PET radiotracer [18F]-P6 selectively targeting COX-1 as a novel biomarker in ovarian cancer: Preliminary investigation

Article abstract of DOI:10.1016/j.ejmech.2014.04.074

Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 inhibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-(5-chlorofuran-2-yl)-5- (fluoromethyl)-4-phenylisoxazole ([^18/19F]-P6) is the most promising derivative [IC₅₀ = 2.0 μM (purified oCOX-1) and 1.37 μM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also prepared and, a method for radio[¹⁸F]chemistry was developed and optimized. The radiochemistry was carried out using a carrier-free K¹⁸F/Kryptofix 2.2.2 complex, that afforded [¹⁸F]-P6 in good radiochemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer [¹⁸F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as compared with the normal leg muscle. Our results suggest that [¹⁸F]-P6 might be an useful radiotracer in preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1.

Full text of DOI:10.1016/j.ejmech.2014.04.074

European Journal of Medicinal Chemistry 80 (2014) 562e568
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
PET radiotracer [18F]-P6 selectively targeting COX-1 as a novel
biomarker in ovarian cancer: Preliminary investigation
,
,
Maria Grazia Perrone a 1, Paola Malerba a 1, Md. Jashim Uddin b, Paola Vitale a,
Andrea Panella a, Brenda C. Crews b, Cristina K. Daniel b, Kebreab Ghebreselasie b,
Mike Nickels c, Mohammed N. Tantawyc, H. Charles Manning c, Lawrence J. Marnett b
,
,
*
Antonio Scilimati a
,
*
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “A. Moro”, Via E. Orabona 4, 70125 Bari, Italy
b A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology,
Vanderbilt Institute of Chemical Biology, Nashville, TN 37232-0146, USA
c Department of Radiology and Radiological Sciences, Vanderbilt Institute of Imaging Sciences, Vanderbilt University School of Medicine, Nashville,
TN 37232-0146, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human
epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a
consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated
COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 in-
hibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-
(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole ([18/19F]-P6) is the most promising derivative
Received 30 September 2013
Received in revised form
23 April 2014
Accepted 25 April 2014
Available online 29 April 2014
[IC50 ¼ 2.0
mM (purified oCOX-1) and 1.37 mM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also
Keywords:
[
Cyclooxygenase (COX)-1
Ovarian cancer
Biomarker
prepared and, a method for radio[18F]chemistry was developed and optimized. The radiochemistry was
carried out using a carrier-free K18F/Kryptofix 2.2.2 complex, that afforded [18F]-P6 in good radio-
chemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer
18F]-P6 radiotracer
[
18F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as
compared with the normal leg muscle. Our results suggest that [18F]-P6 might be an useful radiotracer in
preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1.
Ó 2014 Elsevier Masson SAS. All rights reserved.
OVCAR-3 cell
PET-CT imaging
1. Introduction
human pathologies, including neuroinflammation [5,6] (as the
earliest stage of some neurodegenerative diseases such as Alz-
Cyclooxygenases (COX-1 and COX-2) catalyze the biotransfor-
mation of arachidonic acid into prostanoids [1,2]. The constitutively
expressed isoform COX-1 is responsible for maintaining homeo-
stasis (gastric and renal integrity) and normal production of ei-
cosanoids [3], whereas the inducible isoform COX-2 is implicated in
the synthesis of prostanoids involved in acute and chronic in-
flammatory processes. In addition to its role in inflammatory dis-
orders, COX-2 has an important function in carcinogenesis by
stimulating angiogenesis, tissue invasion, metastasis, and apoptosis
inhibition [4]. Recent reports have proven the COX-1 role in several
heimer- and Parkinson-disease), atherosclerosis [7], endothelial
dysfunction [8], preterm labor [9], pain [10], and carcinogenesis
[11e15]. In particular, COX-1 is overexpressed in various stages
(onset and progression) of human epithelial ovarian cancers, where
it controls the production of prostaglandins and promotes angio-
genic growth factor production [13,16,17]. Moreover, in an in vivo
model obtained with ovarian surface epithelial (OSE) cells allog-
rafted in female nude mice, SC-560 [18], a highly selective COX-1
inhibitor, significantly reduces the tumor growth [19]. On the
other hand, celecoxib, a selective COX-2 inhibitor, has no significant
effect, suggesting that, COX-1 is the primary player for the gener-
ation of prostaglandins in murine epithelial ovarian cancers (EOCs)
[16]. Similar studies, accomplished by treating mice bearing human
ovarian SKOV-3 carcinoma xenografts, have shown that SC-560,
used alone or in combination with the non selective COX inhibitor
* Corresponding authors.
