Synthesis and inhibitory effects of triarylpyrazoles on LPS-induced NO and PGE2 productions in RAW 264.7 macrophages

Article abstract of DOI:10.1007/s00044-017-1923-9

Abstract: The inhibition of nitric oxide and prostaglandin E₂ productions is a very interesting research topic in the field of anti-inflammatory drug development. In the current study, a new series of 1,3,4-triarylpyrazole derivatives was synth

Full text of DOI:10.1007/s00044-017-1923-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:2161–2171
DOI 10.1007/s00044-017-1923-9
ORIGINAL RESEARCH
Synthesis and inhibitory effects of triarylpyrazoles on LPS-induced
NO and PGE productions in RAW 264.7 macrophages
2
1 ●
2,3,4 ●
5,6 ●
5,6 ●
Byung-Jun Park Mohammed I. El-Gamal
Woo-Suck Lee
Ji-Sun Shin
7 ●
5,6 ●
1,8
Kyung Ho Yoo Kyung-Tae Lee
Chang-Hyun Oh
Received: 18 January 2017 / Accepted: 15 May 2017 / Published online: 29 May 2017
Springer Science+Business Media New York 2017
©
Abstract The inhibition of nitric oxide and prostaglandin
E2 productions is a very interesting research topic in the
field of anti-inflammatory drug development. In the current
study, a new series of 1,3,4-triarylpyrazole derivatives was
synthesized and evaluated for their capabilities to inhibit
nitric oxide and prostaglandin E2 productions in
lipopolysaccharide-induced RAW 264.7 macrophages.
Among all the target analogs, the diarylurea hydroxyl
compounds 1f and 1h possessing phenyl and 3-(tri-
fluoromethyl)phenyl terminal moiety, respectively, showed
the highest inhibitory effect on the production of pros-
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-017-1923-9) contains supplementary material,
which is available to authorized users.
taglandin E . Both compounds exerted equal activity to the
2
Byung-Jun Park and Mohammed I. El-Gamal have contributed equally
to this work.
reference compound NS-398 at 3 µM concentration. This
effect was due to inhibition cyclooxygenase-2 enzyme
activity not inhibition of cyclooxygenase-2 protein
expression. The IC50 value of compound 1f against
*
*
Kyung-Tae Lee
ktlee@khu.ac.kr
Chang-Hyun Oh
choh@kist.re.kr
lipopolysaccharide-induced prostaglandin E production in
2
the macrophages was 1.12 μM. In addition, compound 1j
with urea linker, hydroxyl group, and 3,5-bis(tri-
fluoromethyl)phenyl terminal ring was the strongest nitric
oxide inhibitor. Western blot study showed that it exerted
that effect through inhibition of inducible nitric oxide syn-
thase protein expression.
1
Center for Biomaterials, Korea Institute of Science and
Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic
of Korea
2
3
4
5
Department of Medicinal Chemistry, College of Pharmacy,
University of Sharjah, Sharjah 27272, United Arab Emirates
Sharjah Institute for Medical Research, University of Sharjah,
Sharjah 27272, United Arab Emirates
Graphical Abstract
Department of Medicinal Chemistry, Faculty of Pharmacy,
University of Mansoura, Mansoura 35516, Egypt
Department of Pharmaceutical Biochemistry, College of
Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-
gu, Seoul 130-701, Republic of Korea
6
7
8
Department of Life and Nanopharmaceutical Science, College of
Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-
gu, Seoul 130-701, Republic of Korea
Chemical Kinomics Research Center, Korea Institute of Science
and Technology, PO Box 131, Cheongryang, Seoul 130-650,
Republic of Korea
Department of Biomolecular Science, University of Science and
Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333,
Republic of Korea
Keywords Anti-inflammatory Diarylurea Nitric oxide
PGE2 Pyrazole
●
●
●
●

2
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Med Chem Res (2017) 26:2161–2171
Introduction
Inflammation is a complex physiological and pathological
process associated with activation of the local vascular
system, the immune system, and various cells within the
damaged tissue (Coussens and Werb 2002). It is a normal
protective response to tissue injury caused by physical
trauma (cut, burn, or bruise), microbiological agents, nox-
ious chemicals, or even autoimmune disorders. Acute
inflammation is a part of the defense response by the
organisms to remove injurious stimuli, such as pathogens,
irritants, or physical injury, from tissues and to initiate the
healing process. However, persistent and excessive immune
response can promote tissue damage, resulting in chronic
inflammation. This chronic inflammation is a part of many
human diseases including arteriosclerosis (Qui et al. 2006),
inflammatory bowel disease (Lee et al. 2010), arthritis
Fig. 1 Structures of celecoxib and the target compounds 1a–k
fluidics organizer, Waters 2545 binary gradient module,
Waters reagent manager, Waters 2767 sample manager,
TM
Sunfire
C18 column (4.6 × 50 mm, 5 μm particle size);
Solvent gradient = 95% A at 0 min, 1% A at 5 min; solvent
A: 0.035% trifluoroacetic acid (TFA) in water; solvent B:
1
0
.035% TFA in CH OH; flow rate of 3.0 mL/min. H-
3
(
Hochberg 1990), cancer (Sung et al. 2011), and Alzhei-
1
3
nuclear magnetic resonance (NMR) and C-NMR analyses
were carried out using a Bruker ARX-300, 300 MHz
mer’s disease (Sastre et al. 2011). In the inflammatory state,
the activated immune cells such as macrophages, secrete
large amounts of proinflammatory cytokines, nitric oxide
(Bruker Bioscience, Billerica, MA, USA) and a Bruker
ARX-400, 400 MHz (Bruker Bioscience, Billerica, MA,
USA) in NMR solvents containing tetramethylsilane as an
internal standard. All the solvents and reagents were com-
mercially available and used as such without further
purification.
(
NO), and prostaglandin E (PGE ). However, high levels
2 2
of NO and PGE in a chronic inflammation state can result
in various pathological conditions (Yun et al. 1996; Hinz
and Brune 2002). Accordingly, control of the production of
2
NO and PGE in macrophages are currently interesting
2
research topics for the development of new anti-
inflammatory drugs.
