Efficient synthesis and chiral separation of 11C-labeled ibuprofen assisted by DMSO for imaging of in vivo behavior of the individual isomers by positron emission tomography

Article abstract of DOI:10.1016/j.bmc.2011.03.041

The pharmacological mechanisms focusing on chiral isomer of ibuprofen are not fully understood. Only the (S)-isomer of ibuprofen inhibits cyclooxygenases, which mediates the generation of prostanoids and thromboxanes. Consequently, (S)-isomers represent a major promoter of the anti-inflammatory effect, and the effects of the (R)-isomers have not been widely discussed. However, more recently, the cyclooxygenase-independent pharmacological effects of ibuprofen have been elucidated. Pharmacokinetic studies with individual isomers of ibuprofen by positron emission tomography should aid our understanding of the pharmacological mechanisms of ibuprofen. The efficient ¹¹C-labeling of ibuprofen for chiral separation via the TBAF-promoted α-[ ¹¹C]methylation was achieved by using DMSO rather than THF as the reaction solvent. The robust production of the radiochemically labile ¹¹C-labeled ibuprofen ester was realized by the protective effect of DMSO on radiolysis. After intravenous injection of each enantiomer of [ ¹¹C]ibuprofen, significantly high radioactivity was observed in the joints of arthritis mice when compared to the levels observed in normal mice. However, the high accumulation was equivalent between the enantiomers, indicating that ibuprofen is accumulated in the arthritic joints regardless of the expression of cyclooxygenases.

Full text of DOI:10.1016/j.bmc.2011.03.041

Bioorganic & Medicinal Chemistry 19 (2011) 3265–3273
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient synthesis and chiral separation of 11C-labeled ibuprofen assisted
by DMSO for imaging of in vivo behavior of the individual isomers
by positron emission tomography
Tatsuya Kikuchi, Maki Okada, Nobuki Nengaki, Kenji Furutsuka, Hidekatsu Wakizaka, Toshimitsu Okamura,
⇑
Ming-Rong Zhang, Koichi Kato
Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The pharmacological mechanisms focusing on chiral isomer of ibuprofen are not fully understood. Only
the (S)-isomer of ibuprofen inhibits cyclooxygenases, which mediates the generation of prostanoids and
thromboxanes. Consequently, (S)-isomers represent a major promoter of the anti-inflammatory effect,
and the effects of the (R)-isomers have not been widely discussed. However, more recently, the cycloox-
ygenase-independent pharmacological effects of ibuprofen have been elucidated. Pharmacokinetic stud-
ies with individual isomers of ibuprofen by positron emission tomography should aid our understanding
of the pharmacological mechanisms of ibuprofen. The efficient 11C-labeling of ibuprofen for chiral sepa-
Received 25 January 2011
Revised 17 March 2011
Accepted 18 March 2011
Available online 27 March 2011
Keywords:
Radiolabeling
Radiolysis
DMSO
Chirality
NSAIDs
ration via the TBAF-promoted
a
-[11C]methylation was achieved by using DMSO rather than THF as the
reaction solvent. The robust production of the radiochemically labile 11C-labeled ibuprofen ester was
realized by the protective effect of DMSO on radiolysis. After intravenous injection of each enantiomer
of [11C]ibuprofen, significantly high radioactivity was observed in the joints of arthritis mice when com-
pared to the levels observed in normal mice. However, the high accumulation was equivalent between
the enantiomers, indicating that ibuprofen is accumulated in the arthritic joints regardless of the expres-
sion of cyclooxygenases.
Ibuprofen
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
have been found to show a more potent effect than their (S)-isomer
counterparts.4 Hamburger and McCay proposed that radical scav-
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely
used as therapeutic agents for the treatment of fever, pain and
inflammation. NSAIDs are divided into several types based on their
chemical structures. Among the different types of NSAIDs, the
importance of the chiral center of the arylpropionate type (profens)
at the 2-position has been recognized in pharmaceutical sciences.
This is because the (S)-isomer of these profens possesses an
inhibitory effect on cyclooxygenases (COX-1 and/or COX-2) that
mediates the generation of prostanoids and thromboxanes.1,2
Therefore, (S)-isomers represent a major promoter of the anti-
inflammatory effect, and the effects of the (R)-isomers have been
essentially overlooked. More recently, the pharmacological effects
of NSAIDs that are COX-independent have been elucidated. Anti-
tumor activity and the effect towards Alzheimer’s disease involve
COX-independent pathways,3 and the (R)-isomers of some profens
enging activities of ibuprofen (1) gave an additional anti-inflam-
matory effect to the COXs inhibition.5 In addition, Jedlitschky
et al. proposed that the inhibition of transmitter storage and re-
lease in platelets via multidrug resistance associated protein 4 by
NSAIDs may prevent platelet aggregation, besides the inhibition
of COXs.6 Pharmacokinetic studies with individual isomers of pro-
fens in conjunction with current knowledge would facilitate fur-
ther understanding of the pharmacologic effects of NSAIDs and
the development of novel NSAIDs.
A significant number of pharmacokinetic studies have been car-
ried out for either racemic or chiral form of 1; however, there are
no reports based on non-invasive in vivo imaging.7 Positron emis-
sion tomography (PET) provides molecular information, such as
pharmacokinetics of labeled radiopharmaceuticals and their
metabolites.8 Recently, PET has been considered a useful tool in
drug research and development.9 Direct measurement of in vivo
pharmacokinetics by PET can help to advance pharmacological re-
search. Consequently, PET studies of individual stereo-isomers of
profens represent an important subject. Since 1 is a representative
of profens and shows all the pharmacological activities described
Abbreviations: NSAID, non-steroidal anti-inflammatory drug; COX, cyclooxy-
genase; PET, positron emission tomography; ee, enantiomeric excess.
⇑
Corresponding author. Tel.: +81 43 206 4041; fax: +81 43 206 3261.
E-mail address: katok@nirs.go.jp (K. Kato).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.041

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T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
above, we designed a number of PET studies using chirally en-
riched 1 labeled with carbon-11. However, there are no reports
describing the preparation of 11C-labeled stereo-isomers of 1
((R)-[11C]1 and (S)-[11C]1) and the use of these molecules for
in vivo PET.10,11 Thus, individual isomers of 11C-labeled stereo-iso-
mers of 1 were prepared.
