[123I]-celecoxib analogues as SPECT tracers of cyclooxygenase-2 in inflammation

Article abstract of DOI:10.1021/ml100232q

We report the synthesis and evaluation of a series of iodinated celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission computerized tomography (SPECT) imaging agents for the detection of inflammation. The structure-activity relati

Full text of DOI:10.1021/ml100232q

pubs.acs.org/acsmedchemlett
[123I]-Celecoxib Analogues as SPECT Tracers of
Cyclooxygenase-2 in Inflammation
Md. Jashim Uddin,† Brenda C. Crews,† Kebreab Ghebreselasie,† Mohammed N. Tantawy,‡
and Lawrence J. Marnett*,†
†A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry and Pharmacology,
Vanderbilt Institute of Chemical Biology, Center for Molecular Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-0146, United States , and ‡Department of Radiology, Vanderbilt University Institute of
Imaging Science, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States
ABSTRACT We report the synthesis and evaluation of a series of iodinated
celecoxib analogues as cyclooxygenase-2 (COX-2)-targeted single photon emission
computerized tomography (SPECT) imaging agents for the detection of inflammation.
The structure-activity relationship identified 5-(4-iodophenyl)-1-{4-(methylsulfonyl)-
phenyl}-3-(trifluoromethyl)-1H-pyrazole (8) as a promising compound with IC50
values of 0.05 μM against purified COX-2 and 0.03 μM against COX-2 in activated
macrophages. The arylstannane of 8 undergoes facile radio-[123I]-iodination upon
treatment with Na123I/NaI and chloramine T using an EtOAc/H2O two-phase system.
The [123I]-8 was produced in a radiochemical yield of 85% and a radiochemical purity
of 99%. In vivo SPECT imaging demonstrated that the radiotracer was taken up by
inflamed rat paws with an average 1.7-fold enrichment over contralateral noninflamed
paws. This study suggests that conversion of celecoxib into its isomeric iodo-[123I]-
analogues is a useful approach for generating novel and efficacious agents for COX-2-
targeted SPECT imaging of inflammation.
KEYWORDS Iodine-123, celecoxib, iododestannylation, two-phase reaction, cy-
clooxygenase-2 (COX-2), in vivo SPECT imaging
rostaglandins are important biological mediators of
inflammation, originating from the biotransformation
of arachidonic acid catalyzed by cyclooxygenases
recently reported that COX-2-targeted fluorescent imaging
agents can be selectively delivered into inflammatory tissues
and COX-2-expressing tumors in vivo.17 Because an enormous
amount of medicinal chemistry has been conducted to create
COX-2-specific inhibitors, there are numerous classes of poten-
tial building blocks that are available for the preparation of single
photon emission computerized tomography (SPECT) imaging
agents for COX-2.18,19 For the development of COX-2-targeted
SPECT imaging agents, we synthesized a series of derivatives of
celecoxib. Here, we report the synthesis, evaluation, and radio-
labeling of [123I]-celecoxib analogues as selective COX-2 tracers
for SPECT imaging and describe the in vivo delivery of a tracer
that selectively accumulates in the COX-2-expressing carrageenan-
induced rat paw model of acute inflammation.
P
(COX).1 Constitutively expressed COX-1 is found in most
normal tissues, where it modulates homeostatic functions,
such as hemostasis, vascular tone, and cyctoprotection of the
gastric mucosa.2 Inducible COX-2 is expressed in inflammatory
lesions, where it modulates pain, fever, and edema, and in
proliferative lesions where it promotes growth, angiogen-
esis and enhances the metastatic potential of tumor cells.3 The
expression of COX-2 is an early event in tumorigenesis that
plays a role in tumor progression.4 COX-2 mRNA and protein
are detectable in a significant percentage of precursor lesions
(e.g., colon polyps and Barrett's esophagus)5,6 and an even higher
percentage of malignant tumors (e.g., colon adenocarcinoma
and esophageal adenocarcinoma).7,8 Recent work has shown
that selective COX-2 inhibitors are useful in the treatment of
certain human cancers.9,10 Therefore, COX-2 is an attractive
molecular target for detection of cancers by imaging with
radiolabeled COX-2 inhibitors. In fact, syntheses of 18F- and
123I-labeled COX-2 inhibitors as potential imaging agents have
been reported.11 , 1 2 Preliminary characterization of these com-
pounds indicated that they accumulated in COX-2-expressing
macrophages,13-15 but the in vivo uptake in tumors of most of
the agents did not correlate to the presence of COX-2.16 We
The Claisen reaction of iodoacetophenone (A, R1=3-I or 4-I)
with methyltrifluoroacetate in the presence ofsodium methoxide
gave the expected β-diketones B(R1=3-I or 4-I, and R2 =CF3) in
80-88% yield. An ultrasonication-assisted condensation of A
(R1=3-I or 4-I) with either dimethyl- or diethyloxalate afforded
compounds B (R1 =3-I or 4-I, and R2 =CO2Me or CO2Et) in
65-70% yield. Alternatively, the reaction of A (R1=3-I or 4-I)
Received Date: September 29, 2010
Accepted Date: November 10, 2010
Published on Web Date: November 12, 2010
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Scheme 1. Synthesis of Isomeric Iodo Compounds 1-10a
Table 1. In Vitro Purified COX-1 and COX-2 and Lipopolysacca-
ride (LPS)-Activated Macrophage-Like (RAW254.7 Cell) Cellular
COX-2 Enzyme Inhibition Assay Data of Isomeric Iodo Com-
pounds 1-10
IC50(μM)a
RAW
compd
no.
