Synthesis and initial PET imaging of new potential dopamine D3 receptor radioligands (E)-4,3,2-[11C]methoxy-N-4-(4-(2-methoxyphenyl) piperazin-1-yl)butyl-cinnamoylamides

Article abstract of DOI:10.1016/j.bmc.2005.06.055

D₃ receptor radioligands (E)-4,3,2-[¹¹C]methoxy-N-4- (4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4-[¹¹C]MMC, [¹¹C]1a; 3-[¹¹C]MMC, [¹¹C]1b; and 2-[ ¹¹C]MMC, [¹¹

Full text of DOI:10.1016/j.bmc.2005.06.055

Bioorganic & Medicinal Chemistry 13 (2005) 6233–6243
Synthesis and initial PET imaging of new potential
dopamine D3 receptor radioligands (E)-4,3,2-[11C]methoxy-N-
4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides
Mingzhang Gao, Bruce H. Mock, Gary D. Hutchins and Qi-Huang Zheng*
Department of Radiology, Indiana University School of Medicine, 1345 West 16th Street, L-3 Room 202, Indianapolis, IN 46202, USA
Received 2 June 2005; revised 23 June 2005; accepted 23 June 2005
Available online 8 August 2005
Abstract—D3 receptor radioligands (E)-4,3,2-[11C]methoxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4-
[11C]MMC, [11C]1a; 3-[11C]MMC, [11C]1b; and 2-[11C]MMC, [11C]1c) were synthesized for evaluation as novel potential positron
emission tomography (PET) imaging agents for brain D3 receptors. The new tracers 4,3,2-[11C]MMCs were prepared by O-
[11C]methylation of corresponding precursors (E)-4,3,2-hydroxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides
(4,3,2-HMCs) using [11C]methyl triflate and isolated by the solid-phase extraction (SPE) purification procedure with 40–65% radio-
chemical yields, decay corrected to end of bombardment (EOB), and a synthesis time of 15–20 min. The PET dynamic studies of the
tracers [11C]1a–c in rats were performed using an animal PET scanner, IndyPET-II, developed in our laboratory. The results show
that the brain uptake sequence was 4-[11C]MMC > 3-[11C]MMC > 2-[11C]MMC, which is consistent with their in vitro biological
properties. The initial PET blocking studies of the tracers 4,3,2-[11C]MMCs with corresponding pretreatment drugs (E)-4,3,2-meth-
oxy-N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoylamides (4,3,2-MMCs, 1a–c) had no effect on 4,3,2-[11C]MMCs-PET
rat brain imaging. These results suggest that the localization of 4,3,2-[11C]MMCs in rat brain is mediated by nonspecific processes,
and the visualization of 4,3,2-[11C]MMCs-PET in rat brain is related to nonspecific binding.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
be visualized by PET with the tracer 3-N-[11C]methyl-
spiperone ([11C]NMSP).4 There is a growing interest in
developing positron-labeled D2 and D3 receptor antago-
nistsas in vivo markers for use in PET, and many attempts
have been made in this area.5–7 However, so far only
[11C]raclopride has been extensively used in human clini-
cal PET studies, because of its high affinity and great spec-
ificity for binding to cerebral dopaminergic D2 receptors.8
We have developed an improved synthetic approach for
the production of [11C]raclopride routinely used in our
PET center for human and animal studies,9,10 and we
are interested in the development of PET D3 receptor
radioligands. (E)-4,2-Methoxy-N-4-(4-(2-methoxyphe-
nyl)piperazin-1-yl)butyl-cinnamoylamides (4-MMC, 1a,
Ki/D3 0.38 nM, Ki/D2 12 nM, ratio D2/D3 32; 2-MMC,
1c, Ki/D3 0.56 nM, Ki/D2 14 nM, ratio D2/D3 25) are
two new high-affinity D3 receptor antagonists recently
developed by Hackling et al.1 Both compounds possess
the combination of favorable pharmacokinetics and
nanomolar Ki binding affinity for D3 receptor, and O-
methyl positions amenable to labeling with carbon-11.
These same properties are often beneficial in a diagnostic
radiotracer. We investigated whether 11C-labeled analogs
of 4-MMC and 2-MMC, (E)-4,2-[11C]methoxy-N-4-(4-
The neurotransmitter dopamine is implicated in a number
of physiological and pathophysiological processes, which
regulate brain functions like motion, emotion, and cogni-
tion.1 Five subtypes of brain dopamine receptors have
been classified into two major classes: the D1-like receptors
including D1 and D5 receptors and D2-like receptors
including D2, D3, and D4 receptors. The dopamine D2-like
receptor subtypes, D and D3 receptors, are recognized as
2
potential therapeutic targets for the treatment of various
neurological and psychiatric disorders such as Parkin-
sonꢀs disease, Huntingtonꢀs disease, and schizophrenia.2
An in vivo biomedical imaging technique, positron emis-
sion tomography (PET), coupled with appropriate recep-
tor radioligands, has become a clinically valuable and
accepted diagnostic tool to image brain diseases.3 The
dopamine D2-like receptor was the first neuroreceptor to
Keywords: (E)-4,3,2-[11C]Methoxy-N-(4-4-(2-methoxyphenyl)butyl-ci-
nnamoylamides; Radioligands; Positron emission tomography; Brain;
D3 receptors.
