGDE),
as a PET tracer for group II mGluRs and its preliminary biological
evaluation in Sprague–Dawley rats to imagine group II mGluRs.
Ornstein et al. had reported a series of aryl-substituted carboxy-
cyclopropylglycine derivatives as the potent and selective antago-
nists of group II mGluRs,15 in which three of these compounds, 1, 2,
and 3 (Fig. 2 and Table 1), were resolved into their four constituent
isomers (R,S,S-, S,S,S-, S,R,R- and R,R,R-isomer). They found that
affinity and functional activity for group II mGluRs reside solely
in the S,S,S-isomers of compounds 1–3, in which the binding affin-
ity of the S,S,S-isomer surpass that of the corresponding isomer
mixture by factors of 3.4, 7.4 and 6.9, respectively. LY341495 (1-
SSS) was the most potent compound in this series and readily pen-
etrated into the brain, however, it is impossible to introduce car-
bon-11 or fluorine-18 radiolabel into it (along with compounds 2
and 3) without structural alterations. We therefore selected com-
pound 4 as a lead to PET ligand based on its affinity and easy radio-
labelling, in which its S,S,S-isomer will be radiolabelled with
carbon-11. Although the IC50 value was available only for the mix-
ture of 4, it was anticipated the radiolabeled S,S,S-isomer of 4
would be more potent than its isomeric mixtures by a similar fac-
tor as observed for compounds 1–3. Moreover, since the amino dia-
cid analog is very polar we also adopted a prodrug approach to
improve CNS exposure and pharmaceutical properties of the PET
tracer. As Figure 3 shows that the designed prodrug as methyl es-
ters 5-SSS (CMGDE) has better calculated physical properties com-
pared to the parent compound 4-SSS (CMG), in which the tPSA
value drops from 109.85 to 87.85 and the clogP value increase
from À0.52 to 1.51.
tion with NaOH. The crude product was purified by semi-prepara-
tive HPLC23 to give 76 mCi of pure [11C]CMG (radiochemical purity
>95%). Total synthesis time was about 45 min. Identity of the la-
beled compound was confirmed by co-injection of the product
[
11C]CMG with the cold compound 4-SSS (CMG). Radiosynthesis
of the prodrug [11C]CMGDE was carried out in two steps.24 Com-
pound 6 was reacted with [11C]methyl iodide in DMSO at 120 °C
in the presence of aqueous K2CO3 to afford the labeled intermedi-
ate 7. It was critical to use K2CO3 as the base in this reaction, which
promoted the reaction but not hydrolyzed the ester groups. The
labeling reaction was monitored by the analytical HPLC.23 The
intermediate 7 was purified by semi-preparative HPLC, which
was then deprotected with trifluoroacetic acid (TFA) in CH2Cl2 at
room temperature for 12 min. After neutralized by K2CO3 solution,
the solvent was removed under reduced pressure. The reaction
mixture was purified by the semi-preparative HPLC to give
4.4 mCi of [11C]CMGDE with a radiochemical purity of more than
96%. Figure 4 exhibits the HPLC chromatogram of the purified
product [11C]CMGDE. The two-steps synthesis took about 70 min.
MicroPET imaging studies in the male Sprague–Dawley rats25
showed that there was no brain penetration when injected with
[
[
11C]CMG (Fig. 5). However, the studies showed that the prodrug
11C]CMGDE readily penetrated into the brain and upon entering
into brain the radiotracer26 generated from [11C]CMGDE had fast
reversible binding in several cortical areas, hippocampus and stri-
atum, the sites, which are known to express group II mGluRs27
(Figs. 6 and 7). The maximum accumulation (2.5–3.5% of the in-
jected dose per cm3) was observed 2 min after administration
(Fig. 6). To confirm the binding specificity, a selective antagonist
for group II mGluRs, LY 34149528, was used as a block agent. The
results showed 20–30% decrease of [11C]CMGDE binding in several
brain areas excluding olfactory area, which might be affected by
the nonspecific accumulation in the Harderian glands (Fig. 7).
The highest activity averaged at the time interval 20–40 min was
in the striatum, where the highest ‘‘blocking’’ of 31.5 3.2% was
also observed. Pre-injection of LY341495 induced 29.5 4.2%
Radiosynthesis of [11C]CMG and [11C]CMGDE is shown in
Scheme 1. [11C]CMG was synthesized in one pot.21 Carbon-11
methylation of the phenolic hydroxyl in 622 was achieved by heat-
ing 6 and [11C]methyl iodide at 100 °C in DMSO and 5 N NaOH, in
which two ester groups were also hydrolyzed. In situ addition of
hydrochloric acid removed the Boc group, followed by neutraliza-
Figure 5. Distribution of [11C]CMG (0.4 mCi) in a Sprague–Dawley rat at 20–25 min after administration.

