Design of fluorine-containing 3,4-diarylfuran-2(5H)-ones as selective COX-1 inhibitors

Article abstract of DOI:10.1021/ml500344j

We report the design and synthesis of fluorine-containing cyclooxygenase-1 (COX-1)-selective inhibitors to serve as prototypes for the development of a COX-1-targeted imaging agent. Deletion of the SO₂CH₃ group of rofecoxib switches the compound from a COX-2- to a COX-1-selective inhibitor, providing a 3,4-diarylfuran-2(5H)-one scaffold for structure-activity relationship studies of COX-1 inhibition. A wide range of fluorine-containing 3,4-diarylfuran-2(5H)-ones were designed, synthesized, and tested for their ability to selectively inhibit COX-1 in purified protein and human cancer cell assays. Compounds containing a fluoro-substituent on the C-3 phenyl ring and a methoxy-substituent on the C-4 phenyl ring of the 3,4-diarylfuran-2(5H)-one scaffold were the best COX-1-selective agents of those evaluated, exhibiting IC₅₀s in the submicromolar range. These compounds provide the foundation for development of an agent to facilitate radiologic imaging of ovarian cancer expressing elevated levels of COX-1.

Full text of DOI:10.1021/ml500344j

Letter
pubs.acs.org/acsmedchemlett
Design of Fluorine-Containing 3,4-Diarylfuran-2(5H)‑ones as
Selective COX‑1 Inhibitors
Md. Jashim Uddin, Anna V. Elleman, Kebreab Ghebreselasie, Cristina K. Daniel, Brenda C. Crews,
Kellie D. Nance, Tamanna Huda, and Lawrence J. Marnett*
A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology, Vanderbilt
Institute of Chemical Biology, Center for Molecular Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School
of Medicine, Nashville, Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: We report the design and synthesis of fluorine-containing
cyclooxygenase-1 (COX-1)-selective inhibitors to serve as prototypes for the
development of a COX-1-targeted imaging agent. Deletion of the SO2CH3
group of rofecoxib switches the compound from a COX-2- to a COX-1-
selective inhibitor, providing a 3,4-diarylfuran-2(5H)-one scaffold for
structure−activity relationship studies of COX-1 inhibition. A wide range
of fluorine-containing 3,4-diarylfuran-2(5H)-ones were designed, synthe-
sized, and tested for their ability to selectively inhibit COX-1 in purified
protein and human cancer cell assays. Compounds containing a fluoro-
substituent on the C-3 phenyl ring and a methoxy-substituent on the C-4 phenyl ring of the 3,4-diarylfuran-2(5H)-one scaffold
were the best COX-1-selective agents of those evaluated, exhibiting IC50s in the submicromolar range. These compounds provide
the foundation for development of an agent to facilitate radiologic imaging of ovarian cancer expressing elevated levels of COX-1.
KEYWORDS: Cyclooxygenase-1 (COX-1), rofecoxib, furanone, structure−activity relationship, imaging
he cyclooxygenase enzymes (COX-1 and COX-2), which
Tcatalyze the first two steps in the biosynthesis of
prostaglandins from arachidonic acid, are the primary targets
of the nonsteroidal anti-inflammatory drugs, such as
indomethacin, ibuprofen, and naproxen. The inducible isoform,
COX-2, is strongly expressed in response to inflammatory and
mitogenic stimuli, leading to the widely accepted belief that this
enzyme plays an important role in inflammation and carcino-
genesis.1 However, growing evidence suggests that the
constitutively expressed COX-1 also contributes to some
disease processes, including neuroinflammation, thrombosis,
and some cancers.2−6 Of the cancers reported to overexpress
COX-1, the strongest case has been made for epithelial ovarian
cancer. Indeed, recent evidence suggests that COX-1
contributes to the pathophysiology of ovarian cancer and that
COX-1 inhibition may have both preventive and therapeutic
benefits in this disease.7−11
reported.17 These studies provided proof-of-concept for COX-1
targeting in ovarian cancer; however, it has been difficult to
achieve adequate potency, selectivity, and pharmacokinetic
properties for in vivo imaging using the P6 scaffold.18 To date,
only a very few COX-1-selective inhibitors have been reported.
