5-(5-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin- 2-yl)-1H-indole as a potential PET radioligand for imaging cerebral α7-nAChR in mice

Article abstract of DOI:10.1016/j.bmc.2012.04.056

The radiosynthesis and in vivo evaluation of 5-(5-(6-[¹¹C] methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole [ ¹¹C]rac-(1), a potential PET tracer for α7 nicotinic acetylcholine receptors (α7-nAChR), are described. Syntheses of the nonradioactive standard rac-1 and corresponding desmethyl precursor 7 were achieved in several reaction steps. Radiomethylation of 7 with [ ¹¹C]CH₃I afforded [¹¹C]rac-1 in an average radiochemical yield of 30 ± 5% (n = 5) with high radiochemical purity and an average specific radioactivity of 444 ± 74 GBq/μmol (n = 5). The total synthesis time was 30 min from end-of-bombardment. Biodistribution studies in mice showed that [¹¹C]rac-1 penetrates the blood-brain barrier and specifically labels neuronal α7-nAChRs.

Full text of DOI:10.1016/j.bmc.2012.04.056

Bioorganic & Medicinal Chemistry 20 (2012) 3698–3702
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-(5-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole
as a potential PET radioligand for imaging cerebral a7-nAChR in mice
⇑
Yongjun Gao , Hayden T. Ravert, Heather Valentine, Ursula Scheffel, Paige Finley, Dean F. Wong,
Robert F. Dannals, Andrew G. Horti
Division of Nuclear Medicine, Department of Radiology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287-0816, USA
a r t i c l e i n f o
a b s t r a c t
The radiosynthesis and in vivo evaluation of 5-(5-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-
Article history:
yl)pyridin-2-yl)-1H-indole [11C]rac-(1), a potential PET tracer for
a
7 nicotinic acetylcholine receptors
7-nAChR), are described. Syntheses of the nonradioactive standard rac-1 and corresponding desmethyl
precursor 7 were achieved in several reaction steps. Radiomethylation of 7 with [11C]CH3I afforded
11C]rac-1 in an average radiochemical yield of 30 5% (n = 5) with high radiochemical purity and an
average specific radioactivity of 444 74 GBq/ mol (n = 5). The total synthesis time was 30 min from
end-of-bombardment. Biodistribution studies in mice showed that [11C]rac-1 penetrates the blood–brain
barrier and specifically labels neuronal 7-nAChRs.
Received 16 February 2012
Revised 19 April 2012
Accepted 27 April 2012
Available online 4 May 2012
(
a
[
l
Keywords:
Positron emission tomography
Radioligand
a
Ó 2012 Elsevier Ltd. All rights reserved.
Nicotinic acetylcholine receptors
a7-nAChR
1. Introduction
clo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole ((1R,5R)-1, A-859261)
and 5-(5-((1S,5S)-6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)
A large body of experimental evidence associates cerebral nico-
tinic acetylcholine receptors (nAChRs) with the pathophysiology of
a variety of disorders of central nervous system (CNS) disorders.1–4
pyridin-2-yl)-1H-indole ((1S,5S)-1) exhibit highest
a
7-nAChR
binding affinities (Ki = 0.5 and 0.6 nM,19 respectively,
a7 Ki dis-
placement of [3H]-A-585539 ([3H]-(S,S)-2,2-dimethyl-5-(6-phen-
ylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane iodide) from
membrane enriched fractions of rat brain minus cerebellum or
cortex; n P 3; SEM <10%.) (Fig. 1). A-859261 and analogs showed
no interaction at 5HT3 receptors. In the CEREP profiling,
Because of the importance of
treatment of various central disorders including schizophrenia,5,6
synthesis and pre-clinical examination of 7-nAChR subtype selec-
a7-nAChR as potential drug target for
a
tive compounds receives substantial attention in industry and aca-
demia.7–10
A-859261 exhibited none or weak interaction up to 10
lM. Also,
Substantial effort of PET researchers in the last decade has been
devoted to imaging nAChRs in human brain. This imaging work in
turn is linked to the development of PET radioligands for nAChR.
Several PET radioligands for the major cerebral nAChR subtype,
A-859261 exhibited low activity at the hERG channel, its
a
4b2-nAChR, are currently available for human and animal studies
(see for review11,12). In contrast, exploration of PET radioligands for
NH
N
N
N
imaging the second major cerebral nicotinic receptor subtype a7-
CH3
nAChR so far has been less successful (see for review13,14). Recent
studies revealed new interesting compounds,15–17 but more qual-
rac-1
ity radioligands for PET imaging of
discovered.
