Synthesis and preliminary in vitro biological evaluation of new carbon-11-labeled celecoxib derivatives as candidate PET tracers for imaging of COX-2 expression in cancer

Article abstract of DOI:10.1016/j.ejmech.2011.05.024

The enzyme cyclooxygenase-2 (COX-2) is overexpressed in a variety of malignant tumors. This study was designed to develop new radiotracers for imaging of COX-2 in cancer using biomedical imaging technique positron emission tomography (PET). Carbon-11-labeled celecoxib derivatives, [¹¹C]4a-c and [¹¹C]8a-d, were prepared by O-[¹¹C] methylation of their corresponding precursors using [¹¹C]CH₃OTf under basic conditions and isolated by a simplified solid-phase extraction (SPE) method in 52 ± 2% (n = 5) and 57 ± 3% (n = 5) radiochemical yields based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 277.5 ± 92.5 GBq/μmol (n = 5). The IC₅₀ values to block COX-2 for known compounds celecoxib (4d), 4a and 4c were 40, 290 and 8 nM, respectively, and preliminary findings from in vitro biological assay indicated that the synthesized new compounds 4b and 8a-d display similar strong inhibitory effectiveness in the MDA-MB-435 human cancer cell line in comparison with the parent compound 4d. These results encourage further in vivo evaluation of carbon-11-labeled celecoxib derivatives as new potential PET radiotracers for imaging of COX-2 expression in cancer.

Full text of DOI:10.1016/j.ejmech.2011.05.024

European Journal of Medicinal Chemistry 46 (2011) 4760e4767
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and preliminary in vitro biological evaluation of new carbon-11-labeled
celecoxib derivatives as candidate PET tracers for imaging of COX-2 expression
in cancer
,
Mingzhang Gao a, Min Wang a, Kathy D. Miller b, Qi-Huang Zheng a
*
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-208, Indianapolis, IN 46202, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The enzyme cyclooxygenase-2 (COX-2) is overexpressed in a variety of malignant tumors. This study was
designed to develop new radiotracers for imaging of COX-2 in cancer using biomedical imaging technique
positron emission tomography (PET). Carbon-11-labeled celecoxib derivatives, [11C]4aec and [11C]8aed,
were prepared by O-[11C] methylation of their corresponding precursors using [11C]CH3OTf under basic
conditions and isolated by a simplified solid-phase extraction (SPE) method in 52 ꢀ 2% (n ¼ 5) and 57 ꢀ 3%
(n ¼ 5) radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB). The
overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at
Received 20 February 2011
Received in revised form
2 May 2011
Accepted 10 May 2011
Available online 20 May 2011
Keywords:
end of synthesis (EOS) was 277.5 ꢀ 92.5 GBq/ mol (n ¼ 5). The IC50 values to block COX-2 for known
m
Carbon-11-labeled celecoxib derivatives
Positron emission tomography (PET)
Cyclooxygenase-2 (COX-2)
Radiotracers
compounds celecoxib (4d), 4a and 4c were 40, 290 and 8 nM, respectively, and preliminary findings from
in vitro biological assay indicated that the synthesized new compounds 4b and 8aed display similar strong
inhibitory effectiveness in the MDA-MB-435 human cancer cell line in comparison with the parent
compound 4d. These results encourage further in vivo evaluation of carbon-11-labeled celecoxib deriva-
tives as new potential PET radiotracers for imaging of COX-2 expression in cancer.
Cancer imaging
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Pfizer. COX-2 represents an attractive target for the development of
therapeutic agents. However, it is important to note the significant
Cyclooxygenase (prostaglandin endoperoxide synthase or COX)
is the key enzyme involved in the biosynthesis of prostaglandin,
prostacyclin and thromboxane from arachidonic acid [1,2]. COX
contains three isoforms: COX-1, COX-2 and COX-3 [3]. COX-1 is
present in healthy tissues and responsible for the thrombogensis
and the homeostasis. COX-2 is almost undetectable under physio-
logic conditions and mainly induced by inflammatory stimuli.
COX-3 is located in the central nervous system and could be the
pharmacological target of acetaminophen. COX-2 plays an impor-
tant role in a variety of diseases or disease processes including
cancer, cardiovascular and neuroscience diseases [4e8]. The
discovery of the COX-2 enzyme in the early 1990s has resulted in
the development of COX-2 selective inhibitors [9]. Great efforts
have been made in the structureeactivity relationship (SAR)
studies [10]. The representative COX-2 inhibitor is celecoxib. It is
asulfa non-steroidal anti-inflammatory drug (NSAID) marketed by
gastrointestinal side effects of some COX-2 selective inhibitors [11].
For example, rofecoxib (Vioxx) has been withdrawn from the
market due to its increased risk of serious cardiovascular diseases.
To improve the therapy with fewer side effects, recently a novel
series of celecoxib derivatives have been designed and synthesized
as more potent anti-inflammatory agents [12e14]. COX-2 also
provides a particularly attractive target for the development of
imaging agents, since in vivo imaging of COX-2 expression would be
useful for early detection and for monitoring the progression and
treatment efficacy for the diseases in oncology, cardiology and
neurology [15]. In this regard, several COX-2 inhibitors such as
celecoxib and rofecoxib have been radiolabeled by carbon-11,
fluorine-18, and iodine-123/125 and evaluated as potential
tracers for biomedical imaging techniques positron emission
tomography (PET) and single photon emission computed tomog-
raphy (SPECT) [16e22]. However, there is still a requirement for
continued in vivo investigation using superior agents to establish
the clinical utility of this strategy [10]. In addition, most of these
COX-2 radioligands are labeled with SPECT isotope I-123/125 and
PET isotope F-18. Recent efforts have turned to develop selective
* Corresponding author. Tel.: þ1 317 278 4671; fax: þ1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.024

M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
4761
COX-2 radioligands labeled with another positron emitting radio-
nuclide C-11, because some of F-18 radioligands resulted in de[18F]
fluorination in vivo [20]. We are interested in the development
of enzyme-based and/or receptor-based PETcancer imaging agents.