Asymmetric synthesis is a well-established research area and
versatile methods of enantioselective synthesis of arylpropionate-
type NSAIDs including 1 have been developed under non-labeling
conditions.12 However, the time and radioactive constraints of
short-lived positron-emitting carbon-11 (T1/2 = 20.3 min) restricts
the available 11C-labeling agent and reaction operations, thereby
limiting the introduction of current methods to produce chiral
additional problem.16 Takashima-Hirano et al. reported that 11C-la-
beled methyl ester analogs of some profens were radiolysis labile
under the high radioactivity level and the rank of 11C-labeled ibu-
profen methyl ester ([11C]4) was the highest by their criteria.10
Although the method for the a
-[11C]methylation of 3 was different,
a similar issue was considered in our case. It was unclear how
much radiolysis of [11C]4 influenced the isolated radioactivity of
the final products and it should appear en route to the preparation
of racemic [11C]1 during our multi-step synthesis. A treatment that
would suppress the radiolysis of [11C]4 without influencing the en-
tire (R)- or (S)-[11C]1 synthesis was necessary. We proposed the
suppression of radiolysis using the solvent effect. Here, we de-
scribed the successful efficient production of racemic [11C]1 via
[
11C]1. Asymmetric14C-labeling synthesis of(R)-[14C]1 was reported
TBAF-mediated a
-[11C]methylation and chiral separation of both
by Zhang et al.13 Enantioselective
a
-[14C]methylation using
enantiomers of [11C]1 by chiral HPLC with a reversed stationary
phase. Remarkable solvent effects, which were obtained by the
replacement of THF with DMSO, are also described. In addition,
we present preliminary results showing the accumulation of the
individual stereo-isomers of [11C]1 in the joints of arthritis mice
measured by PET.
iodo[14C]methane to a 4-methyl-5phenyl-2-oxazolidinone imide
derivative was the key step of this synthesis and (R)-[14C]1 was ob-
tained in 47% overall yield with more than 98% enantiomeric excess
(ee). Although iodo[14C]methane in the synthetic scheme could be
replaced by iodo[11C]methane (2) formally, a longer reaction time
at low temperature for
a
-[14C]methylation and isolation before
the removal of the chiral auxiliary would be difficult to translate
for the synthetic labeling of short-lived carbon-11.
2. Material and methods
2.1. Chemicals
Chiral separation by high-performance liquid chromatography
(HPLC) is a useful strategy for the preparation of both enantiomers,
and the molar scale of 11C-labeling is suitable for a semi-prepara-
tive HPLC approach.14 Each PET tracer should be synthesized for
every PET experiment. This is because of the short-lived radioac-
tive character of the PET tracer. Therefore, this feature can offset
the disadvantage of chiral separation by HPLC where only less than
half of the product is usable. However, there are additional aspects
required before introducing this technology into PET tracer synthe-
sis as compared with non-labeling syntheses. Usually, normal-
phase HPLC is used for the separation of versatile chiral molecules.
Conversely, reversed-phase HPLC is preferable for the separation
step of PET tracer syntheses by semi-preparative HPLC. In addition,
the isolated fraction should be administered to animals or humans
after evaporation, and this condition limits the usable buffer sys-
tems. As a result, chiral separation has not frequently been intro-
duced into PET tracer syntheses.15
(4-Isobutylphenyl)acetic acid was purchased from Toronto Re-
search Chemicals Inc. (North York, ON, Canada) via WAKO Pure
Chemical Industries Ltd (Osaka, Japan). (S)- and (R)-1 were pur-
chased from Tokyo Chemical Industry Ltd (Tokyo, Japan) and Enzo
Life Science International Inc. (Plymouth Meeting, PA, USA),
respectively. Ibuprofen, tetrabutylammonium bifluoride, 1.0 M
solution of TBAF in THF and DBU were purchased from Tokyo
Chemical Industry Ltd (Tokyo, Japan). Methyl phenylacetate (5),
triethylamine and 2-phenyl-propanoic acid were purchased from
WAKO Pure Chemical Industries Ltd (Osaka, Japan). Methyl 2-phe-
nyl-propanoate (6), 3 and 4 were prepared by refluxing a MeOH
solution of the corresponding acids in the presence of catalytic
amounts of H2SO4.
2.2. Equipment
Hence the radioactivity of each chiral product should be less
than half following chiral separation, and efficient production of
racemic [11C]1 was necessary. Consequently, a method which
yields racemic [11C]1 with high efficiency from the starting 11C-
labeling agent was needed. We recently developed the synthesis
HPLC separations were achieved with a pump (PU-1580, JASCO,
Tokyo, Japan), UV detector (UV-970M, JASCO, Tokyo, Japan), a man-
ual injector (Rheodyne (IDEX Health & Science LLC, Tokyo, Japan),
of [11C]1 via TBAF-promoted -[11C]methylation of methyl (4-iso-
a
20 lL loop), and a NaI(Tl) well-type scintillation detector with an
ACE Mate Amplifier and BIAS supply (925-SCINT, ORTEC, Oak
Ridge, TN, USA) for radioactivity detection. Data acquisition and
interpretation were performed with EZChrom Elite (Scientific Soft-
ware Inc., San Ramon, CA, USA). The enantiomeric purity (% ee) was
measured by an analytical HPLC system with a pump (PU-2089
plus, JASCO, Tokyo, Japan) and the same radiodetector described
butylphenyl)acetate (3) using 2 in THF with high total radiochem-
ical conversion (Scheme 1).11 The remote-controlled synthesis,
which is crucial for efficient production, was successful for this
method. We considered that the first step toward the chiral sepa-
ration of [11C]1 was solved. However, radiolysis of 11C-labeled
products with high radioactivity and high specific activity is an
O
O
iodo[11C]methan(2)
THF, rt, 30s
+ TBAF
OMe
OMe
11C]4; 89 3.3%
*
3
(20 µmol)
20 µmol
[
aq. NaOH
O
MeOH
80 °C, 3min
OH
* = 11
C
*
[
11C]1; 77 4.9%
Scheme 1. Synthesis of [11C]1 via TBAF-promoted -[11C]methylation of 3 using 2 in THF.
a

T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
3267
above. The remote-controlled system for radiolabeling under high
radioactivity level was composed in-house. Inversion of (R)-[11C]1
to (S)-[11C]1 in the blood of mice was quantified by the same HPLC
system, except for the use of a high sensitive positron detector
(Ohyo Koken Kogyo Co. Ltd, Tokyo, Japan).
(500
l
L) was placed at 0 °C. After evaporating THF, an aqueous
57% HI solution (400
lL) was added to the vessel. The resulting
mixture was heated to 150 °C to produce iodo[11C]methane (2).