*
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278
9711; e-mail: qzheng@iupui.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.055

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M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
(2-methoxyphenyl)piperazin-1-yl)butyl)-cinnamoylamides
(4-[11C]MMC, [11C]1a; 2-[11C]MMC, [11C]1c), could be
used to map brain D3 receptors in vivo. As part of our
efforts to evaluate potential brain-imaging agents, we
synthesized 4-[11C]MMC ([11C]1a); (E)-3-[11C]methoxy-
N-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinna-
moylamide (3-[11C]MMC, [11C]1b); and 2-[11C]MMC
([11C]1c), and performed initial PET imaging studies of
the tracers [11C]1a–c in rat brain.
The synthesis of the precursors (E)-4,3,2-hydroxy-N-4-
(4-(2-methoxyphenyl)piperazin-1-yl)butyl-cinnamoyla-
mides (4,3,2-HMCs, 1d–f), and reference standards
(E)-4,3,2-methoxy-N-4-(4-(2-methoxyphenyl)piperazin-
1-yl)butyl-cinnamoylamides (4,3,2-MMCs, 1a–c), as
indicated in Scheme 1, was performed using a modifica-
tion of the literature procedure.1 Commercially available
N-(4-bromobutyl)phthalimide (2) was reacted with 1-(2-
methoxyphenyl)piperazine (3) to give an intermediate
(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)phthalimide
(4), which was used for next step reaction without fur-
ther purification. The intermediate 4 was reacted with
hydrazine hydrate to provide 4-(4-(2-methoxyphe-
nyl)piperazin-1-yl)butylamine (5) in 92% yield. There
were two methods to prepare the precursors and refer-
ence standards. Method A: 4,3,2-methoxycinnamic
acids (6a–c) and 4,3,2-hydroxycinnamic acids (6d–f)
2. Results and discussion
2.1. Chemistry, radiochemistry, and lipophilicity
The synthetic approach for 4,3,2-[11C]MMCs ([11C]
1a–c) is shown in Scheme 1.
O
N
CH3CN, K2CO3
reflux
OMe
N
N
N
Br
O
OMe
N
HN
O
O
2
3
4
i). H2NNH2, MeOH, reflux
ii). HCl, reflux
N
O
OMe
SOCl2
reflux
N
R
OH
6
H2N
5
O
R
6d-f, TEA, DCC
CH2Cl2
TEA, CH2Cl2
Cl
7
Method A
Method B
O
N
N
OMe
NH
R
R=4-OCH3, 1a, 4-MMC
R=3-OCH3, 1b, 3-MMC
R=2-OCH3, 1c, 2-MMC
R=4-OH, 1d, 4-HMC
R=3-OH, 1e, 3-HMC
R=2-OH, 1f, 2-HMC
O
N
N
OMe
NH
HO
R=4-OH, 1d, 4-HMC
R=3-OH, 1e, 3-HMC
R=2-OH, 1f, 2-HMC
11CH3OTf, TBAH
CH3CN
O
N
N
OMe
NH
11CH3O
R=4-O11CH3, [11C]1a, [11C]4-MMC
R=3-O11CH3, [11C]1b, [11C]3-MMC
R=2-O11CH3, [11C]1c, [11C]2-MMC
Scheme 1. Synthetic approach for the tracers 4,3,2-[11C]MMCs ([11C]1a–c).

M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
6235
were treated with thionyl chloride to produce corre-
sponding 4,3,2-methoxycinnamoyl chlorides (7a–c) and
4,3,2-hydroxycinnamoyl chlorides (7d–f), which were
reacted with compound 5 in triethylamine (TEA) and
dichloromethane to give the reference standards 1a–c
and precursors 1d–f for radiolabeling. Method B: Com-
pounds 6d–f were directly reacted with compound 5 in
TEA and 1,3-dicyclohexylcarbodiimide (DCC) to afford
the precursors 1d–f. The chemical yields of the reference
standards using Method A are moderate, and the chem-
ical yields of the precursors using Method B are higher
than using Method A.
sors 1d–f and reference standards 1a–c were >95%. The
radiochemical purities of target radiotracers [11C]1a–c
were >99%, and the chemical purities of target radiotra-
cers [11C]1a–c were >93%. The specific radioactivity of
target radiotracers [11C]1 and [11C]2 was >1 Ci/lmol at
end of synthesis (EOS). Retention times in the analytical
HPLC system were: tR 1d = 4.13 min, tR 1e = 3.74 min,
tR
1f = 3.46 min;
tR
[11C]1a = 6.75 min,
tR
[11C]1b = 6.48 min, tR [11C]1c = 6.05 min.
The ability of a D3 receptor radioligand to penetrate the
blood–brain barrier (BBB) could be due, at least in part,
to its lipophilicity, and thus we were interested in identify-
ing an analog that was significantly lipophilic brain tracer.
We calculated lipophilicity coefficients (logP) of the trac-
ers 4,3,2-[11C]MMCs based on their retention times that
were measured by C-18 HPLC method.14 The calculation
results showed that the logP values of the tracers 4,3,2-
[11C]MMCs are 2.86, 2.81, and 2.71, respectively, that
is, the lipophilicity sequence is 4-[11C]MMC > 3-
[11C]MMC > 2-[11C]MMC. Therefore, we assume the
ability to penetrate the BBB is 4-[11C]MMC > 3-
[11C]MMC > 2-[11C]MMC, and 4-[11C]MMC is the most
promising candidate for use as a potential brain imaging
agent in a series of 4,3,2-[11C]MMCs compounds.