Synthesis of 4-(3-(3-chloro-5-methoxyphenyl)-1H-
pyrazol-4-yl)pyridine (6)
In this study, we have synthesized a series of new com-
pounds possessing 1,3,4-triarylpyrazole scaffold for the
purpose of anticancer screening (will be carried out later and
the results will be published in due course). Due to the
presence of a vicinal diaryl heterocycle moiety which has
been reported in selective cyclooxygenase (COX)-2 inhibi-
tors such as celecoxib (Fig. 1), we decided to test their anti-
inflammatory activity. Several pyrazole derivatives including
celecoxib have been reported as anti-inflammatory agents
It was synthesized by the previously reported 4-step method
(Bennett et al. 2007; Choi and Oh 2009; El-Gamal et al.
2011). It was obtained as a white to off-white solid.
4-[3-(3-Chloro-5-methoxyphenyl)-1-(3-nitrophenyl)-1H-
pyrazol-4-yl]pyridine (7)
(
Norgard et al. 2004; Keche et al. 2012; El-Sayed et al. 2012;
Ragab et al. 2013; Malvar et al. 2014; Kurumbail et al.
996). The anti-inflammatory activities of our target com-
A mixture of compound 6 (0.5 g, 1.7 mmol), 1-iodo-3-
nitrobenzene (0.9 g, 3.5 mmol), K CO (0.7 g, 5.2 mmol),
2
3
1
CuI (0.033 g, 0.2 mmol), and L-proline (0.04 g, 0.2 mmol) in
DMSO (7 mL) was heated at 90 °C under nitrogen atmo-
sphere for 8 h. The reaction mixture was cooled and then
extracted between ethyl acetate (15 mL) and saturated saline
pounds 1a–k were evaluated through measuring their effects
as inhibitors of lipopolysaccharide (LPS)-induced NO and
PGE productions in RAW 264.7 macrophages.
2
(15 mL). The organic phase was washed with saturated
saline solution (three times) and dried using anhydrous
sodium sulfate. After evaporation of the organic solvent, the
residue was purified by column chromatography (silica gel,
Experimental
General
hexane-ethyl acetate 1:2 v/v) to yield compound 7 (0.55 g,
1
5
9%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ
6
Mass spectra (MS) were determined by liquid
chromatography–mass spectrometry (LC-MS) analysis
using the following system: Waters 2998 photodiode array
detector, Waters 3100 mass detector, Waters SFO system
9.28 (s, 1H), 8.79 (d, 1H, J = 2.0 Hz), 8.60 (d, 2H, J = 4.4
Hz), 8.46 (d, 1H, J = 8.2 Hz), 8.24 (d, 1H, J = 8.2 Hz), 7.87
(t, 1H, J = 8.2 Hz), 7.39 (d, 2H, J = 4.2 Hz), 7.16–7.14 (m,
2H), 6.99 (s, 1H), 3.75 (s, 3H).

Med Chem Res (2017) 26:2161–2171
2163
4
1
-[3-(3-Chloro-5-methoxyphenyl)-1-(3-aminophenyl)-
H-pyrazol-4-yl]pyridine (8)
1-(3-(3-(3-Chloro-5-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl)phenyl)-3-(3,4-dichlorophenyl)urea (1b)
1
A mixture of the nitro compound 7 (0.5 g, 1.2 mmol) and
palladium over carbon 5% (0.5 g) in anhydrous tetra-
hydrofuran (5 mL) under hydrogen (50 psi) was stirred at
room temperature for 2 h. The mixture was filtered through
celite and the filtrate was evaporated under reduced pressure
White solid; H NMR (400 MHz, DMSO-d ) δ 9.14 (s, 1H),
6
9.06 (s, 1H), 8.95 (s, 1H), 8.56 (d, 2H, J = 6.0 Hz), 8.16 (s,
1H), 7.90 (d, 1H, J = 2.4 Hz), 7.57–7.51 (m, 2H),
7.48–7.40 (m, 2H), 7.38–7.35 (m, 3H), 7.13–7.10 (m, 2H),
6.95 (dd, 1H, J = 2.0 Hz, 1.2 Hz), 3.74 (s, 3H); C NMR
(100 MHz, DMSO-d ) δ 160.6 (C=O), 152.8 (C–OMe),
1
3
1
to give compound 8 (0.4 g, 87%) as an oil. H NMR (300
6
MHz, CDCl ) δ 8.56 (d, 2H, J = 4.6 Hz), 8.07 (s, 1H),
150.4 (pyridyl C–N–C), 148.7 (pyrazolyl C-3′), 141.1
(pyridyl C-4′), 140.3 (central phenyl Ar–C), 140.2 (Cl,
OMe-phenyl Ar–C), 139.9 (Cl, OMe-phenyl Ar–C), 135.6
(terminal phenyl Ar–C), 134.5 (terminal phenyl Ar–C),
131.5 (pyrazolyl C-5′), 131.0 (central phenyl Ar–C), 130.5
(terminal phenyl Ar–C), 129.7 (terminal phenyl Ar–C),
123.8 (terminal phenyl Ar–C), 123.2 (central phenyl Ar–C),
3
7
=
.26–7.18 (m, 5H), 7.05 (d, 1H, J = 6.7 Hz), 6.92 (d, 2H, J
1.9 Hz), 6.64 (d, 1H, J = 6.4 Hz), 3.72 (s, 3H); C NMR
1
3
(
75 MHz, CDCl ) δ 160.3 (C=O), 150.0, 149.2, 147.8,
3
1
1
40.5, 140.4, 135.1, 135.0, 130.4, 127.3, 122.9, 121.0,
20.0, 114.6, 113.8, 112.6, 108.8, 106.0, 55.6 (OMe); LC-
+
+
+
MS: 378.89 (M + 2), 377.90 (M +1), 376.91 (M ).
