Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [ 11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation

Article abstract of DOI:10.1016/j.bmc.2011.03.020

Synthesis of [¹¹C]celecoxib, a selective COX-2 inhibitor, and [¹¹C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was

Full text of DOI:10.1016/j.bmc.2011.03.020

Bioorganic & Medicinal Chemistry 19 (2011) 2997–3004
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib
and its major metabolite [11C]SC-62807 and in vivo PET evaluation
Misato Takashima-Hirano a, Tadayuki Takashima a, Yumiko Katayama a, Yasuhiro Wada a,
Yuichi Sugiyama b, Yasuyoshi Watanabe a, Hisashi Doi a, Masaaki Suzuki a,
⇑
a RIKEN Center for Molecular Imaging Science (CMIS), Kobe, Japan
b Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Synthesis of [11C]celecoxib, a selective COX-2 inhibitor, and [11C]SC-62807, a major metabolite of
celecoxib, were achieved and the potential of these PET probes for assessing the function of drug
transporter in biliary excretion was evaluated. The synthesis of [11C]celecoxib was achieved in one-pot
by reacting [11C]methyl iodide with an excess of the corresponding pinacol borate precursor using
Article history:
Received 26 January 2011
Revised 8 March 2011
Accepted 9 March 2011
Available online 13 March 2011
Pd2(dba)3, P(o-tolyl)3, and K2CO3 (1:4:9) in DMF. The radiochemical yield of
[
11C]celecoxib was
mol
63 23% (decay-corrected, based on [11C]CH3I) (n = 7) with a specific radioactivity of 83 23 GBq/
l
Keywords:
(n = 7). The average time of synthesis from end of bombardment including formulation was 30 min with
>99% radiochemical purity. [11C]SC-62807 was synthesized from [11C]celecoxib by further rapid oxidation
in the presence of excess KMnO4 with microwave irradiation. The radiochemical yield of [11C]SC-62807
was 55 9% (n = 3) (decay-corrected, based on [11C]celecoxib) with a specific radioactivity of 39 4 GBq/
Drug transporter
Biliary excretion
PET (positron emission tomography)
Radiochemistry
Celecoxib
l
mol (n = 3). The average time of synthesis from [11C]celecoxib including formulation was 20 min and the
radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [11C]celecoxib and
SC-62807
[
11C]SC-62807 showed largely different excretion processes, and consequently, [11C]SC-62807 was rap-
idly excreted via hepatobiliary excretion without further metabolism. [11C]SC-62807 was shown to have
a high potential as a PET probe for evaluating drug transporter function in biliary excretion.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyr-
azole-1-yl]benzenesulfonamide) is
a selective cyclooxygenase
It is now accepted that drug transporters play important roles in
tissue distribution and excretion of drugs and their metabolites.
Clinical studies have shown that variation in drug transporter
activity caused by genetic polymorphisms or drug–drug interac-
tions can affect the variability in therapeutic efficacy and the inci-
dence of adverse effects.1,2 The information on the functional
characteristics of drug transporters in vivo allows improvements
in drug delivery or drug design by targeting specific transporter
proteins.3 Accordingly, the methods that allow quantitative
estimation of the tissue concentration of the drugs in vivo have
been required for the investigation of such variations in the tissue
distribution or disposition processes of drugs.
(COX)-2 inhibitor that has analgesic and anti-inflammatory
effects in patients with rheumatoid arthritis, but has no effect
on COX-1 activity at therapeutic plasma concentrations.8 In
humans, celecoxib is extensively metabolized in the liver via
sequential two-step oxidative pathways, initially to a hydroxy-
methyl metabolite (SC-60613), and upon subsequent further oxi-
dation to a carboxylic acid metabolite (SC-62807) (Fig. 1).9,10 The
majority of celecoxib is excreted into the bile as SC-62807. In this
context, Wu et al. reported that SC-62807 is a substrate of drug
transporters, such as Organic Anion Transporting Polypeptide
1B1 (OATP1B1) and Breast Cancer Resistance Protein (BCRP),
which presumably mediate its hepatobiliary transport.11 There-
Positron emission tomography (PET) is a powerful and widely
accepted noninvasive method for molecular imaging in living
systems.4–6 The high sensitivity and exceptional spatial-temporal
resolution of PET make it particularly useful for in vivo estimation
of the function of drug transporters in various tissues.7
fore, celecoxib or SC-62807 radiolabeled with a short-lived
positron-emitting radionuclide could be a potential PET probe
for evaluating the function of these drug transporters in hepatob-
iliary excretion.
Celecoxib and SC-62807 have two readily accessible possible
positions for 11C and 18F radiolabeling, and Prabhakaran et al.
reported the synthesis of both
[ [
11C]celecoxib and 18F]cele-
⇑
Corresponding author. Address: RIKEN Center for Molecular Imaging Science
(CMIS), 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Tel.: +81 78 304 7130; fax: +81 78 304 7131.
coxib.12,13 However, chemical and metabolic instabilities derived
from [18F]defluorination on the benzylic position of [18F]celecoxib
resulted in undesirable bone imaging due to
[
18F]fluorine
E-mail address: masaaki.suzuki@riken.jp (M. Suzuki).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.020

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M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
Figure 1. Two-step metabolic pathway from celecoxib to SC-62807.
generation. Therefore, 11C-labeling of celecoxib would be prefera-
ble to 18F-labeling.
