Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

Article abstract of DOI:10.1016/j.bmc.2008.07.016

The radiosynthesis and radiopharmacological evaluation of 1-[¹¹C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [¹¹C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [¹¹C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25 GBq/μmol at the end-of-synthesis within 35 min. The radiotracer [¹¹C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [¹¹C]5 was characterized in male Wistar rats. Compound [¹¹C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.

Full text of DOI:10.1016/j.bmc.2008.07.016

Bioorganic & Medicinal Chemistry 16 (2008) 7662–7670
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation in vitro and in vivo of a 11C-labeled
cyclooxygenase-2 (COX-2) inhibitor
*
Frank Wuest , Torsten Kniess, Ralf Bergmann, Jens Pietzsch
Institute of Radiopharmacy, Research Center Dresden-Rossendorf, PO Box 510119, 01314 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
The radiosynthesis and radiopharmacological evaluation of 1-[11C]methoxy-4-(2-(4-(methanesulfo-
nyl)phenyl)cyclopent-1-enyl)-benzene [11C]5 as novel PET radiotracer for imaging of COX-2 expression
is described\. The radiotracer was prepared via O-methylation reaction with [11C]methyl iodide in 19%
Article history:
Received 17 March 2008
Revised 30 June 2008
Accepted 4 July 2008
Available online 10 July 2008
decay-corrected radiochemical yield at a specific activity of 20–25 GBq/lmol at the end-of-synthesis
within 35 min. The radiotracer [11C]5 was evaluated in vitro using various pro-inflammatory and tumor
cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribu-
tion of compound [11C]5 was characterized in male Wistar rats. Compound [11C]5 was rapidly metabo-
lized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were
demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake
of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be
blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioac-
tivity uptake in the tumor was not COX-2 mediated.
Keywords:
Positron emission tomography
Cyclooxygenase
COX-2 inhibitor
Carbon-11
Inflammation
Cancerogenesis
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and pain, it is well documented that especially COX-2 is overex-
pressed in many human cancer entities such as colorectal, gastric,
Cyclooxygenases (COXs) control the complex conversion of ara-
chidonic acid to prostaglandins and thromboxanes, which trigger
as autocrine and paracrine chemical messengers with many phys-
iological and pathophysiological responses.1–3 The COXs exist in
two distinct isoforms, a constitutive form (COX-1) and an inducible
form (COX-2). More recently, a novel COX-1 splice variant termed
as COX-3 has been reported.4 COX-1 and COX-2 share the same
substrates, produce the same products, and catalyze the same reac-
tion using identical catalytic mechanisms. The X-ray crystal struc-
ture of both enzymes suggests that the proteins are very similar in
their tertiary conformation.5,6 The amino acids which serve as sub-
strate binding pocket and catalytic site are nearly identical in both
enzymes.7–9 The COX-1 enzyme is expressed in resting cells of
most tissues, functions as a housekeeping enzyme, and is responsi-
ble for maintaining homeostasis (gastric and renal integrity) and
normal production of eicosanes.10 COX-2 is predominantly found
in brain and kidney while being virtually absent in most other tis-
sues. However, COX-2 expression is significantly up-regulated as
part of various acute and chronic inflammatory conditions. COX-
2 expression can be induced in fibroblast, epithelial, endothelial,
macrophage, and smooth muscle cells in response to growth fac-
tors, cytokines, and proinflammatory stimuli, and expression is
usually transient.11 Besides being associated with inflammation
and breast cancer.12,13 COX-2 enzyme is assumed to play an impor-
tant role in carcinogenesis by stimulating angiogenesis, tissue
invasion, metastasis, and apoptosis inhibition. COX-2 expression
is stimulated by a number of inflammatory cytokines, growth fac-
tors, oncogenes, lipopolysaccharides, and tumor promoters.14
Since the discovery of the COX-2 enzyme in the early 1990s,
much efforts have been made in the development of COX-2 selec-
tive inhibitors. Because of the structural similarities of the COX-1
and COX-2 enzyme, the search for selective inhibitors for COX-2
versus COX-1 represents a formidable challenge. Based on the com-
mon diaryl heterocycle platform, as initially described for DuP 697
as a COX-2 selective inhibitor, a large array of different compounds
has been reported as selective COX-2 inhibitors. A selection of dif-
ferent selective COX-2 inhibitors, such as DuP 697, celecoxib, rofec-
oxib, and valdecoxib, is depicted in Figure 1.
Currently, an exact and accurate assessment of COX-2 expres-
sion levels and/or activity in organs or tissues can only be achieved
by laborious analyses ex vivo. Moreover, instability of COX-2
mRNA and protein leads to further difficulties in terms of the inter-
vals between tissue sampling and time of analysis. In this line, non-
invasive monitoring of COX-2 functional expression by means of
nuclear molecular imaging techniques like positron emission
tomography (PET) and single photon emission computed tomogra-
phy (SPECT) provides unique opportunities to obtain data on COX-
2 expression levels during disease progression and the potential
role of COX-2 in diseases. Moreover, the development and in vivo
* Corresponding author. Tel./fax: +49 351 260 27 60.
E-mail address: f.wuest@fz-rossendorf.de (F. Wuest).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.016

F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
7663
SO2NH2
F
H3C
MeO2S
H2NO2S
MeO2S
N
N
S
O
N
O
O
H3C
Valdecoxib
CF3
Br
Celecoxib
Rofecoxib
DuP-697
Figure 1. Selection of selective COX-2 inhibitors.
study of appropriately radiolabeled selective COX-2 inhibitors
would provide pharmacological data, which may help to under-
stand their exact physiological actions and metabolic pathways.
Over the last decade, various attempts have been made to
develop radiotracers for COX-2 imaging with PET and SPECT. As a
result, several 18F- and 11C-labeled analogues of DuP-697,15 cele-
coxib,16,17 rofecoxib,18–20 valdecoxib,18,21 and other compounds
containing a heterocycle core structure22–24 have been synthesized
and, in some cases, radiopharmacologically evaluated in vivo as
potential PET radiotracers for imaging of COX-2. Moreover, some
reports have also described the synthesis of 99mTc- and 123I-labeled
analogues of celecoxib25,26 as potential SPECT imaging agents.
However, all reports have revealed that to date no satisfactory nu-
clear probe for in vivo imaging of COX-2 expression is available.