To radiolabel therapeutic agents as diagnostic agents for
noninvasive imaging of cancer and monitoring of therapeutic effi-
cacy, we report the design, synthesis, labeling and preliminary
in vitro biological evaluation of a series of celecoxib derivatives
with nanomolar IC50 values, 4-(5-(2-[11C]methoxyphenyl)-3-(tri-
yield. If free base 3 was used instead of 3ꢁHCl salt, additional HCl (1.4
equivalent to 3) was needed, since HCl was necessary for the
regioselective transformation reaction. Both 3 and 3ꢁHCl were
commercially available, and the yield of 4aed was not significantly
different by using either 3 or 3ꢁHCl as the starting material. The
standard compounds 4aec were des-methylated using sodium
ethanethiolate (EtSNa) to afford des-methyl precursors 5aec in
85 ꢀ 5% (n ¼ 3) yield.
Synthesis of celecoxib derivatives, methyl 3-(trifluoromethyl)-
1H-pyrazol-1-yl)benzoates 8aed and their corresponding acid
precursors 7aed, is outlined in Scheme 2 using a modification of
the previously reported procedures [14,22e24]. Diketone 2aed
was converted into 3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoic
acid precursors 7aed in 83 ꢀ 3% (n ¼ 3) yield through the regio-
selective transformation reaction with 4-hydrazinylbenzoic acid (6)
in similar method used to prepare compound 4aed. Then the
methylation of precursors 7aed with SOCl2 in methanol yielded the
standard compounds 8aed in 90 ꢀ 5% (n ¼ 3) yield.
fluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
([11C]4a),
4-(5-(3-[11C]methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl)benzenesulfonamide ([11C]4b), 4-(5-(4-[11C]methoxyphenyl)-3-
(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide ([11C]4c),
[
11C]methyl 4-(5-(2-methoxyphenyl)-3-(trifluoromethyl)-1H-pyr-
azol-1-yl)benzoate ([11C]8a), [11C]methyl 4-(5-(3-methoxyphenyl)-
3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoate ([11C]8b), [11C]me-
thyl 4-(5-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzoate ([11C]8c), and
[
11C]methyl 4-(5-(p-tolyl)-3-(trifluoro-
methyl)-1H-pyrazol-1-yl)benzoate ([11C]8d), as new candidate PET
tracers for imaging of COX-2 expression in cancer.
2.2. Radiochemistry
Synthesis of carbon-11-labeled celecoxib derivatives, [11C]4aec
and [11C]8aed, is indicated in Scheme 3. Phenolic hydroxyl precur-
sors 5aec and acid precursors 7aed were labeled by a reactive [11C]
methylating agent, [11C]methyl triflate ([11C]CH3OTf) [27,28] pre-
pared from [11C]CO2, under basic conditions (2 N NaOH) in acetoni-
trile through the O-[11C]methylation and isolated by a simplified
solid-phase extraction (SPE) method [22,29e31] to provide target
tracers[11C]4aec and[11C]8aed in 52ꢀ 2%(n ¼ 5)and57 ꢀ3%(n ¼ 5)
radiochemical yields, respectively, decay corrected to end of
bombardment (EOB), based on [11C]CO2. The O-[11C]methylation of
acid precursors 7aed gave slightly higher decay-corrected radio-
chemical yields of [11C]8aed than that of [11C]4aec through the O-
2. Results and discussion
2.1. Chemistry
The synthetic strategy is that celecoxib was used as a model
compound for the design of a novel series of celecoxib derivatives
[14,22e24]. Celecoxib contains a fairly rigid core structure with three
apparent interaction points: R1, R2, and R3, as indicated in Fig. 1. We
designed new celecoxib derivatives with slight chemical modifica-
tions in the R1 and R3 positions and without any change in the R2
position. These newly designed target compounds were expected to
be potent COX-2 inhibitors with comparable IC50 values to their
parent compound celecoxib and improved metabolic properties,
favorable lipophilicity and O-methyl position amenable to labeling
with carbon-11 [22]. Moreover, we and other group have reported
a methyl ester prodrug of matrix metalloproteinase (MMP) inhibitor
has better uptake in tumor than its acid parent drug [25,26].
Synthesis of celecoxib (4d) and its derivatives, benzenesulfona-
mide compounds 4aec and their corresponding phenolic hydroxyl
precursors 5aec, is shown in Scheme 1 using a modification of the
previously reported procedures [14,22e24]. Claisen condensation of
acetophenone 1aed with trifluoroacetic acid ethyl ester gave
diketone 2aed in 92 ꢀ 2% (n ¼ 3) yield using an improved method
which employed base NaH to replace base NaOMe [24]. Diketone
2aed were then reacted with 4-hydrazinylbenzenesulfonamide (3)
hydrochloride in the solvent ethanol through a regioselective
transformation reaction to give compounds 4aed in 85 ꢀ 5% (n ¼ 3)
[
11C]methylation of phenolic hydroxyl precursors 5aec, because acid
precursors were easier for O-[11C]methylation than phenolic
hydroxyl precursors. The large polarity difference between the
phenolic hydroxyl or acid precursor and the corresponding labeled
O-methylated ether or ester product permitted the use of SPE tech-
nique for purification of the labeled product from the radiolabeling
reaction mixture. Either a C-18 Plus Sep-Pak cartridge or a semi-prep
C-18 guard cartridge column was used in SPE purification technique.
No different results were found by using either Sep-Pak cartridge or
guard cartridge column, because they follow the same principal of
the purification. The only difference is that a guard column is much
expensive than a Sep-Pak cartridge, thus, a cartridge is disposable
(single use) and a column needs repeat use. For tracer development,
both column and cartridge can be used for the same function.