1H-pyrazol-1-yl]-1-phenylsulfonamide, (2) was achieved as
described in Scheme 1. 4-[5-(4-Bromophenyl)-3-(trifluoromethy)-
1H-pyrazol-1-yl]-1-phenylsulfonamide (5) was prepared from
1-(4-bromophenyl)-4,4,4-trifluorobutane-1,3-dione and hydrazine
(4), which was generated from aniline (3) in one-pot based on
previously described method with minor modifications.12,23 Com-
pound 5 was converted into 2 using bis(pinacolato)diboron and
Pd catalyst in DMSO.24
The palladium(0)-mediated rapid C–[11C]methylation reaction
developed by our group and based on the use of [11C]CH3I and trib-
utyl stannyl substrate is used in the synthesis of [11C]celecoxib
(Scheme 1).14 Protection of the sulfonamide group is necessary to
promote such a reaction. We recently reported a new type of palla-
dium(0)-mediated rapid C–[11C]methylation that uses an organobo-
ron compound.15 This reaction proceeds in the presence of
Pd2(dba)3, P(o-tolyl)3, and K2CO3 in DMF for 5 min under mild tem-
peratureand withouttheuse ofamicrowave.16,17 Ingeneral, organo-
boron compounds exhibit higher reactivity in the coupling reaction
than organostannyl compounds,18,19 and are also less toxic. Accord-
ingly, the synthesis of [11C]celecoxib reported draws extensively
from our brief report15 and advances made since its publication.
SC-62807 has a benzoic acid structure that can be labeled with
11C. Efficient 11C labeling of a benzoic acid structure has been re-
ported that involves: (1) [11C]CO insertion to the corresponding
aryl halide,20 (2) reaction of the Grignard reagent with
Synthesis of [11C]celecoxib was previously reported by Prabhak-
aran et al.,12 using an organotin compound with a sulfonamide
group protected by a dimethoxytrityl (DMT) group as a precursor
for radiolabeling. The authors succeeded in synthesizing [11C]cele-
coxib by the rapid methylation reaction using a DMT-protected
stannyl precursor and [11C]CH3I in the presence of Pd2(dba)3 and
P(o-tolyl)3 in DMF at 135 °C for 5 min followed by deprotection of
the DMT group in the presence of trifluoroacetic acid (TFA) at
60 °C for 5 min. Prabhakaran et al. also reported the reaction be-
tween the organostannyl-precursor without the protecting group
and CH3I in the presence of Pd2(dba)3, P(o-tolyl)3, CuCl, and K2CO3
in DMF, but the reaction yielded starting material together with
undesired destannylated product.25 We concluded that in the syn-
thesis of a short-lived PET probe, a direct one-step reaction without
deprotection is necessary to reduce the total synthesis time. In a
context related approach, we employed the rapid C–[11C]methyla-
tion reaction recently developed by our group that uses an organo-
boron precursor. Thus, [11C]celecoxib was synthesized by treating
pinacol borate precursor (2) with [11C]CH3I in DMF in the presence
of Pd2(dba)3/P(o-tolyl)3/K2CO3 (1:4:9) at 65 °C for 4 min (Scheme
[
11C]CO2,21 and (3) coupling of the [11C]cyanide ion and the
corresponding aryl halide, and hydrolysis.22 Here, we explored
the novel possibility of 11C labeling using a combination of rapid
C–[11C]methylation and rapid oxidation, starting with a common
organoboron precursor to carry out the sequential transformation
of [11C]celecoxib to [11C]SC-62807.
This report describes the highly efficient synthesis of [11C]cele-
coxib and as an expanding of rapid C–[11C]methylation, the synthe-
sis of [11C]SC-62807 by applying it to rapid C–[11C]carboxylation.
In addition, we describe evaluation of the in vivo behavior of each
11C-labeled compound in rats using PET to determine whether
these radiotracers enable the visualization of hepatobiliary excre-
tion for the quantitative assessment of drug transporter function.
2). This reaction produced
a 98% analytical yield by HPLC
(Fig. 2A). The total synthesis time was 30 min from the end of bom-
bardment (EOB). The average decay-corrected radiochemical yield
(DCY) based on [11C]CH3I was 63 23% (n = 7), and the specific
radioactivity at the end of synthesis was 84 23 GBq/
with >98% radiochemical purity (Fig. 2B). The chemical identity of
11C]celecoxib was confirmed by co-injection with the reference
lmol (n = 7)
2. Results and discussion
[
standard celecoxib in analytical HPLC and it showed the same
retention time peaks on the UV and the radioactive chromatograms.
Thus, we efficiently synthesized [11C]celecoxib from the corre-
sponding pinacol borate precursor without the protection of a sul-
fonamide group.
2.1. Synthesis of [11C]celecoxib and [11C]SC-62807 PET probes
The synthesis of the pinacol borate precursor, (4-[5-[4-(4,4,5,5-
tetramethyl-[1.3.2]dioxaborolan-2-yl)phenyl]-3-trifluoromethyl-
Scheme 1. Synthesis of the boron precursor for [11C]celecoxib. Reagents and conditions: (a) NaNO2, concd HCl, ꢀ5 °C, 15 min; (b) SnCl2ꢁH2O, ꢀ20 °C, 1 h; (c) compound 3,
C2H5OH, reflux, 16 h, 75% (overall in three steps); (d) bis(pinacolato)diboron, PdCl2(dppf)ꢁCH2Cl2, AcOK, DMSO, 80 °C, 16 h, 78%.