E-mail addresses: larry.marnett@vanderbilt.edu (L.J. Marnett), antonio.
scilimati@uniba.it (A. Scilimati).
1
These authors equally contributed to the project.
http://dx.doi.org/10.1016/j.ejmech.2014.04.074
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
563
ibuprofen, significantly reduces tumor growth and angiogenesis
[19,20]. As a consequence, COX-1 might be an ideal target for
theranostic investigations of human epithelial ovarian cancers.
Unfortunately, treatment of these neoplastic diseases is still limited
[20] because of the lack of a non-invasive and effective imaging
agent for early diagnosis. So, development of a COX-1-targeted
Positron Emission Tomography (PET) radiotracer is a matter of
extreme importance.
As a continuation of our previous investigations [21e24], herein
we report the synthesis, the biochemical and cellular evaluation of
a
P6 [3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole] [21]
derived [18F]-fluorinated COX-1 inhibitor, as a COX-1-targeted PET
imaging agent. In addition, we describe the radiochemical syn-
thesis of the promising compound [18F]-P6 and its in vivo delivery
into human OVCAR-3 (ovarian cancer) tumor xenografts expressing
elevated levels of COX-1.
2. Results and discussion
We designed fluorinated COX-1-targeted PET imaging agents
based on the chemical structure of P6, a highly selective COX-1
inhibitor (COX-1 IC50 ¼ 0.5
mM, COX-2 IC50 > 100 mM, measured
by human whole blood assay) [21,25], and identified a novel fluo-
rinated-P6 as a potent and selective COX-1 inhibitor. A one-pot
procedure towards the isoxazole scaffold, involves the reaction of
arylnitrile oxides and the enolates of 3-aryl-2-propanones [23e26].
Fig. 1. [19F]-P6 IC50 values determination: (a) inhibition of in vitro purified oCOX-1 and
mCOX-2; (b) inhibition assay of OVCAR-3 intracellular hCOX-1 performed in the
In
phenylisoxazole ([19F]-P6) was prepared starting from P6, first
converted into 5-bromomethyl-3-(5-chlorofuran-2-yl)-4-
particular,
3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-
presence of 8 m
M [1-14C]-AA and increasing amount of [19F]-P6.
phenylisoxazole (Br-P6) by bromination with N-bromosuccini-
mide (NBS) in the presence of azobisisobutyronitrile (AIBN) as a
radical initiator. Then, Br-P6 reacted with anhydrous tetrabuty-
lammonium fluoride (TBAF) to afford the target [19F]-P6. The latter
was also prepared starting from the alcohol HO-P6 [23,25] previ-
ously transformed into its tosylate OTs-P6 by reaction with Ts2O in
the presence of Et3N. The tosylate derivative was then treated with
K19F/Kryptofix for the radiomimetic approach (Scheme 1).
The IC50 values for inhibition of purified mCOX-2 and oCOX-1
enzymes by [19F]-P6 were determined by a thin layer chromatog-
raphy (TLC) assay (Fig. 1a) [27].
Hematin-reconstituted murine COX-2 (66 nM) or ovine COX-1
(44 nM) in 100 mM TriseHCl, pH 8.0 containing 500 M phenol
was treated with several concentrations of inhibitor (0e66 M) at
25 ꢀC for 17 min and 37 ꢀC for 3 min followed by metabolism of
[1-14C]-arachidonic acid (50 M) for 30 s at 37 ꢀC. In this assay,
19F]-P6 showed selective COX-1 inhibitory activity.
The ability of the promising fluorinated P6 analogue to inhibit
COX-1 activity in intact cells was also evaluated by using the human
ovarian cancer cell line OVCAR-3 (Fig. 1b) [13,16,28], in which high
levels of COX-1 expression and no detectable COX-2 were found by
m
m
m
[
Scheme 1. Synthesis of 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole,
TBAF ¼ Tetrabutylammonium fluoride; Ts2O ¼ p-Toluenesulfonic anhydride; Et3N ¼ Triethylamine.
[
19F]-P6. NBS
¼
N-bromosuccinimide; AIBN
¼
2,20-Azobis(2-methylpropionitrile);

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
Western Blotting Assay [16]. [19F]-P6, incubated with OVCAR-
3 cells in the presence of [1-14C]-arachidonic acid, significantly
reduced intracellular COX-1-mediated AA metabolism (hCOX-1
IC50 ¼ 1.37
mM).
Furthermore, [19F]-P6 up to 100
mM did not affect at all the
OVCAR-3 cell viability within 48 h.
