Trifluoromethyl fluorocoxib A detects cyclooxygenase-2 expression in inflammatory tissues and human tumor xenografts

Article abstract of DOI:10.1021/ml400485g

Fluorocoxib A is an effective COX-2-targeted optical imaging agent, used for in vivo detection of inflammatory tissues and premalignant and malignant tumors that express elevated levels of COX-2 (Uddin et al. Cancer Res. 2010, 70, 3618-3627). In an effort to discover novel optical probes for COX-2, a trifluoromethyl analogue of fluorocoxib A (CF₃-fluorocoxib A) was synthesized and evaluated for its ability to inhibit COX-2 in vitro purified enzyme and human cancer cell lines. Kinetic analysis revealed that CF ₃-fluorocoxib A is a slow, tight binding inhibitor of COX-2 that exhibits low nanomolar inhibitory potency. While CF₃-fluorocoxib A and fluorocoxib A are similar in structure, CF₃-fluorocoxib A shows improved potency in inhibition of wtCOX-2 and with a series of site-directed COX-2 mutants. After intraperitoneal injection, selective uptake of CF ₃-fluorocoxib A is detected in inflamed mouse paws compared to noninflamed contralateral paws by optical imaging, and uptake is blocked by pretreatment with the COX-2 inhibitor, celecoxib. Selective uptake is also detected in the COX-2-positive human tumor xenografts (1483 HNSCC) as compared with the COX-2-negative tumor xenografts (HCT116) in an in vivo nude mouse tumor model. These in vitro and in vivo studies suggest that binding to COX-2 is the major determinant of uptake of CF₃-fluorocoxib A into the inflamed tissues and tumor xenografts. Thus, this new COX-2-targeted imaging probe should find utility in the detection and evaluation of COX-2 status in naturally occurring malignancies.

Full text of DOI:10.1021/ml400485g

Technology Note
pubs.acs.org/acsmedchemlett
Trifluoromethyl Fluorocoxib A Detects Cyclooxygenase‑2 Expression
in Inflammatory Tissues and Human Tumor Xenografts
Md. Jashim Uddin,* Brenda C. Crews, Imran Huda, Kebreab Ghebreselasie, Cristina K. Daniel,
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: Fluorocoxib A is an effective COX-2-targeted optical imaging agent, used for in vivo detection of inflammatory
tissues and premalignant and malignant tumors that express elevated levels of COX-2 (Uddin et al. Cancer Res. 2010, 70, 3618−
3627). In an effort to discover novel optical probes for COX-2, a trifluoromethyl analogue of fluorocoxib A (CF3-fluorocoxib A)
was synthesized and evaluated for its ability to inhibit COX-2 in vitro purified enzyme and human cancer cell lines. Kinetic
analysis revealed that CF3-fluorocoxib A is a slow, tight binding inhibitor of COX-2 that exhibits low nanomolar inhibitory
potency. While CF3-fluorocoxib A and fluorocoxib A are similar in structure, CF3-fluorocoxib A shows improved potency in
inhibition of wtCOX-2 and with a series of site-directed COX-2 mutants. After intraperitoneal injection, selective uptake of CF3-
fluorocoxib A is detected in inflamed mouse paws compared to noninflamed contralateral paws by optical imaging, and uptake is
blocked by pretreatment with the COX-2 inhibitor, celecoxib. Selective uptake is also detected in the COX-2-positive human
tumor xenografts (1483 HNSCC) as compared with the COX-2-negative tumor xenografts (HCT116) in an in vivo nude mouse
tumor model. These in vitro and in vivo studies suggest that binding to COX-2 is the major determinant of uptake of CF3-
fluorocoxib A into the inflamed tissues and tumor xenografts. Thus, this new COX-2-targeted imaging probe should find utility in
the detection and evaluation of COX-2 status in naturally occurring malignancies.