purified
COX-1
purified
COX-2
264.7
cell COX-2
1
2
3
4
5
6
7
8
9
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
0.08
3.80
0.56
0.26
>4
0.04
NT
NT
2.70
NT
>4
NT
0.32
0.05
>4
NT
0.03
NT
10
3.00
0.03
NT
celecoxib
NT
a IC50 values were determined by incubating several concentrations
of inhibitors in DMSO with purified murine COX-2 (66 nM) and ovine
COX-1 (44 nM) for 20 min followed by treatment with 1-14C-AA (50 mM)
at 37 °C for 30 s. Assays were run in duplicate. NT, not tested.
(IC50=0.56 μM). A further increase in COX-2 potency was ob-
served when the CO2Me group of 3 was replaced with a CO2Et
group (4, COX-2 IC50=0.26 μM). Replacement of the CO2Me
group of3 or4 with a propionic acid(R2 = C2H4CO2H) moiety
afforded inactive compounds, 5 and 6. However, isomeric
iodination or replacement of the SO2NH2 group of compound 1
with a SO2Me group increased the COX-2 inhibitory potency
and selectivity as exhibited by compound 8(R3 =SO2Me, COX-2
IC50=0.05 μM). A complete loss of poor inhibition was observed
when the CF3 group of 7 or 8 was replaced with a CO2Me group
(compounds 9 and 10).
The ability of promising compounds to inhibit COX-2 in
intact cells was assayed in the RAW264.7 murine macro-
phage-like cell line.21 The RAW264.7 cells were treated with
lipopolysaccharide (200 ng/mL) and γ-interferon (10 U/mL)
for 7 h to induce COX-2 expression and then treated with vehicle
or the test compounds at several concentrations. The IC50 values
for inhibition of COX-2 by compounds 1 and 8 were 0.04 and
0.03 μM, respectively (Ta bl e 1 ). Among these compounds, 8
showed the most potent inhibitory activity against COX-2 in
cultured inflammatory cells without inhibition of COX-1 at con-
centrations up to 5 μM. Thus, compound 8 was selected for
radio-123I-iodination. The remaining isomeric iodo compounds
that have low to moderate COX-2 inhibitory potency and selec-
tivity in the purified COX enzyme assay were not tested for their
inhibitory activity against COX-2 enzyme in the activated intact
RAW264.7 cell line assay.
a Reagents and conditions: (a) 25% NaOMe/MeOH, methyl t-butyl
ether, 25 °C, 48 h, or 25% NaOMe/MeOH, ultrasound, 45 °C, 16 h, or
LDA, succinic anhydride, THF, -78 °C. (b) R3-Ph-NHNH2 HCl, MeOH,
3
reflux 16 h, or R3-Ph-NHNH2 HCl, TEA, MeOH, reflux 16 h.
3
with succinic anhydride in the presence of lithium diisopropyla-
mide (LDA) proceeded smoothly to afford B (R1=3-I or 4-I, and
R2=C2H4CO2H) in 68-72% yield. Condensation of compounds
B (R1=3-I or 4-I, and R2 = C2H4CO2H) with R3-Ph-NHNH2 HCl
3
(R3=SO2NH2 or SO2Me) afforded the respective pyrazole prod-
ucts, 1-10, in 76-84% yield (Scheme 1). The 1,5-regioisomers
were generated almost exclusively by carrying out the reaction in
the presence of the hydrochloride salt of the substituted phenyl-
hydrazine in refluxing ethanol.18 However, when required, the
1,5-diarylpyrazoles were separated from the minor 1,3-diaryl-
pyrazole isomers by flash chromatography.