The radiosynthesis of
[ a
18F]-P6 was accomplished, on
commercially available GE Tracerlab FXFN automated reaction
module, by a 18F-nucleophilic substitution on the tosylate precursor
(OTs-P6) performed in the presence of K18F/Kryptofix
2.2.2 (Scheme 2).
This reaction was preceded by a radiomimetic synthesis of [19F]-
P6 in which it was obtained in 50% yield (Scheme 1).
Then, the ability of [18F]-P6 to target COX-1 in human ovarian
tumor xenografts in female nude mice was evaluated. The novel
radiotracer was administered by intra-peritoneal injection, then
the mice were sacrificed by isoflurane overdose. The OVCAR-3 tu-
mors were removed and weighed, and the radioactivity associated
with each tumor was counted with a well gamma counter. After
normalization of uptake (Bq/g), the OVCAR-3 tumors showed
approximately an 8-fold higher uptake of the radiotracer compared
with normal leg muscle (Fig. 2, n ¼ 3, p ¼ 0.01), thus suggesting a
selective uptake of the positron emitting agent [18F]-P6 in the tu-
mor expressing COX-1-targeted due to the presence of COX-1
protein in OVCAR-3 cell induced-tumor.
Fig. 2. In vivo uptake in tumors. Female nude mice bearing OVCAR-3 xenografts were
dosed with compound [18F]-P6 (7.4 MBq, intraperitoneal injection). Then, the animals
were sacrificed by isoflurane overdose. The OVCAR-3 tumor and normal leg muscles
were removed and weighed, and radioactivity associated with each tissue was counted
in a well gamma counter. The plot shows the increased radiotracer (Bq/g) in COX-1-
expressing OVCAR-3 tumors versus normal leg muscle (n ¼ 3, p ¼ 0.01) (*statistical
significance).
Moreover, the PET and CT images were co-registered to each
other on the basis of bed position and even thought [18F]-P6
radiotracer uptake in the OVCAR 3-induced tumors was 0.35% of
the injected dose/g, PET imaging did not show a marked selective
uptake. The different results might be due to the lower in vivo PET
detector sensitivity than that of Geiger counter in measuring tissue
extract radioactivity (Fig. 3). This would also indicate that not
enough material has been delivered to the tumor and that greater
radiotracer amount or more potent COX-1 inhibitors are needed.
Further and deeper studies (dynamic PET imaging recordings) are
ongoing.
In vivo stability of compound
[
19F]-P6 was evaluated by
analyzing the tissue samples on an LCeESI-MS system. The nude
mice bearing OVCAR-3 xenografts were dosed with intraperitoneal
(i.p.) injection of [19F]-P6. The intact parent compound [19F]-P6
was identified in the tumors on an average of 63 pmol/g tissue
(n ¼ 4) (Fig. 4). A minimal or no amount of the compound [19F]-P6
was detected in the normal leg muscles. Also, no defluorinated
metabolite (compound HO-P6) was detected in any of the tissue
samples analyzed, suggesting that compound [19F]-P6 remained
intact in the biological environment for long enough period of time
to be taken up by the COX-1-expressing tumors (Fig. 4).
3. Conclusions
Fig. 3. In vivo PET imaging of COX-1eexpressing tumor by [18F]-P6. (a) Coronal view,
(b) Sagittal view. Tumor-bearing female nude mice were dosed by i.p. injection with
A novel compound [18F]-P6 was identified as a promising
lead for a COX-1-targeted PET imaging agent. The synthetic
procedure to prepare [19F]-P6, and OTs-P6 for the incorporation
compound [18F]-P6 (100
mL, 7.4 MBq, intraperitoneal injection) under anesthesia. At
4 h post injection, the animals were imaged in the microPET/CT instrument (30-min
acquisition).
Scheme 2. Radiosynthesis of [18F]-P6. DMSO ¼ dimethylsulfoxide.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
565
4.1.1. Materials
Tetrahydrofuran (THF) from commercial source was purified by
distillation (twice) from sodium wire under nitrogen. Dichloro-
methane and DMF from commercial source were purified by
distillation from CaH2 under nitrogen atmosphere. Standardized
(2.5 M) n-butyllithium in hexanes was purchased from Aldrich
Chemical Co. and its titration was performed with N-pivaloyl-o-
toluidine [29]. Triethylamine from commercial source was purified
by distillation from CaH2 under reduced pressure and nitrogen
atmosphere. All other chemicals and solvents were commercial
grade further purified by distillation or crystallization prior to use.