Recently, various 1,2-diarylcyclopentenes have been reported
as high affinity and selective COX-2 inhibitors.27,28 The determined
IC50 values in a COX-2 enzyme binding assay were in the low nano-
molar range while showing high COX-1/COX-2 selectivity ratios
favoring binding toward the COX-2 enzyme. Among various 1,2-
diarylcyclopentenes as selective COX-2 inhibitors, compounds con-
taining a 4-methoxyphenyl substituent showed especially high
affinity and selectivity toward the COX-2 enzyme (Fig. 2).28
Both compounds possess high affinity for COX-2, and especially
the compound containing a methyl sulfone substitution pattern
shows a favorable COX-1/COX-2 selectivity profile (COX-2/COX-1
ratio: 2000). Moreover, the methoxy group makes this compound
especially attractive for isotopic labeling with the short-lived pos-
itron emitter 11C (t1/2 = 20.4 min) through a methylation reaction
of the corresponding desmethyl precursor with [11C]methyl iodide.
Here, we report on the radiosynthesis and radiopharmacological
evaluation of 1-[11C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)-
cyclopent-1-enyl)-benzene ([11C]5) as potential nuclear imaging
probe for monitoring COX-2 expression in vivo by means of PET.
The radiopharmacological characterization of [11C]5 involved cell
uptake studies, metabolism and biodistribution studies in normal
rats, and small animal PET studies in NMRI nu/nu mice bearing
the human colorectal adenocarcinoma tumor HT-29.
2. Results
2.1. Chemistry and radiochemistry
The synthesis of the appropriate desmethyl compound 6 as the
labeling precursor for incorporating the 11C label and substance 5
as the reference compound is given in Figure 3.
The synthesis of 1,2-diarylcyclopentenes was conveniently car-
ried out via sequential Suzuki cross-coupling reactions starting
from commercially available 1,2-dibromocyclopentene 1. The
sequential Suzuki cross-coupling reactions were performed in a bi-
layer system (toluene/Na2CO3-solution) at elevated temperatures
using commercially available boronic acids 2 and 4 as the coupling
partners, and Pd(PPh3)4 as the catalyst. In the first step of the reac-
tion sequence, the 4-methoxyphenyl substituent was introduced.
For this purpose, two equivalents of 1,2-dibromocyclopentene 1
were used in the cross-coupling reaction with 4-methoxyphenyl
boronic acid 2 to minimize the formation of the corresponding
symmetrically bis-coupled product. Following this procedure, the
mono-substituted cross-coupling product 3 could be isolated in
SO2NH2
SO2Me
MeO
MeO
IC50 (COX-2): 0.005 µM
IC50 (COX-1): 9.9 µM
COX-1/COX-2 = 2000
IC50 (COX-2): 0.002 µM
IC50 (COX-1): 0.2 µM
COX-1/COX-2 = 100
Figure 2. High affinity selective COX-2 inhibitors containing a cyclopentene core
structure.28
MeO
Br
MeO2S
Br
MeO
2
B(OH)2
B(OH)2
4
Br
Pd(PPh3)4, 2 M Na2CO3,
toluene, EtOH, reflux, 24 h
Pd(PPh3)4, 2 M Na2CO3,
toluene, EtOH, reflux, 24 h
3
1
62%
79%
OMe
OH
BBr3
MeO2S
MeO2S
CH2Cl2, -78 ºC
73%
6
5
Figure 3. Synthesis of labeling precursor 6 and reference compound 5.

7664
F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
79% yield after purification by means of flash chromatography. A
second Suzuki cross-coupling reaction of equimolar amounts of
High radioactivity uptake was found in the liver, pancreas, and
white adipose tissue. Moreover, the radiotracer seems readily to
penetrate the blood–brain barrier as shown by the high initial
brain uptake of 0.59 0.07%ID/g at 5 min after radiotracer injec-
tion. Except for white adipose tissue, radioactivity was cleared
from all organs and tissues within 60 min after radiotracer admin-
istration. The determined biodistribution profile suggests a hepa-
tobiliary elimination of the radiotracer, and the overall tissue
distribution of compound [11C]5 is consistent with the high lipo-
philicity of the radiotracer (logP = 4.2) as determined on the basis
of ACDLabÒ predictions.
mono-substituted compound
3 with 4-(methylsulfonyl)phenyl
boronic acid 4 afforded the desired 1,2-diarylcyclopentene 5 as
the reference compound in 62% isolated yield. Standard treatment
of compound 5 with BBr3 in methylene chloride at À78 °C led to
cleavage of the methoxy group resulting in the formation of com-
pound 6 as the desired labeling precursor in 73% isolated yield.
The radiosynthesis of compound [11C]5 was accomplished
through an O-methylation reaction of desmethyl precursor 6
with [11C]methyl iodide in a remotely controlled synthesis module
(Fig. 4).
[
11C]Methyl iodide was transferred in a stream of nitrogen into
2.4. Metabolite analysis
the reaction vessel containing the sodium salt of desmethyl precur-
sor 6 in DMF and 5 N NaOH at À20 °C. The formation of the sodium
salt of compound 6 could easily be monitored by the appearance of
a yellow color. After completion of the [11C]MeI transfer, the reac-
tion vessel was sealed and heated at 60 °C for 3 min. The reaction
mixture was diluted with eluent, and the mixture was transferred
from the reaction vessel onto a semi-preparative C-18 column. The
fraction eluting at 8–9 min was collected, diluted with water, and
passed through a C-18 Sep-Pak Light cartridge. The cartridge was
washed with water and the desired radiotracer [11C]5 was eluted
with ethanol. Addition of 0.9% saline gave a final 5% EtOH solution
of compound [11C]5 in saline suitable for subsequent in vitro and in
vivo experiments. Radiotracer [11C]5 was obtained in a radiochem-
ical yield of 19% (decay-corrected, based upon [11C]CO2) after HPLC
Metabolite analysis of arterial blood samples with radio-HPLC
indicated that compound [11C]5 was rapidly metabolized to a sin-
gle more polar metabolite in rat and mouse plasma. Metabolism of
compound [11C]5 in mouse plasma was significantly more rapid
compared with metabolism in rat plasma. The amount of the polar
metabolite steadily increased overtime. Conversely, the amount
of intact compound [11C]5 decreased, being 97% at 1 min, 57% at
5 min, 35% at 10 min, 26% at 20 min, 22% at 30 min, and 18%
at 60 min in rat plasma, and 95% at 1 min, 40% at 5 min, 17% at
10 min, 8% at 20 min, 5% at 30 min, and 2% at 60 min in mouse
plasma (Fig. 6).
2.5. In vivo small animal PET imaging in HT-29 tumor-bearing
mice
purification at a specific radioactivity of 20–25 GBq/lmol at the
end-of-synthesis within 35 min. The radiochemical purity ex-
ceeded 95%.
