Synthesis and evaluation in monkey of two sensitive 11C-labeled aryloxyanilide ligands for imaging brain peripheral benzodiazepine receptors in vivo

Article abstract of DOI:10.1021/jm0707370

We sought to develop ¹¹C-labeled ligands for sensitive imaging of brain peripheral benzodiazepine receptors (PBR) in vivo. Two aryloxyanilides with high affinity for PBR were identified and synthesized, namely, N-acetyl-N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline (3, PBR01) and N-(2-methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide (10, PBR28). 3 was hydrolyzed to 4, which was esterified with [¹¹C]iodomethane to provide [¹¹C]3. The O-desmethyl analogue of 10 was converted into [¹¹C]10 with [¹¹C]iodomethane. [¹¹C]3 and [¹¹C]10 were each injected into monkey to assess their brain kinetics with positron emission tomography (PET). After administration of either radioligand there was moderately high brain uptake of radioactivity. Receptor blocking and displacement experiments showed that a high proportion of this radioactivity was bound specifically to PBR. In monkey and rat, 3 and 10 were rapidly metabolized by ester hydrolysis and N-debenzylation, respectively, each to a single polar radiometabolite. [¹¹C]3 and [¹¹C]10 are effective for imaging PBR in monkey brain. [¹¹C]10 especially warrants further evaluation in human subjects.

Full text of DOI:10.1021/jm0707370

J. Med. Chem. 2008, 51, 17–30
17
Synthesis and Evaluation in Monkey of Two Sensitive 11C-Labeled Aryloxyanilide Ligands for
Imaging Brain Peripheral Benzodiazepine Receptors In Vivo
Emmanuelle Briard, Sami S. Zoghbi, Masao Imaizumi, Jonathan P. Gourley, H. Umesha Shetty, Jinsoo Hong, Vanessa Cropley,
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 June 22, 2007
We sought to develop 11C-labeled ligands for sensitive imaging of brain peripheral benzodiazepine receptors
(PBR) in vivo. Two aryloxyanilides with high affinity for PBR were identified and synthesized, namely,
N-acetyl-N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline (3, PBR01) and N-(2-methoxybenzyl)-N-(4-phe-
noxypyridin-3-yl)acetamide (10, PBR28). 3 was hydrolyzed to 4, which was esterified with [11C]iodomethane
to provide [11C]3. The O-desmethyl analogue of 10 was converted into [11C]10 with [11C]iodomethane.
[11C]3 and [11C]10 were each injected into monkey to assess their brain kinetics with positron emission
tomography (PET). After administration of either radioligand there was moderately high brain uptake of
radioactivity. Receptor blocking and displacement experiments showed that a high proportion of this
radioactivity was bound specifically to PBR. In monkey and rat, 3 and 10 were rapidly metabolized by ester
hydrolysis and N-debenzylation, respectively, each to a single polar radiometabolite. [11C]3 and [11C]10
are effective for imaging PBR in monkey brain. [11C]10 especially warrants further evaluation in human
subjects.
Introduction
The “peripheral benzodiazepine receptor” (PBRa), recently
called the “translocator protein (18 kDa)”,1 is wholly distinct
structurally, functionally, and pharmacologically from the well-
known classes of brain receptors that bind both GABA and
synthetic benzodiazepines (e.g., diazepam). Originally discov-
ered as a diazepam binding site in kidney,2 PBR is an
evolutionarily well-conserved and highly hydrophobic 18 kDa
protein that is associated with the voltage-dependent ion channel
and adenine nucleotide transporter at the contact site between
outer and inner mitochondrial membranes.3 PBRs are also
expressed on plasma membranes in erythrocytes.4 At mitochon-
dria, the PBR protein is arranged in five transmembrane
domains5 forming a cavity that likely contributes to a perme-
ability transition core.6 PBRs have many putative functions, such
as cholesterol transport from the outer to inner mitochondrial
membrane7,8 and involvement in regulation of cell death,3 cell
Figure 1. Structures of some PBR ligands. Asterisks mark positions
proliferation,9 and ion transport.10,11 PBRs are widely distributed
in which ligands have been labeled with either carbon-11 or fluorine-
throughout the body.12 The glands and gonads are particularly
18.
rich, while the kidney, lung, and heart have intermediate levels,
in several neuropathological conditions and also after experi-
and the liver and brain have low levels.
mental injuries to the central nervous system.15 Evidence
PBRs are implicated in numerous nervous system disorders
suggests that these increases are mainly associated with activated
such as epilepsy, cerebral ischemia, astrocytoma, nerve injury,
microglia and to a lesser extent with astrocytes. The prototypical
and neurodegenerative diseases.13,14 Brain PBR density increases
PBR-selective radioligand, the isoquinoline carboxamide
[11C]PK 11195, initially as its racemate16 and later as its single
* To whom correspondence should be addressed. Phone: 301-594-5986.
Fax: 301-480-5112. E-mail: pikev@mail.nih.gov.
R-enantiomer17 (Figure 1), has long been used with positron
aAbbreviations: D, dopaminergic; DAA1106, N-(2,5-dimethoxybenzyl)-
emission tomography (PET) to visualize such changes in vivo
N-(5-fluoro-2-phenoxyphenyl)acetamide; DAT, dopamine transporter; EOS,
(for a recent comprehensive review, see Venneti et al.18).
end of synthesis; ERP, end of radionuclide production; FDA, Food and
Drug Administration; [18F]FEDAA1106, O-[18F]2-(2-fluoroethyl)desmethyl-
DAA1106; [18F]FMDAA1106, O-[18F]2-fluoromethyldesmethyl-DAA1106;
GABA, γ-aminobutyric acid; H, histaminergic; HEPES, 4-(2-hydroxyeth-
yl)piperazineethanesulfonic acid; IND, investigational new drug; M, mus-
carinic; PET, positron emission tomography; PBR, peripheral benzodiaz-
epine receptor; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-
3-isoquinoline carboxamide; RCY, decay-corrected radiochemical yield;
NET, noradrenaline transporter; rt, room temperature; SERT, serotonin
transporter; SR, specific radioactivity; SUV, standardized uptake value;
5-HT, serotonin.
Examples include imaging of elevated PBR in dementia,19
herpes encephalitis,20 and multiple sclerosis.21,22 Nonetheless,
[11C]PK 11195 has many limitations on its sensitivity and
accuracy for measuring PBR increases. In human subjects these
include low brain uptake,22 low sensitivity (i.e., a low ratio of
PBR-specific to nonspecific binding), extensive metabolism to
several unidentified less lipophilic radiometabolites,23–25 that
have a possible undesirable ability to enter brain, and finally
10.1021/jm0707370 This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society
Published on Web 12/08/2007

18 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Briard et al.
variable binding to blood proteins.26 [11C]PK 11195 shows
similarly moderate brain uptake, low sensitivity, and extensive
metabolism in animals (mouse,23 rat,27 pig,28 baboon,29 and
rhesus monkey30). All these factors contribute to difficulty in
robustly quantifying regional variations in PBR density in human
and animal subjects with [11C]PK 11195; therefore, sophisticated
biomathematical efforts have become necessary to try to
surmount these issues with this radioligand.31–33
Scheme 1. Synthesis of Ligand 3, Precursor 4, and Radioligand
[11C]3 a
Recently, new structural classes of high-affinity PBR ligands
have been discovered, opening up opportunities for developing
improved PET radioligands for PBR. These include quinoline
carboxamides, benzothiazepines, benzoxazepines, indoleaceta-
mides, pyrazolopyrimidines, vinca alkaloids, and aryloxyanil-
ides.34 Subsequently, many candidate PBR radioligands for PET
imaging have been proposed and assessed in animals, including
[11C]DAA1106,35–37[18F]FMDAA1106,38,39[18F]FEDAA1106,38,39
[11C]DPA-713,40 [11C]VC195,41 and [11C]vinpocetine42 (Figure
1). Although some of these radioligands appear promising, for
many of them data in human subjects are so far sparse or
nonexistent and preclude a final conclusion on their utility for
measuring PBR in clinical situations. Exceptionally, two of the
more promising PET radioligands, based on aryloxyanilides,
have very recently entered study in human subjects, namely,
[11C]DAA110643 and [18F]FEDAA1106.44 Even so, these
radioligands may turn out to have less than ideal kinetic
properties that ultimately limit their effective application for
quantitative studies. We have sought to develop alternative
sensitive brain-penetrant PBR ligands from the aryloxyanilide
class, as we now report here.
a Reagents, conditions, and yields: (i) 2-phenoxyaniline, MeOH, rt, 24 h,
then NaBH4, rt, 1 h, yield 65%; (ii) MeCOCl, Et3N, CH2Cl2, rt, 1 h, yield
71%; (iii) KOH, MeOH, rt, 1 h, yield 85%; (iv) DMF, (tBu)4NOH, MeOH,
11CH3I, rt, ∼9 min, RCY 18% from [11C]carbon dioxide.
Scheme 2. Synthesis of Ligand 10 and Metabolite 8 a
Specifically, we report the radiosyntheses and evaluations of
11C-labeled versions of an especially high-affinity ligand,
namely, N-acetyl-N-(2-methoxycarboxybenzyl)-2-phenoxya-
niline (3, PBR01) (IC50 ) 0.0643 nM)45 and another less
lipophilic alternative, namely, N-(2-methoxybenzyl)-N-(4-phe-
noxypyridin-3-yl)acetamide (10, PBR28) (IC50 ) 0.658 nM).46
Ligands were simply labeled by alkylation of a prepared
desmethyl precursor with [11C]iodomethane, itself generated
from cyclotron-produced [11C]carbon dioxide. The PBR binding
affinities in rat, monkey, and human tissue homogenates, brain
uptake and distribution in vivo, and metabolite analysis of [11C]3
and [11C]10 are reported here. [11C]3 and [11C]10 are each found
to be sensitive radioligands for imaging PBR in monkey brain,
and [11C]10 in particular poses high potential for measuring
PBR in human subjects with PET.
a Reagents, conditions, and yields: (i) Fe powder, HCl in aqueous EtOH,
reflux, 3 h, 74%; (ii) AcCl, Et3N, CH2Cl2, rt, 1 h, 55%; (iii) 2-methoxy-
benzaldehyde, toluene, reflux, 24 h, then NaBH4, MeOH, rt, 1 h, 90%; (iv)
Ac2O, AcOH, reflux, 2 h, 84%.
Scheme 3. Synthesis of Phenol Precursor 13 and [11C]10 a
Results
Chemistry. Ligands 3 and 10 were obtained efficiently and
in useful yields from commercial materials based on general
synthetic methods45,46 (Schemes 1 and 2). Ligand 3 was
converted by simple hydrolysis into the acid precursor 4 that
was required for the synthesis of [11C]3 (Scheme 1). The phenol
precursor 13 required for the synthesis of [11C]10 was prepared
de novo in three steps from commercial materials (Scheme 3).
Metabolite 8 was obtained by simple acetylation of the amine
7 with acetyl chloride.
Ligand Pharmacological Assays and Screen. The affinities
of ligand 3 for PBR in rat, monkey, and human mitochondrial
fractions were high and similar to those of DAA1106, while
those of ligand 10 were marginally lower, especially for rat
tissue (Table 1). The affinity of PK 11195 was significantly
lower than for the aryloxyanilides in monkey and human.
Ligands 3, 10, and DAA1106 all showed a small interspecies
variability in affinity for PBR, with that for human tissue being
somewhat lower than for monkey or rat. Values for the affinities
a Reagents, conditions, and yields: (i) Ac2O, pyridine, rt, overnight, 95%;
(ii) 7, toluene, reflux, 24 h, then NaBH4, MeOH, reflux, 3 h, 25%; (iii)
Ac2O, AcOH, reflux, 3 h, then KOH, MeOH, rt, 3 h, 63%; (iv) (tBu)4NOH,
MeOH, 11CH3I, rt, ∼7 min, RCY 25% from [11C]carbon dioxide.
of ligands 3, 10, and DAA1106 for rat tissue PBR were broadly
consistent with those previously found.45,46 Ligand 10 showed
a Ki of >10 µM at several central benzodiazepine (GABAA)
receptors (R1ꢀ1γ2, R2ꢀ2γ2, R5ꢀ2γ2, and R6ꢀ2γ2 subtypes) and at
10 µM concentration was found to cause less than 50%
displacement of reference ligand from 5-HT1A,1B,1D,1E,2A-C,3,5A,6,7
,
R1A,1B,2A-C, ꢀ1-3, D1-4, H1-4, M2,5, DAT, NET, and SERT sites.
A full displacement study revealed a Ki of 2.2 µM at κ opiate
receptors.
Computation of cLogD and cLogP and Measurement
of LogD. The measured LogD values and computed cLogP and
cLogD values of 3, 10, and PK 11195 are shown in Table 1.

