Synthesis and Characterization of Fluorine-18-Labeled N-(4-Chloro-3-((fluoromethyl- d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain

Article abstract of DOI:10.1021/acs.jmedchem.0c00201

We have synthesized and characterized [¹⁸F]-N-(4-chloro-3-((fluoromethyl-d₂)thio)phenyl)-picolinamide ([¹⁸F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [

Full text of DOI:10.1021/acs.jmedchem.0c00201

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Article
Synthesis and Characterization of Fluorine-18 labeled N-(4-Chloro-3-
(fluoromethyl-d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain
(
Junfeng Wang, Xiying Qu, Timothy M. Shoup, Gengyang Yuan, Sepideh Afshar, Chuzhi Pan, Aijun Zhu, Ji-
Kyung Choi, Hye Jin Kang, Pekka Poutiainen, Georges ElFakhri, Zhaoda Zhang, and Anna-Liisa Brownell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00201 • Publication Date (Web): 21 Feb 2020
Downloaded from pubs.acs.org on February 23, 2020
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Synthesis and Characterization of Fluorine-18 labeled N-(4-Chloro-3-((fluoromethyl-
d )thio)phenyl)picolinamide for Imaging of mGluR4 in Brain
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Junfeng Wang, Xiying Qu, Timothy M. Shoup, Gengyang Yuan, Sepideh Afshar, Chuzhi
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Pan,1,2 Aijun Zhu, Ji-Kyung Choi, Hye Jin Kang, Pekka Poutiainen, Georges ElFakhri,
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Zhaoda Zhang * and Anna-Liisa Brownell *
1Gordon Center for Medical Imaging, Massachusetts General Hospital and Harvard Medical
School, 125 Nashua Street, Suite 660, Boston, MA 02114
2The Third Affiliated Hospital of Sun Yat-sen University, 510630, China
3Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and
Harvard Medical School, 149 Thirteenth Street, Suite 2301 Charlestown, MA 02129
4Department of Pharmacology, University of North Carolina Chapel Hill School of Medicine,
Chapel Hill, North Carolina 27514, United States
5University Central Hospital of Kuopio, 70210 Kuopio, Finland
&
Equal contribution
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ABSTRACT
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We have synthesized and characterized [ F]-N-(4-chloro-3-((fluoromethyl-d )thio)phenyl)-
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picolinamide ([ F]15) as a potential ligand for the PET imaging of mGluR4 in the brain.
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Radioligand [ F]15 displays CNS drug-like properties, including mGluR4 affinity, potent
mGluR4 PAM activity and selectivity against other mGluRs, as well as sufficient metabolic
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stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [ F]15 was 11.6
± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol.
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Ex vivo biodistribution studies showed reversible binding of [ F]15 in all investigated tissues
including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague-Dawley
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rats showed that [ F]15 accumulates in the brain regions known to express mGluR4. Pretreatment
with the unlabeled mGluR4 PAM compounds 13 (methylthio analog) and 15 showed significant
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dose-dependent blocking effects. These results suggest that [ F]15 is a promising radioligand for
PET imaging mGluR4 in the brain.
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INTRODUCTION
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting more
than six million people worldwide.1-2 PD results from the progressive loss of dopaminergic
neurons in the Substantia Nigra pars compacta (SNpc), causing dysfunction of the basal ganglia
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(
BG) motor circuit.3-4 The current pharmacotherapy aims to replace missing dopamine by using
the dopamine precursor levodopa (L-DOPA). This treatment provides symptomatic relief and is
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successful in the early PD medication period, however, as the disease progresses L-DOPA
becomes less effective and produces debilitating side effects such as L-dopa-induced dyskinesia
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LID).6-7 Hence the development of a disease modifying therapy for PD remains a challenge for
researchers.
Over the last decade, metabotropic glutamate receptor 4 (mGluR4) has gained interest due to
the potential benefits in treating numerous neuronal diseases such as PD, LID and other disorders.6,
8-9
The mGluR4 receptor is expressed at multiple synapses in desired regions of the BG motor
circuit, mainly localized presynaptically in the striatum, hippocampus, thalamus, and
cerebellum.10-12 As a group III mGluR, mGluR4 interacts with the Gai/o subunit of G-protein that
negatively couples with adenylate cyclase to inhibit cAMP dependent signal pathways.1, 13 Its
activation reduces presynaptic neurotransmitter release, subsequently, decreasing output of the
indirect pathway. This is a promising strategy to normalize the BG circuitry in PD and ultimately
reduce or eliminate PD symptoms.3, 5, 8, 10 Consequently, this approach has opened a new avenue
for developing nondopaminergic treatments for PD and for identifying novel disease modifying
therapeutics.
