Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for CRF1 receptors

Article abstract of DOI:10.1016/j.bmc.2006.02.019

Synthesis and evaluation of [O-methyl-¹¹C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [¹¹C]SN003 ([¹¹C]6), as a PET imaging agent for CRF₁ recepto

Full text of DOI:10.1016/j.bmc.2006.02.019

Bioorganic & Medicinal Chemistry 14 (2006) 4029–4034
Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for
CRF1 receptors
a
a
a
J. S. Dileep Kumar,a,b, Vattoly J. Majo, Gregory M. Sullivan, Jaya Prabhakaran,
*
Norman R. Simpson,c Ronald L. Van Heertum,c J. John Manna,b,c and Ramin V. Parseya,b
aDepartment of Psychiatry, Columbia University College of Physicians and Surgeons, New York, USA
bDivision of Neuroscience, New York State Psychiatric Institute, New York, NY 10032, USA
cDepartment of Radiology, Columbia University College of Physicians and Surgeons, New York, USA
Received 17 November 2005; revised 31 January 2006; accepted 6 February 2006
Available online 10 March 2006
Abstract—Synthesis and evaluation of [O-methyl-11C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-
[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [11C]SN003 ([11C]6), as a PET imaging agent for CRF1 receptors, in baboons is described.
4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5), the precursor molecule for
the radiolabeling, was synthesized from 2,4-dichloro-6-methyl-3-nitropyridine in seven steps with 20% overall yield. The total time
required for the synthesis of [11C]SN003 is 30 min from EOB using [11C]methyl triflate in the presence of NaOH in acetone. The yield
of the synthesis is 22% (EOS) with >99% chemical and radiochemical purities and a specific activity of >2000 Ci/mmol. PET studies
in baboon show that [11C]6 penetrates the BBB and accumulates in brain. No detectable specific binding was observed, likely due to
the rapid metabolism or low density of CRF1 receptors in primate brain.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
mood disorders, and neurodegenerative diseases such
as Alzheimer’s disease and Parkinson’s disease, but
direct evidence from in vivo studies is lacking.5–11
Corticotropin, releasing factor (CRF or CRH) is a nat-
urally occurring 41-amino acid residue neuropeptide
that regulates the hypothalamic–pituitary–adrenal
(HPA) axis and is a neurotransmitter.1 CRF modulates
the endocrine, autonomic, behavioral, and immune
responses to stress.1 CRF acts on two subtypes (CRF1
and CRF2) and a CRF binding protein (CRF-BP).2
CRF receptors are G-protein-coupled receptors
(GPCR) and both the subtypes are found in the central
nervous system (CNS), CRF1 is found in cerebral
cortex, cerebellum, olfactory bulb, medial septum, hip-
pocampus, amygdala, and pituitary.3 CRF2 is predomi-
nantly limited to lateral septum, choroid plexus, and
hypothalamus, but is widely expressed in peripheral tis-
sues, including the heart, gastrointestinal tract, lung,
skeletal muscle, and vasculature.4 CRF-BP is widely dis-
tributed in CNS including hippocampus and amygdala.4
Elevated expression of CRF1 is implicated in the patho-
genesis of neuropsychiatric disorders such as anxiety,
Development of a high-specific activity radiolabeled,
pharmacologically selective CRF1 receptor antagonist
for positron emission tomography (PET) would make
it possible to quantify binding to CRF1 receptors in vivo,
and permit us to study the pathophysiology of depres-
sion, anxiety, and neurodegenerative diseases. Currently
there is no imaging agent available for the in vivo mea-
surement of CRF1 receptor in humans.12–17 A number
of non-peptide CRF1 receptor antagonists that can spe-
cifically and selectively block the CRF1 receptor subtype
have been recently identified. To be effective in vivo,
these molecules must have receptor subtype specificity
(a high CRF1/CRF2 affinity ratio), aqueous solubility,
good oral bioavailability, and rapid permeability across
the blood–brain barrier (BBB).18 We have selected
SN003 as a potential PET ligand based on its high bind-
ing affinity (Ki = 4 nmol/L) and in vivo effect on CRF1
receptors.19 [3H]SN003 binding to rat cortex and human
cell membranes demonstrates KD values of 4.8 and
4.6 nM, respectively, and Bmax values of 0.142 and
7.42 pmol/mg protein.19 Moreover, the distribution of
[3H]SN003 binding sites is consistent with the expression
Keywords: [11C]Carbon; Radiotracer; Brain; Baboon.
