General method for the C-labeling of 2-arylpropionic acids and their esters: Construction of a PET tracer library for a study of biological events involved in COXs expression

Article abstract of DOI:10.1002/chem.200903044

Cyclooxygenase (COX) is a critical enzyme in prostaglandin biosynthesis that modulates a wide range of biological functions, such as pain, fever, and so on. To perform in vivo COX imaging by positron emission tomography (PET), we developed a method to incorporate ¹¹C radionuclide into various 2-arylpropionic acids that have a common methylated structure, particularly among nonsteroidal antiinflammatory drugs (NSAIDs). Thus, we developed a novel ¹¹C-radiolabeling methodology based on rapid C[¹¹C] methylation by the reaction of [¹¹C]CH₃I with enolate intermediates generated from the corresponding esters under basic conditions. One-pot hydrolysis of the above [¹¹C]methylation products also allows the synthesis of desired ¹¹C-incorporated acids. We demonstrated the utility of this method in the syntheses of six PET tracers, [ ¹¹C]Ibuprofen, [¹¹C]Naproxen, [¹¹C] Flurbiprofen, [¹¹C]Fenoprofen, [¹¹C]Ketoprofen, and [ ¹¹C]Loxoprofen. Notably, we found that their methyl esters were particularly useful as proradiotracers for a study of neuroinflammation. The microPET studies of rats with lipopolysaccharide (LPS)-induced brain inflammation clearly showed that the radioactivity of PET tracers accumulated in the inflamed region. Among these PET tracers, the specificity of [ ¹¹C]Ketoprofen methyl ester was demonstrated by a blocking study. Metabolite analysis in the rat brain revealed that the methyl esters were initially taken up in the brain and then underwent hydrolysis to form pharmacologically active forms of the corresponding acids. Thus, we succeeded in general ¹¹C-labeling of 2-arylpropionic acids and their methyl esters as PET tracers of NSAIDs to construct a potentially useful PET tracer library for in vivo imaging of inflammation involved in COXs expression.

Full text of DOI:10.1002/chem.200903044

DOI: 10.1002/chem.200903044
11
General Method for the C-Labeling of 2-Arylpropionic Acids and
Their Esters: Construction of a PET Tracer Library for a Study of
Biological Events Involved in COXs Expression
Misato Takashima-Hirano, Miho Shukuri, Tadayuki Takashima, Miki Goto,
Yasuhiro Wada, Yasuyoshi Watanabe, Hirotaka Onoe, Hisashi Doi, and
Masaaki Suzuki*[a]
Abstract: Cyclooxygenase (COX) is a
critical enzyme in prostaglandin bio-
synthesis that modulates a wide range
of biological functions, such as pain,
fever, and so on. To perform in vivo
COX imaging by positron emission to-
[11C]methylation products also allows
the synthesis of desired 11C-incorporat-
ed acids. We demonstrated the utility
of this method in the syntheses of six
clearly showed that the radioactivity of
PET tracers accumulated in the in-
flamed region. Among these PET trac-
ers, the specificity of [11C]Ketoprofen
methyl ester was demonstrated by a
blocking study. Metabolite analysis in
the rat brain revealed that the methyl
esters were initially taken up in the
brain and then underwent hydrolysis to
form pharmacologically active forms of
the corresponding acids. Thus, we suc-
ceeded in general 11C-labeling of 2-ar-
ylpropionic acids and their methyl
esters as PET tracers of NSAIDs to
PET
tracers,
[11C]Ibuprofen,
[11C]Naproxen,
[11C]Flurbiprofen,
mography (PET), we developed
a
[11C]Fenoprofen, [11C]Ketoprofen, and
[11C]Loxoprofen. Notably, we found
that their methyl esters were particular-
ly useful as proradiotracers for a study
of neuroinflammation. The microPET
studies of rats with lipopolysaccharide
(LPS)-induced brain inflammation
method to incorporate 11C radionuclide
into various 2-arylpropionic acids that
have a common methylated structure,
particularly among nonsteroidal anti-
inflammatory drugs (NSAIDs). Thus,
we developed a novel 11C-radiolabeling
methodology based on rapid C-
[11C]methylation by the reaction of
[11C]CH3I with enolate intermediates
generated from the corresponding
esters under basic conditions. One-pot
construct
a potentially useful PET
Keywords: anti-inflammatory
drugs · C–C coupling · cyclooxyge-
tracer library for in vivo imaging of in-
flammation involved in COXs expres-
sion.
nase
· inflammation · positron
emission tomography
hydrolysis
of
the
above
Introduction
on cyclooxygenase (COX), which is a critical enzyme in con-
verting arachidonic acid into prostaglandins and thrombox-
anes. Mainly, two distinct isoforms, COX-1 and COX-2, are
known as a constitutive and an inducible form, respectively.
Recently, a novel COX-1 splice variant termed as COX-3
has also been reported.[2] COX-1 enzyme exists in most
mammalian cells including the endothelium, stomach, and
kidney, and is responsible for maintaining homeostasis. In
contrast, COX-2 is found in the brain and kidney, it is pri-
marily induced in response to cytokines, mitogens, and en-
dotoxins in a variety of cell types including macrophages
and tumors. Therefore, the quantification of COX-2 in many
disease processes has great potential as a useful biomarker
for early diagnosis, a monitor of disease progression, and an
indicator of effective medical treatment.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among
the most widely prescribed drugs worldwide, because they
are the first choice in the treatment of rheumatic disorders
and other degenerative inflammatory diseases.[1] One of the
pharmacological actions of NSAIDs is an inhibitory effect
[a] M. Takashima-Hirano, M. Shukuri, T. Takashima, M. Goto, Y. Wada,
Prof. Dr. Y. Watanabe, Dr. H. Onoe, Dr. H. Doi, Prof. Dr. M. Suzuki
RIKEN Center for Molecular Imaging Science (CMIS)
6-7-3 Minatojima-minamimachi, Chuo-ku
Kobe, Hyogo, 650-0047 (Japan)
Fax : (+81)78-304-7131
E-mail: masaaki.suzuki@riken.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903044.
