Pyrimidine-based fluorescent COX-2 inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1039/c6ob00493h

The cyclooxygenase-2 (COX-2) enzyme is overexpressed in a variety of cancers and mediates inflammatory processes that aid the growth and progression of malignancies. Three novel and selective fluorescent COX-2 inhibitors have been designed and synthesized on the basis of previously reported pyrimidine-based COX-2 inhibitors and the 7-nitrobenzofurazan fluorophore. In vitro evaluation of COX-1/COX-2 isozyme inhibition identified N-(2-((7-nitro-benzo[c][1,2,5]oxadiazol-4-yl)amino)propyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoro-methyl)-pyrimidin-2-amine (6) as a novel potent and selective COX-2 inhibitor (IC₅₀ = 1.8 μM). Lead compound (6) was further evaluated for its ability to selectively visualize COX-2 isozyme in COX-2 expressing human colon cancer cell line HCA-7 using confocal microscopy experiments.

Full text of DOI:10.1039/c6ob00493h

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Page 1 of 20
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Pyrimidine-based fluorescent COX-2 inhibitors:
Synthesis and biological evaluation
Ole Tietza,1 Jatinder Kaura,b,1 Atul Bhardwaja,b; Frank R. Wuest*a,b
aDepartment of Oncology, Cross Cancer Institute, University of Alberta, 11560 University
Avenue, T6G 1Z2, Edmonton, AB, Canada.
bDepartment of Pharmacy and Pharmaceutical Sciences, Medical Sciences Building, University
of Alberta, T6G 2H1, Edmonton, AB, Canada.
1 Ole Tietz and Jatinder Kaur equally contributed to this work.
CORRESPONDING AUTHOR: Frank Wuest, Department of Oncology, Cross Cancer Institute,
University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada
Email: wuest@ualberta.ca
Telephone: 780 989 8150
Fax: 780ꢀ432ꢀ8483
EMAIL:
Ole Tietz – ole@ualberta.ca
Jatinder Kaur – kaur2@ualberta.ca
Atul Bhardwaj – abhardwa@ualberta.ca
Frank Wuest – wuest@ualberta.ca
1

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ABSTRACT
The cyclooxygenaseꢀ2 (COXꢀ2) enzyme is overexpressed in a variety of cancers and mediates
inflammatory processes that aid the growth and progression of malignancies. Three novel and
selective fluorescent COXꢀ2 inhibitors have been designed and synthesized on the basis of
previously reported pyrimidineꢀbased COXꢀ2 inhibitors and the 7ꢀnitrobenzofurazan
fluorophore. In vitro evaluation of COXꢀ1/COXꢀ2 isozyme inhibition identified Nꢀ(2ꢀ((7ꢀnitroꢀ
benzo[c][1,2,5]oxadiazolꢀ4ꢀyl)amino)propyl)ꢀ4ꢀ[4ꢀ(methylsulfonyl)phenyl]ꢀ6ꢀ(trifluoroꢀmethyl)ꢀ
pyrimidinꢀ2ꢀamine (6) as novel potent and selective COXꢀ2 inhibitor (IC50 = 1.8 µM). Lead
compound (6) was further evaluated for its ability to selectively visualize COXꢀ2 isozyme in
COXꢀ2 expressing human colon cancer cell line HCAꢀ7 using confocal microscopy experiments.
2

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INTRODUCTION
Cancer remains one of the most prevalent causes of death in the western world and contributes
substantially to the pressure on healthcare systems worldwide. Despite significant advances in
survival rates over the past decades, many therapeutic and diagnostic challenges of cancer
remain. Inflammatory processes have been of constant interest in cancer research following their
inclusion as one of the hallmarks of cancer.1 Inflammation is of particular interest since it has
been shown to enable and cause cancer. Inflammatory conditions are known to aid the
development of tumors and act as a primary cause for cancer. A prominent example is
inflammatory bowl disease which can lead to the development of colorectal cancer. More
recently it was discovered that certain cancers develop inflammatory conditions in their tumor
microenvironment even though there is no pathological basis for inflammation, indicating that
inflammation is a driver for carcinogenesis. Inflammatory conditions were shown to influence
cell proliferation and cell survival, angiogenesis, tumor cell migration and metastasis leading to
tumor growth, progression and metastasis.2 Tools that enable researchers to study biochemical
mechanisms that initiate and sustain inflammatory responses in vivo would be of great value in
the field of cancer research and advance our understanding of the multiple roles inflammation
plays in the development and progression of cancer.
