Synthesis and evaluation in monkey of [18F]4-Fluoro- N -methyl- N -(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([ 18F]FIMX): A promising radioligand for PET imaging of brain metabotropic glutamate receptor 1 (mGluR1)

Article abstract of DOI:10.1021/jm4012017

We sought to develop a PET radioligand that would be useful for imaging human brain metabotropic subtype 1 receptors (mGluR1) in neuropsychiatric disorders and in drug development. 4-Fluoro-N-methyl-N-(4-(6-(methylamino) pyrimidin-4-yl)thiazol-2-yl)benzamide (FIMX, 11) was identified as having favorable properties for development as a PET radioligand. We developed a method for preparing [¹⁸F]11 in useful radiochemical yield and in high specific activity from [¹⁸F]fluoride ion and an N-Boc-protected (phenyl)aryliodonium salt precursor (15). In baseline experiments in rhesus monkey, [¹⁸F]11 gave high brain radioactivity uptake, reflecting the expected distribution of mGluR1 with notably high uptake in cerebellum, which became 47% lower by 120 min after radioligand injection. Pharmacological challenges demonstrated that a very high proportion of the radioactivity in monkey brain was bound specifically and reversibly to mGluR1. [¹⁸F]11 is concluded to be an effective PET radioligand for imaging mGluR1 in monkey brain and therefore merits further evaluation in human subjects.

Full text of DOI:10.1021/jm4012017

Article
pubs.acs.org/jmc
Synthesis and Evaluation in Monkey of [18F]4-Fluoro‑N‑methyl‑N‑(4-
(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide ([18F]FIMX): A
Promising Radioligand for PET Imaging of Brain Metabotropic
Glutamate Receptor 1 (mGluR1)
Rong Xu, Paolo Zanotti-Fregonara, Sami S. Zoghbi, Robert L. Gladding, Alicia E. Woock, Robert B. Innis,
and Victor W Pike*
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346A, 10
Center Drive, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: We sought to develop a PET radioligand that
would be useful for imaging human brain metabotropic
subtype 1 receptors (mGluR1) in neuropsychiatric disorders
and in drug development. 4-Fluoro-N-methyl-N-(4-(6-
(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide (FIMX,
11) was identified as having favorable properties for
development as a PET radioligand. We developed a method
for preparing [18F]11 in useful radiochemical yield and in high specific activity from [18F]fluoride ion and an N-Boc-protected
(phenyl)aryliodonium salt precursor (15). In baseline experiments in rhesus monkey, [18F]11 gave high brain radioactivity
uptake, reflecting the expected distribution of mGluR1 with notably high uptake in cerebellum, which became 47% lower by 120
min after radioligand injection. Pharmacological challenges demonstrated that a very high proportion of the radioactivity in
monkey brain was bound specifically and reversibly to mGluR1. [18F]11 is concluded to be an effective PET radioligand for
imaging mGluR1 in monkey brain and therefore merits further evaluation in human subjects.
radioligand have explored several candidates among a variety of
structural classes.17−30 Some of these have been evaluated in
rodents only. A few are reported to have been evaluated in
primates, namely, [11C]JNJ-16567083 ([11C]1), [18F]2,
[11C]MMTP ([11C]3), [18F]MK-1312 ([18F]4), [18F]FPIT
([18F]5), [11C]LY2428703 ([11C]6), [18F]FITM ([18F]7), and
[11C]8 (Figure 1). Only one of these, [11C]6, is known to have
been further evaluated in human. Although [11C]6 showed
promising imaging characteristics in rat,28 this radioligand failed
to give a receptor-specific signal in monkey or human subjects,
primarily because of a significantly lower effective binding
potential (Vs) for human mGluR1 (0.0161) than for rat
mGluR1 (0.683).30 Vs is defined as f P × Bmax/KD, and although
mGluR1 receptor density (Bmax) in human is similar to that in
rat and higher than that in monkey,22,31 the plasma free fraction
(fP) and affinity (1/KD) of 6 are each substantially lower for
human than for either rat or monkey. Among other
radioligands evaluated in monkey, [18F]7 appears to have
been one of the most promising because it displays high affinity
(IC50 = 5.1 nM) and selectivity for binding to mGluR1 as well
as moderate radioactivity uptake in monkey brain with a high
mGluR1-specific signal. Nevertheless, the reported radiosyn-
thesis of [18F]723 requires heating of a base-sensitive nitro
precursor at 180 °C and is known to be challenging.24
INTRODUCTION
■
Glutamate is a major brain excitatory neurotransmitter that acts
on a wide range of receptors that are categorized as either
metabotropic (mGluR) or ionotropic (iGluR).1 Eight subtypes
are recognized for mGluR, and they fall into three groups based
on their amino acid sequences and pharmacology.2,3 mGluR1,
along with the mGluR5 subtype, belong to group I and in
human brain are most abundant in cerebellum, hypothalamus,
thalamus, basal ganglia, and hippocampus.4 They have been
implicated in several neuropsychiatric disorders, including pain,
cerebellar ataxia, extrapyramidal motor dysfunction, fear,
anxiety, mood disorders, excitotoxicity, epilepsy, and schizo-
phrenia.5−7 Consequently, mGluR1 are considered to be a
target for drug development.8−10 A means to image and
quantify human brain mGluR1 with PET would be valuable to
understand better the role of mGluR1 in neuropsychiatric
disorders as well as for developing drugs intended to target
these receptors. However, unlike for human brain mGluR5,11,12
satisfactory radioligands for quantifying human brain mGluR1
with PET are not yet established. Our ultimate goal is to
develop such a radioligand.
