Fluorophore-labeled cyclooxygenase-2 inhibitors for the imaging of cyclooxygenase-2 overexpression in cancer: Synthesis and biological studies

Article abstract of DOI:10.1002/cmdc.201300355

A group of cyclooxygenase-2 (COX-2)-specific fluorescent cancer biomarkers were synthesized by linking the anti-inflammatory drugs ibuprofen, (S)-naproxen, and celecoxib to the 7-nitrobenzofurazan (NBD) fluorophore. In vitro COX-1/COX-2 inhibition studies

Full text of DOI:10.1002/cmdc.201300355

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DOI: 10.1002/cmdc.201300355
Fluorophore-Labeled Cyclooxygenase-2 Inhibitors for the
Imaging of Cyclooxygenase-2 Overexpression in Cancer:
Synthesis and Biological Studies
[
a, b]
[a, b]
[a]
[a]
Atul Bhardwaj,
Jatinder Kaur,
Frank Wuest,* and Edward E. Knaus*
A group of cyclooxygenase-2 (COX-2)-specific fluorescent
cancer biomarkers were synthesized by linking the anti-inflam-
matory drugs ibuprofen, (S)-naproxen, and celecoxib to the 7-
nitrobenzofurazan (NBD) fluorophore. In vitro COX-1/COX-2 in-
hibition studies indicated that all of these fluorescent conju-
gates are COX-2 inhibitors (IC50 range: 0.19–23.0 mm) with
an appreciable COX-2 selectivity index (SIꢀ4.3–444). In this
study the celecoxib–NBD conjugate N-(2-((7-nitrobenzo[c]-
[1,2,5]oxadiazol-4-yl)amino)ethyl)-4-(5-(p-tolyl)-3-(trifluorometh-
yl)-1H-pyrazol-1-yl)benzenesulfonamide (14), which displayed
the highest COX-2 inhibitory potency and selectivity (COX-2
IC =0.19 mm; SI=443.6), was identified as an impending
COX-2-specific biomarker for the fluorescence imaging of
cancer using a COX-2-expressing human colon cancer cell line
(HCA-7).
50
Introduction
The high prevalence of cancer has contributed to a tremendous
socioeconomic burden on healthcare systems worldwide. Ac-
cording to World Health Organization (WHO) statistical data in
ibuprofen (3) have broad inhibitory profiles. This nonselective
inhibition that blocks the expression of both COX isozymes is
responsible for their gastrointestinal (GI), ulcerogenic, hepatic,
[6–10]
2008, 7.6 million people died of cancer, accounting for 13% of
and renal toxicity.
The discovery of the inducible COX-2 iso-
all deaths worldwide. Recognition of the early signs (symp-
toms) of cancer and careful monitoring of chemotherapies can
decrease cancer mortality. It is estimated that 30% of cancer
deaths can be prevented. In this regard, imaging techniques
have played a pivotal role in stimulating the growth and evolu-
tion of disease-specific imaging probes that can be used for
selective detection of disease biomarkers. There has been
a recent increase in interest in the development of molecular
imaging probes that can sense the overexpression of cyclooxy-
genase-2 (COX-2) in cancer cells. The cyclooxygenases (COX-
zyme in the early 1990s lent credence to the idea that selective
inhibition of COX-2 would be an attractive approach for the
safe treatment of inflammatory conditions. The subsequent de-
velopment and US Food and Drug Administration (FDA) appro-
val of selective COX-2 inhibitors (COXIBs, examples shown in
Figure 1) provided new non-ulcerogenic drugs for the safe
treatment of inflammation, arthritis, and moderate-to-severe
[3]
pain. Despite their initial commercial success, the selective
COX-2 inhibitors rofecoxib (Vioxx) and valdecoxib (Bextra) were
withdrawn from the market due to adverse cardiovascular
[11,12]
1
and COX-2) are key isozymes that catalyze the complex bio-
events associated with their use.
Nitric oxide (NO)-releas-
transformation of arachidonic acid into prostaglandins (PGs)
and thromboxanes, which are ultimately responsible for many
ing NSAIDs and selective COX-2 inhibitory prodrugs designed
to circumvent adverse cardiovascular risks have now been de-
[
1,2]
[13–16]
physiological and pathophysiological responses.
The COX-
scribed.
1
isozyme mediates homeostatic functions such as cytoprotec-
In addition to its ability to induce peripheral inflammation,
expression of the COX-2 isozyme is up-regulated in many
human cancers including gastric, breast, lung, colon, esopha-
tion of the gastric mucosa, induction of labor pain, regulation
of renal blood flow, and platelet aggregation. In contrast, the
COX-2 isozyme is mainly responsible for the production of in-
[17–20]
geal, prostate, and hepatocellular carcinomas.