The cyclooxygenase enzyme family consists of two members: cyclooxygenaseꢀ1 (COXꢀ1) and
cyclooxygenaseꢀ2 (COXꢀ2). COX enzymes control the initial step within the prostaglandin
pathway by converting arachidonic acid to prostaglandin H2 (PGH2). PGH2 is the primary
substrate for the synthesis of a variety of prostaglandin signaling molecules, which are employed
as autocrine and paracrine messengers to mediate a number of physiological and
pathophysiological processes through binding to their respective Gꢀprotein coupled receptors.
COXꢀ1 is constitutively expressed in most resting tissues, while COXꢀ2 is an inducible COX
isoform and virtually absent in healthy tissues. COXꢀ1 maintains tissue homeostasis and is
primarily responsible for gastrointestinal and renal integrity.3 COXꢀ2 is expressed in response to
proꢀinflammatory stimuli and its expression is usually transient.4 COXꢀ2 is responsible for the
production of inflammatory prostaglandins, which ultimately induce inflammation, pain and
fever. Consequently, COXꢀ2 is one of the most important therapeutic targets for the treatment of
this pathological conditions.5
3

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However, in view of cardiovascular safety concerns posed by most selective COXꢀ2 inhibitors
previously in clinical use, recent research efforts were focused on the development of nitric
oxide releasing NSAIDs and dual COX/LOX inhibitors.6ꢀ8
COXꢀ2 was shown to be upregulated in many human cancers, including gastric, lung, colon,
esophageal and prostate carcinomas.9ꢀ12 As a result, a number of COXꢀ2 inhibitors have been
13ꢀ15
investigated as anticancer agents
and some were shown to retard or prevent the progression
of colon carcinomas.16ꢀ18 The central role which COXꢀ2 plays in the initiation and regulation of
inflammatory processes, and the observed overexpression of COXꢀ2 in a variety of cancers
represent the rational for targeting the enzyme for cancer diagnosis and therapy. This perspective
has led to the development of various radioactive positron emission tomography (PET)
radiopharmaceuticals for the assessment of COXꢀ2 expression in vivo.19ꢀ21 Radiolabeled COXꢀ2
inhibitors are ideally suited to study inflammation in vivo, but require laborious and expensive
synthesis on a daily basis, due to their short halfꢀlive.
Fluorescence imaging agents on the other hand are cheaper, easier to prepare and have a longer
shelfꢀlive. Advances in chemistry and biological application of optical imaging probes have
extensively been reviewed.22ꢀ23 Fluorescentꢀlabeled COXꢀ2 inhibitors are suitable optical probes
for the detection of COXꢀ2 in cells and animals. Furthermore, they can be used for clinical
imaging of tissues suitable for topical and endoluminal illumination such as esophagus and
colon. A number of research groups are actively engaged in the design and synthesis of novel
fluorescentꢀlabeled COXꢀ2 inhibitors.24ꢀ26 Recently, our lab presented a highly selective
celecoxib derivative conjugated with a 7ꢀnitrobenzofurazan fluorophore (CelecoxibꢀNBD).27
CelecoxibꢀNBD showed high affinity for COXꢀ2 (IC50 = 0.19 µM) and a good selectivity profile
over COXꢀ1 (SI = 443). Furthermore, the fluorescence conjugate showed specific uptake in
COXꢀ2 positive HCAꢀ7 cells while no uptake was observed in COXꢀ2 negative HCTꢀ116 cells,
indicating that celecoxibꢀNBD is COXꢀ2 specific in vitro. This work further showed that the
NBD fluorophore is especially suitable for COXꢀ2 targeting. Previous studies using celecoxibꢀ
rhodamine B conjugates showed lower affinity (IC50 = 3.9 µM) compared to celecoxibꢀNBD and
reduced success in in vitro assays.25
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Another research direction in our lab led to the development of a set of pyrimidine based COXꢀ2
inhibitors to be used as shortꢀlived PET radiotracers for molecular imaging.30 Best inhibitors in
the compound library showed inhibitory potencies for COXꢀ2 greater than inhibitory potencies
displayed by celecoxib in the same assay (IC50 = 5 nM versus IC50 = 40 nM).
In the present study we designed and developed a fluorescent COXꢀ2 imaging probe (6) by using
previously reported pyricoxib as lead structure (Figure 1).28
Figure 1. (Top) Design principle for fluorescentꢀlabeled COXꢀ2 inhibitors based on PET
imaging radiotracers. (Bottom) Structures of celecoxib (1), rofecoxib (2), 18Fꢀlabelled radiotracer
([18F]3), and NBDꢀlabeled compounds (6, 8, 10)
As part of our ongoing research program focused on molecular imaging of COXꢀ2 in cancer,27ꢀ30
we now describe (i) the synthesis of pyrimidineꢀbased fluorescent conjugates (6, 8, 10) and their
evaluation as (ii) in vitro COXꢀ1 and COXꢀ2 enzyme inhibitors, and their suitability as (iii)
fluorescence imaging agents for the selective visualization of COXꢀ2 activity in COXꢀ2
expressing human colon cancer cell line HCAꢀ7.