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As a family C GPCR, the activation of mGluR4 can be accomplished or enhanced by two
different mechanisms: orthosteric agonists or positive allosteric modulators (PAMs). Although
most orthosteric ligands lack clear subtype-selectivity and/or blood–brain barrier (BBB)
penetration, few examples of selective and brain penetrant orthosteric agonists such as LSP4-2022
have been reported.14-15 Since allosteric modulators may offer some benefits such as subtype-
selectivity, retained physiology of the receptor and the saturation effects, substantial effort has
been focused on development of mGluR4 PAMs as new therapeutics for neurological diseases
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such as PD and LID. Hundreds of mGluR4 PAMs have been reported and/or patented since
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009.16-17 Figure 1 shows some representative mGluR4 PAMs, of which compounds 1-10 have
demonstrated antiparkinsonian activity in animal models of PD.18-28 Compound 11 is a potent
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mGluR4 PAM that has been used as the reference compound for mGluR4 PAM activity. Prexton
Therapeutics (Prexton) announced in 2017 the launch of phase II clinical trial of its investigational
drug candidate, PXT002331 (10, Foliglurax), in PD.6
[Figure 1]
An efficient PET radioligand for mGluR4 could be an important tool for understanding the
role of mGluR4 in healthy and disease conditions, and also for the development of new drugs
targeting this receptor.9, 29 PET offers picomolar sensitivity and is a fully translational technique;
therefore, extensive research efforts have been directed towards the development of PET ligands
suitable for in vivo probing of mGluR4 (Figure 2).30-33 In the past, we have studied the
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pharmacokinetics of [ C]4, ([ C]ML-128), [ F]12 ([ F]KALB001), [ C]13 ([ C]mG4P012 or
[11
C]KALB012) and [ C]14 by using PET imaging in rats. PET studies with [ F]12 and [ C]13
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were conducted also in non-human primates. These PET radiotracers have been designed and/or
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developed based on mGluR4 PAMs.
Figure 2]
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Among these PET ligands, the in vitro data revealed that compound 13 has sufficient mGluR4
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binding affinity and selectivity over other mGluRs, as well as a suitable metabolic stability. PET
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studies in rats showed that [ C]13 accumulated rapidly into the brain and had higher uptake,
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slower washout and 25% better contrast than [ C]4, indicating improved imaging characteristics
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as a PET radiotracer for mGluR4. The improved pharmacological properties and the enhanced
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imaging characteristics indicated that [ C]13 is a useful PET radiotracer for mGluR4 in biological
research and drug development.
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Recently [ C]13 (renamed [ C]PXT012253 by Prexton Therapeutics) has been used as a
biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (10,
an mGluR4 PAM), for PD and LID.34-35 The radiotracer [ C]13 was reported to display binding
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in mGluR4-expressing regions in the brain of cynomolgus monkeys. Competition of [ C]13 with
0 showed high specific binding in the total distribution volume, which is useful for target
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occupancy or longitudinal studies. This data further supports [ C]13 as a promising PET
radioligand for mGluR4 in the monkey brain and for further development in human subjects.
Development of a fluorine-18 labeled PET tracer for imaging mGluR4 is important since
fluorine-18 is often the radionuclide of choice for both its physical and nuclear characteristics. Its
half-life is sufficient to carry out relatively extensive imaging protocols when compared to what
is possible with carbon-11. This facilitates kinetic studies and high-quality metabolic and plasma
analysis, while the low positron energy associated with fluorine-18 decay leads to high imaging
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resolution. We reported a fluorine-18 labeled PET tracer for mGluR4 ([ F]12, [ F]KALB001)
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in 2014, which showed good affinity to mGluR4 and sufficient brain uptake ; however, this
phthalimide derivative was not stable under neutral condition and required formulation in an acidic
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buffer (pH 4.0). Further development of mGluR4 PET tracers indicated that [ C]13 had more
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favorable characteristics for imaging and it initiated designing a new fluorine-18 labeled PET
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tracer, [ F]mG4P027 ([ F]15).