120.6 (central phenyl Ar–C), 120.2 (terminal phenyl Ar–C),
General procedure for synthesis of diarylurea
derivatives 1a–e
120.0 (central phenyl Ar–C), 119.0 (Cl, OMe-phenyl
Ar–C), 117.4 (Cl, OMe-phenyl Ar–C), 114.4 (Cl, OMe-
phenyl Ar–C), 113.6 (Cl, OMe-phenyl Ar–C), 112.7 (pyr-
azolyl C-4′), 109.4 (central phenyl Ar–C), 56.1 (OMe); LC-
To a solution of compound 8 (50 mg, 0.1 mmol) in anhy-
drous tetrahydrofuran (THF) (1 mL), a solution of the
appropriate aryl isocyanate (0.1 mmol) in THF (1 mL) was
+
+
MS: 564.9 (M + 2), 563.9 (M + 1).
added dropwise at room temperature under N . The reaction
1-(3-(3-(3-Chloro-5-methoxyphenyl)-4-(pyridin-4-yl)-1H-
2
mixture was stirred at room temperature for 12 h. After
completion of the reaction, the mixture was evaporated
under reduced pressure, and the residue was purified by
column chromatography (silica gel, hexane-ethyl acetate
pyrazol-1-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (1c)
1
White solid; H NMR (400 MHz, DMSO-d ) δ 9.16 (brs,
6
2H), 8.97 (s, 1H), 8.57 (d, 2H, J = 6.0 Hz), 8.17 (s, 1H),
8.03 (s, 1H), 7.62 (d, 1H, J = 8.0 Hz), 7.57–7.47 (m, 3H),
7.45 (s, 1H), 7.38 (d, 2H, 5.6 Hz), 7.32 (d, 1H, J = 7.6 Hz),
2
:1 v/v then switching to 1:5 v/v) to yield the target com-
pound 1a–e.
7
.14 (s, 1H), 7.11 (t, 1H, J = 1.8 Hz), 6.96 (s, 1H), 3.75 (s,
1
3
1
-(3-(3-(3-Chloro-5-methoxyphenyl)-4-(pyridin-4-yl)-1H-
3H); C NMR (100 MHz, DMSO-d ) δ 160.6 (C=O),
6
pyrazol-1-yl)phenyl)-3-phenylurea (1a)
153.0 (C–OMe), 150.4 (pyridyl C–N–C), 148.7 (pyrazolyl
C-3′), 141.1 (pyridyl C-4′), 140.9 (central phenyl Ar–C),
140.2 (Cl, OMe-phenyl Ar–C), 139.9 (terminal phenyl
Ar–C), 135.7 (pyrazolyl C-5′), 134.5 (central phenyl Ar–C),
130.5 (terminal phenyl Ar–C), 130.4 (terminal phenyl
Ar–C), 129.7 (terminal phenyl Ar–C), 123.2 (central phenyl
Ar–C), 122.5 (Cl, OMe-phenyl Ar–C), 120.6 (terminal
phenyl Ar–C), 120.1 (central phenyl Ar–C), 117.4 (CF3),
114.8 (Cl, OMe-phenyl Ar–C), 114.4 (Cl, OMe-phenyl
Ar–C), 113.5 (Cl, OMe-phenyl Ar–C), 112.7 (pyrazolyl C-
4′), 109.4 (central phenyl Ar–C), 56.1 (OMe); LC-MS:
1
White solid; H NMR (400 MHz, DMSO-d ) δ 8.99 (s, 1H),
6
8
1
1
2
6
.96 (s, 1H), 8.73 (s, 1H), 8.57 (d, 2H, J = 6.0 Hz), 8.17 (s,
H), 7.53 (d, 1H, J = 7.6 Hz), 7.49–7.46 (m, 3H), 7.43 (d,
H, J = 4.8 Hz), 7.38 (dd, 2H, J = 4.8 Hz, 1.2 Hz), 7.30 (t,
H, J = 7.8 Hz), 7.11 (s, 1H), 7.11 (t, 1H, J = 2.0 Hz),
.99 (d, 1H, J = 7.2 Hz), 6.97–6.96 (m, 1H), 3.75 (s, 3H);
1
3
C NMR (100 MHz, DMSO-d ) δ 160.6 (C=O), 153.0
6
(
C–OMe), 150.4 (pyridyl C–N–C), 148.7 (pyrazolyl
C-3′), 141.5 (pyridyl C-4′), 140.2 (central phenyl Ar–C),
39.9 (C–Cl), 135.7 (Cl, OMe-phenyl Ar–C), 134.5
terminal phenyl Ar–C), 130.4 (terminal phenyl Ar–C),
29.7 (pyrazolyl C-5′), 129.3 (central phenyl Ar–C), 123.2
terminal phenyl Ar–C), 122.5 (terminal phenyl
+
1
(
1
(
564.0 (M + 1).
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(3-(3-chloro-5-
methoxyphenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl)phenyl)
urea (1d)
Ar–C), 120.6 (terminal phenyl Ar–C), 120.1 (central
phenyl Ar–C), 118.9 (central phenyl Ar–C), 117.1 (Cl,
OMe-phenyl Ar–C), 114.4 (Cl, OMe-phenyl Ar–C),
1
White solid; H NMR (400 MHz, DMSO-d ) δ 9.68 (brs,
6
1
1
13.5 (Cl, OMe-phenyl Ar–C), 112.3 (pyrazolyl C-4′),
2H), 8.95 (s, 1H), 8.56 (d, 2H, J = 5.6 Hz), 8.18 (s, 1H),
8.13 (d, 1H, J = 2.4 Hz), 7.71 (dd, 1H, J = 8.4 Hz, 1.6 Hz),
7.61 (d, 1H, J = 8.8 Hz), 7.55 (d, 1H, J = 4.0 Hz), 7.45
09.1 (central phenyl Ar–C), 56.1 (OMe); LC-MS: 496.0
+
(
M + 1).

2
164
Med Chem Res (2017) 26:2161–2171
(
2
d, 2H, J = 4.4 Hz), 7.38 (d, 2H, J = 6.0 Hz), 7.13–7.11 (m,
H), 6.96 (s, 1H), 3.75 (s, 3H); C NMR (100 MHz,
saturated saline solution, and then dried with anhydrous
Na SO . The organic solvent was evaporated under reduced
1
3
2
4
DMSO-d ) δ 160.6 (C=O), 153.1 (C–OMe), 150.4 (pyridyl
C–N–C), 148.7 (pyrazolyl C-3′), 141.2 (pyridyl C-4′), 140.2
pressure, and the remained crude product was purified by
short column chromatography (silica gel, hexane-ethyl
acetate 1:1 v/v then switching to 1:5 v/v) to yield the tar-
get compound 1f–j.