M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
2999
Scheme 2. Synthesis of [11C]celecoxib and [11C]SC-62807. Reagents and conditions: (a) [11C]CH3I, Pd2(dba)3, P(o-tolyl)3, DMF, 65 °C, 4 min. (b) KMnO4, 0.2 M NaOH, heating,
5 min.
A
B
11C]celecoxib
[
11C]celecoxib
[
UV
UV
Radio
Radio
0
5
10
15
20
0
2
4
6
8
10
Time (min)
Time (min)
Figure 2. (A) HPLC chromatogram before [11C]celecoxib purification; (B) HPLC chromatogram after [11C]celecoxib purification. UV absorbance: 254 nm.
time to 5 min in order to optimize the heating conditions (Table 1).
Consequently, [11C]celecoxib was converted to [11C]SC-62807 in
Table 1
Rapid oxidation reaction conditions. Reaction time = 5 min
32 18% (n = 5) DCY26 based on [11C]celecoxib (HPLC analytical
yield27: 55 30% (n = 5)) by conventional heating at 120 °C. The
process had poor reproducibility, presumably due to the presence
of different concentrations of [11C]celecoxib in each reaction.
Therefore, we used microwave irradiation as an alternative heating
method based on prior experiments in which microwave irradia-
tion tended to enhance reactivity at low concentrations. As
expected, microwave irradiation was extremely effective in
increasing both the yield and the reproducibility of the reaction.
Entry
Temp.a (°C)
HPLC analytical
yieldb (%)
Radiochemical
yieldc (%)
1
2
3
120 (n = 5)
120, microwave (n = 4)
140, microwave (n = 3)
55 30
79 13
32 18
47 11
87
5
55
9
a
Values in parentheses show number of runs.
b
Not decay-corrected, calculated by the peak area ratio of the desired
[
11C]methylated product obtained by radio-HPLC analysis of the reaction mixture.
Decay-corrected, calculated based on [11C]celecoxib.
c
Finally,
[
11C]SC-62807 was efficiently synthesized by rapid
oxidation of [11C]celecoxib at 140 °C for 5 min under microwave
irradiation with 55 9% (n = 3) DCY based on [11C]celecoxib (HPLC
analytical yield: 87 5% (n = 3)) (Fig. 3A). The total synthesis time
was 20 min from the [11C]celecoxib transfer, with a specific
Scheme 2 also illustrates the synthesis of 11C-labeled SC-62807
via [11C]celecoxib based on sequential rapid C–[11C]methylation
and oxidation. [11C]celecoxib was dissolved in 0.2 M NaOH and
transferred to a reaction vessel containing KMnO4 (the amount of
radioactivity at the end of synthesis of 39 4 GBq/
>98% radiochemical purity (Fig. 3B). The chemical identity of
11C]SC-62807 was confirmed by co-injection with the reference
lmol (n = 3)
[
11C]celecoxib transferred by this method was ꢂ20–60% of the syn-
thesized radioactivity). The reaction was carried out by setting the
[
11C]SC-62807
A
B
[
11C]SC-62807
[
UV
UV
Radio
Radio
0
2
4
6
8
10
0
5
10
15
Time (min)
Time (min)
Figure 3. (A) HPLC chromatogram before [11C]SC-62807 purification; (B) HPLC chromatogram after [11C]SC-62807 purification. UV absorbance: 254 nm.

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M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
Figure 4. Automated radiolabeling system designed for sequential two-step-radiosyntehsis of [11C]SC-62807. [1] = reactor for [11C]CH3I production, [2] and [3] = reactor,
[4] = microwave apparatus, [5] = reservoir, [6] = HPLC pump, [7] = column switching, [8] = fraction collector, [9] and [11] = evaporator, [10] and [12] = syringe, [13] = vial.
Figure 5. PET images, blood time–activity curve, and radiometabolite analysis following intravenous administration of [11C]celecoxib to male Sprague–Dawley rats. (A) PET
images of radioactivity in the abdominal region at 1, 2, 5, 10, 20, 40, and 60 min after administration. SUV = standardized uptake value; (B) the time profiles of radioactivity in
the blood were determined by blood sampling over a 60-min period after administration of [11C]celecoxib. The data represent the individual data of two rats; (C)
representative HPLC chromatograms of blood, liver, and bile extracts.
standard SC-62807 in analytical HPLC and it showed the same
retention time peaks on the UV and the radioactive chromato-
2.2. Construction of an automated two-step radiolabeling
system for the synthesis of [11C]-SC-62807
grams. Thus, we successfully synthesized
[
11C]celecoxib and
[
11C]SC-62807 by rapid C–[11C]methylation of the corresponding
Figure 4 shows the radiolabeling system we developed to carry
out two-step synthesis of [11C]SC-62807 using microwave irradia-
tion. The system consists of a reactor for 11CH3I production using
an established method,28 two reactors for cooling and heating,
one reactor for rapid oxidation under microwave irradiation (Initi-
pinacol borate precursor without the protection of a sulfonamide
group, and sequential combination rapid oxidation of the
[
11C]methyl group, respectively. The total time required for this
sequential synthesis was 50 min from EOB.