Arylnitrile oxides were prepared from aldehydes through their
conversion into the corresponding oximes and then into benzo-
hydroximinoyl chlorides [21,23]. These were finally converted into
nitrile oxides, just before use, by treatment with NEt3 at 0 ꢀC fol-
lowed by vacuum filtration to remove the NEt3$HCl from the THF
solution. Oximes, prepared from reaction of aldehydes/EtOH and
NH2OH$HCl/aq. NaOH, had analytical and spectroscopic data
identical to those previously reported or commercially available
[30e34].
Fig. 4. OVCAR-3 tumor and normal leg muscles were removed and amount of com-
pound [19F]-P6 was determined by LCeMS. The plot shows the increased unlabeled
compound [19F]-P6 in COX-1-expressing OVCAR-3 tumors versus normal leg muscle
(n ¼ 4, p ¼ 0.01) (*statistical significance).
4.1.2. Synthesis of 5-(bromomethyl)-3-(5-chlorofuran-2-yl)-4-
phenylisoxazole (Br-P6)
To a solution of 3-(5-chloro-2-furyl)-4-phenyl-5-methylisoxazole
(P6) (0.500 g,1.930 mmol)in anhydrous CCl4 (10 mL), contained in an
argon-flushed, three necked flask equipped with a magnetic stirrer,
an argon inlet and two dropping funnels N-bromosuccinimide
(412 mg, 2.32 mmol) were added. Then AIBN (0.158 g, 0.965 mmol)
was added, and the obtained suspension was stirred overnight at
room temperature. The reaction mixture was kept under reflux for
4 h, and then water was added. The two phases, formed by adding
ethyl acetate, were separated and aqueous layer was extracted three
times with ethyl acetate. The combined organic extracts washed first
with brine and then with 1 M KOH, were dried over anhydrous
Na2SO4. The solvent was evaporated under reduced pressure. Col-
umn chromatography (silica gel, petroleum ether/ethyl
acetate ¼ 10:1) of the residue affords the 5-(bromomethyl)-3-(5-
chlorofuran-2-yl)-4-phenylisoxazole in 95% yield as a solid.
of fluorine-18 to obtain the radiolabeled [18F]-P6 was optimized.
[
19F]-P6 showed a high degree of selectivity towards in vitro and
in vivo COX-1 inhibition and uptake into OVCAR-3 xenografts in
immuno-compromised mice. Its IC50 is 1.37 M in OVCAR-3 cells
and 2 M and >100 M in purified oCOX-1 and mCOX-2 en-
m
m
m
zymes, respectively. A suitable in vivo stability of [19F]-P6 was
found in tumor, moreover no defluorinated derivative was
detected in any analyzed tissue. These preliminary results
indicate that [18F]-P6, a fluorinated COX-1 selective inhibitor,
might be a good radiotracer to detect early stages of EOCs,
further experiments are ongoing to provide more evidences to
certificate [18F]-P6 as a tracer in EOCs.
4. Experimental protocols
4.1. General
4.1.3. 5-(Bromomethyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole
(Br-P6)
Melting points taken on Electrothermal apparatus were uncor-
rected. 1H NMR and 13C NMR spectra were recorded on a Varian-
Mercury 300 MHz, Varian-Inova-400 MHz spectrometer, Bruker-
Aspect 3000 console-500 MHz spectrometer, Bruker AV-I console
operating at 400.13 MHz, or a Bruker DRX console operating at
White solid (95%). FT-IR (neat): 3132, 3053, 2921, 1636, 1517,
1432, 1412, 1372, 1359, 1226, 1127, 1048, 1018, 985, 939, 903, 790,
777, 704 cmꢂ1 1H NMR (400 MHz, CDCl3,
. d): 7.50e7.46 (m, 3H,
aromatic protons); 7.39e7.36 (m, 2H, aromatic protons); 6.31 (d,
J ¼ 3.5 Hz, 1H, furyl proton); 6.15 (d, J ¼ 3.5 Hz, 1H, furyl proton);
600.13 MHz. Chemical shifts are reported in parts per million (
d
).
4.38 (s, 2H, CH2). 13C NMR (100 MHz, CDCl3,
d): 170.3, 163.5, 152.7,
Fluorine spectra were recorded by using CFCl3 as internal standard.
Absolute values of the coupling constant are reported. FT-IR spectra
were recorded on a PerkineElmer 681 spectrometer. GC analyses
were performed by using an HP1 column (methyl siloxane;
143.1, 139.0, 130.2, 129.2, 129.1, 128.1, 118.1, 114.4, 108.2, 55.4, 20.7.
