Single-step high-yield radiosynthesis and evaluation of a sensitive 18F-labeled ligand for imaging brain peripheral benzodiazepine receptors with PET

Article abstract of DOI:10.1021/jm8011855

Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory conditions. Sensitive, easily prepared, and

Full text of DOI:10.1021/jm8011855

688
J. Med. Chem. 2009, 52, 688–699
18
Single-Step High-Yield Radiosynthesis and Evaluation of a Sensitive F-Labeled Ligand for
Imaging Brain Peripheral Benzodiazepine Receptors with PET
Emmanuelle Briard, Sami S. Zoghbi, Fabrice G. Sime´on, Masao Imaizumi, Jonathan P. Gourley, H. Umesha Shetty, Shuiyu Lu,
Masahiro Fujita, 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
ReceiVed September 19, 2008
Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in
their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory
conditions. Sensitive, easily prepared, and readily available radioligands for imaging with positron emission
tomography (PET) are desirable for this purpose. We describe a new 18F-labeled PBR radioligand, namely
[18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]9). [18F]9 was produced easily through
a single and highly efficient step, the reaction of [18F]fluoride ion with the corresponding bromo precursor,
8. Ligand 9 exhibited high affinity for PBR in vitro. PET showed that [18F]9 was avidly taken into monkey
brain and gave a high ratio of PBR-specific to nonspecific binding. [18F]9 was devoid of defluorination in
rat and monkey and gave predominantly polar radiometabolite(s). In rat, a low level radiometabolite of
intermediate lipophilicity was identified as [18F]2-fluoro-N-(2-phenoxyphenyl)acetamide ([18F]11). [18F]9 is
a promising radioligand for future imaging of PBR in living human brain.
Chart 1. Ligands and PET Radioligands Based on the
Aryloxyanilide 1a
Introduction
Peripheral benzodiazepine receptors (PBRa) are located on
heteropolymeric 18 kDa proteins1 and their complexes and have
a uniquely strong avidity for certain benzodiazepines (e.g., 4′-
chlorodiazepam).2 PBR were initially discovered as diazepam
binding sites in kidney3 and are now known to be widespread4
in peripheral organs including not only kidney but also lung,
heart, and endocrine glands. Relatively low levels of PBR are
also found in normal brain.5 The structure and functions of PBR
are wholly distinct from those of the well-known brain benzo-
diazepine receptors that are acted upon by endogenous GABA
or by well-known therapeutic benzodiazepines such as diazepam.
Consequently, it has recently been suggested to rename PBR
as the “translocator protein (18 kDa)” or TP-18.6 PBR are
primarily located at outer mitochondrial membranes1 within
transmembrane channels7 that have many putative functions,
including cholesterol transport,8 porphyrin transport,9 immu-
nomodulation,10 cell proliferation,11 and apoptosis.12 PBR are
present in activated microglia13 and to a lesser extent in
astrocytes,14 and hence PBR concentrations are found to be
elevated in regions of brain inflammation arising from neuro-
degenerative disorders (e.g., multiple sclerosis, Alzheimer’s
disease) or brain trauma (e.g., stroke).15-17 Imaging of PBR in
vivo is therefore recognized as a means to detect and investigate
neuroinflammation.18 The radioligand [11C]PK 1119519 ([N-
methyl-11C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-
isoquinoline carboxamide), preferably as its R-enantiomer,20 has
long been used with positron emission tomography (PET) for
a An asterisk indicates a site in which the ligand has been labeled with
either carbon-11 or fluorine-18.
such investigations21 but is widely recognized to lack both
sensitivity and amenability to quantitation of in vivo binding
parameters.22
Potent ligands for PBR are known from many different
structural classes23 and many have been explored as potential
PET radioligands.22 Aryloxyanilides, related to the high-affinity
and selective PBR ligand, 1 (DAA1106),24 have become an
especially abundant source of candidate radioligands (Chart 1).
PBR radioligands in this class, which have a carbon-11 label
and that are more sensitive and effective than [11C]PK 11195,
are now known, including [11C]1.25 One of the most promising
is [11C]3 ([11C]PBR28).26-29 Nevertheless, the use of such
radioligands is necessarily restricted to imaging facilities either
at or very close to their site of production because of the short
half-life of carbon-11 (t1/2 ) 20.4 min; ꢀ+ ) 99.8%).
Alternative labeling with fluorine-18 (t1/2 ) 109.7 min; ꢀ+
) 97%) opens up several advantages over labeling with carbon-
11. First, high (multi-Ci) activities of fluorine-18, as [18F]fluoride
ion, can be produced from moderate energy cyclotrons by the
18O(p,n)18F reaction on [18O]water.30 Second, the longer half-
life allows transport over considerable distances to remote PET
* To whom correspondence should be addressed. Phone: 301 594 5986.
Fax: 301 480 5112. E-mail: pikev@mail.nih.gov.
a Abbreviations: CA, carrier-added; D, dopaminergic; DAT, dopamine
transporter; GABA, γ-aminobutyric acid; H, histaminergic; HPLC, high-
performance liquid chromatography;
K 2.2.2, 4,7,13,18-tetraoxa-1,10-
diazabicyclo[8.8.8]hexacosane; NCA, no-carrier-added; PET, positron emission
tomography; PBR, peripheral benzodiazepine receptors; RCY, decay-corrected
radiochemical yield; MR, magnetic resonance; NET, noradrenaline transporter;
rt, room temperature; SERT, serotonin transporter; SR, specific radioactivity;
SUV, standardized uptake value; TAC, time-activity curve; VOI, volume of
interest; 5-HT, serotonin.
10.1021/jm8011855 This article not subject to U.S. Copyright. Published 2009 by the American Chemical Society
Published on Web 01/02/2009

A SensitiVe 18F-Labeled Ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 689
Scheme 1. Synthesis of 9 and [18F]9a
a Reaction conditions and yields. (a) 2-phenoxyaniline, MeOH, rt, 24 h; (b) NaBH4, rt, 1 h; 75% for steps a plus b overall; (c) bromoacetyl bromide,
CH2Cl2, Et3N, 0 °C, 1 h; 73%; (d) KF, di(ethylene glycol), 150 °C, 4 h; 95%; (e) [18F]fluoride ion, MeCN-trace water, 18-crown-6, KHCO3, 110 °C, 10 min;
RCY 97%, or [18F]fluoride ion; MeCN, K 2.2.2, K2CO3, 100 °C, 20 min; isolated and formulated RCY, 12.2%.
a
Scheme 2. Synthesis of 11 and [18F]11
centers and also allows longer and more accurate imaging
sessions. Finally, fluorine-18 emits a low-energy positron during
decay, and this permits the high spatial resolution of modern
PET cameras (∼2 mm) to be fully utilized. So far, only two
18F-labeled ligands, [18F]4 ([18F]FMDAA1106) and [18F]5
([18F]FEDAA1106) (Chart 1), have been developed from the
aryloxyanilide class31,32 and evaluated with PET imaging in
animal or human subjects.33 However, each of their preferred
methods of radiosynthesis requires two steps and is overall
inefficient. The single-step labeling of 1 with fluorine-18 in high
radiochemical yield has recently been described.34 No evaluation
of this radioligand in vivo has yet been reported, although the
11C-labeled version has appeared promising in mouse25 and
monkey.35 Recently, another 18F-labeled aryloxyanilide radio-
ligand for PBR, namely [18F]6 ([18F]-FEPPA), was labeled in a
single-step, but its preliminary evaluation in rats was incomplete
a Reaction conditions and yields: (a) bromoacetic anhydride, AcOH, Et3N,
with regard to characterizing its behavior and therefore its
rt, 2 h; 80%; (b) KF, ethylene glycol, 150 °C, 2 h; 65%; (c) [18F]fluoride
potential for PBR imaging in higher species.36 Only a few other
ion, MeCN, K 2.2.2, K2CO3; 160 °C, RCY 26-30%.
18F-labeled PBR ligands are known. One is a close analogue of
[11C]PK 11195, namely [18F]1-(2-fluoro-5-nitrophenyl)-N-meth-
Table 1. Affinities (Ki values) of Ligands for PBR in Brain
yl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([18F]PK
14105),37 which was shown to be a marker of brain ischemic
lesions in rat,38 but which has not been progressed to human
evaluation. Another is a recently reported39 high-affinity [18F]flu-
oropropyl analogue of iodo-PK 11195, whose imaging properties
have not yet been described. Recently, candidate 18F-labeled
ligands from other structural classes (imidazo[1,2-a]pyridines
and pyrazolo[1,5-a]pyrimidines) have been synthesized and
evaluated in rats. However, even the most promising of these
radioligands suffers from extensive metabolism and defluori-
nation.40 Therefore, there is still a clear need for an easily
prepared and effective 18F-labeled ligand for brain PBR imaging.