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 19
Table 1. PBR Lipophilicity, Affinity, Blood Distribution, and Free Fraction (fP) Data for [11C]3, [11C]10, DA1106, and [11C]PK 11195
monkey plasma:
cell distribution (%)
radioligand
cLogP
cLogD
LogDa
Ki for PBR (nM)
monkey fP (%)
[
[
11C]3
4.4
4.4
3.90 ( 0.02
3.01 ( 0.11
ndh
1.40 ( 0.07b 0.254 ( 0.009d 0.0978 ( 0.0058e
2.47 ( 0.39b 0.944 ( 0.101d 0.680 ( 0.027e
0.242 ( 0.016b 0.230 ( 0.011d 0.0726 ( 0.0036e
4.50 ( 0.43b 4.35 ( 0.52d 0.73 ( 0.05e
36.7 ( 4.0: 63.3 ( 4.0c
0.15 ( 0.06c
5.6 ( 0.1g
ndh
11C]10
3.01
4.28
5.28
2.95
4.28
5.28
33.2 ( 2.1: 66.8 ( 2.1f
DAA1106
[
ndh
ndh
11C]PK 11195
3.97 ( 0.18
ndh
a Mean ( SD for six determinations. b For human mitochondrial tissue; mean ( SD for six determinations. PK 11195 was found to have a KD of 5.17
( 3.00 nM (n ) 6) in this assay. c Mean ( SD for four determinations. d For monkey mitochondrial tissue; mean ( SD for six determinations. PK 11195
was found to have a KD of 3.48 ( 1.26 nM (n ) 6) in this assay. e For rat mitochondrial tissue; mean ( SD for six determinations. PK 11195 was found
to have a KD of 0.707 ( 0.361 nM (n ) 6) in this assay. f Mean ( SD for three determinations. g Mean ( SD for nine determinations. h nd ) not determined.
Ligand 10 shows the lower values. Ligands 3 and PK 11195
were found to have similar measured LogD values. The cLogD
value of the acid 15 was found to be -1.52.
Figure 4 shows PET summation images of monkey brains
acquired over a late period after injection of either [11C]3
(summed between 100 and 150 min) or [11C]10 (summed
between 80 and 120 min) and coregistered with MRI scans.
Each radioligand displays a high level of radioactivity in brain
especially in the cerebellum, striatum, and choroid plexus of
the fourth ventricle and displays intermediate levels in frontal,
parietal, temporal, and occipital regions.
Emergence of Radiometabolites in Monkey Blood In
Vivo. During PET experiments with [11C]3 and [11C]10,
recoveries of radioactivity from plasma into supernatant aceto-
nitrile for HPLC analysis averaged 91.0 ( 6.8% (n ) 30) and
95.8 ( 3.3% (n ) 27), respectively. [11C]3 gave only one
radiometabolite in plasma that eluted at the void volume on
reverse-phase HPLC (tR ) 2.1 ( 0.1 min; n ) 29), as did
[11C]10 (radiometabolite tR ) 2.0 ( 0.4 min; n ) 22), while
the parent radioligands eluted in the same analyses at 4.7 (
0.7 and 3.9 ( 0.7 min, respectively.
Synthesis of Radioligands. [11C]3 and [11C]10 were readily
prepared in acceptable isolated and formulated decay-corrected
radiochemical yields from cyclotron-produced [11C]carbon
dioxide (18% and 26%, respectively), and in high chemical and
radiochemical purity (>99%), with specific radioactivity (SR)
in the range 2.04-3.73 Ci/µmol for [11C]3 and 2.28-8.90 Ci/
µmol for [11C]10 at EOS (end of synthesis) (about 30 min from
the start of radiosynthesis). The formulated radioligands were
radiochemically stable for at least 1 h.
Analysis of Radioligand Distribution in Monkey Blood.
The monkey blood distribution and plasma free fraction (fP)
values of [11C]3 and [11C]10 are presented in Table 1. Both
radioligands were distributed mainly to cellular blood elements.
[11C]10 has a much higher fP value than [11C]3.
Stability of Radioligands in Whole Monkey Blood, Rat
Brain Homogenate, and Buffer. Radio-HPLC analysis of
radioactivity extracted from monkey plasma incubated with
either [11C]3 or [11C]10 for >30 min showed [11C]3 and [11C]10
to be 96.5 ( 1.5% (n ) 5) and 95.8 ( 1.6% (n ) 10)
unchanged, respectively. [11C]10 was 99.8% unchanged after
incubation with rat brain homogenate in saline for 2 h at 37
°C. After 2.5 h of storage in sodium phosphate buffer (0.15 M,
pH 7.4), [11C]3 and [11C]10 were 99.9 ( 0.0% (n ) 4) and
99.0 ( 0.2% (n ) 4) unchanged, respectively.
Monkey PET Imaging Experiments. After injection of
[11C]3 into monkey, radioactivity entered the brain well with
peak uptake occurring in all PBR-containing regions at later
than 10 min after injection (Figure 2A). Maximal uptake was
259% SUV in choroid plexus of fourth ventricle. The washout
of radioactivity from all PBR-containing regions was quite
slow. In an experiment in which PBR receptors were blocked
in the same monkey by coadministration of 3, the kinetics
of brain radioactivity was strikingly different. Radioactivity
was rapidly taken into all examined brain regions and to a
much higher level than in the baseline experiment and then
rapidly washed out (Figure 2B). In a displacement experi-
ment, radioactivity in all PBR-containing regions was
substantially reduced after administration of PK 11195
(Figure 2C).
Figure 5 shows the time course of radioactive species in
plasma after injection of monkey with [11C]3 in the baseline
and self-block experiments. The time at which plasma radio-
activity was composed equally of parent radioligand and
radiometabolite was significantly shorter in the self-block
experiment (8.2 min) than in the baseline experiment (20.2 min).
A similar pattern was seen for [11C]10 between the preblock
and baseline experiments (Figure 6).
For [11C]3, the level of parent radioligand in plasma (% SUV)
was substantially higher in the self-block than in the baseline
experiment, especially over the initial 6 min; peak values were
nearly 7-fold higher in the self-block experiment (Figure 7). A
similar pattern was also shown by [11C]10 between the preblock
and baseline experiments (Figure 8).
Radiometabolites in Rat Plasma, Urine, and Brain In
Vivo. At 30 min after administration of either [11C]3 or [11C]10
to rat, recoveries of radioactivity from plasma, urine, and brain
homogenate samples into supernatant acetonitrile for analysis
with reverse-phase radio-HPLC were generally very high (values
are shown in parentheses in Table 2). Recoveries of injected
radioactivity from HPLC were quantitative in all cases. For each
radioligand in all samples, HPLC analyses revealed a single
polar radiometabolite for [11C]3 (tR ) 2.1 min) and for [11C]10
(tR ) 2.2 min) (cf. tR ) 4.8, and 6.5 min for [11C]3 and [11C]10,
respectively). In brain, in each case, parent radioligand was
predominant, while in plasma and urine, radiometabolite was
predominant (Table 2). With reduction of the amount of carrier
in experiments with [11C]10, the percentage of radioactivity in
brain represented by unchanged radioligand increased (Table
2). In an experiment with [11C]10 in a rat that was perfused
with saline during sacrifice, the level of radioactivity represented
by unchanged radioligand (95.2%) was high and fully consistent
with that seen in nonperfused rats.
In a baseline experiment in the same monkey, [11C]10
behaved similarly to [11C]3 except that maximal brain radio-
activity uptake was higher (394% SUV in choroid plexus of
fourth ventricle) (Figure 3A). In another experiment with [11C]10
in the same monkey, in which PBR receptors were preblocked
with DAA1106, the kinetics of brain radioactivity was again
strikingly different. Radioactivity was rapidly taken into all
examined brain regions and to a higher level than in the baseline
experiment and then rapidly washed out to a low level (Figure
3B). In an experiment in which PK 11195 was used as displacing
agent, radioactivity in all PBR-containing regions was rapidly
and substantially reduced (Figure 3C).
LC-MS, MS-MS, and GC-MS of Reference Com-
pounds. LC-MS of ligand 3 (tR ) 14.76 min) gave the
protonated molecule ([M + H]+, m/z 376) and fragment ions