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FULL PAPER
Positron emission tomography (PET) is a powerful nonin-
vasive molecular imaging technique that provides high sensi-
tivity, good spatial resolution, and easy and accurate quan-
tification; PET is frequently used in biological research and
clinical studies.[3] Radionuclides used in PET include short-
lived radioisotopes (11C, 13N, 15O) of the abundant elements
in the structures of biologically active compounds, such as
natural products and drugs, etc. A dose of the labeled com-
pound in medicine is very tiny, namely less than 1/100 of
actual pharmacological dose or 100 mg.[4] Therefore, the ap-
plication of PET in a human microdose study at an early
stage of drug and biomarker development has been expect-
ed.[5] The increasing need for pharmacologically significant
PET tracers requires efficient synthetic strategies for new
molecular tracer designs and advances in radiolabeling
methodology. Carbon-11 (half-life=20.4 min) is one of the
most important isotopes for PET research. In particular, the
incorporation of the [11C]methyl group into organic frame-
works through [11C]carbon–carbon bond formation is one of
the attractive approaches because 1) the [11C]methyl group
can be introduced into a metabolically stable position in the
molecules and, therefore, such a 11C-labeled tracer provides
a highly credible PET image, 2) the methyl group is often
used in drug design to control the lipophilicity of the mole-
cule and the blocking of the metabolic position as the small-
est substituted group containing carbon, and 3) the short
half-life of the 11C-incorporated tracer allows rapid screen-
ing of the PET images and increases progress by several
trials per day.[6]
we describe the efficient general syntheses of 11C-labeled 2-
arylpropionic esters by rapid [11C]methylation through sp3–
sp3-type coupling by the reaction of [11C]CH3I and the corre-
sponding enolates with the aim of realizing COX imaging by
PET. These esters were readily converted to the correspond-
ing acids as NSAID structures by one-pot hydrolysis.[16] The
evaluation of the blood-brain barrier (BBB) permeability of
these NSAIDs PET tracers in rats and in vivo studies of a
rat lipopolysaccharide (LPS)-induced inflammation model
are also described.
Results and Discussions
Chemistry: The 2-arylpropionic acids of six NSAIDs, Ibu-
profen, Naproxen, Flurbiprofen, Fenoprofen, Ketoprofen,
and Loxoprofen, were selected for 11C-labeling. They share
a common chemical structure for [11C]methyl group intro-
Since the discovery of COX-2, the development of drugs
that selectively inhibit this isoform and the development of
corresponding specific PET tracers have become a major
area of pharmaceutical research.[7] During the past decade,
PET tracers corresponding to highly selective and potent
COX-2 inhibitors, such as [18F]desbromo-Dup-697,[8]
[11C]Celecoxib,[9] [11C]Rofecoxib,[10] and [11C]Valdecoxib[11]
have been developed. Additionally, 11C-labeling of diaryl-
substituted imidazole and indole analogues has yielded PET
probes with a high affinity and selectivity for COX-2.[12]
However, to the best of our knowledge, the successful
COX-2 imaging by using these PET tracers has not yet been
reported.[13]
The cause of the adverse side effects of NSAIDs is sus-
pected to be the inhibition of COX-1, a necessary house-
keeping gene that is not elevated during inflammation. In
contrast, COX-2 is normally nondetectable in most tissues,
but is rapidly elevated during inflammation. Thus, the inhib-
ition of COX-2 by NSAIDs is thought to be responsible for
their therapeutic effects. However, the relative biological
contributions of COX-1 and COX-2 isoforms in the mainte-
nance of normal physiological functions and in disease
states are not entirely clear.[14] Therefore, the development
of both nonselective and selective COX inhibitors and the
evaluation of their in vivo behavior by PET imaging could
greatly help to elucidate their physiological actions.
duction, despite their different levels of inhibition of COX-1
and COX-2.[17] All precursors and authentic samples were
prepared according to conventional synthetic methods or
purchased as described in the Experimental Section.[18]
Radiochemistry: Scheme 1 illustrates the syntheses of 11C-la-
beled 2-arylpropionic acids and their esters; the syntheses
were based on rapid C-[11C]methylation by using [11C]CH3I
under basic conditions. In general, sodium hydride was
added to a solution of methyl arylacetate as a precursor in
Scheme 1. Syntheses of 11C-labeled 2-arylpropionic acids and their methyl
esters.
We previously developed a simple method to synthesize
[11C]Celecoxib, a COX-2 inhibitor.[15] In the present report,
Chem. Eur. J. 2010, 16, 4250 – 4258
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4251

M. Suzuki et al.
DMF at room temperature for the deprotonation of the ben-
zylic position. After 10–20 min, [11C]CH3I with He flow
(30 mLminÀ1) was trapped in the mixture and reacted with
the enolate at 308C for 2 min, and then the resulting mix-
ture was quickly subjected to the preparative HPLC opera-
tion because of the rapid completion of the C-
[11C]methylation. When the reaction was continued for a
slightly longer time or at a slightly higher temperature after
[11C]CH3I trapping, the yield of the [11C]methylated com-
pounds was decreased. In the synthesis of [11C]Ketoprofen
methyl ester ([11C]5), when the reaction mixture was heated
at 508C or left for several minutes after [11C]CH3I trapping,
the desired product [11C]5 was gradually decomposed with
time, and some unknown side products appeared (Figure 1).
rification by analytical HPLC. At present, the mechanism of
the radiolytic decomposition and the intrinsic stability of the
methyl esters are not clear. In addition, the corresponding
carboxylic acids, [11C]Ibuprofen ([11C]7), [11C]Naproxen
([11C]8),
[11C]Flurbiprofen
([11C]9),
[11C]Fenoprofen
([11C]10), [11C]Ketoprofen ([11C]11), and [11C]Loxoprofen
([11C]12), were also synthesized in one-pot reactions by the
rapid hydrolysis of each 2-arylACHTUNGTRNEUNG
[11C]propionic acid methyl
ester with 2m sodium hydroxide at 508C for 1 min. This hy-
drolysis completed even at 308C for 1 min. Interestingly,
11C-labeled 2-arylpropionic acids were radiochemically
stable. The physicochemical properties of the labeled com-
pounds are listed in Table 1. All 12 11C-labeled compounds
Table 1. Synthetic results and the stability of 11C-labeled 2-arylopropionic
acids and their methyl esters.