KEYWORDS: Cyclooxygenase-2 (COX-2), CF3-indomethacin, CF3-fluorocoxib A, optical imaging, inflammation, cancer
yclooxygenases (COX) catalyze the biotransformation of
arachidonic acid into a wide variety of prostaglandins,
visualization of COX-2 in inflammatory and malignant lesions.7
Herein, we report the synthesis and evaluation of a
trifluoromethyl analogue of fluorocoxib A as a selective COX-
2 inhibitor in purified protein and cells. We also describe the
enzyme−inhibitor binding kinetics and in vivo delivery of CF3-
fluorocoxib A to inflammatory tissues and human tumor
xenografts (Figure 1).
CF3-indomethacin was synthesized using a Fisher indole
cyclization of 5,5,5-trifluorolevulinic acid lactone with 1-(4-
methoxyphenyl)-1-(4-chlorobenzoyl)hydrazine hydrochloride
under acidic conditions, as described in a previous report.8
C
which are important biological mediators of inflammation.1
COX-1 is constitutively expressed in most normal tissues,
where it performs housekeeping functions, such as maintenance
of vascular tone and cytoprotection of the gastric mucosa.2
COX-2 is an inducible enzyme that is expressed in
inflammation, where it modulates edema and pain, and in
proliferative diseases, where it promotes growth and enhances
metastasis.3 COX-2 overexpression is an early event in
tumorigenesis, and it plays a role in tumor progression.4
Selective COX-2 inhibitors are useful in the treatment of
various cancers.5,6 Therefore, COX-2 can be used as a target for
imaging of inflammation and cancer with fluorescently
conjugated COX-2 inhibitors. We recently reported the
synthesis and evaluation of fluorocoxib A for the selective
Received: November 26, 2013
Accepted: January 23, 2014
Published: January 23, 2014
© 2014 American Chemical Society
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Figure 1. Chemical evolution of COX-2-selective inhibitors from nonsteroidal anti-inflammatory drug indomethacin.
The CF3-indomethacin was then coupled with mono N-tert-
butoxycarbonyl-butylenediamine using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzo-
triazole hydrate, and N,N-diisopropylethylamine to afford t-
butyl 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-
1H-indol-3-yl}acetamido]butylcarbamate (Scheme 1). Treat-
benzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-3-yl}-
acetamide (CF3-fluorocoxib A) (Scheme 1). Synthetic
procedures and the characterization of all the new compounds
are described in the Supporting Information.
CF3-fluorocoxib A was assayed against purified COX-2 or
COX-1 by a previously reported thin layer chromatography
assay.10 We found that CF3-fluorocoxib A is a selective and
potent inhibitor of COX-2. The calculated LogP values of CF3-
fluorocoxib A and fluorocoxib A are 6.96 and 6.34, respectively.
The IC50 value for inhibition of COX-2 is 0.56 μM with no
inhibition of COX-1 up to 25 μM. CF3-fluorocoxib A was
assayed in RAW264.7 murine macrophage-like cells to check
for membrane permeability and subsequent COX-2 inhib-
ition.11 The IC50 value for inhibition of COX-2 by CF3-
fluorocoxib A was 0.08 μM. Further, the ability of CF3-
fluorocoxib A to inhibit COX-2 in 1483 head and neck
squamous cell carcinoma (HNSCC) cells was assayed.7 CF3-
fluorocoxib A was incubated with 1483 HNSCC cells at several
concentrations (0−5 μM) for 30 min followed by the addition
of 10 μM [1-14C]-arachidonic acid (∼55 mCi/mmol). CF3-
Fluorocoxib A inhibited COX-2 with an IC50 value of 0.59 μM.
Nearly all COX inhibitors, whether nonselective, like
indomethacin, or COX-2-selective, like celecoxib, bind with
the enzyme in a noncovalent manner. The one notable
exception is aspirin, which irreversibly inactivates COX-1 and
COX-2 through covalent modification of an active site serine
residue. Indomethacin and celecoxib are examples of slow,
tight-binding COX inhibitors. They rapidly establish an
equilibrium with a loosely bound enzyme−inhibitor complex,
which slowly converts to a much more tightly bound complex
(eq 1). Figure 2a shows the time- and concentration-dependent
inhibition of mCOX-2 by CF3-fluorocoxib A. The time-
dependency of COX-2 inhibition is clearly evident, as it
requires approximately 10 min to achieve maximal inhibition.