Many stud-
[
3–5]
flammatory PGs that induce pain, swelling, and fever.
ies have shown the association between COX-2 overexpression
and the development of cancer. Consequently, several COX-2
Traditional nonsteroidal anti-inflammatory drugs (NSAIDs)
[21–24]
such as acetylsalicylic acid (Aspirin, 1), (S)-naproxen (2), and
inhibitors have been investigated as anticancer agents.
It
has been observed that regular intake of NSAIDs can induce
apoptosis in colon carcinoma cells, retarding cancer progres-
+
+
[
a] Dr. A. Bhardwaj, Dr. J. Kaur, Dr. F. Wuest, Dr. E. E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences
University of Alberta, Edmonton, Alberta, T6G 2E1 (Canada)
E-mail: wuest@ualberta.ca
[25–27]
sion.
Overall, pharmacological studies advocate that over-
expression of the COX-2 isozyme in cancer cells, relative to
normal neighboring tissues where COX-2 is not overexpressed,
could constitute a useful noninvasive diagnostic and therapeu-
tic strategy for the detection of cancer progression and/or
treatment. These perspectives provided the impetus to investi-
gate radioactive positron emission tomography (PET) radio-
eknaus@ualberta.ca
+
+
[
b] Dr. A. Bhardwaj, Dr. J. Kaur
Department of Oncology, Cross Cancer Institute
University of Alberta, Edmonton, Alberta, T6G 1Z2 (Canada)
+
[
] These authors contributed equally to this work.
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furnished the respective ester product 8 or 10. Re-
moval of the Boc protecting group in compounds 8
or 10 by treatment with trifluoroacetic acid (TFA) in
dry dichloromethane at 258C afforded the respective
amino product, which, without any additional purifi-
cation, was allowed to react with NBD chloride in the
presence of dry triethylamine (TEA) in dry THF to fur-
nish the target ibuprofen–NBD conjugate 9 (brown
solid, 64% yield, lem =548 nm) or (S)-naproxen–NBD
conjugate 11 (yellow solid, 62% yield, lem =555 nm).
The
4-(5-para-tolyl-3-trifluoromethylpyrazol-1-yl)
benzene sulfonyl chloride compound 12 was synthe-
sized by using a previously reported synthetic meth-
[
32]
Figure 1. Representative examples of nonselective COX-1/COX-2 inhibitors 1–3 and selec-
tive COX-2 inhibitors 4–6.
odology.
Condensation of this sulfonyl chloride
precursor 12 with N-Boc-ethylenediamine in the pres-
pharmaceuticals for imaging COX-2 expression; such com-
pounds are suitable for in vivo imaging, but they require
[
28,29]
a more challenging radiosynthesis.
Although the discovery of COX-2-specific fluorescent cancer
biomarkers is still in its early stages, Marnett and colleagues re-
cently reported that fluorescence labeling of COX-2 overex-
[30]
pression is a useful technique for the detection of cancer.
Fluorescently labeled COX-2 inhibitors are useful optical
probes for targeted imaging of COX-2 in cells and small ani-
mals, as well as for clinical imaging of tissues suitable for topi-
cal or endoluminal illumination, such as the esophagus and
colon. In this context, we recently reported a celecoxib–rhoda-
mine conjugate 7, which was investigated as a COX-2-imaging
[
31]
cancer biomarker. Unfortunately, the weak COX-2 inhibitory
potency and poor imaging results limited the use of com-
pound 7 as a potential biomarker. As part of this ongoing pro-
gram, we now describe the synthesis of a group of “fluores-
cent conjugates of COX inhibitors” wherein the 7-nitrobenzo-
furazan (NBD) fluorophore is coupled to the anti-inflammatory
drugs ibuprofen, (S)-naproxen, and celecoxib (see structures
and illustration in Figure 2) and their evaluation as in vitro
COX-1 and COX-2 inhibitors, as well as their suitability as fluo-
rescence imaging agents for the selective visualization of COX-
2
activity in HCA-7 cells (COX-2-expressing human colon
cancer cells).
Figure 2. Structures of COX-2-inhibiting fluorescent compounds 7, 9, 11, 14
and 17 (top) and a pictorial representation illustrating the design of fluores-
cent conjugates of COX-2 inhibitors (bottom).