The precursors 1d–f were labeled by [11C]methyl triflate11
through O-[11C]methylation12 under basic conditions
using tetrabutylammonium hydroxide (TBAH) to give
the target tracers 4,3,2-[11C]MMCs ([11C]1a–c). The trac-
ers were isolated by solid-phase extraction (SPE) purifica-
tion.13 The radiochemical yields for target tracers
[11C]1a–c were 40–65%, based on 11CO2, decay corrected
to end of bombardment (EOB). The synthesis time of the
tracers was 15–20 min. The large polarity difference be-
tween the precursors and the labeled methylated products
permitted the use of SPE technique for rapid purification
of labeled products from the radiolabeling reaction mix-
ture. The reaction mixture was diluted with NaHCO3
and loaded onto C-18 cartridge by gas pressure. The car-
tridge column was washed with water to remove unre-
acted [11C]methyl triflate, its precursor, and reaction
solvent. The final labeled product was then eluted with
ethanol. Chemical purity, radiochemical purity, and spe-
cific radioactivity were determined by analytical reversed-
phase HPLC methods. The chemical purities of precur-
2.2. In vivo PET imaging studies
In vivo dynamic PET imaging studies15 of the tracers
4,3,2-[11C]MMCs in young adult female Sprague–Daw-
ley rats were performed in the IndyPET-II scanner16 for
60 min post iv injection of 0.5–1.0 mCi of the tracer in a
rat, and the images are shown in Figures 1–3. All PET
Figure 1. PET/CT overlay images of the tracer 4-[11C]MMC with no blocker (control) and with blocker 4-MMC (3.0 mg/kg) (blocker) in female rats
anesthetized with acepromazine (0.2 mg/kg, i.m.) and torbugesic (0.2 mg/kg, i.m.), administrated with 0.5–1.0 mCi radioactivity, and scanned with
IndyPET-II for 60 min. The images are sagittal views in which the location of the brain is indicated in the circle region.

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M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
Figure 2. PET/CT overlay images of the tracer 3-[11C]MMC with no blocker (control) and with blocker 3-MMC (3.0 mg/kg) (blocker) in female rats
anesthetized with acepromazine (0.2 mg/kg, i.m.) and torbugesic (0.2 mg/kg, i.m.), administrated with 0.5–1.0 mCi radioactivity, and scanned with
IndyPET-II for 60 min. The images are sagittal views in which the location of the brain is indicated in the circle region.
Figure 3. PET/CT overlay images of the tracer 2-[11C]MMC with no blocker (control) and with blocker 2-MMC (3.0 mg/kg) (blocker) in female rats
anesthetized with acepromazine (0.2 mg/kg, i.m.) and torbugesic (0.2 mg/kg, i.m.), administrated with 0.5–1.0 mCi radioactivity, and scanned with
IndyPET-II for 60 min. The images are sagittal views in which the location of the brain is indicated in the circle region.
images are sagittal views and overlaid with l-CT. The
location of the brain is indicated in the circle region.
We have developed a method17 for registration and
fusion of small animal scans acquired with the Indy-
PET-II scanner and the EVS RS-9 CT scanner (En-
hanced Vision System, London, Ontario), which can

M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
6237
provide vital information about the anatomic informa-
tion of tissues and organs in small animals. The l-CT
uses X-rays that pass through the animal to obtain
structural information of the animal. It is an ideal
instrument for biomedical research laboratories to non-
destructively acquire 3-D images of both in vivo and
in vitro specimens. The X-ray radiation that is imposed
on the animal is minimum and does not result in any
physiology change. The registration and fusion of EVS
l-CT with IndyPET-II will help to draw the regions of
interest (ROIs) of the images. All images were acquired
in list-mode and sorted into 15 · 20-s frames, 10 · 60-s
frames, and 9 · 300-s frames. Images were reconstructed
using filtered back projection with a 70% Hanning filter
(4.242 cmÀ1 cutoff frequency). The PET/CT images of
the tracer 4-[11C]MMC from rats 8 to 10 in Figure 1
showed that some brain images are visible with the trac-
er, and some images are not clear. This situation applied
to the PET/CT images of the tracer 3-[11C]MMC from
rats 11 to 13 in Figure 2 and the PET/CT images of
the tracer 2-[11C]MMC from rats 14 to 16 in Figure 3.
Comparing the images of the tracers 4,3,2-[11C]MMCs
in Figures 1–3, we assume the brain uptake sequence
was 4-[11C]MMC > 3-[11C]MMC > 2-[11C]MMC.
neal injection with the drugs 4,3,2-MMCs prior to intra-
venous injection with corresponding tracers 4,3,2-
[11C]MMCs. The blocking images with blockers 4,3,2-
MMCs are shown in Figures 1–3. Similarly, the blocking
studies show that some of the PET/CT brain images of
the tracers 4,3,2-[11C]MMCs with corresponding block-
ers 4,3,2-MMCs from rats 8 to 16 were visible, and some
brain images were not clear as indicated in Figures 1–3.
In comparison of the brain images in the blocking studies
with those in control studies shows that the changes are
no distinct. Therefore, we assume there is not significant
difference between control studies and blocking studies.
The dynamic PET data of the tracers 4-[11C]MMC with
no blocker (control) and with blockers 4-MMC (block-
er) are shown in Figures 4 and 5. The plot in Figure 4 is
the individualized average brain ROI intensity (y-axis)
versus time (x-axis) from injection, which indicated the
kinetics of the tracer 4-[11C]MMC in each individual
animal rats 8, 9, and 10 at each time point in 60 min
of the entire scan time. The plot in Figure 5 is the com-
parison of the accumulated average intensity values (y-
axis) between 4-[11C]MMC with no blocker (control)
and 4-[11C]MMC with blocker 4-MMC (blocker) in
two different experimental animal groups (n = 3) (x-axis)
in 60 min of the entire scan time, which showed that the
tracer 4-[11C]MMC uptake in the brain was changed in
the blocking study. However, a t-test comparing the
means of 4-[11C]MMC with no blocker and with blocker
4-MMC gave a P-value of 0.31, which did not denote
the presence of a statistically significant difference.
