[11C]Cyclopropyl-FLB 457: A PET radioligand for low densities of dopamine D2 receptors

Article abstract of DOI:10.1016/j.bmc.2008.05.039

(S)-5-Bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide (4) has pico-molar in vitro binding affinity to D₂ receptor (K_i (D₂) = 0.003 nM) with lower affinity to D₃ receptor (K_i (D₃) = 0.22 nM). In this study, we describe radiosynthesis of [¹¹C]4 and evaluation of its binding characteristics in post-mortem human brain autoradiography and with PET in cynomolgus monkeys. The ¹¹C labelled 4 was synthesized by using [¹¹C]methyltriflate in a methylation reaction with its phenolic precursor with good incorporation yield (64 ± 11%, DCY) and high specific radioactivity >370 GBq/μmol (>10 000 Ci/mmol). In post-mortem human brain autoradiography [¹¹C]4 exhibited high specific binding in brain regions enriched with dopamine D₂/D₃ receptors and low level of non-specific binding. In cynomolgus monkeys [¹¹C]4 exhibited high brain uptake reaching 4.4% ID at 7.5 min. The binding in the extrastriatal low density D₂-receptor regions; thalamus and frontal, parietal, temporal, and occipital cortex, was clearly visible. Pre-treatment with raclopride (1 mg/kg as tartrate) caused high reduction of binding in extrastriatal regions, including cerebellum. [¹¹C]4 is a promising radioligand for imaging D₂ receptors in low density regions in brain.

Full text of DOI:10.1016/j.bmc.2008.05.039

Bioorganic & Medicinal Chemistry 16 (2008) 6467–6473
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
[11C]Cyclopropyl-FLB 457: A PET radioligand for low densities of dopamine
D2 receptors
a
a
c
c
Anu J. Airaksinen a,b, , Sangram Nag , Sjoerd J. Finnema , Jogeshwar Mukherjee , Sankha Chattopadhyay ,
*
Balázs Gulyás a, Lars Farde a, Christer Halldin a
a Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
b University of Helsinki, Department of Chemistry, Laboratory of Radiochemistry, FI-00014 University of Helsinki, Finland
c Department of Psychiatry and Human Behaviour, University of California-Irvine, Irvine, CA 92697, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 February 2008
Revised 7 May 2008
Accepted 16 May 2008
Available online 20 May 2008
(S)-5-Bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide (4) has pico-
molar in vitro binding affinity to D2 receptor (Ki (D2) = 0.003 nM) with lower affinity to D3 receptor (Ki
(D3) = 0.22 nM). In this study, we describe radiosynthesis of [11C]4 and evaluation of its binding charac-
teristics in post-mortem human brain autoradiography and with PET in cynomolgus monkeys. The 11C
labelled 4 was synthesized by using [11C]methyltriflate in a methylation reaction with its phenolic pre-
cursor with good incorporation yield (64 11%, DCY) and high specific radioactivity >370 GBq/lmol (>10
Keywords:
Dopamine
D2-receptor
Antagonist
PET
000 Ci/mmol). In post-mortem human brain autoradiography [11C]4 exhibited high specific binding in
brain regions enriched with dopamine D2/D3 receptors and low level of non-specific binding. In cynomol-
gus monkeys [11C]4 exhibited high brain uptake reaching 4.4% ID at 7.5 min. The binding in the extrastri-
atal low density D2-receptor regions; thalamus and frontal, parietal, temporal, and occipital cortex, was
clearly visible. Pre-treatment with raclopride (1 mg/kg as tartrate) caused high reduction of binding in
extrastriatal regions, including cerebellum. [11C]4 is a promising radioligand for imaging D2 receptors
in low density regions in brain.
Autoradiography
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
equal affinity to both D2 and D3 receptors and their usage in D2
receptor imaging has been promoted because of the low receptor
Positron emission tomography (PET) has been widely utilized in
visualizing striatal and extrastriatal dopamine D2 receptors using
D2/D3 receptor antagonists such as [11C]raclopride, [11C]FLB 457
and [18F]fallypride (Fig. 1).1–5 These benzamides have provided
deep knowledge in understanding involvement of changes in dopa-
mine neurotransmission in development of neuropsychiatric disor-
ders such as Parkinson’s disease, schizophrenia and depression.6–10
Pico-molar binding affinity D2/D3 receptor ligands [11C]FLB 457
and [18F]fallypride have been used in exploring changes in D2/D3
receptor densities especially in extrastriatal structures in the brain,
for which [11C]raclopride as a moderate affinity ligand does not
provide enough signal for quantitative D2/D3 receptor imaging.