However, for tracer production for PET biological evaluation in
animals and humans, we recommend the use of Sep-Pak cartridge to
avoid potential contaminations in the tracer solution due to the
repeat use of a guard column. The crude reaction mixturewas treated
with sodium bicarbonate and loaded onto the cartridge by gas
pressure. Any non-reacted phenolic hydroxyl or acid precursor was
actually converted to the corresponding sodium salt, and any non-
reacted [11C]CH3OTf was actually hydrolyzed to [11C]CH3OH, which
would not stick to the C-18 Sep-Pak. The cartridge was washed with
water to remove non-reacted [11C]CH3OTf, phenolic hydroxyl or acid
precursor and reaction solvent, and then the final labeled product
was eluted with ethanol. Overall synthesis time was 23 min from
EOB, including approximately 11 min for [11C]CH3OTf production,
5 min for O-[11C]methylation reaction, and 7 min for SPE purification,
evaporation and reformulation. SPE technique is fast, efficient and
convenient and works very well for the O-methylated ether or ester
tracer purification using the phenolic hydroxyl or acid precursor for
radiolabeling [22,29e31]. The radiosynthesis was performed in an
O
H2N
R3
S
O
N
CF3 R2
N
R1
H3C
Celecoxib
Fig. 1. COX-2 inhibitor celecoxib.

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M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
NHNH2
O
O
CF3CO2Et, NaH
THF, 0 oC
EtOH/HCl
reflux
O
H2NO2S
CF3
3
R
or 3 HCl, EtOH, reflux
R
2a, R=2-OMe
2b, R=3-OMe
2c, R=4-OMe
2d, R=4-Me
92 2%
1a, R=2-OMe
1b, R=3-OMe
1c, R=4-OMe
1d, R=4-Me
H2NO2S
H2NO2S
EtSNa, DMF, reflux
N
N
N
N
R
X
CF3
CF3
4a, R=2-OMe
4b, R=3-OMe
4c, R=4-OMe
4d, R=4-Me (Celecoxib)
85 5%
5a, X=2-OH
5b, X=3-OH
5c, X=4-OH
85 5%
Scheme 1. Synthesis of celecoxib (4d) and its derivatives 4aec and 5aec.
automated self-designed multi-purpose 11C-radiosynthesis module,
allowing measurement of specific activity at EOB during synthesis
[32,33]. On line determination of specific activity at EOB is accurate
when HPLC is used as purification method. However, the on-the-fly
technique to determine specific activity at EOB is not applied when
SPE is used as purification method. The specific activity for all 11C-
tracers produced in our PET radiochemistry facility is depended on
two parts: 1) carrier from the 11C gas target, and 2) carrier from the
11C radiolabeled precursor [11C]CH3OTf production system. The 11C
gas target we used is the Siemens RDS-111 Eclipse cyclotron 11C gas
target. The [11C]CH3OTf production system we used is an Eckert &
Ziegler Modular Lab C-11 Methyl Iodide/Triflate module [27]. The
specific activity for our 11C-tracers usually ranges from 370 to 925
determined by analytical HPLC [35]. The chemical purity of the
precursors and reference standards was >97%. The radiochemical
purity of the target tracers was >99% determined by radio-HPLC
through g-ray (PIN diode) flow detector, and the chemical purity of
the target tracers was >95% for [11C]4aec and >96% for [11C]8aed
determined by reversed-phase HPLC through UV flow detector.
Chemical purity of the labeled ester products was slightly higher
than thatof the labeled ether products, because it was easier to purify
the ester from its acid precursor than the ether from its phenolic
hydroxyl precursor through SPE. A representative analytical HPLC
chromatographic profile for [11C]4a is shown in Fig. 2. The retention
time of [11C]4a (tR [11C]4a) in analytical radioactive HPLC (Fig. 2A)
and analytical UV HPLC (Fig. 2B) is 4.48 and 4.33 min, respectively.
GBq/
activity of carbon-11-labeled celecoxib derivatives was estimated in
a range of 185e370 GBq/ mol at the end of synthesis (EOS) based on
mmol at EOB according to our previous works. The specific
2.3. MDA-MB-435 cell proliferation assay
m
other compounds produced in our facility using the same targetry
conditions which have been measured by the on-the-fly technique or
the same SPE purification method [33,34]. The actual measurement
of specific activity at EOS was performed by analytical HPLC [35] and
Compounds 4aec and 8aed were designed and synthesized
through a slight structural modification based on its parent
compound celecoxib (4d) [14,22e24]. The IC50 values against COX-
2 for known compounds 4d, 4a and 4c were 40, 290 and 8 nM,
respectively [23]. Compound 8d is not a new compound, but its
biological activity is not evaluated [36]. Thus, we can assume the
slight structural modification of compounds 4b and 8aed would
calculated. The exact values were 277.5 ꢀ 92.5 GBq/ mol (n ¼ 5),
m
which are in agreement with the estimated values and the “on line”
determined values. Chemical purity and radiochemical purity were
H3COOC
HOOC
NHNH2
SOCl2, MeOH
0 oC to RT
EtOH/HCl
reflux
2a
2b
2c
2d
HOOC
N
6
N
N
N
R
R
CF3
CF3
8a, R=2-OMe
7a, R=2-OMe
7b, R=3-OMe
7c, R=4-OMe
7d, R=4-Me
83 3%
8b, R=3-OMe
8c, R=4-OMe
8d, R=4-Me
90 5%
Scheme 2. Synthesis of celecoxib derivatives 7aed and 8aed.

M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
4763
(p ¼ 0.377) of celecoxib (Fig. 3A). MDA-MB-435 cell proliferation
was inhibited by 160 nM (p ¼ 0.147) of compound 4b (Fig. 3B),
160e320 nM (p* ¼ 0.015) of compound 8a (Fig. 3C), 640 nM
(p ¼ 0.238) of compound 8b (Fig. 3D), 160 nM (p* ¼ 0.014) of
compound 8c (Fig. 3E), and 320 nM (p ¼ 0.152) of compound 8d
(Fig. 3F), respectively. There was a statistically significant difference
in Fig. 3C and E, since p* <0.05. There was not a statistically
significant difference in Fig. 3A, B, Fig. 3D and F, since p > 0.05.
Taken collectively, these results suggest that the new compounds
4b and 8aed were proved to be potent COX-2 inhibitors, and dis-
played similar strong inhibitory effectiveness in the MDA-MB-435
human cancer cell line in comparison with the parent compound
4d, albeit not as effective as 4d, under our assay conditions.
