Radiosynthesis of N-(4-chloro-3-[11C]methoxyphenyl)-2- picolinamide ([11C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4)

Article abstract of DOI:10.1016/j.bmc.2013.07.046

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu₄ positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu₄ PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu ₄ expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([¹¹C]3) as a PET radiotracer for mGlu₄, and characterized its biological properties in Sprague Dawley rats. [¹¹C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [¹¹C]CH ₃I. Total synthesis time was 38 ± 2.2 min (n = 7) from the end of bombardment to the formulation. The radioligand [¹¹C]3 was obtained in 27.7 ± 5.3% (n = 5) decay corrected radiochemical yield based on the radioactivity of [¹¹C]CO₂. The radiochemical purity of [¹¹C]3 was >99%. Specific activity was 188.7 ± 88.8 GBq/mol (n = 4) at the end of synthesis (EOS). PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu₄ modulator (4) to investigate specificity and 3 studies blocking with an mGlu₅ modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [¹¹C]3 (peak activity between 1-3 min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu₄ modulator 4 showed 22-28% decrease of [¹¹C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu₅. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu₄, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.

Full text of DOI:10.1016/j.bmc.2013.07.046

Bioorganic & Medicinal Chemistry 21 (2013) 5955–5962
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosynthesis of N-(4-chloro-3-[11C]methoxyphenyl)-2-picolina-
mide ([11C]ML128) as a PET radiotracer for metabotropic glutamate
receptor subtype 4 (mGlu4)
Kun-Eek Kil, Zhaoda Zhang, Kimmo Jokivarsi, Chunyu Gong, Ji-Kyung Choi, Sreekanth Kura,
⇑
Anna-Liisa Brownell
Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, MA 02129, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu4 positive allosteric
modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu4 PAM to show effi-
cacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging
technique and a powerful tool in neurological research, positron emission tomography (PET) offers a pos-
sibility to investigate mGlu4 expression in vivo under physiologic and pathological conditions. We syn-
thesized a carbon-11 labeled ML128 ([11C]3) as a PET radiotracer for mGlu4, and characterized its
biological properties in Sprague Dawley rats. [11C]3 was synthesized from N-(4-chloro-3-hydroxy-
phenyl)-2-picolinamide (2) using [11C]CH3I. Total synthesis time was 38 2.2 min (n = 7) from the end
of bombardment to the formulation. The radioligand [11C]3 was obtained in 27.7 5.3% (n = 5) decay cor-
rected radiochemical yield based on the radioactivity of [11C]CO2. The radiochemical purity of [11C]3 was
>99%. Specific activity was 188.7 88.8 GBq/mol (n = 4) at the end of synthesis (EOS).
Received 22 May 2013
Revised 17 July 2013
Accepted 27 July 2013
Available online 2 August 2013
Keywords:
[
11C]ML128
PET
Metabotropic glutamate receptor subtype 4
(mGlu4)
Positive allosteric modulator
PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6
studies blocking with an mGlu4 modulator (4) to investigate specificity and 3 studies blocking with an
mGlu5 modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [11C]3
(peak activity between 1–3 min) in several brain areas including striatum, thalamus, hippocampus, cer-
ebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu4 modulator 4
showed 22–28% decrease of [11C]3 accumulation while studies of selectivity showed only minor decrease
supporting good selectivity over mGlu5. Biodistribution studies and blood analyses support fast metab-
olism. Altogether this is the first PET imaging ligand for mGlu4, in which the labeled ML128 was used
for imaging its in vivo distribution and pharmacokinetics in brain.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
mGlu7, and mGlu8, while group I mGluRs include mGlu1 and mGlu5
and group II mGluRs comprise mGlu2 and mGlu3. While group I
In contrast to ionotropic glutamate receptors, which relay excit-
atory glutamate signal to open ion channels directly and instantly,
metabotropic glutamate receptors (mGluRs) transfer glutamate
signal indirectly through GTP-protein (G-protein) to various effec-
tor systems such as adenylate cyclase, phospholipase C, mitogen-
activated protein kinase (MAPK), and phosphoinositide 3 kinase
(PI3K).1 Their signal effects are prolonged and influence on many
signal pathways in normal physiological or pathological condi-
tions. mGluRs belong to family C of G-protein coupled receptors,
and have three subgroups and eight subtypes.2 mGlu receptor sub-
type 4 (mGlu4) belongs to group III mGluRs along with mGlu6,
mGluRs are positively coupled to activate various downstream
effectors and protein kinase pathways, group II and III mGluRs
are negatively coupled, and inhibit the activity of adenylate cy-
clase. But, they activate other signal pathways such as MAPK and
PI3K pathways.3,4
mGluRs contain extracellular region, transmembrane region
and intracellular regions. Extracellular region consists of the Venus
flytrap domain (VFD), the orthosteric binding domain of glutamate,
and cysteine-rich domain which relays the conformational change
induced by glutamate binding at VFD to transmembrane region.5
The transmembrane region contains seven helical domains, and
these domains deliver the conformational change in extracellular
region to intracellular region of mGluRs. The binding locations at
the transmembrane domain render the non-competitive binding
sites for most allosteric modulators of mGluRs.6 Intracellular
⇑
Corresponding author. Tel.: +1 617 726 3807; fax: +1 617 726 7422.