Results and Discussion
Chemistry
ence of dry TEA in dry THF provided the N-Boc-protected cele-
coxib derivative 13. The Boc protecting group was removed by
treatment of 13 with TFA in dry dichloromethane to give an
amino product, which, without further purification, was al-
lowed to react with NBD chloride in the presence of dry TEA at
The methods used to synthesize the target compounds 9, 11,
14, and 17 are illustrated in Scheme 1. 4-Chloro-7-nitro-1,2,3-
benzoxadiazole (NBD chloride, 15) was used as a fluorophore
to prepare fluorescent conjugates of the known anti-inflamma-
tory drugs ibuprofen (compound 9), (S)-naproxen (11), and cel-
ecoxib (14 and 17) through a synthetic strategy similar to that
previously reported by our research group (Scheme 1). Reac-
tion of ibuprofen (3) or (S)-naproxen (2) with N-Boc-ethanola-
mine in the presence of N,N’-dicyclohexylcarbodiimide (DCC)
and 4-dimethylaminopyridine (DMAP) in dry dichloromethane
258C to furnish the celecoxib–NBD conjugate 14 (lem
=
550 nm) as a yellow solid in 69% yield.
[
31]
The precursor compound 4-amino-7-nitrobenzofurazane (16)
required for synthesis of the celecoxib–NBD conjugate 17 was
[33,34]
prepared according to published procedures.
Reaction of
NBD chloride 15 with ammonium hydroxide solution (30% in
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Scheme 1. Reagents and conditions: a),c) DCC, DMAP, dry CH
2
Cl
2
, 258C, argon atmosphere, 3 h; b),d) TFA, dry CH
Cl , 258C, 6 h; NBD-Cl, dry TEA, dry THF, argon
2
O), MeOH, 258C, 24 h; h) NaH, dry THF, compound 12, 0–258C, 2 h.
2 2
Cl , 258C, 6 h; NBD-Cl, dry TEA, dry THF,
argon, 258C, 1 h for compound 9, and 2 h for compound 11; e) dry TEA, dry THF, 258C, 5 h; f) TFA, dry CH
atmosphere, 258C, 30 min; g) NH OH solution (30% in H
2
2
4
water), with methanol as solvent, provided compound 16. Sub-
sequent reaction of 16 with the sulfonyl chloride analogue of
celecoxib (compound 12) in the presence of sodium hydride
and dry THF afforded the target compound 17 (lem =545 nm)
as a yellow solid in 65% yield.
the nonselective COX-1/COX-2 inhibitors ibuprofen and (S)-
naproxen are coupled to the NBD fluorophore via an ethylami-
no spacer, showed greater COX-2 selectivity than the parent
NSAID. This enhanced COX-2 selectivity may be attributed to
3
the larger molecular volumes of conjugates 9 (370.1 ꢁ ) and 11
3
3
(
353.9 ꢁ ) relative to the parent NSAIDs ibuprofen (211.2 ꢁ )
3
and (S)-naproxen (214.0 ꢁ ) (Table 1). Accordingly, the larger
size of the ibuprofen–NBD and (S)-naproxen–NBD conjugates 9
and 11 may hinder their entry into the smaller COX-1 binding
COX-1 and COX-2 inhibition studies
In vitro COX-1/COX-2 inhibition studies showed that com-
3
pounds 9, 11, 14, and 17 are more potent inhibitors of COX-2
site (V=316 ꢁ ) relative to the COX-2 binding site, which is
3
(IC50 range: 0.19–23 mm) than COX-1 (IC50 range: 84–100 mm).
~25% larger (V=394 ꢁ ), resulting in a higher COX-2 selectivity
[
35]
Conjugates 9 and 11, equipped with an NBD fluorophore,
showed higher selectivity for COX-2 than their respective
parent NSAIDs ibuprofen (3) or (S)-naproxen (2) (Table 1). The
fluorescent ibuprofen conjugate 9 showed a higher COX-2 in-
hibitory activity and selectivity (COX-2 IC =1.6 mm, SI>62)
index.
Celecoxib–NBD conjugates 14 and 17 are less potent inhibi-
tors of COX-2 than the parent drug celecoxib (4, COX-2 IC =
5
0
0.07 mm). The X-ray crystal structure of celecoxib bound to the
COX-2 isozyme indicates that the SO NH2 COX-2 pharmaco-
50
2
than the (S)-naproxen conjugate 11 (COX-2 IC =7.9 mm, SI=
phore inserts deep into the secondary pocket of the COX-2
binding site, suggesting that this may be an important deter-
minant of its high COX-2 inhibitory potency and selectivity.