Compound Radioactivity Specific radioactivity
DCY
[%]
Stability[b]
[GBq][a]
[GBqmmolÀ1
]
methyl ester
[11C]1
[11C]2
[11C]3
[11C]4
[11C]5
[11C]6
G
3.1Æ0.8
2.4Æ0.1
5.5Æ0.2
2.9Æ1.9
5.5Æ0.4
1.9Æ0.1
20Æ2.9
32Æ7.6
40Æ1.5
38Æ5.6
47Æ11
26Æ8.7
56Æ17
48Æ5.7
76Æ12
43Æ24
72Æ13
26Æ5.3
C
C
A
A
B
B
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
carboxylic acid
[11C]7
[11C]8
[11C]9
[11C]10
[11C]11
[11C]12
G
4.3Æ0.7
2.7Æ0.6
2.8Æ0.3
3.3Æ2.3
3.0Æ0.6
1.6Æ0.6
25Æ8.5
24Æ1.0
31Æ12
20Æ5.0
30Æ15
22Æ6.9
72Æ15
47Æ12
43Æ6.7
60Æ21
29Æ13
29Æ13
A
A
A
A
A
A
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Data are expressed as mean ÆSD ([11C]1, [11C]2, [11C]3, [11C]4, [11C]7,
[11C]9, [11C]10, [11C]11, and [11C]12, n=3; [11C]5, [11C]6, and [11C]8, n=4).
[a] Radioactivity showing the isolated radioactivity. [b] A: stable, B:
stable in the presence of ascorbic acid, C: stable under the lower current
beam and in the presence of ascorbic acid; DCY: decay-corrected yield
based on [11C]CH3I.
Figure 1. Radiochromatograms of the crude reaction mixture of
[11C]Ketoprofen methyl ester ([11C]5) obtained by the reaction of
[11C]CH3I and methyl (3-benzoylphenyl)acetate, demethylated precursor.
A) [11C]CH3I trapping at 308C for 2 min. B) [11C]CH3I trapping at 308C
for 2 min and reaction at 508C for 4 min.
Finally, [11C]5 was almost completely decomposed after the
reaction for 4 min at 508C (Figure 1B). After extensive ex-
periments, we found that the reaction was completed during
[11C]CH3I trapping at 308C without further reaction time.
This reaction was also proceeded by [11C]CH3I trapping at
À108C. By using these conditions, [11C]Ibuprofen methyl
ester ([11C]1), [11C]Naproxen methyl ester ([11C]2),
[11C]Flurbiprofen methyl ester ([11C]3), [11C]Fenoprofen
methyl ester ([11C]4), and [11C]Loxoprofen methyl ester
([11C]6) were efficiently synthesized. Inexplicably, the com-
pounds, [11C]1, [11C]2, [11C]5, and [11C]6 tended to decom-
pose upon radiolyses. In particular, [11C]1 and [11C]2 were
gradually decomposed during HPLC purification or concen-
tration by evaporator. However, in our experience, we have
found that such radiolyses could be prevented by the use of
[11C]CH3I at less than 15 GBq. The addition of ascorbic acid
as an antioxidant stabilizer to the reaction mixture before
the HPLC purification also effectively prevented the radiol-
ysis.[19] Consequently, we found that the six kinds of 2-aryl-
that we synthesized by the above procedures possessed suffi-
cient radioactivity (1.7–5.5 GBq) for an animal PET study.
The decay-corrected radiochemical yields (DCY) based on
[11C]CH3I were 26–76%. Of these, the yields of [11C]12 and
its methyl ester [11C]6 were relatively low. Such low yields
were presumably due to the co-occurrence of the enolization
at both of the a-positions of the ester and ketone moieties.
We classified isolated [11C] products into three groups based
on their radiochemical stability. [11C]3, [11C]4, [11C]7, [11C]8,
[11C]9, [11C]10, [11C]11, and [11C]12 were stably isolated with-
out the addition of ascorbic acid, and they were categorized
as class A. [11C]5 and [11C]6 in class B required ascorbic acid
for the HPLC purification and evaporation. [11C]1 and
[11C]2 in class C were very unstable, and they required not
only ascorbic acid but also synthetic conditions with the
lower radioactivity (~15 GBq). The reaction with higher ra-
dioactivity (>15 GBq) gave many kinds of products among
which it was too difficult to isolate the desired labeled com-
pound. The chemical and radiochemical purities of the iso-
lated products were greater than 98% because of their un-
ACHTUNGTRENNUNG
[11C]propionic acid methyl esters are stable for 2 h after pu-
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11C-Labeling of 2-Arylpropionic Acids and Their Esters
FULL PAPER
expectedly ready separation from their corresponding deme-
thylated precursors.[20] Here, we consider that such a large
polarity difference between methylated and demethylated
compounds, as indicated in reverse-phase HPLC, may be
caused by the following electronic and structural factors:
The first factor is the ordinary increase of hydrophobicity by
introducing a nonpolar methyl group into an organic frame-
work. The second factor is the decrease of the acidity of the
benzylic proton of methylated compounds that is attributed
À
to the hyperconjugation between the C H s bond of a ben-
zylic position and C=O p*, which is also possible for the
LUMO (p*) of a phenyl moiety. Such hyperconjugation in
methylated products, if any, would also be less favored in
the methylated compound because of steric congestion
emerging from the assumed fully substituted pseudo olefin
structure.
Brain penetration of [11C]5 and [11C]11 evaluated by micro-
PET imaging: PET scans were performed to measure the
uptake of [11C]5 and [11C]11 in the inflamed area of the left
striatum in rats; the inflammation was induced by the injec-
tion of 50 mg of lipopolysaccharide (LPS) one day before
the PET scan, because it was reported to cause the activa-
tion of microglia within 24 h.[21] In this condition, we have
also observed the increase of the COX-2 expression sur-
rounding the LPS injection site by the immunohistochemical
method (data not shown). Radioactivity of [11C]5 after intra-
venous bolus administration was highly accumulated into
the LPS injection site and the surrounding area, whereas ra-
dioactivity of [11C]11 after the administration was not accu-
mulated in the brain including the injected site (Figure 2).