Although a few have been built on benzamide or sulindac
sulfide scaffolds,19−21 most have employed a pyrazole-,
thiazole-, triazole-, or isoxazole-containing diaryl heterocycle
scaffold similar to that of the COX-2-selective inhibitors,
celecoxib, rofecoxib, and valdecoxib (Figure 1).22−28 Here, we
report that the 3,4-diphenylfuran-2(5H)-one obtained from
desulfurization of rofecoxib exhibits a weak COX-1-selective
inhibitory activity. Furthermore, we describe the structure−
activity relationships obtained from the modification of that
We have shown that COX-2-selective inhibitors bearing
fluorescent, 18F, or 123I tags can be used in conjunction with
optical, positron emission tomography (PET), or single-photon
emission computerized tomography imaging modalities,
respectively, to visualize COX-2 expressed in tumors and
inflammatory sites in vivo.12−16 These findings led us to
hypothesize that COX-1 could serve as an imaging target to
detect ovarian cancer, a disease for which better diagnostic
modalities are sorely needed. To that end, selective uptake of
an [18F]-labeled analogue of the COX-1-selective inhibitor P6
(3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole) by
COX-1-expressing ovarian carcinoma xenografts was recently
Figure 1. Nitrogen-containing diaryl heterocyclic class of COX-1-
selective inhibitors.
Received: August 25, 2014
Accepted: October 12, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml500344j | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
scaffold to obtain potent and selective fluorine-containing
COX-1 inhibitors suitable for use as a prototype for the
development of a PET imaging agent.
Table 1. In Vitro Biochemical Properties of 3-Aryl-4-(2-, 3-,
or 4-fluorophenyl)-furan-2(5H)-one Derivatives
The key determinant of the COX-2-selectivity of the diaryl
heterocycle-based COX-2 inhibitors is the presence of a
sulfonamide or a methylsulfone on one of the aromatic rings.
This sulfur-containing functional group inserts into a side-
pocket in the cyclooxygenase active site that is only accessible
in COX-2. Interestingly, the COX-1-selective inhibitor SC-560
was derived from celecoxib via replacement of the sulfonamide
group with a methoxy group.29 Similarly, deletion of the
sulfonylmethyl group of rofecoxib affords 3,4-diphenylfuran-
2(5H)-one (1), which exhibits a weak COX-1 inhibitory
activity, suggesting that it could serve as a scaffold for the
discovery of novel selective COX-1 inhibitors. We employed an
efficient one-pot parallel synthetic method for the synthesis of
fluorinated 3,4-diarylfuran-2(5H)-one derivatives involving
condensation of a group of substituted-phenacyl bromides
with substituted-phenylacetic acids followed by intramolecular
cyclization of the acetate intermediate using 1,8-
diazabicyclo[5.4.0]undec-7-ene (Scheme 1).30 The IC50 values
a
a
no.
R1
R2
oCOX-1 IC50 (μM)
mCOX-2 IC50 (μM)
1
H
H
H
H
H
5.90
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
2
2-F
3-F
4-F
2-F
3-F
4-F
2-F
4-F
4-F
2-F
4-F
2-F
4-F
2-F
4-F
3
4
5
4-CH3
6
4-CH3
7
4-CH3
8
4-CH2OH
4-CH2OH
4-OH
9
10
11
12
13
14
15
16
4-N(CH3)2
4-N(CH3)2
4-Br
4-Br
Scheme 1. One-Pot Synthesis of Fluorine-Containing 3,4-
4-Cl
Diarylfuran-2(5H)-one 1−40 or 3-Pyridyl-4-arylfuran-
4-Cl
a
a
2(5H)-one derivatives 41−48
IC50 values were determined by incubating several concentrations of
inhibitors or DMSO vehicle with purified murine COX-2 (63 nM) or
ovine COX-1 (22.5 nM) for 20 min, followed by treatment with
[1-14C]-arachidonic acid (50 μM) at 37 °C for 30 s. Assays were run in
duplicate.