Recently, a series of biarylsubstituted 3,6-diazabicyclo[3.2.0]
heptanes derivatives was discovered by Abbott Laboratories as
a7-nAChR remains to be
NH
N
N
N
CH3
(1R,5R)-1,A-859261
potent and selective a7-nAChR agonists with low binding affinities
at other cerebral receptors and nicotinic receptor subtypes.18,19
Two members of the series, 5-(5-((1R,5R)-6-methyl-3,6-diazabicy-
NH
N
N
N
CH3
(1S,5S)-1
⇑
Corresponding author. Tel.: +1 410 614 0108; fax: +1 410 614 0111.
E-mail address: ygao5@jhmi.edu (Y. Gao).
Figure 1. Structures of potent and selective a7-nAChR ligands.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.056

Y. Gao et al. / Bioorg. Med. Chem. 20 (2012) 3698–3702
3699
OH
HO B
b
Br
a
NH
N
N
Cl
NH
N
R
N
N
N
N
H
N
N
Cl
Boc
6: R=Boc
7: R=H
Boc
c
5
4
3
2
d
1: R=Me
Scheme 1. Synthesis of 5-(5-(6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)pyridin-2-yl)-1H-indole and its desmethyl precursor. Reagents and conditions: (a) Pd2(dba)3,
BINAP, t-BuONa in toluene, at 85 °C 20 h, 83%; (b) Pd(PPh3)4, dioxane/Na2CO3 aq 2 M) = 4:1, at 115 °C 1.5 h, 77%; (c) CF3CO2H–CH2Cl2, 27%; (d) HCHO, NaBH(OAc)3, CH3CN,
31%.
11CH3I
NH
N
NH
N
N
CH3CN
N
HN
N
11CH3
[
11C]rac-1
7
Scheme 2. Radiosynthesis of [11C]rac-1.
[3H]dofetilide binding Ki is 1.9
that A-859261 readily enters the mouse brain after intraperitoneal
l
M. An in vivo study demonstrated
nous injection of the radioligand (Fig. 2). The highest accumulation
of [11C]rac-1 radioactivity was seen in four brain regions: the fron-
tal cortex, thalamus, striatum, and hippocampus with peak at
30 min post-injection. A gradual decline of radioactivity in these
regions was observed throughout the rest of the experiment. The
rate of uptake and washout of radioactivity in the mouse cerebel-
lum, a region with the lowest concentration of radioactivity, was
more rapid. The regional radioactivity concentrations of [11C]rac-
1 in the mouse brain agreed with in vitro and in vivo data on the
administration and exhibit cognition-enhancing properties that
are attributable to
racemic 1 (rac-1) ( Fig. 1) should also manifest good
a
7-nAChR agonists.19 We hypothesized that
a7-nAChR
binding affinity and could penetrate the blood–brain barrier. Thus
radiolabeled [11C]rac-1 might be suitable for PET imaging the cere-
bral a7-nAChR.
In this study we report the synthesis of rac-1 (Scheme 1), radio-
synthesis of ([11C]rac-1) (Scheme 2) and its in vivo evaluation as a
distribution of a
7-nAChRs.14 The ratios of tissue to cerebellum in-
potential PET radioligand for imaging
a7-nAChR.
creased steadily over the observation period, reaching values of 3
in the frontal cortex at 90 min after injection (Fig. 2).
To investigate the specific binding for
a
7-nAChRs, a blocking
7-nAChRs agonist
2. Results and discussion
2.1. Chemistry
study was performed using the selective
a
PHA543613.22 A blocking dose of PHA543613 significantly inhib-
ited [11C]rac-1 binding in the hippocampus and frontal cortex sug-
gesting that the binding in these regions is specific and it is
As shown in Scheme 1, racemic tert-butyl 3,6-diazabicy-
clo[3.2.0]heptane-6-carboxylate (2) was condensed with 5-bro-
mo-2-chloropyridine (3) via the Buchwald–Hartwig amination
procedure18,20,21 to yield the corresponding intermediate, tert-bu-
tyl 3-(6-chloropyridin-3-yl)-3,6-diazabicyclo[3.2.0]heptane-6-car-
boxylate (4) in high yield. A transformation of 4 under the Suzuki
coupling reaction conditions with indol-5-ylboronic acid (5) gave
6. The N-Boc deprotection of 6 with trifluoroacetic acid (TFA)
yielded the corresponding 7. Reductive methylation of 7 proceeded
well to give the corresponding N-Me analog rac-1.