The radioactivity of [11C]5 in the inflammatory area was
about 9-fold higher than that of [11C]11. Low penetration of
[11C]11 into the brain, which was consistent with a previous
study that used 2-arylpropionic acids,[22] was dramatically
improved by the conversion of carboxylic acid into methyl
ester.[23] As compared with [11C]PK11195, which is one of
the PET tracers widely used for the imaging of activated mi-
croglia that have occurred by neuroinflammation, the SUV
level of [11C]5 in the brain is almost the same, and moreover,
the ratio of radioactivity in the inflammatory region to back-
ground is rather superior.[24] Therefore, [11C]5 could be ap-
plicable for the PET imaging of neuroinflammation.
Figure 2. A) Summated PET images (from 5 to 45 min after the tracer in-
jection) of [11C]5 (left panel) and [11C]11 (right panel) in rat brain inflam-
mation induced by LPS (50 mg) injection into the left striatum. B) Time–
activity curves of [11C]5 (n=2) and [11C]11 (n=3) in the LPS-injected in-
flammatory area (LPS) and the contralateral region (control). Data are
11
11
~
*
*
expressed as mean ÆSD ( : LPS [ C]5, : control [ C]5; : LPS
11
11
~
[ C]11, : control [ C]11).
between the inflammatory region and contralateral region
was observed in the later time point after the injection (Fig-
ure 2B). Thus, [11C]5 showed appropriate behavior as a pro-
radiotracer for the functional imaging in the rat brain.
Screening study of 2-arylACHTNUGRTNEUNG
[11C]propionic acid methyl esters
by microPET imaging: To explore the appropriate PET trac-
ers for neuroinflammation, PET studies by using [11C]1,
[11C]2, [11C]3, [11C]4, [11C]5, and [11C]6 were performed in
the rat model of neuroinflammation. PET images with 2-
arylACHTNUGRTNEUNG
[11C]propionic acid methyl esters demonstrated that all
of the proradiotracers, with the exception of [11C]1, showed
high accumulation in the area of LPS-induced inflammation
(Figure 4). Since the radiochemical stability of [11C]1 was
comparable with that of [11C]2, the lower brain uptake of
[11C]1 might be derived from the differences of pharmacoki-
netic properties, such as metabolic stability and/or affinity
for efflux pumps at the BBB. Regional brain accumulation
of these proradiotracers is shown as a standardized uptake
value (SUV) during 40 min (5–45 min post-injection of the
tracer) in Table 2. The highest accumulation in the inflam-
matory area was observed for [11C]5, followed by [11C]6,
[11C]3, [11C]4, and [11C]2, whereas the ratio to the contrala-
teral lesioned area was the highest for [11C]6 and [11C]2
(about 4-fold).
Measurement of [11C]5 and the radioactive metabolites in
rat brain and blood: Metabolite analyses in rat brain and
blood of the ester as proradiotracers were performed in
normal rats after the administration of [11C]5 (Figure 3).
[11C]5 was rapidly hydrolyzed in rat blood to the pharmaco-
logically active form [11C]11, and the hydrolysis rate of
[11C]5 in rat brain was much slower than that in the blood.
Thus more than 90% of [11C]5 that passed the BBB was hy-
drolyzed to [11C]11 within 5 min after administration.
Though the first hydrolysis step should be considered for
the quantitative kinetic analysis of PET, the hydrolysis rate
of [11C]5 might be fast enough for the functional analysis of
COX imaging in rat brain, because a significant difference
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4253

M. Suzuki et al.
the ex vivo autoradiography
study also revealed the signifi-
cant reduction of increased ra-
dioactivities in the LPS-injected
side of the striatum and cortex
(Figure 5C). Since many inflam-
matory-related events, such as
organ infiltration and invasion
of immune cells, are known to
hardly occur in the early phase
of inflammation, the incomplete
blocking result observed in the
inflammatory area may be de-
rived from large amounts of
nonspecific
accumulation
caused by such events.
Currently, PET and related
biological studies are being con-
ducted with rodents and nonhu-
man primates to determine the
suitable NSAID PET tracers
for in vivo COXs imaging,
which is useful for diagnosis
and drug development for dis-
eases relating to COXs expres-
sion.
Figure 3. Rat brain (left) and blood (right) compositions of Ketoprofen methyl ester ([11C]5, ) and its phar-
*
macological active metabolite, Ketoprofen ([11C]11, ). Data are expressed as mean ÆSD (n=3). Top) Com-
*
position of [11C]5 and [11C]11. Bottom) HPLC analyses by radio detector after 2 min.
Table 2. Regional brain accumulation of the 2-arylACTHNUTRGNEUNG
[11C]propionic acid
methyl esters in the inflammatory area induced by LPS (50 mg) injection
into the left striatum.[a]
SUV
Ratio
LPS injected striatum
contralateral striatum
LPS/control
A
0.51Æ0.07
0.92Æ0.17
0.96Æ0.03
0.93Æ0.17
1.21Æ0.18
1.10Æ0.18
0.30Æ0.04
0.26Æ0.02
0.39Æ0.03
0.39Æ0.16
0.47Æ0.12
0.29Æ0.02
1.70Æ0.24
3.57Æ0.70
2.50Æ0.27
2.57Æ0.63
2.75Æ1.10
3.76Æ0.48
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
[a] The data were expressed as the standardized uptake value (SUV),
normalized for injected radioactivity and body weight. SUV=(radioactiv-
ity per cubic centimeter tissue/injected radioactivity)ꢁgram body weight.
Data are expressed as mean ÆSD ([11C]3 and [11C]5, n=2; [11C]1, [11C]2,
[11C]4, and [11C]6, n=3).
Figure 4. PET images of 2-arylACHTNUGRTNEUNG
[11C]propionic acid methyl esters in rat
brain inflammation induced by LPS (50 mg) injection into the left stria-
tum.