E-mail address: abrownell@partners.org (A.-L. Brownell).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.046

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K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
diseases such as Parkinson’s disease (PD) and movement disor-
ders.11–14 Imaging of presynaptic mGlu4 function might provide
valuable insight to the synaptic transmission of glutamate. More
profoundly, by combining the studies of presynaptic mGlu4 and
postsynaptic mGlu5 it can provide in deep information of synaptic
transmission of glutamate, which will be useful for evaluating neu-
rotransmission related pathological processes. Thus, compounds
that are potent and selective for mGlu4 could provide a valuable
tool to investigate the involvement of these receptors in various
diseases to develop new therapeutics.
Much effort has focused on development of mGluR allosteric
modulators, which target the seven-transmembrane spanning do-
main. Compared to orthosteric compounds, allosteric modulators
often display better selectivity among mGluR subtypes and ade-
quate drug-like properties. Since PHCCC was identified as a selec-
tive mGlu4 PAM, there has been substantial progress in
Scheme 1. Syntheses of reference ML128 (3), [11C]ML128 ([11C]3), its precursor (2),
and blocking agent (4).
15
identifying positive allosteric modulators (PAM) for mGlu4. The
Vanderbilt University group developed a selective mGlu4 PAM, N-
region comprises C-terminus where signal transfers are occurred
by G-protein coupling, kinase pathways, and alternative splicing.
mGlu4 is preferentially localized on presynaptic site. Expression
of mGlu4 based on mRNA studies is most intensive in cerebellar
cortex, and moderately expressed in hippocampus, striatum, olfac-
tory bulb and cerebellum.7–10 mGlu4 participates in presynaptic
neurotransmission of excitatory glutamate signal, and has received
much attention lately due to its implication for various neuronal
(4-chloro-3-methoxyphenyl)picolinamide
(3,
ML128,
VU0361737), which is potent (rEC50 = 110 nM and hEC50 = 240
nM) and centrally penetrate.16 It is the first mGlu4 PAM to demon-
strate efficacy in a preclinical rodent model of motor impairments
associated with PD.16,17 This compound has been recommend as a
molecular probe to investigate the role of selective allosteric acti-
vation of mGlu4 in vitro and in vivo.
Figure 1. (a) UV and radioactivity traces of semi-preparative HPLC; (b) The analytical HPLC chromatogram for co-injection of [11C]3 with the cold reference 3.

K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
5957
To better understand the role of mGlu4 in normal and disease
conditions, we were interested in developing an mGlu4 selective
radiotracer for in vivo study. As a noninvasive medical imaging
technique and a powerful tool in neurological research, positron
emission tomography (PET) offers the possibility to visualize and
analyze the target receptor expression under physiological and
pathophysiological conditions. Moreover, PET tracers serve as
invaluable biomarker during the chemical development of poten-
tial therapeutic drugs. Many PET radioligands have been developed
synthesis time was 38 2.2 min (n = 7) from the EOB to the formu-
lation. The radioligand [11C]3 was obtained in 27.7 5.3% (n = 5)
decay corrected radiochemical yield (RCY) based on the radioactiv-
ity of [11C]CO2.
The chemical identity of the purified product ([11C]3) was con-
firmed by co-injection with the cold reference 3 into radio-HPLC,
and it showed the same peak on the UV chromatogram for both
compounds (Fig. 1b). The time difference between the UV peak
(4.7 min) and the radioactivity peak (5.0 min) in the chromatogram
arose from the line setting between the radioactivity detector and
the UV detector. Radio-TLC also confirmed that reference standard
3 and radioactive spot had same Rf value (Rf = 0.6 with ethyl ace-
ate/hexane = 1:1). The radiochemical purity of [11C]3 was >99%.
Specific activity was 188.7 88.8 GBq/mol (n = 4) at the end of syn-
thesis (EOS). Experimentally determined partition co-efficient
(logD) between pH 7.4 phosphate buffered saline (PBS) and octanol
was 3.09 0.14 (n = 5).