Gaseous 2 was transferred by a N2 gas stream with a flow rate of
30 mL/min and collected by the reaction solvent (0.5–1 mL) in a
glass vessel at room temperature (rt). The preparation time of 2
was around 7 min after the end of bombardment. The solution of
PET scans of mice were performed with an Inveon Dedicated
PET system (Siemens Medical Solutions, Knoxville, TN, USA). Mice
were anesthetized with 2% isoflurane using
a
small animal
2 was divided into 100 lL aliquots and used for the a
-[11C]methyl-
anesthetizer (TK-6, Bio Machinery, Chiba, Japan), and the body
temperature was maintained within the normal range using a
heating pad system (MulꢀTꢀPads with T/Pump, Gaymar Industries
Inc., Orchard Park, NY, USA) during the PET scans. The radioactivity
was obtained with volume of interest (VOI) analysis using the ASI-
Pro VM software (CTI Concorde Microsystems, Knoxville, TN, USA).
The radioactivity of the injected solution and that in the plasma
were measured with a dose calibrator (IGC-7, ALOKA, Tokyo,
Japan).
ation reaction.
2.5. A typical procedure for the reaction yielding [11C]6 in Table
1
A mixture of 5 (200
(200 L) was prepared a few minutes prior to the end of bombard-
ment. A solution of 2 (37–370 MBq, 100 L) was added to the mix-
ture of 5 and TBAF and stored for 30 s at rt. Acetic acid (100 L) was
lL) and TBAF (1.0 M in THF) in a solvent
l
l
l
added to a reaction solution and the resulting mixture was ana-
2.3. Systems for HPLC
lyzed by system A.
HPLC was performed using the following systems.
2.6. Synthesis of [11C]1 by the low radioactivity level in THF
System A: Analytical HPLC of [11C]6 was performed with an
analytical reversed-phase XBridge C18 column (150 ꢁ 4.6 mm,
A mixture of 3 (20
THF, 20 L, 20 mol) was prepared a few minutes prior to the
end of bombardment. A solution of 2 (37ꢂ370 MBq) in THF
(100 L) was added to a mixture of 3 and TBAF and stored for
30 s at rt. To the reaction mixture, a mixture of MeOH (200 L)
and NaOH (1 M, 200 L) was added and the resulting solution
was heated at 80 °C. After 3 min, acetic acid (100 L) was added
lmol) in THF (200 lL) and TBAF (1.0 M in
3.5
lm, Waters Corporation, Milford, MA, USA). Elution was per-
l
l
formed at a flow rate of 0.7 mL/min with MeCN/buffer/AcOH
(60:40:0.2). A 30 mM solution of NH4OAc was used for the buffer.
UV absorption was detected at 254 nm.
l
l
System B: Analytical HPLC of [11C]4 was performed with an ana-
l
lytical reversed-phase XBridge C8 column (150 ꢁ 4.6 mm, 3.5
l
m,
l
Waters Corporation, Milford, MA, USA). Elution was performed at
a flow rate of 0.7 mL/min with MeCN/buffer/AcOH (70:30:0.2). A
30 mM solution of NH4OAc was used for the buffer. UV absorption
was detected at 254 nm.
to a reaction solution and the resulting mixture was analyzed by
system C.
2.7. Synthesis of [11C]1 under the low radioactivity level in
DMSO
System C: Analytical HPLC of [11C]1 was performed with an ana-
lytical reversed-phase XBridge C18 column (150 ꢁ 4.6 mm, 3.5
lm,
Waters Corporation, Milford, MA, USA). Elution was performed at a
flow rate of 0.7 mL/min with MeCN/buffer/AcOH (60:40:0.2). A
30 mM solution of NH4OAc was used for the buffer. UV absorption
was detected at 254 nm.
A mixture of solutions of 3 (10
TBAF (1.0 M in THF, 10 L, 10 mol) was prepared a few minutes
prior to the end of bombardment. A solution of 2 (37ꢂ370 MBq)
in DMSO (100 L) was added to a mixture of 3 and TBAF and stored
for 30 s at rt. For the analysis of 4, the reaction mixture was
quenched by the addition of acetic acid (100 L) and analyzed by
system B. To the unquenched reaction mixture, an aqueous solu-
tion of NaOH (1 M, 300 L) was added and the resulting solution
was heated at 60 °C. After 2 min, acetic acid (100 L) was added
lmol) in DMSO (200 lL) and
l
l
l
System D: Preparative HPLC of [11C]1 was conducted with a
semi-preparative reversed-phase CHIRALPAC OJ-RH column
l
(150 ꢁ 20 mm, 5
l
m, DAICEL Co. Ltd, Tokyo, Japan). Elution was
performed at
a
flow rate of 7.5 mL/min with MeCN/buffer
l
(45:55). A sodium phosphate solution (30 mM, pH 2.5) was used
for the buffer. UV absorption was detected at 254 nm.
l
to a reaction solution and the resulting mixture was analyzed by
system C.
System E: The enantiomeric purity of isolated (R)- or (S)-[11C]1
were determined with an analytical reversed-phase CHIRALPAC
OJ-RH column (150 ꢁ 4.6 mm, 5
l
m, DAICEL Co. Ltd, Tokyo, Japan).
2.8. Synthesis of [11C]1 under the high radioactivity level in
DMSO
Elution was performed at a flow rate of 0.5 mL/min with MeCN/
buffer (50:50). A sodium phosphate solution (20 mM, pH 3) was
used for the buffer. UV absorption was detected at 254 nm.
System F: The specific activities and the radiochemical purities
of isolated (R)- or (S)-[11C]1 were determined with an analytical re-
A mixture of solutions of 3 (10
lmol) in DMSO (300 lL) and
TBAF (1.0 M in THF, 10 L, 10 mol) was prepared a few minutes
l
l
prior to the end of bombardment and put into the reaction vessel.
The 11C-labeled agent 2 was prepared by the above method and
transferred to the reaction vessel containing a solution of 3 and
TBAF by a N2 gas stream with a flow rate of 30 mL/min at rt. After
the radioactivity of the vessel reached a plateau, the gas stream
was suspended. After 30 s, an aqueous solution of NaOH (1 M,
versed-phase XBridge C18 column (50 ꢁ 3.0 mm, 2.5
lm, Waters
Corporation, Milford, MA, USA). Elution was performed at a flow
rate of 1 mL/min with 90% MeCN/buffer (60:40). A mixture of
100 mM of (NH4)3PO4 (pH 2) and 5 mM of C8H17SO4Na was used
for the buffer. UV absorption was detected at 254 nm.