GCeMS (70 eV) m/z (rel. int.): 341 [M (81Br, 37Cl)þ, 26], 339 [M (79Br,
37Cl)þ, 100], 337 [M (79Br, 35Cl)þ, 78], 258 (22), 246 (11), 244 (33),
232 (12), 230 (35), 223 (14), 219 (17), 218 (13), 217 (54), 216 (19),
190 (16), 188 (46), 167 (25), 166 (16), 163 (12), 154 (18), 153 (29), 152
(28), 140 (10), 139 (18), 129 (66), 128 (11), 127 (24), 115 (34), 103
(19), 102 (13), 91 (28), 89 (38), 77 (18), 75 (13), 73 (17), 63 (12), 51
(9). Anal. Calc. for C16H12ClNO3: C, 64.48; H, 3.81; N, 4.41. Found: C,
64.46; H, 3.82; N, 4.46.
30 m ꢁ 0.32 mm ꢁ 0.25
mm film thickness) on a HP 6890 model,
Series II. Thin-layer chromatography (TLC) was performed on silica
gel sheets with fluorescent indicator, the spots on the TLC were
observed under ultraviolet light or were visualized by I2 vapor.
Column chromatography was conducted by using Sorbent silica gel
ꢀ
standard grade, porosity 60 A, particle size 32e63
mm
(230 ꢁ 450 mesh), surface area 500e600 m2/g, bulk density 0.4 g/
mL, pH range 6.5e7.5. GCeMass Spectrometric analyses were per-
formed on an HP 5995C model. MS-ESI analyses were performed on
Agilent 1100 LC/MSD trap system VL or on a Thermo Electron
Surveyor pump and autosampler operated in-line with a Quantum
triple quadrupole instrument in ESI positive or negative ion mode.
Elemental analyses were performed on an Elemental Analyzer
1106-Carlo Erba-instrument.
4.1.4. Synthesis of 5-(fluoromethyl)-3-(5-chlorofuran-2-yl)-4-
phenylisoxazole (19F-P6)
To an anhydrous 1 M solution of TBAF in THF (0.940 mL) kept
under stirring in a round bottom flask at room temperature, 5-
(bromomethyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole (0.117 g,
0.350 mmol) was added. After 24 h, the reaction mixture was
filtered over celite, and the solvent evaporated under reduced
pressure. Column chromatography (silica gel, petroleum ether/

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
ethyl acetate ¼ 8:2) of the residue affords 0.063 g of 5-(fluo-
romethyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole in 65% yield
as a yellow solid.
4.2.1. Synthesis of [18F]-P6 radiotracer
The 18F-radiosynthesis [27] was performed on a commercially
available GE Tracerlab FXFN automated reaction module. The 18F-
fluoride anion (128,020 MBq) was trapped (adsorbed) onto an
anion (K2CO3 contained) exchange resin (QMA-cartridge) and
eluted with a solution of Kryptofix 2.2.2 (12.5 mg) and K2CO3 (7 mg)
in CH3CN/H2O (1:1 v/v) (Scheme 2). The solvent was then evapo-
rated under vacuum with a gentle stream of helium gas (32 kPa),
followed by sequential additions of anhydrous acetonitrile and
further azeotropic drying. Once drying was complete, a solution of
tosylate precursor OTs-P6 (4.4 mg) in anhydrous DMSO (0.5 mL)
was added to the K18F/Kryptofix 2.2.2 complex. The reaction vessel
was then sealed and the reaction mixture was heated with stirring
to 140 ꢀC for a period of 10 min. Purification of the crude product
was accomplished by using semi-prep HPLC (MachereyeNagel
Nucleosil 100-7 C-18 VarioPrep Column 250 ꢁ 16 mm, EtOH/Water
60:40, flow rate 6 mL/min, Rf 12.8 min). The [18F]-P6 compound
peak was collected and diluted with deionized-H2O (100 mL) fol-
lowed by trapping on a C-18 Sep-Pak. The final radiolabeled
product [18F]-P6 was then eluted off of the Sep-Pak directly into the
final vial using ethanol (200 proof) followed by saline (0.9%) (1:9 v/
v, 10 mL), product 11,803 MBq (non-decay corrected), radiochem-
ical yield 18% (decay corrected), radiochemical purity 95% (by
radio-HPLC), Specific activity: 176,305 MBq/mmol. A radio-HPLC
chromatogram of a typical radiosynthesis is shown in Fig. 5.