18,19
for group I mGlus (e.g. [18F]FPEB, [11C]ABP688, etc. for mGlu5
and [11C]YM202074 and [18F]MK1312 for mGlu1),20,21 however,
no PET tracer is available for imaging mGlu4. ML128 is a good can-
didate to be radiolabeled as a PET tracer for mGlu4. Although low
microsomal stability, high clearance and short half-life, ML128
showed some favorable properties for developing a potential PET
radiotracer, which include rapid penetration into rat brain follow-
ing intraperitoneally injection (Tmax for brain: 0.5 h), high brain:-
plasma (B/P) partition co-efficients (B/P = 4.1), good selectivity
over other mGluRs subtypes and the chemical structure of the pre-
cursor to allow fast labeling. On the other hand, the labeled ML128
can be used to imaging its in vivo distribution and pharmacokinet-
ics in brain, which should give further insight into the underlying
pharmacological processes.
22
2.2. The selectivity to mGlu5 and mGlu8
The selectivity to mGlu5 and mGlu8 were studied by intracellu-
lar inositol phosphates (IPs) accumulation assay using CHO cells
that expressed stably mGlu5a or mGlu8. Compound 3 was tested
Here we report on the radiosynthesis of a carbon-11 labeled
mGlu4 PAM (3, ML128, VU0361737) as a PET tracer for mGlu4
and its preliminary biological evaluation in Sprague Dawley rats
to image mGlu4.
at a single concentration (10
antagonist. Testing for antagonism was performed in presence of
the EC50 concentration of glutamate (5 M). The results showed
there was no agonist activity for mGlu5 (ꢀ15% maximal activity
as obtained with maximal agonist concentrations for mGlu5), but
slight agonist activity for mGlu8 (13% of maximal activity). The re-
sults showed 51% of inhibition of receptor activity for mGlu5 and
ꢀ5% for mGlu8 in presence of an EC50 concentration of glutamate.
lM) for activity as an agonist or an
l
2. Results
2.1. Chemistry and radiochemistry
2.3. In vivo characterization of [11C]3
Compounds 2, 3 and 4 were prepared by the amide coupling
reaction between 1 and the corresponding aniline derivatives with
28%, 53%, and 68% yield, respectively. The radioligand [11C]3 was
synthesized by methylation of the precursor 2 with [11C]iodometh-
ane ([11C]CH3I) mediated by 5 M potassium hydroxide (KOH) in
N,N-dimethylformamide (DMF) at 90 °C for 5 min (Scheme 1), in
The PET images indicate that the radiotracer crosses blood brain
barrier (BBB), and high accumulation is observed in hippocampus,
striatum, thalamus, olfactory bulb, and cerebellum (Fig. 2a). The
time-activity curves (TACs) showed fast uptake (peak activity be-
tween 1 and 3 min) followed by fast wash-out within 20 min
throughout the brain. Fast clearance of the radioligand [11C]3 from
the brain agrees with the previous study indicating pharmacoki-
netic half-life of 0.15 h for h for 3 in the brain (Fig. 2b).16
which
[
11C]CH3I was obtained from
[
11C]carbon dioxide
([11C]CO2) via [11C]methane ([11C]CH4). The reaction mixture was
purified by a semi-preparative HPLC, in which an isocratic method
(see Section 2.3.1) was developed to achieve good separation be-
tween the desired product [11C]3 (9.8–10.6 min) and the precursor
2 (5.4–6.0 min) as well as [11C]CH3I (4.8–5.2 min) (Fig. 1). Total
Comparison of [11C]3 distribution in rat brain at the time inter-
val 2–15 min in a control study and after blocking of mGlu4 with
Figure 2. Pharmacokinetics of [11C]3. (a) Representative cumulative PET/CT images of rat brain at the time interval 2-15 min after administration of [11C]3 (24.1 MBq iv). Slice
thickness is 0.625 mm. Str = striatum, Hp = hippocampus, Th = thalamus and Cer = cerebellum. (b) TACs show fast uptake (peak activity between 1 and 3 min) followed by fast
washout throughout the brain.

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K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
100
4.HCl or mGlu5 with 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)-pyri-
dine hydrochloride (MTEP.HCl) is presented in Figure 3(a). mGlu4
blocking conducted by administration of 4.HCl (10 mg/kg ip) as a
blocking agent 30 min before injection of [11C]3 radioactivity
showed 23–28% reduced uptake of [11C]3 in the striatum, hippo-
campus, cerebellum and averaged whole brain. Another blocking
was done to investigate the radioligand’s selectivity to mGlu5 by
using an mGlu5 NAM, MTEP, as a blocking agent (5 mg/kg iv
2 min before injection of [11C]3). The results show that the uptake
of [11C]3 was reduced 1–2% in the striatum and hippocampus and
12% averaged from the whole brain (Fig. 3b).