Here we report the radiosynthesis of a new 18F-labeled PBR
ligand, [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxy-
aniline ([18F]9; Scheme 1) in a single efficient radiosynthetic
step that was easily incorporated into automated radiosynthesis
devices. Our evaluation of [18F]9 in monkey and rat showed
that this radioligand is sensitive for imaging PBR with PET
and has high promise for imaging of PBR in living human brain.
Homogenates from Rat, Monkey, and Human (Determined with [3H]PK
11195 as Radioligand)
PBR affinity (Ki, nM)a
ligand
rat
monkey
human
1
9
0.0726 ( 0.0036
0.180 ( 0.007
0.230 ( 0.011
0.318 ( 0.018
0.242 ( 0.016
0.997 ( 0.070
a Values are mean ( SD (n ) 6).
aniline (7) (Scheme 1). Treatment of 7 with bromoacetyl
bromide in the presence of triethylamine gave the bromo
precursor (8), required for the radiosynthesis of [18F]9. Reference
ligand 9 was prepared by treating 8 with dry potassium fluoride
in di(ethylene glycol) at 150 °C. Similar bromoacetylation of
2-phenoxyaniline followed by fluorination gave the metabolite
11 (Scheme 2).
Binding Assays. The affinities of ligand 9 for PBR in rat,
monkey, and human mitochondrial fractions were high and
similar to those of 1 (Table 1). Ligands 1 and 9 showed a small
interspecies variability in affinity for PBR, with that for human
tissue being somewhat lower than for monkey or rat.
Experimental Radiosynthesis of [18F]9 and [18F]11. Ex-
perimentally, [18F]9 was obtained in almost quantitative (97%)
Results
Chemistry. Condensation of 2-phenoxyaniline with 2,5-
dimethoxybenzaldehyde in methanol at room temperature fol-
lowed by reduction gave N-(2,5-dimethoxybenzyl)-2-phenoxy-

690 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Briard et al.
Distribution of [18F]9 in Monkey Blood and Plasma
Free Fraction. In monkey blood, [18F]9 distributed 93.4 ( 0.3%
to cells and 6.6 ( 0.4% to plasma (n ) 4). The plasma free
fraction (fP) of [18F]9 in two monkeys was 3.5 ( 0.2% (n ) 9)
and 1.34 ( 0.1% (n ) 3) and in human pooled plasma was
0.70 ( 0.03% (n ) 3).
PET Imaging of [18F]9 in Monkeys. After injection of NCA
(no-carrier-added) [18F]9 into monkeys, radioactivity entered the
brain well with peak uptake occurring in all PBR-containing regions
between 27 and 72 min after injection (Figure 2A). Maximal uptake
occurred in choroid plexus of fourth ventricle (350 ( 116% SUV;
n ) 3). Subsequent washout of radioactivity from all PBR-
containing regions was quite slow. In a repeat experiment in one
monkey, in which PBR receptors were preblocked by administra-
tion of 1, the kinetics of brain radioactivity was strikingly different.
Radioactivity was rapidly taken into all examined brain regions,
but to a much higher level than in the baseline experiment, and
then washed out rapidly to a low common level (Figure 2B). In a
displacement experiment in another monkey, administration of 1
at 60 min after radioligand caused a rapid decline in radioactivity
in all PBR-containing regions to a low common level (Figure 2C).
Average PET images of monkey brain acquired over a late
period (60-120 min) after injection of [18F]9 (Figure 3, upper
row) were coregistered with MR images (Figure 3, lower row)
and displayed a high level of radioactivity, especially in the
choroid plexus of fourth ventricle, cerebellum and striatum, and
intermediate levels in frontal, parietal, temporal, and occipital
cortex. Corresponding scans from the preblock experiment
showed a low uniform distribution of radioactivity (Figure 3).
No radioactivity accumulated in bone (skull).
Emergence of Radiometabolites of [18F]9 in Monkey
Plasma In Vivo. After administration of [18F]9 into monkey,
radioactivity (and hence radioligand) cleared rapidly from
plasma (Figure 4). HPLC analyses of plasma from four monkeys
revealed [18F]9 eluting at 6.64 ( 0.25 min, a major radiome-
tabolite at 2.47 ( 0.18 min, and a minor radiometabolite at 4.38
( 0.90 min (n ) 64). The latter was never higher than 4% of
the total plasma radioactivity except in one monkey, where it
reached 8% in one plasma sample at 60 min after injection.
The 64 observations were obtained from three monkeys on
which four studies were performed. Recoveries of radioactivity
from plasma into supernatant acetonitrile for HPLC analysis
averaged 93.4 ( 2.3% (n ) 64). Monkey plasma became
composed equally of radiometabolite and [18F]9 at 31.1 ( 14.7
min (n ) 3) after radioligand injection in baseline experiments
and at the much shorter time of 4.0 min in the preblock
experiment (Figure 5). The concentration of unchanged [18F]9
in plasma (%SUV) was vastly higher in the preblock than in
the baseline experiment, especially over the initial 10 min; in
fact, peak values were nearly 18-fold higher in the preblock
experiment (Figure 6).
Figure 1. Temperature dependence of decay-corrected radiochemical
yields of [18F]9 prepared in anhydrous MeCN (b) or MeCN containing
0.3% v/v H2O (O), and of [18F]11 prepared in anhydrous MeCN within
a microfluidic device using two 2 m-coiled microreactors (9).
decay-corrected radiochemical yield (RCY) from the reaction
of cyclotron-produced [18F]fluoride ion with 8 in pyrex glass
vials.
In the microfluidic apparatus, [18F]9 was produced in higher
RCY in anhydrous acetonitrile than in slightly wet acetonitrile
at temperatures below 90 °C (Figure 1). However, this difference
disappeared when the temperature of the reactor was raised
above 110 °C. RCY reached 85% at 110 °C; further temperature
increase gave no improvement. At a set temperature, excess 8
(i.e., [18F]F- to 8 solution volume ratio ) 1:2) gave much higher
radiochemical yield than excess [18F]F--K+-K 2.2.2 (i.e.,
[18F]F- to 8 solution volume ratio ) 2:1). For example, at 90
°C in anhydrous acetonitrile, RCYs were 77 versus 44%, while
in acetonitrile containing trace water (0.3% v/v), they were 79
versus 29%.
The RCY of [18F]11 in the microfluidic apparatus was
substantially lower than for [18F]9 over the temperature range
30-150 °C under comparable conditions (Figure 1).
Microfluidic Production of [18F]9 for Intravenous
Injection into Rat. RCYs of radiochemically pure [18F]9 ranged
from 69 to 76% for reactions conducted at 110 °C.
Microfluidic Production of [18F]11 for Intravenous
Injection into Rat. RCYs of radiochemically pure [18F]11
ranged from 26 to 30% for reactions conducted at 160 °C.
Automated Production of [18F]9 for Intravenous
Injection into Monkey. [18F]9 was produced automatically
within a lead-shielded hot-cell from high levels of cyclotron-
produced [18F]fluoride ion (∼300 mCi) in either a commercial
apparatus (TRACERlab FXF-N) or a Synthia device41 equipped
with a microwave heater.42 Purification of [18F]9 was achieved
by single-pass HPLC. From the Synthia apparatus, [18F]9 was
obtained in high radiochemical purity (100%) and chemical
purity. Specific radioactivity ranged from 3.4 to 9.0 Ci/µmol at
the end of synthesis at about 180 min from the end of
radionuclide production. The average isolated RCY of [18F]9
was 12.2% (n ) 6).
Measurement of LogD and Computation of cLogD and
cLogP. The measured LogD value and computed cLogP and
cLogD values of 9 were 4.05 ( 0.02 (n ) 6), 4.37 and 4.38,
respectively.
Stability of [18F]9 in Monkey Whole Blood and Plasma,
and in Buffer. After incubation with monkey whole blood and
plasma for at least 45 min at room temperature, [18F]9 was 95.5
( 3.2% (n ) 5) and 97.2 ( 1.0% (n ) 6) unchanged,
respectively. [18F]9 was stable in sodium phosphate buffer (0.15
M, pH 7.4) for longer than 2 h at room temperature.
Stability of [18F]9 to Rat Brain In Vitro. [18F]9 was found
to be >99.2% unchanged when incubated with brain homoge-
nate at 37 °C for up to 60 min. Recoveries of radioactivity from
homogenates for analyses were >82%.