The direct attachment of the bulky fluorescent NBD group
to the sulfonamide nitrogen in conjugate 17 has a deleterious
50
11.7). Among the celecoxib fluorescent conjugates 14 and 17,
compound 14, with an additional ethylamino (CH CH NH)
2
2
spacer between the NBD aryl ring and the sulfonamide nitro-
gen, showed much higher COX-2 potency and selectivity
(
COX-2 IC =0.19 mm, SI=443.6) than compound 17, which
effect on COX-2 potency and selectivity (COX-2 IC =23 mm,
50
50
lacks this spacer (COX-2 IC =23.0 mm, SI>4.3). This ethylami-
no spacer is an important determinant for COX-2 inhibitory po-
SI>4.3) relative to the parent celecoxib, which has a free
sulfonamide group. In comparison, conjugate 14, with an eth-
ylamino spacer between the sulfonamide nitrogen atom and
50
tency and selectivity (14>17). Conjugates 9 and 11, in which
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by confocal microscopy. Good
cellular uptake of conjugate 14
was observed in COX-2-overex-
pressing HCA-7 cells (Figure 3b–
d). No fluorescence was ob-
served after incubation of HCA-7
cells with phosphate-buffered
saline (PBS) control (Figure 3a)
or NBD-Cl (Figure 3 f) under simi-
lar experimental conditions. Pre-
treatment of HCA-7 cells with
the potent and selective COX-2
inhibitor celecoxib (4) at 100 mm
blocked the uptake of the cele-
coxib–NBD conjugate 14 by
HCA-7 cells (Figure 3e), and no
fluorescence labeling of the
COX-2 isozyme was observed.
The results of these imaging ex-
periments indicate that HCA-7
cells, which express high levels
of COX-2, exhibited strong fluo-
rescence labeling by the celecox-
ib–NBD conjugate 14 at a con-
centration of 10 mm. This new
lead compound 14 shows signif-
icant improvements in COX-2 se-
lectivity and fluorescence imag-
Table 1. In vitro COX-1 and COX-2 isozyme inhibition, molecular volume, and logP data.
[
a]
[b]
3
[c]
[d]
Compd
Structure
IC50 [mm]
COX-1
SI
V [ꢁ ]
logP
COX-2
1.6
9
>100
>62.5
370.1
353.9
4.7
11
92.5
7.9
11.7
4.7
5.2
1
4
84.3
0.19
443.6
457.7
1
7
>100
23.0
>4.3
411.7
5.5
[
31]
7
3
2
4
celecoxib–rhodamine B conjugate
ibuprofen
(S)-naproxen
>100
2.9
3.9
1.1
12.4
0.07
>25.5
714.5
211.2
214.0
298.6
8.6
3.4
3.3
3.6
2.6
0.01
110
0.18
7.7
celecoxib
ing properties relative to conju-
[
a] In vitro concentration of test compound required to produce 50% inhibition of ovine COX-1 or human re-
combinant COX-2; results are the average of two determinations acquired using an enzyme immunoassay kit
cat. no. 560131, Cayman Chemicals Inc., Ann Arbor, MI, USA), and the deviation from the mean is <10% of
[31]
gate 7, previously reported.
A
control study performed using
COX-2-negative HCT-116 cells va-
lidated the hypothesis that the
fluorescence imaging of COX-2
expression in HCA-7 colon
(
the mean value. [b] In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50). [c,d] Volume and logP of all mole-
cules were calculated using Molinspiration Cheminformatics software (http://www.molinspiration.com).
the NBD moiety, retained high COX-2 inhibitory potency and
cancer cells with 14 is mediated by COX-2.
selectivity (IC =0.19 mm, SI=443). These differences in COX-2
50
inhibitory potency and selectivity between conjugates 14 and
7 suggest that there are important differences in their bind-
Fluorescence imaging with COX-2-negative HCT-116 cells
1
ing interaction with residues in the COX-2 binding site. LogP
molecular property calculations indicated that conjugates 9
To determine whether the fluorescence labeling of compound
14 depends on COX-2 isozyme expression, a cellular uptake ex-
periment with HCT-116 (COX-2-negative human colon cancer)
cells was performed. DAPI was used as nuclear stain. HCT-116
cells were incubated with compound 14 at either 10 or
100 mm at 378C prior to imaging by confocal microscopy. Im-
portantly, no labeling was observed at either concentration of
compound 14 (Figure 4b,c), and as expected, no fluorescence
labeling was observed after incubation with PBS control (Fig-
ure 4a). These results indicate that the uptake of celecoxib–
NBD conjugate 14 is mediated by COX-2.
(logP=4.7), 11 (logP=4.7), 14 (logP=5.2), and 17 (logP=5.5)
are much more lipophilic than ibuprofen (logP=3.4), (S)-nap-
roxen (logP=3.3), and celecoxib (logP=3.6), as illustrated in
Table 1.