In extrastriatal D2-receptor regions, that is, thalamus, hypothal-
amus, amygdala, substantia nigra, frontal cortex and temporal cor-
tex, D2 receptor density is ten to hundred times lower than in the
high density striatum (measured with D2/D3 antagonist),11 which
sets high demands for selectivity and binding affinity of the radio-
ligand. The generally used D2 receptor antagonists have almost
density of D3 receptor in the brain, and because of the higher occu-
pancy of D3 receptors in vivo by endogenous dopamine.12 How-
ever, the recent advancement of D3 receptor preferring
radioligands has provided new information on the brain distribu-
tion of the receptor and the role of these receptors in drug addic-
tion and in neurological and neuropsychiatric disorders.13–15
While located primarily in limbic brain regions, moderate densities
of D3 receptors are expressed also in thalamus, hypothalamus and
nucleus accumbens, regions overlapping with the low density D2
regions. There are recent findings that D2 and D3 receptors have
a diverse role in progress of schizophrenia, Parkinson’s disease
and in drug abuse.16–18 For discriminating their role in these path-
ological conditions, radioligands purely selective only to one recep-
tor type would be beneficial.
The new cyclopropylmethyl analogue of FLB 457, (S)-N-[(1-
cyclopropylmethyl-2-pyrrolidinyl)methyl]-5-bromo-2,3-dime-
thoxybenzamide (4), was discovered in studies, which focused on
development of D3 receptor selective substituted benzamide
antagonists.19 In the structural series, effect of cyclopropylmethyl
group and substitutions in the aromatic and pyrrolidine ring were
investigated. On the contrary to the expectations, the compound 4
* Corresponding author. Tel.: +358 9 191 50133; fax: +358 9 191 50121.
E-mail address: anu.airaksinen@helsinki.fi (A.J. Airaksinen).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.039

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A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
2.2. Radiosynthesis
[
11C]4 was synthesized with standard methods with minor
modifications as previously published for synthesis of [11C]FLB
457.20,21 The precursor 3 was methylated with [11C]methyl triflate
in room temperature, with good incorporation yield 64 11% (DCY
%). The total synthesis time, including the HPLC purification was
28 min and specific radioactivity at the time of injection was over
370 GBq/lmol (>10 000 Ci/mmol)(Fig. 2). The radiochemical purity
was >99%. The product [11C]4 was confirmed with a co-elution
with the co-injected unlabelled reference (4).
3. Post-mortem human brain autoradiography
Autoradiography with [11C]4 in baseline conditions gave high
binding in regions with high D2 receptor density, caudate head,
putamen and pallidum (Fig. 3). [11C]4 bound also with moderate
level to thalamus, where the D2-receptor expression is lower. Addi-
Figure 1. Chemical structures of some commonly used D2/D3-receptor tracers for
PET and structure of the new [11C]cyclopropylmethyl analogue of FLB 457 ([11C]4).
tion of raclopride (10
blocked the binding.
lM) into the incubation buffer effectively
4. PET measurements
was found to have ten times higher in vitro binding affinity to D2
receptor than the widely used structural analogues FLB 457 and
Total brain uptake of [11C]4 in cynomolgus monkey was high,
reaching 4.4% ID at 7.5 min. Binding in D2-receptor rich striatal re-
gions was highest at the end of the PET measurement, without
reaching equilibrium during the 93 min scanning time (Figs. 4
and 5). The highest level of radioactivity was observed in putamen,
followed by caudate nucleus and pallidum. Region over cerebellum
ratios reached maximum at 84 min, giving ratios of 7.61, 6.60, and
5.40, for putamen, caudate nucleus and pallidum, respectively.
Extrastriatal regions were clearly visible, indicating sufficient bind-
ing affinity also in vivo. The binding was highest in the moderate
density receptor region thalamus, followed by cortical regions with
low D2-receptor density. Region over cerebellum ratios was at
maximum 3.70, 3.24, 2.98, 2.59, and 2.08 for thalamus and frontal,
parietal, temporal, and occipital cortex, respectively.
fallypride, but with
a similar affinity to D3 receptor Ki
(D2) = 0.003 nM, Ki (D3) = 0.22 nM, ratio Ki D2/D3 = 0.014.19 Despite
its early discovery, it has not been radiolabelled and evaluated in
vivo before. Our aim was to radiolabel the new D2 receptor selec-
tive analogue compound 4, and preliminarily evaluate its binding
characteristics in post-mortem human brain and in vivo in cyno-
molgus monkeys.
2. Chemistry
2.1. Synthesis of the precursor
Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) was used as the condensing agent in the
reaction of the substituted benzoic acid (1) with (S)-2-(amino-
methyl)-N-cyclopropylmethylpyrrolidine (2) (Scheme 1). Isolated
yields of the final amide product 3 were modest (40%). It is likely
that the yields may be improved if the phenolic group is protected
during the reaction. The amide 3 was found to be stable over sev-
eral months and stored at 0 to ꢀ20 °C.
Specific binding of [11C]4 to D2/D3 receptors was measured after
pre-treatment with raclopride (Fig. 5). For this preliminary in vivo
evaluation, raclopride was chosen for pre-treatment agent, because
its in vivo behaviour is well characterized. In addition, because it is
non-selective dopamine D2 and D3 receptor antagonist, it would
clearly demonstrate the amount of non-specific binding in low
density D2/D3-receptor brain regions, however, without differenti-
O
OH
BOP, TEA
H2N
OH
+
N
ACN, 24 h, rt.
