Structure-activity relationship of celecoxib and rofecoxib for the membrane permeabilizing activity

Article abstract of DOI:10.1016/j.bmc.2014.02.032

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in terms of their structure-activity relationship, we separated the structures of celecoxib and rofecoxib into three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the membrane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl subgroup of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent or weak membrane permeabilizing activity, respectively. These findings provide important information for design and synthesis of new coxibs with lower membrane permeabilizing activity.

Full text of DOI:10.1016/j.bmc.2014.02.032

Bioorganic & Medicinal Chemistry 22 (2014) 2529–2534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of celecoxib and rofecoxib
for the membrane permeabilizing activity
Naoki Yamakawa a,b, , Koichiro Suzuki a, , Yasunobu Yamashita a, Takashi Katsu c, Kengo Hanaya a,
Mitsuru Shoji a, Takeshi Sugai a, Tohru Mizushima a,
⇑
a Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
b Shujitsu University School of Pharmacy, Okayama 703-8516, Japan
c Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory effect by inhibiting
cyclooxygenase activity. We previously suggested that in addition to cyclooxygenase-inhibition at the
gastric mucosa, NSAID-induced gastric mucosal cell death is required for the formation of NSAID-induced
gastric lesions in vivo. We showed that celecoxib exhibited the most potent membrane permeabilizing
activity among the NSAIDs tested. In contrast, we have found that the NSAID rofecoxib has very weak
membrane permeabilizing activity. To understand the membrane permeabilizing activity of coxibs in
terms of their structure–activity relationship, we separated the structures of celecoxib and rofecoxib into
three parts, synthesized hybrid compounds by substitution of each of the parts, and examined the mem-
brane permeabilizing activities of these hybrids. The results suggest that the sulfonamidophenyl sub-
group of celecoxib or the methanesulfonylphenyl subgroup of rofecoxib is important for their potent
or weak membrane permeabilizing activity, respectively. These findings provide important information
for design and synthesis of new coxibs with lower membrane permeabilizing activity.
Received 21 January 2014
Revised 19 February 2014
Accepted 22 February 2014
Available online 12 March 2014
Keywords:
Celecoxib
Rofecoxib
COX-2 selectivity
Membrane permeabilization
Gastric adverse effect
Ó 2014 Published by Elsevier Ltd.
1. Introduction
COX activity at the gastric mucosa and in inflammatory tissues,
respectively,3,4 selective COX-2 inhibitors (most of which are cox-
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the
most frequently used classes of medicines.1 NSAIDs are inhibitors
of cyclooxygenase (COX), a protein essential for the synthesis of
prostaglandins (PGs), which have a strong ability to induce inflam-
mation. However, NSAID use is associated with gastrointestinal
complications, such as gastric ulcers and bleeding. In the United
States, about 16,500 people per year die as a result of NSAID-asso-
ciated gastrointestinal complications.2 Thus, understanding the
mechanism of NSAID-induced gastric lesions and its application
to design and synthesis of new NSAIDs with reduced adverse ef-
fects on the gastric mucosa is important.
The inhibition of COX by NSAIDs was initially thought to be
responsible for the adverse gastric side effects manifested by such
treatment, because PGs have a strong protective effect on the gas-
tric mucosa. Thus, after the identification of two subtypes of COX
(COX-1 and COX-2), which are responsible for the majority of
ibs, such as celecoxib and rofecoxib) were developed as NSAIDs
with reduced adverse gastric side effects.5–7 However, due to the
observation that rofecoxib was associated with an increased poten-
tial risk of cardiovascular thrombotic events,8,9 this NSAID was
withdrawn from the market. At first, this increased risk was
believed to be due to the class effect of selective COX-2 inhibitors,
because prostacyclin, a potent anti-aggregator of platelets and a
vasodilator, is mainly produced by COX-2.10–12 However, some
clinical studies showed that the potential risk of cardiovascular
thrombotic events was indistinguishable between celecoxib users
and classic NSAID users.13,14 Thus, it is possible that the increased
potential risk of cardiovascular thrombotic events is not due to the
class effect of selective COX-2 inhibitors, but rather is a specific
characteristic of rofecoxib. While mechanisms to explain this
rofecoxib-specific increase in the potential risk of cardiovascular
thrombotic events have been proposed,15–17 a definitive explana-
tion for this increase has not yet been forthcoming.
It is now believed that the inhibition of COX by NSAIDs is not
the sole explanation for the adverse gastric side effects of NSAIDs,
given that the increased incidence of gastric lesions and the
⇑
Corresponding author. Tel.: +81 354002628.
E-mail address: mizushima-th@pha.keio.ac.jp (T. Mizushima).
These two authors contributed to this paper equally.
http://dx.doi.org/10.1016/j.bmc.2014.02.032
0968-0896/Ó 2014 Published by Elsevier Ltd.