Fluorescence imaging of COX-2 expression in HCA-7 colon
cancer cells
Within the group of four fluorescent conjugates (9, 11, 14, and
17), the celecoxib–NBD conjugate 14 was selected for fluores-
cence imaging investigations based on its appreciable in vitro
COX-2 inhibitory potency. A fluorescence labeling experiment
was performed with HCA-7 cells (COX-2-expressing human
colon cancer cells). 4,6-Diamidino-2-phenylindole (DAPI) was
used as a nucleus-specific stain. HCA-7 cells were incubated
with 10 mm celecoxib–NBD conjugate 14 at 378C and imaged
Conclusions
A new group of fluorescent conjugates wherein the nonselec-
tive COX-1/COX-2 inhibitors ibuprofen and (S)-naproxen, or se-
lective COX-2 inhibitor celecoxib, were coupled either directly
or via an ethylamino linker group to an NBD fluorophore were
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Figure 3. Fluorescence labeling of COX-2-expressing (HCA-7) cells: Cells were treated with a) PBS (control) or b)–d) 10 mm celecoxib–NBD conjugate 14. In
panel b), the stained nucleus is shown only (perinuclear staining is not shown); panel c) represents perinuclear staining due to cellular uptake of conjugate
1
4 (nuclear staining not shown); panel d) represents a merged image of both nuclei and perinuclear staining as a result of cellular uptake of conjugate 14.
e) Cells pre-treated with 100 mm celecoxib before treatment with conjugate 14; f) cells treated with 10 mm NBD-Cl. Cells were imaged by confocal microscopy,
and all images at the same scale as indicated in panel a).
Figure 4. Uptake of compound 14 in HCT-116 cells: Cells were treated with a) PBS (control), b) 100 mm celecoxib–NBD conjugate 14, or c) 10 mm conjugate
1
4. Cells were imaged by confocal microscopy, and all images at the same scale as indicated in panel a).
synthesized for biological evaluation. In vitro COX-1/COX-2 iso-
zyme inhibition structure–activity data showed that the ibu-
profen–NBD conjugate 9 is a more selective COX-2 inhibitor
than ibuprofen, the (S)-naproxen–NBD conjugate 11, unlike
naproxen, which is a selective COX-1 inhibitor, exhibited selec-
tive COX-2 inhibition, and the celecoxib–NBD conjugate 14
emerged as a lead compound showing the most potent COX-2
inhibitory potency and selectivity (COX-2 IC =0.19 mm, SI=
50
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4
43.6). Furthermore, fluorescence imaging experiments using
the cancer cell lines HCA-7 (overexpressing COX-2) and HCT-
16 (non-expressing COX-2) identified the celecoxib–NBD con-
6.49 mmol) was added to a solution of compound 10 (500 mg,
1
.33 mmol) in dry CH Cl2 (10 mL), and the reaction mixture was
2
stirred at 258C for 6 h. Excess acid and solvent were removed
under vacuo, and the sample was dried in vacuo overnight. The
amino product obtained, without any further purification, was dis-
solved in dry THF (10 mL) under an argon atmosphere, and to this
solution dry TEA (0.4 mL, 2.86 mmol) and NBD- Cl (220 mg,
1
jugate 14 as a potential fluorescence imaging agent for the la-
beling of overexpressed COX-2 in colon cancer cells.