Candidate ligands for PET imaging of brain receptors should
present a variety of features, among which are, importantly,
high affinity, high selectivity, moderate lipophilicity, and
amenability to labeling with a positron-emitter, usually
carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.7
min).13−16 Previous efforts to develop a suitable mGluR1 PET
Received: August 5, 2013
Published: October 22, 2013
© 2013 American Chemical Society
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with a positron-emitter (Figure 2), namely, either of the two N-
methyl groups for labeling by methylation with [11C]-
iodomethane34 or the aryl fluoro substituent for labeling by
aromatic nucleophilic substitution with [18F]fluoride ion.35 Our
initial attempts at labeling 11 by methylation of either the
secondary desmethyl amido analogue or the N-desmethyl
pyrimidinyl-amino analogue with [11C]iodomethane under
basic conditions did not give satisfactory yields or product
purities (data not shown). Therefore, we focused on achieving
the radiofluorination of 11. We first tried conventional
substitutions in nitro and bromo analogues with [18F]fluoride
ion, but no [18F]11 was formed even under harsh conditions of
high temperature and extended reaction times (data not
shown). Our findings were consistent with the recognized
difficulty of labeling the close analogue 7 (Figure 1) by reaction
of a nitro precursor with [18F]fluoride ion.23,24 The difficulty of
this process for preparing [18F]7 or [18F]11 may be ascribed to
the presence of only a weak electron-withdrawing para-N-
methylamido group on the ring to be labeled with fluorine-18.35
We and others have studied the radiofluorination of
diaryliodonium salts extensively36−40 and have shown that
this methodology enables introduction of cyclotron-produced
no-carrier-added [18F]fluoride ion into aryl rings. Relatively few
structurally complex radioligands have yet been prepared with
this methodology.41−43 Nonetheless, we decided to explore this
method for labeling 11 because of its notable success in labeling
both electron-rich and electron-deficient arenes.36−40 We
considered that the free amino group in 11 would need to be
protected during synthesis of an iodonium salt precursor and
possibly during its radiofluorination to avoid detrimental
interactions of the amino group with [18F]fluoride ion. We
chose Boc as a protecting group because this can be removed
quite quickly in radiosyntheses with fluorine-18, usually under
acidic conditions.44 Treatment of trialkylarylstannanes with
Koser’s reagent (hydroxy(tosyloxy)iodobenzene) has been
shown to be an effective method for producing functionalized
diaryliodonium salts,39−43,45,46 and we considered this method
for generating a desirable precursor. From this method, the
diaryliodonium salt would have phenyl as one of the ring
partners and tosylate as counterion. On the basis of
experimental39,40 and theoretical studies47,48 on the aryl ring
preference in the radiofluorination of diaryliodonium salts, we
expected that radiofluorination would occur not at the phenyl
ring but at the desired more electron-deficient ring. Moreover,
the weakly nucleophilic tosylate ion is well tolerated in the
radiofluorination of diaryliodonium salts.39,40,42
Figure 1. Candidate mGluR1 PET radioligands that have been
evaluated in nonhuman primates.
Moreover, washout of radioactivity from rat or monkey brain
after intravenous administration of [18F]7 is quite slow and may
hinder accurate quantification of mGluR1.23,26 A radioligand
with faster brain kinetics in monkey is desirable.
The N-methyl analogue of 7, which we dub FIMX (11)
(Figure 2), has been reported to have 3-fold higher human
Figure 2. Structure of 11 (FIMX) and sites considered for labeling
with a positron-emitter.
mGluR1 affinity (IC50 = 1.8 nM) and to retain high selectivity
for binding to mGluR1 versus binding to mGluR5 in vitro;32 11
is also metabolized more extensively than 7 in human liver
microsomes,32 which might also promote faster decrease of
nonspecific binding in brain. Moreover, we expected 11, having
an N-methyl group in place of an N-isopropyl group, to have
lower lipophilicity than 7. In view of these properties, we
considered that labeling 11 with carbon-11 or fluorine-18 might
provide an mGluR1 PET radioligand having improved features
over [18F]7, including higher plasma free fraction, higher brain
uptake, greater receptor-specific signal, and faster brain
kinetics.13−16,33 Here, we report our development of a method
for preparing [18F]11 in useful radiochemical yields as well as
an evaluation of this radioligand with PET, showing that
[18F]11 is effective for imaging monkey brain mGluR1 and
therefore warrants further evaluation in human subjects.
The target precursor, 4-((4-(6-(tert-butoxycarbonyl(methyl)-
amino)pyrimidin-4-yl)thiazol-2-yl)(methyl)carbamoyl)phenyl)-
(phenyl)iodonium tosylate (15), was successfully prepared in
five steps from commercially available 4-(6-chloropyrimidin-4-
yl)-N-methylthiazol-2-amine (Scheme 1). Thus, acylation of
this starting material with 4-bromobenzoyl chloride gave 10, as
previously reported.29 Subsequent amination of 10 gave 12 in
quantitative yield, and then treatment of 12 with (Boc)2O in
THF gave 13, also in excellent yield (96%). Treatment of 13
with (Bu3Sn)2 in the presence of tetrakis(triphenylphosphine)-
palladium(0) gave the tributylstannyl compound 14 in
moderate yield (40%). Treatment of 14 with Koser’s reagent
for 4 days at room temperature then gave the desired precursor
15 in 10−30% yield.
RESULTS AND DISCUSSION
Chemistry. The structure of 11 presents three sites that
were initially considered for well-known methods of labeling
Here, we also synthesized 11 for use as a reference
compound in chromatography and for more extensive
pharmacological screening. This synthesis was achieved
■
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a
Scheme 1. Synthesis of 11 and Its Radiolabeling Precursor, 15
a
Reagents and conditions: (a) 4-fluorobenzoylchloride (for 9) or 4-bromobenzoyl chloride (for 10), Et3N, toluene, 100 °C, 4 h to overnight; (b)
MeNH2, K2CO3, 1,4-dioxane, 80 °C, 1.5 h (for 11) or 85 °C, 5 h (for 12); (c) DMAP, (Boc)2O, THF, 70 °C, 1 h; (d) (Bu3Sn)2, Pd(PPh3)4, 1,4-
dioxane, overnight; (e) Koser’s reagent, 4 days.
a
Table 1. Incorporation Yields for Reactions of [18F]Fluoride Ion with 15 under Various Conditions
conditions
time (min)
[18F]F− incorporation (%)
putative [18F]16 [18F]11
entry
energy source
thermal
temp. (°C)
solvent
TEMPO (equiv)
1
2
3
4
5
6
7
8
100
5
DMF
26
39
40
30
9
0
thermal
100
5
DMF
2
2
4
2
2
2
2
3
microwave 100 W
microwave 90 W
microwave 90 W
microwave 90 W
microwave 90 W
microwave 90 W
60−140
61−113
60−149
60−152
60−149
60−152
2
DMF
11
2.5
2
MeCN
DMSO
DMSO
DMSO
0
40
4
3
29
c
b
2
n.m.