Note that the plateau of 15% activity remaining at high CF3-
fluorocoxib A concentrations suggests some reversibility of the
tightly bound enzyme−inhibitor complex. Figure 2b displays a
plot of the observed single exponential rate constants for
inhibition (kobs) as a function of CF3-fluorocoxib A
a
Scheme 1
a
Reagents and conditions (a) H2N-(CH2)4-NH-BOC, 1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzo-
triazole hydrate, N,N-diisopropylethylamine, dimethyl formamide, 25
°C, 16 h; (b) HCl (gas), CH2Cl2, 025 °C 1 h; (c) 5-carboxy-X-
rhodamine N-succinimidyl ester, triethylamine, dimethylsulfoxide, 25
°C, 16 h.
ment of 4-[2-{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoro-
methyl-1H-indol-3-yl}acetamido]butylcarbamate with HCl
(gas) gave N-(4-aminobutyl)-2-{1-(4-chlorobenzoyl)-5-me-
thoxy-2′-trifluoromethyl-1H-indol-3-yl}acetamide hydrochlor-
ide. 5-Carboxy-X-rhodamine N-succinnimidyl ester (5-ROX
NSE)9 was reacted with the free amine of N-(4-aminobutyl)-2-
{1-(4-chlorobenzoyl)-5-methoxy-2′-trifluoromethyl-1H-indol-
3-yl}acetamide hydrochloride in the presence of triethylamine
to give N-{(5-carboxy-X-rhodaminyl)but-4-yl}-2-{1-(4-chloro-
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in dimethyl sulfoxide. After 3 h, the animals were lightly
anesthetized with 2% isoflurane and placed in a Xenogen
IVIS200 optical imaging system. CF3-fluorocoxib A targeted the
inflamed footpad selectively over the contralateral control
footpad (Figure 3a). The dependence of compound uptake on
COX-2 in the inflammatory tissues was confirmed by blocking
the COX-2 active site with celecoxib. We administered
celecoxib dissolved in dimethyl sulfoxide (50 mg/kg, i.p.) at
24 h postcarrageenan and waited 1 h for absorption and
blockage of the COX-2 active site prior to dosing with CF3-
fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3-
fluorocoxib A, we lightly anesthetized the animals with 2%
isoflurane and imaged them using the Xenogen IVIS200
camera. There was no enrichment of CF3-fluorocoxib A in the
inflamed paw compared to the control paw (Figure 3b). Figure
3c displays the relative uptake of CF3-fluorocoxib A in the
inflamed footpad versus the control footpad by image analysis
of the data in Figure 3a (n = 3, p = 0.02). Also, imaging was
performed at 5 and 30 min points, where significant probe
distribution was observed in both paws.
We next evaluated the ability of CF3-fluorocoxib A to target
COX-2 in human tumor xenografts. Female nude mice were
injected in the left hip with COX-2-expressing human 1483
HNSCC cells or in the right hip with COX-2-null human
colorectal carcinoma (HCT116) cells. The tumor xenografts
were allowed to grow to approximately 800−1000 mm3.
Animals were injected (1 mg/kg, i.p.) with CF3-fluorocoxib A.
At 4 h postinjection, the animals were lightly anesthetized with
2% isoflurane and placed in the Xenogen IVIS 200 optical
imaging system. A significant uptake of CF3-fluorocoxib A was
documented in the COX-2-expressing 1483 tumors (Figure
4a), where as only a minimal uptake was observed in the COX-
2-null HCT116 tumors (Figure 4b). Figure 4c displays
quantification of the uptake of CF3-fluorocoxib A in the 1483
and HCT116 tumors obtained from image analysis (n = 4, p =
0.01). This suggests that the difference in uptake of CF3-
fluorocoxib A in 1483 and HCT116 xenografts is due to their
differential in COX-2 expression. A significant peritoneal
accumulation was detected at the earlier time points, which is
due the distribution of the CF3-fluorocoxib A in liver, kidney
with clearance in both urine and feces. CF3−Fluorocoxib A
exhibits promise for in vivo detection of COX-2-expressing
tumors that are deep-seated, such as tumors located in bladder,
colon or intestine using noninvasive endoscopic techniques.