300 lL) was added to the reaction mixture and then mixed by
2.4. Production of 2
the N2 gas stream. The mixture was heated to 60 °C and placed
for 2 min at the same temperature. The reaction mixture was
cooled by the air flow, flowed by the addition of a 9:1 mixture of
[
11C]Carbon dioxide was produced by the 14N(p, 11C nuclear
a)
reaction in a nitrogen gas containing 0.01% oxygen with 18 MeV
protons using the CYPRIS HM-18 cyclotron (Sumitomo Heavy
Industry, Tokyo, Japan). After bombardment, [11C]O2 was trans-
ferred to the reaction vessel where a 0.05 M THF solution of LAH
eluent and H3PO4 (500 l
L). Purification of (R)- or (S)-[11C]1 was
carried out by system D. The desired individual isomers [11C]1
were collected by the flask containing a mixture of EtOH and
Tween 80. The fraction was evaporated and the radioactive residue

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T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
was dissolved in an aqueous Na2HPO4 solution (50 mM, 2 mL) to
neutralization. Enantiomeric purity and specific activity were
determined by system E and system F, respectively.
trate (50 lL) was subjected to HPLC analysis (system E). The recov-
ery of the radioactivity in the filtrate was quantitatively based on
the plasma radioactivity. The peaks of (R)- and (S)-[11C]1 on the
radiochromatogram were confirmed by UV absorption of non-
radioactive 1 coinjected to HPLC. The ratio of the components of
the radioactive compounds was calculated after correcting the
radiochromatogram for baseline noise and decay.
2.9. PET measurements of (R)- and (S)-[11C]1 accumulation in
the joints of arthritis mice
Male mice (BALB/cCr Slc, 6–7 weeks old) used in the present
study were purchased from Japan SLC Inc. (Shizuoka, Japan), and
treated and handled according to the ‘Recommendations for Han-
dling of Laboratory Animals for Biomedical Research’, compiled
by the Committee on Safety and Ethical Handling Regulations for
Laboratory Animal Experiments, National Institute of Radiological
Sciences, Japan.
Anti-collagen antibody induced arthritis model mice were gen-
erated by intravenous injection of an arthritogenic monoclonal
antibody cocktail (1.5 mg), consisting of five monoclonal anti-type
II collagen antibodies (Arthrogen-CIA, Chondrex, Redmond, WA,
USA) followed by the intraperitoneal injection of lipopolysaccha-
3. Results
3.1. Synthesis of [11C]1 under low radioactivity level
The synthesis of [11C]1 should be carried out under the high
radioactivity level. In our previous report, sufficient amounts of
racemic [11C]1 was obtained when starting with ꢃ11 GBq of
[
11C]O2. However, this synthesis approach was not used with a
scale over 15 GBq, which was the standard level of radiolysis in a
previous report.11 No information about the radiolysis of [11C]4
with high radioactivity was available under the TBAF-mediated
a
-[11C]methylation. However, similar radiolysis of [11C]4 was
ride (30 lg) on the third day following the administration of the
antibody. At day 4, after the lipopolysaccharide injection, the mice,
whose arthritis score of each limb was 3 according to the manufac-
turer’s instructions, were used for PET examination as an arthritis
model.
expected and such radiolysis would appear at the end of the
-[11C]methylation of 3 and the beginning of the hydrolysis of
11C]4. The addition of ascorbic acid, ethanol, or formic acid to
a
[
the mixture after the reaction had been carried out was found to
counter radiolysis; however, these treatments retard the TBAF-
mediated [11C]methylation process.17 Ideally the most suitable ap-
proach would involve no addition of reagents to prevent radiolysis,
but rely on the reaction solvent to suppress the radiolysis. Thus, we
explored the possibility of using a different reaction solvent (other
Eight normal mice and four arthritis model mice were used for
PET scanning. The body temperature was maintained within the
normal range using a heating pad system. The phosphate buffered
solutions, which contained (R)- or (S)-[11C]1 at high concentra-
tions, were diluted with saline to appropriate concentrations of
radioactivity for the injection. Each of the (R)- and (S)-[11C]1 spe-
cies (9 to 12 MBq; <0.18 nmol) were administered as a 0.2 mL
intravenous bolus injection to each mouse. The mice were main-
tained under isoflurane anesthesia during the scanning periods.
Data were acquired by the animal tomograph for 30 min following
the injection in 13 frames divided as follows: 5 ꢁ 1 min, 5 ꢁ 2 min
and 3 ꢁ 5 min frames. Without attenuation correction, the data
were reconstructed using Fourier re-binning and filtered back pro-
jection with a Hanning filter cutoff at the Nyquist frequency into
images with a 128 ꢁ 128 ꢁ 159 matrix size and twice zoom, to give
a voxel size of 0.39 ꢁ 0.39 ꢁ 0.80 mm3. After image reconstruction,
VOI covering 3–4 slices were manually placed on a transverse view
of summed PET images and transferred to all of the frames of the
images to generate time–radioactivity concentration (Bq/mL)
curves for heart and bilateral ankles. To compare the accumulation
of the isomers to the joints of arthritis mice rigorously, PET scans of
the isomers were performed with the same arthritis mouse on the
same day. That is, an arthritis mouse was administrated (R)- or (S)-
than THF) of the TBAF-mediated a
-[11C]methylation. The effect of
the radiolysis of [11C]4 was investigated by analyzing the succes-
sive ester hydrolysis of [11C]4 without any additives.
Initially, the
a
-[11C]methylation of 5 was chosen as a model
reaction and explored in several solvents. All reactions in Table 1
were carried out by the addition of a solution of 2 to a mixture
of 5 and TBAF at rt for 30 s. A mixture of 5 and TBAF was prepared
about 10 min before the addition of a solution of 2. As seen in Table
1, TBAF underwent a
-[11C]methylation efficiently in DMSO and the
decay-corrected radiochemical conversion of 5 was more than 98%.
The radiochemical conversion of [11C]6 was similar to that ob-
served in THF (entries 1 and 2). In contrast to the reaction in
THF, a
-[11C]methylation in DMSO led to a reduction in the amount
of both precursor and TBAF while maintaining the radiochemical
conversion of [11C]6 (entries 5–7). The reaction in acetonitrile
was significantly less efficient and in dichloromethane was not
effective under the 11C-labeling conditions (entries 3 and 4).
We applied the conditions of entry 6 for the synthesis of [11C]1
under the small bombardment experiment. The [11C]methylation
of 3 in DMSO gave [11C]4 in 93 1.2% radiochemical conversion
(decay-corrected). The successive alkaline hydrolysis of the methyl
[
11C]1 5 h following the administration of the other isomer.
The statistical analysis of the time–activity curves was per-
formed with the three-way ANOVA followed by the Holm-Sidak
test using SigmaPlot (ver. 11, Systat Software Inc.). The significance
level was set at p <0.01.