Emergence of Radiometabolites of [18F]9 in Rat
Plasma, Brain, and Urine In Vivo. At 30 min after administra-
tion of NCA [18F]9 (1.36 Ci/µmol) to rat (mass dose of 9, 0.85
µg/kg), the recoveries of radioactivity from plasma and brain
homogenate samples into acetonitrile for analyses with HPLC
were very high, namely 92.7% (n ) 2) and 85.1% (n ) 4),
respectively. [18F]9 (tR ) 5.3 min in plasma analyses, 6.5 (
1.2 min in brain analyses) represented 12.5 (n ) 2) and 90.4 (
0.2% (n ) 4) of plasma and brain radioactivity, respectively.

A SensitiVe 18F-Labeled Ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 691
(<5% of total radioactivity) eluted ahead of [18F]9 at tR ) 3.5
min. In this experiment, recoveries of radioactivity into aceto-
nitrile analytes were 93.0 ( 0.8% for plasma and 81.8% for
brain.
In the two rats administered with carrier-added (CA) [18F]9,
unchanged [18F]9 represented 32.6 ( 2.5% of brain radioactivity
and 14.2 ( 1.9% of plasma at time of sacrifice (60 min).
Unchanged [18F]9 represented only 0.2 ( 0.1% of radioactivity
in urine. The remainder of radioactivity in brain, plasma, and
urine was dominated by polar radiometabolite(s). The minor
radiometabolite represented <5, <3, and 9.9 ( 2.2% of
radioactivity in brain, plasma, and urine, respectively. In this
experiment, recoveries of radioactivity into acetonitrile analytes
were 93.2 ( 1.0%, 91 ( 0.7%, and 80.3 ( 3.5% for plasma,
brain, and urine, respectively.
LC-MS-MS Search for Metabolites of 9 Emerging in
Rat Brain, Urine and Plasma in Vivo. Reference ligand 9
eluted with tR ) 12.9 min in LC-MS and showed an [M + 1]+
ion at m/z ) 396 and an adduct at m/z ) 441. MS-MS analysis
of this ion gave product ions m/z 258 and 151.
LC-MS-MS analyses of brain and plasma, sampled at 115
min after administration of 3 to rat, detected unchanged 9.
Unchanged 9 was not detected in urine. A knowledge-based
metabolite search of the total ion chromatograms for brain,
plasma, and urine detected an intense peak at 11.3 min for m/z
186 ion, consistent with [M + H]+ for 2-phenoxyaniline, a
possible metabolite of 9. The MS-MS of this ion gave a product
ion spectrum consisting of m/z 169, 168, 158, 108, and 93, which
exactly matched that of reference 2-phenoxyaniline. This
metabolite was also similarly detected and identified in plasma.
The metabolite that would have been generated solely by
N-deacetylation of 9, namely 7, was not detected in brain, urine
or plasma. No metabolites from possible O-demethylation were
detected.
Emergence of Radiometabolites of [18F]11 in Rat Brain
and Plasma In Vitro. Incubation of the synthesized radiome-
tabolite, [18F]11, with rat brain homogenate or rat whole blood
for 30 min resulted in virtually complete conversion (>99.9%)
into polar radiometabolite(s) that eluted at the solvent front on
reverse phase HPLC.
Emergence of Radiometabolites of [18F]11 in Rat Brain
and Plasma Ex Vivo. At 30 min after administration of [18F]11
to rat, ex vivo analysis showed that radioactivity in brain and
plasma was almost entirely (>99%) the polar radiometabolite(s)
that eluted at the solvent front on reverse phase HPLC.
Evaluation of the Rat Brain Penetration of the
Radiometabolite(s) of [18F]11. The radiometabolite(s) of [18F]11
generated in rat blood in vivo were found to have a moderate
presence in brain at 30 min (130% SUV uncorrected, 120%
corrected for blood) after intravenous injection relative to their
concentrations in blood (196% SUV) or plasma (226% SUV)
at the same time.
Figure 2. Rhesus monkey brain time-activity curves after intravenous
injection of [18F]9 (5.80 mCi with 3.91 µg of 9) alone (A), intravenous
injection of 1 (3 mg/kg) in the same monkey at 24 min before [18F]9
(5.09 mCi, with 4.58 µg of 9) (B), and intravenous injection in a
different monkey of [18F]9 (1.28 mCi, with 1.75 µg of 9) followed at
60 min later with 1 (3 mg/kg) (C). Key: fourth ventricle of choroid
plexus (b), striatum (9), thalamus (0), cerebellum (O), frontal cortex
(×), temporal cortex (4), parietal cortex (*), and occipital cortex (]).
Discussion
Our aim was to develop a sensitive and easily prepared 18F-
labeled ligand for PET imaging of brain PBR. High affinity is
a primary requirement for a PET radioligand to exhibit high
sensitivity.43,44 Many high affinity N-acyl aryloxyanilide ligands,
related to 1, are known for PBR.45-47 The reported structure-
affinity data have indicated that the N-acyl function may be
varied slightly without excessive detriment to binding affinity.
We considered that the N-acyl function might be a favorable
site for single-step introduction of fluorine-18 in our develop-
ment of an 18F-labeled PET ligand. For example, we had recently
The remainder of radioactivity was predominantly polar radi-
ometabolite(s), eluting at the void volume (tR ) 2.0 min). In a
second rat, administered with NCA [18F]9 at a somewhat lower
specific radioactivity (mass dose of 9, 3.78 µg/kg), [18F]9
represented 44.6, 37.6, and 5.7% of radioactivity in plasma at
7.3, 10.9, and 94.3 min, respectively and 39.2% of radioactivity
in brain at time of sacrifice (95 min) (n ) 2 for all measures).
The remainder of radioactivity in plasma and brain was mainly
polar radiometabolite(s) (tR ) 2.0 min). A minor radiometabolite

692 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Briard et al.
Figure 3. (A) In left to right order, average coronal, sagittal, and horizontal PET images of monkey brain, acquired between 60 and 120 min after
intravenous injection of NCA [18F]9 (5.8 mCi) alone (upper row) and in the same monkey after preblock of PBR with 1 (3 mg/kg, iv) given 24 min
before NCA [18F]9 (5.09 mCi) (lower row). (B) The same PET data have been superimposed on the corresponding MR images.
Figure 4. Time course of plasma concentration of total radioactivity
(%SUV) (b) and unchanged [18F]9 (O) after intravenous injection of
NCA [18F]9 into rhesus monkey.
Figure 6. Time course of plasma concentration of [18F]9 (%SUV) after
intravenous injection of [18F]9 in the baseline (O) and preblock
experiment in the same monkey (0).
and above those normally considered ideal for achieving good
brain penetration in PET radioligands without excessive non-
specific binding. Nevertheless, these values were not seen as
problematic for the development of a PET radioligand because
[11C]1 was already known to penetrate brain readily, as were
[11C]2 ([11C]PBR01) and [11C]3,26 which are also effective PBR
radioligands based on a core aryloxyanilide structure (Chart 1).
Therefore, we set out to prepare and test 9 as a potential ligand
for PBR.
Figure 5. Time course of compositions of plasma radioactivity after
The synthesis of 9 was simply accomplished in high yield in
three steps from commercially available materials and proceeded
injection of [18F]9 into the same rhesus monkey under baseline and
PBR preblock conditions. (b) unchanged [18F]9 in baseline experiment;
via the corresponding bromo compound (8) required for
radiolabeling (Scheme 1). Ligand 9 was found to be selective
for PBR50 and to have high affinity for PBR across three species,
namely rat, monkey, and human, with values comparable to
those of the prototypical PBR ligand, 1 (Table 1). Only a small
species variation in affinity was observed.
Experimentally, radioligand [18F]9 was easily prepared in a
single step from the bromo precursor 8 and cyclotron-produced
NCA [18F]fluoride ion under conditions (MeCN, KHCO3, 18-
crown-6, trace water, 110 °C, 10 min) previously established
for this type of radiosynthesis48 (Scheme 1). Almost quantitative
incorporation of [18F]fluoride ion was achieved experimentally.
The labeling reaction was investigated further in a commercial
microfluidic device, which provided for well-controlled condi-
tions (reaction stoichiometry, temperature, and transit time) and
multiple reactions over a short time-span. In this apparatus,
radiochemical yields of [18F]9 reached 85% at 110 °C, whether
(O) radiometabolites in baseline experiment; (9) unchanged [18F]9 in
preblock experiment; (0) radiometabolites in preblock experiment.