80
60
40
20
0
0
10
20
30
40
50
60
Time (min)
Figure 4. Metabolite corrected blood curve after [11C]3 injection.
The concentration of the radioligand in blood plasma was mea-
sured by withdrawing blood from three male rats at three time
points (5, 25, 55 min) after injection of [11C]3. The analysis of blood
plasma revealed fast in vivo metabolism of [11C]3. The results show
that the intact radioligand was rapidly reduced to 30% of initial
amount in 5 min after the iv injection, and then gradually reduced
to 10% over next 50 min (Fig. 4).
1.4
1 min
5 min
10 min
20 min
40 min
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2.4. Ex vivo characterization
The pharmacokinetic property of the radioligand was further
evaluated by ex vivo biodistribution study. Total 15 male rats were
sacrificed in a group of three at five different time points (1, 5, 10,
20, and 40 min) after injection of [11C]3, in which major organs
were harvested and radio-counted. The amount of activity was re-
ported as percentage of injected dose (ID) in unit weight of tissue.
As shown in Fig. 5, the radioactivity was distributed with less than
1% of ID over major organs. However, the uptake in liver and kid-
ney was relatively higher than in other organs what also relates
to fast metabolism.
Brain
Blood
Heart
Lung
Liver
Kidney Muscle
Figure 5. Biodistribution study of [11C]3.
syntheses from commercially available chemicals using amide
bond formation reaction. A rapid methylation on phenolic oxygen
was achieved with [11C]CH3I generated by two step gas-phase
reaction from [11C]CO2. The radiochemistry was completed with
two half-life cycle from the end of bombardment (EOB) to the for-
mulation including HPLC purification. The high RCY was achieved
without significant side reaction under a mild condition. There
was enough separation between 2 and [11C]3 on HPLC, so no
3. Discussion
The current work describes the first PET imaging ligand what
can be used to investigate a role of mGlu4 in glutaminergic neuro-
transmission. Compounds 2, 3 and 4 were prepared by one-step
Figure 3. PET studies of specificity and selectivity of [11C]3. (a) Comparison of [11C]3 distribution in rat brain at the time interval 2–15 min in a control study, after blocking of
mGlu4 with4, HCl(10 mg/kg ip 30 min before [11C]3 injection) and after blocking of mGlu5 with MTEP (5 mg/kg iv 2 min before [11C]3). Coronal images are at the striatal and
hippocampal level while axial and sagittal views at the midstriatal level. (b) The uptake of [11C]3 was reduced by 23–28% in all brain areas with 4.HCl while MTEP induced
blocking was minor.

K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
5959
impurities from side product or precursor affected radiochemical
purity. Radio-TLC and radio-HPLC indicated that the collected
product was radiochemically pure. Co-injection with cold refer-
ence 3 demonstrated that the collected product was authentic
radiolabeled target compound.
normal rats. This is the first report of in vivo PET imaging of mGlu4.
3 can serve as an invaluable lead compound for future innovations
of imaging ligands for mGlu4 to investigate different neurological
conditions.
The PET image of brain with [11C]3 indicated that 3 crossed BBB
which was consistent with previous study.16 The positive brain per-
meability of 3 was also predicted by many theoretical properties
such as clogP, molecular weight (MW), and topological polar surface
area (tPSA). The clogP, MW, and tPSA of 3 were 2.80, 262.7, and 50.7,
respectively,23 and these theoretical properties satisfy the require-
ments as a central nervous system (CNS) drug presented by previous
literatures.24,25 3 also satisfies the number of hydrogen bond (HB)
donors (n = 1), and HB acceptors (n = 3). Actual experimental logD
(3.09 0.14) also indicated that 3 could be a CNS drug.24,25
The PET images of rat brain showed that the radiotracer mainly
occupied hippocampus, striatum, thalamus, olfactory bulb, and
cerebellum. This result agrees with previous studies that report
in vitro mGlu4 distribution in brain.7–10 This is first study to verify
in vivo mGlu4 distribution in the brain with mGlu4 active PET
radiotracer.
5. Experimental
5.1. General
MTEP.HCl was purchased from Abcam PLC (Cambridge, MA). All
other reagents and solvents utilized for experiments were pur-
chased from commercial suppliers (Sigma–Aldrich, TCI America,
VWR and J.T. Baker) and were used without further purification.