H2NO2S
[11C]CH3OTf
CH3CN, 2 N NaOH
N
5a
5b
5c
N
R
CF3
[11C]4a, R=2-O11CH3
[11C]4b, R=3-O11CH3
[11C]4c, R=4-O11CH3
52 2%
H311COOC
3. Conclusions
An efficient and convenient synthesis of new radiotracers,
carbon-11-labeled celecoxib derivatives, has been developed. The
synthetic methodology employed multiple step organic reactions
including Claisen condensation, regioselective transformation,
desmethylation and methylation to prepare a series of new cele-
coxib derivatives, phenolic hydroxyl or acid precursors, and their
corresponding ether or ester standard compounds. The target
tracers were prepared by O-[11C]methylation of their correspond-
ing precursors using a reactive [11C]methylating agent, [11C]CH3OTf,
and isolated by a simplified SPE purification procedure in high
radiochemical yields, short overall synthesis time, and high specific
radioactivities. Cell proliferation assay indicated that the synthe-
sized new compounds 4b and 8aed display similar strong inhibi-
tory effectiveness in the MDA-MB-435 human cancer cell line in
comparison with the parent compound celecoxib. These chemistry
and biology results combined with the reported in vitro and in vivo
biological data [16e22] encourage further in vivo biological eval-
uation of new carbon-11-labeled celecoxib derivatives, as candidate
PET tracers to image COX-2 enzyme in cancer.
[11C]CH3OTf
CH3CN, 2 N NaOH
7a
7b
7c
7d
N
N
R
CF3
[11C]8a, R=2-OMe
[11C]8b, R=3-OMe
[11C]8c, R=4-OMe
[11C]8d, R=4-Me
57 3%
Scheme 3. Synthesis of carbon-11-labeled celecoxib derivatives [11C]4aec and [11C]
8aed.
not significantly change their biological activity in light of the SAR
[23,24]. The preliminary in vitro biological evaluation of the
synthesized new compounds 4b and 8aed were performed via an
MDA-MB-435 cell proliferation assay [37,38] and compared to
celecoxib. MDA-MB-435 was thought to be breast cancer cells in
the past but now is believed to be more closely related to mela-
noma [37]. The results (mean ꢀ SD) for blank, cell control, DMSO
control, and 20e1280 nM of reference compound celecoxib, and
synthesized compounds 4b and 8aed are summarized in Fig. 3.
Error bars show the standard deviation (SD). As indicated in Fig. 3,
proliferation of MDA-MB-435 cells was inhibited by 40 nM
4. Materials and methods
4.1. General
All commercial reagents and solvents were purchased from
SigmaeAldrich and Fisher, and used without further purification.
[
11C]CH3OTf was prepared according to a literature procedure
A
300.00
200.00
100.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00 11.00 12.00
Minutes
B0.050
0.040
0.030
0.020
0.010
0.000
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00 11.00 12.00
Minutes
Fig. 2. A representative analytical HPLC chromatographic profile for the tracer [11C]4a. (A). Analytical radioactive HPLC trace for [11C]4a, tR [11C]4a ¼ 4.48 min (B). Analytical UV
HPLC trace for [11C]4a, tR [11C]4a ¼ 4.33 min.

4764
M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
A
D
B
E
C
F
Fig. 3. MDA-MB-435 cell proliferation assay of celecoxib (A), and new celecoxib derivatives 4b (B) and 8aed (CeF). The X-axis represents blank, cell control, DMSO control and
different concentrations (20e1280 nM) of celecoxib, 4b and 8aed, and the Y-axis represents their average inhibition intensity (mean ꢀ SD) in MDA-MB-435 cells measured under
optical density (OD) 490 nm, which error bars show standard deviation. Statistical analyses indicate p values for celecoxib (A), 4b (B) and 8aed (CeF) are 0.377, 0.147, 0.015, 0.238,
0.014 and 0.152, respectively. Celltiter 96 assay demonstrated that new celecoxib derivatives 4b and 8aed have similar potencies in the MDA-MB-435 human cancer cell line in
comparison with the parent compound celecoxib, albeit not as effective as celecoxib.
[27]. Melting points were determined on a MEL-TEMP II capillary
tube apparatus and were uncorrected. 1H NMR and 13C NMR
spectra were recorded on Varian Gemini 2000 200 MHz FT-NMR
and Bruker Avance II 500 MHz NMR spectrometers using tetra-
methylsilane (TMS) as an internal standard. Chemical shift data
for the proton resonances were reported in parts per million
uniplates (5 ꢂ 10 cm2). Plates were visualized under UV light.
Normal phase flash column chromatography was carried out on
EM Science silica gel 60 (230e400 mesh) with a forced flow of the
indicated solvent system in the proportions described below. All
moisture- and/or airesensitive reactions were performed under
a positive pressure of nitrogen maintained by a direct line from
a nitrogen source. Analytical HPLC was performed using a Prodigy
(ppm,
d scale) relative to internal standard TMS (d 0.0), and
coupling constants (J) were reported in hertz (Hz). Liquid
chromatography-mass spectra (LC-MS) analysis was performed
on an Agilent system, consisting of an 1100 series HPLC connected
to a diode array detector and a 1946D mass spectrometer
configured for positive-ion/negative-ion electrospray ionization.
The high resolution mass spectra (HRMS) were obtained using
a Waters/Micromass LCT Classic spectrometer. Chromatographic
solvent proportions are indicated as volume: volume ratio. Thin-
layer chromatography (TLC) was run using Analtech silica gel GF
(Phenomenex)
5
m
m
C-18 column, 4.6 ꢂ 250 mm2; 3:1:1
CH3CN:MeOH:20 mM, pH 6.7 phosphate (buffer solution) mobile
phase; flow rate 1.5 mL/min; and UV (254 nm) and
g-ray (PIN
diode) flow detectors. C-18 Plus Sep-Pak cartridges were obtained
from Waters Corporate Headquarters, Milford, MA. Semi-prep
C-18 guard cartridge column 1 ꢂ 1 cm, 10
E. S. Industries, Berlin, NJ, and part number 300121-C18-BD.
Sterile Millex-GS 0.22 m vented filter unit was obtained from
Millipore Corporation, Bedford, MA.
mm was obtained from
m

M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
4765
4.2. General procedure for preparation of 4,4,4-trifluoro-1-
(substituted phenyl)butane-1,3-dione (2)
d
3.83 (s, 3H, OCH3), 6.76 (s, 2H, NH2), 6.95 (s, 1H, CH¼), 6.97 (dt,
J ¼ 2.5, 8.5 Hz, 2H, Ph-H), 7.38 (dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H), 7.55 (dt,
J ¼ 2.5, 8.5 Hz, 2H, Ph-H), 7.95 (dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H). MS
(ESI): 398 ([M þ H]þ, 100%).