M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
3001
Figure 6. PET images, time–activity curves, and radiometabolite analysis following intravenous administration of [11C]SC-62807 to male Sprague–Dawley rats. (A) PET
images of radioactivity in the abdominal region at 1, 2, 5, 10, 20, 40, and 60 min after administration. SUV = standardized uptake value; (B) the time profiles of radioactivity in
the blood, liver, kidney, intestine (bile), and urinary bladder were determined by PET imaging and blood sampling over a 60-min period after administration of [11C]SC-62807.
The data represent the mean S.D. (n = 3); (C) representative HPLC chromatograms of blood, liver, bile, and urine extracts.
ator™, Biotage), multiple reservoirs for the addition of reagents,
column switching (six columns available), and four fraction collec-
tors, two of which play the role of evaporators attached to the
formulation system. Any combination of reactors and fraction
collectors can be chosen by changing the connections. This
remote-controlled synthesis apparatus was utilized for the prepa-
ration of [11C]SC-62807 using the rapid sequential C–[11C]methyl-
ation–oxidation reaction. The automated multistep labeling
system could be utilized for the synthesis of a wide range of
11C-incorporated PET probes.
nent found in the bile after administration of [11C]celecoxib. These
pharmacokinetic profiles of [11C]celecoxib will make the analysis
of biliary excretion difficult for the following reasons: (1) since
PET cannot discriminate between [11C]celecoxib and its radiome-
tabolite, [11C]celecoxib PET images of hepatobiliary excretion will
be the result of two different pharmacokinetic functions, metabo-
lism and biliary excretion of [11C]celecoxib, and (2) the very high
concentration of [11C]celecoxib in the blood may affect the tissue
concentration determined by PET image analysis. Therefore,
[
11C]celecoxib should not be used as a PET probe for the evaluating
drug transporter function in biliary excretion.
2.3. PET study and radiometabolite analyzes of [11C]celecoxib
2.4. PET study and radiometabolite analyzes of [11C]SC-62807
Time course maximum intensity projection PET images of the
abdominal region of a rat following administration of [11C]cele-
coxib are shown in Figure 5A. Radioactivity initially localized in
the liver, and subsequently part of the radioactivity moved to the
intestine, although the amount of radioactivity in the entire
abdominal region was relatively high. Radioactivity in the blood
gradually decreased, however, the radioactivity was still higher le-
vel (more than 1% of dose/ml blood) even at the end of the scan
(Fig. 5B). Figure 5C shows representative radiochromatograms of
blood, liver, and bile extracts prepared after administration of
Figure 6A shows time course maximum intensity projection PET
images of radioactivity in the rat abdominal region following
administration of [11C]SC-62807. Radioactivity localized primarily
in the liver and kidneys within 2 min after [11C]SC-62807 adminis-
tration. By 60 min, radioactivity was localized in the intestine (de-
rived from bile excreted into the intestine) and the urinary bladder.
Time–activity curves for blood and tissues in the abdominal re-
gion of rats are shown in Figure 6B. The radioactivity in blood rap-
idly decreased. A maximum of 35 6% and 14 1% of the dose was
distributed in the liver and kidney, respectively, by 2 min post-
administration, at which point the amount of radioactivity began
to decline rapidly. The radioactivity in the intestine (derived from
radioactivity in bile excreted into the intestine) and the urinary
bladder (derived from radioactivity excreted into the urine) in-
creased until 60 min, reaching 58 6% and 22 3% of the dose,
[
11C]celecoxib to rats. At 40 min after administration, [11C]cele-
coxib was detected predominantly in the blood and the liver, and
a very small amount of radiometabolites, [11C]SC-60613 (hydroxy-
methyl form of celecoxib) and [11C]SC-62807 (carboxylic acid
form), were detected in the blood and the liver, respectively. In
contrast, the [11C]SC-62807 radiometabolite was the major compo-

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M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
respectively. Metabolites of [11C]SC-62807 were not detected in
the blood, liver, bile, or urine within 40 min after administration
of [11C]SC-62807 (Fig. 6C). These results show that the radioactiv-
ity of [11C]SC-62807 is rapidly excreted via hepatobiliary and renal
excretion without further metabolism.
spectra (MS) was measured on a ThermoFinnigan LCQ ion trap
mass spectrometer (San Jose, CA, USA) equipped with a turbo ESI
ion source. Carbon-11 was produced by an 14N(p, 11C nuclear
a)
reaction using a CYPRIS HM-12S Cyclotron (Sumitomo Heavy
Industry, Tokyo, Japan). An automated radiolabeling system was
used for heating the reaction mixture, dilution, HPLC injection,
fraction collection, evaporation, and sterile filtration. Purification
with semipreparative HPLC was performed on a GL Science system
(Tokyo, Japan). Microwave irradiation was carried out in a Biotage
Initiator™ (Tokyo, Japan) using a sealed vessel. Radioactivity was
quantified with an ATOMLAB™ 300 dose calibrator (Aloka, Tokyo,
Japan). Analytical HPLC was performed on a Shimadzu system
(Kyoto, Japan) equipped with pumps and a UV detector, and efflu-
ent radioactivity was measured with an RLC700 radio analyzer
(Aloka). The columns used for analytical and semipreparative HPLC
were COSMOSIL C18 MS-II and AR-II (Nacalai Tesque), respectively.