Although, CF3-fluorocoxib A and fluorocoxib A are similar in
structure, dissimilarities or improved properties were observed
for CF3-fluorocoxib A in inhibition assays with a series of site-
directed COX-2 mutants (Table 1).13 The compounds
described earlier by our laboratory were conjugates of the
nonselective NSAID, indomethacin, with fluorophores.7 The
present compound is a conjugate of a COX-2-selective
inhibitor, CF3-indomethacin, with a fluorophore. It possesses
superior selectivity and binding characteristics for COX-2. The
inhibition of COX-2 by fluorocoxib A is due to the key
interactions of the 2′-CH3 group with the residues Ala-527, Val-
349, Ser-530, and Leu-531 that form a small hydrophobic
pocket. Mutation of Val-349 to Ala increases the potency of
fluorocoxib A by ∼2-fold due to enlargement of the pocket,
whereas mutation to Leu reduces the pocket size and decreases
the potency of fluorocoxib A by ∼6-fold. A more intense trend
was observed for CF3-fluorocoxib A, where it showed an
improved potency against Val-349 to Ala mutant and Val-349
to Leu mutant, suggesting that the 2′-trifluoromethyl group of
Figure 2. Kinetics of the time-dependent inhibition of COX-2 by CF3-
fluorocoxib A. The purified COX-2 enzyme was reconstituted with
heme and preincubated with the inhibitor at 37 °C for various times
(0, 0.5, 1, 3, 5, 10, 15, and 30 min) prior to the addition of the
substrate (50 μM). (a) Time-dependent inhibition of COX-2 by CF3-
fluorocoxib A at the indicated concentrations. (b) Secondary plot of
kobs versus inhibitor concentration used to generate values for KI, k2,
and k−2.
concentration, which allows the determination of the
equilibrium constant for initial association (KI = k−1/k1) and
the forward and reverse rate constants for the conversion to the
tightly bound enzyme−inhibitor complex (k2 and k−2,
respectively, eq 2). CF3-fluorocoxib A and fluorocoxib A
demonstrate similar affinities for initial complex formation, as
indicated by their KI values (1.5
0.35 and 1.7
2.3 μM,
respectively). The forward rate constants (k2) are also similar
for these compounds (CF3-fluorocoxib A, k2 = 0.004 s−1;
fluorocoxib A, k2 = 0.005 s−1), and the reverse rate constants
(k−2) are essentially the same (CF3-fluorocoxib A, k−2 = 0.001
s−1; fluorocoxib A, k−2 = 0.001 s−1). The low rate constant for
dissociation of the tightly bound complex is a strong
contributing factor toward the potency of the compounds.
k1
k2
E + I HooI [EI] HooI EI*
k−1
k−2
(1)
where KI = k−1/k1 and
k2*[I]
kobs
=
+ k−2
KI + [I]
(2)
The C57BL/6 mouse footpad model is a well-established
system for the study of COX-2-dependent inflammation. COX-
2-derived prostaglandins have a significant role in the acute
edema induced by carrageenan injection into the paw.12
A
major advantage of this inflammation model is the ability to
image the inflamed mouse footpad in comparison to the
noninflamed contralateral footpad without COX-2 expression.
We injected 50 μL of 1% carrageenan in the rear right footpad
of each mouse (body weight 20−25 g) and waited 24 h for
inflammation to develop. Then, we injected the fluorescent
CF3-fluorocoxib A (1 mg/kg) intraperitoneally (i.p.) dissolved
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ACS Medicinal Chemistry Letters
Technology Note
Figure 3. continued
lightly anesthetized with 2% isoflurane and imaged in the Xenogen
IVIS 200 optical imaging system. There was minimal uptake of CF3-
fluorocoxib A in the inflamed footpad that was comparable to the
noninflamed foot. (c) Quantitation of CF3-fluorocoxib A uptake in
inflamed vs control footpad at 3 h postinjection of the agent (from
data in panel a).