Substituted acetophenone 1 (149 mmol) was dissolved in dry
THF (300 mL) under nitrogen atmosphere and NaH (7.15 g,
298 mmol) was added in portions maintaining the temperature
between ꢃ5 and 0 ꢄC. After stirring at this temperature for 30 min,
ethyl trifluoroacetate (31.75 g, 224 mmol) was added and the
reaction mixture was allowed to stir at room temperature for 6 h.
The reaction mixture was evaporated, added with ice-water, acid-
ified with HCl (1 N) to pH 6, and extracted with EtOAc (3 ꢂ 100 mL).
The combined organic layer was washed with water (50 mL), dried
over MgSO4, and concentrated. The solid residue was washed with
hexane and dried under high vacuum to provide a solid mass,
which was re-dissolved in CH2Cl2, and dried under high vacuum to
give a white solid diketone 2 in 92 ꢀ 2% (n ¼ 3) yield, Rf ¼ 0.22e0.25
(25% EtOAc/Hexanes).
4-(5-(p-Tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzene-
sulfonamide (celecoxib, 4d), mp 157e158 ꢄC 1H NMR (acetone-d6):
d
2.38 (s, 3H, CH3), 4.92 (s, 2H, NH2), 6.74 (s, 1H, CH¼), 7.10 (d,
J ¼ 8.0 Hz, 2H, Ph-H), 7.17 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.46 (dt, J ¼ 2.0,
8.0 Hz, 2H, Ph-H), 7.89 (dt, J ¼ 2.0, 8.0 Hz, 2H, Ph-H), 8.87 (s, 1H,
OH). MS (ESI): 382 ([M þ H]þ, 100%).
4.4. General procedure for preparation of hydroxyphenyl-3-(trifluo-
romethyl)-1H-pyrazol-1-yl)benzenesulfonamide (5)
To a solution of 4 (1.5 mmol) in anhydrous DMF (10 mL) was
added EtSNa (630 mg, 9.0 mmol), and the mixture was heated at
150 ꢄC for 3 h. Then the reaction mixture was evaporated in vacuo,
added EtOAc, washed with NH4Cl solution and water, dried over
Na2SO4, and concentrated. The residue was purified by column
chromatography on silica gel (12% MeOH/CH2Cl2) to afford a white
solid 5 in 85 ꢀ 5% (n ¼ 3) yield, Rf ¼ 0.67e0.70 (10% MeOH/CH2Cl2).
4-(5-(2-Hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (5a), mp 201e203 ꢄC 1H NMR (acetone-d6):
4,4,4-Trifluoro-1-(2-methoxyphenyl)butane-1,3-dione (2a), mp
36e38 ꢄC 1H NMR (CDCl3):
d 3.94 (s, 3H, OCH3), 6.97 (s, 1H, CH]),
6.99 (t, J ¼ 7.5 Hz, 1H, Ph-H), 7.07 (t, J ¼ 7.5 Hz, 1H, Ph-H), 7.53 (dt,
J ¼ 2.0, 7.5 Hz, 1H, Ph-H), 7.96 (dd, J ¼ 2.0, 7.5 Hz, 1H, Ph-H), 15.2 (s,
1H, OH). MS (ESI): 247 ([M þ H]þ, 100%).
4,4,4-Trifluoro-1-(3-methoxyphenyl)butane-1,3-dione (2b), mp
32e34 ꢄC 1H NMR (CDCl3):
d
6.56 (s, 1H, CH]), 7.15 (ddd, J ¼ 1.0,
d
6.72 (s, 2H, NH2), 6.93e6.96 (m, 3H, CH¼ and Ph-H), 7.31e7.33 (m,
2.5, 8.0 Hz, 1H, Ph-H), 7.43 (t, J ¼ 8.0 Hz, 1H, Ph-H), 7.46 (t, J ¼ 2.0 hz,
7.50 (dd, J ¼ 1.0, 7.5 Hz, 1H, Ph-H), 15.1 (s, 1H, OH). MS (ESI): 247
([M þ H]þ, 100%).
2H, Ph-H), 7.53 (dd, J ¼ 2.0, 7.0 Hz, 2H, Ph-H), 7.87 (dd, J ¼ 2.0,
7.0 Hz, 2H, Ph-H). MS (ESI): 384 ([M þ H]þ, 100%). HRMS (ESI): calcd
for C16H13N3O3F3S 384.0630 ([M þ H]þ), found 384.0620.
4,4,4-Trifluoro-1-(4-methoxyphenyl)butane-1,3-dione (2c), mp
4-(5-(3-Hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (5b), mp 131e133 ꢄC 1H NMR (acetone-d6):
48e49 ꢄC 1H NMR (CDCl3):
d 3.90 (s, 3H, OCH3), 6.50 (s, 1H, CH]),
6.96 (d, J ¼ 8.8 Hz, 2H, Ph-H), 7.91 (d, J ¼ 9.0 Hz, 2H, Ph-H). MS (ESI):
d
6.76 (s, 2H, NH2), 6.79e6.83 (m, 2H, Ph-H), 6.90e6.92 (m, 1H, Ph-
247 ([M þ H]þ, 100%).