Conclusion
To construct a PET tracer library, we developed a method
for common and efficient rapid 11C-labeling of 2-arylpro-
pionic acids and their methyl esters through the
[11C]carbon–carbon bond formation; this method provided a
moderate radiochemical yield with high chemical and radio-
chemical purity.[25] Although the pharmacologically active
To determine the binding specificity of 2-aryl-
ACHTUNGTRENNUNG
[11C]propionic acid methyl esters as proradiotracer in the in-
flamed area, we performed blocking experiments of [11C]5
(proradiotracer of [11C]11) by using PET and ex vivo autora-
diography. Simultaneous injection of authentic Ketoprofen
methyl ester (KTP-Me) (10 mgkgÀ1) with [11C]5 resulted in
a significantly reduced accumulation of radioactivities in the
area of LPS-induced inflammation (Figure 5A,B). Images of
[11C]5 (proradiotracer of [11C]11) at 50 min post-injection in
forms, 2-aryl
uptake, we expect that these compounds will be applied to
peripheral imaging. On the other hand, 2-aryl
[11C]propionic
acid methyl esters showed good brain penetration. In addi-
[11C]propionic acids, showed low levels of brain
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
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11C-Labeling of 2-Arylpropionic Acids and Their Esters
FULL PAPER
ACTHNUTRGNEUNG
duced by an 14N(p,a)11C nuclear reaction by using a CYPRIS HM-12S
Cyclotron (Sumitomo Heavy Industry, Tokyo, Japan). An original auto-
mated radiolabeling system consisting of the heating of the reaction mix-
ture, dilution, HPLC injection, fractional collection, evaporation, and
sterile filtration was used for the production of [11C]CH3I and the 11C-la-
beling. Purification with semi-preparative HPLC was performed on a
JASCO system (Tokyo, Japan). Radioactivity was quantified with an
ATOMLABTM300 dose calibrator (Aloka, Tokyo, Japan). Analytical
HPLC was performed on a Shimadzu system (Kyoto, Japan) equipped
with pumps and a UV detector, and the effluent radioactivity was deter-
mined by using a RLC700 radio analyzer (Aloka). The columns used for
the analytical and semi-preparative HPLC were COSMOSIL C18 MS-II
and AR-II (Nacalai Tesque). [11C]CH3I was prepared as previously de-
scribed.[26]
Radiosynthesis of [11C]Ibuprofen methyl ester (1): Sodium hydride
(1 mg) was added to a solution of methyl (4-isobutylphenyl)acetate in an-
hydrous DMF (200 mL) under an Ar atmosphere. [11C]CH3I was trans-
ported by a stream of helium (30 mLminÀ1) and trapped in the mixture
at 308C for 2 min. After the addition of a solution of 25% ascorbic acid
(50 mL), water (400 mL), and acetonitrile (400 mL), the resulting mixture
was injected into preparative HPLC (mobile phase: acetonitrile/10 mm
ammonium formate 65:35; column: COSMOSIL, 5C18-MS-II, 10 (i.d.)ꢁ
250 mm, 5 mm; flow rate: 6 mLminÀ1; UV detection: 195 nm; retention
time: 15 min). The desired fraction was collected into a flask containing
25% ascorbic acid (200 mL) and the organic solvent was removed under
reduced pressure. The desired radiotracer was dissolved in a mixture of
polysorbate 80, propylene glycol, and saline (0.1:1:10 v/v/v, 4 mL). The
total synthesis time including HPLC purification and radiopharmaceuti-
cal formulation for intravenous administration was 28 min. The isolated
radioactivity was 4.0 GBq at the end of synthesis and the specific radioac-
tivity was 23 GBqmmolÀ1. The chemical identity of [11C]Ibuprofen methyl
ester was confirmed by co-injection with the authentic sample of Ibupro-
fen methyl ester on analytical HPLC (mobile phase: acetonitrile/water
70:30; column: COSMOSIL, 5C18-AR-II, 4.6 (i.d.)ꢁ150 mm, 5 mm; flow
rate: 1 mLminÀ1; UV detection: 210 nm; retention time: 5.3 min). The
chemical purity analyzed at 210 nm and the radiochemical purity were
greater than 99%.
Radiosynthesis of [11C]Naproxen methyl ester (2): The radiosynthesis
method was similar to that of [11C]Ibuprofen methyl ester (1).
[11C]Naproxen methyl ester was purified by semi-preparative HPLC
(mobile phase: acetonitrile/water=65:35; column: COSMOSIL, 5C18-
MS-II, 20 (i.d.)ꢁ250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection:
230 nm; retention time: 16 min). The total synthesis time including
HPLC purification and radiopharmaceutical formulation for intravenous
administration was 32 min. The isolated radioactivity was 2.5 GBq at the
end of synthesis, and the specific radioactivity was 37 GBqmmolÀ1. The
chemical identity of [11C]Naproxen methyl ester was confirmed by co-in-
jection with the authentic sample of Naproxen methyl ester on analytical
HPLC (mobile phase: acetonitrile/water 65:35; column: COSMOSIL,
5C18-MS-II, 4.6 (i.d.)ꢁ150 mm, 5 mm; flow rate: 1 mLminÀ1; UV detec-
tion: 230 nm; retention time: 5.5 min). The chemical purity analyzed at
230 nm and the radiochemical purity were greater than 99%.
Figure 5. A) PET images of [11C]5 blocked by excess unlabeled Ketopro-
fen methyl ester (KTP-Me) in rat brain inflammation induced by LPS
(0.5 mg) injection into the left striatum. Unlabeled KTP-Me (10 mgkgÀ1
)
was simultaneously administered with radiotracer. B) Accumulated radi-
oactivity of [11C]5 with simultaneous injection of KTP-Me in the inflamed
area (LPS) and the contralateral region (control). The data are expressed
as SUV. The results are means ÆSD (Vehicle (&), n=5; 10 mg KTP-Me
(&), n=4). *: p<0.05; unpaired t-test. C) Ex vivo autoradiographic
images of [11C]5 at 50 min post-injection. The autoradiographic images
coregistered with their brain-slice photographs.
tion, the metabolite analysis of the ester [11C]5 in the rat
brain showed that it was converted into the pharmacologi-
cally active form [11C]11. The studies of a rat model of LPS-
induced brain inflammation showed that [11C]2, [11C]3,
[11C]4, [11C]5, and [11C]6 were more highly taken up into the
LPS-treated area than the contralateral nontreated area.