*
Corresponding author. Tel.: +1 212 543 1163; fax: +1 212 543
6017; e-mail: dk2038@columbia.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.019

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J. S. Dileep Kumar et al. / Bioorg. Med. Chem. 14 (2006) 4029–4034
pattern of CRF1 receptors in rat brain regions.19 This
attractive in vitro profile coupled with a favorable calcu-
lated lipophilicity of clogP = 3.3 (calculated with ACD/
logP DB program) to penetrate BBB, and a potential
site for [11C]-labeling prompted us to develop
[11C]SN003 as a candidate ligand for the in vivo quanti-
fication of CRF1 receptors.
O
O
N N
N N
N
N
a, b
N
NH
N
NH
2. Results and discussion
2.1. Chemistry
O
11CH3
[11C]6
OH
The synthesis of 4-[1-(1-methoxymethylpropyl)-6-meth-
yl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methyl-
phenol (5), the precursor molecule for the radiolabeling,
and SN003 standard (6) was achieved from 2,4-dichloro-
6-methyl-3-nitropyridine (1) in four steps with an overall
yield of 20% (Scheme 1). Condensation of pyridine 1 with
1-(methoxymethyl)propylamine in the presence of DI-
PEA provided (2-chloro-6-methyl-3-nitropyridin-4-
yl)(1-methoxymethylpropyl)amine (2) in 48% yield.
Stannous chloride mediated reduction of nitro com-
pound 2 provided 2-chloro-N4-(1-methoxymethylpro-
pyl)-6-methylpyridine-3,4-diamine (3) in 96% yield.
Diazotization of diamine 3, followed by acid catalyzed
ring closure, yielded 4-chloro-1-(1-methoxymethylpro-
pyl)-6-methyl-1H-[1,2,3]triazolo-[4,5-c]pyridine (4) in
98% yield.20 The radiolabeling precursor was synthesized
by reacting compound 4 with 4-amino-3-methylphenol
in the presence of NaHMDS as base in 41% yield. Unla-
beled SN003 was synthesized from 4 by coupling with 4-
methoxy-2-methylphenylamine in 51% yield.
5
Scheme 2. Radiosynthesis of [11C]SN003. Reagents and conditions: (a)
5 M NaOH, acetone, [11C]CH3OTf, rt to 60 ꢁC, 4 min; (b) semipre-
parative HPLC purification, 22% (EOS).
optimized using NaOH as the base in THF. Under these
conditions unlabeled SN003 (6) was obtained in 90%
yield. Labeling experiments with precursor 5 using
[11C]MeOTf in acetone using NaOH provided [11C]6.
The radiolabeled product was separated from the reac-
tion mixture by reverse-phase high performance liquid
chromatography (RP-HPLC) with an average yield of
22 4% (EOS, Scheme 2, n = 6) (Scheme 2). The chem-
ical identity of [11C]6 was confirmed by co-injection with
an authentic sample of standard 6 on analytical RP-
HPLC. The specific activity was calculated based on a
standard mass curve using HPLC technique. The chem-
ical and radiochemical purities of [11C]6 was found to be
>99% with a specific activity 2000 400 Ci/mmol (EOB,
n = 6). The average time required for the [11C]-labeling
2.2. Radiochemistry
was 30 min (EOB). The partition coefficient (logPo/w
)
of [11C]6 was determined by standard shake flask meth-
The labeling reaction was initially modeled with non-ra-
dioactive methyl triflate and the reaction condition was
od21 and found to be 3.1.
O
O
3. In vivo imaging in baboon
Cl
As is evident from Figure 1A, the PET studies in baboon
show that the tracer penetrates BBB and accumulates in
brain. The time–activity curves of several brain regions
were examined and no regional specific binding was
detected (Figs. 1B and 2). The regional distribution is
found to be homogeneous across all brain regions after
10 min and a fast washout of radioactivity was observed
(Fig. 1B). The radioactive metabolites were determined
by HPLC technique. Only polar metabolites were found
NH
NH
NO2
Cl
b
a
NH2
Cl
NO2
Cl
N
N
N
1
3
2
O
O
R
N
N N
d/ e
N
c
N
N
H
N
N
O
N
4
Cl
5, R = H
6, R = CH3
Scheme 1. Synthesis of SN003 and radiolabeling precursor. Reagents
and conditions: (a) 1-(methoxymethyl)propylamine, DIPEA, CH3CN,
rt, reflux, 48%; (b) SnCl2Æ2H2O, EtOH, 70 ꢁC, 1h, 96%; (c) NaNO2, aq
CH3COOH, CH2Cl2, 0 ꢁC, 98%; (d) R = H; NaHMDS, THF,
4-amino-3-methylphenol, À78 ꢁC to rt, 41%; (e) R = CH3; NaHMDS,
THF, 4-methoxy-2-methylphenylamine, À78 ꢁC to rt, 51%.