18 (n = 2)
b
2.5
DMSO
0−14
38 (n = 13)
a
b
Amounts of precursor were 1.4−1.9 mg, and reaction volume was 500 μL. Products from reactions 7 and 8 were analyzed on a semipreparative
HPLC column, whereas other reactions were analyzed on an analytical column. n.m. = not measured.
c
through acylation of 4-(6-chloropyrimidin-4-yl)-N-methylthia-
zol-2-amine with p-fluorobenzoyl chloride and subsequent
amination (Scheme 1), as has been described previously but
without full details.32
Radiochemistry. Polar and aprotic solvents, such as
acetonitrile and DMF, have been used successfully for the
radiofluorination of diaryliodonium salts.38−43 We attempted
the synthesis of [18F]11 by treating 15 with cyclotron-produced
[18F]fluoride ion in either of these solvents and also in the
higher-boiling polar aprotic solvent DMSO. Reactions were
conducted in the presence of cryptand (K 2.2.2), K2CO3, and
also in most cases the free-radical scavenger TEMPO (2,2,2-
tetramethylpiperidine 1-oxyl) under various conditions with
either thermal or microwave heating (Table 1). We expected
that such reactions would produce the N-Boc-protected
compound [18F]16 that would then need to be deprotected
under acidic conditions to give [18F]11. TEMPO was often
included because it has been shown to enhance yields in the
radiofluorination of many diaryliodonium salts, presumably by
hindering their free-radical decompositions.50 We found that
TEMPO improved the incorporation of [18F]fluoride ion from
26 to 42% for reactions of 15 under thermal conditions (100
°C bath temperature, 5 min) in DMF (Table 1, entries 1 and
2). The dominant radioactive product from these reactions was
very lipophilic and was presumed to be the expected
Pharmacological Screen of 11. Ligand 11 has been
reported32 to have a high affinity for human mGluR1, with an
IC50 of 1.8 nM. We found 11 to have low affinity for a wide
range of other mainly human receptors, transporters, and
binding sites, as represented by the following high Ki values:
2096 nM for mGluR5 (rat brain), 2207 nM for 5-HT2A, 2536
nM for DAT, 7900 nM for mGluR5, and >10 000 nM for 5-
HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A‑2C, 5-HT3, 5-HT5A, 5-
HT6, 5-HT7, α1A, α1B, α1D, α2A−α2C, β1−β3, rat BZP site, D1−
D5, DAT, DOR, GABAA, H1, H3, KOR, M1−M5, MOR, NET,
NMDA, SERT, TSPO, σ1, and σ2. Thus, 11 has excellent
mGluR1-selectivity, as required for a prospective PET mGluR1
radioligand. Moreover, 11 was found to be an antagonist at
both mGluR1 and mGluR5 when tested for stimulation of
phosphoinositide hydrolysis49 using cloned rat receptors at a
concentration of 10 μM.
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a
Scheme 2. Radiosynthesis of [18F]11
a
Reagents and conditions: (a) [18F]F−, K2CO3/K 2.2.2, DMF, 100 °C, 5 min; (b) [18F]F−, K2CO3/K 2.2.2, DMSO, microwave (90 W), 2−4 min.
Figure 3. PET time-activity curves in selected brain regions of rhesus monkeys administered [18F]11 at baseline (panel A), after preblock of mGluR1
with 17 (panel B), in an experiment in which 17 (3 mg/kg, i.v.) was administered at 30 min (panel C), and in an experiment in which the selective
mGluR5 ligand 18 (5 mg/kg, i.v.) was administered at 30 min (panel D) .
intermediate [18F]16 (Scheme 2). In the presence of TEMPO,
3% of the initial fluorine-18 was incorporated into [18F]11,
showing that some loss of the N-Boc protecting group had
occurred. A microwave-promoted reaction, conducted over the
temperature range 60−140 °C for 2 min in DMF, gave a very
similar yield of [18F]16 to that from thermal conditions (100
°C for 5 min) but also increased the production of the desired
[18F]11 to 11% (Table 1, entry 3). A microwave-promoted
reaction in MeCN over the temperature range 61−113 °C for
2.5 min gave 30% incorporation of [18F]fluoride ion but solely
into [18F]16 (Table 1, entry 4). When using DMSO under
microwave conditions, 49% of the [18F]fluoride ion was
incorporated into organic products, specifically 9% into
[18F]16 and 40% into the desired [18F]11 (Table 1, entry 5).
Thus, DMF and DMSO gave similar incorporations of
[18F]fluoride ion, but in DMSO, the ratio of radioactive
products was reversed in favor of the desired product [18F]11
over the intermediate [18F]16. The effectiveness of the
radiofluorination of 15 in DMSO was unexpected in view of
reports of low yields for the radiofluorination of some other
diaryliodonium salts in DMSO.38,43 An increase of the
microwave reaction time in DMSO to 4 min (two cycles of 2
min) reduced the yields of both [18F]16 and [18F]11 (Table 1,
entry 6). Therefore, we initially selected DMSO as solvent and
microwave heating for 2 min for the production of [18F]11
(Table 1, entry 7). Subsequently, we found that microwave
irradiation for 2.5 min gave appreciably higher incorporations
of [18F]fluoride ion into [18F]11 (38%; Table 1, entry 8), and
these conditions then became standard for the production of
[18F]11 for PET experiments. With these reaction conditions
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followed by purification with single-pass reversed-phase HPLC
(see Figure S3 in the Supporting Information), [18F]11 was
obtained in useful radiochemical yield (20% decay-corrected),
high radiochemical purity (96−100%) free of significant
chemical impurities, and with specific activities of 2.84 2.2
Ci/μmol (n = 7). Formulations of [18F]11 in sterile medium for
injection were radiochemically stable for at least 4 h.