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RESULTS AND DISCUSSION
Chemistry
The synthesis of NBDꢀlabeled compounds 6, 8 and 10 is illustrated in Scheme 1.
o
Scheme 1. Reagents and conditions: (a) NꢀBocꢀpropylenediamine, CH3CN, 140 C, 2 h; (b)
o
o
TFA, dry CH2Cl2, 25 C, 6 h; (c) NBDꢀCl, dry TEA, dry THF, inert atmosphere, 25 C, 2 h; (d)
o
NꢀBocꢀethylenediamine, CH3CN, 140 C, 2 h; (e) NH4OH solution (30 % in H2O), CH3OH,
25 oC, 18 h; (f) compound 4, CH3CN, 140 oC, 2 h.
4ꢀChloroꢀ7ꢀnitroꢀ1,2,3ꢀbenzoxadiazole (NBDꢀchloride) was used to synthesize fluorescent
pyrimidine scaffolds based on previously reported selective COXꢀ2 inhibitor pyricoxib.28,31 The
use of NBD as a fluorophore for COXꢀ2 imaging is well established, and the synthetic strategy
used to develop fluorescent compounds 6, 8 and 10 is similar to methods previously developed in
our lab.25,27
6

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Reaction of previously reported building block 2ꢀ(methylsulfonyl)ꢀ4ꢀ(4ꢀ(methylsulfonyl)ꢀ
phenyl)ꢀ6ꢀ(trifluoromethyl)pyrimidine (4)28,31 with NꢀBocꢀpropylenediamine or NꢀBocꢀ
ethylenediamine in acetonitrile furnished the respective Bocꢀprotected amines 5 and 7 in 58%
and 64% yield, respectively.
Removal of the Boc protecting groups in compounds 5 and 7 by treatment with trifluoroacetic
acid (TFA) in dry dichloromethane at 25ꢁ°C afforded the respective amine products, which were
allowed to react with NBDꢀchloride without further purification. The NBDꢀconjugates 6 and 8
were synthesized in the presence of dry triethylamine (TEA) in dry THF in nitrogen atmosphere
and underwent extensive purification by silica column chromatography and HPLC to obtain the
final products in 25% (compound 6) and 17% (compound 8). The synthesis of compound 10 was
accomplished by converting NBDꢀchloride into the primary amine compound 9 using
ammonium hydroxide solution in methanol and water. The resulting 4ꢀaminoꢀ7ꢀ
nitrobenzofurazan (9) was reacted with building block (4) in acetonitrile and purified by silica
column chromatography to give product 10 in 54% yield as a yellow solid.
COXꢀ1 and COXꢀ2 inhibition studies
Compounds (6, 8 and 10) were evaluated for their in vitro COXꢀ1/COXꢀ2 isozyme inhibitory
potencies. The results are summarized in Table 1.
Table 1. In vitro COXꢀ1 and COXꢀ2 isozyme inhibition data.
IC50 (µM)a
Compound
COX-2 (SI)b
COX-1
>100
COX-2
1.8
>55.5
>16.6
>21.7
>14285.7
192
6
>100
>100
>100
7.7
6.0
4.6
8
10
3
0.007
0.04
1 (celecoxib)
[a]
In vitro concentration of tested compound required to produce 50% inhibition of ovine COXꢀ1 or human
recombinant COXꢀ2; results are the average of two determinations acquired using a COX fluorescence inhibitor
assay (Cayman Chemical, Ann Arbor, USA; catalog #: 700100), and the deviation from the mean is <10% of the
mean value. [b] In vitro COXꢀ2 selectivity index (COXꢀ1 IC50/COXꢀ2 IC50).
7

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In vitro COXꢀ1/COXꢀ2 inhibition studies showed that all three tested compounds (6, 8 and 10)
are more potent inhibitors of COXꢀ2 isoenzyme (IC50 = 1.8ꢀ6.0 µM range) than for COXꢀ1 (IC50
= >100 µM). Among all three NBDꢀlabeled compounds 6, 8 and 10, compound 6 containing a
propylamino spacer between the NBD aryl ring and the pyrimidine moiety showed best COXꢀ2
inhibitory potency and selectivity profile (COXꢀ2 IC50 = 1.8 µM, SI = >55.5) compared to
compounds containing a shorter (compound 8) or no spacer (compound 10). However, inhibitory
potency of compound 6 against COXꢀ2 isozyme is sunstantially lower compared to that of
previously reported pyricoxib (COXꢀ2 IC50 = 0.007 µM) and fluorescentꢀlabeled celecoxibꢀNBD
compound (COXꢀ2 IC50 = 0.19 ꢂM). In comparison to compound 6, compound 8 (COXꢀ2 IC50 =
6.0 ꢂM) and 10 (COXꢀ2 IC50 = 4.6 ꢂM) were identified as less potent COXꢀ2 inhibitors.