Br
OCH3
2
1
H
H
N
N
N
N
O
O
11CH3OTf
NaOH, Acetone
OH
O
11CH3
Br
OCH3
Br
OCH3
3
[
11C]4
Scheme 1. Synthesis of the precursor (3) and radiosynthesis of [11C]4.

A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
6469
Figure 2. Semipreparative HPLC-chromatogram of [11C]4 purification.
found eluting prior to the parent compound and more lipophilic
radioactive metabolites were not observed (Fig. 8). More than
94% of the injected radioactivity was recovered from the HPLC-col-
umn. Radioactivity in pellets after deproteinization with acetoni-
trile was 8.3 1.9%.
5. In vitro lipophilicity and plasma protein binding
Apparent partition coefficient of
[
11C]4 in pH 7.4 was
1.82 0.01. Partition coefficient of [11C]FLB 457 was measured as
a control. The measured partition coefficient of [11C]FLB 457 was
1.33 0.01. The result confirms the predicted increase in lipophil-
icity due to introduction of a cyclopropyl methyl group to the N-
position of the pyrrolidine side chain. The free fraction of [11C]4
in plasma in baseline conditions was 32 2% and after pre-treat-
ment it was 36 1%, giving confirmation that the raclopride pre-
treatment had no significant influence on the free fraction.
Figure 3. Human brain autoradiograms labelled with [11C]4. (A) Baseline condi-
tions. (B) Incubation with raclopride (10 M).
l
6. Discussion
Substituted benzamides containing the N-cyclopropylmethyl
pyrrolidine ring has been synthesized previously and found to be
stable and bound to dopamine receptor subtypes with high binding
affinities.19 These derivatives were synthesized using a coupling
reaction between the acid fluorides/anhydrides with (S)-2-(amino-
methyl)-N-cyclopropylmethylpyrrolidine (2). For this study, in or-
der to prepare the precursor amide 3, which contains a free
phenolic group at the 2-position, we used BOP instead, for assisting
in the coupling of the substituted salicylic acid 1 with (S)-2-(ami-
nomethyl)-N-cyclopropylmethylpyrrolidine (2). Product yields
were somewhat lower compared to the acid fluoride/anhydride
method (40% vs. 70–80%).
ating between these two receptor types. The pre-treatment caused
substantial reduction in binding in extrastriatal regions, but 40% of
the binding in striatal regions remained unblocked. Also 50% of the
[
11C]4 binding in cerebellum was blocked due to the pre-treatment
(Fig. 6).
4.1. Plasma metabolite studies
The HPLC analysis of metabolites in monkey plasma revealed
fastest metabolism of [11C]4 during the first 15 min (Fig. 7). About
60% of the parent compound was left after 45 min observation
time. Two more polar unidentified radioactive metabolites were

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A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
Figure 4. Colour coded PET images showing the distribution of radioactivity in the monkey brain after injection of [11C]4. Injected doses were 56 and 50 MBq, respectively.
(A) Baseline conditions. (B) After pre-treatment with raclopride (1 mg/kg as tartrate). Summation images from 9th- to 93rd-min are shown. Anaesthesia was induced and
maintained by repeated intramuscular injections of a mixture of ketamine hydrochloride (3.75 mg/kg/h) and xylazine hydrochloride (1.5 mg/kg/h).
A
B
500
450
400
350
300
250
200
150
100
50
500
450
400
350
300
250
200
150
100
50
Putamen
Pariet.Cx
Temp.Cx
Occip.Cx
Cerebellum
Caudate nucleus
Pallidum
Thalamus
Front.Cx
0
0
0
20
40
60
80
100
0
20
40
60
80
100
Time (min)
Time (min)
Figure 5. Time activity curves of [11C]4 binding in cynomolgus monkey in baseline conditions (A) and after pre-treatment with raclopride (B).
[11C]4 (Rf = 6.3 min)
180
160
140
120
100
80
100
Metabolite 1 (Rf = 2.9 min)
Metabolite 2 (Rf= 4.7 min)
Baseline
90
80
70
60
50
40
30
20
10
0
Pretreated
60
40
20
0
0
20
40
60
80
100
Time (min)
Figure 6. Effect of raclopride pre-treatment (1 mg/kg) on
cerebellum.
[
11C]4 binding in
0
10
20
30
40
50
Time (min)
The radiolabelled [11C]4 was synthesized via methylation using
Figure 7. Time course for the metabolism of [11C]4 in monkey plasma (n = 3).