The blocking study in PET and ex vivo autoradiography re-
vealed the specificity of [11C]5 (proradiotracer of [11C]11)
for the target regions.
Radiosynthesis of [11C]Flurbiprofen methyl ester (3): The radiosynthesis
method was similar to that of [11C]Ibuprofen methyl ester (1).
[11C]Flurbiprofen methyl ester was purified by semi-preparative HPLC
(mobile phase: acetonitrile/water 75:25; column: COSMOSIL, 5C18-MS-
II, 20 (i.d.)ꢁ250 mm, 5 mm; flow rate: 10 mLminÀ1
; UV detection:
254 nm; retention time: 15 min). The total synthesis time including
HPLC purification and radiopharmaceutical formulation for intravenous
administration was 35 min. The isolated radioactivity was 5.8 GBq at the
end of synthesis, and the specific radioactivity was 41 GBqmmolÀ1. The
chemical identity of [11C]Flurbiprofen methyl ester was confirmed by co-
injection with the authentic sample of Flurbiprofen methyl ester on ana-
lytical HPLC (mobile phase: acetonitrile/water 60:40; column: COSMO-
SIL, 5C18-AR-II, 4.6 (i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV de-
tection: 254 nm; retention time: 5.9 min). The chemical purity analyzed
at 254 nm and the radiochemical purity were greater than 99%.
Experimental Section
Chemistry: All chemicals and solvents were purchased from Sigma–Al-
drich Japan (Tokyo, Japan), Wako Pure Chemical Industries (Osaka,
Japan), Tokyo Kasei Kogyo (Tokyo, Japan), and Nacalai Tesque (Kyoto,
Japan), and were used without further purification. Carbon-11 was pro-
Chem. Eur. J. 2010, 16, 4250 – 4258
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4255

M. Suzuki et al.
Radiosynthesis of [11C]Fenoprofen methyl ester (4): The radiosynthesis
method was similar to that of [11C]Ibuprofen methyl ester (1).
[11C]Fenoprofen methyl ester was purified by semi-preparative HPLC
(mobile phase: acetonitrile/water 70:30; column: COSMOSIL, 5C18-MS-
II, 4.6 (i.d.)ꢁ150 mm, 5 mm; flow rate: 1 mLminÀ1
; UV detection:
210 nm; retention time: 5.8 min). The chemical purity analyzed at 210 nm
and the radiochemical purity of [11C]Ibuprofen were greater than 99%.
Radiosynthesis of [11C]Naproxen (8): The radiosynthesis method was
similar to that of [11C]Ibuprofen (7). [11C]]Naproxen was purified by
semi-preparative HPLC (mobile phase: acetonitrile/10 mm sodium phos-
phate buffer (pH 7.4) 22:78; column: COSMOSIL, 5C18-MS-II, 20 (i.d.)ꢁ
250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection: 200 nm; retention
time: 19 min). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was
37 min. The isolated radioactivity was 2.7 GBq at the end of synthesis
and the specific radioactivity was 30 GBqmmolÀ1. The chemical identity
of [11C]Naproxen was confirmed by co-injection with the authentic
sample of Naproxen on analytical HPLC (mobile phase: acetonitrile/1%
phosphoric acid 50:50; column: COSMOSIL, 5C18-MS-II, 4.6 (i.d.)ꢁ
150 mm, 5 mm; flow rate: 1 mLminÀ1; UV detection: 254 nm; retention
time: 5.2 min). The chemical purity analyzed at 254 nm and the radio-
chemical purity were greater than 99%.
Radiosynthesis of [11C]Flurbiprofen (9): The radiosynthesis method was
similar to that of [11C]Ibuprofen (7). [11C]Flurbiprofen was purified by
semi-preparative HPLC (mobile phase, acetonitrile/10 mm sodium phos-
phate buffer (pH 7.4) 33:67; column: COSMOSIL, 5C18-MS-II, 20 (i.d.)ꢁ
250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection: 254 nm; retention
time: 14 min). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was
40 min. The isolated radioactivity was 3.5 GBq at the end of synthesis
and the specific radioactivity was 47 GBqmmolÀ1. The chemical identity
of [11C]Flurbiprofen was confirmed by co-injection with the authentic
sample of Flurbiprofen on analytical HPLC (mobile phase: acetonitrile/
1% phosphoric acid 50:50; column: COSMOSIL, 5C18-AR-II, 4.6 (i.d.)ꢁ
100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detection: 254 nm; retention
time: 6.0 min). The chemical purity analyzed at 254 nm and the radio-
chemical purity were greater than 99%.
Radiosynthesis of [11C]Fenoprofen (10): The radiosynthesis method was
similar to that of [11C]Ibuprofen (7). [11C]Fenoprofen was purified by
semi-preparative HPLC (mobile phase, acetonitrile/10 mm sodium phos-
phate buffer (pH 7.4) 20:80; column: COSMOSIL, 5C18-MS-II, 20 (i.d.)ꢁ
250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection: 206 nm; retention
time: 16 min). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was
40 min. The isolated radioactivity was 6.0 GBq at the end of synthesis
and the specific radioactivity was 25 GBqmmolÀ1. The chemical identity
of [11C]Fenoprofen was confirmed by co-injection with the authentic
sample of Fenoprofen on analytical HPLC (mobile phase: acetonitrile/
0.1% phosphoric acid 45:55; column: COSMOSIL, 5C18-AR-II, 4.6
(i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detection: 220 nm; re-
tention time: 8.4 min). The chemical purity at 220 nm was more than
98% and the radiochemical purity of [11C]Fenoprofen was greater than
99%.