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N. Yamakawa et al. / Bioorg. Med. Chem. 22 (2014) 2529–2534
decrease in PG levels induced by NSAIDs do not always occur in
parallel.18–20 We proposed that, in addition to COX-inhibition at
the gastric mucosa, NSAID-induced gastric mucosal cell death is re-
quired for the formation of NSAID-induced gastric lesions
in vivo.21,22 Furthermore, we reproduced NSAID-induce cell death
in cultured gastric mucosal cells in vitro22–26 and showed that
the primary target of NSAIDs for the induction of cell death is the
cytoplasmic membrane. Moreover, a close relationship between
membrane permeabilizing activity and cell death-inducing activity
among various NSAIDs was shown.23,25 Thus, decreasing the mem-
brane permeabilizing activity of NSAIDs may be another strategy to
synthesize safer NSAIDs for the gastric mucosa. In fact, we recently
reported that screening for NSAIDs with lower membrane perme-
abilizing activity resulted in the identification of an interesting
new NSAID, fluoro-loxoprofen, which has much lower membrane
permeabilizing and gastric ulcerogenic activities compared with
clinically used NSAIDs.27–31 These results suggest that NSAIDs with
lower membrane permeabilizing activity could be therapeutically
beneficial. Thus, it is important to understand how the membrane
permeabilizing properties of NSAIDs are affected by their struc-
ture–activity relationship.
We previously reported that celecoxib showed the most potent
membrane permeabilizing and cytotoxic activities among the NSA-
IDs we tested.23,25 We also reported that the cytotoxic activity of
rofecoxib is much lower than that of celecoxib.21 As these results
suggested that the membrane permeabilizing activity of rofecoxib
is lower than that of celecoxib, our objective here was to confirm
this hypothesis.
Furthermore, to identify how the structure–activity relationship
of coxibs affects their membrane permeabilizing activity, we
synthesized hybrid compounds from celecoxib and rofecoxib and
examined their membrane permeabilizing activities. The results
suggest that the sulfonamidophenyl subgroup of celecoxib and the
methanesulfonylphenyl subgroup of rofecoxib are important for
determining the membrane permeabilizing activities of these
NSAIDs.
condensation of a phenylacetic acid analog and phenacyl bromide.
The reaction of 13 with 15, 14 with 17 or 14 with 16 in the pres-
ence of triethylamine afforded the phenacyl phenylacetate prod-
ucts 18, 19 or 20, respectively. Treatment of intermediates 18–20
with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) provided 3,4-diphenyl-
2(5H)furanone 21 or target compounds 7 or 8. chlorosulfonylation
of 21 by the reaction with chlorosulfonic acid followed by sulfo-
namidation using ammonium hydroxide gave target compound 6.
The final compounds were characterized by nuclear magnetic
resonance (NMR), infrared spectroscopy (IR), high resolution mass
spectra (HR-MS) and elemental analysis.
3. Results and discussion
The chemical structures of celecoxib and rofecoxib exhibit some
similarities (Fig. 1) and can be divided into three parts (A–C in
Table 1); part A, methylphenyl for celecoxib, phenyl for rofecoxib;
part B, trifluoromethylpyrazole for celecoxib, furanone for rofecox-
ib; part C, sulfonamidophenyl for celecoxib, methanesulfonylphe-
nyl for rofecoxib. Thus, in addition to celecoxib and rofecoxib,
there are six possible combinations of these three parts that could
be used to obtain hybrid compounds of celecoxib and rofecoxib
(compounds 3–8 in Table 1). We synthesized these six compounds
and tested their membrane permeabilizing and COX-inhibitory
activities.
To begin with, we used calcein-loaded liposomes to compare
the membrane permeabilizing activities of celecoxib and rofecoxib.
As calcein fluorescence is very weak at high concentrations due to
self-quenching, the addition of membrane-permeabilizing drugs to
a medium containing calcein-loaded liposomes causes an increase
in fluorescence by diluting the calcein.25 As shown in Figure 2,
celecoxib and rofecoxib increased the calcein fluorescence in a
dose-dependent manner. Compared with celecoxib, however, a
rofecoxib concentration about 100 times higher was required to in-
crease the fluorescence by the same amount. Figure 2 shows that
rofecoxib has a much lower membrane permeabilizing activity
than celecoxib.
2. Chemistry
We next examined the membrane permeabilizing activities of
the six hybrid compounds in a similar manner. As shown in
Figure 3, all of the hybrid compounds increased the calcein fluores-
cence in a dose-dependent manner. To compare the membrane
permeabilizing activity of these compounds, we used the EC50
(half-maximal effective concentration) index, which is defined as
the concentration of each compound required for 50% of the calce-
in in loaded liposomes to be released (Table 2). Comparison of the
EC50 index of 3, 5 and 8 (compounds with one part substitution
from celecoxib) showed that the membrane permeabilizing activ-
ity of 3 was much lower than that of 5 or 8, suggesting that part
The synthetic route for target compounds 3–5 is outlined in
Scheme 1. Pyrazole compounds 3–5 were synthesized by the
condensation of appropriate 1,3-diketones and hydrazine. The
reaction of 4,4,4-trifluoro-1-p-tolylbutane-1,3-dione
9
with
4-methylphenylhydrazine hydrochloride 11, 4,4,4-trifluoro-
1-phenylbutane-1,3-dione 10 with 11, or 10 with 4-sul-
famoylphenylhydrazine hydrochloride 12 afforded target
compounds 3, 4 or 5, respectively.