Table 1
TBAF-mediated
2.10. Determination of the component of radioactive
compounds in the mice plasma after (R)- and (S)-[11C]1
administration
a
-[11C]methylation of 5a
Entry
Solvent
TBAF (
l
mol)
5 (lmol)
RCCb (%)
1c
2
3
4
5
6
7
THF
20
20
20
20
10
10
5
20
20
20
20
10
10
5
95>
98>
13 1.1
0
61 2.6
95>
After intravenous administration of (R)- and (S)-[11C]1
(70–125 MBq; <0.43 nmol) to normal mice (n = 3 each, 20–25 g),
they were decapitated at 30 min. Blood samples (0.5 mL) were col-
lected at the stump to a heparinized tube, and centrifuged at
10,000ꢁg for 10 min to obtain the plasma. The plasma samples
(0.2 mL) were mixed with a twofold volume of ethanol in a tube
followed by centrifugation at 10,000ꢁg for 5 min to deproteinize.
DMSO
MeCN
CH2Cl2
THF
DMSO
DMSO
91
a
Each reaction was carried out more than three times.
b
The radiochemical conversion (RCC) was determined by the radiochromato-
gram of the analytical HPLC following the decay correction.
The supernatant was filtered with a 0.45
lm pore membrane
Ref.11
.
c
(Centricut W-MO, Kurabo Industries Ltd., Osaka, Japan), and the fil-

T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
3269
ester was carried out by the addition of an aqueous solution of
NaOH to the reaction mixture and heated at 60 °C for 2 min. The
reaction gave
11C]1 in 92 2.8% radiochemical conversion
(Scheme 2, in two steps, decay-corrected). The radiochemical con-
versions for both [11C]1 and [11C]4 were improved using the sol-
vent DMSO and smaller starting amounts of TBAF and 3,
compared to the reaction with THF, were used (Schemes 1 and 2).
A
(R)-[11C]1
(S)-[11C]1
7
6
5
4
3
2
1
0
[
1
0.5
0
3.2. Synthesis of racemic [11C]1 under high radioactivity level
and its chiral separation
After successfully synthesizing racemic [11C]1 using DMSO un-
der the low radioactivity level, we applied this method using
DMSO for the efficient synthesis and chiral separation of [11C]1.
Our previous results using THF were not significantly different
from DMSO and thus the synthesis using THF was also investi-
gated. We performed the remote-controlled synthesis of [11C]1
and the radioactivity of [11C]O2 was scaled to >22.2 GBq (calculated
0
10
20
30
Time (min)
B
7
6
5
4
3
2
1
0
1
amount). In both solvent conditions,
ried out by flowing gaseous nitrogen containing 2 into a solution
of 3 at rt. Consequently, during the trapping of 2,
-[11C]methyla-
a
-[11C]methylation was car-
a
0.5
0
tion of 3 underwent simultaneously. The time-course radioactivity
of the solution in the reaction vessel was monitored by a simple
radioactivity sensor.18
In contrast to the synthesis under the low radioactivity level,
low efficiency of radioactivity was observed for the trapping of 2
in THF. After the ester hydrolysis of [11C]4, isolation of (R)- or
(S)-[11C]1 using chiral HPLC was performed. In this case, significant
amounts of radioactive hydrophilic byproducts were observed in
the radiochromatogram (Fig. 1A). As a result of these negative fac-
tors, the radioactivity of the chiral product was less than 185 MBq
(<1% decay uncorrected yield, referred to [11C]O2). On the other
0
10
20
30
Time (min)
Figure 1. Radiochromatograms of preparative HPLC. Radiochromatogram (solid
line) for the reaction mixture of the synthesis of [11C]1 via [11C]methylation in
DMSO (A) and in THF (B). The dotted lines represent the intensity of UV absorption
at 254 nm.
hand, the trapping efficiency of 2 during the
a
-[11C]methylation
in DMSO was consistent with the reaction carried out using
11.1 GBq of [11C]O2. Furthermore, the radiochromatogram of the
reaction mixture following the ester hydrolysis of [11C]4 suggested
significant suppression of the formation of hydrophilic byproducts
(Fig. 1B). As a result, the obtained radioactivity of each isomer was
more than 740 MBq (>3% decay uncorrected yield, referred to
[
11C]O2) and the range of specific activities was 66–289 GBq/
lmol.
The ees were more than 95% for the (R)-isomer and 90% for the (S)-
isomer (Fig. 2). The radiochemical purity of injection solution of
each isomer was always more than 99% even at the end of
in vivo study.
0
0
5
10
15
20
Time (min)
The HPLC conditions for the separation of both isomers using
chiral semi-preparative HPLC were determined as below. Since re-
versed-phase conditions are frequently introduced for PET tracer
syntheses, the choice of a chiral column with a reversed-phase
mode is preferable. We selected the Daicel OJ-RH column
(20 ꢁ 150 mm, 5
lm) for preparative HPLC because this allowed
us to use a mixture of MeCN and phosphate as an eluent without
the addition of transition metal salts that require a further purifi-
cation step.19 Therefore, radioactive products will be administered
only by adjusting the pH of the solution. Thus, chiral separation
conditions for ibuprofen using semi-preparative HPLC were opti-
mized by adjusting the flow rate and the ratio of MeCN and the
buffer of the mobile phase. Optimum conditions that afforded effi-
cient chiral separation for [11C]1 were a 45:55 mixture of MeCN
and phosphate buffer as the eluent with a 7.5 mL flow rate (Fig. 1).
0
0
5
10
15
20
Time (min)
Figure 2. A typical radiochromatogram of isolated (R)- (upper) and (S)-[11C]1
(lower) after correcting for baseline noise and decay. The vertical axes (radioac-
tivity) are presented as a relative value.
3.3. PET measurements of (R)- and (S)-[11C]1 accumulation in
the joints of arthritis mice
2
aq. NaOH
60 °C, 2min
11
11C]1
92 2.8%
4
3
[
C]
[
+ TBAF
DSMO, rt, 30s
93 1.2%
Since 1 has been used for the treatment of inflamed arthritis
and anti-collagen antibody induced arthritis model mice represent
Scheme 2. Syntheses of [11C]1 under the low radioactive level.

3270
T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
a well-established inflammation model, this model was used for
investigating the accumulation of (R)- and (S)-[11C]1 at an inflamed
lesion. PET scans of the isomers were performed with the same
arthritis mouse for rigorous comparison of the accumulation of
the isomers in the joints. PET images after the administration of
the individual isomers to normal and arthritis mice are shown in
Figure 3. In the PET images, the high accumulation of both isomers
in the arthritic joints was observed. The concentration of radioac-
tivity in the hindlimb ankles of mice almost reached a plateau dur-
ing the 30 min of PET scanning (Fig. 4). The accumulation of the
individual isomers in the hindlimb ankles of arthritis mice was
more than twice as high as that of normal mice after 30 min post-
injection (p <0.01). In contrast, comparable levels of the isomers
accumulated in the ankles of arthritis and normal mice (not signif-
icant). The time–activity curves in the heart, which are considered
to reflect the kinetics of radioactivity in the blood because of the
high fraction binding of 1 to albumin,20,21 were similar among all
cases (not significant, Fig. 4). The uptake in other regions of normal
mice such as cerebrum, liver, kidney and bladder were also similar
between the isomers (Supplementary data).