mediated by
a7-nAChRs. As expected, the blockade in the cerebel-
CB
Hipp
Str
FrCtx
Th
6
S.coll
Rest
FrCtx/CB
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5
4
3
2
1
The radiosynthesis of [11C]rac-1 was accomplished via N-meth-
ylation of the nor-methyl precursor 7 with the [11C]CH3I in aceto-
nitrile as shown in Scheme 2 followed by semi-preparative HPLC
purification and C18 solid phase extraction and formulation. The fi-
nal product [11C]rac-1 was prepared with an average radiochemi-
cal yield of 30 5% (n = 5), radiochemical purity greater than 98%
and an average specific radioactivity of 444 74 GBq/lmol mCi/
l
mol (n = 5). The total synthesis time was 30 min from the end-
of-bombardment (EOB). The identity of the radiotracer [11C]rac-1
was confirmed by co-injection with non-radioactive reference
compound rac-1 onto the analytical HPLC system. The final product
0
0
[
11C]rac-1 was formulated as a sterile and apyrogenic solution in
10
20
30
40
50
60
70
80
90 100
saline with 7% ethyl alcohol.
Time, min
Figure 2. Regional brain distribution of [11C]rac-1 in CD1 mice. CB = cerebellum;
Hipp = hippocampus; Str = striatum; FrCtx = frontal cortex; Th = thalamus,
S.coll = superior colliculus; Rest = the rest of the brain) in the baseline study. The
frontal cortex to cerebellum ratio (——) reached a value of 3 at 90 minutes post iv
injection.
2.2. In vivo experiments in CD1 mice
The radioactivity accumulation for [11C]rac-1 was determined
as % ID/g tissue in six regions of the CD1 mouse brain after intrave-

3700
Y. Gao et al. / Bioorg. Med. Chem. 20 (2012) 3698–3702
flash chromatography on silica gel (Hexanes/Ethyl acetate 3:1) to
5
4
3
2
1
0
give product 4 as pale yellow oil (840 mg, 83%). 1H NMR (CDCl3,
400 MHz) d 7.86 (d, J = 3.2 Hz, 1H), 7.17 (d, J = 9.2 Hz, 1H), 7.02
(dd, J = 3.2 Hz, 8.8 Hz, 1H), 4.83 (s, 1H), 4.11 (t, J = 8 Hz, 1H),
3.98–3.89 (m, 1 H), 3.69–3.62 (m, 2H), 3.24–3.17 (m, 1H), 3.03
(dd, J = 6.6 Hz, 10.2 Hz, 1H), 2.90 (dd, J = 4.4 Hz, 10.8 Hz, 1H), 1.45
(s, 9H); 13C NMR (CDCl3, 100 MHz) d 148.6, 144.1, 139.7, 135.2,
124.0, 123.9, 79.9, 65.4, 64.4, 54.5, 53.3, 32.8, 28.5; HRMS calcu-
lated for C15H20ClN3O2 [M+H] m/z = 310.1322; found, 310.1344.
3.1.2. 3-[6-(1H-Indol-5-yl)-3-pyridinyl]-6-Boc-3,6-diazabicyclo
[3.2.0]heptane (6)
A mixture of 4 (392 mg, 1.27 mmol), 5 (316 mg, 1.96 mmol) and
tetrakis(triphenylphosphine)palladium(0) (150 mg, 0.13 mmol)
were dissolved in 1,4-dioxane (12 mL). After 10 min stirring, a
solution of sodium carbonate (519 mg, 4.9 mmol) in water (3 mL)
was added. The mixture was heated to reflux and allowed to react
at 115 °C for 1.5 h. TLC showed the starting material disappeared.