The discovery that addition of acid was unnecessary for
deprotection of [18F]16 to [18F]11 in DMSO under the
preferred microwave conditions greatly simplified the produc-
tion of [18F]11 in our upgraded Synthia51,52 apparatus. We
reasoned that deprotection occurred through pyrolysis and that
this occurred more readily in DMSO than in acetonitrile or
DMF under microwave conditions because the reaction mixture
reached a higher temperature. We confirmed that exposure of
neat solvents in the reactor to the microwave field used in the
radiochemistry (90 W for 2.5 min) increased solvent
temperatures at different rates such that their terminal
temperatures were near their boiling points (189, 152, and 82
°C for DMSO, DMF, and MeCN, respectively) (see Figure S4
in the Supporting Information). We also observed decom-
position of precursor 15 at a temperature (180 °C) close to the
boiling point of DMSO during an attempt to measure its
melting point, consistent with the likely loss of the N-Boc
group with evolution of carbon dioxide.
Figure 4. Brain PET images acquired as summed data from 0−120
min after intravenous injection of rhesus monkey with [18F]11 under
baseline conditions (top row) or under conditions in which the same
monkey was injected with the selective mGluR1 ligand 17 (3 mg/kg,
i.v.) at 5 min before [18F]11 (bottom row). Panels A and D are
transaxial, B and E, coronal, and C and F, sagittal images.
monkey, the mGluR5-selective ligand 3-((2-methyl-4-
thiazolyl)ethynyl)pyridine (MTEP, 18) was given at 30 min
after administration of [18F]11, but this caused no change in
the washout kinetics from any brain region (Figure 3, panel D);
essentially, the data obtained were very similar to that in the
baseline experiment in a different monkey. This experiment
confirmed the specificity of [18F]11 for binding to mGluR1
versus mGluR5 in monkey brain, as expected from the in vitro
binding data determined for cloned human receptors.32 No
significant radioactivity uptake occurred in the skull in any of
the experiments, and radiodefluorination was therefore
negligible (Figure 4).
In Vitro Stability in Whole Blood and Plasma and
Plasma-Free Fraction. [18F]11 was stable in vitro at room
temperature for at least 30 min in monkey whole blood (98.1%
unchanged) and plasma (98.9% unchanged). The plasma-free
fraction (fp) of [18F]11 for rhesus monkey was 0.0123
0.0001 (n = 3). The fp value for human (0.0114 0.0003, n =
3) was very similar and therefore is not expected to adversely
affect the effective binding potential that might be obtained in
PET imaging of human mGluR1 with this radioligand. These fp
values are as would be expected for a compound with this
measured LogD value.33
Lipophilicities of 7 and 11. The cLogD values of 7 and 11
were calculated to be 2.77 and 2.25, respectively. The LogD of
[18F]11 was measured to be 2.52
0.04 (n = 6), which is
within the desired range (2.0−3.0) for favorable radioligand
properties, such as the ability to pass the blood-brain barrier
without consequent high nonspecific binding and a lack of
generation of lipophilic radiometabolites in the periphery or
brain.13−16
PET Experiments in Monkey. Injection of [18F]11 into a
rhesus monkey at baseline resulted in high brain radioactivity
uptake. Regional peak uptakes were consistent with known
mGluR1 density; maximal uptakes were highest in receptor-rich
cerebellum (5.3 SUV at 12 min), moderate in thalamus (2.9
SUV), frontal cortex (2.7 SUV), hippocampus (2.5 SUV),
putamen (2.5 SUV), and anterior cingulate (2.4 SUV) and were
lower in the rest of the brain (Figure 3, panel A). Peak
radioactivity uptakes in all regions were followed by a steady
decrease in radioactivity level, as exemplified by receptor-rich
cerebellum that decreased by 37% to 3.32 SUV at 90 min after
injection and by 47% to 2.8 SUV at the end of the 2 h scan.
This decrease is approximately twice that reported previously
for [18F]7,26 which was 17% at 90 min. In another experiment
in the same monkey in which the selective mGluR1 antagonist
JNJ-1625968553 (17 3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-
yl)-(cis-4-methoxycyclohexyl)-methanone; 3 mg/kg) was ad-
ministered intravenously at 5 min before the injection of
[18F]11, all regional time-activity curves were almost
indistinguishable, with each showing a fast decline from a
quick peak radioactivity level (Figure 3, panel B). The
distribution of radioactivity across brain became low and
uniform (Figure 4). This experiment indicated that a high level
of brain radioactivity in the baseline experiment was due to
specific binding to mGluR1 receptors. In an experiment in a
second monkey, 17 (3 mg/kg) was administered intravenously
at 30 min after administration of [18F]11, and this caused a
rapid decrease in radioactivity at all mGluR1-containing
regions, thereby confirming the reversibility of radioligand
specific binding (Figure 3, panel C). In an experiment in a third
Emergence of Radiometabolites in Plasma. After
intravenous injection of [18F]11 into monkey, radioactivity
decreased rapidly in whole blood (Figure 5). Radioactivity
concentrations in venous plasma were higher than in whole
blood throughout the scanning period. in accord with high
binding to plasma proteins. HPLC analysis of venous plasma
Figure 5. Time courses for total radioactivity in whole blood and
venous plasma and of [18F]11 in venous plasma.