To further assess the feasibility of compound [11C]5 as a PET
radiotracer for imaging COX-2 expressing tumors, small animal
PET imaging was performed in tumor-bearing mice. In the small
animal PET experiments, 30 MBq of [11C]5 was administered intra-
venously over 1 min into the tail vein of NMRI nu/nu mice bearing
human colorectal adenocarcinoma tumor HT-29.35 This equals a
2.2. Cell uptake studies
Several human and murine cell lines were used to study the up-
take of compound [11C]5 in vitro. All cell lines used show constitu-
tive expression of COX-1. To differentiate between the specific
contribution of COX-1 and COX-2 to overall tracer uptake and
intracellular association, unstimulated human (THP-1) and murine
(RAW 264.7) monocytes/macrophages and quiescent murine fibro-
blasts (NIH 3T3) were used as models showing very low baseline
expression of COX-2.29–31 In contrast, human tumor cell lines FaDu
and HT29, respectively, mouse (fibroblast-like) preadipocytes (3T3
L1) and both TPA-stimulated THP-1 and RAW 264.7 cells served as
models for upregulated COX-2 expression.30,32–34 The specificity of
the radiotracer uptake was further tested by performing blocking
total amount of 2–3 lg of compound 5.
PET imaging was performed over 60 min with a dedicated small
animal PET scanner (microPETÒ P4, Siemens CTI Molecular Imaging
Inc., Knoxville) to elucidate the in vivo biodistribution of com-
pound [11C]5 overtime, and to assess accumulation of radioactivity
in the xenotransplanted HT-29 tumor. Figure 7 shows two repre-
sentative PET images of radioactivity distribution at 1 and 50 min
after radiotracer administration.
Accumulation of radioactivity in the HT-29 xenografted tumor
over time was clearly visible. However, attempts to block uptake
in the tumor by pre-injection with nonradioactive compound 5
proved to be unsuccessful. No decrease in radioactivity accumula-
tion was observed (data not shown).
experiments with all cells after preincubation with a 100 lmol/L
solution of the corresponding nonradioactive reference compound
5 for 10 min (Fig. 5).
Time–activity curves representing time–activity concentration
were used to further assess the kinetics of [11C]5 in blood (corrected
for metabolites), muscle, and tumor. The time–activity curves and
the derived increasing tumor-to-muscle ratio are shown in Figure 8.
The obtained time–activity curves confirm rapid clearance of
the radiotracer from the blood and muscle, whereas accumulation
of radioactivity was observed in the tumor until 10 min p.i.,
followed by a slow wash out of radioactivity from tumor tissue.
The tumor-to-muscle ratio is increasing overtime, reaching 1.7 at
60 min after radiotracer injection.
2.3. Biodistribution in normal rats
The distribution of radioactivity in selected tissues and organs
was studied in normal Wistar rats at two different time points (5
and 60 min) after injection of [11C]5. The results are depicted in Ta-
ble 1.
OH
O11CH3
11C]MeI
3. Discussion
[
MeO2S
MeO2S
NaOH, DMF,
3 min, 60 ºC
Among the plethora of different COX-2 inhibitors developed
during the last 15–20 years, most compounds share a common
1,2-diaryl heterocycle structure motif as exemplified for the ap-
proved selective COX-2 inhibitors celecoxib (Celebrex), rofecoxib
(VIOXX), and valdecoxib (Bextra).
11C]5
6
[
Figure 4. Radiosynthesis of COX-2 inhibitor [11C]5.

F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
7665
Figure 5. In vitro study on cellular uptake and intracellular association of compound [11C]5 in human monocytes/macrophages (A), murine monocytes/macrophages (B),
murine fibroblasts/fibroblast-like cells (C), and human tumor cells (D). Results are given as mean SD (n = 4). TPA, stimulation by 64 nM phorbol myristate acetate (TPA) for
72 h; blocked, preincubation (for 10 min) with a 100
lmol/L solution of the corresponding nonradioactive reference compound 5.
Table 1
100
Biodistribution of [11C]5 in selected tissues and organs of normal male Wistar rats
(body weight 179 10 g) after single intravenous application at 5 min and 60 min p.i.
Data are presented as %ID/g, mean SD (4 animals per time point)
90
80
70
60
50
40
30
20
10
0
Time p.i.
5 min
60 min
Rat plasma
Mouse plasma
Blood
Brain
Heart
Lung
Liver
Spleen
Pancreas
Adrenals
Kidneys
Thymus
Muscle
Harderian glands
Brown adipose tissue
White adipose tissue
Femur
0.23 0.08
0.59 0.07
0.50 0.04
0.59 0.09
1.35 0.25
0.32 0.04
0.78 0.12
1.33 0.11
0.62 0.06
0.64 0.13
0.32 0.05
1.06 0.30
1.89 0.65
0.53 0.29
0.34 0.06
0.20 0.03
0.18 0.03
0.11 0.01
0.16 0.03
0.19 0.01
0.76 0.12
0.21 0.01
0.52 0.01
0.57 0.06
0.26 0.01
0.20 0.04
0.11 0.01
0.42 0.07
0.67 0.18
0.52 0.09
0.19 0.05
0.09 0.01
0
10
20
30
40
50
60
Time in min
Figure 6. Intact compound [11C]5 overtime in rat plasma (diamonds) and mouse
plasma (squares).
Testes
aryl substituents proved to be essential for a favorable high selec-
tivity ratio COX-2 versus COX-1.36 Consequently, the development
of radiotracers for imaging COX-2 expression in vivo was also
mainly focused on compounds containing a central heterocyclic
core structure. The preferred radioisotopes for radiolabeling were
The basic 1,2-diaryl heterocycle platform was used for various
structural modifications leading to COX inhibitors with a high
selectivity profile toward the COX-2 enzyme. In this line, a methyl
sulfone or sulfonamide substitution pattern attached to one of the
the short-lived positron emitters 11C (t1/2 = 20.4 min) and 18F (t1/2
=
109.8 min). Radiosyntheses were accomplished either by using one

7666
F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
Figure 7. Representative coronal small animal PET images (maximum intensity projection) of HT-29 tumor (red circle)-bearing mice 1 min (left) and 50 min (right) after
single intravenous application of 30 MBq of [11C]5.
Figure 8. Representative time–activity curves from one animal after single intravenous application of [11C]5. (A) Time-activity concentration (SUV) curves of the blood
(corrected for metabolites), tumor and muscle. (B) Increasing tumor-to-muscle ratio over time.
of the aryl substituents or by utilizing the central heterocycle moi-
ety as the preferred sites for the incorporation of the radiolabel.