Chart 1]
There are three possible positions to introduce fluorine-18: the 6-pyridyl, 4-chloro and 3-
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methylthio moieties (Chart 1). SAR results indicate that 4-fluoro substitution of the chloro-atom
gives reduced binding affinity which follows the order: Cl < H < F < I < Br. On the other hand,
incorporation of a fluorine atom at the 6-pyrindyl position also slightly lowers the affinity and
affects its metabolic stability. Next step was to evaluate the position of 3-phenyl, which is sensitive
to substitutions. Our previous results showed that the 3-fluoromethoxy compound had an improved
affinity compared to that of the 3-methoxy compound. Since the fluoromethylthio group may not
be metabolically stable, we applied a 3-dideuteriumfluoromethylthio moiety to replace the 3-
methylthio group as shown in Chart 1. Deuterium isotope effects have been used to reduce in vivo
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metabolic rates. The incorporation of dideuterium into [ F]15 could reduce the rate of
defluorination initiated by cleavage of the C–H bond without altering the binding affinity to
mGluR4.
Herein, we report our results on the chemical synthesis, in vitro pharmacochemical properties,
radiolabeling, ex vivo biodistribution and PET imaging in rats of this new fluorine-18 labeled PET
ligand for mGluR4.
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RESULTS AND DISCUSSION
Chemistry. The novel unlabeled mGluR4 ligand 15 was prepared to study its in vitro
pharmacochemical properties (Scheme 1). The reaction of methylene-d bis(tosylate) 16 with
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cesium fluoride was carried out in t-amyl alcohol at 80°C to give fluoromethyl-d 4-
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methylbenzenesulfonate 17 in 48% yield. It was found that the choice of solvent used in this
reaction was very important. If the reaction was carried out in acetonitrile, the yield was 5%. The
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thiophenol precursor 18 had been used for the radiolabeling of [ C]13, which was prepared from
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-chloro-nitrobenzene via multi-steps. In order to support the translational study of [ C]13, a
new synthetic route was developed for synthesis of 18 with optimized reaction conditions,
resulting in an improved yield and a more efficient workup procedure.37 The synthesis of
compound 15 was achieved by treating 17 with the thiophenol precursor 18 in 92% yield.
[Scheme 1]
In vitro pharmacochemical properties. To evaluate compound 15, the pharmacochemical
properties including affinity to mGluR4, mGluR4 PAM activity, selectivity to other mGluRs,
lipophilicity, plasma protein binding, metabolic and solution stabilities were determined. In this
study compound 15 was compared to the previously reported mGluR4 PET ligands: compounds
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, 13 and others.
The tritium-labeled radioligand [ H]4 (N-(4-chloro-3-(methoxy-t )phenyl)picolinamide) was
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prepared for competitive binding assay (supporting information).31, 38 Compounds 4, 8, 13 and 15
were characterized with the competitive binding assay using mGluR4 transfected CHO cells by
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increasing the concentration of test compound from 0.01 nM to 10 M in the presence of 2 nM of
[3
H]4, in which the binding affinities to mGluR4 were described as IC values (Table 1).
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[Table 1]
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As displayed in Table 1, compound 15 has a similar IC value (3.5±0.5 nM) as that of 8 (3.2±0.6
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nM) and 13 (3.5±0.7 nM) and has slightly improved affinity compared to 4 (5.1±1.1 nM). The
results also confirmed that compound 15 binds to the same allosteric site of mGluR4 as that of the
other three compounds.
The mGluR4 PAM activity was determined using Promega’s split luciferase based GloSensor
cAMP biosensor assay.39-40 The mGluR4 PAM activity of 15 was evaluated in presence of EC20
concentration of agonist (1 M L-SOP) by measuring changes in intracellular cAMP concentration,
the relevant second messenger mechanism. Compound 11, one of the most potent mGluR4 PAMs,
was used as the reference compound for the assay. As shown in Figure 3, the EC values of 11
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and 15 were 55 nM and 324 nM, respectively, suggesting that 15 is a moderately potent mGluR4
PAM. In the mGluR4 Gi fold shift assay, 15 shifts the agonist (L-SOP) response curves to the left
7.5-fold at 3.0 M in 98.3% Glu Max efficacy.
[Figure 3]
The selectivity of 15 was also analyzed among the various mGluR subtypes, in which the Gq
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coupled receptors (mGluR1 and mGluR5) were tested using Ca mobilization assay and the Gi/o
coupled receptors (mGluR2, mGluR3, mGluR4, mGluR6 and mGluR8) using the cAMP assay.
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Results demonstrated that 15 has selectivity against other mGlu receptors (Table S1) and that 15
has mGluR4 agonist activity (EC = 2.75 M), hence, 15 is an mGluR4 ago-PAM.