The IC50 values for inhibition of purified human COX-2 or
ovine COX-1 by test compounds were determined by a thin-
layer chromatography (TLC) assay.20 Hematin-reconstituted
COX-2 (66 nM) or COX-1 (44 nM) in 100 mM Tris-HCl, pH
8.0, containing 500 μM phenol was treated with several con-
centrations of inhibitors (0-66 μM) at 25 °C for 17 min and
37 °C for 3 min followed by metabolism of 14C-arachidonic acid
(50 μM) for 30 s at 37 °C. Table 1 displays the in vitro COX-1 and
COX-2 inhibition data. We found that the iodo-celecoxib deriv-
ative, 1, which contains an iodo group at the meta-position of
the 5-phenyl substituent, is a potent and selective COX-2
We recently reported an efficient two-phase radioiodination
method that was used in the present case to radiolabel
compound 8.22 The aryltributylstannane 11 of the stable iodo
compound 8 was generated by a palladium-catalyzed deiodos-
tannylation reaction using hexabutylditin and tetrakis-(triphe-
nylphosphine)palladium(0) in refluxing 1,4-dioxane. The radio-
iodination of tributylstannyl derivative 11 was conducted by
inhibitor with a COX-2 IC50 value of 0.08 μM (COX-1 IC50
>
66 μM). When the CF3 group of 1 was replaced by a CO2Me
substituent, the COX-2 inhibitory potency was significantly
decreased (2, COX-2 IC50=3.8 μM). Interestingly, the para-
iodo isomer, 3, showed better potency against purified COX-2
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Scheme 2. Radiosynthesis of [123I]-8a
Figure 1. In vivo SPECT/CT image of a Sprague-Dawley rat foot-
pad at 3 h post tail vein administration of [123I]-8.
a Reagents and conditions: (a) tetrakis-(Triphenylphosphine)palla-
dium(0), bis-tributyltin, 1,4-dioxane, reflux 16 h. (b) Na123I/NaI, chlor-
amine T, EtOAc, H2O, 1 N HCl, 25 °C, 4 min.
electrophilic 123I-iododestannylation in an EtOAc/H2O binary-
phase system using Na123I/NaI, chloramine T, and aqueous 1 N
HCl (Scheme 2). The radiolabeled product was isolated by
collecting the organic layer from the two-phase system and
evaporating the solvent under a flow of argon. The electrophilic
123I species is generated in the water layer at pH 3.5 and rapidly
extracted into the EtOAc layer for subsequent electrophilic aro-
matic substitution reaction. The final product stays in the EtOAc
layer, which lacks the polar byproducts of the reaction to give the
high radiochemical yield and purity of the product. This phase
transfer technology does not require any further chromato-
graphic purification. In a typical experiment, we reacted the
precursor 11 (20 μg in 1.5 mL of EtOAc) with the radioactive
Na123I (17.3 mCi in 500 μL of 0.01 N NaOH) in the presence of
carrier NaI (4.5 μg in 300 μL of H2O), chloramine-T trihydrate
(8.4 μg in 300 μL of H2O), and aqueous 1 N HCl (300 μL). After
the reaction mixture was stirred for 5 min at room temperature,
water (1 mL) followed by EtOAc (1 mL) was added. The organic
layer was collected, washed with water, and evaporated under
argon flow to afford [123I]-8 (product, 14.7 mCi; radiochemical
yield, 85%; and radiochemical purity, 99%). The radiochemical
purity was determined by a radio-TLC scan. The radiotracer
Figure 2. Relative uptake of [123I]-8 in inflamed vs noninflamed
rat footpads at 3 and 6 h postinjection of [123I]-8.
model of inflammation is the ability to image the inflamed
paw in comparison to the noninflamed contralateral footpad,
which does not express COX-2. We injected 100 μL of 1%
carrageenan in the rear right footpad of Sprague-Dawley rats
(325-350 g) andwaited3hfor inflammationto develop. Then,
we tail vein injected [123I]-8 dissolved in a formulation solvent
consisting of dimethyl sulfoxide (10%), ethanol (40%), and
sterile saline (50%) (300 μL, 600;800 μCi) into the anesthe-
tized animals. Three hours later, the rats were reanesthetized
with 2% isoflurane and placed in a BioScan NanoSPECT/CT, and
a 30 min acquisition (24 projections ꢀ 60 s per projection) was
initiated. The images were reconstructed with a resolution of
170 ꢀ 170 ꢀ 184 at 0.4 mm ꢀ 0.4 mm ꢀ 0.4 mm. The SPECT
images were analyzed using AMIDE.24 Compound [123I]-8 tar-
geted the swollen footpad selectively over the contralateral
control footpad (Figure 1). The rats were sacrificed at various
time points postinjection by isoflurane overdose. The hind feet
were removed and weighed, and radioactivity associated with
each footpad was counted with a well γ-counter. Figure 2 dis-
plays the relative uptake of [123I]-8 in the carrageenan-injected
footpad over the control footpad obtained from measurements
of individual footpad radioactivity after removal at two different
time points (3 and 6 h; p = 0.005 at 3 h). The uptake of the
radiotracer in the inflamed paw was 23.5 ( 5% of the injected
dose/g and was 11.5 ( 1% in the noninflamed paw.