6
(
1
Cl, OMe-phenyl Ar–C), 139.9 (terminal phenyl Ar–C),
35.7 (pyrazolyl C-5′), 134.5 (central phenyl Ar–C), 132.4
(
(
(
(
central phenyl Ar–C), 130.4 (terminal phenyl Ar–C), 129.7
terminal phenyl Ar–C), 123.7 (central phenyl Ar–C), 123.2
central phenyl Ar–C), 120.6 (terminal phenyl Ar–C), 120.1
1-(3-(3-(3-Chloro-5-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl)phenyl)-3-phenylurea (1f)
central phenyl Ar–C), 117.5 (CF ), 117.4 (Cl, OMe-phenyl
3
Ar–C), 114.4 (Cl, OMe-phenyl Ar–C), 113.5 (Cl, OMe-
phenyl Ar–C), 112.7 (pyrazolyl C-4′), 109.5 (central phenyl
1
White solid; H NMR (400 MHz, DMSO-d ) δ 10.10 (s,
6
+
Ar–C), 56.1 (OMe); LC-MS: 598.8 (M + 2), 597.8
1H), 8.98 (s, 1H), 8.95 (s, 1H), 8.71 (s, 1H), 8.57 (s, 2H),
8.18 (t, 1H, J = 2.0 Hz), 7.54–7.49 (m, 1H), 7.48 (d, 1H, J
+
(
M + 1).
=
1.2 Hz), 7.46 (d, 1H, J = 0.8 Hz), 7.43 (d, 1H, J = 8.0
1
-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(3-(3-chloro-5-
Hz), 7.39–7.37 (m, 3H), 7.30 (t, 2H, J = 8.0 Hz), 6.99–6.98
(m, 2H), 6.87–6.85 (m, 2H); C NMR (100 MHz, DMSO-
d6) δ 159.0 (C=O), 153.0 (C–OH), 150.2 (pyridyl C–N–C),
1
3
methoxyphenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl)phenyl)
urea (1e)
1
49.0 (central phenyl Ar–C), 141.5 (pyrazolyl C-3′), 140.4
1
White solid; H NMR (400 MHz, DMSO-d ) δ 10.53 (brs,
(pyridyl C-4′), 140.0 (Cl, OH-phenyl Ar–C), 135.6 (Cl,
OH-phenyl Ar–C), 134.2 (terminal phenyl Ar–C), 130.4
(pyrazolyl C-5′), 129.7 (central phenyl Ar–C), 129.3 (cen-
tral phenyl Ar–C), 123.2 (terminal phenyl Ar–C), 122.5
(terminal phenyl Ar–C), 120.0 (central phenyl Ar–C), 119.0
(central phenyl Ar–C), 118.9 (terminal phenyl Ar–C), 117.0
(central phenyl Ar–C), 115.8 (Cl, OH-phenyl Ar–C), 114.5
(Cl, OH-phenyl Ar–C), 112.3 (pyrazolyl C-4′), 109.0
6
2
3
8
H), 8.90 (s, 1H), 8.56 (d, 2H, J = 6.0 Hz), 8.22–8.20 (m,
H), 7.58 (s, 1H), 7.51 (d, 2H, J = 6.4 Hz), 7.43 (d, 1H, J =
.0 Hz), 7.38 (d, 2H, J = 5.6 Hz), 7.13–7.10 (m, 2H), 6.96
s, 1H), 3.74 (s, 3H); C NMR (100 MHz, DMSO-d ) δ
6
60.6 (C=O), 153.4 (C–OMe), 150.3 (pyridyl C–N–C),
48.7 (central phenyl Ar–C), 141.4 (pyrazolyl C-3′), 140.2
pyridyl C-4′), 139.8 (Cl, OMe-phenyl Ar–C), 135.7 (Cl,
OMe-phenyl Ar–C), 134.5 (terminal phenyl Ar–C), 131.2
pyrazolyl C-5′), 130.9 (central phenyl Ar–C), 130.2 (cen-
tral phenyl Ar–C), 129.6 (terminal phenyl Ar–C), 125.2
1
3
(
1
1
(
+
(central phenyl Ar–C); LC-MS: 482.0 (M + 1).
(
(
(
(
terminal phenyl Ar–C), 123.1 (central phenyl Ar–C), 122.5
central phenyl Ar–C), 120.6 (terminal phenyl Ar–C), 120.1
1-(3-(3-(3-Chloro-5-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl)phenyl)-3-(3,4-dichlorophenyl)urea (1g)
central phenyl Ar–C), 118.3 (2 CF ), 117.6 (Cl, OMe-
3
1
phenyl Ar–C), 114.4 (Cl, OMe-phenyl Ar–C), 113.5 (Cl,
OMe-phenyl Ar–C), 112.7 (pyrazolyl C-4′), 109.6 (central
phenyl Ar–C), 56.1 (OMe); LC-MS: 632.9 (M + 2), 631.9
White solid; H NMR (400 MHz, DMSO-d ) δ 10.11
6
(s, 1H), 9.18 (s, 1H), 9.10 (s, 1H), 8.94 (s, 1H), 8.56
(dd, 2H, J = 4.4 Hz, 1.6 Hz), 8.18 (t, 1H, J = 2.0 Hz), 7.91
(d, 1H J = 2.4 Hz), 7.56–7.52 (m, 2H), 7.46 (t, 1H, J = 8.0
Hz), 7.40–7.36 (m, 4 H), 6.98 (t, 1H, J = 1.6 Hz), 6.87
+
+
(
M + 1).
1
3
General procedure for demethylation to the target
(dt, 2H, J = 6.8 Hz, 1.9 Hz);
C NMR (100 MHz,
hydroxyl derivatives 1f–j
DMSO-d ) δ 159.0 (C=O), 152.8 (C–OH), 150.4 (pyridyl
6
C–N–C), 149.0 (central phenyl Ar–C), 141.1 (pyrazolyl C-
3′), 140.3 (pyridyl C-4′), 140.2 (central phenyl Ar–C),
140.0 (Cl, OH-phenyl Ar–C), 135.6 (Cl, OH-phenyl Ar–C),
134.1 (terminal phenyl Ar–C), 131.5 (terminal phenyl
Ar–C), 131.0 (pyrazolyl C-5′), 130.4 (central phenyl Ar–C),
129.7 (terminal phenyl Ar–C), 123.8 (terminal phenyl
Ar–C), 123.1 (terminal phenyl Ar–C), 120.0 (central
phenyl Ar–C), 119.9 (central phenyl Ar–C), 119.0 (terminal
phenyl Ar–C), 118.9 (central phenyl Ar–C), 117.3
(Cl, OH-phenyl Ar–C), 115.8 (Cl, OH-phenyl Ar–C), 114.5
(Cl, OH-phenyl Ar–C), 112.6 (pyrazolyl C-4′), 109.3
Compound 1a–e (0.1 mmol) was dissolved in anhydrous
dichloromethane (1 mL), and boron tribromide (0.08 mL of
a 1 M solution in methylene chloride, 1.2 mmol) was added
thereto dropwise at −78 °C under nitrogen atmosphere. The
reaction mixture was stirred at the same temperature for 30
min. The temperature was raised to room temperature, and
the mixture was stirred overnight at room temperature. The
mixture was quenched with saturated aqueous sodium car-
bonate. Ethyl acetate (15 mL) was added, the mixture was
stirred, and the organic layer was separated. The aqueous
layer was extracted again with ethyl acetate (2 × 10 mL).