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Lipophilicity has a major effect on BBB penetration, ADMET properties and pharmacological
activity. The values of lipophilicity (cLogP) and tPSA for 15 were calculated by using ChemDraw
as 2.95 and 41.46, respectively. The LogD of 15 was 3.82 as measured with a scaled-down shake
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flask method using [ F]15 (Table 2) and is in the range normally considered favorable for a PET
ligand.42
[Table 2]
As the PET tracer is delivered into the bloodstream, it can bind to albumin, 1-acid
glycoprotein and lipoproteins. Plasma protein binding (PPB) reduces the free drug in the
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bloodstream and inhibits BBB penetration. The PPB value was obtained with an ultrafiltration
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assay by using [ F]15. As Table 2 shows, the PPB of 15 was 93.1%, which gives a suitable free
tracer concentration available to cross the BBB.
After iv injection PET tracers encounter plasma decomposition by hydrolytic enzymes in
the blood and are carried into the liver where they face diverse hepatic metabolic reactions such as
phase I oxidations by CYP and flavin monooxygenases (FMO). Unstable compounds often have
high clearance (Cl ) and short half-life (t ) resulting in poor in vivo pharmacokinetics (PK) and
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unsatisfactory pharmacological performance. The in vitro plasma and liver microsomal stability
of 4, 13 and 15 were studied by incubating the compounds in rat serum and rat liver microsomes
as well as NADPH cofactor, respectively, using previously published methods.43-44 Diltiazem and
propranolol were used as co-assay QC controls for plasma and microsomal stability assays,
respectively, to ensure that the assays were operating properly, and that the activity of the plasma
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and microsomes were consistent with established criteria. As Table 3 shows, 4, 13 and 15 are more
stable than diltiazem in rat plasma. The results also show that 15 exhibits excellent microsomal
stability and is more stable than 4 and 13, in which the suitable hepatic clearance was predicted.
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The solution stability of 15 was evaluated at pH 5.0, 7.4 and 9.4, respectively. The results indicate
that 15 is relatively stable in pH ranging from 5.0 to 9.4 (Table 3).
[Table 3]
The in vitro pharmacological studies reveal that compound 15 has many CNS drug-like
properties, including appropriate mGluR4 affinity, potent mGluR4 PAM activity and selectivity
against other mGluRs, and suitable lipophilicity as well as PPB, adequate metabolic stability and
solution stability. Based on these results, compound 15 was selected for radiolabeling and in vivo
evaluation as a potential mGluR4 PET radioligand.
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Radiochemistry. As shown in Scheme 2 the radiolabeling of [ F]15 was carried out in two steps:
1
8
18
18
1) generation of [ F]fluoromethyl-d 4-methylbenzenesulfonate [ F]17 by [ F]fluorination of
2
methylene-d bis(4-methylbenzenesulfonate) 16; 2) the radiolabeling of the thiophenol precursor
2
1
8
1
8 with [ F]17.
[Scheme 2]
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Compound [ F]17 was prepared from the reaction of 16 in acetonitrile and t-butanol with non-
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carrier-added (nca) [ F]fluoride in the presence of tetrabutylammonium bicarbonate at 85 °C for
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18
10 min. The radioactive product was a mixture of [ F]17 and the side product [ F]19, which was
purified by semi-preparative HPLC, eluting with a solution of water and acetonitrile (50:50) at a
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flow rate of 4 mL/min to give [ F]17 in 20% yield. The obtained [ F]17 reacted with the
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thiophenol precursor 18 in DMSO at 150 ˚C for 10 min, resulting the desired product [ F]15 in
over 95% conversion. The radiolabeled product was purified by semi-preparative HPLC, eluting
with 0.1% formic acid solution of water and acetonitrile (40:60) at a flow rate of 4 mL/min to
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produce [ F]15. The final product was obtained after eluting 0.5 mL of ethanol followed by 4.5
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mL of sterile saline through the C Sep-Pak column resulting the final formulation of [ F]15 in
18
saline containing 10% ethanol with 11.6% ± 2.9% radiochemical yield (RCY, n = 7, decay
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corrected) and over 99% radiochemical purity. The total synthesis of [ F]15 took 2.5-3.5 h with
the molar activity 84.1 ± 11.8 GBq/mol (n = 7).