In contrast to our earlier work with celecoxibꢀNBD, where the NBD fluorophore was attached to
structurally unaltered celecoxib at the sulfonamide moiety, here we did alter the structure of
previously reported pyricoxib (3). The fluorophore was inserted to replace the 4ꢀfluorobenzyl
moiety rather than attached to an "intact" antiꢀinflammatory drug as previously reported.24ꢀ27 Our
aim was to synthesize a small fluorescence conjugate that could achieve deeper penetration into
the COXꢀ2 binding pocket due to reduced steric bulk. Compound 10 was designed to meet this
specification, where the 4ꢀfluorobenzyl moiety is replaced with similar sized nitroꢀ
benzoxadiazole moiety. However, the resulting compounds showed a substantial decrease in
COXꢀ2 inhibitory potency (from compound 3 COXꢀ2 IC50 = 0.007 µM to compound 10 COXꢀ2
IC50 = 4.6 µM). We have recently shown that a modest increase in steric bulk at 4ꢀfluorobenzyl
position in pyricoxib, by replacing fluorine with a phenyl, tert.ꢀbutyl or iodine group, had a
substantial negative impact on binding affinity.28,30 Molecular docking studies subsequently
showed that the increased size of iodine lead the substituted benzyl flip into the complete
opposite direction with lead to a less favorable interaction.30 It is likely that the nitroꢀ
benzoxadiazole group is similarly reoriented in the binding pocket causing a loss of affinity.
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Fluorescence imaging of COXꢀ2 expression in HCAꢀ7 colon cancer cells:
From the three fluorescent conjugates (6, 8 and 10), compound 6 was selected for further
investigation using a fluorescence imaging experiment on COXꢀ2ꢀexpressing human colon
cancer HCAꢀ7 cells. 4,6ꢀDiamidinoꢀ2ꢀphenylindole (DAPI) was used as a nucleusꢀspecific stain.
o
HCAꢀ7 cells were incubated with compound 6 (100 µM) at 37 C and cells were imaged by
confocal microscopy. Cellular uptake of compound 6 was observed in COXꢀ2 overexpressing
HCAꢀ7 cells (Figure 2b–d). Cells incubated with phosphateꢀbuffered saline (PBS) under similar
experimental conditions showed no fluorescence staining (Figure 2a). Pretreatment of HCAꢀ7
cells with the potent and selective COXꢀ2 inhibitor celecoxib (1, 5 µM) prevented all uptake of
fluorescence conjugate (6) and no fluorescence labeling of the COXꢀ2 isozyme was observed,
which indicates that celecoxib is able to blocked the COXꢀ2 binding of compound 6 in HCAꢀ7
cells (Figure 2e). We performed the COXꢀ2 fluorescence imaging experiment with compound 6
at different concentrations (1 µM, 50 µM, 100 µM, 200 µM and 500 µM). The results of these
experiments indicate that COXꢀ2 upregulation in HCAꢀ7 cells is best visualized upon incubation
of compound 6 at 100 µM concentration.
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a)
b)
c)
d)
e)
Figure 2. Fluorescence labeling of COXꢀ2 expressing cells (HCAꢀ7 cells): (a) Cells treated with
PBS (control); (b, c, d) cells treated with 100 µM conjugate 6; in figure b) the DAPI stained
nucleus is shown only (perinuclear staining is not shown here); (c) represents perinuclear
staining due to uptake of compound 6 in HCAꢀ7 cells (nuclear staining is not shown here); (d)
represents a merged image of both nuclei and perinuclear staining as a result of uptake of
compound 6 in HCAꢀ7 cells; (e) cells were treated with 5 µM celecoxib 1 before the incubation
with compound 6.
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Fluorescence imaging with COXꢀ2ꢀnegative HCTꢀ116 cells:
To determine whether the fluorescence labeling of HCAꢀ7 with compound 6 depends on COXꢀ2
isozyme expression or not, a fluorescence imaging experiment using HCTꢀ116 (COXꢀ2ꢀnegative
human colon cancer) cells was performed. DAPI was used as nuclear stain. HCTꢀ116 cells were
o
incubated with compound 6 (100 or 500 µM) at 37 C and cells were imaged by confocal
microscopy.
No fluorescence labeling was observed at either concentration of compound 6 (Figure 3b, c), and
no fluorescence labeling was observed after incubation with PBS control as expected (Figure 3a).