4.1.5. 5-(Fluoromethyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole
(
19F-P6)
Yellow solid (65%). Mp 82.9e84.6 ꢀC. FT-IR (neat): 3143, 3059,
2963, 2925, 2853, 1606, 1518, 1447, 1414, 1372, 1261, 1206, 1130,
1093, 1018, 930, 899, 797, 700 cmꢂ1. 1H NMR (400 MHz, CDCl3,
):
d
7.50e7.46 (m, 3H, aromatic protons); 7.39e7.35 (m, 2H, aromatic
protons); 6.37 (d, J ¼ 3.7 Hz, 1H, furyl proton); 6.18 (d, J ¼ 3.7 Hz, 1H,
furyl proton); 5.31 (d, 2H, J ¼ 48 Hz, CH2). 13C NMR (100 MHz, CDCl3,
d
): 162.9 (d, 2JCeF ¼ 16.8 Hz), 152.4 (d, 4JCeF ¼ 3.8 Hz), 142.7, 138.9,
129.9, 129.1, 128.9, 127.5, 119.4 (d, 3JCeF ¼ 5.3 Hz), 114.3, 108.0, 72.2
(d, JCeF ¼ 169.4 Hz). 19F NMR (376 MHz, CDCl3,
d
): ꢂ146.7 (t,
1
J ¼ 48 Hz). GCeMS (70 eV) m/z (rel. int.): 279 [M (37Cl)þ, 35], 277 [M
(
35Cl)þ, 100], 246 (8), 244 (23), 218 (9), 216 (26), 190 (22), 188 (67),
153 (31), 152 (28), 129 (62), 128 (10), 127 (17), 89 (28), 77 (10), 73
(9), 63 (10). Anal. Calc. for C14H9ClFNO2: C, 60.56; H, 3.27; N, 5.04.
Found: C, 60.46; H, 3.82; N, 4.46.
4.1.6. Synthesis of [3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]
methyl-4-methylbenzenesulfonate (OTs-P6)
Triethylamine (0.8 mL, 5.854 mmol) and p-toluensulfonic an-
hydride (954 mg, 2.926 mmol) were added to a solution of com-
pound HO-P6 [23,25] (236 mg, 0.858 mmol) in CH2Cl2 (20 mL) at
4.3. Biology
0
ꢀC contained in a round bottom flask equipped with a magnetic
stirrer. After 2.5 h the reaction was stopped by adding H2O (10 mL)
and the two phases were separated. The aqueous layer was washed
three times with EtOAc. The combined organic extracts were dried
over anhydrous Na2SO4 and the solvent was removed under
reduced pressure. Column chromatography (silica gel, mobile
phase: petroleum ether/ethyl acetate ¼ 8:2) of the residue (370 mg)
afforded the product as a white solid (256 mg, 70% yield).
4.3.1. Human ovarian cancer cell (OVCAR 3) viability assay
Human adenocarcinoma cells NIH:OVCAR-3 (HTB-161) (ATCC,
passage number 8e20, mycoplasma-negative by pcr mycoplasma
detection assay) were grown at 37 ꢀC in a humidified incubator
4.1.7. [3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-yl]methyl-4-
methylbenzenesulfonate (OTs-P6)
White solid (70%). 1H NMR (600 MHz, DMSO-d6,
d): 7.67e7.66
(m, 2H, aromatic protons); 7.44e7.40 (m, 5H, aromatic protons);
7.24e7.23 (m, 2H, aromatic protons); 6.59 (d, J ¼ 3.4 Hz, 1H, furyl
proton); 6.44 (d, J ¼ 3.4 Hz, 1H, furyl proton); 5.12 (s, 2H, CH2OTS);
2.4 (s, 3H, CH3). 13C NMR (600 MHz, DMSO-d6,
d): 161.8,152.4,145.9,
142.5, 137.9, 131.8, 130.7, 130.0, 129.5, 129.3, 128.1, 127.1; 115.2;
109.5; 60.5; 21.5.
4.2. Radiomimetic synthesis of the [19F]-P6
A solution of KF (7.8 mg) in 400
mixture of 29 mg Kryptofix 2.2.2 in 1.5 mL of acetonitrile and K2CO3
1 M (29 L). The reaction mixture was stirred for 5 min at room
mL of water was added to a
m
temperature. The solvents were evaporated using a stream of argon
for 10 min at 85 ꢀC and co-evaporated to dryness with CH3CN
(400 mL for two times) and the solution was stirred at room tem-
perature under a stream of argon for another 20 min at 85 ꢀC to
remove the residual traces of water. To the dried KF/Kryptofix
complex, a solution of 8 mg of the tosylate precursor OTs-P6 in
DMSO (2 mL) was added. The reaction mixture was allowed to react
at 150 ꢀC for 20 min and subsequently cooled to room temperature.
The reaction mixture was diluted with 5 mL of water and extracted
with ethyl acetate (three times). Organic phases were washed with
water for three times and then dried over anhydrous Na2SO4; the
solvent was removed under reduced pressure. Column chroma-
tography (silica gel, mobile phase: hexane/ethyl acetate ¼ 8:2)
afforded the product in 50% yield.