After cooling to room temperature, the mixture was poured into
a 1:1 mixture of water/ethyl acetate (50:50). The organic layer
was separated, and the aqueous layer was further extracted with
30 mL of ethyl acetate twice. The combined organic layers were
dried over Na2SO4, filtered, and concentrated to dryness. The resi-
due was purified by silica gel chromatography (Ethyl acetate/Hex-
anes 1:1 to 1:2) to give product 6 as pale yellow solid (381 mg,
77%). Mp 121–123 °C; 1H NMR (CDCl3, 400 MHz) d 8.55 (s, 1H),
8.27 (s, 1H), 8.21 (s, 1H), 7.81 (br s, 1H), 7.72–7.67 (m, 1H), 7.46
(d, J = 8 Hz, 1H), 7.24–7.22 (m. 1H), 7.09 (br s, 1H), 6.62–6.61 (m,
1H), 4.85 (s, 1H), 4.13 (t, J = 8 Hz, 1H), 4.05 (s, 1H), 3.75 (d,
J = 10.4 Hz, 1H), 3.69 (dd, J = 4.4 Hz, 8.4 Hz, 1H), 3.24–3.18 (m,
1H), 3.07–3.03 (m, 1H), 2.91 (s, 1H), 1.49 (s, 9H). 13C NMR (CDCl3,
100 MHz) d 148.8, 143.3, 135.8, 131.7, 128.3, 124.9, 121.9, 120.8,
120.5, 118.3, 111.2, 103.1, 79.8, 65.5, 64.5, 54.5, 53.2, 32.8, 28.5;
HRMS calculated for C23H26N4O2 [M+H] m/z = 391.2134; found,
391.2146.
CB
FrCtx
Hipp
RB
Figuire 3. At 90 min after injection in controls (open bars) and blocking experi-
ments with the selective 7-nAChRs agonist PHA543613 (0.5 mg/kg, subcutaneous)
a
(black bars), the regional brain uptake of [11C]rac-1 in mice (cerebellum (CB),
frontal cortex (FrCtx), hippocampus (Hipp), and the rest of the brain (RB) were
compared. There was significant blocking in all regions except cerebellum. Data are
mean SD. ⁄P <0.05, significantly different from controls; ⁄⁄P >0.05, insignificantly
different from controls (ANOVA single factor analysis).
lum was minimal since the known density of a7-nAChRs is low in
this region14 (Fig. 3). When the specific binding was estimated by
using the radioactivity concentration in the blocked cerebellum
as a measure of nonspecific binding, the decrease in the frontal cor-
tex was 48% and the decrease in the hippocampus was 33% (Fig. 3).
The regional brain distribution and blockade results suggest
that [11C]rac-1 is a potentially useful radioligand for measuring
a
7-nAChRs in the mouse brain.
3.1.3. 3-[6-(1H-Indol-5-yl)-3-pyridinyl]-3,6-diazabicyclo[3.2.0]
heptane (7)
3. Materials and methods
3.1. Chemistry
TFA (5 mL) was added to a solution of 6 (380 mg, 0.97 mmol) in
CH2Cl2 (10 mL) at 0 °C. The mixture was stirred at 0 °C for 15 min,
then at room temperature for 2 h until TLC showed the starting
material disappeared. The solvent was evaporated off. The residue
was purified by silica gel chromatography using CH2Cl2/MeOH/
NH4OH 9:1:0.1 to give product 7 as pale yellow oil (76 mg, 27%).
1H NMR (CDCl3, 400 MHz) d 8.27–8.25 (m, 2H), 8.19–8.17 (m,
1H), 7.83 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.45
(d, J = 8.8 Hz, 1H), 7.22 (t, J = 2.8 Hz, 1H), 7.12 (dd, J = 3.0 Hz,
9.0 Hz, 1H), 6.61–6.59 (m, 1H), 4.58–4.56 (m, 1H), 3.88 (t,
J = 8 Hz, 1H), 3.78 (d, J = 10 Hz, 1H), 3.72 (d, J = 10.8 Hz, 1H),
3.43–3.35 (m, 2H), 3.13–3.03 (m, 2H), 1.77 (br s, 1H); 13C NMR
(CDCl3, 100 MHz) d 143.7, 135.9, 131.9, 128.3, 124.7, 121.7,
120.9, 120.4, 118.3, 111.1, 103.3, 62.2, 57.5, 54.5, 51.0, 37.6; HRMS
calculated for C18H18N4, [M+H] m/z = 291.1610; found, 291.1628.