There are some key transporters in hepatobiliary transporter
including MRP2 (multidrug resistance-associated protein 2), BCRP,
and OATPs. Some probes such as 99mTc-mebrofenin, N-[11C]acetyl-
leukotriene E4, and (15R)-16-m-tolyl-17,18,19,20-tetranorisocar-
bacyclin (15R-[11C]TIC-Me) for molecular imaging technology have
been reported for evaluating the function of MRP2 or OATPs in
hepatobiliary excretion.29–31 Compared to these probes, the study
using [11C]SC-62807 may have the potentials for evaluating the
other transporter BCRP at least in hepatobiliary transport and renal
excretion. In addition, [11C]SC-62807 is superior to the other
probes in the use of diagnosis because of the rapid excretion with-
out further metabolism, which may enable the functional analysis
more simple. Further investigations using [11C]SC-62807 are ongo-
ing to evaluate the function of these drug transporters using
knockout animal models or specific drug transporter inhibitors.
4.1.1. 4-[5-(4-bromophenyl)-3-(trifluoromethy)-1H-pyrazol-1-
yl]1-phenylsulfonamide (5)
A solution of sodium nitrate (1.38 g, 20 mmol) was added into a
mixture of sulfanilamide (6.8 g, 20 mmol) in concd HCl (10 mL)
and water (2.5 mL) over 15 min at ꢀ5 °C. The mixture was rapidly
added to a cooled (ꢀ20 °C) solution of tin(II) chloride dyhydrate
(10 g, 44 mmol) in conc. HCl (15 mL). The resulting mixture was
stirred for 1 h at room temperature. A solution of 1-(4-bromophe-
nyl)-4,4,4-trifluorobutane-1,3-dione (4.19 g, 14.2 mmol) in ethanol
(20 mL) was added. The mixture was stirred under reflux for 16 h.
It was evaporated under the reduced pressure and the residue was
extracted with ethyl acetate (30 mL, three times). Organic layer
was washed with brine (30 mL), dried over sodium sulfate, filtered,
and evaporated. The crude product was purified by flash chroma-
tography eluting with hexane/ethyl acetate = 2:1 to give the title
compound (4.5 g, 71%) as a white solid. 1H NMR spectrum was
identified with the data of Ref. 12.
3. Conclusion
A novel procedure for the synthesis of a [11C]benzoic acid struc-
ture via sequential rapid C–[11C]methylation and oxidation reac-
tions was established. The first step involves rapid and highly
efficient 11C-labeling of celecoxib from a pinacol boron precursor.
The subsequent rapid oxidation under microwave irradiation
produces
[
11C]SC-62807. Microwave irradiation significantly
enhances both radiochemical yield and reproducibility. The protocol
for this two-step rapid radiosynthesis can be executed in a fully re-
mote-controlled manner by using the radiolabeling system we
developed.
PET image analysis in parallel with radiometabolite analyzes of
rats indicated that [11C]celecoxib is not suitable for evaluating bil-
iary excretion because it shows high blood concentration and its
biliary excretion includes the mixture of two different pharmacoki-
netic functions, metabolism and biliary excretion of [11C]celecoxib.
On the other hand, [11C]SC-62807 enables the visualization of
hepatobiliary excretion as well as renal excretion without further
metabolism, and therefore is a potentially useful PET probe for
the quantitative determination of the drug transporter. Further
evaluation of [11C]SC-62807 in terms of its utility in functional ana-
lyzes of drug transporters in hepatobiliary and renal excretion is in
progress.
4.1.2. 4-[5-[4-(4,4,5,5-Tetramethyl-[1.3.2]dioxaborolan-2-yl)-
phenyl]-3-trifluoromethyl-1H-pyrazol-1-yl]-1-phenylsulf-
onamide (2)