Fig. 5. HPLC chromatogram of
[ [
18F]-P6 compared with cold standard 19F]-P6
(Rf ¼ 12.8 min). The HPLC analysis was performed using MachereyeNagel Nucleosil
100-7 C-18 VarioPrep Column 250 ꢁ 16 mm, mobile phase EtOH/Water ¼ 60:40 and
flow rate ¼ 6 mL/min.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
567
with 5% CO2 in RPMI 1640 þ 10% FBS. Cells were plated in growth
medium at 7000e8000 cells per well (100 L) in 96 well plates
(Sarstedt) and allowed to attach for 24 h. No cells were plated in the
and Accreditation of Laboratory Animal Care (AAALAC)-approved
animal care facility.
m
In vivo stability of compound [19F]-P6 was evaluated by
analyzing the tissue samples on an LCeESI-MS system. A group of
four animals (nude mice) were injected with Human Ovarian
Cancer 3 (OVCAR 3) cells on the left flank subcutaneously. The
tumor xenografts were allowed to grow to approximately
750 mm3 for 2 months. The nude mice bearing OVCAR-3 xeno-
grafts were dosed with intraperitoneal (i.p.) injection of [19F]-P6
(2 mg/kg) dissolved in dimethylsulfoxide (DMSO). Surgery was
performed to collect tumors and normal leg muscles. The tissue
samples were weighed, and snap-frozen at ꢂ80 ꢀC for storage. For
analysis, the frozen tissue was thawed and digested with Pro-
teinase K (Sigma) overnight. An aliquot of the digestion solution
was extracted with 1.2 volumes of EtOAc/n-Hexane (2:1). The
organic layer was removed, dried and reconstituted in MeOH and
water. The unknown samples were quantitated against a 5-point
standard curve, which was subjected to the Proteinase K diges-
tion and liquideliquid extraction. The compounds were separated
in reverse-phase mode on a C18 column (5 ꢁ 0.2 cm, Phenomenex)
using gradient elution. Mobile phase component A was water and
B was acetonitrile, each with 0.1% acetic acid. The gradient used
was 50% B to 80% B over 4 min, followed by a brief hold and return
to initial conditions. The flow rate was 0.30 mL/min. The samples
were analyzed on an LCeESI-MS system (Thermo) and compound
first column. Fresh RPMI 1640 þ 10% FBS þ penicillin/streptomycin
(100
mL) containing the final concentrations of DMSO or test
compound dilutions in DMSO (DMSO final concentration, 0.1%) was
replaced in each well. After 48 h of cell growth with respective
treatments, WST-1 Cell Proliferation Reagent (Roche,11644807001)
was added (10
45e60 min at 37 ꢀC. The formazan crystals were solubilized using
100 L of DMSO and the absorbance values were read (Molecular
mL) to each well, then the plate was incubated for
m
Devices) at 450 nm. Six replicates were used in the calculations per
treatment concentration in duplicate experiments.
4.3.2. [1-14C]-Arachidonic acid TLC COX activity assay by using
purified oCOX-1 and mCOX-2 enzymes
COX inhibition screening assay to evaluate compounds as
competitive inhibitors: time- and concentration-dependent inhi-
bition reactions were performed by pre-incubating inhibitor and
hematin-reconstituted enzyme in 100 mM TriseHCl buffer with
500
m
M phenol for 17 min at room temperature followed by a 3 min
incubation at 37 ꢀC. Following the addition of 5
m
M [1-14C]-AA
(wKm of arachidonate), samples were incubated for 30 s at 37 ꢀC,
and the reactions were then terminated by extraction with diethyl
ether/methanol/citrate (30:4:1). The extracts were analyzed for
substrate consumption by thin-layer chromatography as previously
described [35]. All inhibitor concentrations for 50% enzyme activity
(IC50) were determined by nonlinear regression analysis using
Graphpad Prism software and were the average of at least two in-
dependent experiments. All inhibitors were prepared as stock so-
lutions in dimethylsulfoxide (DMSO), and diluted into reaction
buffer so that the final DMSO concentration was 2.5% in all samples.
Reactions were run with hematin-reconstituted proteins at final
enzyme concentrations adjusted to give approximately 30% sub-
strate consumption. AA was prepared as a stock solution in 0.1 N
NaOH.
[
19F]-P6 was detected via selected reaction monitoring. The intact
parent compound [19F]-P6 was identified in the tumors on an
average of 63 pmol/g tissue (n ¼ 4) (Fig. 4). A minimal or no
amount of the compound [19F]-P6 was detected in the normal leg
muscles. Also, no defluorinated metabolite (compound HO-P6)
was detected in any of the tissue samples analyzed, suggesting
that compound [19F]-P6 remained intact in the biological envi-
ronment for long enough period of time to be taken up by the
COX-1-expressing tumors (Fig. 4).