H), 6.99 (s, 1H, CH¼), 7.24 (t, J ¼ 8.0 Hz, 1H, Ph-H), 7.56 (dt, J ¼ 2.0,
8.5 Hz, 2H, Ph-H), 7.95 (dt, J ¼ 2.0, 8.5 Hz, 2H, Ph-H), 8.79 (s, 1H,
OH). MS (ESI): 384 ([M þ H]þ, 100%). HRMS (ESI): calcd for
C16H13N3O3F3S 384.0630 ([M þ H]þ), found 384.0614.
4,4,4-Trifluoro-1-(4-methylphenyl)butane-1,3-dione (2d), mp
40e42 ꢄC 1H NMR (CDCl3):
d
2.44 (s, 3H, CH3), 6.54 (s, 1H, CH ¼ ),
7.28 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.83 (d, J ¼ 8.0 Hz, 2H, Ph-H). MS (EI):
229 ([M ꢃ H]ꢃ, 91%), 159 (100%).
4-(5-(4-Hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (5c), mp 209e211 ꢄC 1H NMR (acetone-d6):
4.3. General procedure for preparation of 3-(trifluoromethyl)-1H-
pyrazol-1-yl)benzenesulfonamide (4)
d
6.76 (s, 2H, NH2), 6.88 (dd, J ¼ 3.0, 8.0 Hz, 2H, Ph-H), 6.92 (s, 1H,
CH¼), 7.17 (dt, J ¼ 2.5, 8.5 Hz, 2H, Ph-H), 7.54 (dt, J ¼ 2.5, 8.5 Hz, 2H,
Ph-H), 7.94 (dt, J ¼ 2.5, 8.5 Hz, 2H, Ph-H), 8.96 (s, 1H, OH). MS (ESI):
384 ([M þ H]þ, 100%).
4-Hydrazinylbenzenesulfonamide hydrochloride (3ꢁHCl) (982 mg,
4.4 mmol) was added to the stirred solution of diketone2(4.0 mmol) in
EtOH (60 mL). If free base 3 was used to replace 3ꢁHCl, additional HCl
(3 N, 2.0 mL) is needed. The mixture was heated to refluxfor 20h. Then
the reaction mixturewas concentrated invacuo. The residuewasadded
EtOAc, washed with water, dried, filtered, and concentrated. The crude
product was purified by column chromatography on silica gel (12%
MeOH/CH2Cl2) to give a white solid 4 in 85 ꢀ 5% (n ¼ 3) yield,
Rf ¼ 0.78e0.82 (10% MeOH/CH2Cl2).
4.5. General procedure for preparation of 3-(trifluoromethyl)-1H-
pyrazol-1-yl)benzoic acid (7)
Diketone
2 (20 mmol) was added into a solution of
4-hydrazinylbenzoic acid (6) (3.35 g, 22 mmol) in EtOH (250 mL)
and HCl (12 mL, 2 N). The mixture was heated to reflux and stirred
for 20 h. Then the reaction mixture was evaporated under reduced
pressure, extracted by EtOAc (3 ꢂ 150 mL), washed with brine,
dried, filtered, and evaporated to dryness. The residue was purified
by column chromatography on silica gel (5% MeOH/CH2Cl2) to
afford a white solid 7 in 83 ꢀ 3% (n ¼ 3) yield, Rf ¼ 0.25e0.30 (6%
MeOH/CH2Cl2).
4-(5-(2-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (4a), mp 170e172 ꢄC 1H NMR (acetone-d6):
d
3.40 (s, 3H, OCH3), 6.71 (s, 1H, CH¼), 6.92 (s, 2H, NH2), 6.99 (d,
J¼ 8.0Hz,1H, Ph-H),7.08(t,J¼ 7.5Hz,1H, Ph-H), 7.42e7.50(m, 4H, Ph-
H), 7.90 (dd, J ¼ 2.0, 7.5 Hz, 2H, Ph-H). MS (ESI): 398 ([M þ H]þ,100%).
4-(5-(3-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (4b), mp 155e157 ꢄC 1H NMR (acetone-d6):
4-(5-(2-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
d
3.72 (s, 3H, OCH3), 6.77 (s, 2H, NH2), 6.89 (dd, J ¼ 1.0, 2.0 Hz, 1H,
benzoic acid (7a), mp 205e207 ꢄC 1H NMR (acetone-d6):
d 3.40 (s,
Ph-H), 6.90 (t, J ¼ 2.0 Hz, 1H, Ph-H), 7.01 (dt, J ¼ 1.0, 8.0 Hz, 1H, Ph-
H), 7.04 (s, 1H, CH¼), 7.32 (t, J ¼ 8.0 Hz, 1H, Ph-H), 7.57 (dd, J ¼ 2.0,
7.0 Hz, 2H, Ph-H), 7.96 (dd, J ¼ 2.0, 7.0 Hz, 2H, Ph-H). 13C NMR
3H, CH3O), 6.91 (s, 1H, CH¼), 6.98 (d, J ¼ 8.5 Hz, 1H, Ph-H), 7.08 (t,
J ¼ 7.5 Hz, 1H, Ph-H), 7.42e7.48 (m, 4H, Ph-H), 8.0 (d, J ¼ 8.5 Hz, 2H,
Ph-H). MS (ESI): 377 ([M þ H]þ, 100%). HRMS (ESI): calcd for
C19H16N2O3F3 377.1113 ([M þ H]þ), found 377.1110.
(dmso-d6):
d
55.14, 106.45, 114.43, 115.11, 120.22 (q, J ¼ 267 Hz),
121.07, 126.08, 126.79, 129.42, 130.01, 141.08, 142.02 (q, J ¼ 38 Hz),
144.12, 145.04, 159.21. MS (ESI): 398 ([M þ H]þ, 100%). HRMS (ESI):
calcd for C17H15N3O3F3S 398.0786 ([M þ H]þ), found 398.0768.
4-(5-(4-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (4c), mp 156e158 ꢄC 1H NMR (acetone-d6):
4-(5-(3-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzoic acid (7b), mp 141e143 ꢄC 1H NMR (acetone-d6):
d 3.73 (s,
3H, CH3O), 6.87 (d, J ¼ 1.0, 7.0 Hz, 1H, Ph-H), 6.94 (t, J ¼ 2.0 Hz, 1H,
Ph-H), 6.98 (ddd, J ¼ 0.5, 2.5, 8.5 Hz, 1H, Ph-H), 7.04 (s, 1H, CH]),
7.31 (t, J ¼ 8.0 Hz, 1H, Ph-H), 7.51 (dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H), 8.09

4766
M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
(dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H). MS (ESI): 377 ([M þ H]þ, 100%). HRMS
(ESI): calcd for C19H16N2O3F3 377.1113 ([M þ H]þ), found 377.1102.