[
11C]methyl triflate with good incorporation yield and with high
specific radioactivity (>10 000 Ci/mmol). For the structural ana-
logue [11C]FLB 457 the co-injected mass of 0.5
lg has been esti-
[
11C]4 were 0.005 and 0.001
lg for cynomolgus monkeys weight-
mated to cause 5% occupancy in extrastriatal D2/D3 binding sites
ing 2.4 and 4.2 kg, respectively. The low injected mass is not pre-
in humans.22 In our study, in baseline scans, carrier masses of
sumed to significantly influence the [11C]4 binding even in the

A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
6471
post-mortem human autoradiography and in vivo in cynomolgus
monkeys. Analysis of radiolabelled metabolites of [11C]4 from
monkey plasma revealed that only more polar metabolites were
formed, which are unlikely to enter the brain. No clear indication
for higher D2-selectivity over D3-receptors of [11C]4 was demon-
strated in the present study, but the observed high specific bind-
ing in low density D2 regions gives an indication for the high
binding affinity also in vivo. In conclusion, [11C]4 is a potential
radioligand for studying extrastriatal low density D2 regions,
including cerebellum.
[11C]4
Radioactivity
25
20
15
10
5
M 1
M 2
0
0
2
4
6
8
10
Time (min)
8. Experimental
8.1. Chemistry
Figure 8. Representative HPLC chromatogram of radioactive metabolites of [11C]4
in monkey plasma 30 min after tracer injection.
Commercial reagents and solvents (Aldrich Chemical Com-
pany, Milwaukee, WI, USA) of analytical grade were used with-
out further purification. 5-Bromo-2-hydroxy-3-methoxybenzoic
acid (1), (S)-2-(aminomethyl)-N-cyclopropylmethylpyrrolidine
(2) and the reference compound (S)-5-bromo -N-[(1-cyclopro-
pylmethyl-2-pyrrolidinyl) methyl]-2,3-dimethoxybenzamide (4)
were prepared according to previously described methods.19,26
Reaction mixtures and extractions were evaporated in a rotavap-
ory evaporator under reduced pressure as appropriate. Analytical
and preparative thin layer chromatography (TLC) was performed
using Baker-flex silica gel IB-F (Philipsburg, NJ USA) and Alltech
DC-Fertigplatten SIL G-200 UV254 plates (Deerfield, IL, USA),
respectively. Proton nuclear magnetic resonance (NMR,
500 MHz, TMS as internal reference in CDC13) spectra were ob-
tained on a Bruker-Omega 500 spectrometer. Electrospray mass
spectra were obtained on a Model 7250 mass spectrometer
(Micromass LCT).
low density regions. The binding of [11C]4 in extrastriatal regions,
in thalamus and cortical regions, was clearly visible and it exhib-
ited relatively high binding in cerebellum, from which about 50%
was blocked after pre-treatment with raclopride. This result is in
accordance with recent findings using [11C]FLB 457 in rats, where
60% of the total cerebellar binding corresponded to specific bind-
ing.23 Due to the relatively high level of specific binding in cerebel-
lum, the level of specific binding in extrastriatal regions is
underestimated, when cerebellum is used as reference region.
In striatal regions [11C]4 exhibited slow kinetics, which has
been also a disadvantage of other high affinity tracers, such as
[
11C]FLB 457 and [11C]fallypride.5,24 In addition to the high binding
affinity of [11C]4, the slow wash-out may be partly a result of the
relatively high lipophilicity (logD7.4 ([11C]4) = 1.82 0.01) caused
by the additional cyclopropylmethyl group. The lipophilicity of
[
11C]4 may be even underestimated in our experimental setup, be-
cause the measured partition coefficient of the reference com-
pound [11C]FLB 457 was lower to what has been published by
Wilson et al. with the same procedure ([11C]FLB 457
logD7.4 = 1.71 0.02).25
The pre-treatment with raclopride did not completely block
binding of [11C]4 in striatal regions, only 60% reduction in binding
was observed. In post-mortem autoradiography, binding of [11C]4
was totally prevented by 10 mM raclopride, which may be ex-
plained by that in autoradiography, the conditions for washing
and binding competition are more harsh than in vivo. The preli-
minary results of the corresponding 18F-labelled cyclopropyl ana-
All reagents used for radiosynthesis were purchased from
commercial suppliers and used without further purification. Ace-
tone and 0.5 M NaOH solution were from Merck Chemicals Ltd.
HPLC solvents were of HPLC grade from Fisher Scientific.
[
11C]Methane was produced in a GEMS PETtrace cyclotron by
bombardment of a nitrogen gas target containing 10% of H2 with
16 MeV protons (14N(p, 11C reaction). The target gas was irradi-
ated for 20 min with a beam intensity of 35 A. The synthesis
a)
l
and purification of the radiolabelled compounds was performed
in a fully automated methylation system that has been described
earlier.20
gas-phase iodination.