The synthetic route for target compounds 6–8 is outlined in
Scheme 2. Furanone compounds 6–8 were synthesized by the
Scheme 1. Synthesis of pyrazole compounds 3–5.

N. Yamakawa et al. / Bioorg. Med. Chem. 22 (2014) 2529–2534
2531
Scheme 2. Synthesis of furanone compounds 6–8.
er than that of 4 or 7. In this case, part C of rofecoxib (the metha-
nesulfonylphenyl subgroup) was also seemed to be the most
important subgroup determining its low membrane permeabiliz-
ing activity. Thus, part C seems to be important for determining
the permeabilizing activity of these coxibs. The fundamental struc-
tural requirement underlying the ability of molecule to permeabi-
lize membrane is a shape in which clusters of hydrophobic and
hydrophilic parts are spatially organized with appropriate dis-
tance, because this structure enable the hydrophobic part to be lo-
cated near the surface of membrane, resulting in perturbation of
membrane structure. In the present case, because the sulfonamid-
ophenyl subgroup is more hydrophilic than the methanesulfonyl-
phenyl subgroup, the former but not the latter produces
hydrophilic part. The methanesulfonylphenyl subgroup is hydro-
phobic enough to be totally buried within the lipid bilayer struc-
ture and therefore, does not affect the membrane structure
drastically. On the other hand, the sulfonamidophenyl subgroup
is relatively hydrophilic, which allows the compound to face mem-
brane surface, resulting in perturbation of the membrane structure.
Figure 1. Structures of celecoxib and rofecoxib.
C of celecoxib (the sulfonamidophenyl subgroup) is the most
important subgroup determining its high membrane permeabiliz-
ing activity. On the other hand, comparison of the EC50 index of
4, 6 and 7 (compounds with one part substitution from rofecoxib)
revealed that the membrane permeabilizing activity of 6 was high-
Table 1
Structures of celecoxib, rofecoxib and their hybrid compounds (3–8)
A
B
C
A
B
C
Celecoxib
Rofecoxib
3
4
5
6
7
8

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N. Yamakawa et al. / Bioorg. Med. Chem. 22 (2014) 2529–2534
Table 2
Membrane permeabilizing activities and inhibitory activities on COX-1 and COX-2 of
celecoxib, rofecoxib and their hybrid compounds (3–8)
Compounds EC50 (mM)
IC50
(
lM)
COX-1/COX-2 EC50/IC50
Calcein release COX-1 COX-2
Celecoxib
Rofecoxib
0.050
12.6
1.44
1.30
0.12
0.48
6.23
0.024
117
0.07
0.36
0.13
6.84
0.16
>500
0.30
0.11
1670
>1380
>3830
>73
1330
—
0.71
35.0
11.1
0.19
0.75
—
>500
>500
>500
213
>500
>500
385
3
4
5
6
7
8
>1680
>3500
20.8
0.22
The EC50 value for membrane permeabilization (concentration of each compound
required for 50% release of calcein) was calculated based on the data shown in
Figures 2 and 3. The inhibitory effect of each compound on COX-1 and COX-2 was
examined using purified ovine COX-1 and human recombinant COX-2 as described
in the experimental section. The values of IC50 (concentration of each compound
required for 50% inhibition) were estimated from the sigmoid-like dose-response
curve (4-parameter logistic curve model) drawn using logistic-curve fitting soft-
ware (ImageJ 1.43u; National Institutes of Health, USA), and the COX-1/COX-2
ratios of IC50 values were calculated. EC50 for membrane permeabilization/IC50 for
COX-2 inhibition was shown. Values are mean (n = 3).
Figure 2. Membrane permeabilization by celecoxib and rofecoxib. Calcein-loaded
liposomes were incubated for 10 min at 30 °C with the indicated concentration of
each compound. The release of calcein from the liposomes was determined by
measuring fluorescence intensity as described in the experimental section. Triton
X-100 (10 lM) was used to establish the 100% level of calcein release. Values are
mean SD (n = 3).
Among the six hybrid compounds, 4 and 6 can be eliminated as
candidates for future clinical use based on their relatively weak
inhibitory activity on COX-2 (Table 2). On the other hand, 5 and
8 can be eliminated as candidates based on their relatively potent
membrane permeabilizing activity (Table 2). To further compare
the potential value of these compounds, we calculated the value
of the EC50 index for calcein release/IC50 index for COX-2 (Table 2).
Compounds 3 and 7 appear as the most likely selections as candi-
dates for future clinical use based on this index (Table 2). As de-
scribed in the introduction section, rofecoxib was withdrawn
from the market due to an observed increased potential risk for
cardiovascular thrombotic events,8,9 which may not be a drug class
effect but actually something characteristic of rofecoxib alone.