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showed that [18F]11 represented 43 and 31% of total
radioactivity at 12 and 31 min, respectively. Thereafter, until
the end of the 2 h scan there was little further change in the
percentage of radioactivity in plasma represented by [18F]11. At
least five radiometabolites ([18F]A−[18F]E) were detected in
plasma at 60 min after radioligand injection, and these were all
less lipophilic than [18F]11, as judged by their shorter retention
times in reversed-phase HPLC (Figure 6). [18F]A−[18F]C were
HRMS data were acquired at the Mass Spectrometry Laboratory,
University of Illinois at Urbana−Champaign (Urbana, IL) under
electron ionization conditions using a double-focusing high-resolution
mass spectrometer (Micromass, Waters, Milford, MA). Synthesized
compounds were analyzed with LC−MS on an LCQ Deca instrument
(Thermo Fisher Scientific Corp., Waltham, MA). Gradient or isocratic
LC was performed with binary solvents (A/B, 150 μL/min) composed
of water/methanol/acetic acid (90:10:0.5 v/v/v) (A) and methanol/
acetic acid (100:0.5 v/v) (B) on a Luna C18 column (3 μm, 50 mm ×
2 mm; Phenomenex, Torrance, CA). For the analysis of compound 14,
no LC column was used. Following electrospray ionization of the
effluent, ions m/z 150−750 were acquired.
Melting points were measured with a Mel-Temp manual apparatus
(Electrothermal, Thermo Fisher Scientific Corp.).
γ-Radioactivity from 18F was measured with a calibrated dose
calibrator (Atomlab 300, Biodex Medical Systems, Shirley, NY) or a γ-
counter (Wizard 3″, 1480 automatic γ-counter; PerkinElmer,
Waltham, MA). Radioactivity measurements were corrected for
physical decay. All radiochemistry was performed in a lead-shielded
hot cell for personnel protection from radiation.
Radioactive products were separated by HPLC on a Luna C18
column (5 μm, 10 × 250 mm; Phenomenex) eluted with 10 mM
HCOONH4/MeOH at the stated compositions and flow rates. Eluates
were monitored for radioactivity with a pin-diode detector (Bioscan
Inc., Washington, DC) and for UV absorbance at 230 nm with a
System Gold 166 detector (Beckman Coulter Inc., Indianapolis, IN).
The purity of compound 11 was assessed to be >98% by reversed-
phase HPLC on a Luna C18 column (10 μm, 4.6 × 250 mm;
Phenomenex) eluted with 70% A (0.1% TFA(aq))/30% B (MeCN) at
2 mL/min. [18F]11 was analyzed in the same manner. Eluates were
monitored for UV absorbance at 230 nm (System Gold 166 detector,
Beckman) and also when applicable for radioactivity (pin-diode
detector, Bioscan). Samples were injected alone and then coinjected
with the reference nonradioactive compound to check for coelution.
RCYs were calculated for labeled products isolated with HPLC.
Grouped quantitative data are expressed as mean SD.
Figure 6. HPLC analysis of monkey plasma at 60 min after
intravenous injection of [18F]11.
not well resolved from each other, but all were resolved from
[18F]11. The distribution of radioactivity in plasma at this time-
point was estimated to be [18F]A, 19%; [18F]B, 23%; [18F]C,
10%; [18F]D, 1%; [18F]E, 21%; and [18F]11, 26%. Radio-
metabolites were not chemically identified, but their lower
lipophilicities relative to [18F]11 are expected to mitigate
against significant brain entry that might contaminate receptor-
specific signal. In this regard, the time-activity curves for
monkey brain regions following intravenous injection of
[18F]11 show continuous decline throughout the scanning
period (Figure 3) and provide no evidence of significant ingress
of radiometabolites.
Chemistry. N-(4-(6-Chloropyrimidin-4-yl)thiazol-2-yl)-4-fluoro-
N-methylbenzamide (9). 4-Fluorobenzoyl chloride (232 mg, 1.47
mmol) was added to a suspension of 4-(6-chloropyrimidin-4-yl)-N-
methylthiazol-2-amine (222 mg, 0.98 mmol) in toluene (5 mL) and
Et3N (594 mg, 5.88 mmol) under Ar. The mixture was stirred at 100
°C overnight, quenched with water, and finally extracted with CH2Cl2.
The organic layer was dried over Na2SO4 and evaporated to remove
the solvent. Silica gel chromatography (CH2Cl2/Et3N, 1000:1 v/v and
then CH2Cl2/EtOAc, 4:1 v/v) of the residue gave 9 as a light-yellow
CONCLUSIONS
■
The highly selective mGluR1 radioligand [18F]11 may be
produced from [18F]fluoride ion and the N-Boc-protected
diaryliodonium salt precursor 15 in useful radiochemical yield.
Intravenously injected [18F]11 gave high radioactivity uptake in
rhesus monkey brain and a large proportion of this radioactivity
was specifically and reversibly bound to mGluR1 receptors.
[18F]11 appears to give much faster washout kinetics than the
previously reported [18F]7. Thus, [18F]11 is an effective
radioligand for imaging monkey brain with PET and therefore
merits evaluation in human subjects.
1
solid (320 mg, 94%). mp 212−213 °C. H NMR (CDCl3): δ 3.77
(3H, s), 7.20−7.24 (2H, m), 7.62−7.65 (2H, m), 8.04 (1H, d, J = 0.8
Hz), 8.12 (1H, s), 8.94 (1H, d, J = 1.2 Hz). 13C NMR (CDCl3): δ
38.61, 115.91, 116.13, 117.37, 118.82, 130.04, 130.07, 130.17, 130.26,
146.14, 158.71, 160.42, 160.82, 162.25, 163.01, 165.53, 169.66. 19F
NMR ((CD3)2SO): δ −107.25. LC−MS: m/z [M + H]+ 349.1.
HRMS calcd for C15H11N4OFSCl (M+ + H), 349.0326; found,
349.0328.