[
11C]Methyl iodide was prepared from [11C]methane by
11C]Methyl triflate was obtained by
logue of fallypride exhibited high non-specific binding in
a
[
rhesus monkey.19 In addition, the compound showed rapid accu-
mulation of metabolites of the radiotracer. The HPLC-analysis of
sweeping [11C]methyl iodide vapour through a glass column con-
taining silver-triflate-impregnated graphitized carbon (Fluka AG)
and heated at 150–200 °C.20,27
[
11C]4 and its radioactive metabolites in monkey plasma did not
11C]4 was purified in a built-in high performance liquid chro-
reveal any indication on lipophilic radiolabelled metabolites,
which may enter the brain.
[
matography (HPLC) system, consisting of a Gilson 234 autoinjector
(Middleton, MA, USA), a Gilson 304 piston pump, a Waters Porasil
m) and a Gilson 118 UV/VIS
Compound 4 has a higher selectivity to D2-receptors over D3-
receptors in vitro than the commonly used D2/D3 receptor antago-
nists FLB 457 and fallypride. Moderate densities of D3 receptors are
expressed in thalamus and there are estimates that even one third
of the receptor population in pallidum corresponds to D3 recep-
tors.14 The compound [11C]4 did not exhibit any significantly lower
uptake in these regions, showing no clear evidence for higher D2-
selectivity over D3-receptors in vivo. For studying D2 selectivity
of [11C]4 binding, further experiments using D3-selective com-
pounds and comparison to commonly used [11C] labelled D2/D3
receptor ligands are needed.
l
silica column (300 ꢁ 7.8 mm, 10
l
detector (wavelength 254 nm) in a series with a Geiger Müller
(GM) tube for radiation detection. Radiochemical purity of [11C]4
was analyzed on reverse phase HPLC using a Merck-Hitachi L-
7100 Pump, equipped with a Waters
lBondapak C18 column
(300 ꢁ 3.9 mm, 10 m) (Milford, MA, USA) and L-7400 UV-detector,
l
D-7000 interface and Beckman radiodetector (Model 170). The sys-
tem was controlled by Merck-Hitachi Chromatography Data Station
Software D-7000 (version 4.1). Acetonitrile (30%) in 0.01 M H3PO4
was used as mobile phase. Specific radioactivity of [11C]4 was ana-
lyzed with HPLC, consisting of Waters
l-Bondapak C18 column
7. Conclusion
(300 ꢁ 3.9 mm, 10
l
m), Merck-Hitachi L-6200A intelligent pump
and Merck-Hitachi L-4000 UV (254 nm) detector. The mobile phase
was acetonitrile in 0.05 M H3PO4 (18/82) and flow rate was 3 mL/
min. The product eluted at 12–13 min.
The D2 receptor antagonist [11C]4 exhibited high level of spe-
cific binding to brain regions with dopamine D2 receptors in

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A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
8.2. (S)-5-Bromo-N-[(1-cyclopropylmethyl-2-
pyrrolidinyl)methyl]-2-hydroxy-3-methoxybenzamide (3)
sections with a separation of 3.1 mm and a transversal resolution
of about 3.8 mm FWHM (Full Width Half Maximum). The attenua-
tion correction of the data was obtained with three rotating 68Ge
rod sources. Raw PET data were then reconstructed using the stan-
dard filtered back projection consisting of the following recon-
struction parameters: 2 mm Hanning Filter, scatter correction, a
zoom factor of 2.17 and a 128 ꢁ 128 matrix seize.28
To a solution of 5-bromo-2-hydroxy-3-methoxybenzoic acid, 1
(0.25 g, 1 mmol) in anhydrous acetonitrile (5 mL) were added (S)-
2-(aminomethyl)-N-cyclopropylmethylpyrrolidine
2
(0.17 g,
1.1 mmol). This mixture was stirred at room temperature and
BOP reagent (benzotriazol-1-yloxy-tris(dimethylamino)phospho-
nium hexafluorophosphate, 0.44 g, 1 mmol) and triethylamine
(0.4 mL) was added. The reaction mixture was stirred for 24 h. Vol-
atiles were removed in vacuo and the residue was taken in dichlo-
romethane and washed with water and saturated sodium
bicarbonate. The organic layer was separated, dried over magne-
sium sulfate, and filtered. The filtrate was concentrated and subse-
10.2. PET experimental procedure
PET measurements were performed in two female cynomolgus
monkeys (Macaca fascicularis), weighting 2.4 and 4.2 kg. The mon-
keys were supplied by Astrid Fagraeus Laboratory, Swedish Insti-
tute for Infectious Disease Control (SMI), Solna, Sweden. The
study was approved by the Animal Research Ethical Committee
of the Northern Stockholm Region. Anaesthesia was induced and
maintained by repeated intramuscular injections of a mixture of
ketamine hydrochloride (3.75 mg/kg/h, KetalarÒ, Pfizer) and xyla-
zine hydrochloride (1.5 mg/kg/h RompunÒ, Vet. Bayer) for the
duration of each measurement. A head fixation system was used
to secure a fixed position of the monkey head during the PET
experiments.29 Body temperature was maintained by Bair Hug-
ger—Model 505 (Arizant Healthcare Inc., MN) and monitored by
a rectal thermometer (Precision Thermometer, Harvard Apparatus,
MA). Cardiac and respiratory rates were monitored continuously
throughout the experiment (VetGard monitor, Veterinary Market-
ing Systems, Lincoln, NE).