A
mixture of 5 (2.23 g, 5.0 mmol), bis(pinacolato)diboron
(1.40 g, 5.5 mmol), [1,10-bis(diphenylphosphino)ferrocene]dichlo-
ropalladium (122 mg, 0.15 mmol), and potassium acetate (1.47 g,
15 mmol) in anhydrous DMSO (20 mL) was stirred at 80 °C for
3 h. It was partitioned quenched with water (50 mL) and then ex-
tracted with diethylehter (50 mL, two times). Organic layer was
dried over sodium sulfate, filtered, and evaporated. The crude
product was purified by flash chromatography eluting with hex-
ane/ethyl acetate = 4:1 to give the title compound (1.5 g, 40%) as
a white solid. 1H NMR (400 MHz, CDCl3) d: 7.90 (d, J = 8.8 Hz,
2H), 7.79 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.22(d,
J = 8.0 Hz, 2H), 6.80 (s, 1H), 5.04 (br s, 2H), 1.35 (s, 12H). 13C
NMR (100 MHz, CDCl3) d: 145.0, 144.4, 144.0, 142.4, 141.4, 135.3,
131.0, 128.1, 127.6, 125.5, 121.0 (q, J = 268.7 Hz), 106.8, 84.2,
24.6. MS (ESI): m/z 494.35 [M+H]+, 492.40 [MꢀH]ꢀ.
4. Materials and methods
4.1. Chemistry
All chemicals and solvents were purchased from Sigma–Aldrich
Japan (Tokyo, Japan), Wako Pure Chemical Industries (Osaka,
Japan), Tokyo Kasei Kogyo (Tokyo, Japan), Nacalai Tesque (Kyoto,
Japan), and ABX (Radeberg, Germany), and were used without
further purification. Celecoxib, SC-60613, and SC-62807 as cold
standards were purchased from Tronto Research Chemicals (North
York, Canada). Flash chromatography was performed on Teledyne
Isco CombiFlash Companion (Lincoln, USA). Nuclear magnetic res-
onance (NMR) spectra were recorded on JEOL JNM-ECX400P spec-
trometer (Tokyo, Japan) at ambient temperature. The chemical
shifts are expressed in parts per million (ppm) downfield from tet-
ramethylsilane or in ppm relative to CHCl3 (d 7.26 in 1H NMR and
77.0 in 13C NMR). Signal patterns are indicated as follows: s,
singlet; d, doublet; t, tripled; q, quartet; m, multiplet; br, broad sig-
nal. Coupling constants (J values) are given in hertz (Hz). Mass
4.1.3. Synthesis of [11C]celecoxib
[
11C]CO2 was converted to [11C]CH3I by treatment with lithium
aluminum hydride followed by hyrdoiodic acid using an auto-
mated synthesis system.28 11C]CH3I was trapped in a solution of
pinacol borate precursor 2 (3.8 mg, 7.7 mol), Pd2(dba)3 (2.9 mg,
3.2 mol), P(o-tolyl)3 (3.8 mg, 12.7 mol), and K2CO3 (4.0 mg,
28.9 mol) in DMF (400 L) at 30 °C. Next, the mixture was heated
to 65 °C for 4 min and then diluted with CH3CN (700 L) and water
(300 L). After filtration through a 0.2 m PVDF filter (Millipore),
the resulting mixture was injected onto a preparative HPLC column
(AR-II C18, 20 mm i.d. ꢃ 250 mm, 5 m (COSMOSIL, Nacalai Tes-
que) using a mobile phase of CH3CN/water = 70:30 at a flow rate
[
l
l
l
l
l
l
l
l
l

M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
3003
of 10 mL/min with UV detection at 254 nm. The [11C]celecoxib
retention time was 9.6 min. The desired fraction was collected in
a flask and evaporated to dryness.
PET scanner has a spatial resolution of 1.4 mm FWHM at the center
of the field of view, which is 220 mm in diameter with an axial
extent 78 mm in length. After intravenous bolus injection of
For the subsequent oxidation reaction, [11C]celecoxib was dis-
solved in 0.2 M NaOH, while for the isolation and radiochemical
formulation of [11C]celecoxib for use in the PET study, it was recon-
stituted with a mixture of polysorbate 80, propylene glycol, and
saline (0.1:1:10, v/v/v, 4.0 mL). The total synthesis time from EOB
until formulation was 30 min. The decay-corrected radiochemical
yield was 63 23% (n = 7) based on [11C]CH3I with a specific radio-
[
11C]celecoxib (approximately 26–33 MBq per animal) or [11C]SC-
62807 (approximately 11–20 MBq per animal) via
a venous
catheter inserted into the tail vein, a 60-min emission scan was
performed. The chemical amounts of [11C]celecoxib and [11C]SC-
62807 contained in the bolus injections were calculated to be
0.29–1.3 nmol/body (0.11–0.51
lg/body) and 0.20–1.4 nmol/body
(0.10–0.72 g/body), respectively. Arterial blood was sampled via
l
activity of 83 23 GBq/
11C]celecoxib was confirmed by co-injection with reference
standard celecoxib in analytical HPLC (column: AR-II C18
m (COSMOSIL, Nacalai Tesque); mobile
phase: CH3CN/water (pH 7.4) = 65:35; flow rate: 1 mL/min; UV
detection: 254 nm; retention time: 3.8 min) and it showed the
same retention time peaks on the UV and the radioactive chro-
matograms. Both the chemical purity analyzed at 254 nm and
the radiochemical purity were always greater than 98%.
l
mol (n = 7). The chemical identity of
the cannulated femoral artery at the following time points: 10,
20, 30, 40, and 50 s, and 1, 2, 5, 10, 20, 40, and 60 min after admin-
istration of the radiotracers. Blood radioactivity was measured
using a 1470 WIZARDÒ Automatic Gamma Counter (PerkinElmer,
Waltham, MA, USA). Emission data were acquired in list mode,
and the data were reconstructed with standard 2D filtered back
projection (Ramp filter, cutoff frequency of 0.5 cycles per pixel).
Region of interests (ROIs) were placed on liver, intestine, kidney,
or urinary bladder using image processing software (Pmod
ver.3.0, PMOD Technologies Ltd, Zurich, Switzerland). Regional up-
take of radioactivity in the tissue and blood radioactivity were de-
cay-corrected to the injection time and expressed as % dose/tissue
or % dose/ml blood, normalized for injected radioactivity.
[
,
4.6 mm i.d. ꢃ 100 mm, 5
l
4.1.4. Synthesis of [11C]SC-62807
[
11C]celecoxib in 0.2 M NaOH was mixed with potassium per-
manganate (20 mg). The resulting mixture was heated under micro-
wave irradiation to 140 °C for 5 min. The reaction was quenched
with 30% sodium hydrogen sulfite aqueous solution (400
then the mixture was acidified with 2 M HCl (200 L). After dilution
with CH3OH (600 L), the mixture was injected onto a preparative
lL) and
4.4. Radiometabolite analyzes
l
l
Rats were anesthetized with 1.5% isoflurane before administra-
tion of the radiotracers. After intravenous injection of [11C]cele-
coxib (approximately 26–48 MBq per animal) or [11C]SC-62807
(approximately 10–32 MBq per animal), blood, urine, and liver
samples were taken at 10, 20, and 40 min post-administration.