4.5. In vivo PET-CT investigation by [18F]-P6
4.3.3. In vitro OVCAR-3 cell assay
Human ovarian adenocarcinoma cells, OVCAR 3, passage 13e17,
mycoplasma negative by PCR detection method (Sigma VenorGem)
were grown in RPMI 1640 (Invitrogen/Gibco) þ 10% FBS (Atlas) to
70% confluence. Cells were plated in 6-well plates (Sarstedt) and
grown to w60% confluency. Warm HBSS/Tyrodes 1:1 (2 mL) was
replaced in each well, and the cells were treated with inhibitor
We evaluated the ability of [18F]-P6 to target COX-1 in human
ovarian tumor xenografts (Fig. 2). Female nude mice, NU-Fox1nu,
were purchased at 6e7 weeks of age from Charles River Labs.
Human OVCAR-3 cells were trypsinized and resuspended in cold
PBS containing 30% Matrigel such that 1 ꢁ 106 cells in 100
mL were
injected subcutaneously on the left flank. The tumor xenografts
were allowed to grow to approximately 750 mm3. The OVCAR-3
xenografts required 2e3 months of growth. Mice bearing
OVCAR-3 xenografts were dosed with intraperitoneal injection
dissolved in DMSO (0.2e50
37 ꢀC followed by the addition of [1-14C]-arachidonic acid [8
w2035 MBq/mmol, Perkin Elmer] for 30 min at 37 ꢀC. Aliquots
(400 L) were removed and the reactions were terminated by
solvent extraction in 400 L ice-cold Et2O/CH3OH/1 M citrate, pH
mM, final concentration) for 30 min at
m
M,
m
with [18F]-P6 (100
mL, 7.4 MBq), formulated in dimethylsulfoxide
m
(10%), ethanol (40%) and sterile saline (50%). Four hours post-
injection, the animals were anesthetized with 2% isoflurane and
positioned in the microPET Focus 220 (Siemens), and 30 min
acquisition (24 projections ꢁ 60 s per projection) was started. CT
images of the hips were then produced by the microCAT II
(Siemens, Knoxville, TN) at X-ray beam intensity of 500 mA and
tube voltage of 80 kVp. The PET images were reconstructed using
an OSEM3D/MAP algorithm into transaxial slices (128 ꢁ 128 ꢁ 95)
with voxel sizes of 0.095 cm ꢁ 0.095 cm ꢁ 0.08 cm. The PET and CT
images were co-registered to each other on the basis of bed po-
sition. After imaging, the mice were sacrificed by isoflurane over-
dose. The OVCAR 3 tumors were removed and weighed, and
radioactivity associated with each tumor was counted with a well
gamma counter. After normalization of uptake (Bq/g), the OVCAR 3
tumors showed approximately an 8-fold higher uptake of the
radiotracer compared with normal leg muscle as shown in Fig. 2
(n ¼ 3, p ¼ 0.01).
4.0 (30:4:1). The organic phase was spotted on a 20 ꢁ 20 cm TLC
plate (EMD Kieselgel 60, VWR). The plate was developed in EtOAc/
CH2Cl2/glacial AcOH (75:25:1), and radiolabeled prostaglandins
were quantified with a radioactivity scanner (Bioscan, Inc., Wash-
ington, D.C.). The percentage of total products observed at different
inhibitor concentrations was divided by the percentage of products
observed for cells pre-incubated with DMSO.
4.4. In vivo stability of compound [19F]-P6 and its tumor vs normal
leg muscle distribution
Animal studies were conducted in accordance with the stan-
dards of humane animal care described in the NIH Guide for the
Care and Use of Laboratory Animals, using protocols approved by
the Vanderbilt University institutional animal care and use com-
mittee. Animals were housed in an Association for the Assessment

568
M.G. Perrone et al. / European Journal of Medicinal Chemistry 80 (2014) 562e568
growth factor production in ovarian cancer, Cancer Research 63 (2003)
Acknowledgment
906e911.
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This work has been supported by a grant from the National In-
stitutes of Health, USA (CA128323) and Ministero dell’Istruzione
dell’Università e della Ricerca (Italy, PRIN 20097FJHPZ_001), and a
graduate scholarship to PM from University of Bari (Italy). Authors
are grateful to Mr. Donel of the Department of Radiology and
Radiological Sciences for radio-HPLC analysis optimization condi-
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