(30), or 3-chloro-2-fluoro (32) group in the C-3 phenyl ring all
exhibited submicromolar IC50s against COX-1, while residual
activity of COX-2 in the presence of 25 μM of the compounds
was higher than 50% (IC50 > 25 μM). A p-bromo-substituted
compound (29) was also a potent COX-1 inhibitor, but
demonstrated some activity against COX-2, while 3- and 4-
trifluoromethyl-substituted compounds (39 and 40) exhibited
weak COX-1-selective activity, and unsubstituted (25), 2-
fluoro-substituted (26), and 4-fluorophenoxy-substituted (31)
compounds were inactive. Of four compounds bearing no
substituent on the C-4 phenyl ring (17−20), only one, with a
4-fluoro substituent in the C-3 phenyl ring, demonstrated weak
COX-1 inhibitory activity. Two out of five compounds (21−
24) bearing a 4-methyl group in the C-4 ring exhibited selective
COX-1 inhibitory activity with IC50s in the low micromolar
range. These compounds contained 2-fluoro (21) and 4-fluoro
(23) substituents in the C-3 phenyl ring. Compounds bearing a
3-fluoro substituent in the C-3 phenyl ring with 4-cyano (35),
4-ethyl (37), and 4-hydroxyl (38) groups in the C-4 phenyl
ring were selective COX-1 inhibitors with a range of IC50s from
0.4 to 10 μM. A single compound bearing a 4-fluoro group in
the C-4 phenyl ring and a 4-thiomethyl group in the C-3 phenyl
ring (36) was inactive.
a
Reagents and conditions: (a) acetonitrile, triethylamine, room
temperature, 20 min; (b) 1,8-diazabicyclo[5.4.0]undec-7-ene, room
temperature, 20 min.
for inhibition of purified murine COX-2 or ovine COX-1 by
test compounds were determined by a thin layer chromatog-
raphy (TLC)-based assay that measures the conversion of
[1-14C]-arachidonic acid to radiolabeled prostaglandins.13
The first series of compounds that were synthesized by this
approach possessed halogen substituents at the 2-, 3-, or 4-
positions of the C-4 phenyl ring of 3,4-diphenyl-2(5H)-
furanone. Compounds possessing a fluoro substituent at these
positions (compounds 2−4) exhibited no COX inhibitory
activity. Attachment of methyl, hydroxymethyl, methoxy,
dimethylamino, bromo, or chloro substituents to the C-3
phenyl ring of these fluorinated derivatives similarly produced
inactive compounds (compounds 5−16, Table 1). Thus, we
concluded that compounds bearing a fluoro-substituent on the
C-4 phenyl ring of 3,4-diphenyl-2(5H)-furanone are inactive as
COX inhibitors.
The second series of compounds possessed halogen-
containing substituents at the 2-, 3-, or 4-positions of the C-3
phenyl ring and a range of substituents in the para-position of
the C-4 phenyl ring of the scaffold (Table 2). Of these, the
most potent selective COX-1 inhibitors possessed a 4-methoxy
group in the C-4 phenyl ring. Compounds containing this
substituent along with a 3-fluoro (27), 4-fluoro (28), 4-iodo
The third series of compounds possessed a substituted
phenyl ring at the C-4 position and a substituted-4-pyridyl ring
at the C-3 position on the furanone core. Compounds 41
through 48 were synthesized from the reaction of methyl-,
methoxy-, chloro-, or cyano-substituted phenacyl bromides and
2-chloro- or 2-fluoro-4-pyridylacetic acids, followed by a
cyclization reaction. These pyridyl analogues showed COX-1-
selective inhibition with very low levels of potency (Scheme 1
and Table 3).
B
dx.doi.org/10.1021/ml500344j | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
The ability of the promising fluorine-containing furanone
derivatives to inhibit COX-1 and COX-2 in intact cells was
evaluated using COX-1-expressing human ovarian cancer cells
(OVCAR3) and COX-2-expressing human head and neck
squamous cell carcinoma cells (1483 HNSCC).13,17 Selected
compounds were incubated with these cells in the presence of
[1-14C]-arachidonic acid, and COX-mediated formation of
prostaglandin products was monitored by a TLC assay.13,17
Compounds 19, 23, 27, and 28 inhibited COX-1 in OVCAR3
cells but not COX-2 in 1483 HNSCC cells (Table 4). Although
Table 2. In Vitro Biochemical Properties of 3-(2-, 3-, or 4-
Fluorophenyl)-4-arylfuran-2(5H)-one Derivatives
mCOX-2 IC50
a
a
no.
R1
R2
oCOX-1 IC50 (μM)
(μM)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
R
H
H
H
H
2-F
3-F
4-F
>25
>25
6
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.45
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.06
Table 4. In Vitro Inhibition of COX-1 in OVCAR3 and
COX-2 in 1483 HNSCC Cell Line Assay Data of Promising
Compounds
4-OPhF
2-F
>25
1.00
>25
0.95
>25
>25
>25
0.36
0.48
0.12
0.09
>25
0.30
>25
>25
0.47
>25
9.75
1.75
8.80
1.00
>25
CH3
a
a
no.