Besides numerous COX-2 selective inhibitors containing a cen-
tral heterocyclic core structure, various 1,2-diarylcyclopentenes,
as depicted in Figure 2, also showed a remarkable high affinity
and selectivity toward the COX-2 enzyme.27,28 In particular, the
high affinity and selectivity of methyl sulfone-containing com-
pound 5 toward COX-2 within the array of 1,2-diarylcyclopenten-
e-containing compounds prompted us to set up a radiosynthesis of
the corresponding 11C-labeled radiotracer [11C]5. The radiosynthe-
sis was based on standard methylation reaction of desmethyl pre-
cursor 6 with readily available [11C]methyl iodide. The required
labeling precursor 6 and the reference compound 5 were easily
prepared through two consecutive Suzuki cross-coupling reactions
starting from commercially available 1,2-dibromocyclopentene.
The used reaction sequence for the synthesis of compounds 5
and 6 differs significantly from the procedure reported in the liter-
ature. The literature procedure requires an additional oxidation
step of a methyl thioether intermediate to give the desired mono-
substituted methyl sulfone as the coupling partner for the second
Suzuki cross-coupling reaction.28
4-(methyl-sulfonyl)phenyl boronic acid 4 was used as the coupling
partner in the first Suzuki cross-coupling step, only the undesired
symmetrically bis-coupled product could be isolated, even when
an excess of 1,2-dibromocyclopentene 1 was used. Conversely,
the use of 4-methoxyphenyl boronic acid 2 as the coupling partner
in the first Suzuki cross-coupling step with 1,2-dibromocyclopen-
tene 1 gave nicely the desired monosubstituted intermediate 3 in
good isolated 79% yield. Intermediate 3 could easily be converted
into compound 5, which was further reacted to give the labeling
precursor 6. Compounds 5 and 6 were obtained in total chemical
yields of 49% and 36%, respectively.
The radiosynthesis of [11C]5 was performed in a remotely con-
trolled synthesis apparatus (Nuclear Interface, Münster), which al-
lowed the convenient and reproducible performance of the
radiolabeling reaction. [11C]Methyl iodide was prepared starting
from [11C]CO2 according to the ‘wet’ chemistry route.37 Radiolabel-
ing was easily carried out by standard treatment of desmethyl pre-
cursor 6 with [11C]methyl iodide. In a typical experiment 60 GBq of
[
11C]CO2 could be converted into 3.5 GBq of [11C]5 (19% decay-cor-
rected radiochemical yield, based upon [11C]CO2) after HPLC puri-
fication within 35 min. The radiochemical purity exceeded 95%.
We found that the choice of the aryl boronic acid to be attached
first onto the cyclopentene ring via Suzuki reaction proved to be
very important for the success of the entire reaction. Hence, when
The obtained specific radioactivity of 20–25 GBq/
l
mol equals a to-
tal amount of 4.5 to 6
uct solution.
lg/ml of cold compound 5 in the final prod-

F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
7667
Cellular uptake studies revealed a comparable moderate uptake
of compound [11C]5 in all cells showing no or only very low base-
line expression of COX-2. In these cells radiotracer uptake was not
influenced by preincubation with the nonradioactive reference
compound 5. On the other hand, in cells showing high baseline
expression of COX-2 (HT-29, FaDu, and 3T3 L1) or TPA-stimulated
COX-2 expression (THP-1 and RAW 264.7), respectively, overall
radiotracer uptake and intracellular association was markedly
higher. This increment in overall radiotracer uptake and intracellu-
lar association could be substantially reduced by pre-incubation of
the cells with a molar excess of the nonradioactive reference com-
pound 5, indicating high specificity of compound [11C]5 for COX-2
in vitro. Of note, the tumor promoter TPA (12-O-tetradecanoyl-
phorbol-13-acetate) used in the present study was reported to
act as a potent inducer of COX-2 expression in various cell types,
particularly, macrophages and monocytic cells.38
sponding [11C]methylsulfones could easily be performed either
via a two-step in situ deprotection/methylation oxidation proce-
dure19 or via a direct labeling strategy with [11C]methyl iodide
starting from the corresponding sulfinate precursor.20
Dynamic small animal PET studies using compound [11C]5 in a
mouse HT-29 tumor xenograft model revealed accumulation in the
xenotransplanted tumor. Radioactivity accumulation in the tumor
peaked at 10 min followed by a slow wash-out within the time
frame of the study. The accumulation of radioactivity in the tumor
was further confirmed by an increasing tumor-to-muscle ratio
overtime. This observation is consistent with our data showing
comparably high uptake of compound [11C]5 in HT-29 cells in vi-
tro. The human colorectal adenocarcinoma cell line HT-29 was
used as a well characterized model showing increased baseline lev-
els of COX-2 mRNA and protein.41–46 However, intraperitoneal
injection of nonradioactive COX-2 inhibitor 5 at a dose of 5 mg/
kg body weight 10 min prior to radiotracer administration to block
radiotracer uptake in the tumor and to confirm a COX-2-mediated
uptake in vivo was not successful probably due to the too low COX-
2 expression in the HT-29 xenotransplanted tumor. Application of
higher doses of compound 5 (>5 mg/kg), as well as an intravenous
administration of the blocking agent, was not possible due to sol-
ubility problems. Hence, the need for an intraperitoneal adminis-
tration and the use of 5 mg/kg as blocking dose presumably
resulted in limited bioavailability and low total amount of com-
pound 5 to sufficiently block radioactivity uptake in the tumor.
The failure of blocking of radioactivity uptake in the xenografted
tumor is in contrast to the cellular uptake studies in vitro. How-
In the biodistribution study in normal rats, the radiotracer
[
11C]5 showed clearance from all tissues and organs within the
time frame studied, except for white adipose tissue. The radio-
tracer showed a 22% clearance from the blood at 60 min p.i.
reaching 0.18 0.03%ID/g. The remaining radioactivity concentra-
tion of 0.18 0.03%ID/g in the blood at 60 min after radiotracer
administration is in agreement with the observed radioactivity
concentration of other 11C-labeled24 and 18F-labeled COX-2 inhib-
itors16, whereas a 125I-labeled39 and one other 11C-labeled COX-2
inhibitor24 showed a higher radioactivity concentration in the
blood of 0.44% and 1.16%ID/g, respectively, at 60 min p.i. The
highest initial radioactivity uptake after 5 min p.i. of compound
[
11C]5 was observed in the brown adipose tissue (1.89
ever, in these studies a much higher concentration (100 lmol/L)
0.65%ID/g), liver (1.35 0.25%ID/g), adrenals (1.33 0.11%ID/g),
and Harderian glands (1.06 0.30%ID/g). Compared to other tis-
sues and organs, relatively high initial radioactivity concentration
was further found in the pancreas (0.78 0.12%ID/g at 5 min p.i.).