20 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Briard et al.
Figure 3. Brain region time-activity curves (TACs) in rhesus monkey
after injection of [11C]10 alone (baseline experiment) (A), after injection
of DAA1106 preceding [11C]10 at 24 min later (preblock experiment)
(B), and after injection of [11C]10 and then PK 11195 at 45 min later
(displacement experiment) (C). Experiments in parts A and B were
performed in the same monkey (and the same monkey giving the data
shown in parts A and B of Figure 2): 0, cerebellum; 4, occipital cortex;
3, parietal cortex; ], temporal cortex; O, frontal cortex; [, putamen;
1, thalamus; 2, striatum; b, choroid plexus of fourth ventricle.
Figure 2. Brain region time-activity curves (TACs) in rhesus monkey
after injection of [11C]3 alone (baseline experiment) (A), after injection
of 3 with [11C]3 (self-block experiment) (B), and after injection of
[
11C]3 and then PK 11195 at 45 min later (displacement experiment)
(C). Experiments in parts A and B were performed in the same monkey
(and the same monkey giving the data shown in parts A and B of Figure
3): 0, cerebellum; 4, occipital cortex; 3, parietal cortex; ], temporal
cortex; O, frontal cortex; [, putamen; 1, thalamus; 2, striatum; b,
choroid plexus of fourth ventricle.
m/z 302 ([M + H - (CH3 + COOCH3)]+) and 334 ([M + H
- CH2CO]+) in its full scan MS. MS-MS analysis showed
the product ions m/z 302 (100%) and m/z 334 (<10%). LC-MS
of the acid 4 (tR ) 11.52 min) showed m/z 362 ([M + H]+)
and the fragment ions m/z 320 ([M + H - CH2CO]+) and 302
([M + H - (CH3 + COOH])+). MS-MS analysis gave the
same ions (m/z 302, 100%). LC-MS of ligand 10 (tR ) 12.9
min) showed m/z 349 ([M + H]+), and its dissociation in
MS-MS analysis generated major product ions m/z 308, 307,
255, 188, and 121. LC-MS of 8 (tR ) 10.1 min) showed m/z
229 ([M + H]+), and its dissociation yielded the product ions
m/z 187 and 135. GC-MS of 2-methoxybenzoic acid 15 (tR )
8.3 min) showed molecular ion m/z 152 and fragment ions m/z
135, 123, 105, 92, 79, 77, and 63.
Analysis of Rat Brain Extract after Ligand Injection. In
an experiment in which ligand 3 was administered to rat,
LC-MS analysis of brain extract acquired at 95 min showed
a peak (tR ) 14.73 min) for the protonated molecule ([M +
H]+, m/z 376) and fragment ions m/z 334 and 302 assignable
to unmetabolized 3. In the total-ion current, a search for

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 21
Figure 4. Axial, sagittal, and coronal PET summation images of monkey brain coregistered with MRI scans from (top row) a baseline experiment
with [11C]3 and from (bottom row) a baseline experiment with [11C]10. PET data are summed between 100 and 150 min and between 80 and 120
min for [11C]3 and [11C]10, respectively.
Figure 5. Time course of radioactivity composition in plasma after
injecting [11C]3 into rhesus monkey, either alone (baseline experiment)
or after coadministration with 3 (self-block experiment). The experi-
Figure 7. Time course of radioactivity (% SUV) in plasma represented
ments were performed in the same monkey (and the same monkey
by [11C]3 after injection into rhesus monkey under baseline conditions
producing the data shown in Figure 7): O, parent [11C]3 in baseline
(O) and under self-block conditions (0).
experiment; b, radiometabolite in baseline experiment; 0, parent [11C]3
in self-block experiment; 9, radiometabolite in self-block experiment.
The arrows give the times at which radioactivities in plasma are
composed equally of parent radioligand and radiometabolite.
Figure 8. Time course of radioactivity (% SUV) in plasma represented
by [11C]10 after injection into rhesus monkey under baseline condition
Figure 6. Time course of radioactivity composition in plasma after
injecting [11C]10 into rhesus monkey, either alone (baseline experiment)
(O) and under preblock condition (0).
or after pretreatment with DAA1106 (preblock experiment). The
MS-MS analysis showed product ions m/z 320 and 302
(100%). These LC-MS characteristics matched those of the
acid 4.
experiments were performed in the same monkey (and the same monkey
producing the data shown in Figure 8): O, parent [11C]10 in baseline
experiment; b, radiometabolite in baseline experiment; 0, parent
[
11C]10 in preblock experiment; 9, radiometabolite in preblock
In the corresponding experiment with ligand 10, LC-MS
analysis of brain extract acquired at 120 min showed a peak at
tR ) 12.9 min for [M + H]+ (m/z 349) assignable to
unmetabolized 10. MS-MS analysis of this peak showed major
product ions m/z 308, 307, 255, 188, and 121, confirming the
presence of 10 in brain. The LC-MS screen for possible
metabolites of 10 detected a compound (tR ) 10.2 min) with
an intense [M + H]+ ion (m/z 229) that gave fragment ions
experiment. The arrows give the times at which radioactivities in plasma
are composed equally of parent radioligand and radiometabolite.
possible metabolite-specific ions (that might arise from ester
hydrolysis, ring hydroxylation, deacetylation, or dealkylation)
showed m/z 362 ([M + H]+) along with a fragment ion m/z
302. The tR for this component was 11.87 min. In addition,

22 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Briard et al.
Table 2. Radioactivity in Rat Plasma, Urine, and Brain Represented by Unchanged Radioligand at 30 min after Intravenous Administration
radioactivity as unchanged radioligand (%)a
radioligand
injected carrier (µg/kg)
plasma
urine
brain
[
[
[
[
[
11C]3
0.54
2.96
0.06
0.86
4.83
13.1 ( 1.0b (85.5 ( 1.4)b
3.4 ( 1.1b (91.6 ( 0.9)b
4.2d
ndf
ndf
ndf
ndf
83.0 ( 0.7b (86.9)
64.5, 64.5c (87.3)
97.6 d
11C]3
11C]10
11C]10
11C]10
7.0, 7.0c (91.5, 91.6)c
94.4 ( 0.1e (81.2)
2.4, 3.0c (85.3 ( 2.3)b
0.39 ( 0.01 (88.2 ( 8.2)b
89.1, 99.5c (92.3)
a The remainder of radioactivity was the single more polar radiometabolite in each case. Values shown in parentheses are recoveries of radioactivity from
tissue into the analytical sample (%). b Mean ( SD from three HPLC analyses of one sample taken from one rat. c Values from two HPLC analyses of one
sample taken from one rat. d Single determination. e Mean ( SD from four HPLC analyses of one sample taken from one rat. f nd ) not determined.
m/z 187 and 135 in MS-MS. These LC-MS and MS-MS
characteristics matched those of reference 4-phenoxy-3-acety-
laminopyridine (8). No other metabolite was detected by
LC-MS.
10 for PBR in all three species (Table 1). The Ki values of 3
are comparable to those of the prototypical aryloxyanilide PBR
ligand, DAA1106, and lower than the Ki values of PK 11195
in the same assays (Table 1). A small species-dependent
variation in PBR affinity was evident for 3, 10, and DAA1106.
Ligand 10 was found to show excellent PBR selectivity in a
broad pharmacological screen run through the NIMH Psycho-
active Drug Screening Program.
Analysis of Rat Urine after Ligand Injection. In the
experiment in which ligand 3 was administered to rat,
LC-MS analysis of collected urine showed a weak peak for
[M + H]+ (m/z 376) at tR ) 14.74 min. In addition, fragment
ion m/z 302 was present at the same retention time. These
LC-MS characteristics matched those of ligand 3. The
presence of 3 in urine was further confirmed by MS-MS
analysis, which gave the same product-ion spectrum as 3.
The brain metabolite, 4, was not detected in urine. Also, an
extensive search with LC-MS failed to show any other
possible urinary metabolites.
In the corresponding experiment with ligand 10, only a low
concentration of unmetabolized 10 appeared in urine. LC-MS
did not detect the [M + H]+ ion. However, more sensitive
MS-MS showed a peak at tR ) 12.9 min with all the product
ions of 10. LC-MS did detect a peak at tR ) 10.1 min for m/z
229 ion corresponding to the [M + H]+ ion of metabolite 8. In
addition, MS-MS of this peak showed all the product ions of
metabolite 8.
The metabolic N-debenzylation of 10, while generating
metabolite 8, should also yield an equimolar amount of
2-methoxybenzaldehyde (14). Once generated, 14 is likely
to be oxidized rapidly to 2-methoxybenzoic acid (15). To
search for this possible metabolite, urine was extracted into
ethyl acetate and analyzed by GC-MS with electron ioniza-
tion detection. The ion chromatogram showed a molecular
ion m/z 152 at tR ) 8.3 min. The fragment ions in the mass
spectrum were m/z 135, 123, 105, 92, 79, 77, and 63.
Reference 15 showed the same GC-MS and characteristics
as this metabolite. Thus, metabolites 8 and 15 were both
present in rat urine.
For use in 11C-labeling, the acid precursor 4 to [11C]3 was
obtained by simple hydrolysis of 3 (Scheme 1) and the phenol
precursor 13 to [11C]10 by a simple three-step synthesis (Scheme
3). Both radioligands were easily prepared by alkylation with
[11C]iodomethane and obtained pure in adequate radiochemical
yields and specific radioactivities. The LogD values for these
radioligands were then determined experimentally and were in
quite close agreement with those predicted by computation,
again showing ligand 10 to have lower lipophilicity than 3, with
a value lying in the range that is generally considered desirable
for achieving good brain entry.47,48 The formulated radioligands
were radiochemically stable and also stable in monkey plasma
for at least 30 min. In monkey blood, each radioligand was found
to be distributed similarly and heavily into cells (Table 1),
consistent with the known cellular presence of PBR.2 In accord
with its lower lipophilicity, [11C]10 gave a significantly greater
free fraction (fP value) than [11C]3 (Table 1). The higher fP value
of 10 presents the considerable advantage of allowing its
accurate measurement for input into potential biomathematical
models.
Significant species differences in the brain distribution of PBR
exist.49 PBR are known by autoradiography in vitro to be present
in the choroid plexus and cerebellum of rhesus monkey,49 but
little is known concerning their detailed distribution throughout
the remainder of monkey brain. Other PBR radioligands (e.g.,
[11C]DAA1106) have been observed by PET to have high
uptake into the occipital cortex, olfactory bulb, and choroid
plexus of rhesus monkey in vivo.30,36
After intravenous injection of [11C]3 into rhesus monkey, PET
revealed a substantial initial uptake of radioactivity followed
by slow washout from all selected volumes of interest (cerebel-
lum, occipital cortex, parietal cortex, temporal cortex, frontal
cortex, putamen, thalamus, and choroid plexus of the fourth
ventricle) (Figure 2A). After 10 min from radioligand injection,
the highest uptake (∼250% SUV) was observed in the choroid
plexus of the fourth ventricle and in the cerebellum and the
lowest uptake in the frontal cortex. The selectivity of the brain
radioactivity uptake for binding to PBR was tested in self-block
and displacement experiments. In the self-block experiment,
performed in the same monkey as in the baseline experiment,
the uptake of radioactivity into all examined brain regions was
fast and higher than in the baseline experiment and was then
followed by a fast washout of radioactivity from all regions to
the same very low level (Figure 2B). The higher initial uptake
of radioactivity into brain is explained by the blockade of PBR
Discussion
Ligands 3 and 10 appeared attractive for development as
potential PET radioligands for PBR because of their reported
high affinities45,46 and amenability to labeling with carbon-11
through simple reactions with [11C]iodomethane. Moreover, their
computed lipophilicities, as indexed by cLogP and cLogD
values, are lower than the very high values of PK 11195 (cLogP
) cLogD ) 5.28) (Table 1). These properties of 3 and 10 were
expected to be conducive to better brain entry, higher specific
binding, and lower nonspecific binding in vivo than for PK
11195. Ligands 3 and 10 were readily synthesized in a few steps
from commercially available materials according to described
general methods (Schemes 1 and 2).45,46 Each ligand was
assayed for affinity to PBR through determination of Ki values
in binding assays on human, rat, and monkey mitochondrial
tissue with tritiated PK 11195 as radioligand. These assays
showed that ligand 3 has somewhat higher affinity than ligand