3.4. Determination of the components of radioactive
compounds in the plasma of mice after (R)- and (S)-[11C]1
administration
(R)-1 is inverted to the pharmacologically active (S)-isomer in
terms of COX inhibition by 2-arylpropionyl-CoA epimerase in
vivo.22 Therefore, the concentration of (R)- and (S)-[11C]1 in plasma
(R)-[11C]1
(S)-[11C]1
0.7
Normal
SUV
0
Arthritis
Figure 3. A typical swelling of the hindlimb of arthritic mice (left) and PET images after the administration of (R)- (center) and (S)-[11C]1 (right) to normal (upper) and
arthritis mice (lower). Summed images during 0–30 min after the administration are presented.
0.8
0.6
0.4
0.2
0
7
6
5
4
3
2
1
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time after injection (min)
Time after injection (min)
Figure 4. Time–activity curves of the ankle of the hindlimb (left) and the heart (right) after the administration of (R)- (circle) and (S)-[11C]1 (triangle) to normal (open) and
arthritis mice (closed). The vertical axes represent the standardized uptake value (SUV: concentration of radioactivity in the VOI/(dose/body weight)).

T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
3271
Table 2
The fraction of components of radioactive compounds in the mice plasma after
30 min postinjection of the isomers
Injected isomer
Fraction of components (%)
Metabolitesa
(R)-[11C]1
(S)-[11C]1
[
11C]1b
(R)-[11C]1
(S)-[11C]1
25 2.9
34 2.8
23 4.3
0.78 0.55
52 1.9
65 3.2
75 2.9
66 2.8
0
The values are expressed as mean SD (%, n = 3).
a
The fraction of metabolites were calculated based on the whole radiochro-
0
5
10
15
matogram as shown in Figure 5, except for the regions of (R)- and (S)-[11C]1.
Time (min)
b
Sum of the (R)- and (S)-[11C]1.
after the administration of the each isomer was determined. Fol-
lowing 30 min postinjection of (R)- and (S)-[11C]1 to normal mice,
the concentration of radioactivity in the plasma was 8.3 0.059%
dose/mL and 8.9 0.080% dose/mL, respectively. The ratio of com-
ponents of radioactive compounds in the plasma following 30 min
postinjection of the isomers is summarized in Table 2, and the
chromatograms are presented in Figure 5. From the radioactivity
and the ratio of components of [11C]1 in the plasma, the concentra-
tions of [11C]1 (i.e., sum of (R)- and (S)-[11C]1) in the plasma after
30 min postinjection of (R)- and (S)-[11C]1 were calculated as
5.9 0.25% dose/mL and 6.2 0.24% dose/mL, respectively.
0
0
5
10
15
Time (min)
Figure 5. A typical radiochromatogram of radioactive compounds in the plasma
after 30 min postinjection of (R)- (upper) and (S)-[11C]1 (lower) and following the
correction for baseline noise and decay. The vertical axes (radioactivity) are
presented as relative values.
4. Discussion
ꢂ
Higher order fluorides such as HF2ꢂ, H2F3 and additional ones
to 72 3.2% following the addition of TBAF to a mixture of 2 and 5
(Eq. 3). In contrast, the efficient a
-[11C]methylation of 6 occurred in
were formed in the presence of water.23 Those species were weak-
er bases than Fꢂ and the reaction of 2 and 5 by tetrabutylammo-
the presence of smaller amounts of 5 and TBAF in DMSO. We
concluded that the slower conversion of fluoride to higher order
fluorides in DMSO consistently furnishes the sufficient active anion
of 5.
nium bifluoride did not undergo
a
-[11C]methylation in either
DMSO or THF. We considered that higher order fluorides could
be formed from Fꢂ in the presence of an acidic proton of 5 before
ð1Þ
ð2Þ
ð3Þ
the addition of a solution of 2, and therefore retarded the produc-
tion of the active anion of 5 (Eq. 1).24 Additionally, different results
The results of the synthesis of [11C]1 under the high radioactiv-
ity level differed remarkably between DMSO and THF. In contrast
to unsuccessful results in THF, the efficient trapping of radioactiv-
ity under the preparation of [11C]4 and high yield of [11C]1 were ac-
quired even by reducing the amount of 3 in DMSO. According to a
former report, [11C]4 was labile but [11C]1 was resistant to the radi-
olysis.10 Hydrophilic byproducts (ca. 25%) were observed in the
on the a
-[11C]methylation by decreasing both TBAF and 5 in DMSO
and THF were dominated by the reaction rate of the formation of
higher order fluorides. These assumptions were supported by the
result of
conversion was maintained by changing the reaction manner.
The reaction was carried out by the addition of 10 mol of TBAF
to a mixture of 2 and 10 mol of 5 gave 6 with >95% radiochemical
a
-[11C]methylation of 5, in which a high radiochemical
l
reaction mixture of a
-[11C]methylation of 3 in THF under the high
l
radioactivity level. The amounts of those products were insignifi-
cant under the low radioactivity level. We therefore consider that
the different results in this study were derived from the radiolysis
of the ester form [11C]4 and the primary role of DMSO was the sup-
pression of this radiolysis.
conversion (Eq. 2). The results were remarkably different from the
radiochemical conversion of 6 obtained by the addition of 2 to a
solution of 10 lmol of TABF and 5 (Table 1, entry 5). Therefore,
the anion of 5 was generated sufficiently in the initial stages of
the reaction and certain amounts of higher order fluorides were
formed over the time course of the reaction in THF, thereby result-
Many groups have explored the scavenging effect of DMSO for
hydroxyl radicals induced by
ton reaction and photolysis, and reaction pathways have been elu-
cidated (Scheme 3).25 TBAF used in this synthesis was
commercially available TBAFꢀ3H2O solution and the preparation
c-radiolysis, pulse radiolysis, the Fen-
ing in the slower
were obtained by the
chemical conversion of 6 in MeCN gave rise to a dramatic increase
a
-[11C]methylation of 5. Similar observations
a
-[11C]methylation of 5 in MeCN. The radio-
a

3272
T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
albumin and the serum-synovial fluid albumin ratio.29 Further-
•CH3 + CH3SO2H
(CH3)2SO + •OH
(CH3)2SO(OH)•
more, increased blood flow in the arthritic lesion30 and/or elimina-
tion of albumin, which is a carrier of 1,20 from the blood to the
arthritic lesion31 may be involved in the high accumulation of
(CH3)2SO(OH)•
•CH3 + CH3SO2H
11C]1 in the lesion. Although the accumulation mechanism of
11C]1 in the arthritic lesions remains to be investigated, our results
(CH3)2SO + •CH3
•CH2SOCH3 + CH4
[
[
suggest that [11C]1 or its chiral isomers can be used to detect
arthritis in vivo. Recently, administration of (S)-1 for pain relief
rather than racemic 1 use32 and drug–drug interactions between
1 and concomitant medicines such as methotrexate33 were stud-
ied. [11C]1 or its chiral isomers can also be useful for those pharma-
cokinetic studies.