According to this hypothesis, it could be postulated that 3 and 7
might have fewer adverse effects associated with their use. Never-
theless, because the mechanism underlying the increased cardio-
vascular thrombotic events remains to be elucidated, the
potential risk of these compounds has not yet been tested without
a large-scale clinical study.
Figure 3. Membrane permeabilization by pyrazole compounds 3–5 and furanone
compounds 6–8. Experiments and data analysis were performed as described in the
legend of Fig. 2. Values shown are mean SD (n = 3).
The results presented in Figures 2 and 3 thus provide important
information for the design and synthesis of new coxibs with lower
membrane permeabilizing activity.
The ionization of these compounds would not be related to their
membrane permeabilizing activities. This is because pKa values of
all compounds (celecoxib, rofecoxib, 3– 8) are higher than 10 and
membrane permeabilizing assay was performed under the condi-
tions of pH = 6.8. Furthermore, such ionization would increase
the osmotic pressure outside vesicles and thus, would not stimu-
late the release of calcein from vesicles.
4. Conclusion
We here found that rofecoxib has very weak membrane perme-
abilizing activity compared with celecoxib. Furthermore, analysis
of the membrane permeabilizing activities of hybrid compounds
derived from celecoxib and rofecoxib suggested that the sulfon-
amidophenyl subgroup of celecoxib and the methanesulfonylphe-
nyl subgroup of rofecoxib are important for their potent or weak
membrane permeabilizing activity, respectively.
The inhibitory effects on COX-1 and COX-2 of these compounds
were compared by using the IC50 (half-maximal inhibitory concen-
tration) index, which is defined as the concentration of each com-
pound required for 50% inhibition of each enzyme. The IC50 values
for COX-1 and COX-2 of celecoxib and rofecoxib were roughly sim-
ilar to those reported previously,32 and the IC50 values for COX-1 of
all the hybrid compounds were relatively high (Table 2). With the
exception of 4 and 6, the IC50 values for COX-2 of the hybrid com-
pounds were within the range seen for celecoxib and rofecoxib,
and all the hybrid compounds (except 6) showed COX-2 selectivity
(Table 2). Since both 4 and 6 are compounds with one part substi-
tution from rofecoxib, the structure of rofecoxib rather than that of
celecoxib appears to be sensitive to modification in relation to
COX-2 inhibitory activity.
5. Experimental section
5.1. Chemistry
All solvents and reagents were purchased from Tokyo Chemical
Industry Co., Ltd (Tokyo, Japan) or Wako Pure Chemical Industries
(Tokyo, Japan) and used without further purification. Fourier trans-
form IR spectra were recorded as films with NaCl plates on a JASCO
FT/IR-480 spectrophotometer. 1H NMR and 13C NMR spectra were
recorded on VARIAN 400- or 500-MR spectrometer (Agilent Thech-
nologies Japan, Tokyo, Japan) operating at 400 MHz, in a ca. 2%
solution of CDCl3 or DMSO-d6. Coupling constant (J) values are esti-
mated in hertz (Hz) and spin multiples are given as s (singlet), d

N. Yamakawa et al. / Bioorg. Med. Chem. 22 (2014) 2529–2534
2533
(double), m (multiplet), and br (broad). Mass spectra were detected
with an electrospray ionization time-of-flight (ESI-TOF) mass spec-
trometer (Bruker MicroTOF, Bruker, Bremen, Germany) in the neg-
ative mode. The progress of all reactions was monitored by thin-
nyl-H3, -H4, -H5), 7.45 (d, J = 8.5, 2H, 4-sulfonamidophenyl-H2, -
H6), 7.88 (d, J = 8.5, 2H, 4-sulfonamidophenyl-H3, -H5); HR-ESI-
TOF/MS (negative, m/z): 368.0622 ([MÀH]À, Calcd for C16H12F3N2-
O2S: 368.0681). Anal. Calcd for C16H13F3N2O2S: C, 52.17; H, 3.56;
N, 11.41. Found: C, 52.12; H, 3.45; N, 11.28. 1H NMR spectral data
for 5 were consistent with reported results.35,36
layer chromatography (TLC) with silica gel glass plates (60 F254
)
(Merck Ltd, Tokyo, Japan), and spots were visualized with ultravi-
olet (UV) light (254 nm) and stained with 5% ethanolic phospho-
molybdic acid. Column chromatography was performed using
Silica gel 60 N (Kanto Chemical Co., Tokyo, Japan). Elemental anal-
yses were performed for C, H and N (Central Service Research Cen-
ter, Keio University) and were within 0.4% of the theoretical
values. Melting points (mp) were obtained using a Yanaco melting
point apparatus MP-J3 (Yanaco, Kyoto, Japan) without correction.
Celecoxib and rofecoxib were from LKT Laboratories Inc. Egg phos-
phatidylcholine (PC) was from Kanto Chemicals Co. (Tokyo, Japan).