Figure 1. PET images of [11C]SN003 in baboon. (A) Sagittal view of
early frames (sum of first 20 min); (B) later frames (sum of last 40 min).

J. S. Dileep Kumar et al. / Bioorg. Med. Chem. 14 (2006) 4029–4034
4031
2
1.8
1.6
1.4
1.2
THA
HIP
OCC
CER
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
Time (min.)
Figure 2. Time–activity curves of the radioactivity in baboon after the injection of [11C]SN003. CER, cerebellum; OCC, occipital cortex; THA,
thalamus; HIP, hippocampus.
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
90
100
Time (min.)
Figure 3. Unmetabolized parent fraction of [11C]SN003 in baboon plasma. Filled circles represent the mean fraction in six determinations. Error bars
are standard deviations.
in baboon plasma and the % of unmetabolized fractions
were 96%, 77%, 24%, 15%, 10%, and 8% at 2, 4, 12, 30,
60, and 90 min, respectively (Fig. 3). The metabolite
data indicate that [11C]6 undergoes rapid metabolism
in baboon that would contribute to its poor specific
binding.
In vivo PET imaging studies in baboon revealed that
[11C]SN003 penetrated the BBB, but no regional varia-
tion in total binding was observed possibly due to rapid
metabolism or the small number of CRF1 receptors in
baboons.
5. Materials and methods
5.1. General
4. Conclusion
The radiosynthesis of [11C]SN003, a CRF1 antagonist,
has been achieved. Total time required for the synthesis
of [11C]SN003 is 30 min from EOB using [11C]methyl tri-
flate in acetone, with a 22% yield at EOS based on
[11C]MeOTf. The chemical and radiochemical purities
are >99% and the specific activity is >2000 Ci/mmol.
The commercial chemicals used in the synthesis were
purchased from Sigma–Aldrich Chemical Co., Fisher
Scientific Inc. or Lancaster and were used without fur-
ther purification. Melting points were determined on a
Fisher Scientific Melting point apparatus. 1H NMR

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J. S. Dileep Kumar et al. / Bioorg. Med. Chem. 14 (2006) 4029–4034
spectra were recorded on a Bruker PPX 300 and
400 MHz spectrometer. Spectra were recorded in CDCl3
or CD3OD and chemical shifts (d) are reported in parts
per million (ppm) relative to tetramethylsilane (TMS).
The mass spectra were recorded on JKS-HX 11UHF/
HX110 HF Tandem Mass Spectrometer in the fast atom
bombardment (FAB+) mode. Semipreparative HPLC
analyses were performed using a Waters 1525 HPLC
system, Phenomenex, Prodigy ODS(3) 10 · 250 mm,
10 mm, 10 lm column using 40:60 (acetonitrile/0.1 M
ammonium formate solution), 10 ml/min flow rates
and for analytical studies Phenomenex, Prodigy
ODS(3) 4.6 · 250 mm, 5 lm column using 40:60 (aceto-
nitrile/0.1 M ammonium formate solution), 2 mL/min as
flow rate were used. Flash chromatography was per-
formed on silica gel (Fisher 200–400 mesh) using the sol-
vent system indicated in the experimental procedure for
each compound. [11C]Methyl triflate was synthesized in
the Radioligand Laboratory of Columbia University
by transferring [11C]methyl iodide through a glass col-
umn containing silver triflate (AgOTf) at 200 ꢁC. The
radiochemical and chemical purities were analyzed by
RP-HPLC with PDA and NaI detectors. Partition coef-
ficient determination was performed with a Packard
Instruments Gamma Counter (Model E5005). Metabo-
lite analyses were performed using Phenomenex (C18,
10 · 250 mm, 5 lm) column using a mobile phase
40:60 (acetonitrile/0.1 M ammonium formate solution).