Radiosynthesis of [11C]Ketoprofen (11): The radiosynthesis method was
similar to that of [11C]Ibuprofen (7). [11C]Ketoprofen was purified by
semi-preparative HPLC (mobile phase: acetonitrile/10 mm sodium phos-
phate buffer (pH 7.4) 30:70; column: COSMOSIL, 5C18-MS-II, 20 (i.d.)ꢁ
250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection: 254 nm; retention
time: 9.7 min). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was
28 min. The isolated radioactivity was 3.8 GBq at the end of synthesis
and the specific radioactivity was 40 GBqmmolÀ1. The chemical identity
of [11C]Ketoprofen was confirmed by co-injection with the authentic
sample of Ketoprofen on analytical HPLC (mobile phase: acetonitrile/
0.1% phosphoric acid 40:60; column: COSMOSIL, 5C18-AR-II, 4.6
(i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detection: 254 nm; re-
tention time: 6.2 min). The chemical purity at 254 nm and the radiochem-
ical purity were greater than 99%.
II, 20 (i.d.)ꢁ250 mm, 5 mm; flow rate: 10 mLminÀ1
; UV detection:
206 nm; retention time: 17 min). The total synthesis time including
HPLC purification and radiopharmaceutical formulation for intravenous
administration was 41 min. The isolated radioactivity was 4.8 GBq at the
end of synthesis and the specific radioactivity was 43 GBqmmolÀ1. The
chemical identity of [11C]Fenoprofen methyl ester was confirmed by co-
injection with the authentic sample of Fenoprofen methyl ester on analyt-
ical HPLC (mobile phase: acetonitrile/water 60:40; column: COSMOSIL,
5C18-AR-II, 4.6 (i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detec-
tion: 220 nm; retention time: 5.8 min). The chemical purity analyzed at
220 nm and the radiochemical purity were greater than 99%.
Radiosynthesis of [11C]Ketoprofen methyl ester (5): The radiosynthesis
method was similar to that of [11C]Ibuprofen methyl ester (1).
[11C]Ketprofen methyl ester was purified by semi-preparative HPLC
(mobile phase: acetonitrile/water 70:30; column: COSMOSIL, 5C18-MS-
II, 20 (i.d.)ꢁ250 mm, 5 mm; flow rate: 10 mLminÀ1
; UV detection:
254 nm; retention time: 12 min). The total synthesis time including
HPLC purification and radiopharmaceutical formulation for intravenous
administration was 28 min. The isolated radioactivity was 6.1 GBq at the
end of synthesis and the specific radioactivity was 63 GBqmmolÀ1. The
chemical identity of [11C]Ketoprofen methyl ester was confirmed by co-
injection with the authentic sample of Fenoprofen methyl ester on analyt-
ical HPLC (mobile phase: acetonitrile/water 60:40; column: COSMOSIL,
5C18-AR-II, 4.6 (i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detec-
tion: 254 nm; retention time: 5.8 min). The chemical purity analyzed at
254 nm and the radiochemical purity were greater than 99%.
Radiosynthesis of [11C]Loxoprofen methyl ester (6): The radiosynthesis
method was similar to that of [11C]Ibuprofen methyl ester (1).
[11C]Loxoprofen methyl ester was purified by semi-preparative HPLC
(mobile phase: acetonitrile/10 mm ammonium formate 50:50; column:
COSMOSIL, 5C18-MS-II, 10 (i.d.)ꢁ250 mm, 5 mm; flow rate: 6 mLminÀ1
;
UV detection: 220 nm; retention time: 10 min). The total synthesis time
including HPLC purification and radiopharmaceutical formulation for in-
travenous administration was 36 min. The isolated radioactivity was
2.2 GBq at the end of synthesis, and the specific radioactivity was
28 GBqmmolÀ1. The chemical identity of [11C]Loxoprofen methyl ester
was confirmed by co-injection with the authentic sample of Loxoprofen
methyl ester on analytical HPLC (mobile phase: acetonitrile/water 45:55;
column: COSMOSIL, 5C18-MS-II, 4.6 (i.d.)ꢁ150 mm, 5 mm; flow rate:
1 mLminÀ1; UV detection: 220 nm; retention time: 11.6 min). The chemi-
cal purity analyzed at 220 nm and the radiochemical purity were greater
than 99%.
Radiosynthesis of [11C]Ibuprofen (7): Sodium hydride (1 mg) was added
to a stirred solution of methyl (4-isobutylphenyl)acetate in anhydrous
DMF (200 mL) under an Ar atmosphere. [11C]CH3I was transported by a
stream of helium (30 mLminÀ1) and trapped in the mixture at room tem-
perature for 2 min. A total of 300 mL of 2m sodium hydroxide was added
to the reaction mixture, and then it was heated at 508C for 1 min. After
addition of a solution of 10% formic acid in acetonitrile (300 mL), the
mixture was diluted with 50% acetonitrile in water (300 mL). The given
reaction mixture was injected into preparative HPLC (mobile phase: ace-
tonitrile/10 mm sodium phosphate buffer (pH 7.4) 34:66; column: COS-
MOSIL, 5C18-MS-II, 20 (i.d.)ꢁ250 mm, 5 mm; flow rate: 10 mLminÀ1
;
UV detection: 195 nm; retention time: 16 min). The desired fraction was
collected into a flask containing 25% ascorbic acid (200 mL), and the or-
ganic solvent was removed under the reduced pressure. The desired radi-
otracer was dissolved in a mixture of polysorbate 80, propylene glycol,
and saline (0.1:1:10 v/v/v, 4 mL). The total synthesis time including
HPLC purification and radiopharmaceutical formulation for intravenous
administration was 33 min. The isolated radioactivity was 5.0 GBq at the
end of synthesis, and the specific radioactivity was 34 GBqmmolÀ1. The
chemical identity of [11C]Ibuprofen was confirmed by co-injection with
the authentic sample of Ibuprofen on analytical HPLC (mobile phase:
acetonitrile/0.1% phosphoric acid 60:40; column: COSMOSIL, 5C18-MS-
Radiosynthesis of [11C]Loxoprofen (12): The radiosynthesis method was
similar to that of [11C]Ibuprofen (7). [11C]Loxoprofen was purified by
semi-preparative HPLC (mobile phase: acetonitrile/10 mm sodium phos-
phate buffer (pH 7.4) 20:80; column: COSMOSIL, 5C18-MS-II, 20 (i.d.)ꢁ
4256
www.chemeurj.org
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Chem. Eur. J. 2010, 16, 4250 – 4258

11C-Labeling of 2-Arylpropionic Acids and Their Esters
FULL PAPER
250 mm, 5 mm; flow rate: 10 mLminÀ1; UV detection: 220 nm; retention
time: 16 min). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was
42 min. The isolated radioactivity was 2.0 GBq at the end of synthesis
and the specific radioactivity was 28 GBqmmolÀ1. The chemical identity
of [11C]Loxoprofen was confirmed by co-injection with the authentic
sample of Loxoprofen on analytical HPLC (mobile phase: acetonitrile/
0.1% phosphoric acid 60:40; column: COSMOSIL, 5C18-AR-II, 4.6
(i.d.)ꢁ100 mm, 5 mm; flow rate: 1 mLminÀ1; UV detection: 220 nm; re-
tention time: 6.6 min). The chemical purity at 220 nm and the radiochem-
ical purity were greater than 99%.