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 23
in peripheral organs, leading to a higher plasma level of
radioligand, as confirmed by measurements in blood (see below).
In the displacement experiment, administration of PK 11195 at
45 min after the radioligand caused a progressive reduction of
radioactivity from all examined brain regions to a low level
(∼100% SUV) at 120 min (Figure 2C), thereby showing that
the majority of radioactivity in brain was specifically bound to
PBR before displacement. These data confirmed the high
selectivity of the radioactivity in the brain for binding to PBR
and also the relatively very low level of nonspecific binding in
the baseline experiment.
The results obtained with PET for [11C]10 in monkey were
similar to those for [11C]3 except that brain uptake of radioac-
tivity in the baseline experiment was appreciably higher in the
same monkey (Figure 3A). Maximal uptake was about 400%
SUV in the choroid plexus of the fourth ventricle. This uptake
far exceeds the uptake of [11C]PK 11195 in normal rhesus
monkey brain (maximum of about 130% SUV).36 In the
preblock experiment, with DAA1106 as blocking agent, the
observed kinetics were similar to those in the self-block
experiment with [11C]3, again showing increased initial uptake
of radioactivity and fast washout from all regions (Figure 3B).
In another PET experiment, administration of PK 11195 at 45
min after radioligand resulted in a sharp decline of radioactivity
from all examined brain regions to a low level, showing that
the majority of radioactivity in brain had been bound to PBR
(Figure 3C).
Each radioligand provided similar PET images of summed
radioactivity distribution in brain in axial, sagittal, and coronal
sections which, on coregistrations with MRI scans, matched with
the limited previous information30,36,49 on the regional distribu-
tion of PBR in rhesus monkey brain (Figure 4). Thus, high
radioactivity uptake was seen in known PBR-rich regions such
as cerebellum, occipital cortex, and choroid plexus of the fourth
ventricle.
It is noted that the determination of the ratios of PBR specific
binding to nonspecific binding achieved in monkey brain with
either [11C]3 or [11C]10 cannot be assessed by simple inspection
of the regional time-activity curves, since no reference region
devoid of PBR that might represent nonspecific binding was
identified. Moreover, the plasma input function of unchanged
radioligand is highly dependent on the degree to which
peripheral and brain PBR are occupied by carrier ligand or other
blocking/displacing agent (see below); a peripheral blockade
of [11C]10 binding to PBR in peripheral organs, such as kidney,
spleen, and lungs, by PK 11195 has recently been demon-
strated.50 Full biomathematical analysis of brain data in
conjunction with the measured plasma input function of
unchanged radioligand is required to determine specific binding
in any brain region. Such detailed analyses for [11C]351 and
[11C]1052 have revealed a very high level of specific binding
(>90%) for these radioligands in monkey brain.
radioactivity in plasma by 90 min. The time taken for radiome-
tabolite to equal parent radioligand in plasma was faster in the
preblock experiment (6.0 min) than in the baseline experiment
(18.5 min) conducted in the same monkey (Figure 6). These
findings again appear consistent with the much greater early
level of parent radioligand in plasma in the preblock experiment
than in the baseline experiment (Figure 8).
Experiments were performed in rats to obtain more detailed
information on the metabolism of the two radioligands. Each
radioligand was found to be metabolized to a single polar
radiometabolite as in monkey. At 30 min after the injection of
high specific radioactivity [11C]3 or [11C]10 into rats, the parent
radioligand represented 13.1% or 4.2% of radioactivity in
plasma, respectively (Table 2). These measures were appreciably
lower (3.4% and 2.7%, respectively) when the radioligands were
administered with much higher amounts of carrier ligand (Table
2). Again, a greater availability of the radioligands in plasma
to metabolizing enzymes due to partial blockade of PBRs by
the higher amounts of carrier may account for this difference.
After administration of radioligand at high specific radioactivity
(low carrier), the radioactivity in brain at 30 min was predomi-
nantly parent radioligand (83% for [11C]3 and 97.6% for
[11C]10) (Table 2). For radioligands administered at low specific
radioactivity (high carrier) the percentage of radioactivity present
as parent radioligand in brain was lower. This was especially
so for [11C]3. In three rats given [11C]10 with different carrier
doses there was a clear trend of reduced amount of radiome-
tabolite in brain with increased carrier dose. Even at very high
carrier dose the proportion of radioactivity in brain represented
by [11C]10 was high at 90%. The effect of increasing carrier
on reducing the proportion of radioactivity in brain represented
by parent radioligand was probably due to (i) less PBR-specific
radioligand binding in brain and (ii) more rapid appearance of
radiometabolite in plasma, each due to partial blockade of PBR.
In one experiment with [11C]10, in which the rat was perfused
with saline during sacrifice, the composition of radioactivity in
brain (97.8% [11C]10) was similar to that expected in a
nonperfused rat given the same amount of carrier. Hence, we
conclude that radioactivity in blood did not greatly affect the
measurements in nonperfused rats. In the high carrier experiment
with [11C]10, nearly all the radioactivity found in urine was
radiometabolite (Table 2).
Experiments were performed in rats to elucidate the metabolic
pathways of [11C]3 and [11C]10. LC-MS and MS-MS analyses
of rat brain extracts after intravenous administration of 3
detected 3 plus the acid 4 from its hydrolysis. In urine, only
the parent ligand was detected. No metabolites from other
conceivable routes of metabolism were detected in either brain
or urine. The identified hydrolytic route of metabolism of 3,
acting on [11C]3, would first give [11C]methanol as radiome-
tabolite, which would then be converted rapidly into [11C]carbon
dioxide and be expired. Hence, [11C]3 is not expected to give
radiometabolite that might accumulate in the brain to confound
accurate quantitation of PBR.
In monkey, [11C]3 was found to be almost completely
metabolized to a single polar radioactive metabolite by 120 min
(Figure 5). The time taken for radiometabolite in blood to equal
parent radioligand was faster in the self-block experiment (8.2
min) than in the baseline experiment (20.2 min) (Figure 5). This
difference might be explained by the higher level of parent
radioligand in plasma in the self-block than in the baseline
experiment during the early phase of the experiments (Figure
7), and therefore the greater early availability of parent
radioligand to metabolizing enzymes.
LC-MS and MS-MS analyses of rat brain extracts and urine
after the administration of 10 detected 10 and also 4-phenoxy-
3-acetylaminopyridine arising (8) from N-debenzylation. This
route of metabolism has been observed for structurally related
compounds, such as [18F]FEDAA1106.39 The N-debenzylation
of [11C]10 should also give [11C]2-methoxybenzaldehyde
([11C]14) in equimolar amount to 8. [11C]14 would be expected
to be oxidized rapidly to [11C]2-methoxybenzoic acid ([11C]15)
(Scheme 4). After the administration of 10 to rat, 15 was
detected in urine, confirming N-debenzylation to be the main
In monkey, [11C]10 was also found to be metabolized to a
single polar radiometabolite, which represented almost all