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1958–1962
1961
Figure 6. Coronal and axial slices of [11C]CMGDE (0.4 mCi) distribution in the rat brain from 1 min till 40 min after administration. Color-coded images are normalized to
each other and correspond the acquisition time of 1 min at the same midbrain level (coronal slice at bregma À1.6 mm; axial slice at bregma À5.4 mm).
decrease in the accumulation of [11C]CMGDE in the whole brain
determined at 20–40 min after administration of radioactivity.
In summary, this is the first successful approach to develop PET
imaging ligand for group II mGluRs. The prodrug approach had
greatly improved brain penetration and the receptor targeting. These
data might provide a foundation for future development of specific
PET imaging ligands for group II mGluRs and other subtype selective
radioligands. Optimization of the reaction conditions for preparation
of [11C]CMGDE is under way to reduce total synthesis time.
Acknowledgment
This work was supported by the NIH-NIBIB R01EB-012864 and
NIH-NIMH R01MH-091684 to A-LB.
Figure 7. Control PET imaging studies illustrate high accumulation of [11C]CMGDE
in several areas of rat brain. Blocking studies with LY341495 verify specific binding.
Mean and SEM of the accumulation are presented for 6 control rats and 5 rats after
blocking with LY341495 at the time interval of 20–40 min after administration of
radioactivity. Wbr = whole brain, Hc = hippocampus, Th = thalamus, Str = striatum,
CgC = cingulate cortex, MC=motor cortex, Ce = cerebellum, Olf = olfactory area.
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with saline containing 5% ethanol to give 4.4 mCi of
(radiochemical purity >96%).
[
11C]CMGDE
25. microPET imaging studies were conducted in male Sprague–Dawley rats
(weight 250–300 g, Charles River Laboratories, Wilmington, MA). [11C]CMG
was studied in 5 rats. [11C]CMGDE was tested on eleven rats including six
control studies and five blocking. Care was performed in accordance with the
guidelines of the Committee of Animals of The Massachusetts General Hospital.
After the rat was anesthetized (1.0–1.5% isoflurane with 1 L/min of O2 flow),
the tail vein was catheterized for administration of the radiotracer and/or
blocking agent. The rat was placed ventrally into the imaging position at the
center of the imaging field, where the spatial resolution is 1.8 mm (microPET
scanner, P4, Concorde Microsystems, Knoxville, TN). Heart rate was monitored
over the whole imaging time using
a Heska Vet/Ox Plus 4800 monitor.
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21. 11CH3I was bubbled through a 5-ml reaction vial charged with 6 (0.15 ml,
Transmission imaging (9 min) was done using a rotating point source of
cobolt-57 before administration of the radioactivity to obtain data for
attenuation correction. Subsequently, [11C]CMGDE (0.4–0.5 mCi) was injected
into the tail vein and volumetric dynamic data were acquired for 60 min. To
investigate specific binding the mGluR2/3 antagonists, LY341495 (1–1.50 mg/
kg, iv) was injected 1 min before administration of the radiotracer.
2.7 mg) in DMSO (0.25 ml) and 5 N NaOH (5 ll) at room temperature. The
mixture was heated at 100 °C for 7 min and then 0.7 ml of 6 N HCl was added,
and the mixture was heated at 100 °C for 5 min. After the reaction was cooled
to ambient temperature, 0.9 ml of 5 N NaOH was added to neutralize the
mixture. The reaction mixture was then subject to semi-preparative HPLC
purification with MeCN and water (v/v 50:50) as the mobile phase. The product
was collected between retention time of 3 min and 6 min monitored by a
radiation detector, the solvents were evaporated, and the product was then
formulated with saline containing 5% ethanol. After filtration the activity was
76 mCi (radiochemical purity >95%).
26. The active species were not identified, in which [
11C]CMG or/and the
corresponding monoesters were expected to form after the ester groups
were hydrolyzed in vivo.
27. Shigemoto, R.; Mizuni, N. In Handbook of Chemical Neuroanatomy; Bjorklund, A.,
Hokfelt, T., Eds.; Elsevier Science: Amsterdam, The Netherlands, 2000; Vol.18.
28. LY341495 was purchased from Tocris Cookson, Inc. The powder was dissolved
into the sterile water with a little 0.1 N NaOH. The pH was adjusted to 7 with
0.1 N HCl.