Ex vivo Biodistribution Studies. Ex vivo biodistribution studies were carried out in normal male
Sprague Dawley rats to quantify accumulation of the tracer in different organs, as well as determine
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metabolic pathways. These studies showed reversible binding of [ F]15 in all investigated tissues
with maximum uptake between 10 and 20 min except in the kidneys, where excretion of urine can
affect tissue activity samples. Liver had the highest accumulation of 1.33±0.12 % ID/g at 20 min
followed by kidneys 0.93±0.57 % ID/g at 30 min, lungs 0.58±0.17 % ID/g at 20 min, heart
0
.50±0.08 % ID/g at 20 min and brain 0.41±0.18 % ID/g at 10 min. (Figure 4).
[Figure 4]
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PET imaging. For in vivo characterization with PET imaging, [ F]15 was synthesized nine times
and 30 studies were conducted in male Sprague−Dawley rats comprising 15 baseline studies and
9
blocking studies by using 13 as a blocking agent with doses of 1, 2 or 3 mg/kg and 6 self-blocking
studies by using “cold” 15 as blocking agent with the same doses as 13. These studies demonstrate
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that [ F]15 crosses BBB and accumulates in the brain areas known to express mGluR4 (Figure
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). Time-activity curves (TACs) show fast reversible binding in the striatum, thalamus, cerebellum
and cortex (Figure 6). The maximum average uptake at the time interval of 1-10 min after
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administering of the ligand was in the thalamus, 1.77±0.26 % ID/cc. The binding of [ F]15 in the
rat brain was blocked in the whole brain (22-25%) by using 13 and even dose dependently in
specific brain areas like striatum, 18, 20 and 28% using doses of 1, 2 or 3 mg/kg. Self-blocking
with the same doses of 15 had similar blocking effect with the dose of 2 mg/kg but was
significantly lower with 1 mg/kg and higher with 3 mg/kg compared to blocking with 13 (Figure
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). This PET tracer will be further developed in preclinical research with an aim for translational
studies.
[Figure 5]
[
Figure 6]
Figure 7]
[
SUMMARY
In this study, we have designed, synthesized and characterized a new fluorine-18 labeled PET
ligand for imaging mGluR4 in the brain. This radioligand displays CNS drug-like properties,
including good mGluR4 affinity, potent mGluR4 PAM activity and selectivity against other
mGluRs, suitable lipophilicity as well as PPB, adequate metabolic and solution stability. The
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radiolabeling of [ F]15 was carried out in two steps. The PET imaging studies in male
18
Sprague−Dawley rats demonstrate that [ F]15 crosses BBB and accumulates the brain areas
known to express mGluR4. Pretreatment with the unlabeled compounds 13 and 15 show the
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significant dose-dependent blocking effects. The results suggest that [ F]15 is a promising
candidate for PET imaging of mGluR4 in the brain and for translational studies in PD and/or other
disorders.
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EXPERIMENTAL SECTION
Animal procedures. The animal studies were approved and done under strict supervision of
Subcommittee on Research Animals of the Massachusetts General Hospital and Harvard Medical
School and performed in accordance with the Guide of NIH for the Care and Use of Laboratory
Animals.
Materials and Methods. All reagents and starting materials were obtained from the commercial
sources including Sigma-Aldrich (St. Louis, MO), Thermo Fisher Scientific, Oakwood Products,
Inc., Matrix Scientific and used as received. The reactions were monitored by TLC using a UV
lamp monitored at 254 nm. If necessary, the reactions were also checked by LC−MS using the
Agilent 1200 series HPLC system coupled with a multiwavelength UV detector and a model 6310
ion trap mass spectrometer (Santa Clara, CA) equipped with a Luna C column (Phenomenex,
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8
100 × 2 mm, 5 , 100 Å). The RP-HPLC was carried out by using a 7-min gradient method (LC-
Method 1): eluent A: 0.1% formic acid/ H O; eluent B: 0.1% formic acid/CH CN; gradient: 5% B
2
3
to 95% B from 0 to 3 min, 95% B from 3 to 4.5 min, 95% to 5% B from 4.5 to 5 min, 5% B from
5 to 7 min; flow rate at 0.7 mL/min. The silica gel used in flash column chromatography was from
Aldrich (Cat. 60737, pore size 60 Å, 230-400 mesh). The products were identified by LC−MS as
1
13
19
well as H NMR, C NMR and F NMR using a Varian 500 MHz spectrometer. All NMR samples
were dissolved in chloroform-d (CDCl ) containing tetramethylsilane as a reference standard.