24 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Scheme 4. Metabolism of [11C]10 in Rat
Briard et al.
General Methods. γ-Radioactivity from 11C was measured with
a calibrated dose calibrator (Atomlab 300, Biodex Medical Systems)
or for low levels (<1 µCi) with a well-type γ-counter (model 1080
Wizard, Perkin-Elmer) having an electronic window set between
360 and 1800 keV. 3H was measured with a liquid scintillation
counter (Tri-Carb, Perkin-Elmer). 11C radioactivity measurements
were corrected for background and physical decay.
1H NMR (400 MHz) and 13C NMR (100 MHz) 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 tetramethylsilane.
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 Urbana–
Champaign (Urbana, IL), under electron ionization conditions using
a double-focusing high-resolution mass spectrometer (Autospec,
Micromass Inc.) with samples introduced through a direct insertion
probe.
route of metabolism of 10. [11C]15 would not be expected to
accumulate in the brain because at physiological pH it will be
almost fully ionized (pKa ≈ 4.1; ref 53) and have a very low
LogD (calculated as -1.52). Moreover, we established that
[11C]10 was stable in rat brain homogenate for 2 h, showing
that radiometabolite was not generated in the brain but in the
periphery. Hence, this radiometabolite should not prove to be
troublesome for the quantitation of brain PBR with [11C]10.
The findings on routes of metabolism in rats are not
necessarily transferable to monkeys or humans. Nevertheless,
since one single polar radiometabolite was found for each
radioligand in rat and rhesus monkey, it is very likely that the
same routes of metabolism occur in these species.
LC-MS (general method A) was performed on a LCQ Deca
instrument (Thermo Fisher Scientific; Waltham, MA) equipped with
a reverse-phase HPLC column (Synergi Fusion-RP, 4 µm, 150 mm
× 2 mm, Phenomenex; Torrance, CA) The instrument was set up
to perform electrospray ionization (spray voltage 5 kV, nitrogen
sheath flow 65 units, auxiliary gas flow 10 units, capillary voltage
35 V, and capillary temperature 260 °C). In the full scan MS
acquisition, the instrument was set up to detect ions m/z 150-750.
For the characterization of synthesized compounds the column was
eluted at 150 µL/min either isocratically or with a gradient of a
combination of water/methanol/acetic acid (90:10:0.5 by volume)
and methanol/acetic acid (100:0.5 v/v). For the analysis of parent
ligand and metabolites extracted from biological material the LC
column was equilibrated with 80% mobile phase A (10 mM aqueous
HCOONH4) and 20% mobile phase B [MeCN/H2O, 90:10 v/v
containing 10 mM HCOONH4]. The extracted sample was injected
into the LC-MS instrument and eluted at 150 µL/min with a
mixture of 80% A and 20% B for 2 min followed by a linear
gradient reaching 20% A and 80% B over 8 min which was then
held for 6 min. The LC effluents were diverted from waste into
the electrospray module of the LC-MS at 2.5 min after the start
of each analysis. At the end of each run, the mobile-phase
composition was returned to 80% A and 20% B at 250 µL/min
over 1 min. The column was allowed to equilibrate for 3 min before
the start of another analysis.
Conclusions
Both [11C]3 and [11C]10 showed more favorable properties
than [11C]PK 11195 for imaging PBR in monkey brain,
including higher brain entry, higher PBR-specific signal, low
nonspecific binding, and metabolism limited to the production
of a single polar radiometabolite. Overall [11C]10 may be
regarded as a marginally more promising PET radioligand than
[11C]3 because of its superior brain entry, higher specific signal,
and easily measurable free fraction in blood. This conclusion
is also supported by rigorous kinetic analyses of [11C]351 and
[11C]10.52 [11C]10 is already beginning to find application in
rats54 and warrants further evaluation in human subjects, which
is now in progress under an FDA-approved exploratory IND.
For LC-MS-MS (general method B), the molecular ion was
isolated with 1.5 amu width and dissociated in the ion trap at a
specific collision energy level.
Experimental Section
GC-MS analyses (general method C) were performed on a
Polaris Q instrument (Thermo Fisher Scientific Corp.) equipped
with a capillary GC column (J & W DB-5MS, 30 m × 0.252 mm
× 0.25 µm, Agilent Technologies). The helium flow rate was set
at 1 mL/min, injector temperature to 220 °C, and transfer line to
250 °C. The sample was injected splitless (for 1 min) at a column
temperature of 60 °C. After 1 min the temperature was increased
to 180 °C at a rate of 20 °C/min and held at this temperature for
5 min. Subsequently, the column was returned to the initial
temperature. A full scan acquisition (m/z 60-400) was performed
in positive-ion, electron-ionization mode of operation. The source
temperature of the mass spectrometer was 250 °C.
Materials. Poly(ethyleneimine) and PK 11195 [1-(2-chlorophe-
nyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide] were
purchased from Sigma (St. Louis, MO). DAA1106 (N-(2,5-
dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide) was syn-
thesized from commercially available 2,5-difluoronitrobenzene
according to the route described previously.45 An ethanolic solution
of [N-methyl-3H]PK 11195 was purchased from Perkin-Elmer
(Wellesley, MA) at a specific activity of 83.5 Ci/mmol. The
radiochemical purity of [3H]PK 11195 was verified to be >97%
by instant thin layer chromatography (ITLC-SGl layers; Pall
Gelman, East Hills, NY) with the use of three mobile phases
(CHCl3/MeOH, 95:5 v/v; EtOAc/hexane, 80:20 v/v; MeOH/H2O/
Et3N, 97:3:0.1, by volume). [11C](R)-PK 11195 was prepared via
methylation of (R)-desmethyl-PK 11195 (from ABX, Radeberg,
Germany) with [11C]iodomethane (see below), essentially according
to a previously described method17. 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 Animals55 and the
National Institute of Mental Health Animal Care and Use Com-
mittee. Human brain tissue was obtained from the Clinical Brain
Disorders Branch, National Institute of Mental Health, and experi-
ments with this material were performed under the regulations of
the Ethics Committee of the National Institutes of Health.
Analytical radio-HPLC of [11C]3 and [11C]10 plus their radi-
ometabolites from samples of biological material (general method
D) was performed on a Nova-Pak C18 column (4 µm, 100 mm ×
8 mm, Waters Corp.) housed in a radial compression module (RCM
100). Mobile phases for the analyses consisted of MeOH/H2O/Et3N
(80:20:0.1 by volume for [11C]3 and its metabolites and 75:25:0.1
by volume for [11C]10 and its metabolites) at a flow rate of 1.5
mL/min unless otherwise stated. That all radioactivity had eluted
from the column during each analysis was shown by subsequent
injection of methanol (2 mL) and observation that no more
radioactivity was eluted. The same HPLC method was applied to
determine the radiochemical purities of [11C]3 and [11C]10 preced-

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 25
ing lipophilicity determinations and for the determination of
radiochemical stability in various media (see below).
Melting points were measured with a Mel-Temp manual melting
point apparatus (Electrothermal, Fisher Scientific) and were
uncorrected.
171.62, 172.18. LC-MS, m/z 362.13 (M+ + H). HRMS calcd for
C22H20NO4 (M+ + H), 362.1392; found, 362.1408.
4-Chloro-3-nitropyridine (5).59 4-Hydroxy-3-nitropyridine (5.0
g, 36 mmol) was added to a stirred slurry of phosphorus pentachlo-
ride (8.3 g, 40 mmol) and phosphoryl chloride (2 mL, 21.5 mmol)
and held at 60 °C. The mixture solidified. The temperature was
raised to 140 °C and held for 6 h. Volatile material was then
removed in vacuo at room temperature and the residue poured onto
ice–water. Sodium carbonate was added dropwise while maintaining
the solution at 0 °C until the mixture was basic. The mixture was
extracted with diethyl ether, and the organic layers were filtered
through Celite and dried over magnesium sulfate. Evaporation of
solvents at room temperature gave a residue of 5 (4.9 g, 86%) that
was stored under nitrogen in a freezer preceding rapid use in the
next step. 1H NMR (CDCl3): δ 7.65 (1H, d, J ) 5.4 Hz), 8.80
(1H, d, J ) 5.4 Hz), 9.24 (1H, s).
Methyl 2-Formylbenzoate (1).56,57 To a solution of 2-carboxy-
benzaldehyde (4.0 g, 26.6 mmol) and potassium carbonate (11 g,
80 mmol) in acetone (50 mL) was added iodomethane (1.66 mL,
26.7 mmol) at room temperature. The mixture was refluxed under
nitrogen for 4 h, cooled to room temperature, filtered, and
concentrated. The residue was extracted into chloroform, washed
successively with water, saturated sodium bicarbonate solution, and
saturated brine, and then dried over magnesium sulfate to give 1
(3.4 g, 78%). Mp 50 °C. Lit. mp 50 °C.56 1H NMR (CDCl3): δ
3.98 (3H, s), 7.63-7.67 (2H, m), 7.92-7.99 (2H, m), 10.62 (1H,
s). 13C NMR (CDCl3): δ 52.79, 128.44, 130.39, 132.41, 132.99,
134.44, 137.70, 166.77, 192.16. GC-MS m/z 165.05 (M + H)+.
N-(2-Methoxycarbonylbenzyl)-2-phenoxyaniline (2). A solu-
tion of 1 (0.89 g, 5.4 mmol) and 2-phenoxyaniline (1.0 g, 5.4 mmol)
in methanol (6 mL) was stirred for 24 h at room temperature. Then
sodium borohydride (0.76 g, 20 mmol) was added slowly and the
mixture stirred for 1 h. Acetic acid (5% v/v, 16 mL, 14 mmol) was
added dropwise to the mixture, which was then stirred at room
temperature for 30 min and extracted thrice with ethyl acetate. The
organic layers were combined, washed with saturated sodium
bicarbonate solution and then brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. Flash chromatography of the
residue on silica gel (hexane/EtOAc, 97:3 v/v) gave 2 (1.17 g, 65%).
3-Nitro-4-phenoxypyridine (6).46 To a solution of phenol (3.1
g, 33 mmol) in THF (10 mL) was added a solution (1 M) of
potassium tert-butoxide (33 mmol) in THF (33 mL) at room
temperature. Then a solution of 5 (4.9 g, 31 mmol) in THF (20
mL) was slowly added. The mixture was stirred for 1 h at room
temperature and then poured into ice–water, extracted with ethyl
acetate, washed with saturated brine, dried over magnesium sulfate,
and finally concentrated. The residue was crystallized from pentane
to give 6 (6.3 g, 93%). Mp 75 °C. Lit. mp 73-75 °C.46 1H NMR
(CDCl3): δ 6.88 (1H, d, J ) 5.7 Hz), 7.25-7.65 (5H, m), 8.65
(1H, d, J ) 5.7 Hz), 9.24 (1H, s). 13C NMR (CDCl3): δ 111.90,
120.93, 126.75, 130.66, 136.75, 147.47, 152.74, 154.57, 158.24.
LC-MS m/z 217.3 (M+ + H). HRMS calcd for C11H9N2O3 (M+
+ 1), 217.0613; found, 217.0612.
1
Mp 72-74 °C. H NMR (CDCl3): δ 3.82 (3H, s), 4.73 (2H, s),
4.96 (1H, s br), 6.60-6.67 (2H, m), 6.83-7.08 (4H, m), 7.25-7.35
(4H, m), 7.44-7.49 (2H, m), 7.94 (1H, d, J ) 7.7 Hz). 13C NMR
(CDCl3): δ 46.44, 52.04, 112.01, 116.90, 117.13, 119.66, 122.57,
124.97, 126.97, 128.73, 128.82, 129.66, 131.11, 132.36, 140.29,
141.42, 142.87, 157.72, 167.72. LC-MS, m/z 334.14 (M+ + H).
HRMS calcd for C21H20NO3 (M+ + H), 334.1443; found, 334.1442.
4-Phenoxy-3-pyridinamine (7). In a 100 mL two-necked flask
fitted with a reflux condenser were placed 6 (6.6 g, 31 mmol), iron
powder (325 mesh, 5.2 g, 93 mmol), and aqueous ethanol (50%, 6
mL). The mixture was heated to boiling, and hydrochloric acid
(37%, 0.35 mL) in aqueous ethanol (50%, 2 mL) was added slowly.
The mixture was then refluxed for 3 h. Then the hot solution was
made just alkaline by adding aqueous sodium hydroxide solution
(0.5 M, 8 mL), cooled, and filtered over Celite. Solvent was
evaporated off. The residue was dissolved in dichloromethane and
washed with sodium bicarbonate solution. The organic layer was
dried over magnesium sulfate and evaporated to dryness to give 7
N-Acetyl-N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline
(3).58 Acetyl chloride (0.26 g, 3.3 mmol) was slowly added to a
solution of 2 (1.0 g, 3.0 mmol) and triethylamine (0.3 g, 3.0 mmol)
in dichloromethane (5 mL) at 0 °C. The mixture was stirred for
1 h at room temperature, poured into water, and extracted with
dichloromethane. The organic layers were combined and washed
successively with hydrochloric acid (0.5 M), saturated sodium
bicarbonate solution, and saturated brine, and then dried over
magnesium sulfate and concentrated in vacuo. The crude product
was purified with flash chromatography on silica gel (hexane/
EtOAc, 70:30 v/v) to give 3 (0.98 g, 71%). Mp 80 °C. Lit. mp
78-80 °C.45,58 1H NMR (CDCl3): δ 1.98 (3H, s), 3.71 (3H, s),
5.19 (1H, d, J ) 15.3 Hz), 5.50 (1H, d, J ) 15.6 Hz), 6.83-7.41
1
as a dark-red oil (4.2 g, 74%). H NMR (CDCl3): δ 3.88 (2H, s),
6.57 (1H, d, J ) 5.1 Hz), 7.05-7.10 (2H, m), 7.18-7.27 (1H, m),
7.37-7.45 (2H, m), 7.89 (1H, d, J ) 5.4 Hz), 8.16 (1H, s). 13C
NMR (CDCl3): δ 110.97, 119.89, 124.87, 130.10, 134.09, 138.06,
140.85, 151.10, 154.56. HRMS calcd for C11H11N2O (M+ + 1),
187.0871; found, 187.0864.
N-Acetyl-N-3-(4-phenoxy)pyridine (8). Acetyl chloride (66 µL,
0.82 mmol) was added dropwise to a solution of 7 (0.14 g, 0.76
mmol) plus triethylamine (0.13 mL, 0.92 mmol) in CH2Cl2 (2 mL)
cooled in a dry ice bath. The cold bath was removed and the solution
stirred at room temperature for 1 h. Then the reaction mixture was
concentrated under vacuum, and the residue was poured into water
(10 mL) and extracted with AcOEt (2 × 10 mL). The combined
organic layers were washed with saturated sodium bicarbonate
solution and saturated brine, dried over MgSO4, filtered, and
concentrated under vacuum to obtain an oil that was purified on
silica gel (hexane/AcOEt, 6:4 to 4:6) to provide 8 as fine needles
(m, 11H), 7.62 (1H, d, J ) 7.5 Hz), 7.78 (1H. d, J ) 7.5 Hz). 13
C
NMR (CDCl3): δ 22.18, 49.48, 51.94, 118.39, 119.38, 123.31,
124.12, 126.95, 129.13, 129.75, 129.89, 130.10, 130.24, 130.31,
132.05, 133.11, 138.57, 153.49, 155.73, 167.81, 171.26. LC-MS,
m/z 376.15 (M + H)+. HRMS calcd for C23H21NO4 (M+ + H),
376.1549; found, 376.1549. LC (Luna C-18 column, 5 µm, 10 mm
× 250 mm, Phenomenex; MeCN/0.1 M HCOONH4, 70:30 v/v; 2
mL/min; λ ) 254 nm): tR ) 4.72 min, purity 100%.
N-Acetyl-N-(2-carboxybenzyl)-2-phenoxyaniline (4). Com-
pound 3 (0.40 g, 1.06 mmol) was treated with potassium hydroxide
in methanol (5% w/v) at room temperature for 1 h. The mixture
was concentrated in vacuo, poured into water, and extracted with
ethyl acetate. The organic layers were combined, washed with
hydrochloric acid (0.5 M), saturated sodium bicarbonate and
saturated brine, and then dried over magnesium sulfate, filtered,
and concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel to give 4 (0.33 g, 85%). Mp 142-144
1
(95 mg, 55%). Mp 74-76 °C. H NMR (CDCl3) δ 2.27 (3H, s),
6.59 (d, J ) 5.56 Hz, 1H), 7.11 (d, J ) 8.54 Hz, 2H), 7.3 (t, J )
7.44 Hz, 1H), 7.46 (t, J ) 7.0 Hz, 2H), 7.69 (s, 1H), 8.19 (d, J )
5.52 Hz, 1H). 13C NMR (CDCl3): δ 24.60, 109.50, 120.70, 125.62,
125.97, 130.43, 142.77, 145.78, 153.06, 153.55, 168.22. LC-MS
m/z 229 (M + H)+.
N-(2-Methoxybenzyl)-4-phenoxypyridin-3-amine (9). A mix-
ture of 7 (0.8 g, 4.3 mmol) and 2-methoxybenzaldehyde (0.58 g,
4.3 mmol) in toluene (10 mL) was heated to reflux. A Dean–Stark
trap was used to collect azeotropically removed water. After 24 h
the mixture was cooled to room temperature and concentrated in
vacuo to remove toluene. The residue was dissolved in methanol
(20 mL) and cooled to 0 °C. Sodium borohydride (0.64 g, 17.2
1
°C. H NMR (CDCl3): δ 2.00 (3H, s), 5.07 (1H, d, J ) 15.6 Hz),
5.42 (1H, d, J ) 15.3 Hz), 6.85-7.46 (11H, m), 7.60 (d, 1H, J )
7.5 Hz), 7.88 (d, 1H, J ) 7.5 Hz). 13C NMR (CDCl3): δ 22.09,
50.29, 118.60, 119.36, 123.53, 124.25, 127.23, 129.44, 129.88,
129.96, 130.02, 130.97, 132.52, 133.20, 138.60, 153.29, 155.67,