The free fractions and metabolites were measured using
Packard Instruments Gamma Counter (Model E5005).
sion was filtered to remove the solids, the residue was
washed with EtOAc, and the aqueous layer was extract-
ed with EtOAc (3·20 mL). The combined organic layer
was washed with brine, dried (MgSO4), and concentrat-
ed. Recrystallization of the crude residue from hexane:
Et2O gave the desired amine 3 as a colorless solid
(520 mg, 96% yield). Mp: 127 ꢁC; 1H NMR (CDCl3,
300 MHz): d 6.30 (s, 1H), 4.41 (br s, 1H), 3.52–3.42
(m, 3H), 3.38 (s, 3H), 2.38 (s, 3H), 1.80–1.56 (m, 2H),
0.99 (t, J = 7.4 Hz, 3H). HRMS calcd for C11H19ClN3O
(MH+): 244.1217; found: 244.1203.
5.2.3. 4-Chloro-1-(1-methoxymethylpropyl)-6-methyl-1H-
[1,2,3]triazolo[4,5-c]pyridine (4). NaNO2 (92 mg,
1.34 mmol) was added portionwise to a solution of the
amine 3 (182 mg, 0.75 mmol) in aqueous acetic acid
(2 mL, 50%) and dichloromethane (2 mL) at 0 ꢁC. After
stirring for 30 min at 0 ꢁC, the reaction mixture was al-
lowed to warm to rt and was stirred for further 1 h. The
solution was then cooled and neutralized with solid
NaHCO3. Water (4 mL) was then added and the aque-
ous layer was extracted with EtOAc. The combined
organic layer was washed with brine, dried (MgSO4),
and concentrated to give the product 4 as a viscous li-
quid (187 mg, 98%), which was used for the next step
without further purification. 1H NMR (CDCl3,
300 MHz): d 7.25 (s, 1H), 4.81–4.69 (m, 1H), 3.92 (dd,
J = 9.9, 8.0 Hz, 1H), 3.83 (dd, J = 4.2, 9.9 Hz, 1H),
3.26 (s, 3H), 2.68 (s, 3H), 2.30–2.22 (m, 1H), 2.19–2.06
(m, 1H), 0.86 (t, J = 7.4 Hz, 3H); HRMS calcd for
C11H15ClN4O (MH+): 255.1013; found: 255.1028.
5.2. Chemistry
5.2.4. 4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]-
triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5). A
solution of the aryl chloride 4 (73 mg, 0.28 mmol) was dis-
solved in THF (2 mL) and treated with NaHMDS
(650 lL, 0.65 mmol, 1.0 M in THF, dropwise addition)
at À78 ꢁC. A solution of 4-amino-3-methylphenol
(60 mg, 0.49 mmol) in THF (1 mL) was then introduced
dropwise and the reaction mixture was allowed to warm
to rt and was stirred for further 2 h. Water was added
and the mixture was extracted with EtOAc (3· 5 mL).
The combined organic layer was washed with brine, dried
over MgSO4, concentrated under reduced pressure with-
out heating, and column chromatographed (30% EtOAc
in hexanes) to yield the product 5a as a colorless solid
(40 mg, 41%); Mp 135–137 ꢁC; 1H NMR (CDCl3,
300 MHz): d 7.43 (d, J = 9.3 Hz, 1H), 6.52–6.43 (m,
4H), 4.65–4.57 (m, 1H), 3.87 (dd, J = 9.9, 7.9 Hz, 1H),
3.75 (dd, J = 4.7, 9.9 Hz, 1H), 3.22 (s, 3H), 2.44 (s, 3H),
2.19 (s, 3H), 2.12–1.97 (m, 2H), 0.79 (t, J = 7.4 Hz, 3H).
HRMS calcd for C18H24N5O2 (MH+): 342.1930; found:
342.1933.