Therefore, we conclude that there was no significant
blocking in the data set, the uptakes of 4-[11C]MMC
in rat brain might be the result of nonspecific binding,
and the visualization of 4-[11C]MMC-PET on rat brain
is associated with nonspecific binding.
In vivo competitive inhibition studies were performed to
assess the in vivo specificity of 4,3,2-[11C]MMCs to brain
D3 receptors and whether the tracer distribution is sus-
ceptible to indirect pharmacological or pharmacody-
namic effects that could complicate uptake site
measurements. Competition or ꢁblockingꢀ study was car-
ried out by pretreating groups of animals with drugs,
unlabeled D3 receptor antagonists, which compete, or
are believed to compete for the same binding site that
tracer interacts with in the animal brain. For the blocking
imaging studies,15 the rats were pretreated by intraperito-
0.26
0.13
0
RAT8 control
RAT9 control
RAT10 control
RAT8 blocker
RAT9 blocker
RAT10 blocker
0
20
40
60
Time (minutes)
Figure 4. The kinetics of the tracer 4-[11C]MMC in each rat brain with no blocker (control) and with blocker 4-MMC (blocker) showed the
individualized average brain region of interest intensity versus time from injection in 60 min of entire scan time.

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M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
Similarly, the dynamic PET data of the tracer 3-
[11C]MMC with no blocker (control) and with blocker
3-MMC (blocker) are shown in Figures 6 and 7. The
plot in Figure 6 is the individualized average brain
ROI intensity (y-axis) versus time (x-axis) from injec-
tion, which indicated the kinetics of the tracer 3-
[11C]MMC in each individual animal rats 11, 12, and
13 at each time point in 60 min of the entire scan time.
The plot in Figure 7 is the comparison of the accumulat-
ed average intensity values (y-axis) between 3-
[11C]MMC with no blocker (control) and 3-[11C]MMC
with blocker 3-MMC (blocker) in two different experi-
mental animal groups (n = 3) (x-axis) in 60 min of the
entire scan time, which showed that the tracer 3-
[11C]MMC uptake in the brain was changed in the
blocking study. However, a t-test comparing the means
of 3-[11C]MMC with no blocker and with blocker 3-
MMC gave a P value of 0.79, which did not denote
the presence of a statistically significant difference.
Therefore, we conclude that there was no significant
blocking in the data set, the uptakes of 3-[11C]MMC
in rat brain might be the result of nonspecific binding,
and the visualization of 3-[11C]MMC-PET on rat brain
is associated with nonspecific binding.
10
10
4-MMC
CONTROL
3-MMC
CONTROL
5
5
4-MMC
BLOCKER
3-MMC
BLOCKER
0
0
EXPERIMENTAL GROUPS
EXPERIMENTAL GROUPS
Figure 5. Comparison of the tracer 4-[11C]MMC uptake in rat brain in
the absence and presence of blocker 4-MMC (control group and
blocker group).
Figure 7. Comparison of the tracer 3-[11C]MMC uptake in rat brain in
the absence and presence of blocker 3-MMC (control group and
blocker group).
0.18
0.09
0
RAT11 control
RAT12 control
RAT13 control
RAT11 blocker
RAT12 blocker
RAT13 blocker
0
20
40
60
Time (minutes)
Figure 6. The kinetics of the tracer 3-[11C]MMC in each rat brain with no blocker (control) and with blocker 3-MMC (blocker) showed the
individualized average brain region of interest intensity versus time from injection in 60 min of entire scan time.

M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
6239
Likewise, the dynamic PET data of the tracer 2-
[11C]MMC with no blocker (control) and with blocker
2-MMC (blocker) are shown in Figures 8 and 9. The
plot in Figure 8 is the individualized average brain
ROI intensity (y-axis) versus time (x-axis) from injec-
tion, which indicated the kinetics of the tracer 2-
[11C]MMC in each individual animal rats 14, 15, and
16 at each time point in 60 min of the entire scan time.
The plot in Figure 9 is the comparison of the accumulat-
ed average intensity values (y-axis) between 2-
[11C]MMC with no blocker (control) and 2-[11C]MMC
with blocker 2-MMC (blocker) in two different experi-
mental animal groups (n = 3) (x-axis) in 60 min of the
entire scan time, which showed that the tracer 2-
[11C]MMC uptake in the brain was changed in the
blocking study. However, a t-test comparing the means
of 2-[11C]MMC with no blocker and with blocker 2-
MMC gave a P-value of 0.29, which did not denote
the presence of a statistically significant difference.