Arrows indicate the times at which [18F]9 and its radiometabolite were
equally present in these experiments.
shown that an N-fluoroacetyl group can be labeled efficiently
with fluorine-18 by nucleophilic substitution in the correspond-
ing N-bromoacetyl compound with NCA [18F]fluoride ion.48 We
considered that simply switching the position of the fluorine
atom in 1 (Chart 1) from the aryl ring to the N-acetyl group
might provide a high affinity ligand for PBR. Furthermore, this
switch was expected and then computed to have minimal effect
on ligand lipophilicity, which is a key property influencing
blood-brain barrier penetration, level of nonspecific binding,
blood-protein binding, and metabolism.43,44,49 Thus, cLogP and
cLogD values for N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-
phenoxyaniline (9) were computed to be 4.37 and 4.38 and
almost identical to those of 1 (each 4.27). These values are high

A SensitiVe 18F-Labeled Ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 693
reactions were performed in dry or slightly wet acetonitrile
(Figure 1). The tolerance of a low level of water in this reaction
suggests the precursor is highly reactive toward hydrated
[18F]fluoride ion.51
In a second PET experiment in one monkey, a high dose of
1 was given before administration of [18F]9 to presaturate26 brain
PBR. In this experiment, early maximal uptake of radioactivity
across all inspected brain regions was substantially higher
(Figure 2B) than that seen in the baseline experiment in the
same monkey (Figure 2A). Uptake in all regions was followed
by rapid continuous decline of radioactivity to a very low level
(Figure 2B), indicating low and rapidly reducing nonspecific
binding of radioactive species in brain.
Average PET images obtained under baseline conditions and
coregistered with MR images detail the distribution of radio-
ligand throughout monkey brain and feature especially high
uptake in choroid plexus (Figure 3, upper row). Corresponding
scans from the preblock experiment showed a low uniform
distribution of radioactivity (Figure 3, lower row). Neither set
of images showed a substantial uptake of radioactivity into skull
(bone). Hence, [18F]9 does not defluorinate significantly in
monkey. This may be a considerable advantage of [18F]9 over
the previously reported radioligand, [18F]4 (Chart 1), which is
rapidly defluorinated in mouse.32
An attractive feature of the radiosynthesis of [18F]9 in the
microfluidic device is the very low amount of precursor (40 µg
each run) that was required in the reaction, which in turn
diminished the amount of nonradioactive impurities that were
produced. Reverse phase HPLC analysis of [18F]9 product with
eluate monitored for absorbance at 230 nm revealed very few
nonradioactive impurities, which were all at very low level.
Hence, the microfluidic device was readily applied to producing
low activities of radiochemically pure [18F]9 for intravenous
injection into rats. Similarly, useful activities of the later-
discovered metabolite, [18F]11, were produced in the microf-
luidic apparatus for injection into rats. However, radiochemical
yields of [18F]11 were lower than for [18F]9 under comparable
reaction conditions (Figure 1).
Radiochemistry was also readily adapted for the automated
production of [18F]9 from high activities of [18F]fluoride ion,
first in a commercially available apparatus (TRACERlab FXF-N
;
Administration of the PBR-selective ligand 1 to a monkey at
60 min after [18F]9 caused an almost immediate and rapid
decrease in radioactivity to a low common level in all inspected
brain regions (Figure 2C). This confirmed the high selectivity
of [18F]9 for binding to PBR in brain and the rapid washout of
nonspecific binding. Because administration of 1 displaced a
high proportion of radioactivity (e.g., up to 90% in choroid
plexus, Figure 2C), it appears that nonpharmacologically active
radiometabolite(s) could account for only a small proportion
of radioactivity in monkey brain at 60 min. This observation is
consistent with our ability to quantify the binding of [18F]9 to
monkey brain PBR.50
The combined PET data imply a very high ratio of PBR-
specific to nonspecific binding for [18F]9 in monkey baseline
experiments, and point to the high sensitivity of this radioligand.
Accurate quantification of the specific binding of [18F]9 requires
sophisticated biomathematical modeling of the acquired PET
data plus an accompanying measured arterial input function.50
It should be noted that our previous PET studies with [11C]3
have revealed that even the low density of PBR in normal rhesus
monkey brain54 is about 20-fold higher than in normal human
brain.28,29 Our recent quantitative PET studies in normal
monkey50 and human (Fujimura et al., submitted) with [18F]9
also show this species difference. Hence, in human subjects,
even small increases in [18F]9 binding to PBR, as may be
expected at inflammatory lesions, should be easily detectable
and quantifiable against a low background.
An understanding of the metabolism of a candidate PET
radioligand can be important in establishing its worth for
quantitative imaging. Ideally, the candidate radioligand should
not give rise to radiometabolites that can enter brain significantly
to sully the identity of the acquired PET signal. After admin-
istration of [18F]9 alone to monkey, radioactivity in plasma
reduced quite slowly, while a decreasing proportion of this
radioactivity was unchanged radioligand (Figure 4). The re-
mainder of the radioactivity was composed mainly of a polar
radiometabolite, as evidenced by its elution at the solvent front
on reverse phase HPLC, plus a minor radiometabolite of
intermediate retention time and lipophilicity. Half of the
radioactivity in plasma became radiometabolite at, on average,
31 min after radioligand injection (Figure 5).
GE Medical Systems) under thermal labeling conditions and
second within a Synthia device41 equipped with a microwave-
heated reactor.42 Compound 8 was required to be of high purity
and dryness for production of [18F]9; we found 8 could be kept
in this condition for several months by storage in a desiccator
at room temperature. Single pass reverse phase HPLC was used
to separate [18F]9. Formulated [18F]9 was found to be radio-
chemically stable for at least 4 h. Stringent demands for
radiochemical purity, chemical purity and specific radioactivity
in [18F]9 were met with the Synthia process, which is now being
used to produce [18F]9 for human use under an exploratory
Investigational New Drug application from the U.S. Food and
Drug Administration.
The single-step high-yield radiosynthesis of [18F]9 is a
significant advantage over the radiosyntheses of the other two
evaluated 18F-labeled PBR radioligands known from the ary-
loxyanilide class, [18F]4 and [18F]5, which preferably require
two radiochemistry steps.31 The alternative single-step radio-
syntheses of these two radioligands gave erratic yields. The
nonisolated radiochemical yield of [18F]9 also exceeds that of
[18F]1 (46%).34
The lipophilicity (LogD7.4) of 9 was measured with [18F]9
and found to be very close to the computed value (4.05 vs. 4.37).
[18F]9 was found to be stable in whole monkey blood and plasma
in vitro, in rat brain homogenate, and also in buffer. The
distribution of [18F]9 in blood strongly favored uptake into cells.
Much of this uptake is likely to be specific binding to PBR
present in many blood cells,52 including monocytes, lympho-
cytes, platelets, and erythrocytes.53 The plasma free fraction (fP)
in monkey and human was low but still measurable with
accuracy, as would be required for successful biomathematical
modeling of acquired PET data.44
We assessed the radioligand behavior of [18F]9 in rhesus
monkeys with PET. In three baseline experiments in which NCA
[18F]9 was given alone, radioactivity was avidly taken into brain
(Figure 2A). Maximal uptake occurred in choroid plexus of
fourth ventricle, a region known to have high PBR density
relative to other brain regions from in vitro autoradiography.54
Progressively lower uptakes were seen in putamen, thalamus,
cerebellum, and cortical regions. This distribution was similar
to those seen in rhesus monkey for the PBR radioligands, [11C]2
and [11C]3.26 Radioactivity in all regions was well-retained
throughout the experiments.
Measurements of unchanged [18F]9 in plasma revealed the
vastly increased availability of unchanged [18F]9 in blood under
the preblock condition compared to that under the baseline

694 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Scheme 3. Summary of Metabolism of [18F]9 in rat in vivo
Briard et al.
condition, especially during the early phase after radioligand
injection (Figure 6). This increase is concluded to be due to
the blockade of radioligand binding to abundant peripheral PBR
receptors. A similar large increase in plasma radioligand
concentration under PBR preblock conditions has been ob-
served26 for the related 11C-labeled radioligand, [11C]3. The
preblock conditions, used in that study and here, have been
shown with PET to block PBR receptors in the periphery (e.g.,
in kidney and lung).27 Hence, the early higher brain uptakes of
radioactivity in the preblock experiment (Figure 2B) than in
the baseline experiment (Figure 2A) are explained by a much
higher input of radioactivity into brain.