Figure 4. In vivo labeling of COX-2-expressing cancer by CF3-
fluorocoxib A. Female nude mice bearing COX-2-expressing 1483
HNSCC and COX-2-null HCT116 tumors were dosed with CF3-
fluorocoxib A (1 mg/kg, i.p.). The animals were imaged at 4 h
postinjection of CF3-fluorocoxib A in a Xenogen IVIS200 optical
imaging instrument. (a) A significant uptake of CF3-fluorocoxib A was
documented in the COX-2-expressing 1483 tumors (arrow). (b)
Minimal uptake of CF3-fluorocoxib A was documented in the COX-2-
negative HCT116 tumors. (c) Quantitation of CF3-fluorocoxib A
uptake in COX-2-expressing 1483 tumors and COX-2-negative
HCT116 tumors at 4 h postinjection of the agent.
Figure 3. In vivo labeling of COX-2 expression in C57BL/6 mouse
footpad inflammation by CF3-fluorocoxib A. The inflammation was
developed by injecting 50 μL 1% carrageenan into the right hind
footpad. (a) At 24 h postcarrageenan, the mouse was injected with
CF3-fluorocoxib A (1 mg/kg, i.p.). At 3 h postinjection of CF3-
fluorocoxib A, a mouse was lightly anesthetized with 2% isoflurane and
imaged in the Xenogen IVIS 200 optical imaging system. A significant
uptake of CF3-fluorocoxib A was documented in the inflamed footpad.
(b) At 24 h postcarrageenan, the mouse was predosed with celecoxib
(50 mg/kg, i.p.) 1 h before the administration of CF3-fluorocoxib A (1
mg/kg, i.p.). At 3 h postinjection of CF3-fluorocoxib A, the mouse was
CF3-fluorocoxib A binds with these residues more tightly then
that of the 2′-methyl group of fluorocoxib A. Improved
properties of CF3-fluorocoxib A were also observed with Ser-
530 to Ala mutant, Arg-120 to Ala mutant, or Val-89 to Trp/
Ser-119 to Trp double mutant, suggesting that the 2′-
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Table 1. Inhibition of Wild-Type Ovine COX-1, Wild-Type
Mouse COX-2, and Mouse COX-2 Mutants by Fluorocoxib
A and CF3-Fluorocoxib A
ACKNOWLEDGMENTS
We are grateful to Dr. Carol Rouzer for critical reading and
editing of this manuscript.
■
wild-type or mutant
enzymes
fluorocoxib A
a
CF3-fluorocoxib A
a
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(IC50 μM)
(IC50 μM)
■
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V349A
0.70
>25
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In summary, CF3-fluorocoxib A has been synthesized and
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optical imaging. The fluorescent CF3-fluorocoxib A inhibited
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inflammatory and cancer cells. CF3-fluorocoxib A is a slow and
tight binding inhibitor of COX-2 with similar binding kinetics
for COX-2 as the parent fluorocoxib A. Although CF3-
fluorocoxib A and fluorocoxib A share similar structural
features, CF3-fluorocoxib A is a more potent inhibitor of
wtCOX-2 and of a series of COX-2 mutants. CF3-fluorocoxib A
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determinant of uptake and retention of CF3-fluorocoxib A in
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ASSOCIATED CONTENT
* Supporting Information
■
S
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.
AUTHOR INFORMATION
Corresponding Author
*(M.J.U.) Tel: 615-343-7326. Fax: 615-343-7534. E-mail:
jashim.uddin@vanderbilt.edu.
■
Funding
This work was supported by a grant from the National
Institutes of Health and National Cancer Institute (CA136465,
CA128323, CA128323-4, and CA128323-5).
Notes
The authors declare no competing financial interest.
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