•CH3 + •CH3
C2H6
Scheme 3. Reaction pathways of DMSO and hydroxyl radicals.
of the reaction solutions were carried out under atmospheric
conditions. Therefore, hydroxyl radicals could be formed under
the
a
-[11C]methylation condition of 3; however, such radicals
should be formed to a much lower level. On the other hand, 1 is
considered to play a role as a radical scavenger or as an anti-oxi-
dant to protect against oxidation of biogenic substances.26 In addi-
tion, the presence of ibuprofen radicals under oxidative conditions
was reported by spin trapping experiments.5 Recently, von Gunten
et al. reported the rate constant for the reaction of 1 and hydroxyl
radicals.27 They described that most of the products obtained by
the reaction with hydroxyl radicals were phenolic compounds;
however, structural information was not presented.28 As compared
to their reports, the expected concentrations of hydroxyl radicals in
our conditions were lower. Thus, [11C]1 was also reactive against
hydroxyl radicals; however, the radiolysis of [11C]1 was insignifi-
cant in this study. We summarized these results as follows. The
radiolysis of [11C]4 was promoted by the presence of hydroxyl rad-
icals, and the ester form [11C]4 was more reactive to these radicals
than the acid form [11C]1. The solvent effect by DMSO was effective
in suppressing this type of radiolysis.
Finding an expedient system for HPLC separation of distinct
substrates is necessary in PET tracer syntheses. Reversed-phase
preparative HPLC was selected as the appropriate method to purify
PET tracers. In contrast, chiral separation is usually performed
using normal phase under non-labeling conditions. The use of a
normal phase is technically burdensome because tedious replace-
ment of the solution in the synthetic module should be carried
out before and after every synthesis. In contrast, the buffer system
used for reversed-phase chiral separation frequently contains
perchrolate or copper ions. These conditions are also not preferable
to PET tracer syntheses, because further treatments are required to
remove those ions before administration.15 Chiral separation by a
regularly used buffer system such as phosphate or acetate was nec-
essary and we explored HPLC conditions using a mixture of MeCN
and sodium phosphate. Finally, we achieved an acceptable resolu-
tion for the preparation of individual isomers of [11C]1 and the iso-
lated fraction could be administered by adjusting the pH with the
addition of basic phosphate buffer.
5. Conclusion
Efficient production of [11C]1 was realized via TBAF-mediated
a
-[11C]methylation of 3 in DMSO. The solvent effects of DMSO
for the suppression of radiolysis of [11C]4 and the behavior of the
fluoride anion were remarkable. Individual isomers (R)-[11C]1
and (S)-[11C]1 were obtained by chiral separation using reversed-
phase HPLC with high ee. The PET studies of individual isomers
represent opportunities to investigate profens, including 1, in more
detail by molecular imaging technologies.
Acknowledgements
We thank the technical team of the Cyclotron Section and
Radiopharmaceuticals Section of the National Institute of Radiolog-
ical Sciences for their support during cyclotron operation and the
production of radioisotopes. This work was supported in part by
The Nakatomi Foundation and an Intramural Research Grant from
the National Institute of Radiological Sciences.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.041.
References and notes
1. Adams, S. S.; Bresloff, P.; Mason, C. G. J. Pharm. Pharmacol. 1976, 28, 256.
2. Shen, T. Y. Angew. Chem., Int. Ed. Engl. 1972, 11, 460.
3. (a) Tegeder, I.; Pfeilschifter, J.; Geisslinger, G. FASEB J. 2001, 15, 2057; (b)
Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.; Pietrzik, C. U.; Findlay, K.
A.; Smith, T. E.; Murphy, M. P.; Bulter, T.; Kang, D. E.; Marquez-Sterling, N.;
Golde, T. E.; Koo, E. H. Nature 2001, 414, 212; (c) Zhou, Y.; Su, Y.; Li, B.; Liu, F.;
Ryder, J. W.; Wu, X.; Gonzalez-DeWhitt, P. A.; Gelfanova, V.; Hale, J. E.; May, P.
C.; Paul, S. M.; Ni, B. Science 2003, 302, 1215.
4. Quann, E. J.; Khwaja, F.; Zavitz, K. H.; Djakiew, D. Cancer Res. 2007, 67, 3254.
5. Hamburger, S. A.; McCay, P. B. Free Radical Res. Commun. 1990, 9, 337.
6. Jedlitschky, G.; Tirschmann, K.; Lubenow, L. E.; Nieuwenhuis, H. K.; Akkerman,
J. W. N.; Greinacher, A.; Kroemer, H. K. Blood 2004, 104, 3603.
7. (a) Fornasini, G.; Monti, N.; Brogin, G.; Gallina, M.; Eandi, M.; Persiani, S.; Bani,
M.; Della Pepa, C.; Zara, G.; Strolin Benedetti, M. Chirality 1997, 9, 297; (b) Li, C.;
Benet, L. Z.; Grillo, M. P. Chem. Res. Toxicol. 2002, 15, 1480; (c) Zheng, C.; Hao,
H.; Wang, G.; Sang, G.; Sun, J.; Li, P.; Li, J. Eur. J. Drug Metab. Pharmacokinet.
2008, 33, 45.
8. Phelps, M. E. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9226.
9. Lappin, G. R. C. Garner, Nat. Rev. Drug Disc. 2003, 2, 233.
10. Takashima-Hirano, M.; Shukuri, M.; Takashima, T.; Goto, M.; Wada, Y.;
Watanabe, Y.; Onoe, H.; Doi, H.; Suzuki, M. Chemistry 2010, 16, 4250.
11. Kato, K.; Kikuchi, T.; Nengaki, N.; Arai, T.; Zhang, M.-R. Tetrahedron Lett. 2010,
51, 5908.
12. (a) Oppolzer, W.; Rosset, S.; De Brabander, J. Tetrahedron Lett. 1997, 38, 1539;
(b) Noyori, R. Angew. Chem., Int. Ed. 2002, 41, 2008; (c) Knowles, W. S. Angew.