26 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Briard et al.
mmol) was added portionwise. The mixture was then stirred for
1 h at room temperature with further additions of sodium borohy-
dride until reaction was shown to be complete by TLC. Acetic acid
(5%, 16 mL) was added dropwise to the mixture, which was then
stirred at room temperature for 30 min and extracted thrice with
ethyl acetate. The organic layers were combined, washed with
saturated sodium bicarbonate solution and brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel to give 9 as a
138.70, 150.28, 155.27, 155.66. LC-MS m/z 293.12 (M+ + H).
HRMS calcd for C18H17N2O2 (M+ + H), 293.1290; found,
293.1279.
N-(2-Hydroxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide
(13). Compound 12 (0.3 g, 1 mmol) was refluxed with acetic
anhydride (0.19 mL, 2 mmol) in acetic acid (3 mL) at reflux for
3 h. Solvent was removed under vacuum. The residue was basified
with aqueous sodium hydroxide (1 M) and extracted with ethyl
acetate. The organic layer was washed successively with water,
saturated sodium bicarbonate solution, and brine, dried over
magnesium sulfate, filtered, and concentrated. This crude product
was treated with potassium hydroxide in methanol (5% w/v, 5 mL)
at room temperature for 3 h. The mixture was concentrated in vacuo,
poured into water, and extracted with ethyl acetate. The combined
organic layers were washed successively with hydrochloric acid
(0.5 M), saturated sodium bicarbonate solution and saturated brine,
then dried over magnesium sulfate, filtered, and concentrated in
vacuo to give 13 (0.21 g, 63%). Mp 131-133 °C. 1H NMR
(CDCl3): δ 2.02 (3H, s), 4.81 (2H, s), 6.62-6.80 (5H, m), 6.94
(1H, d, J ) 8.1 Hz), 7.19-7.29 (2H, m), 7.36-7.42 (2H, m), 8.30
(1H, m), 8.40 (1H, d, J ) 5.7 Hz), 9.26 (1H, s). 13C NMR (CDCl3):
δ 21.70, 50.81, 110.66, 117.78, 119.37, 120.72, 121.55, 126.29,
127.91, 130.39, 131.30, 150.90, 151.54, 152.74, 156.15, 160.61,
173.76. LC-MS m/z 335.1 (M+ + H). HRMS calcd for
C20H19N2O3 (M+ + H), 335.1396; found, 335.1396.
1
red oil (1.2 g, 90%). H NMR (CDCl3): δ 3.83 (3H, s), 4.45 (2H,
s), 4.68 (1H, br s), 6.53 (1H, d, J ) 5.4 Hz), 6.87-6.95 (2H, m),
7.02-7.07 (m, 2H), 7.16-7.42 (m, 5H), 7.84 (1H, d, J ) 5.7 Hz),
8.09 (1H, s). 13C NMR (CDCl3): δ 43.05, 55.28, 110.33, 110.38,
119.89, 120.57, 124.74, 126.54, 128.64, 128.93, 130.05, 130.11,
134.12, 135.70, 139.53, 151.02, 154.85, 157.44. LC-MS m/z
307.14 (M+ + H). HRMS, calcd for C19H19N2O2 (M+ + H),
307.1447; found, 307.1446.
N-(2-Methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide
(10).46 Compound 9 (0.60 g, 20 mmol) was dissolved in glacial
acetic acid (10 mL), and then acetic anhydride (1.9 mL, 20 mmol)
was added dropwise at room temperature. The reaction mixture
was stirred for 2 h at reflux. Acetic acid was removed under reduced
pressure. Sodium hydroxide solution (1 M) was added to the residue
and the product extracted with ethyl acetate (3 × 10 mL). The
organic layers were combined, washed with cold water, dried over
magnesium sulfate, filtered, and concentrated. The residue was
purified by flash chromatography on silica gel (CH2Cl2/MeOH, 95:5
v/v) to give 10 (0.57 g, 84%). Mp 89-91 °C. Lit. mp 92.5–93.0
°C.46 1H NMR (CDCl3): δ 1.99 (3H, s), 3.59 (3H, s), 4.90 (1H, d,
J ) 14.1 Hz), 5.16 (1H, d, J ) 14.1 Hz), 6.57 (1H, d, J ) 5.6 Hz),
6.76 (1H, d, J ) 8.2 Hz), 6.87-6.95 (m, 3H), 7.20-7.31 (2H, m),
7.38-7.45 (3H, m), 8.22 (1H, s), 8.29 (1H, d, J ) 5.6 Hz). 13C
NMR (CDCl3): δ 22.32, 46.10, 55.06, 110.21, 110.39, 120.57,
120.73, 124.76, 125.92, 128.76, 129.05, 130.31, 131.39, 150.45,
Radiosynthesis of [11C]Iodomethane. No-carrier-added [11C]car-
bon dioxide (∼1.4 Ci) was produced in a target of nitrogen gas
(∼225 psi) containing oxygen (1%) via the 14N(p,R)11C reaction
induced by irradiation with a 16 MeV proton beam (45 µA) for 40
min from
a PETrace cyclotron (GE; Milwaukee, WI).
[11C]Iodomethane was produced within a lead-shielded hot-cell
from the [11C]carbon dioxide via reduction to [11C]methane and
iodination with a MeI MicroLab apparatus (GE; Milwaukee, WI).
Radiosynthesis of [11C]3. At about 3 min before the end of
radionuclide production (ERP), the acid precursor 4 (1 mg, 3.0
µmol) was dissolved in DMF (80 µL). At 1 min before ERP this
solution was treated with tetra-n-butylammonium hydroxide in
methanol (1.0 M, 2.8 µL) and loaded into a 2 mL stainless steel
loop60 of a commercially available radiomethylation apparatus
(Bioscan; Washington, DC) housed within a lead-shielded hot cell.
The precursor was conditioned by passage of helium (12 mL/min)
through the loop for 10 min. [11C]Iodomethane was then swept
into the loop in a stream (12 mL/min) of helium until radioactivity
in the loop was maximized (about 4 min after release of
[11C]iodomethane from the MeI MicroLab apparatus). Radiom-
ethylation was allowed to proceed for 5 min at room temperature.
Crude [11C]3 was flushed from the loop in the HPLC mobile phase
(2 mL, MeCN, 10 mM HCOONH4, 65:35 v/v) and purified with
HPLC on an Ultrasphere C-18 column (5 µm, 10 mm × 250 mm,
Beckman) eluted with the mobile phase at 8 mL/min with the eluate
monitored for radioactivity (pin diode, Bioscan) and absorbance at
254 nm (System Gold 166, Beckman). [11C]3 (tR ) 4.8 min) was
collected, concentrated to dryness under reduced pressure and heat
(80 °C), formulated in sterile physiological saline (0.9% w/v, 10
mL) containing ethanol (5% v/v), and finally passed through a sterile
filter (0.2 µm pore size, Millex-GV, Millipore; Bedford, MA) into
a sterile, pyrogen-free dose vial.
151.70, 153.33, 157.63, 160.84, 170.67. LC-MS 349.15 (M+
+
H). HRMS calcd for C21H21N2O3 (M+ + 1), 349.1552; found,
349.1543. LC (Onyx C-18 column, 4.6 mm × 100 mm, Phenom-
enex; MeCN/0.01 M HCOONH4, 30:70 v/v; 3 mL/min; λ ) 220
nm): tR ) 5.91 min, purity 99.85%.
2-Acetoxybenzaldehyde (11). To a solution of salicylaldehyde
(2.0 g, 16 mmol) in pyridine (10 mL) at 0 °C was added acetic
anhydride (1.7 mL, 18 mmol). The mixture was stirred at room
temperature overnight, poured into water, and extracted with ethyl
acetate. The organic layers were combined, washed with water,
hydrochloric acid (10%) and saturated brine, and then dried over
magnesium sulfate, filtered, and concentrated to give 11 (2.5 g,
95%). 1H NMR (CDCl3): δ 2.39 (3H, s), 7.15-7.89 (4H, m), 10.1
(1H, s). 13C NMR (CDCl3): δ 20.78, 123.42, 126.06, 131.28,
134.61, 135.28, 151.10, 169.26, 188.83.
2-((4-Phenoxypyridin-3-ylamino)methyl)phenol (12). A mix-
ture of 7 (2.0 g, 10 mmol) and 11 (1.76 g, 10 mmol) in toluene (10
mL) was heated to reflux. A Dean–Stark trap was used to collect
the azeotropically removed water. After 24 h the mixture was cooled
to room temperature and concentrated in vacuo to remove toluene.
The residue was dissolved in methanol (20 mL) and cooled to 0
°C. Sodium borohydride (1.51 g, 40 mmol) was added portionwise,
the ice-bath removed, and the solution stirred for 3 h at reflux.
After dropwise addition of acetic acid (5%, 16 mL), the mixture
was stirred at room temperature for 30 min and then extracted thrice
with ethyl acetate. The organic layers were combined, washed with
saturated sodium bicarbonate solution and brine, and then concen-
trated in vacuo. Methanol was added and the obtained precipitate
filtered off and dried in vacuo to give 12 (0.78 g, 25%). Mp
The radiochemical purity, SR, and chemical purity of formulated
[11C]3 were assessed by analytical HPLC of a 0.1 mL aliquot on
a C-18 Luna column (4.6 mm × 100 mm) eluted with MeCN/10
mM HCOONH4 (70:30 v/v) at 2 mL/min, with eluate monitored
for radioactivity (pin diode, Bioscan) and absorbance at 254 nm
(System Gold 166, Beckman) (tR of [11C]3 ) 5.0 min).
Radiosynthesis of [11C]10. At about 3 min before ERP, the
phenol precursor 13 (1 mg, 3.0 µmol) was treated with tetra-n-
butylammonium hydroxide in methanol (0.5 M, 4 µL). This solution
was loaded into the loop of stainless steel tubing (internal volume,
2 mL) of a commercially available radiomethylation apparatus
(Bioscan) housed within a lead-shielded hot cell. The loop was then
conditioned by flushing with helium (12 mL/min) for 10 min.
[11C]Iodomethane was swept into the loop in a stream (12 mL/
1
186-187 °C (dec). H NMR (DMSO-d6): δ 4.35 (2H, d, J ) 8.4
Hz), 5.91 (1H, t, J ) 8.4 Hz), 6.54 (1H, d, J ) 6.8 Hz), 6.74 (1H,
t, J ) 7.2 Hz), 6.83 (1H, d, J ) 8.1 Hz), 7.02-7.26 (5H, m),
7.42-7.49 (2H, m), 7.70 (1H, d, J ) 5.4 Hz), 7.86 (1H, s), 9.61
(1H, s). 13C NMR (DMSO-d6): δ 41.43, 111.12, 115.50, 119.21,
120.02, 125.00, 125.50, 128.13, 128.52, 130.66, 133.89, 136.02,