The results of this experiment in COXꢀ2ꢀnegative HCTꢀ116 cells validated the hypothesis that
the fluorescence labelling of HCAꢀ7 colon cancer cells with compound 6 is due to COXꢀ2
specific binding of fluorescence conjugate.
a)
c)
b)
Figure 3. Uptake of compound 6 in HCTꢀ116 cells: cells were treated with a) PBS (control), b)
compound 6 at 500 µM concentration and c) compound 6 at 100 µM concentration.
11

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CONCLUSIONS
A group of novel fluorescent conjugates have been synthesized, wherein a pyrimidine scaffold
was coupled either directly or via linker to the NBD fluorophore. In vitro COXꢀ1/COXꢀ2
inhibition studies indicated that all three compounds are selective inhibitors of COXꢀ2. Lead
compound 6 was evaluated for fluorescent COXꢀ2 visualization in human colon cancer cells.
Fluorescence labelling experiments in HCAꢀ7 cells and HCTꢀ116 indicated that compound 6 is
capable to label the COXꢀ2 enzyme in human colon cancer cells. In contrast to earlier work, here
we did not link the fluorophore to an "intact" antiꢀinflammatory drug, but rather attempted to
transform our previously reported highly potent and efficient COXꢀ2 radiotracer 3 into a
fluorescent probe by replacing a part of the molecule with the fluorophore. The resulting
compounds showed a substantial decline in COXꢀ2 potency but lead compound 6 still showed
success in selectively labelling COXꢀ2 at a concentration of 100 µM. Future inquiry might look
into attaching the NBD fluorophore at the methyl sulfone/sulfonamide moiety of an "intact"
pyricoxib scaffold at achieve an improved activity and sensitivity profile.
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MATERIALS AND METHODS
General
All reagents and solvents were obtained from SigmaꢀAldrich, unless otherwise stated and used
without further purification. Nuclear magnetic resonance spectra were recorded on a 600 MHz
Burker unit. 1HꢀNMR and 13CꢀNMR chemical shifts are recorded in ppm relative to
tetramethylsilane (TMS). 19FꢀNMR chemical shifts are recorded in ppm relative to
trichlorofluoromethane. Mass spectra were obtained using am Agilent Technologies 6220 oaTOF
instrument. Column chromatography was conducted using Merck silica gel (mesh size 230–400
ASTM). Thinꢀlayer chromatography (TLC) was performed on Merck silica gel Fꢀ254 aluminum
plates, with visualization under UV light (254 nm).
Chemistry
2ꢀ(Methylsulfonyl)ꢀ4ꢀ(4ꢀ(methylsulfonyl)ꢀphenyl)ꢀ6ꢀ(trifluoromethyl)pyrimidine (4) was preꢀ
pared according to literature procedure.27,31
N-(N-Boc-aminopropyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine
(5).
2ꢀ(Methylsulfonyl)ꢀ4ꢀ(4ꢀ(methylsulfonyl)ꢀphenyl)ꢀ6ꢀ
(trifluoromethyl)pyrimidine (4) (100 mg, 0.26 mmol) was dissolved in 2 ml CH3CN and NꢀBocꢀ
propylenediamine (228 µl, 1.3 mmol) was added. The reaction mixture heated in a sealed tube at
o
140 C for 2 h and cooled to room temperature thereafter. 20 ml of 1N HCl was added, the
product extracted with 3 x 20 ml ethyl acetate and used without further purification. The product
1
was obtained as a white solid (72 mg, 58 % yield). HꢀNMR (600 MHz, CDCl3): 1.38 (s, 9H,
C(CH3)3); 1.72ꢀ1.76 (m, 2H, CH2); 3.04 (s, 3H, SO2CH3); 3.19 (m, 2H, NHCH2); 3.54ꢀ3.58 (m,
2H, NHCH2); 4.90 (s, 1H, NH); 5.82 (s, 1H, NH); 7.19 (s, 1H, Hꢀ5 of pyrimidine); 8.01 (m, J =
8.4 Hz, 2H, sulfonylphenyl Hꢀ2, Hꢀ6); 8.16 (m, J = 8.4 Hz, 2H, sulfonylphenyl Hꢀ3, Hꢀ5). LRꢀ
MS: 497.2 [M+Na].
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N-(2-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propyl)-4-[4-(methylsulfonyl)phenyl]-6-
(trifluoromethyl)-pyrimidin-2-amine (6). Trifluoroacetic acid (TFA) (50 ꢂL, 0.65 mmol) was
added to a solution of compound 5 (50 mg, 0.11 mmol) in dry CH2Cl2 (5 mL), and the reaction
mixture was stirred at 25ꢁ°C for 6 h.