5.1.4. General procedure for preparation of compounds 21, 7
and 8
To a stirred solution of phenylacetic acid (13 or 14) and trieth-
ylamine in dry CH3CN, phenacyl bromide (15–17) in dry CH3CN
was added dropwise at room temperature. The reaction mixture
was stirred for 1 h and was concentrated in vacuo. The resulting
residue was re-dissolved in AcOEt (50 mL) and washed with 1 M
HCl (20 mL). The organic fraction was dried over Na2SO4 and fil-
tered. The filtrate was evaporated under reduced pressure to give
crude product (18–20) that was used in the next step without fur-
ther purification.
DBU (1.0 equiv) in dry CH3CN (2 mL) was added dropwise to a
stirred solution of the crude intermediate (18–20, 1.0 equiv) in
dry CH3CN (8 mL) at 0 °C. After stirring at 0 °C for 15 min, the mix-
ture was poured into dilute HCl solution and the product was ex-
tracted with AcOEt. Evaporation of the solvent and purification of
the residue by silica gel chromatography (n-hexane/AcOEt, 2:1)
yielded the furanone compounds 21, 7 or 8.
5.1.1. General procedure for preparation of compounds 3–5
Phenylhydrazine hydrochloride (11 or 12) was added to a stir-
red solution of the dione (9 or 10) in ethanol (30 mL), and the mix-
ture was refluxed for 20 h. After cooling to room temperature, the
reaction mixture was concentrated in vacuo. The resulting residue
was dissolved in AcOEt (50 mL) and washed with brine. The organ-
ic fraction was dried over Na2SO4 and filtered. The filtrate was con-
centrated in vacuo and the residue was purified by silica gel
chromatography (n-hexane/AcOEt, 2:1) to afford pyrazole com-
pounds 3–5.
5.1.4.1.
3-Phenyl-4-(4-sulfonamidophenyl)-2(5H)-furanone
5.1.2. 1-(4-Methanesulfonylphenyl)-5-p-tolyl-3-
(trifluoromethyl)-1H-pyrazole (3)
Compound 3 was synthesized from 9 (500 mg, 2.2 mmol,
1.0 equiv) and 11 (774 mg, 3.5 mmol, 1.6 equiv). Colorless nee-
(6). Compound 6 was prepared by chlorosulfonylation in chloro-
form and sulfonamide formation using ammonium hydroxide in
ethanol of 21 that was obtained from phenylacetic acid (13, 0.5 g,
3.7 mmol, 1.0 equiv) and 4-sulfonamidophenacyl bromide (15,
1.03 g, 3.7 mmol, 1.0 equiv) via the intermediate 18 by the method
described previously.36 Colorless needle-like crystals (yield 22%,
dle-like crystals (yield 35%); mp 125.1–126.1 °C; IR (film)
m:
1160, 1325 (SO2), 2930 (C-H), 3015 (Ar-H) cmÀ1 1H NMR (CDCl3,
;
500 MHz) d: 2.36 (s, 3H, Ar-CH3), 3.04 (s, 3H, SO2CH3), 6.72 (s,
1H, pyrazole-H4), 7.10 (d, J = 8.0, 2H, p-tolyl-H3, -H5), 7.16 (d,
J = 8.0, 2H, p-tolyl-H2, -H6), 7.52 (d, J = 8.0, 2H, methanesulfonyl-
phenyl-H2, -H6), 7.91 (d, J = 8.0, 2H, methanesulfonylphenyl-H3,
-H5); HR-ESI-TOF/MS (negative, m/z): 379.0706 ([MÀH]À, Calcd
for C18H14F3N2O2S: 379.0728). Anal. Calcd for C18H15F3N2O2S: C,
56.84; H, 3.97; N, 7.36. Found: C, 56.64; H, 3.75; N, 7.20. IR and
three steps); mp 248.2–249.5 °C; IR (film)
m
;
: 1145, 1320 (SO2),
1740 (C@O), 3030 (Ar-H), 3230 (N-H) cmÀ1
1H NMR (DMSO-d6,
400 MHz) d: 5.42 (s, 2H, CH2), 7.35–7.53 (m, 5H, phenyl-H), 7.42
(br s, 2H, NH2), 7.52 (d, J = 8.6, 2H, 4-sulfonamidophenyl-H2, -
H6), 7.85 (d, J = 8.6, 2H, 4-sulfonamidophenyl-H3, -H5); HR-ESI-
TOF/MS (negative, m/z): 314.0495 ([MÀH]À, Calcd for C16H12NO4S:
314.0487). Anal. Calcd for C16H13NO4S: C, 60.94; H, 4.16; N, 4.44.
Found: C, 61.01; H, 4.02; N, 4.30. IR and 1H NMR spectral data
for 6 were consistent with reported results.37,38
1H NMR spectral data for
results.33,34
3 were consistent with reported
5.1.3. 1-(4-Methanesulfonylphenyl)-5-phenyl-3-
(trifluoromethyl)-1H-pyrazole (4)
Compound 4 was synthesized from 10 (500 mg, 2.3 mmol,
1.0 equiv) and 11 (567 mg, 2.5 mmol, 1.6 equiv). Colorless nee-
5.1.4.2.