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 27
min) of helium until radioactivity in the loop was maximized (about
4 min after release of [11C]iodomethane from the MeI MicroLab
apparatus). Radiomethylation was allowed to proceed for 3 min at
room temperature. Crude [11C]10 was flushed from the loop with
HPLC mobile phase (2 mL, MeCN:10 mM HCOONH4, 47: 53,
v/v) and purified with HPLC on a Luna C-18 column (10 µm, 10
mm × 250 mm, Phenomenex) eluted with mobile phase at 8 mL/
min, with eluate monitored for radioactivity and absorbance at 254
nm. [11C]10 (tR ) 7.5 min) was collected, concentrated to dryness
by rotary evaporation under reduced pressure and heat (80 °C),
formulated in sterile physiological saline (0.9% w/v; 10 mL)
containing ethanol (5% v/v), and finally passed through a sterile
filter (0.2 µm pore size, Millex-GV, Millipore) into a sterile,
pyrogen-free dose vial.
Specific radioactivity, chemical purity, and radiochemical purity
of [11C]10 were assessed by HPLC of a 0.1 mL aliquot of
formulated [11C]10 on a monolithic Onyx C-18 column (4.6 mm
× 100 mm) eluted with MeCN/10 mM HCOONH4 (30:70 v/v) at
6 mL/min with eluate monitored for radioactivity and absorbance
at 220 nm (tR of [11C]10 ) 3.0 min).
concentration of radioligand, 0.38 nM). Different amounts of test
ligand in buffer (100 µL) were placed in eight tubes. For
determination of nonspecific binding to brain tissue, buffer (100
µL) containing the highest inhibitor concentration (set between 0.1
and 0.3 µM) was placed in the 9th tube, and for determination of
nonspecific binding to the filter, buffer (900 µL) was placed in the
10th tube. Buffer (100 µL) was placed in the 11th tube for
determination of total binding. Buffer (1.0 mL) alone was placed
in a 12th tube to provide a measure of background radioactivity.
The assay was initiated by adding mitochondrial fraction (800 µL)
to all tubes except the 10th and 12th tubes. The mixtures were
incubated for 2 h at 4 °C and then with a cell harvester (Brandel)
rapidly filtered through a GF/B filter (Whatman) that had been
presoaked with 0.5% polyethyleneimine and washed thrice with
ice-cold buffer (3 mL). Radioactivity on each filter was counted.
Triplicate curves (nine tubes per curve) were determined in each
of two independent experiments. For each test compound, self-
displacement of [3H]PK 11195 with PK 11195 was used as a
control. IC50 values were calculated with GraphPad Prism version
4.03 (GraphPad Software; San Diego, CA) with “one site competi-
tion” curve-fitting. Ki values were calculated according to the Cheng
and Prusoff equation:63
Computation of cLogP and cLogD and Measurement of
LogD. cLogP and cLogD (at pH 7.4) values for 3, 10, 15, PK
11195, and DAA1106 were computed with the program Pallas 3.0
for Windows (CompuDrug; S. San Francisco, CA).
IC50
Ki )
The initial radiochemical purities of samples of [11C](R)-PK
11195, [11C]3, and [11C]10 to be used in the determination of LogD
values were measured with radio-HPLC (general method C). LogD
values were then measured by mixing a solution of 11C-labeled
ligand (∼300 µCi) in saline (17 µL) containing ethanol (5% w/v)
with sodium phosphate buffer (0.15 M, pH 7.4, 17 mL). Aliquots
(1 mL) were added to each of six tubes and extracted with n-octanol
(that had been pre-equilibrated with the phosphate buffer) by
vortexing for 1 min. Each tube was then centrifuged for 1 min,
and the phases were separated. Samples of each phase (200 µL)
were counted for radioactivity, as previously described,61 except
that each of the remaining aqueous phases was also submitted to
radio-HPLC analysis (general method C) to obtain the proportion
of radioactivity present as “unchanged radioligand”. The latter was
used to correct the γ counts to obtain the true radioligand counts
(corrected cpmaq phase). (Such a correction was not applied to the
octanol phase, since the measurement of the relatively very high
activity of this phase, due to the extracted lipophilic radioligand,
would not be prone to significant error from a very minor level of
radioactive impurity.) Two more tubes containing phosphate buffer
(0.1 M) and radioligand also served as a storage medium, usually
for the duration of the study (3-4 h), after which the radiochemical
purity and specific concentration of the radioligand were assayed
to assess radiochemical stability and adsorption to the tube,
respectively. LogD was calculated as log(cpmorganic phase/corrected
cpmaq phase).
[L]
1 +
KD
where [L] is the concentration (0.38 nM) and KD the equilibrium
dissociation constant of the reference radioligand ([3H]PK 11195).
The latter was determined with “Scatchard analysis of homologous
displacement” from multiple runs of PK 11195 with self-displace-
ment in each tissue type.
Ligand 10 was also submitted to the National Institute of Mental
Health Psychoactive Drug Screening Program (NIMH-PDSP) for
assessment of binding affinity against a wide range of other
receptors and binding sites (GABAA subtypes, 5-HT1A-E,2A-C,3,5A,6,7
,
R
1A,1B,2A-C, ꢀ1–3, κ opiate, D1–5, DAT, SERT, NET, H1-4, and M2-5).
Detailed assay protocols are available at the NIMH-PDSP Web site
(http://pdsp.cwru.edu).
Monkey PET Imaging Experiments. Three male rhesus
monkeys (Macaca mulatta, 9-15 kg) were used for these PET
experiments, which were radioligand alone (baseline experiments),
receptor block experiments, or radioligand displacement experi-
ments. A single monkey was used for four of the experiments,
namely, the baseline and block experiments with [11C]3 and
[11C]10. In the self-block experiment with [11C]3, 3 (∼1 mg/kg
iv) was given with the radioligand, and in the preblock experiment
with [11C]10, the PBR-selective ligand DAA1106 (2.83 mg/kg iv)
was given 24 min before the radioligand. Different monkeys were
used for the two displacement experiments. In the displacement
experiment with [11C]3, PK 11195 (1 mg/kg) was given by bolus
injection over 2 min at 45 min after radioligand injection, and in
the displacement experiment with [11C]10, PK 11195 (5 mg/kg)
was given by bolus injection over 2 min at 45 min after radioligand
injection. Displacing agents were prepared for administration in
these experiments by dissolution in ethanol (1 mL) plus Cremophor
EL (30 mg) and then dilution to 10 mL with saline for injection.
For each PET experiment, the monkey was initially anesthetized
with ketamine and then maintained under anesthesia with isoflurane
(1.5-2.05%). An intravenous perfusion line, filled with saline (0.9%
w/v), was used for bolus injection of [11C]3 (3.76-4.93 mCi; SR
at time of injection, 2.04-3.73 Ci/µmol; injected mass of carrier
0.38-0.87 µg) or [11C]10 (5.03-7.49 mCi; SR at time of injection,
2.28-8.90 Ci/µmol; injected mass of carrier 0.20-1.14 µg). PET
serial dynamic images were obtained on an Advance (GE Medical
Systems) or HHRT (Siemens/CPS; Knoxville, TN) PET camera.
Scans were obtained for up to 125 min in up to 33 frames. 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, cerebel-
lum, and choroids plexus on the fourth ventricle. Radioactivity was
Ligand Pharmacological Assay and Screen. Rat brain mito-
chondrial fraction was prepared as described previously.62 Briefly,
male Sprague-Dawley rats (300-450 g) were anesthetized by
inhalation of isoflurane (5%) in oxygen and sacrificed by decapita-
tion. Excised brain was quickly cooled over ice, weighed, and
homogenized in cold sucrose solution (0.32 M) in HEPES buffer
(10 mM, pH 7.4, 10 mL). The homogenate was centrifuged at 900g
for 10 min at 4 °C. The supernatant was collected and then
centrifuged at 9000g for 10 min. The resulting pellet was suspended
in HEPES buffer (50 mM, pH 7.4, 10 mL) and then centrifuged at
12000g for 10 min at 4 °C. This pellet was suspended in HEPES
buffer (50 mM, pH 7.4), distributed into aliquots each at a
concentration of 160 mg/mL of the original brain tissue weight,
and stored at -70 °C until use. 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.
Inhibition of [3H]PK 11195 binding to the rat brain mitochondrial
fraction by DAA1106, 3, or 10 was assessed at 4 °C in HEPES
buffer (50 mM, pH 7.4). [3H]PK 11195 (70 × 103 dpm) in buffer
(100 µL) was added to each of 11 borosilicate tubes (final