Most chemicals were purchased from Sigma–Aldrich (Milwau-
kee, WI) and used without further purification. Compound 2 was
obtained from Astatech (Bristol, PA) and PHA543613 hydrochloride
was purchased from Tocris. Column flash chromatography was car-
ried out using E. Merck silica gel 60F (230–400 mesh). Analytical
thin layer chromatography (TLC) was performed on plastic sheets
coated with Silica Gel 60 F254 (0.25 mm thickness, Macherey-Na-
gel). 1H NMR and 13C NMR (nuclear magnetic resonance) spectra
were recorded with a Varian-400 MHz in CDCl3. The spectra were
acquired at 293 K. The chemical shifts were recorded on the ppm
scale and were referenced to the internal standard trimethylsilane
(TMS) at dH 0 ppm. Coupling constant (J) values were given in
Hertz. Multiplicity was defined by s (singlet), d (doublet), t (trip-
let), and m (multiplet).
3.1.4. 5-(5-(6-Methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)
pyridin-2-yl)-1H-indole (rac-1)
3.1.1. 3-(6-Chloro-3-pyridinyl)-3,6-diazabicyclo[3.2.0]-heptane-
6-carboxylate tert-butyl ester (4)
To a solution of 7 (75 mg, 0.25 mmol) in 37% HCHO (0.6 mL) and
CH3CN (0.6 mL) was added NaBH(OAc)3 (0.11 g, 0.52 mmol). This
mixture was stirred at ambient temperature for 4 h and then
quenched with NaHCO3 (0.25 g). CHCl3 (15 mL) was added, the lay-
ers were separated and the aqueous layer was extracted with
CHCl3 (3 ꢀ 15 mL). The combined organic layers were dried over
Na2SO4, concentrated under reduced pressure and purified via
flash column chromatography on silica gel (CH2Cl2/MeOH/NH4OH
15:1:0.1) to give the product rac-1 as yellow oil (22 mg, 31%). 1H
NMR (CDCl3, 400 MHz) d 8.20 (d, J = 2.8 Hz, 1H), 8.18–8.16 (m,
A
mixture of tert-butyl 3,6-diazabicyclo[3.2.0]heptane-6-
carboxylate (0.648 g, 3.27 mmol), 5-bromo-2-chloropyridine
(0.738 g, 3.83 mmol), tris(dibenzylideneacetone)dipalladium(0)
[Pd2(dba)3] (66 mg, 0.072 mmol), racemic 2,20-bis(diphenylphos-
phino)-1,10-binaphthyl (rac-BINAP) (135 mg, 0.22 mmol), sodium
tert-butoxide (t-BuONa) (0.367 g, 3.82 mmol) in toluene (3 mL)
was heated at 85 °C for 20 h. The mixture was cooled and concen-
trated under reduced pressure. The crude material was purified by

Y. Gao et al. / Bioorg. Med. Chem. 20 (2012) 3698–3702
3701
1H), 7.83 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.44 (d,
J = 8.8 Hz, 1H), 7.22 (t, J = 2.8 Hz, 1H), 7.06 (dd, J = 2.8 Hz, 8.4 Hz,
1H), 6.61–6.59 (m, 1H), 3.95–3.92 (m, 1H), 3.80 (dd, J = 2 Hz,
10 Hz, 1H), 3.73 (d, J = 10.8 Hz, 1H), 3.24–3.37 (m, 3H), 3.17–3.15
(m, 1H), 2.99 (dd, J = 4 Hz, 10.8 Hz, 1H), 2.42 (s, 3H), 1.69 (br s,
1H); 13C NMR (CDCl3, 100 MHz) d 143.2, 135.4, 132.0, 128.3,
124.6, 121.1, 120.9, 120.3, 118.2, 111.1, 103.2, 74.8, 70.2, 59.5,
injected dose per gram of tissue (% ID/g tissue) was calculated.
All animal protocols were approved by the Animal Care and Use
Committee of the Johns Hopkins University.
3.3.2. Blocking dissection studies in mice
In vivo blocking studies were performed by subcutaneous
administration of PHA543613 (0.5 mg/kg) followed 15 min later
by an intravenous injection of the radiotracer (3.7 MBq in 0.2 mL
53.7, 43.1, 33.1; HRMS calculated for
C19H20N4, [M+H] m/
z = 305.1766; found, 305.1785.
saline; specific radioactivity ꢂ185 GBq/
lmol). The blocker was dis-
solved in a vehicle solution (saline/alcohol 9:1) and administered
in a volume of 0.1 mL. Control animals were injected with 0.1 mL
of the vehicle solution. Ninety minutes after administration of
the radiotracer, brain tissues were harvested, and the radioactivity
content was determined. There were three animals per time-point
in the baseline and blockade cohorts.