5.2.1. 2-Chloro-6-methyl-3-nitropyridin-4-yl-(1-meth-
oxymethylpropyl)amine (2). A solution of the aryl
chloride 1 (1.68 g, 8.14 mmol) and 1-(methoxymeth-
yl)propylamine (840 mg, 8.14 mmol) in CH3CN
(15 mL) was treated with DIPEA (1.7 mL, 9.77 mmol,
dropwise addition) at room temperature (rt). The reac-
tion mixture was stirred at rt for 12 h and then heated
to reflux for 3 h. Excess CH3CN was removed under
reduced pressure and the residue was dissolved in
CH2Cl2 (30 mL), washed with water (20 mL). The aque-
ous layer was extracted with dichloromethane (2·
20 mL) and the combined organic layer was dried over
MgSO4, concentrated under high vacuum, and column
chromatographed (75:25 hexane/EtOAc) to yield the
product 2 as a pale yellow solid (1.07 g, 48%). Mp
63 ꢁC; 1H NMR (CDCl3, 300 MHz)
d 6.75 (d,
J = 7.5 Hz, 1H), 6.49 (s, 1H), 3.62–3.52 (m, 1H), 3.44 (d,
J = 4.8 Hz, 2H), 3.36 (s, 3H), 2.42 (s, 3H), 1.83–1.68 (m,
1H), 1.66–1.52 (m, 1H), 0.98 (t, J = 7.4 Hz, 3H); HRMS
calcd for C11H17ClN3O3 (MH+): 274.0958; found:
274.0961.
5.2.5. (4-Methoxy-2-methylphenyl)-[1-(1-methoxymethyl-
propyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine
(6). A solution of the aryl chloride 4 (33 mg, 0.13 mmol)
was dissolved in THF (2 mL) and treated with NaH-
MDS (280 lL, 0.28 mmol, 1.0 M in THF) at À78 ꢁC.
A solution of 4-methoxy-2-methylphenylamine (35 mg,
0.26 mmol) in THF (1 mL) was then introduced drop-
wise and the reaction mixture was allowed to warm to
rt and was stirred for further 2 h. Water was added
5.2.2. 2-Chloro-N4-(1-methoxymethylpropyl)-6-methyl-
pyridine-3,4-diamine (3). A solution of the nitro com-
pound 2 (608 mg, 2.22 mmol) was dissolved in dry
ethanol (5 mL) and treated portionwise with SnCl2Æ2-
H2O (2.50 g, 11.10 mmol) at rt. The reaction mixture
was heated at 70 ꢁC for 1 h, concentrated under reduced
pressure, and neutralized with saturated NaHCO3 solu-
tion, and ethyl acetate was added. The resultant suspen-

J. S. Dileep Kumar et al. / Bioorg. Med. Chem. 14 (2006) 4029–4034
4033
and the mixture was extracted with EtOAc (3·5 mL).
The combined organic layer was washed with brine,
dried over MgSO4, concentrated, and column chro-
matographed (25% EtOAc in hexanes) to yield the prod-
uct 6 as a viscous liquid (23 mg, 51%); 1H NMR
(CDCl3, 400 MHz) d 8.06 (d, J = 9.6 Hz, 1H), 7.33 (br
s, 1H), 6.83–6.80 (m, 2H), 6.61 (s, 1H), 4.73–4.66 (m,
1H), 3.94 (dd, J = 9.9, 7.9 Hz, 1H), 3.84 (d, J = 4.8 Hz,
1H), 3.82 (s, 3H), 3.29 (s, 3H), 2.49 (s, 3H), 2.38 (s,
3H), 2.24–2.12 (m, 1H), 2.11–2.00 (m, 1H), 0.84 (t,
J = 7.4 Hz, 3H). HRMS calcd for C19H26N5O2 (MH+):
356.2087; found: 356.2097.
used for hydration and radiotracer injection. An arterial
line was placed for obtaining arterial samples for deter-
mination of the input function for quantification pur-
poses. In each scanning session, the head was
positioned so that the brain was in the center of the field
of view, and a 10 min transmission scan was performed
before the first tracer injection. For each scan
5.0 0.5 mCi of [11C]6 was injected as an iv bolus and
emission data were collected for 120 min in 3D mode
with the following time frames: 2 · 0.5, 3 · 1, 5 · 2,
4 · 4, and 9 · 10 min. Plasma samples were taken every
10 s for the first 2 min, using an automatic system, and
thereafter manually for a total of 34 samples over 2 h.
5.3. Radiochemistry
5.6. Determination of unchanged radioligand in plasma
Radiosynthesis of [11C]SN003: [11C]MeOTf22 was
trapped into an acetone (400 lL) solution containing
0.5 mg of 5 and 10 lL of 5 N NaOH at rt for 5 min.