3
Chemical shifts were expressed as ppm and calculated downfield or upfield from the NMR signal
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of reference standard. J was expressed as Hz, and its splitting patterns were reported as s, d, t, q,
or m. HRMS was acquired using a DART-SVP ion source (IonSense, Saugus, MA) attached to a
JEOL AccuTOF 4G LC-plus mass spectrometer (JEOLUSA,Peabody, MA) in positive-ion mode
from Prof. Peter Caravan’s Laboratory. Unless otherwise specified, the purity of all new
compounds was over 95% determined by HPLC.
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Chemistry. Fluoromethyl-d 4-methylbenzenesulfonate (17). To the solution of methylene-d2
2
bis(tosylate) (16, 9.01g, 25.2 mmol) in tert-amyl alchol (150.0 mL) was added CsF (3.83 g, 25.2
mmol). The mixture was stirred at 80 ˚C for 2.0 h. A large amount of white solid precipitated out.
After filtration, the filtrate was condensed under vacuum. The resulting residue was
chromatographed on silica gel by eluting with ethyl acetate and hexane (1:30 to 1:15) to afford 17
1
as a colorless oil (2.49 g, 48%) and 3.02 g of starting material 16 was recovered. H NMR (500
1
3
MHz, CDCl ) δ 7.83 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.46 (s, 3H). C NMR (126
3
MHz, CDCl ) δ 145.6, 133.8, 129.9 (2C), 127.9 (2C), 97.6 (dp, J = 26.5 Hz, J = 229.3 Hz), 21.7.
3
1
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F NMR (470 MHz, CDCl ) δ -154.5 (p, J = 9.4 Hz). LC-MS (method 1): t = 3.67 min, (ESI)
3
R
+
m/z calcd. for C H D FNO S 224.0; found 224.0 [M + NH ] .
8
22
2
3
4
N-(4-Chloro-3-((fluoromethyl-d )thio)phenyl)picolinamide (15). To the solution of N-(4-chloro-
2
3
-mercaptophenyl)picolinamide hydrogen chloride salt (18, 150.0 mg, 0.5 mmol) and 17 (154.5
mg, 0.75 mmol) in acetonitrile (7.0 mL) were added K CO (690 mg, 5.0 mmol) and KI (83.0 mg,
2
3
0
.5 mmol). The resulting mixture was heated to reflux for 2 h. The solvent was removed under
vacuum and the residue was dissolved in DCM (20 mL) and water (20 mL). The water phase was
further washed with DCM twice (20 mL). The organic layer was combined, dried over anhydrous
Na SO and concentrated under vacuum. The residue was chromatographed on silica gel by eluting
2
4
1
with EtOAc and hexane (1:3) to afford the title product as a white powder (137.2 mg, 92%). H
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NMR (500 MHz, CDCl ) δ 10.08 (s, 1H), 8.62 (ddd, J = 4.7, 1.6, 0.9 Hz, 1H), 8.29 (dt, J = 7.8,
3
1
.0 Hz, 1H), 7.92 (td, J = 7.7, 1.7 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.51 (ddd, J = 7.6, 4.7, 1.2
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Hz, 1H), 7.44 (dd, J = 8.7, 2.3 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H). F NMR (470 MHz, CDCl ) δ -
3
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13
1
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50.9 (dt, J = 16.2, 8.2 Hz). C NMR (126 MHz, CDCl ) δ 162.1, 149.3, 148.0, 137.8, 137.3,
3
34.3, 130.2, 128.62 (d, J = 2.4 Hz), 126.7, 122.5, 120.6, 119.5, 85.8 (dp, J = 24.0, 217.2 Hz). LC-
MS (method A1): t = 4.05 min, (ESI) m/z calcd. for C H D ClFN OS 299.0; found 298.9 [M +
R
13
9
2
2
+
+
H] . HRMS (m/z) calcd. for C H D ClFN OS 299.0390; found 299.0388 [M + H] .