1
.1 mmol) were added. The reaction mixture was stirred at 258C
for 1 h, H O was added, and the mixture was extracted with CH Cl
2
Experimental Section
2
2
(
3ꢂ25 mL). The combined organic extracts were washed with
General
brine and dried over anhydrous Na SO . Removal of the solvent in
2
4
Melting points were measured with a Thomas–Hoover capillary ap-
paratus and are uncorrected. H and C NMR spectra were mea-
sured on a Bruker AM 600 NMR spectrometer using CDCl3 or
vacuo furnished a yellow liquid which was further purified by
column chromatography using EtOAc/hexane (1:2, v/v) as eluent to
1
13
give the target compound 11 in 62% yield as a yellow solid; mp:
1
[
D ]DMSO as solvent. Chemical shifts are given in parts per million
6
135–1368C; H NMR (600 MHz, CDCl ): d=1.48 (d, J=7.2 Hz, 3H,
3
with tetramethylsilane (TMS) as an internal reference. MS data
were recorded on a Waters Micromass ZQ 4000 mass spectrometer
using ESI mode. The purity of the compounds was established by
elemental analyses, which were performed for C, H, and N by the
Microanalytical Service Laboratory, Department of Chemistry, Uni-
versity of Alberta. Compounds showed a single spot on Macherey–
Nagel Polygram Sil G/UV254 silica gel plates (0.2 mm) using a low,
medium, and highly polar solvent system, and no residue remained
after combustion, indicating a purity >98%. Column chromatogra-
CH ), 3.54–3.59 (m, 2H, CH CH NH), 3.76–3.79 (m, 1H, CHCH ), 3.84
3
2
2
3
(s, 3H, OCH ), 4.21–4.25 (m, 1H, OCHH’), 4.54–4.58 (m, 1H, OCHH’),
3
5.77 (d, J=9.0 Hz, 1H, H-5 of NBD), 5.94 (brs, 1H, NH), 6.83 (d, J=
2.4 Hz, 1H, naphthyl H-5), 6.97 (dd, J=9.0, 2.4 Hz, 1H, naphthyl H-
7), 7.15 (d, J=1.8 Hz, 1H, naphthyl H-1), 7.36 (d, J=8.4 Hz, 1H,
naphthyl H-4), 7.40–7.44 (m, 2H, naphthyl H-3, H-8), 8.04 ppm (d,
13
J=9.0 Hz, 1H, H-6 of NBD); C NMR (150 MHz, CDCl ): d=17.96
3
(CH ), 42.97 (CH NH), 45.36 (CHCH ), 55.33 (OCH ), 61.80 (OCH ),
3
2
3
3
2
98.50 (NBD C5), 105.26 (naphthyl C5), 119.40 (naphthyl C7), 123.10
(NBD C7), 125.62 and 125.67 (naphthyl C1, C3), 127.16 (naphthyl
C4), 128.52 (naphthyl ArC), 128.74 (naphthyl C8), 133.55 and 134.78
(naphthyl ArC), 135.72 (NBD C6), 143.22, 143.41 and 143.88 (NBD
ArC), 157.83 (naphthyl C6), 174.86 ppm (CO); Fluorescence (1%
phy was performed on a Combiflash R system using a gold silica
f
column. All other reagents, purchased from the Aldrich Chemical
Co. (Milwaukee, WI, USA), were used without further purification.
Compounds 8, 10, 12, 13, and 16 were synthesized by using previ-
[31,32]
+
ously reported procedures.
DMSO in PBS): lem =555 nm; ESIMS: 437 [M+H] ; Anal. calcd for
C H N O : C 60.55, H 4.62, N 12.84, found: C 60.51, H 4.78, N
22
20
4
6
2
-(4-Isobutylphenyl)propionic
acid
ester
2-(7-nitrobenzo-
(9): TFA (0.5 mL,
21.0
1
2.54; [a]
= +63.54 (0.500, CHCl3).
D
[1,2,5]oxadiazol-4-ylamino)ethyl
6
.49 mmol) was added to a solution of compound 8 (500 mg,
1
.43 mmol) in dry CH Cl2 (10 mL), and the reaction mixture was
2
N-(2-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)-4-(5-(p-
tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide
stirred at 258C for 6 h. Upon completion of the reaction (TLC moni-
toring), excess acid and solvent was removed under vacuum, and
the residue was dried in vacuo overnight. The amino product ob-
tained, without further purification, was dissolved in dry THF
(14): TFA (0.5 mL, 6.49 mmol) was added to a solution of com-
pound 13 (500 mg, 0.95 mmol) in dry CH Cl (10 mL), and the reac-
2
2
tion mixture was stirred at 258C for 6 h. Excess TFA and solvent
were removed under vacuo, and the sample was dried in vacuo
overnight. The amino product obtained, without any further purifi-
cation, was dissolved in dry THF (10 mL) under an argon atmos-
phere, and to this solution dry TEA (0.375 mL, 2.69 mmol) and
NBD-Cl (200 mg, 1.0 mmol) were added. The reaction mixture was
(
10 mL) under an argon atmosphere, and then a solution of NBD-
Cl (200 mg, 1.0 mmol) in dry TEA (0.4 mL, 2.86 mmol) was added.