quently
purified
using
preparative
TLC
(silica
gel,
dichloromethane: methanol, 9:1) to provide 3 in 40% yield. 1H
NMR d ppm 8.20 (br, 1 H, CONH), 7.4 (d, 1H), 7.0 (d, 1H), 3.8 (s,
3H, OCH3), 3.1–1.5 (m, 11H), 0.30 (5H, c-C3H5). Electrospray Mass
spectra (m/z, %) 383, 385 ([M+H]+, 100%).
8.3. (S)-5-Bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)
methyl]-2-[11C]methoxy-3-methoxy-benzamide, [11C]4
(S)-5-Bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-
2-hydroxy-3-methoxybenzamide (3, 0.5 mg, 1.3
solved in acetone (400 L) and 0.5 M NaOH (6
l
l
mol) was dis-
L, 3 mol) was
l
l
added. The mixture was bubbled with [11C]methyl triflate at room
temperature. The product was purified with the semi-preparative
HPLC system with CH2Cl2/MeOH/TEA (95/5/0.5, pH 8.0) as eluent
with flow 2 mL/min from 0 to 4 min and 4 mL/min after that.
Retention time of the product was 6.3 min. The collected fraction,
containing the purified product, was evaporated to dryness and
formulated in 7 mL of phosphate buffer (pH 7.4) and 3 mL of
30% EtOH in propylene glycol (v/v). Incorporation yield was
64 11%, with the total synthesis time of 28 min. Specific radioac-
Both monkeys participated in one baseline and one pre-treat-
ment measurement, respectively. In the first measurement,
56.2 0.2 MBq of [11C]4 was injected as a bolus during 5 s into a
sural vein and radioactivity in the brain was measured according
to a pre-programmed sequence of frames up to 93 min after the
injection of the tracer. Injected mass in the baseline experiments
was 0.005 and 0.001 lg, respectively. In the second measurement
1.0 mg/kg of raclopride-tartrate was injected intravenously to the
tivity at the time of injection was >370 GBq/
l
mol (>10,000 Ci/
same monkey 35 min before injection of [11C]4 (52.5 2.5 MBq, in-
mmol). Purity was analyzed with the analytical HPLC system with
flow 3 mL/min. The product was identified with a co-injection of
the reference. Retention time of the product was 4.18 min. Radio-
chemical purity was >99%.
jected mass 0.01 and 0.002 lg) and the radioactivity in the brain
was measured as previously described.
10.3. Quantitative image analysis
PET summation images representing radioactivity measured
from 9 to 93 min after an intravenous injection of [11C]4 were gen-
erated. The baseline PET images were transformed into a standard
anatomical space using the monkey version of the Human Brain At-
las developed at Karolinska Institute.30 The transformation matrix
generated on this image was applied to all the frames from the cor-
responding and the consecutively performed pre-treatment PET
study.
9. Post-mortem human brain autoradiography
Horizontal whole hemisphere post-mortem human brain sec-
tions of 100 lm thickness from the level of striatum were incu-
bated for 20 min at room temperature with [11C]4 (approx.
30 MBq) in Tris-buffer (50 mM, pH 7.4), containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, Pargylin (10
lM), and 0.1%
acetic acid. The D2/D3 antagonist raclopride (10 M) was included
l
A standard template of ROIs was generated on an average mon-
key MRI scan and applied to each PET study. ROIs were delineated
for the putamen, caudate nucleus, pallidum, thalamus, frontal,
occipital, parietal and temporal cortex, cerebellum, and whole
brain. The ROI of the cerebellum excluded the vermis and white
matter.
All calculations were based on the assumption that radioactivity
in brain represents unchanged radioligand. The fraction of [11C]4 in
whole brain relative to the total amount of radioligand injected
was calculated and plotted versus time after injection. The radioac-
tivity concentration in the ROI for the whole brain was multiplied
with the whole brain ROI volume (ꢂ65 mL), divided by the radio-
activity injected and multiplied by 100 to obtain the percentage.
Regional radioactivity was normalized to injected activity and
body weight by use of % of standard uptake value (%SUV = (% injec-
tion dose/cm3 brain) ꢁ body weight (g)). The cerebellum was used
as a reference region for non-displaceable [11C]4 binding. The radio-
in the incubation medium to determine the level of non-specific
binding. After incubation, the sections were washed with ice-cold
Tris buffer (50 mM, pH 7.4) for 2 ꢁ 2 min, briefly dipped into ice-
cold distilled water and dried with warm air flow. Phosphor imager
plates (BAS-IP SR 2025, Fuji Photo Film, Japan) were used for detec-
tion of the radioactivity distribution. The plates were exposed for
20 min, scanned and analyzed with Fuji BAS-5000 image reader,
running with Fuji Film Multi Gauge software.