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Radiochemistry Procedure. [ F]Fluoride was generated by a Siemens Eclipse HP 11 MeV
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cyclotron (Malvern, PA) using O-enriched water (Isoflex Isotope, San Francisco, CA) with
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proton bombardment. Fluorine-18 labeling of [ F]15 was accomplished in two steps. First,
18
[ F]fluoride in 18O-enriched water was passed through a QMA Sep-Pak Cartridge (Waters,
18
Milford, MA) to trap [ F]fluoride ions, which was washed off by 0.5 mL of the aqueous solution
of tetrabutylammonium hydrogen carbonate (75 mM, from ABX advanced biochemical
compounds). Acetonitrile (1.0 mL) was added to the solution and the solvents were evaporated at
1
15 °C in a stream of nitrogen. In order to remove water completely, 1.0 mL of acetonitrile was
added and evaporated in a stream of nitrogen three more times. To the residue containing
[18
F]fluoride was added 16 (12.0 mg) in 0.7 mL of acetonitrile and t-BuOH (4:1). The resulting
solution was heated to 85 °C for 10 min and then cooled to room temperature followed by addition
of 1.5 mL of water. The mixture was then purified by a semi-preparative HPLC (Waters 4000
system equipped with an Xbridge BEH C OBD column: 130 Å, 5 µ, 10 × 250 mm) by eluting
18
with a solution of water and acetonitrile (50:50) at a flow rate of 4 mL/min to give the fractions
18
containing [ F]17. The combined fraction was diluted with water to 40 mL and loaded on a C
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Sep-Pak column. The column was further dried through a stream of nitrogen for 20-30 min. [ F]17
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was washed off the C Sep-Pak column by 0.7 mL of dry DMSO to a reaction vessel containing
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.0 mg of 18 and cesium carbonate (3.0 - 5.0 mg). The resulting mixture was heated to 150 ˚C for
10 min and then cooled to rt, followed by addition of the HPLC eluents (2.5 mL, 0.1% formic acid
solution of water and acetonitrile 40:60). The mixture was then purified using the semi-preparative
HPLC (Waters 4000 system equipped with an Xbridge BEH C OBD column: 130 Å, 5 µ, 250 ×
0
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0 mm) by eluting with a 0.1% formic acid solution of water and acetonitrile (40:60) at a flow rate
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of 4 mL/min. The fraction containing [ F]15 was diluted with water to 40 mL and loaded on a C
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Sep-Pak column to give the final formulation of [ F]15 in saline containing 10% ethanol with
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1.6% ± 2.9% radiochemical yield (RCY, n = 7, decay corrected). The purity of [ F]15 was over
99% that was analyzed by an analytical HPLC (Waters 2487 series equipped with a UV detector
and a BIOSCAN radioactivity detector and an ACE 5 C -AR column: 250 × 10 mm, 5 μ). Identity
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of [ F]15 was confirmed by co-injection of the cold compound 15 in HPLC analysis. The total
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synthesis of [ F]15 took 2.5-3.5 h with the molar activity 84.1 ± 11.8 GBq/µmol (n =7).
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Determination of Log D. An aliquot (10 μL, 74 kBq) of [ F]15 was added to a test tube
containing 2.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The test tube was
mixed by vortex for 2 min and then centrifuged for 2 min to fully separate the aqueous and organic
phase. The samples taken from the octanol layer (0.1 mL) and the aqueous layer (1.0 mL) were
saved for radioactivity measurement. An additional aliquot of the octanol layer (2.0 mL) was
carefully transferred to a new test tube containing 0.5 mL of octanol and 2.5 mL of phosphate
buffer solution (pH 7.4). The previous procedure (vortex mixing, centrifugation, sampling, and
transfer to the next test tube) was repeated until six sets of aliquot samples had been prepared. The
radioactivity of each sample was measured using PerkinElmer Wizard2 2480 gamma-counter. The
log D of each set of samples was calculated as the following:
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Log D = Log (radioactivity of octanol layer × 10 / radioactivity of PBS layer)
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.4
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Plasma Protein Binding Assay. An aliquot of radiolabeled compound [ F]15 in saline (10 μL,
7
4 kBq) was added to a sample of human plasma (0.5 mL). The mixture was gently mixed by
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repeated inversion and incubated for 10 min at room temperature. Following incubation, a small
sample (10 μL) was removed to determine the total radioactivity in the plasma sample (A ; A =
T
T
Abound + Aunbound). The upper part of the Centrifree tube was discarded, and an aliquot (10 μL) from
the bottom part of the tube was removed to determine the amount of unbound radioactivity
(Aunbound) that passed through the membrane (molecular weight cutoff 30 kD). The radioactivity of
each sample was measured using PerkinElmer Wizard2 2480 gamma-counter. Plasma protein
binding was derived by the following equation: %unbound= Aunbound x 100/AT.