The reaction was allowed to proceed with stirring at 258C for 1 h,
H O was added, and the mixture was extracted with CH Cl (3ꢂ
2
2
2
2
5 mL). The combined organic extracts were washed with brine
prior to drying over anhydrous Na SO . Removal of the solvent in
vacuo furnished a yellow liquid which was further purified by
column chromatography using EtOAc/hexane (1:2, v/v) as eluent to
give the target compound 9 in 64% yield as a brown solid; mp:
2
4
stirred at 258C for 30 min, H O was added, and the mixture was ex-
2
tracted with CH Cl2 (3ꢂ25 mL). The combined organic extracts
2
were washed with brine and dried over anhydrous Na SO . Remov-
2
4
1
al of the solvent in vacuo furnished a yellow liquid which was fur-
ther purified by column chromatography using EtOAc/hexane (1:1,
9
0–928C; H NMR (600 MHz, CDCl ): d=0.77 (d, J=6.6 Hz, 6H,
3
(
CH ) CHCH ), 1.41 (d, J=7.2 Hz, 3H, CHCH ), 1.67–1.72 (m, 1H,
3 2 2 3
v/v) as eluent to give the title compound 14 in 69% yield as
(
CH ) CHCH ), 2.30 (d, J=7.2 Hz, 2H, (CH ) CHCH ), 3.61–3.66 (m,
3 2 2 3 2 2
1
a yellow solid; mp: 154–1558C; H NMR (600 MHz, [D ]DMSO): d=
6
3
1
6
H, CH CH NH+CHCH ), 4.28–4.31 (m, 1H, OCHH’), 4.42–4.46 (m,
2 2 3
2
.29 (s, 3H, CH ), 3.11–3.14 (m, 2H, SO NHCH ), 3.34–3.38 (m, 2H,
3
2
2
H, OCHH’), 6.03 (d, J=9.0 Hz, 1H, H-5 of NBD), 6.20 (brs, 1H, NH),
.93 and 7.05 (two d, J=7.8 Hz, 2H each, phenyl H-2, H-6 and H-3,
NHCH ), 6.36 (d, J=8.4 Hz, 1H, H-5 of NBD), 7.19–7.21 (m, 5H, pyra-
2
13
zole H-4, 4-methylphenyl H-3, H-5, H-2, H-6), 7.55 (d, J=8.4 Hz, 2H,
sulfonylphenyl H-2, H-6), 7.86 (d, J=8.4 Hz, 2H, sulfonylphenyl H-3,
H-5), 8.34 ppm (d, J=9.0 Hz, 1H, H-6 of NBD); C NMR (150 MHz,
CDCl ): d=18.25 (CH CH), 22.30 [(CH ) CHCH ], 30.15 [CH(CH ) ],
4
9
and C2, C6), 136.10 (NBD C6), 137.02 (phenyl C1), 141.04 (phenyl
C4), 143.44, 143.73 and 144.17 (NBD ArC), 174.99 ppm (CO); Fluo-
3
3
3 2
2
3 2
H-5), 8.05 (brs, 1H, NH), 8.53 (d, J=8.4 Hz, 1H, H-6 of NBD),
3.02 [CH CH(CH ) ], 44.86 (CH NH), 45.03 (CHCH ), 61.78 (OCH ),
2 3 2 2 3 2
8.79 (NBD C5), 124.10 (NBD C7), 126.93 and 126.51 (phenyl C3, C5
13
9
.34 ppm (brs, 1H, NH); C NMR (150 MHz, [D ]DMSO): d=20.74
6
(
CH ), 40.72 (SO NHCH ), 43.04 (CH NH), 99.10 (NBD C5), 106.18
3 2 2 2
1
(CH, pyrazole), 121.24 (q, JC,F =267 Hz, CF ), 121.61 (NBD C7),
3
À
125.25 (4-methylphenyl C1), 126.23 (sulfonylphenyl C2, C6), 127.71
sulfonylphenyl C3, C5), 128.71 (4-methylphenyl C2, C6), 129.38 (4-
methylphenyl C3, C5), 137.86 (NBD C6), 139.09 (ArC), 140.14 (ArC),
rescence (1% DMSO in PBS): lem =548 nm; ESIMS: 411 [MÀH] ;
(
Anal. calcd for C H N O : C 61.15, H 5.87, N 13.58, found: C 61.22,
2
1
24
4
5
H 5.88, N 13.65.
2
1
41.66 (ArC, N-pyrazole), 142.25 (q, JC,F =36 Hz, pyrazole C3),
2
-(6-Methoxynaphthalen-2-yl)propionic acid 2-(7-nitrobenzo-
144.00, 144.43 and 145.03 (NBD ArC), 145.23 ppm (pyrazole C5);
Fluorescence (1% DMSO in PBS): lem =550 nm; ESIMS: 588 [M+
[1,2,5]oxadiazol-4-ylamino)ethyl ester (11): TFA (0.5 mL,
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 109 – 116 114

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+
À1
H] ; Anal. calcd for C H F N O S: C 51.11, H 3.43, N 16.69, S 5.46,
n-propyl gallate as anti-fade and DAPI (50 mgmL ). The cells were
2
5
20
3
7
5
found: C 51.19, H 3.54, N 16.66, S 5.49.
imaged using corresponding lasers for visualizing DAPI (blue nucle-
ar staining) and FITC (green emission) with a Plan-Apochromat
N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)-4-(5-(p-tolyl)-3-(trifluoro-
methyl)-1H-pyrazol-1-yl)benzenesulfonamide (17): NaH (31 mg,
4
0X/1.3 oil DIC M27 lens on a Zeiss LSM 710 AxioObserver confocal
laser scanning microscope. Imaging experiments were carried out
two times using different batches of cells.