The preblock of all PBR receptors also influenced the
emergence of radiometabolite in monkey plasma in that the time
taken for radiometabolite to reach half of the radioactivity
content was greatly reduced, for example, in one of the studied
monkeys from 24 to 4 min (Figure 5). Similar effects of PBR
blockade have been observed for [11C]2 and [11C]3.26 This effect
may be due to early greater availability of parent radioligand
to metabolizing enzyme(s) under the PBR preblock condition,
i.e., under the preblock condition, the [18F]9 is not protected
from metabolism by its specific binding to PBR receptors in
brain and periphery.
Experiments were performed in rats to gain greater insight
into the metabolism of [18F]9. Radiometabolites appeared quite
rapidly in rat plasma after the intravenous administration of
[18F]9. As in monkey, these radiometabolites consisted mainly
of polar radiometabolite eluting at the solvent front on reverse
phase HPLC and a minor radiometabolite eluting ahead of the
unchanged [18F]9. In urine, these radiometabolites were abun-
dant, whereas parent radioligand was relatively very low. [18F]9
and its radiometabolites were found in brain, with their relative
proportions dependent on time from injection and the specific
radioactivity of the administered radioligand. For the adminis-
tration of high specific radioactivity radioligand with brain
measured at 30 min, the proportion of radioactivity in brain
represented by unchanged [18F]9 was high (>90%). This is an
encouraging finding for imaging in higher species and human
subjects, which are generally considered to be less rapidly
metabolizing. In more detailed studies, we have found that the
uptake of [18F]9 in monkey50 and human brain (Fujmura Y. et
al., submitted) may indeed be quantified satisfactorily with
compartmental modeling and that the ingress of radiometabolites
into brain is not greatly problematic for such quantitation. At
later times, radiometabolites represented an increasing percent-
age of radioactivity in brain. This was also the case for [18F]9
administered at lower specific radioactivity. An interpretation
of this finding is that brain PBR are less available for binding
unchanged [18F]9 when at low specific radioactivity, while the
nonspecific binding of unchanged [18F]9 remains low. Also, as
previously mentioned, [18F]9 appears to be less protected from
peripheral metabolism at lower specific radioactivity; this results
in greater early input of radiometabolites into brain.
Our ex vivo search for the metabolites of 9 in rat with LC-
MS-MS detected unchanged 9 in plasma and brain but not urine,
and also 2-phenoxyaniline in plasma, brain, and urine. No other
possible metabolites were detectable, including 7, which would
need to arise by a single step of deacylation. Therefore, we
hypothesized that 2-phenoxyaniline was generated by deben-
zylation of 9 to compound 11 followed by deacylation (Scheme
3). In accord with this hypothesis, [18F]9 would give [18F]11 as
a radiometabolite (Scheme 3). This hypothesis was strongly
supported when synthesized 11 and [18F]11 were found to
coelute with the low level intermediate radiometabolite of [18F]9
on reverse phase HPLC. We therefore further investigated the
behavior of [18F]11 in rat blood and brain homogenate in vitro
and in rat in vivo. [18F]11 was found to be almost completely
metabolized to polar radiometabolite(s) when incubated for 30
min with rat whole blood or rat brain homogenate. At 30 min
after administration of [18F]11 to rat in vivo, polar radiome-
tabolite(s) also emerged rapidly in plasma, brain, and urine. We
postulate that the polar radiometabolite(s) arise first through
deacylation of [18F]11 to polar [18F]fluoroacetate. [18F]Fluoro-
acetate has been proposed as a radiotracer for prostate tumors55
and is known to be further metabolized to [18F]fluoride ion in
mouse and rat but not baboon.56 The polar radiometabolites of
[18F]9 seen in monkey and in rat are therefore likely to be
[18F]fluoroacetate and/or its radiometabolites. We found a low
level of radioactivity in rat brain (120% SUV) at 30 min after
the isolated polar radiometabolite(s) of [18F]11 from rat were
injected into another rat.
Conclusions
Compound 9 is a selective ligand for PBR with high affinity
across three species, rat, monkey, and human. Radioligand [18F]9
is easily prepared in high radiochemical yield and specific
radioactivity from the easily obtained bromo precursor, 8. [18F]9
is avidly taken up by monkey brain and provides a high ratio
of PBR-specific to nonspecific binding. In monkey and in rat,
[18F]9 shows no evidence of significant defluorination and gives
mainly polar radiometabolite. [18F]9 is therefore a promising

A SensitiVe 18F-Labeled Ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 695
aqueous NaHCO3 and then brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified on silica gel
(hexane, EtOAc, 98:2 v/v) to give 7 (1.35 g, 75%). 1H NMR
(CDCl3) δ 3.69 (3H, s), 3.70 (3H, s), 4.34 (2H, s), 4.70 (1H, s),
6.25-7.05 (10H, m), 7.25-7.32 (3H, m). 13C NMR (CDCl3) δ
43.08, 55.69, 111.07, 112.15, 112.18, 114.87, 116.88, 117.13,
119.64, 122.51, 125.09, 128.47, 129.66, 140.64, 142.95, 151.51,
153.56, 157.83. LC-MS, m/z ) 336.0 [M + H]+. HRMS, m/z )
336.1586 [M + H]+, calcd for C21H22NO3 [M+ + H], 336.1600.
N-Bromoacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (8).
Bromoacetyl bromide (0.288 mL; 3.3 mmol) was slowly added to
a solution of 7 (1.0 g; 3.0 mmol) and triethylamine (0.5 mL; 3.3
mmol) in dichloromethane (5 mL) at 0 °C. The mixture was stirred
for 1 h at room temperature and then poured into water and
extracted with dichloromethane. The combined organic layers were
washed successively with hydrochloric acid (0.5 M), saturated
NaHCO3 solution, and saturated brine and then dried over MgSO4
and concentrated to give 8 as a heavy oil (1.0 g, 73%); TLC (silica
gel; hexane/EtOAc, 60:40 v/v, Rf ) 0.38). Repeated recrystallization
of the oil was from methanol-water (95/5 v/v) and finally absolute
radioligand for the future study of PBR in human subjects.
Preliminary evaluation of this radiotracer in human subject has
been performed under an exploratory-IND from the FDA
(Fujimura et al. submitted).
Experimental Section
Materials. Poly(ethyleneimine) and PK 11195 were purchased
from Sigma (St. Louis, MO). 1 was synthesized from commercially
available 2,5-difluoronitrobenzene, as described previously.45 An
ethanolic solution of [N-methyl-3H]PK 11195 (>97% radiochemical
purity; 83.5 Ci/mmol) was purchased from Perkin-Elmer (Wellesley,
MA, USA). Other chemicals were purchased from Aldrich Chemical
Co. (Milwaukee, WI) and used as received. All animals used in
this study were handled in accordance with the Guide for the Care
and Use of Laboratory Animals57 and the National Institute of
Mental Health Animal Care and Use Committee. Human brain
tissue was obtained from the Clinical Brain Disorders Branch,
National Institute of Mental Health, and experiments with this
material were performed under the regulations of the Ethics
Committee of the National Institutes of Health.
1
ethanol gave 8 as thin colorless needles; mp, 72 °C. H NMR
General Methods. γ-Radioactivity from 18F was measured with
a calibrated dose calibrator (Atomlab 300; Biodex Medical Systems,
USA) or for low levels (<1 µCi) with a well-type γ-counter (model
1080 Wizard; Perkin-Elmer; Boston, MA) having an electronic
window set between 360 and 1800 keV. 18F radioactivity measure-
ments were corrected for background and physical decay. All
radiochemistry with fluorine-18 was performed in lead-shielded hot-
cells. 3H was measured with a liquid scintillation counter (Tri-Carb;
(CDCl3) δ 3.53 (3H, s), 3.67 (3H, s), 3.79 (2H, d, J ) 2.7 Hz),
4.69 (1H, d, J ) 14.7 Hz), 5.19 (1H, d, J ) 14.7 Hz), 6.65-6.75
(m, 2H), 6.83-7.02 (m, 4H), 7.11-7.27 (m, 2H), 7.30-7.38 (m,
2H). 13C NMR (CDCl3) δ 27.74, 46.89, 55.72, 55.81, 111.49,
113.88, 115.95, 118.16, 119.39, 123.14, 124.28, 125.78, 129.65,
129.94, 130.35, 131.51, 151.83, 153.53, 155.66, 166.91. LC-MS
[M + H]+, 456.08; HRMS, m/z ) 456.0810 [M + H]+, calcd for
C23H23BrNO4 [M + H]+ 456.0810. Purity by LC: 100%.
,
1
Perkin-Elmer). H- (400 MHz), 13C- (100 MHz), and 19F-NMR
N-Fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (9).