Therefore, we conclude that there was no significant
blocking in the data set, the uptakes of 2-[11C]MMC
in rat brain might be the result of nonspecific binding,
and the visualization of 2-[11C]MMC-PET on rat brain
is associated with nonspecific binding.
their in vitro biological properties (4-MMC, Ki/D3
0.38 nM, Ki/D2 12 nM, ratio D2/D3 32; 3-MMC, N/A;
and 2-MMC, Ki/D3 0.56 nM, Ki/D2 14 nM, ratio D2/
D3 25) that 4-MMC is the most potent D3 receptor
antagonist in 4,3,2-MMCs.1 The previously described
lipophilicity information also partially accounts for this
in vivo imaging result, since the ability to penetrate the
BBB is 4-[11C]MMC > 3-[11C]MMC > 2-[11C]MMC. A
more likely explanation is difference in metabolism of
4,3,2-[11C]MMCs. The ease of metabolism with O-de-
methylation mechanism18 for 4,3,2-[11C]MMCs is 2-
[11C]MMC > 3-[11C]MMC > 4-[11C]MMC as indicated
in Figure 11, therefore, 2-[11C]MMC appears to have a
10
2-MMC
CONTROL
5
2-MMC
BLOCKER
The comparison of the accumulated average intensity in
rat brain in the tracers 4,3,2-[11C]MMCs in three differ-
ent experimental animal groups (control) in 60 min of
the entire scan time is shown in Figure 10. A t-test com-
paring the means of 4-[11C]MMC with 3-[11C]MMC,
4-[11C]MMC with 2-[11C]MMC, and 3-[11C]MMC
with 2-[11C]MMC gave a P-value of 0.20, 0.12, and
0.72, respectively, which did not denote the presence
of a statistically significant difference. From Figure 10,
the brain uptake sequence was 4-[11C]MMC > 3-
[11C]MMC > 2-[11C]MMC, which is consistent with
0
EXPERIMENTAL GROUPS
Figure 9. Comparison of the tracer 2-[11C]MMC uptake in rat brain in
the absence and presence of blocker 2-MMC (control group and
blocker group).
0.2
0.1
0
RAT14 control
RAT15 control
RAT16 control
RAT14 blocker
RAT15 blocker
RAT16 blocker
0
20
40
60
Time (minutes)
Figure 8. The kinetics of the tracer 2-[11C]MMC in each rat brain with no blocker (control) and with blocker 2-MMC (blocker) showed the
individualized average brain region of interest intensity versus time from injection in 60 min of entire scan time.

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M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
10
4. Experimental
4.1. General
All commercial reagents and solvents were used without
further purification unless otherwise specified. The
[11C]methyl triflate was made according to the literature
procedure.11 Melting points were determined on a
MEL-TEMP II capillary tube apparatus and were uncor-
rected. 1H NMR spectra were recorded on a Bruker QE
300 NMR spectrometer using tetramethylsilane (TMS)
as an internal standard. Chemical shift data for the proton
resonances were reported in parts per million (d) relative
to the internal standard TMS (d 0.0). Low-resolution
mass spectra (LRMS) were obtained using a Bruker Biflex
III MALDI-TOF mass spectrometer, and high-resolution
mass spectra (HRMS) measurements were obtained using
a Kratos MS80 mass spectrometer, in the Department of
Chemistry at Indiana University. Chromatographic sol-
vent proportions are expressed on a volume:volume basis.
Thin-layer chromatography was run using Analtech silica
gel GF uniplates (5 · 10 cm2). Plates were visualized by
UV light. Normal phase flash chromatography was car-
ried out on EM Science silica gel 60 (230–400 mesh) with
a forced flow of the indicated solvent system in the pro-
portions described below. All moisture-sensitive reac-
tions were performed under a positive pressure of
nitrogen maintained by a direct line from a nitrogen
source.
4-MMC
3-MMC
2-MMC
5
0
Experimental Groups
Figure 10. Comparison of the tracers 4,3,2-[11C]MMCs uptake in rat
brain in control groups.
faster washout without longer retention in the rat brain
than does 3-[11C]MMC and 4-[11C]MMC.
3. Conclusion
The synthetic procedures that provide new tracers 4,3,2-
[11C]MMCs have been well developed. The initial
PET imaging studies of the tracers 4,3,2-[11C]MMCs
showed that these three tracers had a certain uptake in
the rat brain. Comparative studies of the images and
the average intensity in the rat brain of 4,3,2-[11C]MMCs
showed the brain uptake sequence was 4-[11C]MMC > 3-
[11C]MMC > 2-[11C]MMC. However, the results
from the blocking studies by intraperitoneal injection
with pretreatment drugs 4,3,2-MMCs prior to intrave-
nous injection with corresponding tracers 4,3,2-
[11C]MMCs showed that no specific binding is present
in all of the three tracers. These results suggest that local-
ization of 4,3,2-[11C]MMCs in rat brain is mediated by
non-specific processes, and the visualization of 4,3,2-
[11C]MMCs-PET in rat brain is related to nonspecific
binding.
Analytical HPLC was performed using a Prodigy (Phe-
nomenex) 5 lm C-18 column, 4.6 · 2À50 mm; 3:1:3
CH3CN–MeOH–20 mM, pH 6.7 KHPO4 (buffer solu-
tion) mobile phase, flow rate 1.5 mL/min, and UV
(240 nm) and c-ray (NaI) flow detectors. Semi-prep C-
18 guard cartridge column 1 · 1 cm was obtained from
E. S. Industries, Berlin, NJ, and part number 300121-
C18-BD 10l. Sterile Millex-GS 0.22 lm vented filter unit
was obtained from Millipore Corporation, Bedford, MA.
4.2. Synthesis of precursors and reference standards
4.2.1.4-(4-(2-Methoxyphenyl)piperazin-1-yl)butylamine(5).