The combined ethyl acetate extracts were washed with
+
(central phenyl Ar–C); LC-MS: 551.6 (M + 2), 550.6
+
(M + 1).

Med Chem Res (2017) 26:2161–2171
2165
1
-(3-(3-(3-Chloro-5-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
(pyrazolyl C-4′), 109.7 (central phenyl Ar–C); LC-MS:
618.1 (M + 1).
+
pyrazol-1-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (1h)
1
White solid; H NMR (400 MHz, DMSO-d ) δ 10.10 (brs,
Synthesis of N-(3-(3-(3-chloro-5-methoxyphenyl)-4-
6
1
5
7
H), 9.15 (d, 2H, J = 6.8 Hz), 8.95 (s, 1H), 8.57 (d, 2H, J =
.6 Hz), 8.18 (s, 1H), 8.03 (s, 1H), 7.62 (d, 1H, J = 8.0 Hz),
.55 (t 1H, J = 6.4 Hz), 7.51–7.40 (m, 3H), 7.38 (d, 2H, J
(pyridin-4-yl)-1H-pyrazol-1-yl)phenyl)benzamide (1k)
A mixture of the amine compound 8 (50 mg, 0.1 mmol),
benzoic acid (25 mg, 0.2 mmol), HOBt (36 mg, 0.3 mmol),
and EDCI (38 mg, 0.2 mmol) in DMF (1.0 mL) was cooled
to 0 °C under nitrogen atmosphere. Triethylamine (0.03 mL,
0.2 mmol) was added to the reaction mixture at the same
temperature. The mixture was then stirred at 80 °C for 12 h.
The reaction mixture was cooled and then partitioned
between saturated saline (10 mL) and ethyl acetate (10 mL).
The organic layer was separated, and the aqueous layer was
extracted again with ethyl acetate (3 × 5 mL). The organic
extract was washed with saturated saline (3 × 15 mL), and
dried using anhydrous sodium sulfate. The organic solvent
was evaporated, and the crude residue was purified by
column chromatography (silica gel, hexane-ethyl acetate
=
5.6 Hz), 7.33 (d, 1H, J = 7.6 Hz), 6.98 (s, 1H), 6.86 (dd,
1
3
2
1
1
H, J = 4.8 Hz, 2.0 Hz); C NMR (100 MHz, DMSO-d ) δ
59.0 (C=O), 153.0 (C–OH), 150.4 (pyridyl C–N–C),
49.0 (pyrazolyl C-3′), 141.1 (pyridyl C-4′), 140.9 (central
6
phenyl Ar–C), 140.2 (Cl, OH-phenyl Ar–C), 140.0 (term-
inal phenyl Ar–C), 135.6 (pyrazolyl C-5′), 134.2 (central
phenyl Ar–C), 130.5 (terminal phenyl Ar–C), 130.4
(
terminal phenyl Ar–C), 129.7 (terminal phenyl Ar–C),
1
1
23.1 (central phenyl Ar–C), 122.5 (Cl, OH-phenyl Ar–C),
20.0 (Cl, OH-phenyl Ar–C), 119.0 (CF ), 118.8 (Cl, OH-
3
phenyl Ar–C), 117.4 (Cl, OH-phenyl Ar–C), 115.8 (Cl,
OH-phenyl Ar–C), 114.8 (central phenyl Ar–C), 114.5 (Cl,
OH-phenyl Ar–C), 112.6 (pyrazolyl C-4′), 109.3 (central
+
1
phenyl Ar–C); LC-MS: 550.1 (M + 1).
1:1 v/v) to yield the target compound as a white solid;. H
NMR (400 MHz, DMSO-d ) δ 10.48 (brs, 1H), 8.97 (s,
6
1
H), 8.55 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 8.45 (t, 1H, J = 2.0
1
-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(3-(3-chloro-5-
Hz), 7.99 (d, 2H, J = 6.8 Hz), 7.83 (dd, 1H, J = 8.0 Hz, 1.2
Hz), 7.65 (dd, 1H, J = 8.0 Hz, 1.6 Hz), 7.62–7.58 (m, 1H),
hydroxyphenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl)phenyl)
urea (1i)
7.55–7.51 (m, 3H), 7.36 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 6.94
1
3
(s, 1H), 3.72 (s, 3H); C NMR (100 MHz, DMSO-d ) δ
1
6
White solid; H NMR (400 MHz, DMSO-d ) δ 10.32–10.28
6
166.2 (C=O), 160.6 (C–OMe), 150.4 (pyridyl C–N–C),
148.8 (pyrazolyl C-3′), 140.9 (pyridyl C-4′), 140.2 (C–Cl),
139.7 (Cl, OMe-phenyl Ar–C), 135.6 (terminal phenyl
(
8
=
7
m, 3H), 8.90 (s, 1H), 8.53 (dd, 2H, J = 4.6 Hz, 1.4 Hz),
.18–8.15 (m, 2H), 7.74 (d, 1H, J = 8.8 Hz), 7.59 (d, 1H, J
8.8 Hz), 7.52–7.46 (m, 3H), 7.43 (d, 1H, J = 8.0 Hz),
.38 (dd, 2H, J = 4.4 Hz, 1.6 Hz), 6.75–6.74 (m, 3H); LC-
Ar–C), 135.1 (terminal phenyl Ar–C), 134.5 (pyrazolyl C-
′), 132.3 (central phenyl Ar–C), 130.3 (central phenyl
5
+
+
MS: 585.1 (M + 2), 584.1 (M + 1).