The progress of the reaction was monitored with TLC, upon completion excess acid and solvent
was removed under vacuum, and the residue was dried in vacuo overnight. The deprotected
product was dissolved in dry THF (2 mL) under a nitrogen atmosphere, without further
purification, and a solution of NBDꢀCl (20 mg, 0.10 mmol) in dry TEA (50 ꢂL, 0.36 mmol) was
added. The reaction was stirred at 25ꢁ°C for 1 h, H2O added, and the mixture extracted with
CH2Cl2 (3×10 mL). The combined organic extracts were washed with brine prior to drying over
anhydrous Na2SO4. Solvent was removed in vacuo and the product was purified by column
chromatography using a gradient of EtOAc/hexane running from 10% EtOAc to 50%. The
product thus obtained was not of a sufficient purity to be taken forward into in vitro studies.
Final purification was conducted using semiꢀpreparative HPLC using an isocratic solvent
mixture of 70/30 CH3CN / H2O, a flow rate of 3 ml/min on Phenomenex LUNA® C18 column
(100 Å, 250 × 10 mm, 10 ꢂm) and a Gilson 322 Pump module fitted with a 171 Diode Array
1
detector. The product was obtained as a brown solid (15 mg, 25 % yield). HꢀNMR (600 MHz,
d6ꢀDMSO): 1.92ꢀ1.96 (m, 2H, CH2); 3.26 ꢀ 3.30 (m, 2H, NHCH2); 3.30 (s, 3H, SO2CH3); 3.58 ꢀ
3.62 (m, 2H, NHCH2); 6.36 (d, J = 8.4 Hz, 1H, Hꢀ5 of NBD); 7.62 (s, 1H, Hꢀ5 of pyrimidine);
7.95 (d, J = 8.4 Hz, 2H, sulfonylphenyl Hꢀ2, Hꢀ6); 8.13 (d, J = 8.4 Hz, 2H, sulfonylphenyl Hꢀ3,
Hꢀ5); 8.39 (d, J = 8.4 Hz, 1H, Hꢀ6 of NBD); 9.5 (s, 1H, NH). 19FꢀNMR (560 MHz, d6ꢀDMSO): ꢀ
13
69.1 (s, 3F, CF3). C NMR (d6ꢀDMSO, 150 MHz): δ: 28.08 (CH2), 39.01 (CH2), 39.09 (CH2),
43.82 (CH3), 101.94 (pyrimidine ArCH), 102.08 (NBD ArCH), 120.28 (q, 1JC–F = 275 Hz, CF3),
127.93 (2 x ArCH of sulfonylphenyl), 128.64 (2 x ArCH of sulfonylphenyl), 130.10 (ArC),
138.17 (ArCH), 141.11 (NBD ArC), 143.42 (ArC), 144.42 (ArC), 144.84 (ArC), 145.63 (ArC),
2
156.94 (q, JC–CF = 36 Hz), 162.81 (pyrimidine ArC), 165.46 (pyrimidine ArC). HRMS (ESI)
calculated for C21H18F3N7O5S [MꢀH]ꢀ 536.0964; found [MꢀH]ꢀ 536.0969.
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N-(N-Boc-aminoethyl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine
(7).
2ꢀ(Methylsulfonyl)ꢀ4ꢀ(4ꢀ(methylsulfonyl)ꢀphenyl)ꢀ6ꢀ
(trifluoromethyl)pyrimidine (4) (100 mg, 0.26 mmol) was dissolved in 2 ml CH3CN and NꢀBocꢀ
ethylenediamine (200 µl, 1.3 mmol) added. The reaction mixture heated in a sealed tube at
140 oC for 2 h and cooled to room temperature thereafter. 20 ml of 1N HCl were added, the
product extracted with 3 x 20 ml ethyl acetate and used without further purification. The product
1
was obtained as a white solid (76 mg, 64 % yield). HꢀNMR (600 MHz, CDCl3): 1.35 (s, 9H,
C(CH3)3); 3.06 (s, 3H, SO2CH3); 3.34ꢀ3.38 (m, 2H, NHCH2); 3.58ꢀ3.62 (m, 2H, NHCH2); 4.9 (s,
1H, NH); 5.8 (s, 1H, NH); 7.23 (s, 1H, Hꢀ5 of pyrimidine); 8.01 (d, J = 8.4Hz, 2H,
sulfonylphenyl Hꢀ2, Hꢀ6); 8.16 (d, J = 8.4Hz, 2H, sulfonylphenyl Hꢀ3, Hꢀ5). LRꢀMS: 483.1
[M+Na].
N-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)-4-[4-(methylsulfonyl)phenyl]-6-
(trifluoromethyl)-pyrimidin-2-amine (8).