The clearance of radioactivity in the pancreas was lower (33%)
compared to other tissues and organs with high initial radiotracer
uptake (64% for brown adipose tissue; 44% for liver, and 57% for
adrenals).
The radiotracer seems to readily penetrate the blood–brain bar-
rier resulting in an initial radioactivity concentration in the brain of
0.59 0.07%ID/g after 5 min p.i. However, this initial high brain up-
take decreased significantly to 0.11 0.01%ID/g after 60 min p.i.
representing an 80% clearance. Although the brain is known as
an organ where COX-2 is constitutively expressed, in our study
only little retention of radioactivity was found in the brain after
60 min p.i. This finding is consistent with other reports in the liter-
ature showing also only little radioactivity concentration (0.04% to
0.15%ID/g) in the brain at 60 min after radiotracer administration
for other 11C-labeled COX-2 inhibitors.24 The kidneys are also
known to express COX-2 at high levels.40 However, radioactivity
concentration in the kidney was rapidly cleared overtime, being
0.62 0.06%ID/g at 5 min p.i. and 0.26 0.01%ID/g at 60 min p.i.
The observed kidney clearance agrees with results reported in
the literature using other 11C-labeled COX-2 inhibitors.24
Metabolite analysis of arterial blood samples from rat and
mouse showed that compound [11C]5 was rapidly metabolized.
The fast metabolism of the radiotracer was more pronounced in
mice, leading to a fraction of intact parent compound in mouse
plasma as little as 17% at 10 min after radiotracer administration.
In mice, compound [11C]5 was almost completely metabolized at
later time points, resulting in only 8% of intact radiotracer at
20 min, 5% at 30 min, and 2% at 60 min. The fast metabolism of
the radiotracer suggests the incorporation of the radiolabel into a
different, metabolically more stable position of the molecule. In
this line, the methylsulfone moiety represents an alternative func-
tional group suitable for the incorporation of 11C via a methylation
reaction with [11C]methyl iodide. The radiosynthesis of corre-
of compound 5 as blocking agent was used. The overall organ-spe-
cific in vivo distribution of compound [11C]5 in mice as observed in
the small animal PET studies was comparable with the results from
the biodistribution studies in rats.
4. Conclusion
In this report, we have described the radiosynthesis and radio-
pharmacological evaluation of a 11C-labeled COX-2 inhibitor con-
taining a 1,2-diarylcyclopentenes structure as a novel lead for
COX-2 selective PET radiotracers. Radiotracer [11C]5 was fully char-
acterized in vitro and in vivo, revealing rapid metabolism in rat plas-
ma, and more pronounced, in mouse plasma. The high metabolic
degradation of compound [11C]5 in the animal experiments and
the different COX-2 expression patterns in the in vivo and in vitro
experiments may explain the large differences of the results ob-
tained in vitro and in vivo. In terms of radiotracer metabolism, intro-
duction of the 11C label into the methylsulfone group should be
envisaged to improve the metabolic profile of the radiotracer. The
in vitro results indicate that compound [11C]5 may have the poten-
tial as a COX-2 PET radiotracer to allow the quantification of COX-
2 expression related to tumorigenic processes, for example, in colo-
rectal cancer. However, the contribution of tumor cells and proin-
flammatory cells like macrophages to the overall uptake of
compound [11C]5 in tumors, tumor environment and/or inflamma-
tory lesions has still to be further elucidated. Moreover, for further
studies of COX-2 expression in vivo, compounds with higher affinity
in combination with very high-specific activity should be developed.
5. Experimental
5.1. General
All commercial reagents and solvents were used without further
purification unless otherwise specified. Nuclear magnetic resonance
spectra were recorded on a Varian Unity 300 MHz spectrometer.

7668
F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
1H-NMR chemical shifts were given in ppm and were referenced
with the residual solvent resonances relative to tetramethylsilane
(TMS). Mass spectra were obtained on a Quattro/LC mass spectrom-
eter (MICROMASS) by electrospray ionization.
remotely controlled synthesis apparatus by Nuclear Interface
(Münster). [11C]Methyl iodide was prepared according to Crouzel
and co-workers.37 Radio-HPLC analyses were carried out with
Discovery C18 column (Supelco 4.6 Â 150 mm, 5
lm) using an
Flash chromatography was conducted according to Still et al.47
using MERCK silica gel (mesh size 230–400 ASTM). Thin-layer
chromatography (TLC) was performed on Merck silica gel F-254
aluminum plates with visualization under UV (254 nm).
indicated isocratic eluent with a flow rate of 1.0 ml/min. The prod-
ucts were monitored by an UV detector L4500 (Merck-Hitachi) at
240 nm and by
(X-RAYTEST).
c-detection with a scintillation detector GABI
5.2. Chemical synthesis
5.3.1. 1-[11C]Methoxy-4-(2-(4-(methanesulfonyl)phenyl)-
cyclopent-1-enyl)-benzene [11C]5
5.2.1. 1-(2-Bromocyclopent-1-enyl)-4-methoxybenzene (3)
To a solution of 1.2-dibromocyclopentene 1 (1.0 g. 4.40 mmol)
and 4-methoxyphenyl boronic acid 2 (336.3 mg, 2.20 mmol) in tol-
uene (30 mL), EtOH (30 mL), and 2 M Na2CO3 (30 mL) was added
Pd(PPh3)4 (127.2 mg, 0.11 mmol). The mixture was refluxed over-
night under a nitrogen atmosphere. After the addition of water
(250 mL) the mixture was extracted with EtOAc. The solvent was
evaporated and the residue was purified by flash chromatography
(50% EtOAc/petrol ether) to give 440 mg (79%. based upon
4-(methoxyphenyl boronic acid) of compound 3 as an oil. 1H
NMR (CDCl3. 400 MHz): d 2.06 (quint., J = 7.6 Hz, 2 H, CH2). 2.74
(t, J = 7.6 Hz, 2H, CH2). 2.85 (t, J = 7.6 Hz, 2H, CH2). 3.82 (s, 3H,
CH3). 6.90 and 7.58 (2d of AA’BB’ system, J = 8.8 Hz, 4H, Ar-H).