ponents by using an HPLC system (Shimadzu Corporation, Kyoto,
Japan) with a coupled NaI(Tl) positron detector UG-SCA30 (Universal
Giken, Kanagawa, Japan) to measure intact radiotracer and its acid form.
A fast-gradient condition created with two switching pumps was used to
analyze the samples. An Atlantis T3 column (4.6 (i.d.)ꢁ50 mm; Waters,
Milford, MA, USA) was used as a reverse-phase analytical column, and
a flow rate of 2.0 mLminÀ1 of methanol/water (28:72, v/v) containing
10 mm ammonium acetate (pH 7.2) was the initial condition used. After
0.3 min of the sample injection, the ratio of acetonitrile/water was
changed to 80:20 (v/v) linearly for 2.2 min and maintained for the next
2.0 min. The columns were then washed with acetonitrile/water (28:72, v/
v) containing 10 mm ammonium acetate (pH 7.2). The elution was moni-
tored by UV absorbance at 254 nm and coupled NaI positron detection.
The amount of radioactivity associated with intact radiotracer and its
acid form was calculated as a percentage of the total amount of radioac-
tivity.
Experimental animals: The animals were kept in a temperature- and
light-controlled environment and had ad libitum access to standard food
and tap water. All experimental protocols were approved by the Ethics
Committee on Animal Care and Use of the Center for Molecular Imag-
ing Science in RIKEN, and were performed in accordance with the Prin-
ciples of Laboratory Animal Care (NIH publication no. 85–23, revised
1985).
Generation of neuroinflammation in rats: Male Sprague-Dawley rats
(CLEA Japan, Tokyo, Japan) that weighed approximately 300 g received
an injection of lipopolysaccharides (LPS) from Escherichia coli 026:B6
(Sigma, St. Louis, MO, USA) into the left striatum. Briefly, animals were
anesthetized with 50 mgkgÀ1 sodium pentobarbital and stereotaxically in-
jected with LPS into the left striatum with a Hamilton syringe (anterior:
+0.2, lateral: +3.2, ventral: À5.5 mm from bregma). Animals were re-
turned to their home cages after the surgery and were housed for 1 day
following LPS injection.
Acknowledgements
This work was supported in part by a consignment expense for the Mo-
lecular Imaging Program on “Research Base for Exploring New Drugs”
from the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan. We would like to thank Mr. M. Kurahashi (Sumitomo
Heavy Industry Accelerator Service Ltd.) for operating the cyclotron,
Ms. K. Tokuda (RIKEN) for supporting rat PET studies, Ms. E. Hayashi-
naka (RIKEN) for supporting the reconstruction of PET images, and Dr.
M. Murai, Mr. K. Noutomi, and Ms.Y. Katayama (RIKEN) for support-
ing the metabolite analyses.
PET studies: Rats were anesthetized with a mixture of 1.5% isoflurane
and nitrous oxide/oxygen (7:3) and then placed on the PET scanner
gantry (microPET Focus 220, Siemens Co., Knoxville, TN, USA). The
PET scanner has a spatial resolution of 1.4 mm in FWHM at the center
of the field of view at 220 mm in diameter and an axial extent at 78 mm
in length. After intravenous bolus injection of 2-aryl
[11C]propionic acid
ACHTUNGTRENNUNG
or 2-aryl
ACHTUNGTRENNUNG
means of a venous catheter inserted into the tail vein, a 45 min emission
scan was performed. In the blocking experiment, unlabeled compound
(10 mgkgÀ1) was simultaneously injected with radiotracers. Emission data
were acquired in list mode, and the data were reconstructed with stan-
dard 2D filtered back projection (Ramp filter, cutoff frequency at 0.5
cycles per pixel). Regions of interest (ROI) were placed on the LPS-in-
jected side and the contralateral side of striatum by using image process-
ing software (Pmod ver.3.0, PMOD Technologies Ltd, Zurich, Switzer-
land) with reference to the rat MRI. Regional uptake of radioactivity in
the brain was decay-corrected to the injection time and expressed as the
standardized uptake value (SUV), normalized for injected radioactivity
and body weight.
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Ex vivo autoradiography: Fifty minutes after tracer injection, rats were
euthanized and were perfused with saline under deep anesthesia with
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[20] All products were isolated as a racemic mixture. Generally, the S
enantiomer of 2-arylproiponic acid is pharmacologically active in
terms of the cyclooxygenase inhibition. However, the majority of 2-
arylpropionic acids are prepared in a racemic mixture because an
isomerase enzyme (2-arylpropionyl-CoA epimerase) exists in vivo
system that converts the inactive R enantiomer into the active S
form, see: a) C. Reichel, R. Brugger, H. Bang, G. Geisslinger, K.
Brune, Mol. Pharmacol. 1997, 51, 576–582; exceptionally, a few in-
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Received: November 5, 2009
Revised: February 4, 2010
Published online: March 10, 2010
4258
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