NMR spectra were recorded on Varian 500 MHz spectrometer (Agi-
lent Technology, Santa Clara, CA). All chemical shifts (d) were re-
ported in parts per million (ppm) downfield or upfield from the
standard (tetramethylsilane for 1H and 13C; trichlorofluorometh-
ane for 19F). 1H NMR data are reported as follows: chemical shift,
multiplicity
(s = singlet,
d = doublet,
dd = double-doublet,
ddd = double-double-doublet, t = triplet, br = broad,) coupling con-
stant (Hz), and integration. High resolution mass spectrum (HRMS)
was obtained by The Small Molecule Mass Spectrometry Facility at
Harvard University using Agilent 6210 Time-of-Flight LC/MS. The
melting point was measured by MP50 melting point system (Met-
tler Toledo LLC, Columbus, OH) and was uncorrected. LC–MS spec-
tra were obtained on Agilent 1200 series HPLC coupled with 6310
ion trap mass spectrometer (Agilent Technology, Santa Clara, CA),
in which an Agilent Eclipse XDB C8 analytical column
TACs of regions of interest (ROIs) revealed transient uptake of
[
11C]3 followed by rapid clearance in the brain. Furthermore, no
particular regions displayed either retention or slow wash-out in
the brain. Such phenomena can be explained that efflux from brain
was more dominant than actual specific binding to mGlu4. In spite
of fast removal from brain regions, blocking study with 4.HCl
(10 mg/kg ip) proved moderate reduction of binding in the brain,
in which
4 had been reported to have high PAM activity
(150 ꢁ 4.6 mm, 5
lm) was used. Flash chromatography was con-
ducted on CombiFlashÒ Companion (Teledyne Isco, Lincoln, NE).
(rEC50 = 80 nM) against rat mGlu4 and to be centrally
penetrating.19 Because the dose of blocking agent was not opti-
mized in this study the partial blocking effect might be attributed
to insufficient saturation in target binding site. Moreover, since 3
has fast metabolism, it might occupy the target site only short per-
iod. Therefore, blocking effect might not be prolonged after PET
radiotracer was injected.
The in vivo study with MTEP.HCl demonstrated minor blocking
effect with [11C]3. According to our in vitro study with mGlu5, 3 had
no agonist activity, but minor antagonist activity. Therefore, [11C]3
can be considered to bind specifically to mGlu4.
As it was expected from previous experiment, 3 was featured by
fast metabolism.16 The previous literature showed that the half-life
of 3 in plasma was 1.9 h after ip injection, while our blood metab-
olite study indicated that [11C]3 was reduced to 30% within 5 min
after iv injection (Fig. 4).16 Difference in pharmacokinetic charac-
teristics might come from different injection methods. Fast metab-
olism was also observed in biodistribution studies (Fig. 5). The
relative high ratio of carbon-11 species in liver and kidney re-
flected high metabolic activity. The metabolically unstable meth-
oxy group might contribute to fast pharmacokinetic half-life of 3.
Based on above results, further development based on 3 is
essential to improve pharmacokinetic features and binding proper-
ties to mGlu4. Such a structural modification must accommodate
following features. (i) Considering carbon-11 half-life and PET
scanning time, the metabolic half-life has to be at least 30 min.
(ii) New compound should display better binding property toward
mGlu4, and increased selectivity to other mGluRs subtypes. (iii)
The structural modification does not affect the brain penetrability.
It will be useful to evaluate [11C]3 with animal models of different
neurological disorders.
5.2. Chemistry
5.2.1. N-(4-Chloro-3-hydroxyphenyl)-2-picolinamide (2)
To a solution of 2-picolinic acid (1, 0.857 g, 6.97 mmol), N-
hydroxybenzotriazole hydrate (HOBtꢂH2O, 1.066 g, 6.97 mmol),
diisopropylethylamine (DIPEA, 1.80 g, 13.93 mmol) and N-(3-
methylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(EDC.HCl, 2.0 g, 10.45 mmol) in dry 1,4-dioxane (80 mL) was added
5-amino-2-chlorophenol (1.20 g, 8.36 mmol) at room temperature.
The mixture was heated to 50 °C and stirred overnight. To the reac-
tion mixture was added 300 mL of dichloromethane (DCM), and
sequentially washed with water (3 ꢁ 100 mL). After the organic
layer was separated and concentrated, the reaction mixture was
purified by silica-gel chromatography (hexane/ethyl acetate: from
95/5 to 30:/70) to give the product as a pale-yellow solid (0.482 g,
1.94 mmol, 27.8% yield). mp 140–142 °C. 1H NMR (500 MHz,
CDCl3) d ppm 10.02 (s, 1H), 8.62 (d, J = 5.0 Hz, 1H), 8.30 (d,
J = 8.0 Hz, 1H), 7.92 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.64 (d,
J = 1.5 Hz, 1H), 7.50 (dd, J = 7.0, 5.0 Hz, 1H), 7.31 (d, J = 8.5 Hz,
1H), 7.27 (d, J = 2.5 Hz, 1H), 5.68 (bs, 1H); 13C NMR (125 MHz,
DMSO-d6) d ppm 163.02, 154.39, 150.91, 149.32, 139.37, 138.96,
130.71, 127.82, 123.15, 116.13, 112.88, 109.11. LC–MS, calcd for
C
12H9N2O2Cl: 248.04; obsd: 249.0 [MH]+. HRMS m/z calcd for C12-
H9ClN2O2 (MH+), 249.0425; found 249.0437.