4-(5-(4-Methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
Siemens RDS-111 Eclipse cyclotron. The target gas consisted of 1%
oxygen in nitrogen purchased as a specialty gas from Praxair,
Indianapolis, IN. Typical irradiations used for the development
benzoic acid (7c), mp 206e208 ꢄC 1H NMR (acetone-d6):
d
3.82 (s,
were 50 mA beam current and 15 min on target. The production run
3H, CH3O), 6.95 (s, 1H, CH]), 6.97 (d, J ¼ 8.5 Hz, 2H, Ph-H), 7.27 (d,
J ¼ 9.0 Hz, 2H, Ph-H), 7.49 (d, J ¼ 8.5 Hz, 2H, Ph-H), 8.08 (d,
J ¼ 9.0 Hz, 2H, Ph-H). MS (ESI): 377 ([M þ H]þ, 100%). HRMS (ESI):
calcd for C19H16N2O3F3 377.1113 ([M þ H]þ), found 377.1111.
4-(5-(p-Tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoic acid
produced approximately 25.9 GBq of [11C]CO2 at EOB. The phenolic
hydroxyl or acid precursor 5a, 5b, 5c, 7a, 7b, 7c, or 7d (0.1e0.3 mg)
was dissolved in CH3CN (300
mL). To this solution was added 2 N
NaOH (2 L). The mixture was transferred to a small reaction vial.
m
No-carrier-added (high specific activity) [11C]CH3OTf (13.9 GBq)
that was produced by the gas-phase production method [27]
within 11 min from [11C]CO2 (25.9 GBq) through [11C]CH4 (21.8
GBq) and [11C]CH3Br (13.9 GBq) with silver triflate (AgOTf) column
was passed into the reaction vial at room temperature until
radioactivity reached a maximum (2 min), and then the reaction
vial was isolated and heated at 80 ꢄC for 3 min. The contents of the
reaction vial were diluted with NaHCO3 (0.1 M, 1 mL). The reaction
vial was connected to either a C-18 Plus Sep-Pak cartridge or
a semi-prep C-18 guard cartridge column. The labeled product
mixture solution was passed onto the cartridge for SPE purification
by gas pressure. The cartridge was washed with H2O (2 ꢂ 3 mL), and
the aqueous washing was discarded. The product was eluted from
the cartridge with EtOH (2 ꢂ 3 mL), and then passed onto a rotatory
evaporator. The solvent was removed by evaporation (3 min) under
vacuum. The final volume of ethanol after evaporation was w1 mL.
The labeled product [11C]4a, [11C]4b, [11C]4c, [11C]8a, [11C]8b, [11C]
8c, or [11C]8d was reformulated with saline (10 mL), sterile-filtered
(7d), mp 210e212 ꢄC 1H NMR (acetone-d6):
d 2.34 (s, 3H, CH3), 6.99 (s,
1H, Ph-H), 7.21e7.25 (m, 4H, Ph-H), 7.49 (dt, J ¼ 2.0, 8.5 Hz, 2H, Ph-H),
8.07 (dt, J ¼ 2.0, 8.5 Hz, 2H, Ph-H). MS (ESI): 361 ([M þ H]þ, 100%).
4.6. General procedure for preparation of methyl 3-
(trifluoromethyl)-1H-pyrazol-1-yl)benzoate (8)
To a solution of 7 (1.0 mmol) in MeOH (10 mL) was slowly added
SOCl2 (0.60 g, 5.0 mmol) at 0 ꢄC. Then the reaction mixture was
stirred for 15 h at room temperature. The reaction was quenched
with saturated aqueous NaHCO3, and reaction mixture was
concentrated in vacuo, extracted with EtOAc, washed with brine,
and dried over MgSO4. The organic layer was concentrated, and
residue was purified by column chromatography on silica gel (10%
EtOAc/Hexanes) to obtain 8 in 90 ꢀ 5% (n ¼ 3) yield, Rf ¼ 0.77e0.80
(1:2 EtOAc/Hexanes).
Methyl 4-(5-(2-methoxyphenyl)-3-(trifluoromethyl)-1H-pyr-
azol-1-yl)benzoate (8a), white solid, mp 112e114 ꢄC 1H NMR
through a sterile vented Millex-GS 0.22
mm cellulose acetate
(acetone-d6):
d
3.39 (s, 3H, CH3O), 3.88 (s, 3H, CH3O), 6.91 (s, 1H,
membrane and collected into a sterile vial. Total radioactivity
(5.9e6.4 GBq for [11C]4a-c and 6.4e7.1 GBq for [11C]8a-d) was
assayed and the total volume (10e11 mL) was noted for tracer dose
dispensing. The overall synthesis time including SPE purification
and reformulation was 23 min. The radiochemical yields decay
corrected to EOB, from [11C]CO2, were 52 ꢀ 2% (n ¼ 5) for [11C]4a-c
and 57 ꢀ 3% (n ¼ 5) for [11C]8a-d, respectively. Retention times in
the analytical HPLC system were: tR 5a ¼ 2.87 min, tR 4a ¼ 4.48 min,
CH]), 6.98 (d, J ¼ 8.5 Hz, 1H, Ph-H), 7.06 (t, J ¼ 8.5 Hz, 1H, Ph-H),
7.42e7.49 (m, 4H, Ph-H), 8.00 (dd, J ¼ 2.0, 7.0 Hz, 2H, Ph-H). 13C
NMR (CDCl3):
d 52.28, 54.85, 107.14, 111.36, 118.30, 120.19 (q,
J ¼ 267 Hz), 121.01, 123.46, 129.15, 130.20, 131.06, 131.47, 141.86,
143.38 (q, J ¼ 38 Hz), 143.88, 156.24, 166.23. MS (ESI): 363
([M þ H]þ, 100%). HRMS (ESI): calcd for C18H14N2O3F3 363.0957
([M þ H]þ), found 363.0946.