The liver was removed quickly and then homogenized. To sample
bile, the bile duct was cannulated before administration of the
radiotracer, and bile was collected over the periods 0–10, 10–20,
and 20–40 min post-administration. A two-fold volume of CH3CN
was added to an aliquot of each sample and the resulting mixture
was centrifuged at 12,000 rpm for 2 min at 4 °C. The supernatant
was diluted with HPLC mobile phase and analyzed for intact radio-
tracers and metabolites using a Shimadzu HPLC system coupled to
a NaI(Tl) positron detector UG-SCA30 (Universal Giken, Kanagawa,
HPLC column (AR-II (COSMOSIL), C18, 10 mm i.d. ꢃ 250 mm, 5
lm
with a mobile phase consisting of CH3CN and 0.2% HCOOH (60:40)
and a flow rate of 6 mL/min with UV detection at 254 nm. The
[
11C]SC-62807 retention time was 4.4 min. The desired fraction
was collected in a flask and the organic solvent was removed under
reduced pressure.
For the radiochemical formulation of [11C]SC-62807 for use in
PET analyzes, it was reconstituted with a mixture of polysorbate
80, propylene glycol, and saline (0.1:1:10, v/v/v, 2.0 mL). The total
synthesis time until formulation from [11C]celecoxib was 20 min,
thus, the total synthesis time from EOB was about 50 min. The de-
cay-corrected radiochemical yield was 55 9% (n = 3) with specific
radioactivity of 39 4 GBq/
11C]SC-62807 was confirmed by co-injection with the reference
standard celecoxib in analytical HPLC (column: AR-II, 4.6 mm i.d. ꢃ
100 mm, 5 m; mobile phase: CH3CN and 0.2% HCOOH = 50:50;
lmol (n = 3). The chemical identity of
[
Japan). Chromatographic separation was carried out using
a
4.6 mm i.d. ꢃ 50 mm Waters Atlantis T3 column (Waters, Milford,
MA). The flow was 2.0 mL/min at an initial condition of 80% solvent
A (5% CH3CN in 10 mM CH3COONH4) and 20% solvent B (90%
CH3CN in 10 mM CH3COONH4). Analytes were eluted using the fol-
lowing gradient conditions: 0–0.5 min: 20% solvent B in solvent A;
0.5–2.5 min: 20–100% solvent B in solvent A; 2.5–4 min: 100% sol-
vent B. Following analyte elution, the column was returned to 20%
solvent B in solvent A over 2 min. The elution was monitored by UV
absorbance at 254 nm and coupled with NaI positron detection.
The amount of radioactivity associated with each intact radiotracer
and its metabolite was calculated as a percentage of the total
amount radioactivity.
l
flow rate: 1 mL/min; UV detection: 254 nm) and it showed the same
retention time peaks on the UV and the radioactive chromatograms.
The [11C]SC-62807 retention time was 4.2 min. Both the chemical
purity analyzed at 254 nm and the radiochemical purity were great-
er than 99%.
4.2. Experimental animals
Male Sprague–Dawley (SD) rats weighing 210–290 g
(7–8 weeks old, n = 2 or 3) were purchased from Japan SLC Inc.
(Shizuoka, Japan). The animals were kept in a temperature- and
light-controlled environment and had ad libitum access to stan-
dard food and tap water. All experimental protocols were approved
by the Ethics Committee on Animal Care and Use of the Center for
Molecular Imaging Science in RIKEN, and were performed in accor-
dance with the Principles of Laboratory Animal Care (NIH publica-
tion No. 85-23, revised 1985).
Acknowledgments
This work was supported in part by a consignment expense for
the Molecular Imaging Program on ‘‘Research Base for Exploring
New Drugs’’ from the Ministry of Education, Culture, Sports, Sci-
ence and Technology (MEXT) of Japan. We thank Mr. Masahiro
Kurahashi (Sumitomo Heavy Industry Accelerator Service Ltd) for
operating the cyclotron, Takeshi Ito and Tomohide Handa (Daini-
hon Seiki Co., Ltd) for design and production of the radiolabeling
system, and Satoru Tanabe and Koji Yokota (Biotage, Japan) for
incorporating the microwave apparatus into our system.
4.3. PET studies
Rats were anesthetized with a mixture of 1.5% isoflurane and ni-
trous oxide/oxygen (7:3) and then placed on the PET scanner gan-
try (MicroPET Focus 220, Siemens Co., Ltd, Knoxville, TN, USA). The

3004
M. Takashima-Hirano et al. / Bioorg. Med. Chem. 19 (2011) 2997–3004
15. Doi, H.; Ban, I.; Nonomiya, A.; Sumi, K.; Kuang, C.; Hosoya, T.; Tsukada, H.;
Supplementary data
Suzuki, M. Chem. Eur. J. 2009, 15, 4165.
16. Hostetler, E. D.; Terr, G. E.; Burns, H. D. J. Labelled Compd. Radiopharm. 2005, 48,
629.