3.2. Radiochemistry
The semi-preparative high performance liquid chromatography
(HPLC) system consisted of a Waters model 610 pump, a Valco
injector, a Varian Prostar 325 LC detector set to 254 nm, a Bioscan
Flow-Count PMT radioactivity detector. Analytical HPLC was per-
formed using a Varian Prostar 210 pump with a Prostar 410 Auto-
sampler, a Varian Prostar 325 LC detector set to 254 nm, and a
Bioscan Flow-Count PMT radioactivity detector. All HPLC were re-
corded and analyzed with Varian Galaxie Chromatography Data
System software (version 1.9.302.952). A dose calibrator (Capintec
15R) was used for all radioactivity measurements. [11C]Methyl io-
dide was prepared from 11CO2 using a Tracerlab FX MeI module
(General Electric) and a PETtrace biomedical cyclotron (General
Electric).
4. Conclusion
In summary, 5-(5-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]hep-
tan-3-yl)pyridin-2-yl)-1H-indole ([11C]rac-1), a radioligand with
a
7-nAChRs binding affinity in the subnanomolar range, has been
synthesized. In the mouse brain, [11C]rac-1 specifically accumu-
lated in the frontal cortex and hippocampus, regions with an ele-
vated density of
a7-nAChRs, whereas the uptake in cerebellum a
region with low densities of
a
7-nAChRs, was non-specific. The re-
sults of cerebral distribution of [11C]rac-1 in CD1 mice suggest that
the PET radioligand should be further studied with PET imaging in
non-human primates.
3.2.1. Synthesis of 5-(5-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]
heptan-3-yl)pyridin-2-yl)-1H-indole ([11C]rac-1)
Precursor 7 (0.5 mg) was dissolved in 200
tonitrile, capped in
small V-vial and cooled to ꢁ40 °C.
11C]Methyl iodide was swept by argon flow into the solution. After
the radioactivity reached a plateau, the vial was assayed in the
dose calibrator and then heated at 80 °C for 5 min. Water
lL of anhydrous ace-
Acknowledgments
a
[
The authors would like to thank Dr. William H. Bunnelle (Ab-
bott Laboratories) for helpful discussions and Mrs. Judy Buchanan
for editorial assistance. This research was supported in part by
the Division of Nuclear Medicine of Johns Hopkins University
School of Medicine and by NIH Grants DA020777 and MH079017
(A.H.).
(200
l
L) was added and the solution was injected onto the semi-
preparative HPLC column (Phenomenex Luna C-18 10
l
m column,
semi-preparative 10 ꢀ 250 mm, 32/68/0.2 v/v/v CH3CN/0.1 M
aqueous ammonium formate/Et3N, at a flow-rate of 10 mL/min).
The retention time of 7 was 3.92 min. The product [11C]rac-1 peak,
having a retention time of 8.85 min, was collected into a flask con-
taining 50 mL water. The mixture was transferred through a
Waters C-18 Sep-Pak Plus. The product was eluted with 1 mL eth-
anol into a vial and diluted with 14 mL of 0.9% saline. The final
product [11C]rac-1 was then analyzed by analytical HPLC (Phenom-
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enex Luna C-18 10
l
m columns, analytical 4.6 ꢀ 250 mm, 32/68/
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and the specific radioactivity at the end of synthesis. The total syn-
thesis time was 30 min from EOB with an average radiochemical
yield of 30% and an average specific radioactivity of 444 GBq/
lmol
(non-decay corrected from the end of 11CH3I synthesis).
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3.3. In vivo experiments
3.3.1. Baseline dissection studies in mice
CD1 mice (all males, 23–28 g) from the Charles River Laborato-
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animals were sacrificed by cervical dislocation at various times fol-
lowing injection of [11C]rac-1 (0.1 mCi in 0.2 mL saline, specific
radioactivity ꢂ5000 mCi/
lmol) into a lateral tail vein. The brains
were rapidly removed and dissected on ice. The brain regions of
interest were weighed and their radioactivity content was deter-
mined in a c-counter with a counting error below 3%. Aliquots of
the injectate were used as standards and their radioactivity con-
tent was counted along with the tissue samples. The percent of
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