28 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Briard et al.
normalized for injected dose and monkey weight by expression as
percent standardized uptake value [% SUV ) (% injected dose per
gram) × (body weight in grams)].
In one experiment with [11C]10, three urine samples (100 µL)
were each placed in acetonitrile (700 µL) along with 10 (10 µg)
and further processed for HPLC analysis in the same manner as
plasma samples.
Excised brains were weighed, placed in acetonitrile (2 mL), and
counted for radioactivity. Each brain was then homogenized along
with carrier [3 or 10, ∼50 µg]. Water (0.5 mL) was then added,
and the brain was further homogenized and centrifuged. Precipitates
and supernatant liquids were counted for radioactivity.
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 (General Electric Medical Systems;
Waukesha, WI). PET and NMR images were coregistered with
SPM2 software (Wellcome Department of Cognitive Neurology,
London, U.K.).
Stability of Radioligand in Monkey Whole Blood and Rat
Brain Homogenate In Vitro. [11C]3 or [11C]10 was incubated for
45 min in monkey whole blood (1 mL). A sample of whole blood
(0.45 mL) was removed, added to acetonitrile (0.7 mL), and
centrifuged. Then the supernatant liquid was analyzed by reverse-
phase radio-HPLC (general method C) as described previously in
detail.64
One healthy rat (472 g) was killed and the brain excised and
placed on ice immediately. The brain was homogenized intermit-
tently in 2-fold ice-cold saline (0.9% w/v). [11C]10 (0.5 mCi, 0.5
mL in formulation vehicle) was added to the brain homogenate
and placed in a reciprocating water bath at 37 °C. The mixture
was sampled (100 µL) after 2 h. The samples were deproteinated
with MeCN, diluted in water, centrifuged, and analyzed with radio-
HPLC as described above.
The prepared samples of supernatant liquids from plasma, urine,
and brain were each analyzed by reverse-phase radio-HPLC (general
method D). Mobile phase flow rates were 2.0 and 1.5 mL/min for
the analyses of radiometabolites from [11C]3 and [11C]10,
respectively.
Analysis of Rat Brain and Urinary Metabolites of 3 and
10 by LC-MS-MS and GC-MS. LC-MS and MS-MS data
on reference compounds 3, 4, 8, and 10 and GC-MS data on
reference compound 15 were obtained by analysis of acetonitrile
solutions (10 µg/mL) by general methods A, B, and C, respectively.
A Sprague-Dawley rat (261 g) was placed under anesthesia with
1.5% isoflurane in oxygen and injected through the penile vein over
9 min with 3 (1.4. mg) in 10% aqueous ethanol in saline (0.9%
w/v, 0.9 mL) containing Cremophor EL (30 mg, BASF Ludwig-
shafen; Germany). The urethra was clamped, and every 30 min
the rat was injected with saline (0.9% w/v, 0.1 mL). After 95 min
the bladder was exposed and urine (2.0 mL) withdrawn. The rat
was then sacrificed by injection of a saturated solution of potassium
chloride. The brain was excised, placed in acetonitrile (3 mL),
homogenized with a tissue Tearor (model 985–370, Biospec
Products Inc.), added to water (0.5 mL), and rehomogenized. The
homogenate was centrifuged at 9400g for 10 min and the clear
supernatant liquid decanted off. The precipitate was rehomogenized
with acetonitrile (3 mL) and water and centrifuged as before. The
supernatant liquids were combined. Urine and brain extracts were
stored at -70 °C for later LC-MS (general method A) and
LC-MS-MS analysis (general method B). This procedure was
repeated in a healthy rat (363 g) with injection of 10 (1.33 mg)
over 3 min and exposure of the bladder after 2 h.
Stored urine samples (250 µL) were prepared for LC-MS
analysis (general method A) and LC-MS-MS analysis (general
method B) by centrifugation (10000g, 5 min). The supernatant liquid
was transferred to an autosampler vial and a sample (2 µL) injected
into the LC-MS instrument. The stored extract of brain in
acetonitrile was prepared for LC-MS analysis (general methods
A and B) by evaporation in a SpeedVac apparatus (model SPD
131 DDA, Thermo Fisher Scientific), reconstitution of the residue
in acetonitrile-water (1:1 v/v, 200 µL), and centrifugation. A
sample (8 µL) of the clear supernatant liquid was injected into the
LC-MS instrument.
Stored urine samples (100 µL), from the experiment with 10
only, were also prepared for GC-MS analysis (general method
C). First, they were treated in a glass tube with sodium hydroxide
solution (0.1 M, 100 µL) at 50 °C for 30 min. Then each sample
was cooled, acidified with acetic acid (20 µL), and extracted with
ethyl acetate (2 mL). Following centrifugation (500g, 2 min), the
organic layer was transferred to another tube. The aqueous layer
was extracted once more, and the combined ethyl acetate extracts
were evaporated to dryness. The residue was vortexed with ethyl
acetate (200 µL) and water (30 µL). Then the organic layer was
transferred to an autosampler vial. A sample (1 µL) was injected
into the GC-MS instrument.
Stored rat brain extracts (1 mL) from the experiment with 10
were prepared for GC-MS analysis (general method C) by
evaporation to dryness in a SpeedVac apparatus, mixing of the
residue with ethyl acetate (300 µL) and water (50 µL), and finally
centrifugation. A sample (1 µL) of the organic layer was analyzed.
The urinary and brain metabolites of 3 or 10 were analyzed with
LC-MS (general method A) and MS-MS (general method B).
The molecular ion was dissociated in the ion trap at a collision
energy level of 24% (for 3 and metabolites) or 40% (for 10 and
metabolites).
Binding of [11C]3 and [11C]10 to Monkey Plasma
Proteins.65 The radioligand was added to pooled monkey plasma
(0.5 mL), placed at the top of an “Amicon” Centrifree filter, and
filtered by ultracentrifugation at 5000g. All components of the filter
units were counted for radioactivity to allow calculation of the
percentage of radioligand bound to plasma proteins.
Analysis of Radiometabolites from Monkey Blood. During
monkey PET scans, blood samples were drawn periodically from
the femoral artery and collected in heparin-treated Vacutainer tubes.
The samples were centrifuged, and the plasma was separated. A
plasma sample of known volume (50–200 µL) was measured for
radioactivity. Another sample of plasma of known volume (300-450
µL) was mixed with acetonitrile (0.7 mL) and then centrifuged.
The supernatant liquid was analyzed by radio-HPLC (general
method C) eluted at a flow rate of 2.0 mL/min. The composition
of decay-corrected radioactivity in plasma, in terms of percentages
of radioligand and radiometabolites, was calculated from the
acquired data.
Analysis of Metabolites from Rat Plasma, Urine, and
Brain. All radioligands used in these experiments were of >99.99%
radiochemical purity. Rats (265–312 g) were placed under 1.5%
isoflurane anesthesia and injected intravenously over 3 min with
[11C]3 (0.995 mCi, SR ) 2.42 Ci/µmol, carrier dose 0.54 µg/kg;
or 1.43 mCi, SR ) 682 mCi/µmol, carrier dose 2.96 µg/kg) or
with [11C]10 (1.37 mCi, SR ) 1.83 Ci/µmol, carrier dose 0.86 µg/
kg; or 2.34 mCi, SR ) 540 mCi/µmol, carrier dose 4.83 µg/kg; or
0.472 mCi, SR ) 2.16 Ci/µmol, carrier dose 0.06 µg/kg). A blood
sample (∼2 mL) was drawn at 30 min after each injection, and the
rats were sacrificed by intravenous injection of a saturated solution
of potassium chloride. The brain was excised, and for the experi-
ment with [11C]10 a sample of urine (1.2 mL) was also withdrawn
from the urinary bladder with a hypodermic needle. The brain from
another rat that had been injected with [11C]10 (0.277 mCi, SR )
1.68 Ci/µmol, carrier dose 0.13 µg/kg) was excised after its
perfusion with saline (0.9% w/v, 30 mL) until the perfusate ran
clear and then analyzed as for other brain samples. Samples of
plasma, urine, and brain were then prepared for HPLC analysis as
follows.
Plasma was separated from cells by centrifugation of the blood
sample at 1800g for 1.5 min, and three aliquots (100 µL for [11C]3
or 450 µL for [11C]10) were each placed in acetonitrile (500 µL
for [11C]3, 700 µL for [11C]10) along with added carrier (3 µg of
3 or 10 µg of 10), mixed thoroughly, and centrifuged at 9400g for
2 min. Precipitates were counted for radioactivity to allow calcula-
tion of radioactivity recovery into supernatant liquids. Water (100
µL) was added to the supernatant liquid of each sample, which
was further mixed thoroughly and counted for radioactivity.

11C-Labeled Aryloxyanilide Ligands for Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 29
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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. We thank
the NIH PET Department for fluorine-18 production and
successful completion of the scanning experiments, PMOD
Technologies for providing the image analysis software, and
the NIMH Psychoactive Drug Screening Program (PDSP) for
performing assays. The PDSP is directed by Bryan L. Roth,
Ph.D., with project officer Jamie Driscol (NIMH), at the Uni-
versityofNorthCarolinaatChapelHill(ContractNO1MH32004).
We also thank Dr. Fabrice G. Siméon (NIMH) for useful
contributions.
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