Methyl 4-(5-(3-methoxyphenyl)-3-(trifluoromethyl)-1H-pyr-
azol-1-yl)benzoate (8b), yellowish liquid. 1H NMR (acetone-d6):
tR
[
11C]4a ¼ 4.48 min; tR 5b ¼ 2.68 min, tR 4b ¼ 4.51 min, tR
[
[
[
[
[
[
11C]
11C]
11C]
11C]
11C]
11C]
4b ¼ 4.51 min; tR 5c ¼ 2.65 min, tR 4c ¼ 4.44 min, tR
4c ¼ 4.44 min; tR 7a ¼ 4.43 min, tR 8a ¼ 7.50 min, tR
8a ¼ 7.50 min; tR 7b ¼ 4.69 min, tR 8b ¼ 8.20 min, tR
8b ¼ 8.20 min; tR 7c ¼ 4.68 min, tR 8c ¼ 7.91 min, tR
8c ¼ 7.91 min; and tR 7d ¼ 3.10 min, tR 8d ¼ 5.14 min, tR
8d ¼ 5.14 min.
d
3.71 (s, 3H, CH3O), 3.92 (s, 1H, CH3O), 6.75e6.78 (m, 3H, CH] and
Ph-H), 6.91 (dt, J ¼ 1.0, 7.5 Hz, 1H, Ph-H), 7.23 (t, J ¼ 8.0 Hz, 1H, Ph-
H), 7.40 (dd, J ¼ 2.0, 7.0 Hz, 2H, Ph-H), 8.02 (dd, J ¼ 2.0, 7.0 Hz, 2H,
Ph-H). 13C NMR (CDCl3):
d 52.45, 55.35, 106.40, 114.52, 115.02,
120.16 (q, J ¼ 267 Hz), 121.31, 125.05, 129.90, 130.10, 130.19, 130.60,
142.76, 143.74 (q, J ¼ 38 Hz), 144.92, 159.82, 166.16. MS (ESI): 363
([M þ H]þ, 100%). HRMS (ESI): calcd for C18H14N2O3F3 363.0957
([M þ H]þ), found 363.0940.
4.8. MDA-MB-435 cell proliferation assay
Methyl 4-(5-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyr-
azol-1-yl)benzoate (8c), yellowish liquid. 1H NMR (acetone-d6):
MDA-MB-435 cell proliferation assay was performed following
a literature procedure [37,38]. Briefly, cell proliferation was per-
d
3.71 (s, 3H, CH3O), 3.92 (s, 1H, CH3O), 6.70 (s, 1H, CH]), 6.84 (dt,
formed using Celltiter96 kits. For proliferation assays, 100 mL of
J ¼ 2.0, 9.0 Hz, 2H, Ph-H), 7.12 (dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H), 7.38 (dt,
medium containing 5000 MDA-MB-435 human cancer cells were
plated in a 96-well flat-bottom microplate (Becton Dickinson &
Co., Lincoln Park, NJ) and were incubated at 37 ꢄC for 24 h in
the presence of 20e1280 nM of reference compound celecoxib,
and synthesized compounds 4b and 8aed in DMSO. Following
J ¼ 2.0, 9.0 Hz, 2H, Ph-H), 8.02 (dt, J ¼ 2.0, 9.0 Hz, 2H, Ph-H). 13C
NMR (CDCl3):
d
52.47, 55.43, 105.88, 114.44, 120.23 (q, J ¼ 267 Hz),
121.26, 125.10, 129.81, 130.32, 130.62, 142.91, 143.72 (q, J ¼ 38 Hz),
144.99, 160.45, 166.22. MS (ESI): 363 ([M þ H]þ, 100%). HRMS (ESI):
calcd for C18H14N2O3F3 363.0957 ([M þ H]þ), found 363.0941.
treatment, 20 mL of the reagent, the tetrazolium compound MTS
Methyl
4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt] (Promega, Madison, WI),
was added to each well, and incubation was continued at 37 ꢄC for
1 h. The absorbance was measured at a wavelength of 490 nm.
Control cells were not incubated with celecoxib, 4b and 8aed.
Each series was performed in triplicate.
benzoate (8d), yellowish liquid. 1H NMR (acetone-d6):
d 2.36 (s, 3H,
CH3O), 3.92 (s, 1H, CH3O), 6.73 (s, 1H, CH]), 7.09 (d, J ¼ 8.0 Hz, 2H,
Ph-H), 7.14 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.39 (dd, J ¼ 1.5, 7.0 Hz, 2H, Ph-H),
8.02 (dd, J ¼ 1.5, 7.0 Hz, 2H, Ph-H). MS (ESI): 347 ([M þ H]þ, 100%).
4.7. General procedure for preparation of carbon-11-labeled
celecoxib derivatives [11C]4a-c and [11C]8aed
4.9. Statistical methods
[
11C]CO2 was produced by the 14N(p,
a
)
11C nuclear reaction in the
Statistical analysis was performed using the Student’s t-test for
paired data to determine the significance of differences between
small volume (9.5 cm3) aluminum gas target provided with the

M. Gao et al. / European Journal of Medicinal Chemistry 46 (2011) 4760e4767
4767
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Acknowledgments
This work was partially supported by the Breast Cancer Research
Foundation and Indiana Genomics Initiative (INGEN) of Indiana
University, which is supported in part by Lilly Endowment Inc. The
authors would like to thank Dr. Bruce H. Mock and Barbara E. Glick-
Wilson for their assistance in production of [11C]CH3OTf. 1H NMR
and 13C NMR spectra were recorded on a Bruker Avance II 500 MHz
NMR spectrometer in the Department of Chemistry and Chemical
Biology at Indiana University Purdue University Indianapolis
(IUPUI), which is supported by an NSF-MRI grant CHE-0619254. The
referees’ criticisms and editor’s comments for the revision of the
manuscript are greatly appreciated.
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