A mixture of N-(4-bromobutyl)phthalimide (2) (21 mmol,
O
N
N
OMe
NH
11CH3O
R=4-O11CH3, [11C]1a, [11C]4-MMC
R=3-O11CH3, [11C]1b, [11C]3-MMC
R=2-O11CH3, [11C]1c, [11C]2-MMC
O-Demethylation
O
N
N
OMe
NH
HO
R=4-OH, 1d, 4-HMC
R=3-OH, 1e, 3-HMC
R=2-OH, 1f, 2-HMC
Figure 11. Potential metabolic pathways of the tracers 4,3,2-[11C]MMCs ([11C]1a–c).

M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
6241
5.92 g), 1-(2-methoxyphenyl)piperazine (3) (20 mmol,
3.84 g), and K2CO3 (50 mmol, 6.91 g) in acetonitrile or ace-
tone (60 mL) was heated to reflux for 8 h. After further
addition of compound 2 (1 mmol, 0.27 g), the mixture
washeated for another4 h. Thehot suspension was filtrated
and the residue was washed with acetone three times. The
filtrates were concentrated under reduced pressure to give
an intermediate 4-(4-(2-methoxyphenyl)piperazin-1-yl)bu-
tyl)phthalimide (4), which was not separated and directly
used for next step reaction. Compound 4 (18 mmol,
7.08 g) and hydrazine hydrate (22 mmol, 1.07 g) in metha-
nol (30 mL) were heated to reflux for 2 h. To the hot solu-
tion was added 2 N HCl (20 mL), and reflux was
continuedforanother1 h. After coolingtoambienttemper-
ature, the mixture was filtrated and the filtrate was evapo-
rated to dryness. This residue was suspended in water and
alkalized with 2 N NaOH. Extraction with ethyl acetate
or CH2Cl2 yielded compound 5 (4.36 g, 92%) as a low melt-
ing point and pure enough white solid, which can be further
purified by column chromatography (10:89:1 CH3OH–
CH2Cl2–NH3ÆH2O), Rf = 0.22 (10:89:1 CH3OH–CH2Cl2–
NH3ÆH2O). 1H NMR (300 MHz, CDCl3): d 1.25–1.58 (m,
4H, CH2CH2), 2.15 (s, 2H, CH2), 2.43 (t, J = 6.40 Hz,
2H, 1-Pipera-CH2), 2.67 (s, 4H, Pipera-H), 2.74 (s, 2H,
NH2), 3.10 (s, 4H, Pipera-H), 3.86 (s, 3H, CH3O), 6.85–
7.01 (m, 4H, Ph–H).
4.2.2.1. (E)-4-Methoxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1a). Method A, 78%
yield, mp: 137–139 ꢁC. H NMR (300 MHz, CDCl3): d
1.64 (s, 4H, CH2CH2), 2.46 (s, 2H, 1-Pipera-CH2), 2.68
(s, 4H, Pipera-H), 3.16 (s, 4H, Pipera-H), 3.41 (s, 2H,
CH2CONH), 3.81 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
6.27 (d, J = 15.6 Hz, 1H, CHCO), 6.55 (s, 1H, Ph–H),
6.86–6.93 (m, 5H, Ph–H), 7.01(s, 1H, NH), 7.41 (d, J =
7.60 Hz, 2H, Ph–H), 7.55 (d, J = 15.6 Hz, 1H, Ph–CH).
1
4.2.2.2. (E)-3-Methoxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1b). Method A, 74%
yield, mp: 71–73 ꢁC. 1H NMR (300 MHz, CDCl3): d
1.65 (s, 4H, CH2CH2), 2.46 (t, J = 7.62 Hz, 2H, 1-Pi-
pera-CH2), 2.67 (s, 4H, Pipera-H), 3.13 (s, 4H, Pipera-
H), 3.42 (t, J = 5.88 Hz, 2H, CH2CONH), 3.84 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.38 (d, J = 15.4 Hz, 1H,
CHCO), 6.71 (s, 1H, NH), 6.85–7.08 (m, 7H, Ph–H),
7.22–7.27 (m, 1H, Ph–H), 7.50 (d, J = 15.4 Hz, 1H, Ph–
CH). LRMS (CI, m/z): 423 ([M+H]+, 52%), 261 (100%).
HRMS (CI, m/z): calcd for C25H34N3O3 423.2599. Found
423.2512.
4.2.2.3. (E)-2-Methoxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1c). Method A, 75%
1
yield, mp: 136–138 ꢁC. H NMR (300 MHz, CDCl3): d
1.65 (s, 4H, CH2CH2), 2.49 (t, J = 6.62 Hz, 2H, 1-Pi-
pera-CH2), 2.70 (s, 4H, Pipera-H), 3.13 (s, 4H, Pipera-
H), 3.42 (d, J = 5.15 Hz, 2H, CH2CONH), 3.82 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.38 (d, J = 15.4 Hz, 1H,
CHCO), 6.84–6.97 (m, 6H, Ph–H and NH), 6.98–7.03
(m, 1H, Ph–H), 7.26–7.32 (m, 1H, Ph–H), 7.43 (d,
J = 7.36 Hz, Ph–H), 7.82 (d, J = 15.4 Hz, 1H, Ph–CH).
LRMS (CI, m/z): 423 ([M+H]+, 13.2%), 178 (100%).
HRMS (CI, m/z): calcd for C25H34N3O3 423.2599. Found
423.2519.