Chem., Int. Ed. 2002, 41, 1999; (d) Goossen, L. J. Angew. Chem., Int. Ed. 2002, 41,
3775; (e) Alper, H.; Hamel, N. J. Am. Chem. Soc. 1990, 112, 2803; (f) Uemura, T.;
Zhang, X.; Matsumura, K.; Sayo, N.; Kumobayashi, H.; Ohta, T.; Nozaki, K.;
Takaya, H. J. Org. Chem. 1996, 61, 5510; (g) Chen, A.; Ren, L.; Crudden, C. M. J.
Org. Chem. 1999, 64, 9704; (h) Li, X.; List, B. Chem. Commun. 2007, 1739; (i)
Smith, C. R.; RajanBabu, T. V. Org. Lett. 2008, 10, 1657; (j) Friest, J. A.; Maezato,
Y.; Broussy, S.; Blum, P.; Berkowitz, D. B. J. Am. Chem. Soc. 2010, 132, 5930; (k)
Fadel, A. Synlett 1992, 48.
The efficient synthesis of [11C]1 allowed sufficient preparation
of (R)- and (S)-[11C]1 for the investigation of the in vivo kinetics
of each isomer by PET. We investigated the accumulation of the
individual stereo-isomer of [11C]1 in the joints of arthritis mice
using PET as a preliminary experiment. The (S)-1 isomer is known
to inhibit COX activity, whereas the (R)-1 is inactive.1 Therefore, if
COXs were involved in the accumulation of [11C]1 in the arthritic
joints, the uptake of the (S)-isomer in the lesion would be higher
than that of the (R)-isomer. However, the different accumulation
levels of each isomer in the joints were not observed in both nor-
mal and arthritis mice throughout entire time course of the study.
The (R)-[11C]1 remained in the plasma at the end of the PET scan
and the kinetics of each isomer in the plasma was comparable.
Consequently, the accumulation mechanism of [11C]1 in the ar-
thritic lesions was considered to be independent of COX expression
in the joints. This result was consistent with a previous report; Cox
et al. reported that the steady-state distribution of isomers of 1
into synovial fluid is modulated by the binding of the isomers to

T. Kikuchi et al. / Bioorg. Med. Chem. 19 (2011) 3265–3273
3273
13. Zhang, Y.; Hong, Y.; Huang, C. C.; Belmont, D. J. Labelled Compd. Radiopharm.
2006, 49, 237.
25. (a) Veltwisch, D.; Janata, E.; Asmus, K.-D. J. Chem. Soc., Perkin Trans. 2 1980, 146;
(b) Eberhardt, M. K.; Colina, R. J. Org. Chem. 1988, 53, 1071; (c) Woodward, J. R.;
Lin, T.-S.; Sakaguchi, Y.; Hayashi, H. J. Phys. Chem. A 2000, 104, 557; (d) Koulkes-
Pujo, A. M.; Barat, F.; Mialocq, J. C.; Sutton, J. J. Photochem. 1973/74, 2, 439.
26. Zapolska-Downar, D.; Zapolska-Downar, A.; Bukowska, H.; Gałka, H.;
Naruszewicz, M. Life Sci. 1999, 65, 2289.
27. Huber, M. M.; Canonica, S.; Park, G. Y.; von Gunten, U. Environ. Sci. Technol.
2003, 37, 1016.
28. (a) von Gunten, U. Water Res. 2003, 37, 1443; (b) Pan, X.-M.; Schuchmann, M.
N.; von Sonntag, C. J. Chem. Soc., Perkin Trans. 2 1993, 289; (c) Mvula, E.;
14. Maier, N. M.; Franco, P.; Lindner, W. J. Chromatogr., A 2001, 906, 3.
15. Drandarov, K.; Schubiger, P. A.; Westera, G. Appl. Radiat. Isot. 2006, 64, 1613.
16. (a) MacGregor, R. R.; Schlyer, D. J.; Fowler, J. S.; Wolf, A. P.; Shiue, C. Y. J. Nucl.
Med. 1987, 28, 60; (b) Scott, P. J.; Hockley, B. G.; Kung, H. F.; Manchanda, R.;
Zhang, W.; Kilbourn, M. R. Appl. Radiat. Isot. 2009, 67, 88.
17. (a) Fukumura, T.; Nakao, R.; Yamaguchi, M.; Suzuki, K. Appl. Radiat. Isot. 2004,
61, 1279; (b) Fawdry, R. M. Appl. Radiat. Isot. 2007, 65, 1193; (c) Jacobson, M. S.;
Dankwart, H. R.; Mahoney, D. W. Appl. Radiat. Isot. 2009, 67, 990.
18. Due to the high background radioactivity, precise measuring of radioactivity by
simple sensor was difficult.
Schuchmann, M. N.; von Sonntag, C. J. Chem. Soc., Perkin Trans.
2 2001,
264.
19. The web site of DAICEL CHEMICAL INDUSTRY Ltd shows the chiral separation
data. See http://www.daicelchiral.com/appguide/index.htm. Their service
section informed us the chiral separation of ibuprofen using CHIRALCEL OJ-
RH by analytical scale.
29. Cox, S. R.; Gall, E. P.; Forbes, K. K.; Gresham, M.; Goris, G. J. Clin. Pharmacol.
1991, 31, 88.
30. Clavel, G.; Marchiol-Fournigault, C.; Renault, G.; Boissier, M. C.; Fradelizi, D.;
Bessis, N. Ann. Rheum. Dis. 2008, 67, 1765.
20. (a) Itoh, T.; Maruyama, J.; Tsuda, Y.; Yamada, H. Chirality 1997, 9, 354; (b) Itoh,
T.; Saura, Y.; Tsuda, Y.; Yamada, H. Chirality 1997, 9, 643.
21. Subramanian, K. M. J. Nucl. Med. 1991, 32, 480.
22. Reichel, C.; Brugger, R.; Bang, H.; Geisslinger, G.; Brune, K. Mol. Pharmacol.
1997, 51, 576.
23. Sun, H.; DiMagno, S. G. J. Am. Chem. Soc. 2005, 127, 2050.
24. Adams, D. J.; Clark, J. H.; Nightingale, D. J. Tetrahedron 1999, 55, 7725.
31. Wunder, A.; Müller-Ladner, U.; Stelzer, E. H.; Funk, J.; Neumann, E.; Stehle, G.;
Pap, T.; Sinn, H.; Gay, S.; Fiehn, C. J. Immunol. 2003, 170, 4793.
32. (a) Kaehler, S. T.; Phleps, W.; Hesse, E. Inflammopharmacology 2003, 11, 371; (b)
Moore, R. A.; Derry, S.; McQuay, H. J. Cochrane Database Syst. Rev. 2009, 23, 12.
33. El-Sheikh, A. A. K.; van den Heuvel, J. J. M. W.; Koenderink, J. B.; Russel, F. G. M.
J. Pharmacol. Exp. Ther. 2007, 320, 229.