A mixture of 8 (0.80 g; 1.7 mmol), dry potassium fluoride (0.25 g;
4.3 mmol), and di(ethylene glycol) (5 mL) was stirred, heated
rapidly to 150 °C, and kept at this temperature for 4 h. The reaction
mixture was then cooled, diluted with water (10 mL), and extracted
with ethyl acetate (3 × 10 mL). The combined organic layers were
washed with sodium bicarbonate solution (5% w/v; 10 mL), dried
over MgSO4, filtered, concentrated, and purified on silica gel with
hexane/ethyl acetate to give 9 as a viscous pink oil (0.6 g, 95%).
(376.49 MHz) spectra were recorded at room temperature on an
Avance-400 spectrometer (Bruker; Billerica, MA). 1H and 13C
chemical shifts are reported in δ units (ppm) downfield relative to
the chemical shift for tetramethylsilane and 19F chemical shifts
relative to CFCl3. Abbreviations br, s, d, t, and m denote broad,
singlet, doublet, triplet, and multiplet, respectively.
High resolution mass spectra (HRMS) were acquired from the
Mass Spectrometry Laboratory, University of Illinois at Urbanas
Champaign (Urbana, IL) under electron ionization conditions using
a double-focusing high-resolution mass spectrometer (Autospec;
Micromass Inc., USA).
LC-MS and LC-MS-MS for compound characterization and for
the analyses of biological samples were performed on a LCQ Deca
instrument (Thermo Fisher Scientific Corp., Waltham, MA) equipped
with a Synergi Fusion-RP column (4 µm, 150 mm × 2 mm;
Phenomenex, Torrance, CA), as described previously.26 For
metabolite identification, the chromatogram was searched for all
ions in the m/z range 150-750, to cover all ions from possible
metabolites.
1
TLC (silica gel, hexane-EtOAc, 60:40 v/v, Rf ) 0.27). H NMR
(CDCl3) δ 3.50 (3H, s), 3.67 (3H, s), 4.71 (2H, d, J ) 47.2 Hz),
4.79 (1H, d, J ) 14.2 Hz), 5.15 (1H, d, J ) 14.2 Hz), 6.66-7.36
(12H, m). 13C NMR (CDCl3) δ 46.18, 55.67, 55.79, 77.99, 79.75,
111.50, 113.89, 116.61, 118.15, 119.38, 123.22, 124.33, 125.55,
129.46, 129.88, 129.95, 130.39, 151.99, 153.49, 153.79, 155.56,
166.99. 19F NMR (CFCl3) δ: - 227.10. LC-MS, m/z ) 396.15 [M
+ H]+. HRMS, m/z ) 396.1616 [M + H]+, calcd C23H23FNO4 [M
+ H]+, 396.1611. Purity by LC: 100%.
2-Bromo-N-(2-phenoxyphenyl)acetamide (10). Bromoacetic
anhydride was added dropwise to a solution of 2-phenoxyaniline
(1.0 g, 5.4 mmol) in glacial acetic acid (5 mL) and the reaction
mixture stirred for 2 h at room temperature. Acetic acid was
removed under reduced pressure. Aqueous NaOH (5 mL, 1 M) was
added to the residue and the product was extracted with dichlo-
romethane (3 × 10 mL). The organic layer was washed with water,
dried over MgSO4, filtered, and concentrated under vacuum. Flash
chromatography on silica gel (hexane/ethyl acetate, 80:20 v/v) gave
[18F]9 and its radiometabolites from samples of biological
material were analyzed with HPLC on a Nova-Pak C18 column (4
µm, 100 mm × 8 mm; Waters Corp., USA) housed in a radial
compression module (RCM 100) and eluted with MeOH:H2O:Et3N
(75:25:0.1 by vol.) at 1.5 mL/min. Elution of all radioactivity from
the column during each analysis was confirmed by subsequent
injection of methanol (2 mL) and observation of no more
radioactivity elution. The same HPLC method was applied to
determine the radiochemical purities of [18F]9 preceding lipophilicity
determinations and for the determination of radiochemical stability
in various media (see below).
1
10 (1.31 g; 80%) as a heavy colorless oil. H NMR (CDCl3) δ:
3.98 (s, 2H), 6.89 (dd, 1H, J1 ) 8.12, J2 ) 1.44 Hz), 7.04 (m, 3H),
7.14 (m, 2H), 7.36 (dt, 2H, J1 ) 7.48 and J2 ) 1.08 Hz), 8.39 (dd,
1H, J1 ) 8.08 and J2 ) 1.56 Hz), 8.79 (s, 1H). 13C NMR (CDCl3)
δ: 28.01, 116.47, 116.94, 119.08, 122.46, 122.53, 123.54, 127.45,
128.44, 1244.37, 154.72, 161.83. LC-MS, m/z ) 306 [M + H]+.
HRMS (m/z, ESI); [M + H]+ calcd for C14H13NO2Br 306.0130,
found 306.0116.
Melting points were measured with a Mel-Temp manual melting
point apparatus (Electrothermal; Fisher Scientific), and were
uncorrected.
N-(2,5-Dimethoxybenzyl)-2-phenoxyaniline (7).47 A solution
of 2,5-dimethoxy-benzaldehyde (0.9 g; 5.4 mmol) and 2-phenoxya-
niline (1.0 g; 5.4 mmol) in methanol (6 mL) was stirred for 24 h
at room temperature. Then sodium borohydride (0.75 g; 20 mmol)
was added slowly and the mixture stirred for 1 h. Aqueous acetic
acid (16 mL; 5% v/v) was added dropwise. The mixture was stirred
at room temperature for 30 min and then extracted thrice with
EtOAc. The combined organic layers were washed with saturated
2-Fluoro-N-(2-phenoxyphenyl)acetamide (11). A mixture of
compound 10 (0.5 g, 1.63 mmol) and dry potassium fluoride (0.24
g, 4.1 mmol) was suspended in ethylene glycol (5 mL). The
suspension was stirred and heated rapidly to 150 °C and kept at
this temperature for 2 h. The reaction mixture was cooled, diluted
with water (10 mL), and extracted with toluene (2 × 10 mL). The
organic layer was washed with sodium bicarbonate solution (5%

696 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Briard et al.
w/v; 10 mL), dried over MgSO4, filtered, and concentrated.
Chromatography on silica gel (hexane/ethyl acetate, 90:10 to 80:
20 v/v) gave 11 (260 mg, 65%) as a peach-colored powder; mp:
collected reaction mixture was purified by reverse phase HPLC
(conditions identical to the analytical conditions described above)
and the [18F]9 fraction (6-7.5 min) was collected (∼1.5 mCi). The
process was repeated thrice, and the combined [18F]9 solution
diluted with water (15 mL), passed into a reverse phase column
(3-mL size, SPEC C18AR; Varian, USA), and washed with water
(10 mL). [18F]9 was eluted from the C18AR column with ethanol
(250 µL) and diluted with saline (1.8 mL) to give [18F]9 for injection
in saline containing ethanol (10% v/v).
Microfluidic Production of [18F]11 for Intravenous
Injection into Rats. The procedure for the production of [18F]9
was modified to use 10 as precursor. [18F]11 was separated on a
Luna C18 column (250 mm × 4.6 mm; Phenomenex) eluted with
a gradient of 25 mM HCOONH4-MeCN increasing from 50%
MeCN to 60% over 15 min and then maintained at 60% for 4 min
(tRs 12.0 and 15.2 min for [18F]11 and 10, respectively).
1
75-77 °C. H NMR (CDCl3) δ: 4.95 (d, 2H, J ) 47.3 Hz), 6.88
(dd, 1H, J1 ) 8.08, J2 ) 1.48 Hz), 7.05 (m, 3H), 7.15 (m, 2H),
7.37 (dt, 2H, J1 ) 7.48 and J2 ) 2.45 Hz), 8.45 (dd, 1H, J1 ) 8.04
and J2 ) 1.64 Hz), 8.59 (s, 1H). 13C NMR (CDCl3) δ: 79.92 (d,
JC-F ) 188.46 Hz), 117.48, 118.55, 120.75, 123.70, 123.87, 124.64,
128.07, 129.76, 145.84, 155.93, 165.29 (d, JCO-F ) 161.16 Hz).
19F-NMR (CFCl3) δ: -222.18. LC-MS, m/z ) 246 [M + H]+.
HRMS (m/z, ESI); [M + H]+ calcd for C14H13NO2F 246.0925,
found 246.0930.