3-(4-Methylphenyl)-4-(4-methanesulfonylphenyl)-
2(5H)-furanone (7). Compound 7 was synthesized via the inter-
mediate 19 from 4-methylphenylacetic acid (14, 0.5 g, 3.7 mmol,
1.0 equiv) and 4-methanesulfonylphenacyl bromide (17, 1.03 g,
3.7 mmol, 1.0 equiv). Yellow needle-like crystals (yield 67%, two
dle-like crystals (yield 50%); mp 135.2–136.1 °C; IR (film)
m:
1162, 1320 (SO2), 2935 (C-H), 3020 (Ar-H) cmÀ1 1H NMR (CDCl3,
;
steps); mp 174.5–175.4 °C; IR (film)
m
: 1150, 1320 (SO2), 1750
500 MHz) d: 3.04 (s, 3H, SO2CH3), 6.77 (s, 1H, pyrazole-H4), 7.22–
7.23 (m, 2H, phenyl-H2, -H6), 7.35–7.41 (m, 3H, phenyl-H3, -H4,
-H5), 7.51 (d, J = 8.5, 2H, methanesulfonylphenyl-H2, -H6), 7.91
(d, J = 8.5, 2H, methanesulfonylphenyl-H3, -H5); 13C NMR (CDCl3,
500 MHz) d: 44.28, 106.6, 125.5, 128.4, 128.7, 129.0, 129.5, 139.8,
143.2, 144.0, 144.3, 145.1; HR-ESI-TOF/MS (negative, m/z):
365.0550 ([MÀH]À, Calcd for C17H12F3N2O2S: 365.0572). Anal.
Calcd for C17H13F3N2O2S: C, 55.73; H, 3.58; N, 7.65. Found: C,
55.58; H, 3.60; N, 7.44.
(C@O), 3040 (Ar-H), 2930 (C-H) cmÀ1
;
1H NMR (CDCl3, 400 MHz)
d: 2.38 (s, 3H, Ar-CH3), 3.07 (s, 3H, SO2CH3), 5.17 (s, 2H, CH2),
7.20 (d, J = 8.1, 2H, p-tolyl-H3, -H5), 7.28 (d, J = 8.2, 2H, p-tolyl-
H2, -H6), 7.52 (dd, J = 6.8, 2.0, 2H, methanesulfonylphenyl-H2, -
H6), 7.92 (dd, J = 6.8, 2.0, 2H, methanesulfonylphenyl-H3, -H5);
HR-ESI-TOF/MS (negative, m/z): 327.0718 ([MÀH]À, Calcd for
C
18H15O4S: 327.0691). Anal. Calcd for C18H16O4S: C, 65.84; H,
4.91. Found: C, 66.12; H, 5.00. IR and 1H NMR spectral data for 7
were consistent with reported results.39
5.1.3.1. 1-(4-Sulfonamidophenyl)-5-phenyl-3-(trifluoromethyl)-
1H-pyrazole (5). Compound 5 was synthesized from 10 (500 mg,
2.3 mmol, 1.0 equiv) and 12 (560 mg, 3.5 mmol, 1.6 equiv). Color-
less needle-like crystals (yield 66%); mp 164.1–165.2 °C; IR (film)
5.1.4.3.
3-(4-Methylphenyl)-4-(4-sulfonamidophenyl)-2(5H)-
furanone (8). Compound 8 was synthesized via the intermediate
20 from 4-methylphenylacetic acid (14, 0.5 g, 3.7 mmol, 1.0 equiv)
and 4-sulfonamidophenacyl bromide (16, 1.03 g, 3.7 mmol,
1.0 equiv). Yellow needle-like crystals (yield 67%, two steps); mp
m
: 1165, 1325 (SO2), 3025 (Ar-H), 3680 (N-H) cmÀ1 1H NMR
;
(CDCl3, 400 MHz) d: 4.92 (br s, 2H, NH2), 6.75 (s, 1H, pyrazole-
H4), 7.22–7.24 (m, 2H, phenyl-H2, -H6), 7.32–7.40 (m, 3H, phe-
218.5–219.2 °C; IR (film)
m
: 1140, 1325 (SO2), 1735 (C@O), 3025
(Ar-H), 3220 (N-H), 2925 (C-H)cmÀ1
;
1H NMR (DMSO-d6,

2534
N. Yamakawa et al. / Bioorg. Med. Chem. 22 (2014) 2529–2534
3. Kujubu, D. A.; Fletcher, B. S.; Varnum, B. C.; Lim, R. W.; Herschman, H. R. J. Biol.
Chem. 1991, 266, 12866.
4. Xie, W. L.; Chipman, J. G.; Robertson, D. L.; Erikson, R. L.; Simmons, D. L. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 2692.
5. Silverstein, F. E.; Faich, G.; Goldstein, J. L.; Simon, L. S.; Pincus, T.; Whelton, A.;
Makuch, R.; Eisen, G.; Agrawal, N. M.; Stenson, W. F.; Burr, A. M.; Zhao, W. W.;
Kent, J. D.; Lefkowith, J. B.; Verburg, K. M.; Geis, G. S. JAMA 2000, 284, 1247.