4.2.2. General procedure for the preparation of amides
(E)-4,3,2-methoxy-N-4-(4-(2-methoxyphenyl)piperazin-1-
yl)butyl-cinnamoylamides (4,3,2-MMCs, 1a–c) and (E)-
4,3,2-hydroxy-N-(4-(4-(2-methoxyphenyl)piperazin-1-
yl)butyl)-cinnamoylamides (4,3,2-HMCs, 1d–f). Method
A (1a–f): The 4,3,2-methoxycinnamic acids (6a–c) or
4,3,2-hydroxycinnamic acids (6d–f) (0.06 mol) were
treated with thionyl chloride (20 mL) and two drops of
N,N-dimethylformamide and then heated at reflux for
2–3 h. The excess thionyl chloride was removed by evap-
oration under reduced pressure. Residual thionyl chlo-
ride was removed by co-evaporation with anhydrous
benzene (30 mL) to afford the corresponding 4,3,2-meth-
oxycinnamoyl chlorides (7a–c) and 4,3,2-hydroxycinna-
moyl chlorides (7d–f). A mixture of compound 5
(2 mmol, 0.526 g), TEA (6 mmol, 0.84 mL), and dry
CH2Cl2 (15 mL) was cooled down to 0 ꢁC and was stir-
red under nitrogen for 5 min. Then the corresponding
cinnamoyl chloride (7) (2.2 mmol) was slowly added
and the stirring continued for another 4–15 h. The sol-
vent was removed under reduced pressure. The mixture
was added to water and was extracted with CH2Cl2. The
crude product was purified by silica gel column chroma-
tography (6:94 CH3OH–CH2Cl2) to give pure products
1a–f, Rf = 0.63–0.67 (1:9 CH3OH–CH2Cl2).
4.2.2.4. (E)-4-Hydroxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1d). Method A, 47%
yield; Method B, 76% yield, mp: 124–126 ꢁC. 1H
NMR (300 MHz, CDCl3): d 1.47 (s, 4H, CH2CH2),
2.32 (s, 2H, 1-Pipera-CH2), 2.94 (s, 4H, Pipera-H),
3.16 (d, J = 5.11 Hz, 2H, CH2CONH), 3.34 (s, 4H, Pi-
pera-H), 3.76 (s, 3H, OCH3), 6.37 (d, J = 15.4 Hz, 1H,
CHCO), 6.71 (s, 1H, NH), 6.76–6.95 (m, 6H, Ph–H),
7.27 (d, J = 16.1 Hz, 1H, Ph–CH), 7.35 (d,
J = 8.82 Hz, 2H, Ph–H), 7.97 (t, J = 5.51 Hz, NH),
9.81(s, 1H, OH). LRMS (CI, m/z): 410 ([M+H]+,
12.2%), 405 (100%). HRMS (CI, m/z): calcd for
C24H32N3O3 410.2444. Found 410.3913.
4.2.2.5. (E)-3-Hydroxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1e). Method A, 43%
1
Method B (1d–f): A mixture of 4,3,2-hydroxycinnamic
acids (6d–f) (2 mmol, 0.328 g), TEA (4 mmol, 0.41 g),
and compound 5 (2 mmol, 0.526 g) in dry CH2Cl2
(25 mL) was stirred at room temperature for 10 min under
nitrogen. Then DCC (2.6 mmol, 0.536 g) was added and
the stirring continued for another 10–20 h. The mixture
was filtrated, and the residue was washed with water.
The filtrate was evaporated to dryness, which was purified
by column chromatography (6:94 CH3OH–CH2Cl2) to
give pure products 1d–f, Rf ꢀ 0.63 (1:9 CH3OH–CH2Cl2).
yield; Method B, 52% yield, mp: 130–132 ꢁC. H NMR
(300 MHz, CDCl3): d 1.61 (s, 4H, CH2CH2), 2.50 (t,
J = 6.62 Hz, 2H, 1-Pipera-CH2), 2.75 (s, 4H, Pipera-H),
3.14 (s, 4H, Pipera-H), 3.36 (t, J = 5.61 Hz, 2H,
CH2CONH), 3.84 (s, 3H, OCH3), 6.31 (d, J = 15.4 Hz,
1H, CHCO), 6.76–7.03 (m, 7H, Ph–H), 7.16 (t,
J = 7.70 Hz, 1H, NH), 7.25 (s, 1H, Ph–H), 7.53 (d,
J = 15.4 Hz, 1H, Ph–CH). LRMS (CI, m/z): 410
([M+H]+, 20%), 406 (100%). HRMS (CI, m/z): calcd for
C24H32N3O3 410.2444. Found 410.3915.

6242
M. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6233–6243
4.2.2.6. (E)-2-Hydroxy-N-(4-(4-(2-methoxyphenyl)pip-
erazin-1-yl)butyl)-cinnamoylamide (1f). Method A, 36%
yield; Method B, 80% yield, mp: 167–169 ꢁC. H NMR
injection of 0.5–1.0 mCi of the tracer, and frame dura-
tions were defined as 300 s for the entire 3600-s scan.
1
(300 MHz, CDCl3): d 1.63 (s, 4H, CH2CH2), 2.19 (s,
1H, OH), 2.48 (s, 2H, 1-Pipera-CH2), 2.73 (s, 4H, Pi-
pera-H), 3.13 (s, 4H, Pipera-H), 3.39 (s, 2H, CH2CONH),
3.86 (s, 3H, OCH3), 6.61 (d, J = 15.4 Hz, 1H, CHCO),
6.80–7.01 (m, 7H, Ph–H), 7.17 (t, J = 7.48 Hz, 1H, NH),
7.28–7.39 (m, 1H, Ph–H), 7.91 (d, J = 15.4 Hz, 1H, Ph–
CH). LRMS (CI, m/z): 410 ([M+H]+, 28%), 137 (100%).