Ex vivo Studies of Biodistribution. For biodistribution studies the normal male Sprague Dawley
rats were anesthetized with isoflurane/nitrous oxide (1-1.5% isoflurane) to install catheter into the
18
tail vain for administration of radioactivity ([ F]15, 22±3 MBq, iv, mass 0.072±0.09 µg). Total
of 15 rats in deep anesthesia (isoflurane 4% and cervical dislocation) were sacrificed in a group of
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three at five different time points (5, 10, 20, 30, and 60 min) after injection of [ F]15. Major
organs including lung, heart, liver, spleen, kidney, muscle, midbrain, cortex, cerebellum and blood
were harvested to determine radioactivity. The tissue samples were weighted, and the radioactivity
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was measured with the standard samples of [ F]15 using Wizard2 2480, Perkin Elmer, CA.
Radioactivity of the tissue samples was determined as percent of the injected activity per gram of
the tissue.
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PET Imaging of [ F]15 in Rats. Altogether 24 normal male Sprague Dawley rats were used for
the imaging studies comprising 15 baseline studies and 15 blocking studies. Rats were
anaesthetized with isoflurane/nitrous oxide (1.0-1.5% isoflurane, with oxygen flow of 1-1.5 L/min),
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and catheter was installed into tail vein for the administration of [ F]15. Dynamic volumetric PET
data was acquired with a PET-CT scanner for 60 min (Triumph-II, Tri-Foil Imaging, Northridge,
CA). The vital signals such as heart rate and respiration rate were monitored during scanning
period. PET data acquisition was started immediately after administration of radioactivity with the
dose range of 18-34 MBq (mass 0.059±0.111 µg) depending on the size of the rat followed by CT
imaging to obtain anatomical information and data for attenuation correction of PET data. PET
data was processed by using maximum-likelihood expectation-maximization (MLEM) algorithm
with 30 iterations to dynamic volumetric images, and corrected for uniformity, scatter, and
attenuation. The CT data was processed by the modified Feldkamp algorithm using matrix
volumes of 512×512×512 and pixel size of 170 μm. Co-registration of CT and PET images and
analysis of PET images were carried out using PMOD3.2 software (PMOD Technology, Zurich,
Switzerland).
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For blocking studies, the same scanning protocols were used as for the baseline studies.
For mGluR4 blocking experiments, 1, 2 or 3 mg/kg of 13 or 15 dissolved in a saline solution with
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0% DMSO, 5% Tween-20 and 85% PBS was injected 1 min before iv administration of [ F]15.
ASSOCIATED CONTENT
Supporting Information
The in vitro assays including the radioligand replacement assay, the mGluR functional assays
and metabolic as well as solution stability assays are described in the Supporting Information
that is available free of charge on the ACS Publications website at DOI:
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AUTHOR INFORMATION
Corresponding Authors
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*A-L.B.: phone, 617-744-3725; e-mail: abrownell@mgh.harvard.edu
*Z.Z.: phone, 617-643-4887; e-mail: zzhang9@mgh.harvard.edu
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to acknowledge supporting research grants R01NS10064, R01EB021708
and R01EB012864 and the grants 1S10RR023452-01 and 1S10OD025234-01 for the imaging
instrumentation and characterization of the organic compounds. As well the authors like to thank
the technical staffs at the cyclotron facility at Massachusetts General Hospital.
mGluR1-6 and mGluR8 agonist and antagonist functional data as well as mGluR4 PAM activity
were generously provided by the National Institute of Mental Health's Psychoactive Drug
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Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is
Directed by Bryan L. Roth (mailto:bryan_roth@med.unc.edu) at the University of North Carolina
at Chapel Hill and Project Officer Jamie Driscoll (mailto:jdrisco1@mail.nih.gov) at NIMH,
Bethesda MD, USA. For experimental details please refer to the PDSP web site
https://pdspdb.unc.edu/pdspWeb/ (https://pdspdb.unc.edu/pdspWeb/).
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ABBREVIATIONS USED
Cl , the intrinsic clearance; G , adenylate cyclase inhibitory G-protein; L-SOP, L-Serine-O-
int
i
phosphate; TAC, time-activity curve; %ID/g, percentage of injected dose per gram of wet tissue.
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4. Houston, J. B., Utility of in vitro drug metabolism data in predicting in vivo metabolic
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5. Di, L.; Kerns, E. H.; Chen, H.; Petusky, S. L., Development and application of an automated
solution stability assay for drug discovery. J. Biomol. Screening 2006, 11, 40-47.
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Figure 1. Representative mGluR4 PAMs.
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Figure 2. PET tracers for mGluR4. We have previously synthesized and reported the tracers
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