5.2.2. N-(4-Chloro-3-methoxyphenyl)-2-picolinamide (3)
3 was synthesized in a similar procedure as described for 2 by
using 4-chloro-3-methoxyaniline (400 mg, 2.54 mmol),
(312 mg, 2.54 mmol), HOBtꢂH2O (389 mg, 2.54 mmol), DIPEA
(0.66 g, 5.08 mmol), EDC.HCl (730 mg, 3.81 mmol) and anhydrous
1,4-dioxane (30 mL). 3 was obtained as a pale yellow solid
(325 mg, 1.34 mmol, 53% yield); mp: 120 °C. 1H NMR (500 MHz,
CDCl3) d ppm 10.07 (s, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.29 (d,
J = 8.0 Hz, 1H), 7.92 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.81 (d,
1
4. Conclusion
This paper reports the radiosynthesis of [11C]3, a new carbon-11
labeled PET radiotracer for mGlu4 and its characterization in

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K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
J = 2.0 Hz, 1H), 7.50 (dd, J = 7.0 Hz, 5.0 Hz, 1H), 7.34 (d, J = 8.5 Hz,
1H), 7.09 (dd, J = 9, 2.5 Hz, 1H), 3.97 (s, 3H). 13C NMR (125 MHz,
CDCl3) d ppm 162.41, 155.68, 149.85, 148.37, 138.17, 137.97,
130.47, 127.01, 122.72, 117.80, 112.45, 104.37. LC–MS, calcd for
of water and passed through a C18 Sep-PakÒ Plus cartridge (Waters
Corp.), and the cartridge was washed with additional 5 mL of
water. The [11C]3 was then eluted from cartridge with 1 mL of eth-
anol and passed through a sterile filter (0.22 lm, EMD Millipore
C
13H11N2O2Cl: 262.05; obsd: 263.0 [M+H]+.
Corp.). The final product was diluted with 9 mL of 0.9% saline to
make 10% ethanol solution in saline for animal injection. Total syn-
thesis time was 38 2.2 min (n = 7) from the EOB to the formula-
tion. The radioligand [11C]3 was obtained in 27.7 5.3% (n = 5)
decay corrected RCY based on [11C]CO2.
5.2.3. N-(3-(Difluoromethoxy)phenyl)-2-picolinamide (4)
4 was synthesized in a similar procedure as described for 2 by
using 3-(difluoromethoxy)aniline (1.0 g, 6.28 mmol), 1 (0.773 g,
6.28 mmol), HOBtꢂH2O (0.962 g, 6.28 mmol), DIPEA (1.62 g,
12.56 mmol), EDC.HCl (1.81 g, 9.42 mmol) and anhydrous 1,4-
dioxane (80 mL). Compund 4 was obtained as an off-white solid
(1.12 g, 4.27 mmol, 68.0% yield). mp: 84 °C. 1H NMR (500 MHz,
CDCl3) d ppm 10.09 (s, 1H), 8.62 (d, J = 4.9 Hz, 1H), 8.30 (d,
J = 7.8 Hz, 1H), 7.91 (t, J = 8.8 Hz, 1H), 7.76 (s, 1H), 7.49 (m, 2H),
7.36 (t, J = 8.3 Hz, 1H), 6.90 (d, J = 1.5 Hz, 1H), 6.57 (t,
J = 73.85 Hz, 1H); 13C NMR (125 MHz, CDCl3) d ppm 162.22,
152.18 (t, J = 2.94 Hz), 148.09, 139.51, 138.47, 130.54, 127.13,
123.07, 116.78, 116.33 (t, J = 257.8 Hz), 115.42, 111.20; 19F NMR
(470 MHz, CDCl3) d ppm ꢀ80.0; LC–MS, calcd for C13H10F2N2O2:
264.07; obsd: 265.0 [M+H]+.