Ar–C), 129.7 (terminal phenyl Ar–C), 128.9 (terminal
phenyl Ar–C), 128.2 (central phenyl Ar–C), 123.1 (central
phenyl Ar–C), 120.6 (terminal phenyl Ar–C), 120.2 (central
phenyl Ar–C), 119.1 (Cl, OMe-phenyl Ar–C), 114.4 (Cl,
OMe-phenyl Ar–C), 114.0 (Cl, OMe-phenyl Ar–C), 113.6
1
-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(3-(3-chloro-5-
hydroxyphenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl)phenyl)
urea (1j)
(pyrazolyl C-4′), 111.3 (central phenyl Ar–C), 56.1 (OMe).
1
White solid; H NMR (400 MHz, DMSO-d ) δ 10.09 (brs,
6
1
2
H), 9.49 (brs, 1H), 9.36 (brs, 1H), 8.96 (s, 1H), 8.57 (d,
H, J = 4.8 Hz), 8.17 (brs, 2H), 7.67 (s, 1H), 7.58 (d, 1H, J
Biological evaluation
=
7.2 Hz), 7.50–7.44 (m, 2H), 7.38 (d, 2H, J = 5.6 Hz),
Cell culture and sample treatment, nitrite determination,
1
3
6
.98 (s, 1H), 6.87–6.84 (m, 2H); C NMR (100 MHz,
PGE assay, and MTT assay of cell viability were carried
2
DMSO-d ) δ 159.0 (C=O), 152.9 (C–OH), 150.4 (pyridyl
C–N–C), 149.0 (pyrazolyl C-3′), 142.2 (pyridyl C-4′), 140.8
out following the procedure reported in the literature (Jang
et al. 2014).
6
(Cl, OH-phenyl Ar–C), 140.2 (Cl, OH-phenyl Ar–C), 140.0
(terminal phenyl Ar–C), 135.6 (pyrazolyl C-5′), 134.2
(central phenyl Ar–C), 131.4 (central phenyl Ar–C), 130.5
(terminal phenyl Ar–C), 129.7 (terminal phenyl Ar–C),
Western blot
TM
RAW 264.7 cells were resuspended in PRO-PREP pro-
1
1
1
23.1 (central phenyl Ar–C), 122.4 (central phenyl Ar–C),
20.0 (terminal phenyl Ar–C), 119.0 (central phenyl Ar–C),
tein extraction solution (Intron Biotechnology, Seoul,
Korea) and incubated for 20 min at 4 °C. The cell debris
was removed by microcentrifugation, and the supernatants
were quickly frozen. The protein concentration was
18.7 (2 CF ), 117.8 (Cl, OH-phenyl Ar–C), 115.8 (Cl,
3
OH-phenyl Ar–C), 114.5 (Cl, OH-phenyl Ar–C), 113.0

2
166
Med Chem Res (2017) 26:2161–2171
determined using the Bio-Rad protein assay reagent
according to the manufacture’s instruction. The cell proteins
were electroblotted onto a PVDF membrane after separation
on 8–12% SDS-polyacrylamide gel electrophoresis. The
immunoblot was incubated with a blocking solution (5%
skim milk) at room temperature for 1 h, followed by incu-
bation overnight with a primary antibody at 4 °C. The blots
were washed four times with a Tween 20/Tris-buffered
saline (T/TBS) and incubated with a 1:2000 dilution of
horseradish peroxidase-conjugated secondary antibody at
room temperature for 2 h. The blots were again washed
three times with T/TBS, and then developed by enhanced
chemiluminescence (GE healthcare, WI, USA).
100 μM arachidonic acid and allowed to proceed for 2 min.
The reaction was terminated by addition of HCl solution
containing SnCl . The COX activity assay directly mea-
2
sures PGF2α produced by SnCl reduction of COX-derived
2
PGH . The prostanoid product concentration was analyzed
2
using EIA. Dup-697 (10 μM) was used as a reference
COX-2 inhibitor.
Results and discussion
Chemistry
The target compounds 1a–k were synthesized through the
pathway illustrated in Scheme 1. Heating 3,5-dichlor-
obenzoic acid (2) with sodium methoxide (3 eq.) in hex-
amethylphosphoramide (HMPA) followed by acidification
with HCl produced 3- chloro-5-methoxybenzoic acid (3).
The acid 3 was esterified using methanol in the presence of
acetyl chloride to produce the corresponding methyl ester 4
(Takahashi et al. 1985). Treatment of the ester 4 with 4-
COX-2 enzyme activity assay
Compounds 1f and 1h were evaluated for potency and
selectivity of inhibition in vitro using COX Inhibitor
Screening Assay (Cayman, MI, USA). Recombinant COX-
2
1
proteins were pre-incubated with compounds 1f or 1h for
0 min in 37 °C. The reaction was started by the addition of
Scheme 1 Reagents and conditions: a sodium methoxide, HMPA,
15–120 °C, 15 h, 54%; b acetyl chloride, CH OH, rt, 15 h, 89%; c 4-
picoline, LHMDS, THF, rt, overnight, 77%; d DMF-DMA, rt, 18 h; e
hydrazine monohydrate, C OH, rt, overnight, 72%; f 1-iodo-3-
nitrobenzene, K
10% Pd/C, THF, rt, 2 h, 87%; h appropriate aryl isocyanate, THF, rt,
12 h, 24~85%; i BBr , CH Cl , −78 °C, 30 min; rt, 1 h, 24–89%; j
benzoic acid, HOBt, EDCI, TEA, DMF, 80 °C, 12 h, 65%
2 3 2
CO , CuI, L-proline, DMSO, 90 °C, 8 h, 59%; g H ,
1
3
3
2
2
2 5
H

Med Chem Res (2017) 26:2161–2171
2167
Table 1 Structures of the target compounds and their yield
percentages
picoline in THF in the presence of lithium bis(trimethylsi-
lyl)amide yielded the pyridyl ketide derivative 5. Treatment
of compound 5 with dimethylformamide dimethyl acetal
(
DMF-DMA) followed by treatment with hydrazine
monohydrate led to formation of the cyclized 3,4-dia-
rylpyrazole intermediate 6 (Bennett et al. 2007; Choi and
Oh 2009; El-Gamal et al. 2011). N-Arylation of compound
6
using 1-iodo-3-nitrobenzene in the presence of potassium
carbonate, copper(I)iodide, and L-proline as catalysts pro-
duced the m-nitrophenyl compound 7. The nitro group of
compound 7 was reduced to amino using Pd/C in H2
atmosphere. The amine compound 8 was reacted with the
appropriate aryl isocyanate to produce compounds 1a–e.