At the end of the trapping, the reaction mixture was
heated on a water bath at 60 ꢁC for 4 min and then
directly injected into a semipreparative RP-HPLC and
eluted with acetonitrile/0.1 M ammonium formate solu-
tion (50:50) at a flow rate of 10 mL/min. The product
fraction with a retention time of 10–11 min based on
c-detector was collected, diluted with 100 mL of deion-
ized water, and passed through a classic C-18 Sep-Pak
cartridge. Reconstitution of the product in 1 mL of
absolute ethanol afforded [11C]SN003 (67% yield based
on [11C]CH3OTf at EOB and 22% based on EOS). A
portion of the ethanol solution was analyzed by analyt-
ical HPLC (Mobile phase: acetonitrile/0.1 M ammoni-
um formate solution 60:40, flow rate: 2 mL/min,
retention time: 6.4 min) to determine the specific activity
and radiochemical purity.
The percentage of radioactivity in plasma as unchanged
[11C]6 was determined by HPLC method. Blood samples
were taken at 2, 6, 12, 30, 60, and 90 min after radioac-
tivity injection for metabolite analyses. The supernatant
liquid (0.5 mL) obtained after centrifugation of the
blood sample at 2000 rpm for 1 min was transferred
(0.5 mL) into a tube and mixed with acetonitrile
(0.7 mL). The resulting mixture was vortexed for 10 s
and centrifuged at 14,000 rpm for 4 min. The superna-
tant liquid (ꢀ1 mL) was removed, the radioactivity mea-
sured in a well-counter, and the majority (0.8 mL) was
subsequently injected onto the HPLC column and eluted
with a mobile phase acetonitrile/0.1 M ammonium for-
mate (60:40) solution at 2 mL/min (Phenomenex C18,
10 · 250 mm, 5 lm). The metabolite and free fractions
were collected using a Bioscan gamma detector and all
the acquired data then subjected to correction for back-
ground radioactivity and physical decay to calculate the
percentage of [11C]SN003 in the plasma at different time
points.
5.4. Partition coefficient measurement
Partition coefficient (logP) of the [11C]6 was measured
by mixing 0.1 mL of the radioligand formulation with
5 g each of 1-octanol and freshly prepared PBS buffer
(pH 7.4) in a culture tube.21 The culture tube was shaken
mechanically for 5 min followed by centrifugation for
5 min. Radioactivity per 0.5 g each of 1-octanol and
aqueous layer was measured using a well counter. The
partition coefficient was determined by calculating the
ratio of counts/g of 1-octanol to that of buffer. 1-Octa-
nol fractions were repeatedly portioned with fresh buffer
to get consistent values for partition coefficient. All the
experimental measurements were performed in
triplicate.
5.7. Image processing and analysis
The PET data were reconstructed with attenuation cor-
rection using the transmission data, and scatter correction
was done using model-based scatter correction.23 The
reconstruction filter and estimated image filter were
Shepp 0.5, the axial (Z) filter was all pass 0.4, and the
zoom factor was 4.0. The final image resolution at the
center of the field of view was 5.1 mm FWHM.24
A
T1-weighted magnetic resonance image (MRI) of the
animal’s head was acquired on a GE 1.5-T Signa Advan-
tage system. Regions of interests were drawn on the MRI
using the MEDX software (Sensor Systems, Inc., Sterling,
VA). ROIs included cerebellum, hippocampus, occipital
cortex, and thalamus. The PET data were co-registered
to the MRI using the software AIR,25 and time–activity
curves (TACs) were generated for each ROI.
5.5. PET studies in baboons
A series of PET studies were done on two baboons (Pa-
pio anubis) with an ECAT EXACT HR+ scanner (Sie-
mens, Knoxville, TN). Each animal was scanned on
six separate occasions, with two tracer injections on each
occasion. For each scanning session, the fasted animal
was immobilized with ketamine (10 mg/kg, im) and
anesthetized with 1.5–2.0% isoflurane via an endotrache-
al tube. Core temperature was kept constant at 37 ꢁC
with a heated water blanket. An iv infusion line with
0.9% NaCl was maintained during the experiment and
Acknowledgments
This work was supported by a research grant from the
National Institutes of Health (R21 MH066620). The
authors also thank Ms. Julie Arcement and Mr. Scott
Shepherd for their assistance in the radiolabeling and
metabolite analyses studies.

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Eckelman, W. C.; Jacobson, A. E.; Rice, K. C. Bioorg.
Med. Chem. Lett. 2001, 11, 331–333.
References and notes
15. Hsin, L.-W.; Webser, E. L.; Chrousos, G. P.; Gold, P. W.;
Eckleman, W. C.; Contoreggi, C.; Rice, K. C. J. Labelled
Compd. Radiopharm. 2000, 43, 899–908.