[
11C]Methyl iodide was transferred in a stream of nitrogen into
the reaction vessel containing phenol 6 (1.0 mg) in DMF (400
lL)
and 5 N NaOH (30 L) at room temperature. The formation of the
l
corresponding phenolate occurred in situ immediately after the
addition of NaOH to compound 6 at room temperature. After com-
pletion of the [11C]methyl iodide transfer, the reaction vessel was
sealed and heated at 60 °C for 3 min. The reaction mixture was di-
luted with eluent (1 mL), and the mixture was transferred from the
reaction vessel onto a semi-preparative C-18 column (Nucleodur
ISIS. Macherey-Nagel, C18, 250 Â 10 mm, 5
lm) using CH3CN/
H2O (70:30) as the mobile phase at a flow rate of 5 mL/min. The de-
sired product eluted between 8 and 9 min. The fraction containing
the 11C-labeled compound [11C]5 was diluted with 30 ml of water
and passed through a SPE-cartridge RP-18E (40–63 lm). The prod-
5.2.2. 1-Methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-
1-enyl)-benzene (5)
uct was eluted from the cartridge with EtOH (1 mL), and the final
product solution was prepared by the addition of isotonic sodium
chloride (0.9%) solution (9.0 mL]). An aliquot was taken for quality
control using radio-HPLC (SUPELCO Discovery C18, 150 mm Â
To a solution of 1-(2-bromocyclopent-1-enyl)-4-methoxyben-
zene 3 (375 mg. 1.48 mmol) and 4-(methylsulfonyl)phenyl boronic
acid 4 (296 mg, 1.48 mmol) in toluene (20 mL), ethanol (20 mL),
and 2 M Na2CO3 (20 mL) was added Pd(PPh3)4 (87 mg,
0.075 mmol). The mixture was refluxed for 8 h under a nitrogen
atmosphere. After the addition of water (150 mL), the mixture
was extracted with EtOA. After removal of the solvent under re-
duced pressure, the residue was purified by flash chromatography
(50% EtOAc/petrol ether) to give 300 mg (62%) of the desired
product 5 as a solid. Mp 128–130 °C; 1H NMR (CDCl3. 400 MHz):
d 2.07 (quint., J = 7.3 Hz, 2H, CH2). 2.89 (t, J = 7.3 Hz, 4H, CH2).
3.04 (s, 3H, CH3), 3.79 (s, 3H, CH3). 6.77 and 7.07 (2d of AA’BB’ sys-
tem, J = 8.8 Hz, 4H, Ar-H). 7.35 and 7.75 (2d of AA’BB’ system,
J = 8.5 Hz, 4H, Ar-H). Low-resolution mass spectrometry electron-
spray ionization (ESI+): 351.4 [M+Na].
4.6 mm, 5 lm, acetonitrile/water (70:30), flow rate: 1 ml/min,
tR = 5.1 min). Compound [11C]5 was obtained in 19% decay-cor-
rected radiochemical yield (based upon [11C]CO2). The radiochem-
ical purity exceeded 95%, and the specific activity was determined
to be 20–25 GBq/lmol at the end-of-synthesis.
5.4. Cell uptake studies
Uptake experiments for in vitro evaluation of compound [11C]5
were performed in the following cells: THP-1 (human monocyte/
macrophage line; DSMZ ACC 16), RAW 264.7 (mouse monocyte/
macrophage line; ATCC TIB-71), NIH 3T3 (mouse fibroblast line;
ECACC 93061524), 3T3-L1 (mouse preadipocyte (fibroblast-like)
line; ECACC 86052701), FaDu (human head and neck squamous
cell carcinoma line; ATCC HTB-43), and HT-29 (human colorectal
adenocarcinoma line; ATCC HTB-38). Cells were routinely culti-
vated in RPMI 1640 medium or McCoy medium (HT-29) supple-
mented with 10% (v/v) heat-inactivated fetal calf serum (FCS),
5.2.3. 4-[2-(4-Methanesulfonyl-phenyl)-cyclopent-1-enyl]-
phenol (6)
1-Methoxy-4-(2-(4-(methane-sulfonyl)phenyl)cyclopent-1-enyl)-
benzene 5 (170 mg, 0.52 mmol) was dissolved in dry methylene
chloride (2 mL). The solution was cooled to À78 °C and 1 M BBr3-
solution in methylene chloride (0.6 mL, 0.6 mmol) was added.
The resulting brown-colored solution was warmed up to ambient
temperature and stirring was continued for 1 h. The solution was
re-cooled to À78 °C and MeOH (1 mL) was added. The green solu-
tion was stirred for an additional hour at 0 °C. The solvent was
evaporated, and the residue was purified by flash chromatography
(40% EtOAc/petrol ether) to give 120 mg (73%) of the desired com-
pound 6 as a solid. Mp 154–156 °C; 1H NMR (CDCl3, 400 MHz): d
2.07 (quint., J = 7.3 Hz, 2H, CH2). 2.89 (t, J = 7.3 Hz, 4H, CH2). 3.04
(s, 3H, CH3). 6.70 and 7.03 (2d of AA’BB’ system, J = 8.3 Hz, 4H,
Ar-H). 7.35 and 7.75 (2d of AA’BB’ system, J = 8.5 Hz, 4H, Ar-H).
Low-resolution mass spectrometry electronspray ionization
(ESI+): 337.3 [M+Na].
penicillin (100 U/mL), streptomycin (100 lg/mL), and glutamine
(2 mM) at 37 °C and 5% CO2 in a humidified incubator. Proinflam-
matory stimulation (transformation to macrophages) of THP-1 and
RAW 264.7 cells was performed by adding 64 nM phorbol myris-
tate acetate (TPA) for 72 h. For the uptake studies, cells were
seeded in 24-well plates at a density of 5 Â 104 cells/mL and grown
to confluence. Unstimulated THP-1 suspension cells were centri-
fuged and resuspended in fresh RPMI medium containing 10%
FCS in 24-well plates at a density of 5 Â 105 cells/mL. The cell tra-
cer uptake experiments using compound [11C]5 (1 MBq/mL; spe-
cific activity at application time, 12.1 2.1 GBq/lmol) were
performed in quadruplicate in medium both at 4 and 37 °C for 5,
10, 15, 30, 45, and 60 min. For blocking studies, the cells were
preincubated for 10 min with
a 100 lmol/L solution of the
corresponding nonradioactive reference compound 5. After the tra-
cer uptake was stopped with 1 mL ice-cold PBS, the cells were
washed three times with PBS and dissolved in 0.5 mL NaOH
(0.1 M containing 1% (w/v) sodium dodecylsulfate). The radioactiv-
ity in the cell extracts was measured with a Cobra II gamma
5.3. Radiochemistry
[
11C]CO2 was produced by the 14N(p,
a)
11C nuclear reaction on a
IBA CYCLONE 18/9 cyclotron. The synthesis was carried out in a

F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
7669
counter (Canberra-Packard, Meriden, CT, USA). Total protein con-
centration in the samples was determined by the bicinchoninic
acid method (BCA; Pierce, Rockford, Ill, USA) using bovine serum
albumin as protein standard. Uptake data for all experiments are
0.949 by 1.212 mm and the resolution was around 1.85 mm. No
correction for recovery and partial volume effects was applied.