EXPERIMENTAL SECTION
4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-
2-yl)benzamide (11). A mixture of 9 (400 mg, 1.2 mmol), K2CO3
(330 mg, 2.4 mmol), and MeNH2 solution in ethanol (33%; 2.51 mL)
in 1,4-dioxane (7 mL) was stirred at 80 °C for 1.5 h until TLC showed
all starting material had reacted. The mixture was cooled to room
temperature, quenched with water, and extracted four times with
EtOAc. The organic layer was dried over MgSO4, and the solvent was
removed under reduced pressure. The residue was redissolved in
MeOH, precipitated with EtOAc followed by hexane, and then filtered
to provide an almost white solid, which upon silica gel chromatog-
raphy (CH2Cl2/MeOH, 20:1 v/v) gave 11 as a white solid (98 mg,
29%). mp 187−189 °C. 1H NMR ((CD3)2SO): δ 2.85 (3H, d, J = 4.4
Hz), 3.64 (3H, s), 7.12 (1H, bs), 7.37−7.42 (2H, m), 7.54 (1H, bs),
7.77−7.81 (2H, m), 7.99 (1H, s), 8.45 (1H, s). 13C NMR
((CD3)2SO): δ 27.13, 38.12, 115.48, 115.70, 130.39, 130.48, 130.78,
130.81, 147.43, 158.33, 159.84, 162.02, 163.45, 164.49, 169.11. 19F
■
Materials. 4-(6-Chloropyrimidin-4-yl)-N-methylthiazol-2-amine
was purchased from Aquila Pharmatech LLC (Waterville, OH). The
blocking agents 17 and 18 were purchased from Tocris Bioscience
(Bristol, UK) and EMD Millipore (Billerica, MA), respectively. Other
chemicals were purchased from Sigma-Aldrich Chemical Co.
(Milwaukee, WI) and used as received.
General Methods. 1H (400.13 MHz), 13C (100.62 MHz), and 19
F
(376.46 MHz) NMR spectra were recorded at room temperature on
an Avance-400 spectrometer (Bruker, Billerica, MA). Chemical shifts
are reported in δ units (ppm) downfield relative to the chemical shift
for TMS. Abbreviations s, d, t, and bs denote singlet, doublet, triplet,
and broad singlet, respectively. TLC was performed on silica gel layers
(0.2 mm with fluorescent indicator; Polygram SIL G/UV254; Grace
Davison Discovery Sciences; Deerfield, IL); compounds were
visualized under UV light (λ = 254 nm).
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NMR ((CD3)2SO): δ −109.04. LC−MS: m/z [M + H]+ 344.2.
HRMS calcd for C16H15N5OFS (M+ + H), 344.0981; found, 344.0979.
4-Bromo-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-
2-yl)benzamide (12). A mixture of 1029 (40 mg, 98 μmol), K2CO3
(27 mg, 197 μmol), and MeNH2 solution in ethanol (33%; 0.35 mL)
in 1,4-dioxane (3 mL) was stirred at 80 °C for 5 h. The mixture was
then cooled to room temperature, quenched with water, and extracted
three times with EtOAc. The organic layers were dried over MgSO4,
and solvent was removed under reduced pressure. Silica gel
chromatography (CH2Cl2/MeOH, 15:1 v/v) of the residue gave 12
as a white solid (40 mg, 100%). mp 218−220 °C. 1H NMR
((CD3)2SO): δ 2.85 (3H, d, J = 4.8 Hz), 3.62 (3H, s), 7.12 (1H, bs),
7.54 (1H, bs), 7.64−7.67 (1H, m), 7.75−7.78 (2H, m), 7.99 (1H, s),
8.45 (1H, s). 13C NMR ((CD3)2SO): δ 38.00, 115.62, 124.38, 129.73,
131.56, 133.53, 147.41, 159.71, 169.10. LC−MS: m/z [M + H]+ 404.2.
HRMS calcd for C16H15N5OSBr (M+ + H), 404.0181; found,
404.0185.
tert-Butyl-6-(2-(4-bromo-N-methylbenzamido)thiazol-4-yl)-
pyrimidin-4-yl(methyl)carbamate (13). A mixture of 12 (500 mg,
1.98 mmol), DMAP (890 mg, 9.9 mmol), and (Boc)2O (1.0 g, 6
mmol) in THF (30 mL) was stirred at 70 °C for 1 h until TLC
showed all starting material had reacted. The solvent was removed
under reduced pressure to give crude product that was then diluted
with water and extracted with EtOAc. The organic layer was dried with
MgSO4 and evaporated to dryness. Silica gel chromatography
(hexane/EtOAc, 4:1 v/v) of the residue gave 13 as a white solid
(600 mg, 96%). mp 176−178 °C. 1H NMR (CDCl3): δ 1.59 (9H, s),
3.49 (3H, s), 3.75 (3H, s), 7.46−7.49 (2H, m), 7.64−7.67 (2H, m),
8.01 (1H, s), 8.55 (1H, d, J = 1.2 Hz), 8.90 (1H, d, J = 1.2 Hz). 13C
NMR (CDCl3): δ 28.28, 33.31, 38.51, 82.56, 109.25, 116.82, 125.70,
129.28, 132.01, 133.10, 147.94, 153.61, 157.60, 158.71, 160.32, 161.89,
169.51. LC−MS: m/z [M + H]+ 503.9. HRMS calcd for
C21H23N5O3SBr (M+ + H), 504.0705; found, 504.0707.
Institute of Mental Health (NIMH) Psychoactive Drug Screening
Program (PDSP). The efficacy of 11 at mGluR1 and mGluR5 was also
determined at 10 μM concentration. Detailed protocols can be found
on the PDSP Web site at http://pdsp.med.unc.edu.
Radiochemistry. Radionuclide Production. No-carrier-added
(NCA) [18F]fluoride ion was produced with the 18O(p,n)18F reaction
by irradiating 18O-enriched water (95 atom %; 1.8 mL) with a beam of
protons (14.1 MeV; 20−25 μA) from a PETtrace cyclotron (GE
Medical Systems, Milwaukee, WI).