16. Kumar, J. S. D.; Majo, V. J.; Prabhakaran, J.; Simpson,
N. R.; Van Heertum, R. L.; Mann, J. J. J. Labelled
Compd. Radiopharm. 2003, 46, 1055–1065.
17. Kumar, J. S. D.; Majo, V. J.; Simpson, N. R.; Prabhak-
aran, J.; Van Heertum, R. L.; Mann, J. J. J. Labelled
Compd. Radiopharm. 2004, 47, 971–976.
1. Owens, M. J.; Nemeroff, C. B. Pharmacol. Rev. 1991, 43,
425–473.
2. Chalmers, D. T.; Lovenberg, T. W.; Grigoriadis, D. E.;
Behan, D. P.; De Souza, E. B. Trends Pharmacol. Sci.
1996, 17, 166–172.
3. Gilligan, P. J.; Robertson, D. W.; Zaczek, R. J. Med.
Chem. 2000, 43, 1641–1660.
4. De Souza, E. B. Psychoneuroendocrinology 1995, 20, 789–
819.
18. Jagoda, E.; Contoreggi, C.; Lee, M. J.; Kao, C. H.; Szajek,
L. P.; Listwak, S.; Gold, P.; Chrousos, G.; Greiner, E.;
Kim, B. M.; Jacobson, A. E.; Rice, K. C.; Eckelman, W.
J. Med. Chem. 2003, 46, 3559–3562.
5. O’brien, D.; Skelton, K. H.; Owens, M. J.; Nemeroff, C. B.
Hum. Psychopharmacol. Clin. Exp. 2001, 81–87.
6. Skutella, T.; Probst, J. C.; Renner, U.; Holsboer, F.; Behl,
C. Neuroscience 1998, 85, 795–805.
19. Zhang, G.; Huang, N.; Li, Y.-W.; Qi, X.; Marshall, A. P.;
Yan, X.-X.; Hill, G.; Rominger, C.; Prakash, S. R.;
Bakthavatchalam, R.; Rominger, D. H.; Gilligan, P. J.;
Zaczek, R. J. Pharm. Exp. Ther. 2003, 305, 57–69.
20. Arvanitis, A.G.; Gilligan, P.J.; Beck, J.P.; Bakthavatcha-
lam, R. PCT Int. Appl. 1999, WO 9911643.
21. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilv, J. N.; Houle, S.
Appl. Radiat. Isot. 2001, 54, 203–208.
22. Jewett, D. M. Nucl. Med. Biol. 2001, 28, 733–734.
23. Watson, C. C.; Newport, D.; Casey, M. E. Aix-les-bains,
France, 1995; pp 215–219.
7. Mitchell, A. J. Neurosci. Biobehav. Rev. 1998, 22, 635–
651.
8. Sajdyk, T. J.; Schober, D. A.; Gehlert, D. R.; Shekhar, A.
Behav. Brain Res. 1999, 100, 207–215.
9. Behan, D. P.; Grigoriadis, D. E.; Lovenberg, T.; Chal-
mers, D.; Heinrichs, S.; Liaw, C.; De Souza, E. B. Mol.
Psychiatry 1996, 1, 265–277.
10. De Souza, E. B.; Whitehouse, P. J.; Kuhar, M. J.; Price,
D. L.; Vale, W. W. Nature 1986, 319, 593–595.
11. Saunders, J.; Williams, J. Prog. Med. Chem. 2003, 41, 195–
247.
24. Mawlawi, O.; Martinez, D.; Slifstein, M.; Broft, A.;
Chatterjee, R.; Hwang, D. R.; Huang, Y.; Simpson, N.;
Ngo, K.; Van Heertum, R.; Laruelle, M. J. Cereb. Blood
Flow Metab. 2001, 21, 1034–1057.
12. Holsboer, F. CNS Spectr. 2001, 6, 590–594.
13. Martarello, L.; Kilts, C. D.; Ely, T.; Owens, M. J.;
Nemeroff, C. B.; Camp, M.; Goodman, M. M. Nucl. Med.
Biol. 2001, 28, 187–195.
25. Woods, R. P.; Cherry, S. R.; Mazziotta, J. C. J. Comput.
Assist. Tomogr 1992, 16, 620–633.
14. Tian, X.; Hsin, L.-W.; Webster, E. L.; Contoreggi, C.;
Chrousos, G. P.; Gold, P. W.; Habib, K.; Ayala, A.;