Trifluoroacetic acid (TFA) (50 ꢂL, 0.65 mmol) was added to a solution of compound 7 (50 mg,
0.11 mmol) in dry CH2Cl2 (5 mL), and the reaction mixture was stirred at 25ꢁ°C for 6 h. The
progress of the reaction was monitored by TLC, upon completion excess acid and solvent was
removed under vacuum, and the residue was dried in vacuo overnight. The deprotected product
was dissolved in dry THF (2 mL) under a nitrogen atmosphere, without further purification, and
a solution of NBDꢀCl (20 mg, 0.10 mmol) in dry TEA (50 ꢂL, 0.36 mmol) was added. The
reaction was stirred at 25ꢁ°C for 1 h, H2O added, and the mixture extracted with CH2Cl2
(3×10 mL). The combined organic extracts were washed with brine prior to drying over
anhydrous Na2SO4. Solvent was removed in vacuo and the product purified by column
chromatography using a gradient of EtOAc/hexane running from 10% EtOAc to 50%. The
product thus obtained was not of a sufficient purity to be taken forward into in vitro studies.
Final purification was conducted using semiꢀpreparative HPLC using an isocratic solvent
mixture of 70/30 CH3CN / H20, a flow rate of 3 ml/min on Phenomenex LUNA® C18 column
(100 Å, 250 × 10 mm, 10 ꢂm) and a Gilson 322 Pump module fitted with a 171 Diode Array
1
detector. The product was obtained as a brown solid (10 mg, 17 % yield). HꢀNMR (600 MHz,
d6ꢀDMSO): 3.30 (s, 3H, SO2CH3); 3.30 ꢀ 3.34 (m, 2H, NHCH2); 3.79 ꢀ 3.83 (m, 2H, NHCH2);
6.57 (d, J = 8.4 Hz, 1H, Hꢀ5 of NBD); 7.69 (s, 1H, Hꢀ5 of pyrimidine); 7.91 (d, J = 8.4 Hz, 2H,
15

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sulfonylphenyl Hꢀ2, Hꢀ6); 8.19 (d, J = 8.4 Hz, 2H, sulfonylphenyl Hꢀ3, Hꢀ5); 8.45 (d, J = 8.4 Hz,
19
13
1H, Hꢀ6 of NBD); 9.5 (s, 1H, NꢀH). FꢀNMR (560 MHz, d6ꢀDMSO)): ꢀ69.09 (s, 3F, CF3). C
NMR (d6ꢀDMSO, 150 MHz): δ: 43.01 (CH3), 49.00 (CH2), 49.46 (CH2), 102.37 (pyrimidine
1
ArCH), 102.73 (NBD ArCH), 121.62 (q, JC–F = 275 Hz, CF3), 127.55 (2 x ArCH of
sulfonylphenyl), 127.72 (2 x ArCH of sulfonylphenyl), 128.20 (ArC), 135.64 (ArCH), 140.89
2
(NBD ArC), 141.20 (ArC), 143.07 (ArC), 144.45 (ArC), 145.59 (ArC), 156.84 (q, JC–CF = 36
Hz), 159.94 (pyrimidine ArC), 165.63 (pyrimidine ArC). LRꢀMS: 546.1 [M+Na]. HRMS (ESI)
calculated for C20H16F3N7O5S [MꢀH]ꢀ 522.0807; found [MꢀH]ꢀ 522.0816.
4-Amino-7-nitrobenzofurazan (9).
NBDꢀNH2 was prepared according to literature procedure.25 The product was obtained as a
1
yellow solid (126 mg, 70 % yield). HꢀNMR (600 MHz, CDCl3): 5.60 (s, 2H, NH2); 6.34 (d, J =
8.4 Hz, 1H, Hꢀ5 of NBD); 8.41 (d, J = 8.4 Hz, 1H, Hꢀ6 of NBD). LRꢀMS: 203.0 [M+Na].
N-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-
pyrimidin-2-amine
(10).
2ꢀ(methylsulfonyl)ꢀ4ꢀ(4ꢀ(methylsulfonyl)ꢀphenyl)ꢀ6ꢀ
(trifluoromethyl)pyrimidine (4) (50 mg, 0.13 mmol) and 4ꢀaminoꢀ7ꢀnitrobenzofurazan (9) were
o
dissolved in 1 ml CH3CN. The reaction mixture heated in a sealed tube at 140 C for 2 h and
cooled to room temperature thereafter. 10 ml of 1N HCl were added, the product extracted with
3 x 10 ml ethyl acetate and purified by column chromatography eluting at 30% EtOAc / hexane.