10. PET measurements
10.1. PET system
The radioactivity distribution was measured using a Siemens
ECAT EXACT HR PET camera system (CTI/Siemens, Knoxville, TN)
in three-dimensional mode, which measures radioactivity in 47

A. J. Airaksinen et al. / Bioorg. Med. Chem. 16 (2008) 6467–6473
6473
activity in the cerebellum was accordingly used as an approximate
Acknowledgments
for free and non-specifically bound radioligand concentration in
brain. The time curve for specific [11C]4 binding to D2 receptors in
high-density brain regions was defined as the difference between
the total radioactivity concentration in a ROI and the cerebellum.
We thank Phong Truong, Siv Eriksson, and Dr. Baogang Xue for
technical assistance. A part of this work was supported by grant
from Academy of Finland (decision number: 115385) and grant
number R01EB006110 from the National Institute of Biomedical
Imaging and Bioengineering (USA).
10.4. Plasma metabolite studies
For the analysis of radioactive metabolites in monkey plasma,
blood samples of 2.0 mL were obtained from the femoral vein of a
monkey 4, 15, 30, and 45 min after injection of the tracer. Fractions
of radioactivity in the monkey plasma that corresponded to un-
changed [11C]4 and radioactive metabolites were determined using
a modification of a gradient method described by Halldin et al.31
Shortly, the blood samples were centrifuged at 2000g for 2 min,
and the plasma (0.5 mL) was mixed with acetonitrile (0.7 mL) and
centrifuged at 2000g for 2 min. The supernatant of the acetonitrile
References and notes
1. Halldin, C.; Farde, L.; Högberg, T.; Mohell, N.; Hall, H.; Suhara, T.; Karlsson, P.;
Nakashima, Y.; Swahn, C.-G. J. Nucl. Med. 1995, 36, 1275.
2. Olsson, H.; Halldin, C.; Swahn, C.-G.; Farde, L. . L. J. Cereb. Blood Flow Metab.
1999, 19, 1164.
3. Okauchi, T.; Suhara, T.; Maeda, J.; Kawabe, K.; Obayashi, S.; Suzuki, K. Synapse
2001, 41, 87.
4. Mukherjee, J.; Christian, B. T.; Dunigan, K. A.; Shi, B.; Narakyanan, T. K.; Satter,
M.; Mantil, J. Synapse 2002, 46, 170.
5. Mukherjee, J.; Shi, B.; Christian, B. T.; Chattopadhyay, S.; Narayanan, T. K.
Bioorg. Med. Chem. 2004, 12, 95.
6. Antonini, A.; Scwarz, J.; Oertel, W. H.; Pogarell, O.; Leenders, K. L. Mov. Disord.
1997, 12, 33.
7. Thobois, S.; Jahanshahi, M.; Pinto, S.; Frackowiak, R.; Limousin-Dowsey, P.
Neuroimage 2004, 23, 1.
8. Hirvonen, J.; van Erp, T. G. M.; Huttunen, J.; Någren, K.; Huttunen, M.; Aalto, S.;
Lönnqvist, J.; Kaprio, J.; Cannon, T. D.; Hietala, J. Psychiatry Res. Neuroimaging
2006, 146, 13.
9. Talvik, M.; Nordström, A.-L.; Okubo, Y.; Olsson, H.; Borg, J.; Halldin, C.; Farde, L.
Psychiatry Res. Neuroimaging 2006, 148, 165.
10. Mayer, J. H.; McNeely, H. E.; Sagrati, S.; Boovariwala, A.; Martin, K.; Verhoeff, N.
P. L. G.; Wilson, A. A.; Houle, S. Am J. Psychiatry 2006, 163, 1594.
11. Kessler, R. M.; Whetsell, W. O.; Ansari, M. S.; Votaw, J. R.; de Paulis, T.;
Clanton, J. A.; Schmidt, D. E.; Mason, N. S.; Manning, R. G. Brain Res. 1993,
609, 237.
12. Schotte, A.; Janssen, P. F. M.; Gommeren, W.; Luyten, W. H. M. L.; van
Gompel, P.; Lesage, A. S.; de Loore, K.; Leysen, J. E. Psychopharmacology 1996,
124, 57.
13. Narendran, R.; Slifstein, M.; Guillin, O.; Hwang, Y.; Hwang, D.-R.; Scher, E.;
Reeder, S.; Rabiner, E.; Laruelle, M. Synapse 2006, 60, 485.