Binding Assays of 9. Crude mitochondrial fractions from rat
whole brain were prepared as described previously.58 Monkey
mitochondrial fraction from frozen samples of the temporal and
parietal lobes, and human mitochondrial fraction from frozen brain
tissue samples were prepared in the same manner. Competitive
binding assays were performed with [3H]PK 11195 as reference
radioligand as described previously.26
Automated Production of [18F]9 for Intravenous Injection
into Monkey. Productions were performed automatically within a
lead-shielded hot-cell.
Production of [18F]Fluoride Ion. NCA [18F]fluoride ion was
produced with the 18O(p,n)18F reaction by irradiation of [18O]water
(95 atom %; 1.8 mL) with protons (14.1 MeV, 20-25 µA)
generated with a PETtrace cyclotron (GE, Milwaukee, WI).
Generally, this method produced [18F]fluoride ion with a specific
radioactivity exceeding 10 Ci/µmol.
A commercial automated apparatus, namely the TRACERlab
FXF-N (GE Medical Systems, USA), was used initially. This
apparatus is designed for performance of single-step reactions with
[18F]fluoride ion and for purification and formulation of radioactive
product. In this apparatus, aqueous [18F]fluoride ion (∼300 mCi)
was dried by cycles of addition and evaporation of acetonitrile and
complexed with K+-K+-2.2.2. This complex was reacted with
precursor 8 (0.8-1.2 mg) at 100 °C for 20 min. [18F]9 was purified
by injection (5 mL sample loop) into a semipreparative Luna C18
column (10 µm, 10 mm × 250 mm; Phenomenex) eluted at 4.5
mL/min with a mixture of (A) 50 mM ammonium formate (pH 6)
and (B) 50 mM ammonium formate in MeCN (25:75, v/v)
according to the program 57% B for 2 min, increasing to 62% B
over 8 min and slow increase to 67% B for 40 min. The fraction
containing [18F]9 (tR ) 25 min) was collected in water (100 mL).
This solution was passed through a C-18 Sep-Pak and the trapped
[18F]9 eluted with ethanol (1 mL) into a sterile flask loaded with
saline (9 mL).
Experimental Radiosynthesis of [18F]9 and [18F]11. A glass
reaction vial (5 mL; V-vial) was loaded with an aqueous solution
(10 µL) of KHCO3 (0.7 mg; 7 µmol) plus a solution of 18-crown-6
(1.7 mg; 6.4 µmol) in acetonitrile (0.15 mL). [18F]Fluoride ion (<2
mCi) in [18O]water (20 µL) was added and the solvent evaporated
under reduced pressure (controlled with a bleed of nitrogen) while
heating at 110 °C (oil bath). Water was removed during three cycles
of addition-evaporation of acetonitrile (0.3 mL each cycle). A
solution of water (1 µL; 56 µmol) in acetonitrile (0.5 mL) was
added to the dry residue, followed by addition of 8 (3 mg; 6.6 µmol)
in acetonitrile (0.15 mL). The reaction vial was sealed, heated at
110 °C for 10 min, and then cooled to room temperature before
analysis by radio-HPLC.48
The production of [18F]9 was later performed in a Synthia
apparatus adapted to use microwave irradiation for the labeling
reaction. A microwave reactor (model 521; Resonance Instruments
Inc., Skokie, IL) was securely mounted onto a Synthia MK II
laboratory system. The time and power control were located outside
a lead-shielded hot-cell and linked to the cavity through an RF
coaxial cable. The reaction V-vial (1 mL volume, Alltech Associ-
ates, Deerfield, IL) was equipped with a screw-on cap and septum
(Tuf-Bond Teflon/silicone; Pierce Biotechnology Inc., Rockford,
IL). The septum was pierced with a vent needle that was connected
to a glass vial (20 mL) in order to collect evaporated solvents and
also to a charcoal trap to retain any break-through of volatile
radioactive species. Liquid handling was achieved with a Gilson
Aspec autoinjector/dispenser, which forms part of the Synthia
apparatus. Other operations of the radiosynthesis and purification
procedures were controlled by a Visual Chemistry-based recipe.
Heating time and power input were controlled independently.
[18F]Fluoride ion (∼300 mCi) in 18O-enriched water (350-500
µL) was dried in the V-vial containing K 2.2.2 (5 mg), K2CO3 (0.5
mg) in acetonitrile/water (9:1 v/v, 100 µL). Microwave heating (90
W in 3 pulses of 2 min, 130 °C) was applied under N2 gas flow
(200 mL/min) to speed up the removal of the azeotropic
water-acetonitrile mixture. The heating cycle was repeated twice,
and each time fresh acetonitrile (500 µL) was added. Precursor 8
(∼0.85 ( 0.1 mg) in acetonitrile (to reach a concentration of 1
mg/mL) was introduced into the V-vial and heated at 70 °C (15-35
W in two pulses, each of 1 min). After cooling, the reaction mixture
was diluted with mobile phase (0.75 mL) and injected onto a Luna
C18 column (3 µm, 250 mm × 10 mm; Phenomenex) eluted at 3
mL/min with a mixture of A (water) and B (acetonitrile) according
to the program: 30% B for 3 min, then 47% B over 4 min and
holding to 47% B over 80 min ([18F]9, tR ) 78 min). Formulation
of [18F]9 was performed as described above.
This labeling reaction was further investigated in a commercial
microfluidic device (NanoTek; Advion, Louisville, TN), which
provides for well-controlled stoichiometry between precursor 8 and
[18F]fluoride ion at set flow rates and temperatures. NCA [18F]fluo-
ride ion in [18O]water was first adsorbed onto a small MP-1 cartridge
(ORTG, TN), then released with a solution of K2CO3 (5.5 mg/mL)
and K 2.2.2 (30 mg/mL) in MeCN-H2O (9:1 v/v; 150 µL) into a
5-mL V-vial. The solution was dried by two cycles of azeotropic
evaporation with acetonitrile at 100 °C. The dry [18F]F--K+-2.2.2
solution and precursor 8 (1.0 mg), both in acetonitrile (255 µL),
were loaded into separate storage loops of the microreactor
apparatus. For reaction condition optimization, 10 µL of solution
from each loop was infused into the coiled microreactor (length,
2 m × 2 m; i.d., 100 µm) at different flow rates and temperatures.
The reactor was heated for at least 5 min before the reagents were
dispensed so that the reaction temperature was as close as possible
to the heater temperature. The reaction was quenched by diluting
the reactor output with H2O-MeCN mixture (1:1 v/v; 1 mL) at room
temperature. A portion (50-70 µL) of the diluted solution was
analyzed by radio-HPLC with a Luna hexyl-phenyl column (3 µm,
100 Å, 150 mm × 4.6 mm; Phenomenex) eluted with aqueous
ammonium formate (10 mM, in H2O-MeCN, 40: 60 v/v) at 1 mL/
min. [18F]9 eluted at 7.2 min. RCYs were calculated from the radio-
HPLC analyses and were validated by collecting and measuring
the corresponding HPLC fraction in selected samples. The reaction
of 10 with [18F]fluoride ion to produce [18F]11 was investigated in
the same manner.
Microfluidic Production of [18F]9 for Intravenous Injection
into Rats. In the Nanotek apparatus, an acetonitrile solution (20
µL) of [18F]F--K+-2.2.2 (∼1.2 mCi/mL), prepared as described
above, was dispensed from one reagent loop and an acetonitrile
solution of precursor 8 (4.2 mg/mL; 20 µL) from the other, each at
10 µL/min, into the microreactor (length, 4 m) at 110 °C. The

A SensitiVe 18F-Labeled Ligand
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 697
The radiochemical purity and specific radioactivity of the
formulated [18F]9 were calculated by injecting a sample of the
radioactive solution (0.2-0.5 mCi) into a Luna hexyl phenyl column
(3 µm, 150 mm × 4.6 mm; Phenomenex) eluted with MeCN-10
mM ammonium formate (60:40 v/v) at 1 mL/min ([18F]9, tR ) 6.7
min). The absorbance response (at 230 nm) was calibrated with
respect to mass of 9.
Stability of [18F]9 to Rat Brain In Vitro. Brains from two rats
were excised. The brain (1.44 g) from one rat was homogenized in
saline (1 mL) plus acetonitrile (2 mL), doped with [18F]9 (2 µCi),
rehomogenized, and centrifuged. The other brain (1.70 g) was
homogenized in saline (3 mL), doped with [18F]9 (5 µCi), and
incubated at 37 °C. Samples (200 µL; n ) 6) were removed from
the incubate periodically up to 60 min, placed in acetonitrile (700
µL), and centrifuged. Each supernatant liquid was analyzed with
radio-HPLC. The percentages of radioactivity represented by [18F]9
and its radiometabolites were calculated.