17. Hamill, T. J.; Krause, S.; Ryan, C.; Bonnefous, C.; Govek, S.; Seiders, T. J.; Cosford,
N. D. P.; Roppe, J.; Kamenecka, T.; Patel, S.; Gibson, R. E.; Sanabria, S.; Riffel, K.;
Eng, W.; King, C.; Yang, Z.; Green, M. D.; O’Malley, S. S.; Hargreaves, R.; Burns,
H. D. Synapse 2005, 56, 205.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.020.
References and notes
18. Aldous, D. J.; Bower, S.; Moorcroft, N.; Todd, M. Synlett 2001, 150.
19. Morris, G. A.; Nguyen, S. T. Tetrahedron Lett. 2001, 42, 2093.
20. Långström, B.; Itsenko, O.; Rahman, O. J. Labelled Comp. Radiopharm. 2007, 50,
794.
21. Långström, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; Någren, K.;
Rimland, A.; Svärd, H. J. Nucl. Med. 1987, 28, 1037.
22. Ponchant, M.; Hinnen, F.; Demphel, S.; Crouzel, C. Appl. Radiat. Isot. 1997, 48,
755.
23. Ando, K.; Kato, T.; Kawai, A.; Nonomura, T. WO9964415, 1999.
24. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
25. Further details concerning with the yield, purity, etc. have not been reported in
Ref. 6.
1. Shitara, Y.; Sato, H.; Sugiyama, Y. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 689.
2. Maeda, K.; Sugiyama, Y. Drug Metab. Pharmacokinet. 2008, 23, 223.
3. Willmann, J. K.; van Bruggen, N.; Dinkelborg, L. M.; Gambhir, S. S. Nat. Rev. Drug.
Disc. 2008, 7, 591.
4. Ametamey, S. M.; Honer, M.; Schubiger, P. A. Chem. Rev. 2008, 108, 1501.
5. Fowler, J. S.; Wolf, A. P. Acc. Chem. Res. 1997, 30, 181.
6. Miller, P. W.; Long, N. J.; Vilar, R.; Gee, A. D. Angew. Chem., Int. Ed. 2008, 47,
8998.
7. Riemann, B.; Schäfers, K. P.; Schober, O.; Schäfers, M. Q. J. Nucl. Med. Mol.
Imaging 2008, 52, 215.
8. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;
Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
9. Paulson, S. K.; Zhang, J. Y.; Breau, A. P.; Hri bar, J. D.; Liu, N. W. K.; Jessen, S. M.;
Lawal, Y. M.; Cogburn, J. N.; Gresk, C. J.; Markos, C. S.; Maziasa, T. J.;
Schoenhard, G. L.; Burton, E. G. Drug. Metab. Dispos. 2000, 28, 514.
10. Paulson, S. K.; Zhang, J. Y.; Jessen, S. M.; Lawal, Y.; Liu, N. W. K.; Dudkowski, C.
M.; Wang, Y.-F.; Chang, M.; Yang, D.; Findlay, J. W. A.; Berge, M. A.; Markos, C.
S.; Breau, A. P.; Hribar, J. D.; Yuan, J. Xenobitica 2000, 30, 731.
11. Wu, C.; Kusuhara, H.; Sugiyama, Y.; Takashima, T.; Takashima-Hirano, M.; Doi,
H.; Suzuki, M.; Watanabe, Y. Jpn. Phamacol. Ther. 2009, 37, S37.
12. Prabhakaran, J.; Majo, V. J.; Simpson, N. R.; Van Heertum, R. K.; Mann, J. J.;
Kumar, J. S. D. J. Labelled Compd. Radiopharm. 2005, 48, 887.
13. Prabhakaran, J.; Underwood, M. D.; Parsey, R. V.; Arango, V.; Majo, V. J.;
Simpson, N. R.; Heertum, R. V.; Mann, J. J.; Kumar, J. S. D. Bioorg. Med. Chem.
2007, 15, 1802.
26. Decay-corrected yield (DCY) in the oxidation reaction was calculated based on
the radioactivity of
[
[ a part of
11C]celecoxib. In oxidation reaction,
11C]celecoxib which was isolated in the rapid C–[11C]methylation reaction
was used, therefore, DCY based on [11C]CH3I in oxidation reaction was not able
to calculate.
27. HPLC analytical yield indicates the reaction efficiency. The reaction efficient is
calculated by the peak area ratio of the desired [11C]methylated product
obtained by a radio-HPLC analysis of the reaction mixture. See Ref. 32.
28. Långström, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; Nagren, K.;
Rimland, A.; Svard, H. J. Nucl. Med. 1987, 28, 1037.
29. Ghibellini, G.; Leslie, E. M.; Pollack, G. M.; Brouwer, L. R. Pharm. Res. 2008, 25,
1851.
30. Guhlmann, A.; Krauss, K.; Oberdorfer, F.; Siegel, T.; Scheuber, P. H.; Muller, J.;
Csuk-Glanzer, B.; Ziegler, S.; Ostertag, H.; Keppler, D. Hepatology 1995, 21,
1568.
31. Takashima, T.; Nagata, H.; Nakae, T.; Cui, Y.; Wada, Y.; Kitamura, S.; Doi, H.;
Suzuki, M.; Maeda, K.; Kusuhara, H.; Sugiyama, Y.; Watanabe, Y. J. Pharmacol.
Exp. Ther. 2010, 37, 314.
14. Suzuki, M.; Doi, H.; Björkman, M.; Andersson, Y.; Långström, B.; Watanabe, Y.;
Noyori, R. Chem. Eur. J. 1997, 3, 2039.
32. Suzuki, M.; Doi, H. J. Synth. Org. Chem. Jpn. 2010, 68, 1195.