HRMS (CI, m/z): calcd for C24H32N3O3 410.2444. Found
410.3913.
4.4.2. Image registration and fusion. After sedation, the
subject is strapped to a scanning bed, which is mounted
on the EVS l-CT imaging stage for CT image acquisition.
Following the CT scan, the bed is moved from the EVS
l-CT imaging stage to the IndyPET-II imaging stage.
Both stages have identical mounts with precision pins so
that the bed position is reproducible. A six-parameter rig-
id body registration transform of the PET image to the CT
image space is calculated using PET and CT images of a
registration phantom consisting of parallel and perpen-
dicular tubes filled with a mixture of [18F]FDG and
iodinated contrast agent. Transformation parameters
were calculated using an algorithm that finds the ends of
the tubes and calculates a geometric transform using the
locations of the end points in the two image pairs. This
method also provides a measure of fiducial registration
error. Typically, the overall fiducial registration error is
less than 0.4 mm. The phantom derived registration
transformation parameters are applied to the animal CT
and PET data sets toregister the anatomical and function-
al image data sets for further evaluation.
4.3. Synthesis of target tracers
4.3.1. Typical experimental procedure for the radiosyn-
thesis of (E)-4,3,2-[11C]methoxy-N-4-(4-(2-methoxyphe-
nyl)piperazin-1-yl)butyl-cinnamoylamides (4-[11C]MMC,
[11C]1a; 3-[11C]MMC, [11C]1b; 2-[11C]MMC, [11C]1c).
The precursor (1d, 1e, or 1f) (0.6–1.0 mg) was dissolved
in CH3CN (500 lL). To this solution, tetrabutylammo-
nium hydroxide (TBAH) (5 lL, 1 M solution in metha-
nol) was added. The mixture was transferred to a small
volume, three-necked reaction tube. [11C]Methyl triflate
was passed into the air-cooled reaction tube at À15 to
À20 ꢁC, which was generated by a Venturi cooling de-
vice powered with 100 psi compressed air, until radioac-
tivity reached a maximum (ꢁ3 min), then the reaction
tube was heated at 70–80 ꢁC for 2 min. The contents
of the reaction tube were diluted with NaHCO3 (1 mL,
0.1 M). This solution was passed onto a C-18 cartridge
by gas pressure. The cartridge was washed with H2O
(2 · 3 mL), and the aqueous washing was discarded.
The product was eluted from the column with EtOH
(2 · 3 mL), and then passed onto a rotatory evaporator.
The solvent was removed by evaporation under high
vacuum. The labeled product [11C]1a, [11C]1b, or
[11C]1c was formulated with saline (1–3 mL), sterile-fil-
tered through a sterile vented Millex-GS 0.22 lm cellu-
lose acetate membrane, and collected into a sterile vial.
Total radioactivity was assayed, and total volume was
noted. The overall synthesis time was ꢁ20 min. The de-
cay-corrected yield, from 11CO2, was 40–65%, and the
radiochemical purity was >99% by analytical HPLC.
4.4.3. Blocking IndyPET-II imaging of the tracers 4,3,2-
[11C]MMCs with corresponding cold drugs 4,3,2-MMCs.
For the blocking experiments, the rats (250–300 g) were
pretreated by intraperitoneal injection with drugs 4,3,2-
MMCs (3.0 mg/kg) prior to intravenous injection of cor-
responding tracers 4,3,2-[11C]MMCs in the tail vein. After
tracer administration, the animals were handled as de-
scribed above.
4.5. Statistical analysis
The uptake differences between control group and block-
er group for the tracers 4,3,2-[11C]MMCs, 4-[11C]MMC
with 3-[11C]MMC, 4-[11C]MMC with 2-[11C]MMC, and
3-[11C]MMC with 2-[11C]MMC in control group in ani-
mals were examined for statistical significance using the
Studentꢀs t-test. A P-value less than 0.05 denoted the pres-
ence of a statistically significant difference.
Acknowledgments
4.4. PET imaging of the tracers in the Sprague–Dawley
female rats
This work was partially supported by the Indiana 21st
Century Research and Technology Fund and the Indiana
Genomics Initiative (INGEN) of Indiana University,
which is supported in part by Lilly Endowment Inc. The
authors would like to thank Winston Baity, Terry
McBride, Tanya Martinez, and Kristan Boling for their
assistances in animal studies, and Drs. Evan Morris and
Karmen Yoder for their helpful advice on PET data anal-
ysis. The refereeꢀs criticisms and editorꢀs comments for the
revision of the manuscript are greatly appreciated.
4.4.1. Dynamic IndyPET-II imaging of the tracers 4,3,2-
[11C]MMCs in the rats. All animal experiments were
performed under a protocol approved by the Indiana
University institutional animal care and use committee.
The IndyPET-II scanner designed and developed by
Rouze and Hutchins16 was used for these studies. The
female Sprague–Dawley rat (250–300 g) was anesthe-
tized with acepromazine (0.2 mg/kg, i.m.) and torbugesic
(0.2 mg/kg, i.m.). Dose in the range of 0.5–1.0 mCi of
4-[11C]MMC, 3-[11C]MMC or 2-[11C]MMC was admin-
istered intravenously to the rat via the tail vein. The
l-PET images of the tracers were acquired in Indy-
PET-II scanner by the ordered subsets expectation
maximization (OSEM) using six subsets/four iterations
for 60 min dynamic scans from a rat post-intravenous
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