Preparation of [18F]Fluoride Ion Reagent. [18F]Fluoride ion
reagent was prepared for reactions as follows. A glass V-vial (5 or 1
mL) equipped with a screw-on cap and liner (20/400 PTFE/silicone;
Alltech Associates, Deerfield, IL) was loaded with an aliquot of a
previously prepared solution (50 μL) of K2CO3 (10 mg) and K 2.2.2
(60 mg) in MeCN/H2O (9:1, v/v; 2 mL). NCA [18F]fluoride ion
(10−114 mCi) in [18O]H2O (15−600 μL) and then anhydrous
MeCN (700 μL) were added to the vial. The solvent was evaporated
off at 110 °C under a nitrogen stream (200 mL/min) vented at a
reduced pressure. This azeotropic drying process was repeated three
times.
Experimental Radiochemistry. Reactions were conducted by
adding a solution of precursor 15 (1.5−1.9 mg) in MeCN, DMF, or
DMSO (0.5 mL) with or without TEMPO (0.6−2.2 mg) to the
[18F]fluoride ion reagent, which was then heated thermally (100 °C)
for 5 min or with microwaves (90 W; 60−150 °C) for a specified time
between 2 and 4 min. At the end of the reaction, the mixture was
cooled and analyzed by HPLC. This was performed either on a
Gemini-NX C18 column (5 μm, 4.6 × 250 mm; Phenomenex) eluted
at 2 mL/min with a mixture of 50 mM NH4OAc (A) and MeOH (B),
with B starting at 10%, increased to 80% over 6 min, kept at 80% for 6
min, increased to 100% over 1 min, and finally kept at 100% for 6 min,
or on a Luna C18 column (5 μm, 10 × 250 mm; Phenomenex) eluted
at 5 mL/min with a mixture of 10 mM HCOONH4 (C) and MeOH
(B), with B started at 10%, increased to 60% over 20 min, and finally
held at 60% for 25 min. Incorporation radiochemical yields were
calculated from the HPLC radiochromatograms without decay-
correction.
tert-Butyl-methyl(6-(2-(N-methyl-4-(tributylstannyl)benzamido)-
thiazol-4-yl)pyrimidin-4-yl)carbamate (14). Bu6Sn2 (633 mg, 1.09
mmol) and Pd(PPh3)4 (100 mg, 0.086 mmol) were added to a
solution of 13 (500 mg, 0.99 mmol) in 1,4-dioxane (15 mL). The
mixture was degassed for 30 min and heated at 85 °C for 5.5 h. The
solvent dioxane was then removed under reduced pressure. Silica gel
chromatography (hexane/EtOAc, 20:1 and then 6:1 v/v) of the
Production of NCA [18F]11. Semiautomated radiosynthesis was
conducted in an upgraded Synthia radiochemistry apparatus51 that had
also been adapted to use microwave irradiation for the labeling
reaction.52 A solution of precursor 15 (1.4−1.9 mg) and TEMPO
(0.7−2.2 mg) in DMSO (500 μL) was added to a still warm (∼80 °C)
radioactive V-vial containing dried [18F]fluoride ion reagent (135−260
mCi). The uncooled vial was transferred immediately to a microwave
reactor (model 521; Resonance Instruments Inc., Skokie, IL) and
heated (90 W, 2.5 min), giving an average final temperature of 179 °C.
This reaction mixture was then cooled to rt, diluted with water (500
μL), and separated by HPLC on a semipreparative size HPLC column,
as described under Experimental Radiochemistry. Within a TRACER-
1
mixture then gave 14 as a colorless oil (280 mg, 40%). H NMR
(CDCl3): δ 0.90 (9H, t, J = 7.2 Hz), 1.09−1.13 (6H, m), 1.32−1.37
(6H, m), 1.53−1.59 (15H, m), 3.49 (3H, s), 3.78 (3H, s), 7.50−7.52
(2H, m), 7.59−7.61 (2H, m), 8.00 (1H, s), 8.55 (1H, d, J = 1.2 Hz),
8.91 (1H, d, J = 1.2 Hz). 13C NMR (CDCl3): δ 9.70, 13.67, 27.34,
28.28, 29.04, 33.29, 38.59, 82.54, 109.30, 116.66, 126.63, 133.57,
136.55, 147.13, 147.81, 153.61, 157.59, 158.84, 160.61, 161.89, 170.81.
LC-MS: m/z [M + H]+ 716.0. HRMS calcd for C33H50N5O3SSn (M+
+ H), 716.2656; found, 716.2665.
lab
box (GE, Milwaukee, WI), the fraction containing [18F]11
(4-((4-(6-(tert-Butoxycarbonyl(methyl)amino)pyrimidin-4-yl)-
thiazol-2-yl)(methyl)carbamoyl)phenyl)(phenyl)iodonium tosylate
(15). Koser’s reagent (28 mg, 70 μmol) was added to a solution of
14 (50 mg, 70 μmol) in CH2Cl2 (2 mL). The mixture was stirred
under Ar for 4 days, and then CH2Cl2 was removed under reduced
pressure. The residue was then dissolved in MeOH. Et2O was added,
and the precipitate was filtered to give 15 as a white solid (12 mg,
FX−FN
(tR = 33 min) was collected in a glass vial containing HPLC water (30
mL) and passed into a C18 Sep-pak (Waters Corp.) that had been
activated by flushing first with ethanol (10 mL) and then water (10
mL). [18F]11 was eluted from the Sep-pak with EtOH (1 mL)
followed by saline for injection (2 mL) through a sterile filter (0.22
μm, Millipore MP; Waters Corp.) and into a sterile vial containing
saline for injection (8 mL), with the vial vented through another sterile
filter (0.22 μm; Millipore GV; Waters Corp.). The identity of [18F]11
(tR = 6.8 min) was confirmed by analytical radio-HPLC on a Luna C18
column (10 μm, 4.6 × 250 mm, Phenomenex) eluted at 2 mL/min
with a mixture (3:7, v/v) of MeCN and TFA(aq) (0.1% v/v) as well as
by LC−MS of associated carrier (m/z = 344 [M + 1]+). The
radiochemical purity of [18F]11 was 96−100%. The radiochemical
stability of the formulated dose was tested over 4 h at rt. The average
decay-corrected radiochemical yield (RCY) of [18F]11 was 20 6.2%
(n = 7) from [18F]fluoride ion. The average specific activity of [18F]11
was 2.84 2.20 Ci/μmol (n = 7) at the end of the synthesis.