6. Bombardier, C.; Laine, L.; Reicin, A.; Shapiro, D.; Burgos, V. R.; Davis, B.; Day, R.;
Ferraz, M. B.; Hawkey, C. J.; Hochberg, M. C.; Kvien, T. K.; Schnitzer, T. J. N. Engl.
J. Med. 2000, 343, 1520.
400 MHz) d: 2.32 (s, 3H, Ar-CH3), 3.32 (s, 3H, SO2CH3), 5.36 (s, 2H,
CH2), 7.22 (d, J = 8.0, 2H, p-tolyl-H3, -H5), 7.24 (d, J = 8.0, 2H, p-to-
lyl-H2, -H6), 7.44 (brs, 2H, NH2), 7.54 (d, J = 8.8, 2H, 4-sulfonamid-
ophenyl-H2, -H6), 7.80 (d, J = 8.8, 2H, 4-sulfonamidophenyl-H3, -
H5); HR-ESI-TOF/MS (negative, m/z): 328.0694 ([MÀH]À, Calcd
for C17H14NO4S: 328.0644). Anal. Calcd for C17H15NO4S: C, 61.99;
H, 4.59; N, 4.25. Found: C, 62.05; H, 4.68; N, 4.42.
7. FitzGerald, G. A.; Patrono, C. N. Engl. J. Med. 2001, 345, 433.
8. Mukherjee, D.; Nissen, S. E.; Topol, E. J. JAMA 2001, 286, 954.
9. Mukherjee, D. Biochem. Pharmacol. 2002, 63, 817.
5.2. Membrane permeability assay
10. McAdam, B. F.; Catella, L. F.; Mardini, I. A.; Kapoor, S.; Lawson, J. A.; FitzGerald,
G. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 272.
11. Catella, L. F.; McAdam, B.; Morrison, B. W.; Kapoor, S.; Kujubu, D.; Antes, L.;
Lasseter, K. C.; Quan, H.; Gertz, B. J.; FitzGerald, G. A. J. Pharmacol. Exp. Ther.
1999, 289, 735.
12. Belton, O.; Byrne, D.; Kearney, D.; Leahy, A.; Fitzgerald, D. J. Circulation 2000,
102, 840.
13. Garcia Rodriguez, L. A.; Gonzalez-Perez, A. BMC Med. 2005, 3, 17.
14. Kearney, P. M.; Baigent, C.; Godwin, J.; Halls, H.; Emberson, J. R.; Patrono, C. BMJ
2006, 332, 1302.
15. Oitate, M.; Hirota, T.; Koyama, K.; Inoue, S.; Kawai, K.; Ikeda, T. Drug Metab.
Dispos. 2006, 34, 1417.
16. Oitate, M.; Hirota, T.; Murai, T.; Miura, S.; Ikeda, T. Drug Metab. Dispos. 2007, 35,
1846.
17. Oitate, M.; Hirota, T.; Takahashi, M.; Murai, T.; Miura, S.; Senoo, A.; Hosokawa,
T.; Oonishi, T.; Ikeda, T. J. Pharmacol. Exp. Ther. 2007, 320, 1195.
18. Ligumsky, M.; Golanska, E. M.; Hansen, D. G.; Kauffman, G. J. Gastroenterology
1983, 84, 756.
19. Ligumsky, M.; Sestieri, M.; Karmeli, F.; Zimmerman, J.; Okon, E.; Rachmilewitz,
D. Gastroenterology 1990, 1245.
20. Lichtenberger, L. M. Biochem. Pharmacol. 2001, 61, 631.
21. Tomisato, W.; Tsutsumi, S.; Hoshino, T.; Hwang, H. J.; Mio, M.; Tsuchiya, T.;
Mizushima, T. Biochem. Pharmacol. 2004, 67, 575.
22. Aburaya, M.; Tanaka, K.; Hoshino, T.; Tsutsumi, S.; Suzuki, K.; Makise, M.;
Akagi, R.; Mizushima, T. J. Biol. Chem. 2006, 281, 33422.
23. Tanaka, K.; Tomisato, W.; Hoshino, T.; Ishihara, T.; Namba, T.; Aburaya, M.;
Katsu, T.; Suzuki, K.; Tsutsumi, S.; Mizushima, T. J. Biol. Chem. 2005, 280, 31059.
24. Tsutsumi, S.; Gotoh, T.; Tomisato, W.; Mima, S.; Hoshino, T.; Hwang, H. J.;
Takenaka, H.; Tsuchiya, T.; Mori, M.; Mizushima, T. Cell Death Differ. 2004, 11,
1009.
Permeabilization of calcein-loaded liposomes was assayed as
described previously,25 with some modifications. Liposomes were
prepared using the reversed-phase evaporation method. Egg phos-
phatidylcholine (PC) (10 lmol, 7.7 mg) was dissolved in chloro-
form/methanol (1:2, v/v), dried, dissolved in 1.5 mL of diethyl
ether and added to 1 mL of 100 mM calcein-NaOH (pH 7.4). The
mixture was then sonicated to obtain a homogenous emulsion.