5.3.2. Quality control (QC) analysis
The chemical identity of [11C]3 was confirmed by co-injection
with the cold (i.e. nonradioactive) reference compound 3 into
radio-HPLC (Column: Agilent Eclipse XDB C18 analytical column,
150 ꢁ 4.6 mm,
5 lm; eluent: CH3CN/0.1 M HCO2NH4 solu-
tion = 60:40; flow rate: 1 mL/min) and it showed the same peaks
on the UV and the radioactive chromatograms. The purity of
[
11C]3 was obtained by the radio-HPLC analysis in the same condi-
tion described above. The radiochemical purity of [11C]3 was >99%.
Radio-TLC was also performed. An aliquot of [11C]3 and the cold
reference compound 3 were spotted on a TLC plate, and developed
in a TLC chamber with eluent (ethyl acetate/hexane = 1:1). Their Rf
values were checked by radio-TLC scanner and UV lamp,
respectively.
5.2.4. N-(3-(Difluoromethoxy)phenyl)-2-picolinamide
hydrochloride salt (4.HCl)
To a solution of 4 (0.100 g, 0.379 mmol) in DCM at 0 °C was
added 2 M HCl in ether (0.95 ml, 1.90 mmol) dropwise, and stirred
for 15 min. The solution was warmed up to room temperature, and
stirred for 30 min before the solvent was removed by filtration. The
corresponding salt (4.HCl) was obtained as white solid (0.100 g,
79% yield).
5.3.3. Measurement of specific activity
Two aliquots (10 l
L) of [11C]3 were taken for determination of
the specific activity. An aliquot of [11C]3 was injected into analyti-
cal HPLC using the same method as in QC analysis, and the other
aliquot was saved and its radioactivity was measured and cor-
rected to end of synthesis (EOS). The amount of injected [11C]3
5.3. Radiochemistry
was calculated and expressed as lmol using a HPLC calibration
curve developed from the reference 3 based on the five different
[
11C]CO2 was produced by Siemens Eclipse HP 11 MeV cyclotron
concentrations (5, 10, 50, 100, and 500 ppm). Specific activity of
(Malvern, PA) using nitrogen target containing 2% oxygen with
proton bombardment. [11C]CH3I was synthesized by TRACERlab
FX MeI (GE Healthcare, Waukesha, WI), and [11C]3 was synthesized
by TRACERlab FX M synthesis module (GE Healthcare). The radio-
chemical purity and specific activity of final radioactive product
was measured by Agilent 1200 Series HPLC equipped with UV
spectrometer monitored at 254 nm and Carroll and Ramsey Model
105S Single Channel Radiation Detector (Berkeley, CA) in which an
Agilent Eclipse XDB C18 analytical column (150 ꢁ 4.6 mm, 5 m) or a
[ lmol (n = 4) at the EOS.
11C]3 was 188.7 88.8 GBq/
5.4. Measurement of logD
The logD value of [11C]3 was determined by using previously
published method.26 One test tube containing 2.5 mL of PBS and
2.5 mL of octanol and five test tubes containing 2.5 mL of PBS
and 0.5 mL of octanol were prepared. After aliquot of [11C]3 was
added to first test tube containing 2.5 mL of PBS and 2.5 mL of oct-
anol, the test tube was vortexed for 2 min, and centrifuged over
8000 rpm for 2 min. After 0.1 mL of octanol and 1.0 mL of PBS were
taken from the test tube, 2.0 mL of octanol was transferred to next
test tube. Same procedure was repeated until six sets of samples
were obtained. Each set of samples was counted by 2480 Wizard2
Automatic Gamma Counter (PerkinElmer, Waltham, MA). The logD
value was calculated by the equation: log(decay corrected radioac-
tivity in octanol layer ꢁ 10/decay corrected radioactivity in PBS).
Gemini-NX C18 semi-preparative column (250 ꢁ 10 mm, 5
lm,
Phenomenex Inc., Torrance, CA) was used. The radiochemical pur-
ity was also verified by Bioscan Inc AR-2000 radio-TLC imaging
scanner (Washington DC) using Silicycle silica gel 60-F254 TLC
plates (Quebec, Canada). Radioactivity was quantified with a
CRCÒ-25PET dose calibrator (Capintec Inc., Ramsey, NJ).