14. Seeman, P.; Wilson, A.; Gmeiner, P.; Kapur, S. Synapse 2006, 60, 205.
15. Rabiner, E. A.; Raymond, R.; Diwan, M.; McCormick, P.; Wilson, A. A.; Nobrega,
J. J. Nucl. Med. 2007, 48(Suppl. 2), 113P.
plasma mixture (1.1 mL) was injected to
lBondapak C18
(300 ꢁ 7.8 mm, 10 m) HPLC column and analyzed with a gradient
l
of acetonitrile (A) and phosphoric acid (0.01 M) (B) with a pro-
gramme: 0 min (A/B) 10:90, 5 min (A/B) 80:20, 6.5 min (A/B) 10:90.
Flow was 6 ml/min. Radioactivity of the injected supernatant, pro-
tein pellet, the residual in the HPLC syringe and the collected radio-
activity from the HPLC were measured with a NaI well counter for
calculation of total recovery of the metabolite analysis.
11. In vitro lipophilicity and plasma protein binding
Lipophilicity of [11C]4 in pH 7.4 (logD7.4) was determined with a
shake-flask method.25 Partition coefficient of [11C]FLB 457 was
measured as a standard. In short, the tracer solution (40 ll) was
added to a separation funnel, containing 1-octanol (10 mL) and
phosphate buffer (10 ml, pH 7.4). The flask was shaken 3 min and
the aqueous layer discarded. Another portion of phosphate buffer
(10 mL) was added and the procedure was repeated. Four portions
(2 mL each) of the 1-octanol layer were pipetted to four test tubes
and phosphate buffer (2 mL to each) was added. The tubes were
vortexed for 2 min and centrifuged 4 min in 2000g. Samples
(0.5 mL) were pipetted from the both the layers for counting. The
partition coefficient (D) was calculated using
16. Vogel, M.; Busse, S.; Freyberger, H. J.; Grabe, H. J. Med. Hypotheses 2006, 67,
354.
17. Joyce, J. N.; Millan, M. J. Drug Discovery Today 2005, 10, 917.
18. Heidbreder, C. A.; Gardner, E. L.; Zheng-Xiong, X.; Panayotis, K. T.; Mugnaini,
M.; Hagan, J. J.; Ashby, C. R., Jr. Brain Res. Rev. 2005, 49, 77.
19. Yang, Z.-Y.; J. Mukherjee J. Med. Chem. Res. 1999, 9, 1.
20. Sandell, J.; Langer, O.; Larsen, P.; Dollé, F.; Vaufrey, F.; Demphel, S.; Crouzel, C.;
Halldin, C. J. Labelled Compd. Radiopharm. 2000, 43, 331.
21. Lundkvist, C.; Sandell, J.; Någren, K.; Pike, V. W.; Halldin, C. J. Labelled Compd.
Radiopharm. 1998, 41, 545.
22. Olsson, H.; Halldin, C.; Farde, L. Neuroimage 2004, 22, 794.
23. Asselin, M.-C.; Montgomery, A. J.; Grasby, P. M.; Hume, S. P. J. Cereb. Blood Flow
Metab. 2007, 27, 378.
24. Olsson, H.; Farde, L. Neuroimage 2001, 14, 936.
25. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Radiat. Isot. 2001,
54, 203.
26. Hogberg, T.; de Paulis, T.; Johansson, L.; Kumar, Y.; Hall, H.; Ogren, S. O. J. Med.
Chem. 1990, 33, 2305.
27. Jewett, D. M. Appl. Radiat. Isot. 1992, 43, 1383.
28. Wienhard, K.; Dahlbom, M.; Eriksson, L.; Michel, C.; Bruckbauer, T.; Pietrzyk,
U.; Heiss, W. D. T. J. Comput. Assist. Tomogr. 1994, 18, 110.
29. Karlsson, P.; Farde, L.; Halldin, C.; Swahn, C.; Sedvall, G.; Foged, C.; Hansen, K.
T.; Skrumsager, B. Psychopharmacology 1993, 113, 149.
30. Roland, P. E.; Zilles, K. Trends Neurosci. 1994, 17, 458.
31. Halldin, C.; Swan, C.-G.; Farde, L.; Sedvall, G. In PET for Drug Development and
Evaluation; Comar, D., Ed.; Kluwer: Dordrecht, 1995; pp 55–65.
½cpm=mLꢃoctanol
¼ D
ð1Þ
½cpm=mLꢃbuffer
Plasma protein binding of [11C]4 was measured in duplicate in
baseline conditions and after pre-treatment with raclopride. Mon-
key plasma (500
with [11C]4 solution (100
room temperature for 10 min. Samples (20
tion mixture were measured with a well counter. After the incuba-
tion, 100 L portions of the incubation mixtures were pipetted into
l
L) or 0.9% NaCl-solution as a control was mixed
L, approx. 10 MBq) and incubated in
L) from each incuba-
l
l
l
ultra-filtration tubes (Millipore Centrifree YM-30) and centrifuged
for 10 min in 2000 rpm and 5 min in 3000 rpm (Sigma-202, Axel
Johnson Lab System). Samples (20 lL) from each filtrate were
pipetted for counting. The results were corrected for the mem-
brane binding as measured with the control samples.