1
21%). mp decomposed above 180 °C. H NMR ((CD3)2SO): δ 1.54
(9H, s), 2.28 (3H, s), 3.39 (3H, s), 3.57 (3H, s), 7.10−7.12 (2H, d, J =
8 Hz), 7.46−7.48 (2H, d, J = 8 Hz), 7.56−7.59 (2H, t, J = 7.6 Hz),
7.69−7.73 (1H, t, J = 7.6 Hz), 7.83−7.85 (2H, d, J = 8.4 Hz), 8.22
(1H, s), 8.31−8.33 (2H, d, J = 7.6 Hz), 8.40−8.42 (2H, d, J = 8.4 Hz),
8.48 (1H, s), 8.93 (1H, s). 13C NMR ((CD3)2SO): δ 20.75, 27.75,
33.01, 37.96, 66.32, 82.45, 108.07, 116.60, 117.64, 118.45, 125.46,
128.00, 130.48, 131.86, 132.24, 135.26, 135.36, 137.52, 140.81, 145.76,
146.71, 152.87, 157.67, 157.98, 159.90, 161.26, 168.56. LC−MS: m/z
[M + H]+ 628.0. HRMS calcd for C27H27N5O3SI (M+ + H), 628.0879;
found, 628.0880.
Comprehensive Screening of 11. Ligand 11 was screened for
binding selectivity for mGluR1 versus a wide range of other mainly
human brain binding sites, transporters, and receptors by the National
Calculation and Measurement of LogD. cLogD (at pH 7.4)
values for 7 and 11 were computed with Pallas 3.7 software
(CompuDrug, Bal Harbor, FL). The value for the distribution
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coefficient of [18F]11 between n-octanol and sodium phosphate buffer
(0.15 M, pH 7.4) was determined as described previously.33
AUTHOR INFORMATION
Corresponding Author
*Phone + 301 594 5986. Fax +301 480 5112. E-mail: pikev@
mail.nih.gov.
■
Monkey PET Experiments. Monkey experiments were performed
in compliance with the animal care requirements of our institution.
Three rhesus monkeys (Macaca mulatta, 13.3, 7.0, and 8.1 kg) were
used to acquire four PET scans. In a baseline experiment, [18F]11 was
injected intravenously into the monkey (13.3 kg) as a bolus. In a
receptor preblock experiment in the same monkey on a separate day,
the selective mGluR1 ligand 17 (3 mg/kg) was administered
intravenously 5 min before [18F]11. A challenge experiment was
performed in a second monkey in which 17 (3 mg/kg) was
administered intravenously at 30 min after [18F]11 injection. In an
experiment in a third monkey (8.1 kg), the selective mGluR5 ligand 18
(5 mg/kg) was administered intravenously at 30 min after injection of
[18F]11. The injected activities were 4.6−6.58 mCi, and the specific
activities at the time of injection were 1.67−4.18 Ci/μmol.
PET Scans. PET images were acquired with a microPET Focus 220
scanner (Siemens Medical Solution, Knoxville, TN) for 120 min in 33
frames, with scan durations ranging from 30 s to 5 min. Anesthesia was
initiated with ketamine (10 mg/kg, i.m.) and then maintained with
1.6% isoflurane and 98.4% O2. The position of the head was fixed
using a stereotaxic frame. Electrocardiogram, body temperature, heart,
and respiration rates were measured throughout the experiment. All
PET images were corrected for attenuation and scatter.
Image Analysis. Images were reconstructed using Fourier rebinning
plus two-dimensional filtered back-projection. PET images were
coregistered to a standardized monkey MRI template using SPM5
(Wellcome Trust Centre, London, UK). A set of 34 predefined brain
regions of interest from the template were then applied to the
coregistered PET image to obtain regional decay-corrected time−
activity curves. Cerebellar gray matter, which is not included in the
template, was delineated semiautomatically using isocontour regions of
interest. Uptake of radioactivity in each region of interest was
expressed in units of standardized uptake value, where SUV = [(%
injected activity/cm3) × body weight (g)]/100.
Analysis of Radiometabolites in Plasma. Venous blood
samples were collected at 10, 30, 60, and 120 min after intravenous
injection of [18F]11 (5.24 mCi; 1.4 nmol/kg) into one monkey (7.0
kg). Plasma was separated and analyzed with radio-HPLC on a
Novapak C18 column (4 μm, 100 × 8 mm; Waters Corp.) housed in a
radial compression module (RCM-100) and eluted at 2.0 mL/min
with MeOH/H2O/Et3N (65:35:0.1 v/v/v), as described previously.54
Radioactivity levels in different blood components were expressed as
SUV.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is support by the Intramural Research Program of
NIMH. We thank the NIH Clinical PET Center (Director Dr.
P. Herscovitch) for radioisotope production and the PDSP for
performing binding assays. The PDSP is directed by Bryan L.
Roth, Ph.D., with project officer Jamie Driscol (NIMH), at the
University of North Carolina at Chapel Hill (contract no.
NO1MH32004).
ABBREVIATIONS USED
■
BZP, benzodiazepine; Boc, tert-butyloxycarbonyl; H, histamine;
K 2.2.2, kryptofix 2.2.2; KOR, κ-opiate receptor; MOR, μ-
opiate receptor; PDSP, Psychoactive Drug Screening Program;
PTFE, poly(tetrafluoroethylene); NCA, no-carrier-added;
NET, noradrenalin transporter; NIMH, National Institute of
Mental Health; RCY, decay-corrected radiochemical yield;
SUV, standardized uptake value; TEMPO, 2,2,6,6-tetramethyl-
piperidine 1-oxyl; TSPO, translocator protein 18 kDa
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