OVCAR3 COX-1 IC50 (μM)
1483 HNSCC COX-2 IC50 (μM)
CH3
3-F
19
23
27
28
30
2.80
0.78
0.18
0.36
>4
>5
>5
>5
>5
>5
CH3
4-F
CH3
4-OPhF
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CF3
2-F
3-F
a
IC50 values were determined as described previously13,17 for
4-F
OVCAR3 or 1483 HNSCC cells.
4-Br
4-I
4-OPhF
2-F, 3-Cl
3-F
compound 30 inhibited COX-1 in the purified protein assay, it
did not inhibit COX-1 in OVCAR3 cells. The remaining fluoro-
compounds in Table 2 that exhibit low to moderate COX-1
inhibitory potency and selectivity in the purified COX enzyme
assay were not evaluated in the OVCAR3 or 1483 HNSCC cell
line assays.
Compound 27 was the most potent of those tested against
COX-1 in OVCAR3 cells. We further characterized this
compound to determine whether its inhibitory potency is
time-dependent. In the standard TLC assay, which includes a
20 min preincubation, 27 exhibited an IC50 of 0.36 μM.
Elimination of the preincubation resulted in only a small
change in potency (IC50 of 1.25 μM). Thus, 27 may be an
example of a rapid reversible inhibitor of COX-1. We also
evaluated the effect of plasma proteins on inhibitor potency in
the OVCAR3 cell assay, demonstrating a mild loss of potency
when cells were treated with 27 in the presence of 10% FBS
(IC50 of 0.87 μM) as compared to its potency in the absence of
serum (IC50 of 0.18 μM).
OCF3
CN
3-F
3-F
SCH3
CH2CH3
OH
4-F
3-F
3-F
OCH3
OCH3
SO2CH3
3-CF3
4-CF3
H
a
IC50 values were determined by incubating several concentrations of
inhibitors or DMSO vehicle with purified murine COX-2 (63 nM) or
ovine COX-1 (22.5 nM) for 20 min followed by treatment with
[1-14C]-AA (50 μM) at 37 °C for 30 s. Assays were run in duplicate.
Compound R is rofecoxib.
Table 3. Biochemical Properties of 3-(2-Chloro or 2-
Fluoropyridin-4-yl)-4-arylfuran-2(5H)-one Derivatives
In conclusion, we describe the SAR of a series of COX-1-
selective small molecules, which indicates that the regiochem-
ical disposition of alkyl, thioalkyl, alkoxy, phenoxy, trifluor-
omethyl, halo, or other substituents on the 3,4-diphenylfuran-
2(5H)-one core controls COX inhibitory activity, selectivity,
and potency. In general, 4-methoxy substitution on the C-4
phenyl ring combined with 3- and 4-substitution with fluorine-
containing substituents in the C-3 phenyl ring was the most
productive approach to potent and selective COX-1 inhibitors
that may serve as prototypes for PET imaging agents. Further
work will be required to develop the radiochemistry to
incorporate an [18F] label and evaluate the compounds as in
vivo imaging agents.
a
a
no.
R1
R2
oCOX-1 IC50 (μM)
mCOX-2 IC50 (μM)
41
42
43
44
45
46
47
48
OCH3
OCH3
Cl
F
4.60
4.40
>25
>25
>25
>25
>25
>25
>25
>25
>25
Cl
Cl
F
Cl
15.70
5.00
>25
CH3
CH3
CN
Cl
F
F
4.40
3.00
CN
Cl
a
ASSOCIATED CONTENT
* Supporting Information
Full synthetic procedures and analytical and spectral character-
ization data of the synthesized compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
IC50 values were determined by incubating several concentrations of
■
S
inhibitors or DMSO vehicle with purified murine COX-2 (63 nM) or
ovine COX-1 (22.5 nM) for 20 min followed by treatment with
[1-14C]-AA (50 μM) at 37 °C for 30 s. Assays were run in duplicate.
C
dx.doi.org/10.1021/ml500344j | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
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AUTHOR INFORMATION
Corresponding Author
*Phone: 615-343-7329. Fax: 615-343-7534. E-mail: larry.
marnett@vanderbilt.edu.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This study is supported by research grants from the National
Institutes of Health (CA89450, CA136465, and S10
RR019022).
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Spectroscopic analysis of all new molecules was conducted in
the Small Molecule NMR Facility and Mass Spectrometry
Research Center at the Vanderbilt Institute of Chemical
Biology.
ABBREVIATIONS
COX, cyclooxygenase; PET, positron emission tomography;
TLC, thin layer chromatography
■
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