The image files were processed using the ASIPro software
(SIEMENS, CTI Concorde Microsystems Inc., Knoxville) and the
ROVER software (ABX medical software development, Radeberg,
Germany). A summed image from 30 to 60 min p.i. was used to
define the regions of interest (ROI). The data were normalized
to the injected radioactivity by using 11C standards from the
expressed as percent of injected dose per
protein).
lg protein (%ID/lg
5.5. Biodistribution studies in rats
injection solution measured in a
c-well counter (Isomed 2000,
All animal experiments were carried out according to the guide-
lines of the German Regulations for Animal Welfare. The protocol
was approved by the local Ethical Committee for Animal Experi-
ments. The Wistar rats were housed under standard conditions
with free access to standard food and tap water. Five animals (body
weight 122 16 g) for each time point were intravenously injected
with approximately 20 MBq of [11C]5 in 0.5 mL saline with 10%
ethanol. Animals were sacrificed at 5 and 60 min post injection.
Organs and tissues of interest were rapidly excised, weighed, and
the radioactivity was determined using a Cobra II gamma counter
(Canberra-Packard, Meriden, CT, USA). The activity in the selected
tissues and organs was expressed as percent injected dose per
gram tissue (%ID/g). The activity of the tissue samples was
decay-corrected and calibrated by comparing the counts in tissue
with the counts in aliquots of the injected tracer that had been
measured in the gamma counter at the same time. Values are
quoted as means standard deviation (mean SD) for a group of
four animals.
Germany) cross calibrated to the PET scanner and expressed
in percent of injected dose per cubic centimeter (%ID/cm3). In
the blocking experiments, compound 5 (5 mg/kg body weight)
was injected intraperitoneally 10 min prior to radiotracer
application.
Acknowledgments
The authors are grateful to Mareike Barth, Regina Herrlich, An-
drea Suhr, Stephan Preusche, and Tilow Krauss for their excellent
technical assistance.
References
1. Kurumbail, R. G.; Kiefer, J. R.; Marnett, L. J. Curr. Opin. Struct. Biol. 2001, 11, 752–
760.
2. Marnett, L. J. Curr. Opin. Chem. Biol. 2000, 4, 545–552.
3. Fitzpatrick, F. A. Curr. Pharm. Des. 2004, 10, 577–588.
4. Warner, T. D.; Mitchell, J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13371–
13373.
5. Picot, D.; Loll, P. J.; Garavito, R. M. Nature 1994, 367, 243–249.
6. Luong, C.; Miller, A.; Barnett, J.; Chow, J.; Ramesha, C.; Browner, M. F. Nat.
Struct. Biol. 1996, 3, 927–933.
7. Marnett, L. J.; Rowlinson, S. W.; Goodwin, D. C.; Kalgutkar, A. S.; Lanzo, C. A. J.
Biol. Chem. 1999, 274, 22903–22906.
5.6. Metabolite analysis
The radiotracer [11C]5 was injected intravenously into male
Wistar rats (30 MBq) or HT-29 tumor-bearing mice (20 MBq) un-
der isoflurane (1.5%) oxygen anesthesia. Blood samples from the
right femoral artery were taken using a catheter at 3, 5, 10, 30,
and 60 min after injection. Plasma was separated by centrifugation
(3 min, 11,000g) followed by precipitation of the plasma proteins
with ice-cold methanol (1.5 parts per 1 part plasma) and centrifu-
gation (3 min, 11,000g), and the supernatants were analyzed by
HPLC. The radio-HPLC system (Agilent 1100 series) applied for
metabolite analysis was equipped with UV detection (210 nm)
and an external radiochemical detector (Canberra-Packard, Radi-
omatic Flo-one Beta 150TR) with PET flow cell. Analysis was per-
8. Smith, W. L.; Garavito, R. M.; DeWitt, D. L. J. Biol. Chem. 1996, 271, 33157–
33160.
9. Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125–140.
10. Smith, W. L. In Handbook of Eicosanoids: Prostaglandins and Related Lipids;
Willis, A. L., Ed.; CRC Press: Boca Raton, FL, 1987; pp 175–184.
11. Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125–140.
12. Xu, X. C. Anticancer Drugs 2002, 13, 127–137.
13. Jiménez, P.; García, A.; Santander, S.; Piazuelo, E. Curr. Pharm. Des. 2007, 13,
2261–2273.
14. Brown, J. R.; DuBois, R. N. J. Clin. Oncol. 2005, 23, 2840–2855.
15. de Vries, E. F.; van Waarde, A.; Buursma, A. R.; Vaalburg, W. J. Nucl. Med. 2003,
44, 1700–1706.
16. McCarthy, T. J.; Sheriff, A. U.; Graneto, M. J.; Talley, J. J.; Welch, M. J. J. Nucl. Med.
2002, 43, 117–124.
17. Prabhakaran, J.; Underwood, M. D.; Parsey, R. V.; Arango, V.; Majo, V. J.;
Simpson, N. R.; Van Heertum, R.; Mann, J. J.; Kumar, J. S. Bioorg. Med. Chem.
2007, 15, 1802–1807.
formed on a Zorbax 300SB-C18 (250 Â 9.4 mm; 5
lm) column
with acetonitrile/water (70/30) as the eluent at a flow rate of
2 mL/min.
18. Wuest, F.; Höhne, A.; Metz, P. Org. Biomol. Chem. 2005, 3, 503–507.
19. Majo, V. J.; Prabhakaran, J.; Simpson, N. R.; Van Heertum, R. L.; Mann, J. J.;
Kumar, J. S. Bioorg. Med. Chem. Lett. 2005, 15, 4268–4271.
20. de Vries, E. F.; Doorduin, J.; Dierckx, R. A.; van Waarde, A. Nucl. Med. Biol. 2008,
35, 35–42.