1
.29 mmol) was added to a solution of 16 (200 mg, 1.11 mmol) in
dry THF (5 mL) at 08C. This solution was stirred for 15 min, com-
pound 12 (534 mg, 1.33 mmol) in anhydrous THF (10 mL) was
added dropwise, and the reaction was allowed to proceed with
stirring at 258C for 2 h. The reaction mixture was quenched with
HCT-116 cells: HCT-116 cells (ATCC) were cultured at 378C in a hu-
midified atmosphere of 5% CO (v/v), using DMEM/F12 medium
2
supplemented with 10% FBS (Gibco), 2 mm l-glutamine (Invitro-
gen), and 1% antibiotic/antimycotic (Invitrogen). Cell growth
medium was changed every other day. Cells were treated with
0.25% trypsin/1 mm EDTA (Invitrogen) for ~5 min at room temper-
ature to dissociate cells from the culture flask, and rinsed with PBS
once after harvesting. Cells were resuspended in fresh growth
saturated aqueous NaHCO and extracted with EtOAc (3ꢂ25 mL).
3
The combined organic extracts were washed with brine, dried with
Na SO and then filtered. Removal of the solvent in vacuo fur-
2
4
nished a brown liquid which was further purified by column chro-
matography using EtOAc/hexane (2:1, v/v) as eluent to give the
6
target compound 17 in 65% yield as a yellow solid; mp: 190–
medium and seeded into a six-well plate at 1.5ꢂ10 cells per well.
1
1
918C; H NMR (600 MHz, [D ]DMSO): d=2.29 (s, 3H, CH ), 6.72 (d,
After removing media, cells were treated with celecoxib–NBD con-
jugate at 100 or 10 mm using the procedure mentioned above.
Imaging experiments were carried out two times using different
batches of cells.
6
3
J=9.0 Hz, 1H, H-5 of NBD), 7.16–7.17 (m, 5H, pyrazole H-4, 4-meth-
ylphenyl H-3, H-5, H-2, H-6), 7.47 (d, J=8.4 Hz, 2H, sulfonylphenyl
H-2 and H-6), 7.92 (d, J=8.4 Hz, 2H, sulfonylphenyl H-3, H-5),
13
8
.35 ppm (d, J=9.0 Hz, 1H, H-6 of NBD); C NMR (150 MHz,
[
1
D ]DMSO): d=21.23 (CH ), 106.38 (NBD C5), 107.35 (CH, pyrazole),
6
3
1
21.24 (q, J =267 Hz, CF ), 122.68 (NBD C7), 125.78 (4-methyl- Acknowledgements
C,F
3
phenyl C1), 126.37 (sulfonylphenyl C2, C6), 127.93 (sulfonylphenyl
C3, C5), 129.18 (4-methylphenyl C2, C6), 129.85 (4-methylphenyl
C3, C5), 137.18 (NBD C6), 139.50 (ArC), 141.29 (ArC), 142.52 (q,
We are grateful to the Canadian Institutes of Health Research
Grant no. MOP-14712 to E.E.K.) and the Dianne and Irving
(
2
JC,F =36 Hz, pyrazole C3), 143.81 (ArC, N-pyrazole), 145.18, 145.69
Kipnes Foundation. F.W. and J.K. thank the Alberta Cancer Foun-
dation for a postdoctoral fellowship.
and 149.38 (NBD ArC), 152.34 ppm (pyrazole C5); Fluorescence (1%
À
DMSO in PBS): lem =545 nm; ESIMS: 543 [MÀH] ; Anal. calcd for
C H F N O S: C 50.74, H 2.78, N 15.44, S 5.89, found: C 50.70, H
2
3
15
3
6
5
2
.66, N 15.41, S 5.80.
Keywords: cancer
·
cyclooxygenases
·
enzymes
·
fluorescence · inhibitors · medicinal chemistry
Cyclooxygenase inhibition assays
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: September 5, 2013
Revised: October 8, 2013
Published online on October 31, 2013
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 109 – 116 116