5.3.1. Synthesis of N-(4-Chloro-3-[11C]methoxyphenyl)-2-
picolinamide ([11C]3)
[
11C]CO2 produced by cyclotron was reduced to [11C]methane
5.5. Intracellular inositol phosphate accumulation assay for
([11C]CH4) by ShimaliteÒ nickel (Shinwa Chemical, Japan) at
390 °C, which was converted into [11C]CH3I in gaseous iodination
condition at 720 °C. The resultant [11C]CH3I was transferred in a
stream of nitrogen and trapped in a reaction vessel containing
mGlu5 and mGlu8
22
mGlu5 and mGlu8 were stably expressed in CHO cells. Cultured
in 96-well plates to confluency (3 to 4 days), the cells were incu-
bated overnight in glutamine-free culture medium supplemented
with 18.5 kBq myo-[3H]inositol (NEN) to label the cell membrane
phosphoinositides. After washing the cultures, incubations with 3
the precursor 2 (1.5 mg) and 5 M KOH (3.2 lL) in DMF (0.3 mL)
at 0 °C. After completion of the [11C]CH3I transfer, the reaction ves-
sel was sealed and heated at 90 °C for 5 min. After heating, the
reaction mixture was diluted with 1 mL of HPLC eluent, and in-
jected into radio-HPLC (Phenomenex Gemini-NX C18 semi-pre-
(10 lM) were carried out for 45 min at 37 °C in Locke’s buffer
(156 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1 mM MgCl2,1.3 mM
CaCl2, 5.6 mM glucose, and 20 mM HEPES, pH 7.4) containing
20 mM LiCl to block the degradation of IPs. The reaction was termi-
nated by aspiration, and IPs were extracted with 0.1 M HCl. The
separation of [3H]IPs was performed by anion exchange
parative column, 250 ꢁ 10 mm,
5 lm) using CH3CN/0.1 M
HCO2NH4 solution (45/55) as the mobile phase at a flow rate of
5 mL/min. The desired product was eluted between 10.4 and
11.0 min. The fraction containing [11C]3 was diluted with 20 mL

K.-E. Kil et al. / Bioorg. Med. Chem. 21 (2013) 5955–5962
5961
chromatography and determined by a liquid scintillation counter.
3 was tested at a single concentration (10 M) for activity as an
agonist or an antagonist. Testing for antagonism was performed
in presence of the EC50 concentration of glutamate (5 M). Each
water solution co-solvent by gradually increasing acetonitrile from
0% to 100%. The fractions of each sample were collected in eight
test tubes. The radioactivity of supernatant and eluted portion
(seventh test tube) of [11C]3 were counted by 2480 Wizard2 Auto-
matic Gamma Counter.
l
l
compound was tested in duplicate in two separate experiments
performed on different cell passages. In addition to the tested com-
pounds each 96-well plate contained points for determination of
basal activity, maximal agonist stimulation, agonist EC50 concen-
trations (i.e., concentration–response isotherm), and the IC50 con-
centration of a known antagonist for purposes of positive control
and for activity calculations. The reported results for each com-
pound were calculated for agonists as the percent of maximal
activity (as obtained with maximal agonist concentrations) and
for antagonist as the percent of inhibition of receptor activity (in
presence of an EC50 concentration of glutamate).
5.8. Biodistribution study
Total of 15 Sprague Dawley rats (male, weight 300–500 g) were
used in this study. [11C]3 (27.4–48.1 MBq/animal) was injected
into the 15 rats, and three of them were sacrificed at each of five
different time points (1, 5, 10, 20, and 40 min after administration
of [11C]3). Blood, brain, heart, lung, liver, muscle, and kidney were
harvested, weighed, and counted in 2480 Wizard2 Automatic
Gamma Counter. Radioactivity of the samples was corrected for
decay and injected activity and expressed as percent injection dose
per gram tissue (%ID/g tissue).
5.6. In vivo characterization of [11C]3
Altogether twenty normal Sprague Dawley rats (male, 300–
Acknowledgments
500 g) were used to investigate in vivo imaging characteristics of
[
11C]3. Eleven rats were used for control studies, six rats were used
The authors like to thank NIMH Psychoactive Drug Screening
Program and the research team at the Chapel Hill, North Carolina
for functional assays of 3.22 The authors like to thank also the
cyclotron personnel at Martinos Biomedical Imaging Center at
Massachusetts General Hospital for technical support for radiosyn-
thesis. No other potential conflict of interest was declared.
to investigate specificity and three rats to investigate selectivity of
[
11C]3. All animal studies were performed by the guidance of the
National Institute of Health guide for the care and use of laboratory
animals and, approved and supervised under the subcommittee on
research animals of the Harvard Medical School and Massachusetts
General Hospital. For the imaging studies rats were anesthetized
with isoflurane/nitrous oxide (1.0–1.5% isoflurane, with oxygen
flow of 1–1.5 L/min) and the tail vein was catheterized for admin-
istration of the imaging ligand ([11C]3). The rats were adjusted into
the scanner for imaging position (Triumph II Preclinical Imaging
System, Gamma Medica-IDEAS, Northridge, CA). The vital signs
such as heart rate and/or breathing were monitored throughout
the imaging. Data acquisition of 60 min was started from the injec-
tion of radioligand [11C]3 (22.2–37 MBq iv). To investigate specific-
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[
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