1
The product was obtained as a yellow brownish solid (34 mg, 54% yield). HꢀNMR (600 MHz,
d6ꢀDMSO): 3.34 (s, 3H, SO2CH3); 8.17 (d, J = 8.4 Hz, 2H, sulfonylphenyl Hꢀ2, Hꢀ6); 8.40 (s,
1H, Hꢀ5 of pyrimidine); 8.43 (d, J = 8.4 Hz, 1H, Hꢀ6 of NBD); 8.65 (d, J = 8.4 Hz, 2H,
19
sulfonylphenyl Hꢀ3, Hꢀ5); 8.90 (d, J = 8.4 Hz, 1H, Hꢀ5 of NBD); 11.83 (s, 1H, NH). FꢀNMR
13
(560 MHz, d6ꢀDMSO): ꢀ68.35 (s, 3F, CF3). C NMR (d6ꢀDMSO, 150 MHz): δ: 48.09 (CH3),
1
108.48 (pyrimidine ArCH), 113.01(NBD ArCH), 121.10 (q, JC–F = 275 Hz, CF3), 128.15 (2 x
ArCH), 129.13 (ArC), 129.38 (2 x ArCH), 136.53 (NBD ArC), 136.60 (ArCH), 139.85 (NBD
2
ArC), 144.06 (ArC), 144.15 (ArC), 146.31(ArC), 156.77 (q, JC–CF = 36.1 Hz), 159.64
(pyrimidine ArC), 166.45 (pyrimidine ArC). HRMS (ESI) calculated for C18H11F3N6O5S [MꢀH]ꢀ
479.0385; found [MꢀH]ꢀ 479.0392.
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Cyclooxygenase inhibition assay
The ability of celecoxib (1) and new compounds (6, 8 and 10) to inhibit ovine COXꢀ1 and
recombinant human COXꢀ2 was determined using a COX fluorescence inhibitor assay (Cayman
Chemical, Ann Arbor, USA; catalog #: 700100) according to the manufacturers protocol.
Compounds were assayed in a concentration range of 10−9 M to 10−3 M.
Cell culture and cell imaging studies
HCA-7 cells. HCAꢀ7 colony 29 cells (Sigma Aldrich, 02091238) were used for fluorescence
imaging of COXꢀ2 overꢀexpression. The cells were cultured in T75 flasks using DMEM/F12
(1:1) medium supplemented with 10% (v/v) fetal bovine serum (GIBCO, 12483), 2 mM Lꢀ
glutamine (GIBCO, 25030), 1% penicillin/streptomycin and 20 mM HEPES buffer (GIBCO,
15630) and were kept in a 37°C humidified incubator with a supply of 5% CO2 in air. After the
cells were 80% confluent, they were harvested using 0.25% trypsinꢀEDTA (GIBCO, 25200) and
plated onto sterilized glass cover slips placed into a 6 well cell culture plate at a density of
200,000 cells/well. Cells were washed twice using PBS prior to permeabilization with 0.5%
Triton Xꢀ100 supplemented PBS (pH 7.4) for 5 min. Fixed and permeabilized cells were washed
thrice with PBS before the addition of PBS as control, compound 6 (100 µM), or a mixture of
100 µM celecoxib and 100 µM compound 6 to the cover slips placed in a cell culture plate,
respectively. This set up was placed for incubation at 37°C for 1 hour. Thereafter, the cells were
washed with 0.1% Triton Xꢀ100 supplemented PBS followed by three washes with PBS. PBS
rinsed coverslips were then mounted onto microscopy slides using 30 µL drops of poly vinyl
alcohol based mounting media supplemented with 0.1% nꢀpropyl gallate as antiꢀfade and DAPI
(50 µg/ml).
Cells were imaged using corresponding lasers for visualizing DAPI (blue nuclear staining) and
FITC (green emission) with a PlanꢀApochromat 40X/1.3 Oil DIC M27 lens on a Zeiss LSM 710
AxioObserver confocal laser scanning microscope.
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HCT-116 cells. HCTꢀ116 cells (ATCC) were cultured at 37°C in a humidified atmosphere of 5%
(v/v) CO2, using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS)
(Gbico®), 2mM Lꢀglutamine (Invitrogen) and 1% antibiotic/antimycotic (Invitrogen). Cell
growth medium was changed every other day. Cells were treated with 0.25% Trypsin/1mM
EDTA (Invitogen) for about 5 min at room temperature to dissociate from the culture flask, and
rinsed with PBS once after harvesting. Cells were resuspended in fresh growth medium and
seeded to the 6ꢀwell plate at 1.5x106 cells per well. After removing media, cells were treated
with 100 µM and 500 µM of compound 6 respectively using the procedure mentioned above.
ACKNOWLEDGMENTS
The authors would like to acknowledge the Dianne and Irving Kipnes Foundation, the Canadian
Institute for Health Research (CIHR and the National Science and Engineering Research Council
of Canada (NSERC) for supporting this work. F.W. and J.K. thank the Alberta Cancer
Foundation (ACF) for a postdoctoral fellowship.
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