The methoxy group of compounds 1a–e was demethylated
in using boron tribromide to obtain the corresponding
hydroxyl analogs 1f–j. Compound 1k was synthesized
through condensation of the amino compound 8 with ben-
zoic acid in presence of 1-hydroxybenzotriazole (HOBt), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and
triethylamine (TEA). The structures of the target com-
pounds and their yield percentages are illustrated in Table 1.
1
2
a
Compound no.
R
R
Yield (%)
Log P
5.38
b
1
a
CH
3
3
24
b
1
b
CH
60
6.50
6.30
b
1
c
CH
3
62
Biological screening
b
1
1
d
e
CH
CH
3
3
85
6.86
7.22
Inflammation is a normal protective immune response
against tissue damages that occur due to external factors in
the body (Qui et al. 2006). But the inflammatory response
can also cause considerable damage to the host. The
microbial components such as LPS can induce the pro-
duction of COX-2, inducible nitric oxide synthase (iNOS),
and pro-inflammatory cytokines in the macrophages. In
b
82
particular, if large amounts of NO and PGE secreted by
2
c
1
f
H
H
24
5.12
6.23
activated immune cells in the inflammatory condition, they
can induce several pathological conditions. So inhibition of
the production of inflammatory mediators is a potential
avenue for treatment of acute and chronic inflammations.
The target compounds 1a–k were assessed for their abilities
to inhibit the production of inflammatory mediators, NO
c
1g
1h
89
and PGE , in LPS-induced RAW 264.7 macrophages
2
c
(
Moncada et al. 1991; Kim et al. 2008; Shin et al. 2014).
H
H
62
6.04
6.06
N -(1-Iminoethyl)-L-lysine (L-NIL) and N-(2-cyclohex-
6
yloxy-4-nitrophenyl)methanesulfonamide (NS-398) were
utilized as reference compounds for screening of NO and
PGE production inhibitions, respectively. The cytotoxic
c
2
1
i
79
effects of the triarylpyrazoles 1a–k on RAW 264.7 mac-
rophages were also evaluated using the MTT assay to test
whether the inhibitory effects on the productions of NO and
PGE were caused by non-specific cytotoxicity (Table 2).
2
All the target compounds showed IC50 values ≥5.86 µM.
Compounds 1b and 1e did not inhibit 50% of RAW 264.7
macrophages growth up to 100 µM. After that, the target
compounds were tested for NO and PGE2 production
inhibitions at 5 and 3 µM, respectively.

2
168
Med Chem Res (2017) 26:2161–2171
Table 1 continued
compounds 1a, 1c, and 1d. So the phenolic OH group could
be essential for anti-inflammatory activity of this series of
compounds. It may act as hydrogen bond donor at the
receptor site. Or its steric and/or electronic properties, which
are different from those of the methoxy group, can
strengthen the affinity at the receptor site. In addition, the
increased lipophilicity seems to be unfavorable for PGE2
production inhibitory activity.
Compound 1k with amide linker was synthesized and
biologically tested in order to compare the effect of the
linker on the biological activity. It was found that the urea
compound 1a was more active than the amide analog 1k in
terms of NO production inhibition. This can be rationalized
that the longer urea linker may help the compound fit
appropriately at the receptor site. Or the extra terminal NH
group of the urea linker can make additional hydrogen bond
1
2
a
Compound no.
R
R
Yield (%)
Log P
6.96
c
1
j
H
72
d
1
k
CH
3
65
5.71
(s) at the receptor site. Any or both of these effects can help
enhance the compound affinity at the receptor site, and
hence higher activity. Due to the non-promising results of
compound 1k as NO production inhibitor, it was not tested
a
b
c
d
Calculated by ChemDraw Professional 15.0 software
Synthesized in step “h” as illustrated in Scheme 1
Synthesized in step “i” as illustrated in Scheme 1
Synthesized in step “j” as illustrated in Scheme 1
for PGE production inhibition.
2
The effect of the terminal aryl moiety on the biological
activity was also investigated. Upon comparing the results
of the methoxy compounds 1a–e on NO production inhi-
bitions, the mono-substituted phenylurea derivative 1a was
the most active than the other di- or tri-substituted phenyl
derivatives 1b–e. So the steric/electronic factor(s) may play
a role in these activity differences. But upon comparing the
hydroxyl derivatives 1f–j, compound 1j possessing 3,5-bis
Table 2 Cytotoxicty (IC50, MTT assay) of the target compounds 1a–
k over RAW 264.7 cells
a
Compound no.
IC50 (µM)
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
23.42 ± 0.87
>100
(trifluoromethyl)phenyl terminal moiety showed the best
activity. Its IC50 value over NO production was 4.35 µM. So
that bulky terminal moiety may be optimal for activity.
Since it is well-known that iNOS catalyzes the oxidative
deamination of L-arginine to produce NO (Moncada et al.
23.26 ± 0.99
82.12 ± 6.31
>100
15.86 ± 0.64
19.32 ± 1.38
18.71 ± 0.56
10.29 ± 0.79
19.46 ± 1.21
48.38 ± 2.21
>3
1991), we determined the effect of compound 1j on LPS-
g
h
i
induced iNOS protein expression using Western blot.
Compound 1j significantly inhibited LPS-induced iNOS
expression, suggesting the inhibitory action of compound 1j
on NO was due to inhibition on iNOS protein expression
j
k
(Fig. 2).
NS-398
Regarding the effect on PGE production inhibition, the
2
a
best results were obtained with compounds 1a and 1f with
phenyl ring, and compound 1h possessing 3-(tri-
fluoromethyl)phenyl terminal moiety. Compounds 1f and
Data are presented as the means ± SD of three independent
experiments
1h showed almost the same inhibition percentage as NS-
The target pyrazole compounds 1a–k exerted varying
398 and Celecoxib against PGE at 3 µM concentration. So
2
inhibitory effects on the LPS-induced NO and PGE pro-
both phenyl and m-(trifluoromethyl)phenyl terminal rings
2
ductions (Table 3). Compounds 1h–j possessing m-hydro-
xyl group on the aromatic ring on position 3 of the pyrazole
ring showed higher inhibitory effect on NO production than
the corresponding methoxy analogs 1c–e. Similarly, the
phenolic derivatives 1f, 1h, and 1i were more active as
are the optimum for PGE production inhibitory effect of
this series of compounds. During the inflammatory process,
2
biosynthesis of PGE requires transformation of arachidonic
2
acid by COX-2 to PGH , which is subsequently converted
2
by microsomal PGE synthases (mPGES-1) (Shin et al.