The diethyl ether solvent was removed and the resulting suspen-
sion of liposomes was centrifuged and washed twice with fresh
buffer A (10 mM phosphate buffer (Na2HPO4/NaH2PO4) (pH 6.8)
containing 150 mM NaCl) to remove untrapped calcein. The final
liposome precipitate was re-suspended in 5 mL buffer A. A 30 lL
aliquot of this suspension was diluted with buffer A to 20 mL
and the diluted suspension was then incubated at 30 °C for
10 min in the presence of each compound. The release of calcein
from liposomes was determined by measuring the fluorescence
intensity at 520 nm (excitation at 490 nm). The EC50 value was
estimated from non-linear regression plots with the average of
triplicate experiments for each compound; Triton X-100 (10
was used to establish the 100% level of calcein release.
lM)
5.3. COX-inhibition assay
25. Tomisato, W.; Tanaka, K.; Katsu, T.; Kakuta, H.; Sasaki, K.; Tsutsumi, S.;
Hoshino, T.; Aburaya, M.; Li, D.; Tsuchiya, T.; Suzuki, K.; Yokomizo, K.;
Mizushima, T. Biochem. Biophys. Res. Commun. 2004, 323, 1032.
26. Tomisato, W.; Tsutsumi, S.; Rokutan, K.; Tsuchiya, T.; Mizushima, T. Am. J.
Physiol. Gastrointest. Liver Physiol. 2001, 281, G1092.
27. Yamakawa, N.; Suemasu, S.; Matoyama, M.; Kimoto, A.; Takeda, M.; Tanaka, K.;
Ishihara, T.; Katsu, T.; Okamoto, Y.; Otsuka, M.; Mizushima, T. J. Med. Chem.
2010, 53, 7879.
The inhibitory effect of each compound on COX-1 and COX-2
activity was examined using an enzyme immunoassay (EIA) kit
(Cayman Chemical, Ann Arbor, MI, USA), including purified ovine
COX-1 and human recombinant COX-2 according to the manufac-
turer’s procedures.
28. Yamakawa, N.; Suemasu, S.; Matoyama, M.; Tanaka, K.-I.; Katsu, T.; Miyata, K.;
Okamoto, Y.; Otsuka, M.; Mizushima, T. Bioorg. Med. Chem. 2011, 19, 3299.
29. Yamakawa, N.; Suemasu, S.; Okamoto, Y.; Tanaka, K.; Ishihara, T.; Asano, T.;
Miyata, K.; Otsuka, M.; Mizushima, T. J. Med. Chem. 2012, 55, 5143.
30. Suemasu, S.; Yamakawa, N.; Ishihara, T.; Asano, T.; Tahara, K.; Tanaka, K.;
Matsui, H.; Okamoto, Y.; Otsuka, M.; Takeuchi, K.; Suzuki, H.; Mizushima, T.
Biochem. Pharmacol. 2012, 84, 1470.
31. Yamakawa, N.; Suemasu, S.; Watanabe, H.; Tahara, K.; Tanaka, K.; Okamoto, Y.;
Ohtsuka, M.; Maruyama, T.; Mizushima, T. Drug Metab. Pharmacokinet. 2013,
28, 118.
Acknowledgments
This work was supported by Grants-in-Aid of Scientific Re-
search from the Ministry of Health, Labour, and Welfare of Japan,
Grants-in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan, and
Grants-in-Aid of the Japan Science and Technology Agency.
32. Praveen Rao, P. N.; Amini, M.; Li, H.; Habeeb, A. G.; Knaus, E. E. J. Med. Chem.
2003, 46, 4872.
33. Abdellatif, K. R.; Chowdhury, M. A.; Dong, Y.; Velazquez, C.; Das, D.; Suresh, M.
R.; Knaus, E. E. Bioorg. Med. Chem. 2008, 16, 9694.
34. Ahlström, M. M.; Ridderström, M.; Zamora, I.; Luthman, K. J. Med. Chem. 2007,
50, 4444.
35. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. d.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.;
Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
Supplementary data
Supplementary data (1H NMR spectra of final new compounds
for 4 and 8) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2014.02.032. These
data include MOL files and InChiKeys of the most important com-
pounds described in this article.
36. Gosselin, F.; O’Shea, P. D.; Webster, R. A.; Reamer, R. A.; Tillyer, R. D.;
Grabowski, E. J. J. Synlett 2006, 3267.
References and notes
37. Uddin, M. J.; Rao, P. N. P.; Knaus, E. E. J. Heterocycl. Chem. 2003, 40, 861.
38. Zarghi, A.; Rao, P. N.; Knaus, E. E. J. Pharm. Pharm. Sci. 2007, 10, 159.
39. Abdellatif, K. R.; Huang, Z.; Chowdhury, M. A.; Kaufman, S.; Knaus, E. E. Bioorg.
Med. Chem. Lett. 2011, 21, 3951.
1. Smalley, W. E.; Ray, W. A.; Daugherty, J. R.; Griffin, M. R. Am. J. Epidemiol. 1995,
141, 539.
2. Singh, G. Am. J. Med. 1998, 105, 31S.