21. Toyokuni, T.; Kumar, J. S.; Walsh, J. C.; Shapiro, A.; Talley, J. J.; Phelps, M. E.;
Herschman, H. R.; Barrio, J. R.; Satyamurthy, N. Bioorg. Med. Chem. Lett. 2005,
15, 4699–4702.
22. Fujisaki, Y.; Kawamura, K.; Wang, W. F.; Ishiwata, K.; Yamamoto, F.; Kuwano,
T.; Ono, M.; Maeda, M. Ann. Nucl. Med. 2005, 19, 617–625.
23. Tian, H.; Lee, Z. J. Label. Compd. Radiopharm. 2006, 49, 583–593.
24. Tanaka, M.; Fujisaki, Y.; Kawamura, K.; Ishiwata, K.; Qinggeletu;
Yamamoto, F.; Mukai, T.; Maeda, M. Biol. Pharm. Bull. 2006, 29, 2087–
2094.
25. Yang, D. J.; Bryant, J.; Chang, J. Y.; Mendez, R.; Oh, C. S.; Yu, D. F.; Ito, M.;
Azhdarinia, A.; Kohanim, S.; Edmund Kim, E.; Lin, E.; Podoloff, D. A. Anticancer
Drugs 2004, 15, 255–263.
26. Kabalka, G. W.; Mereddy, A. R.; Schuller, H. M. J. Label. Compd. Radiopharm.
2005, 48, 295–300.
27. Reitz, D. B.; Li, J. J.; Norton, M. B.; Reinhard, E. J.; Collins, J. T.; Anderson, G. D.;
Gregory, S. A.; Koboldt, C. M.; Perkins, W. E.; Seibert, K., et al J. Med. Chem.
1994, 37, 3878–3881.
28. Li, J. J.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Collins, J. T.; Garland, D. J.;
Gregory, S. A.; Huang, H. C.; Isakson, P. C.; Koboldt, C. M., et al J. Med. Chem.
1995, 38, 4570–4578.
29. Bagga, D.; Wang, L.; Farias-Eisner, R.; Glaspy, J. A.; Reddy, S. T. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 1751–1756.
5.7. Small animal PET imaging
General anesthesia of tumor-bearing mice was induced with
inhalation of 3% isoflurane in oxygen, and was maintained with
1.5% isoflurane in oxygen and subsequently fixed in prone position
on the microPETÒ bed. In the PET experiments, 30 MBq of [11C]5 in
0.5 mL of saline was administered intravenously as a bolus via a
tail vein.
PET imaging [11C]5 in HT-29 tumor-bearing mice was per-
formed over 60 min with a microPETÒ P4 scanner (Siemens
CTI Molecular Imaging Inc., Knoxville). Data acquisition was
performed in 3D list mode. A transmission scan was carried
out prior to the injection of [11C]5 using a 137Cs point source.
Emission data were collected continuously for 60 min after
injection of [11C]5. The list mode data were sorted into 4 sino-
grams for the 30 min frames and for the dynamic study using a
framing scheme of 12 Â 10, 6 Â 30, 5 Â 300, and 9 Â 600 s
frames. The data were attenuation corrected and the static
30 min frames (30 to 60 min p.i.) were reconstructed by filtered
back projection using a ramp filter. The pixel size was 0.949 by

7670
F. Wuest et al. / Bioorg. Med. Chem. 16 (2008) 7662–7670
30. Goppelt-Struebe, M.; Schaefer, D.; Habenicht, A. J. Br. J. Pharmacol. 1997, 122,
619–624.
31. Mbonye, U. R.; Wada, M.; Rieke, C. J.; Tang, H. Y.; Dewitt, D. L.; Smith, W. L. J.
Biol. Chem. 2006, 281, 35770–35778.
32. Lev-Ari, S.; Strier, L.; Kazanov, D.; Madar-Shapiro, L.; Dvory-Sobol, H.; Pinchuk,
I.; Marian, B.; Lichtenberg, D.; Arber, N. Clin. Cancer Res. 2005, 11, 6738–6744.
33. Yan, H.; Kermouni, A.; Abdel-Hafez, M.; Lau, D. C. J. Lipid Res. 2003, 44, 424–429.
34. Murakami, A.; Takahashi, D.; Kinoshita, T.; Koshimizu, K.; Kim, H. W.; Yoshihiro,
A.; Nakamura, Y.; Jiwajinda, S.; Terao, J.; Ohigashi, H. Carcinogenesis 2002, 23,
795–802.
39. Kuge, Y.; Katada, Y.; Shimonaka, S.; Temma, T.; Kimura, H.; Kiyono, Y.; Yokota,
C.; Minematsu, K.; Seki, K.; Tamaki, N.; Ohkura, K.; Saji, H. Nucl. Med. Biol. 2006,
33, 21–27.
40. Harris, R. C. J. Am. Soc. Nephrol. 2000, 11, 2387–2394.
41. Koki, A. T.; Masferrer, J. L. Cancer Control 2002, 9, 28–35.
42. Hwang, J. T.; Kim, Y. M.; Surh, Y. J.; Baik, H. W.; Lee, S. K.; Ha, J.; Park, O. J. Cancer
Res. 2006, 66, 10057–10063.
43. Sano, H.; Kawahito, Y.; Wilder, R. L.; Hashiramoto, A.; Mukai, S.; Asai,
K.; Kimura, S.; Kato, H.; Kondo, M.; Hla, T. Cancer Res. 1995, 55, 3785–
3789.
35. Haase, C.; Bergmann, R.; Oswald, J.; Zips, D.; Pietzsch, J. Anticancer Res. 2006,
26, 3527–3533.
44. Kargman, S. L.; O’Neill, G. P.; Vickers, P. J.; Evans, J. F.; Mancini, J. A.; Jothy, S.
Cancer Res. 1995, 55, 2556–2559.
36. Herschman, H. R.; Talley, J. J.; DuBois, R. Mol. Imaging Biol. 2003, 5, 286–303.
37. Crouzel, C.; Langström, B.; Pike, V. W.; Coenen, H. H. Appl. Radiat. Isot. 1987, 38,
601–603.
38. Mestre, J. R.; Mackrell, P. J.; Rivadeneira, D. E.; Stapleton, P. P.; Tanabe, T.; Daly,
J. M. J. Biol. Chem. 2001, 276, 3977–3982.
45. Petersen, S.; Haroske, G.; Hellmich, G.; Ludwig, K.; Petersen, C.; Eicheler, W.
Anticancer Res. 2002, 22, 1225–1230.
46. Wang, L.; Chen, W.; Xie, X.; He, Y.; Bai, X. Exp. Oncol. 2008, 30, 42–
51.
47. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–2925.