2014). To determine the mechanisms by which compound
PGE production inhibitors than the corresponding methoxy
2

Med Chem Res (2017) 26:2161–2171
Table 3 Inhibitory effects of
2169
a
Compound no.
% Inhibition
the target compounds 1a–k on
LPS-induced NO and PGE
RAW 264.7 cells
2
in
NO production at 5 µM
PGE production at 3 µM
2
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
34.22 ± 1.81
3.21 ± 0.09
53.45 ± 4.86
b
–
12.74 ± 0.99
15.57 ± 0.71
24.29 ± 1.27
12.74 ± 1.03
0.71 ± 0.08
36.11 ± 2.38
13.89 ± 1.64
–b
c
92.68 ± 6.17 (1.12 ± 0.34)
g
h
i
55.56 ± 3.83
20.71 ± 1.03
25.88 ± 1.12
53.42 ± 1.91 (4.35 ± 0.62)
2.50 ± 0.07
92.41 ± 6.25
50.58 ± 4.32
c
j
50.74 ± 4.86
k
–
c
L-NIL (40 µM)
75.21 ± 3.67 (28.27 ± 0.13)
–
d
NS-398 (3 µM)
–
93.27 ± 5.58 (6.75 ± 0.70)
a
Values represent means ± S.D. of three independent experiments
No inhibition at less than the cytotoxic concentration
Numbers in parenthesis are IC50 values in μM
b
c
d
Numbers in parenthesis are IC50 values in nM
PGE2 production through inhibition of COX-2 enzyme
activity.
Conclusion
A series of 1,3,4-triarylpyrazole derivatives was synthesized
and studied in order to explore the relationship between
their structures and their antiinflammatory activity. The
hydroxyl compounds were generally more active than the
corresponding methoxy derivatives. The urea moiety was
found optimum linker for activity. The phenyl, m-(tri-
fluoromethyl)phenyl, and 3,5-bis(trifluoromethyl)phenyl
rings were the best terminal moieties. These together can
represent the pharmacophore of this series of compounds.
The highest PGE2 production inhibition activities were
obtained by compounds 1f and 1h. Both of them exerted
that effect due to inhibition of COX-2 enzyme activity not
COX-2 protein expression. Both compounds possess phe-
nolic hydroxyl group and urea linker. Their terminal moi-
eties are phenyl and m-(trifluoromethyl)phenyl rings,
respectively. They showed almost equal inhibitory effect on
Fig. 2 The inhibitory effect of compound 1j on LPS-induced iNOS
expression in RAW 264.7 cells. Total cellular proteins were obtained
from cells stimulated with LPS (1 μg/mL) for 24 h in presence or
L
absence of 1j (a) or -NIL (b). iNOS was detected by Western blot
using specific antibody. β-actin was used as an internal control. The
experiments were repeated three times, and similar results were
obtained
1
f and 1h blocked the PGE production in macrophages, we
2
first examined expression of both COX-2 and mPGES-1 by
Western blot. As shown in Fig. 3a, compounds 1f and 1h
had no effect on LPS-induced COX-2 and mPGES-1
protein expression. The COX-2 enzyme activity is extre-
mely important for PGE production, hence we next studied
2
the effects of compounds 1f and 1h on COX-2 enzyme
activities by performing screening assays using purified
COX-2 enzymes. We found that compounds 1f and 1h
exhibited inhibitory effect to COX-2 activity in a dose-
dependent pattern. Compound 1f was more potent than
compound 1h. Dup697 was used as a positive control for
inhibition of COX-2 enzyme activity (Fig. 3b). These result
indicated that compounds 1f and 1h reduced LPS-induced
PGE production as NS-398 at the same concentration, 3
2
µM. In addition, the urea derivative 1j containing hydroxyl
group and 3,5-bis(trifluoromethyl)phenyl terminal ring
showed the highest inhibitory effect on NO production, and
this was due to inhibition of iNOS protein expression as
concluded from the Western blot study. Further lead

2
170
Med Chem Res (2017) 26:2161–2171
Fig. 3 Effect of compounds 1f and 1h on LPS-induced iNOS and
mPGES expression in RAW 264.7 cells and COX-2 enzyme activities.
a Total cellular proteins were obtained from cells stimulated with LPS
three times, respectively, and similar results were obtained. b
Recombinant COX-2 enzyme was in vitro treated with the indicated
concentrations of 1f or 1h for 10 min. Dup-697 (10 μM) was used as a
positive COX-2 inhibitor controls. Values shown are means ± SD of
three independent experiments; ***p < 0.001 vs. the 100% activity
group
(
1 μg/mL) for 24 h in presence or absence of 1f or 1h. COX-2 and
mPGES-1 were detected by Western blot using specific antibodies. β-
actin was used as an internal control. The experiments were repeated
El-Sayed MA-A, Abdel-Aziz NI, Abdel-Aziz AA-M, El-Azab AS,
ElTahir KEH (2012) Synthesis, biological evaluation and mole-
cular modeling study of pyrazole and pyrazoline derivatives as
selective COX-2 inhibitors and anti-inflammatory agents. Part 2.
Bioorg Med Chem 20:3306–3316
optimization is required for development of more active and
more potent analogs based on the structure–activity rela-
tionship study.
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Acknowledgements This work was supported by Korea Institute of
Science and Technology (KIST), KIST Project (2E24680).
Compliance with ethical standards
Jang H-L, El-Gamal MI, Choi H-E, Cho H-Y, Lee K-T, Oh C-H
(
2014) Synthesis of tricyclic fused coumarin sulfonates and their
inhibitory effects on LPS-induced nitric oxide and PGE pro-
ductions in RAW 264.7 macrophages. Bioorg Med Chem Lett
4:571–575
Conflict of interest The authors declare that they have no competing
interests.
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