[
123I]-8coeluted with an unlabeled standard. The specific activity
of [123I]-8 was 491 Ci/mmol, which was calculated based on the
final radiotracer obtained from the organic layer of the iodina-
tion reaction as compared with the added carrier in the reaction
(specific activity = 0.01473 Ci/0.00003 mmol).
The rat paw model is well-documented for the role of COX-
2-derived prostaglandins as a major driving force for the
acute edema that results after carrageenan injection into the
paw.23 One of the significant advantages of this animal
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The importance of COX-2 in the uptake of isotope into the
inflamed footpad was probed by blocking the COX-2 active site
with an excess of unlabeled 8. We administered the nonradioac-
tive compound 8 at a dose of 55 mg/kg (ip) at 2 h postcarra-
geenan and waited 1 h forabsorption and blockage of the COX-2
active site prior to dosing with [123I]-8 (∼1 mCi, tail vein). At 3 h
postinjection of [123I]-8, we euthanized the animals, removed
the hind paws, and measured the radioactivity of the individual
paws in the well counter. There was no increase of radiotracer in
the inflamed footpad as compared to the noninflamed control
footpad (calculated fold increase = 1.0 ( 0.2).
In summary, isomeric iodo analogues of celecoxib were
generated and radioiodinated such that they retain the ability
of the parent celecoxib to inhibit COX-2 selectively in purified
enzyme as well as in live inflammatory cells (e.g., compound 1
or 8). A striking observation from this study is that replacement
of the p-tolyl ring with a p-iodophenyl ring, as well as substitution
of the p-SO2NH2 group of celecoxib with a p-SO2Me group,
generates compounds like 1 or 8 that are highly potent and
selective COX-2 inhibitors. It is likely that the [123I]-substituent in
compound 8 and the Me group in celecoxib are bioisoteric. This
observation, coupled with the structural flexibility revealed in
the present study, suggests that isomeric iodinated analogues of
celecoxib are efficacious COX-2 inhibitors, and these com-
pounds can be efficiently labeled with [123I] for use in COX-2-
targeted SPECT imaging.
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SUPPORTING INFORMATION AVAILABLE Full synthetic
procedures and analytical and spectral characterization data of
the synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(14) Ishikawa, T.; Jain, N. K.; Taketo, M. M.; Herschman, H. R.
Imaging Cyclooxygenase-2 (Cox-2) Gene Expression in Liv-
ing Animals with a Luciferase Knock-in Reporter Gene. Mol.
Imaging Biol. 2006, 8, 171–187.
AUTHOR INFORMATION
Corresponding Author: *Tel: 615-343-7329. Fax: 615-343-
7534. E-mail: larry.marnett@vanderbilt.edu.
(15) McCarthy, T. J.; Sheriff, A. U.; Graneto, M. J.; Talley, J. J.; Welch,
M. J. Radiosynthesis, In Vitro Validation, and In Vivo Evalua-
tion of 18F-Labeled COX-1 and COX-2 Inhibitors. J. Nucl. Med.
2002, 43, 117–124.
Funding Sources: This work has been supported by grants from
the National Institutes of Health (CA128323 and CA89450).
(16) Schuller, H. M.; Kabalka, G.; Smith, G.; Meredy, A.; Akula, M.;
Cekanova, M. Detection of Overexpressed COX-2 in Precan-
cerous Lesions of Hamster Pancreas and Lungs by Molecular
Imaging: Implications for Early Diagnosis and Prevention.
ChemMedChem 2006, 1, 603–610.
(17) Uddin, M. J.; Crews, B. C.; Blobaum, A. L.; Kingsley, P. J.; Gorden,
D. L.; McIntyre, J. O.; Matrisian, L. M.; Subbaramaiah, K.;
Dannenberg, A. J.; Piston, D. W.; Marnett, L. J. Selective Visua-
lization of Cyclooxygenase-2 in Inflammation and Cancer by
Targeted Fluorescent Imaging Agents. Cancer Res. 2010, 70,
3618–3627.
ACKNOWLEDGMENT We are grateful to Jeffrey A. Clanton of
the Department of Radiology and Radiological Sciences for assis-
tancewith radiochemical syntheses. We are also grateful to Dr. Carol
A. Rouzer of the Vanderbilt Institute for Chemical Biology for critical
reading of this manuscript.
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