Computation of cLogP and cLogD, and Measurement of
LogD. cLogP and cLogD (at pH ) 7.4) values for 1 and 9 were
computed with the program Pallas 3.0 for Windows (CompuDrug;
S. San Francisco, CA).
The LogD value of [18F]9 was measured as the log of its
distribution coefficient between n-octanol and sodium phosphate
buffer (0.15 M, pH 7.4), as described previously in detail for other
radioligands.26
Emergence of Radiometabolites of [18F]9 in Rat Plasma,
Urine and Brain In Vivo. Four rats (277-623 g) were anesthetized
by inhalation of 1.5% isoflurane in oxygen and maintained at 37
°C with a heating pad. Two of the rats were each injected with a
bolus of formulated NCA [18F]9 (0.812 or 1.32 mCi; specific
radioactivity >1.39 Ci/µmol), and the other two with a bolus of
CA [18F]9 (0.86 or 0.89 mCi) of known specific radioactivity (∼0.13
Ci/µmol). The CA doses were formulated in saline (2.0 mL)
containing ethanol (10% v/v) and Tween 80 (5% w/v) In the CA
experiment, expression of urine was assured by ip administration
of saline (5 mL) at 30 min before [18F]9. Blood samples were drawn
from each rat at intervals for radio-HPLC analysis of harvested
and deproteinized plasma. Each rat was then sacrificed at a set time
after injection (30 and 95 min for the two rats administered NCA
[18F]9 and 60 min for those administered CA [18F]9). Urine was
collected at 60 min for radio-HPLC analysis from the two rats
administered CA [18F]9. The brain from each rat was excised,
homogenized in an ice-bath in saline (1 mL) plus acetonitrile (2
mL), further homogenized after addition of water (0.5 mL), and
finally centrifuged. The supernatant liquids were analyzed with
radio-HPLC. The percentages of radioactivity represented by [18F]9
and its radiometabolites were calculated for each brain, urine, and
plasma analyte.
LC-MS-MS Search for Metabolites of 3 Emerging in Rat
Brain, Urine and Plasma in Vivo. Three healthy rats (480 ( 203)
were placed under isoflurane (1.5% in oxygen) anesthesia and
injected through the penile vein over 9 min with compound 9 (2.26
( 0.76 mg) in saline (0.9 mL) containing ethanol (10% v/v) and
either Cremophor EL (30 mg) or Tween 80 (30 mg). The urethras
were clamped and every 30 min the rats were intravenously
administered with saline (0.1 mL). After 60 or 115 min, blood and
urine samples (1.0-2.0 mL) were collected and the rats were
sacrificed by injection of saturated KCl solution or increased
anesthesia. The brains were immediately excised and each homog-
enized in acetonitrile (3.0 mL) with a “Tissue Tearor”. Water (500
µL) was added and the tissue rehomogenized before centrifugation
at 10000g for 10 min. The clear supernatant liquid was collected
and the precipitate rehomogenized and centrifuged as before. The
supernatant liquid was added to that previously collected and stored
at - 70 °C preceding LC-MS-MS analysis (see General Methods).
Urine samples were stored and analyzed in the same way. Collected
blood samples were stored similarly and, preceding analysis, plasma
was harvested and deproteinized with acetonitrile.
PET Experiments with [18F]9 in Monkeys. Three male rhesus
(Mucacca mulatto) monkeys (11-15 kg) were scanned at baseline
for up to 300 min to measure uptake of radioactivity into brain
and to determine activity distribution after bolus intravenous
injection of [18F]9 (2.76-5.80 mCi; dose of 9, 0.068-0.276 µg/
kg). A preblock experiment was also performed in one of the
monkeys (14 kg) in which 1 (3 mg/kg, iv) was administered at 24
min before [18F]9 (5.09 mCi; 0.323 µg/kg of 9) and the monkey
imaged for 120 min from radioligand injection. One of the monkeys
(15 kg) was also scanned for 240 min from the intravenous injection
of a bolus of [18F]9 (1.28 mCi; dose of 9, 0.12 µg/kg) with 1 (3
mg/kg, iv) administered at 60 min from the start of the scan.
For each scanning session, the monkey subject was immobilized
with ketamine and maintained under anesthesia with 1.6% isoflurane
in oxygen. An intravenous perfusion line filled with saline (0.9%
w/v) was used for bolus injection of [18F]9. PET serial dynamic
images were obtained on an Advance (GE Medical Systems WI)
or High Resolution Research Tomograph (Siemens/CPS, Knoxville,
TN) PET camera. Decay-corrected time-activity curves (TACs)
were obtained for irregular volumes of interest (VOIs), selected
from frontal cortex, temporal cortex, parietal cortex, occipital cortex,
striatum, thalamus, cerebellum, and to choroid plexus on fourth
ventricle. Radioactivity was normalized for injected dose and
monkey weight by expression as % standardized uptake value
[%SUV ) (% injected dose per g) × body weight in g].
MRI and Image Fusion. All monkeys had T1-weighted
magnetic resonance imaging (TR/TE/x ) 24 ms/3 ms/300), acquired
on a 1.5-T Horizon instrument (GE Medical Systems, Waukesha,
WI). PET and MR images were coregistered with SPM2 software
(Wellcome Department of Cognitive Neurology, London, UK).
Stability of [18F]9 in Monkey Whole Blood and Plasma in
Vitro, and in Buffer. [18F]9 was incubated for 45 min in whole
monkey blood (1 mL). A sample (0.5 mL) was removed, added to
acetonitrile (0.8 mL), and centrifuged. Then the supernatant liquid
was analyzed by reverse phase radio-HPLC (see General Methods).
The stability of [18F]9 to incubation in sodium phosphate buffer
(0.15 M, pH 7.4) for 2 h at room temperature was also assessed by
HPLC.
Emergence of Radiometabolites of [18F]11 in Rat Brain
and Plasma In Vitro. Two rats (497 and 449 g) were anesthetized
with 1.5% isoflurane in oxygen. Whole blood was drawn from each
rat by cardiac puncture and then the rat sacrificed by excess
anesthesia. The brain of one rat was excised and placed on ice
without perfusion. The brain of the second rat was perfused with
heparinized ice-cold saline until perfusate ran clear and then placed
on ice. Each brain was separately homogenized in saline (3 mL)
while on ice. [18F]11 (∼10 µCi) in formulation vehicle (100 µL)
was then added to each brain homogenate, which was then incubated
at 37 °C for 60 min and then analyzed by radio-HPLC.
Plasma Protein Binding of [18F]9.59 The radioligand was added
to pooled human plasma, placed at the top of an “Amicon”
Centrifree filter unit (200 µL/unit) and filtered by ultra centrifugation
at 5000g. Then all components of filter units were counted for
radioactivity. The distribution of [18F]9 between the plasma and
the cellular component of the blood was determined in vitro by
incubating [18F]9 with monkey whole blood, centrifugation, and
measuring radioactivity associated with plasma and cells.
Emergence of Radiometabolites of [18F]9 in Monkey
Plasma In Vivo. During each of four PET scans (three baseline
and one preblock), blood samples were drawn periodically from
the monkey femoral artery and collected in heparin-treated Vacu-
tainer tubes. The samples were centrifuged and the plasma
separated. A sample of plasma (1 mL) was mixed with acetonitrile
(1.5 mL) and centrifuged. The supernatant liquid was analyzed with
radio-HPLC (see General Methods). The percentages of [18F]9 and
its radiometabolites were calculated.
Emergence of Radiometabolites of [18F]11 in Rat Brain
and Plasma Ex Vivo. One rat (429 g) was anesthetized with 1.5%
isoflurane in oxygen and then maintained at 37 °C with a heating
pad. The rat was injected with [18F]11 (74.6 µCi) in formulation
vehicle (1 mL) through the penile vein over 4 min. After 30 min,
blood was withdrawn via cardiac puncture into a heparinized
syringe. The brain was excised, measured for radioactivity in a

698 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
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γ-counter, and then homogenized in acetonitrile (2 mL) with a
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Acknowledgment. This study was supported by the Intra-
mural Research Program of the National Institutes of Health,
specifically the National Institute of Mental Health (project no.
Z01-MH-002793). We thank Kimberley Jenko for assistance
on experiments in rats. We also thank the NIH PET Department
for fluorine-18 production and successful completion of the
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Supporting Information Available: HPLC chromatograms
verifying the purities of compounds 8-11. This material is available
free of charge via the Internet at http://pubs.acs.org.
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