Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.bmcl.2019.126884

In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E₂ (PGE₂) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE₂ production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE₂ inhibitors with IC₅₀ values of 2.94 μM and 4.21 μM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages.

Full text of DOI:10.1016/j.bmcl.2019.126884

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide
and PGE2 productions in murine RAW 264.7 macrophages
Mahmoud M. Gamal El-Dina, Mohammed I. El-Gamalb,c,d, Mohammed S. Abdel-Maksouda,
Huijeong Leee, Jungseung Choie, Tae-Woo Kimf,g, Ji-Sun Shinf, Hwi-Ho Leef,g, Hee-Kwon Kimh,i,
⁎
⁎
Kyung-Tae Leef,g, , Daejin Baeke,
a Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC (ID: 60014618)), Dokki-Giza 12622, Egypt
b Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates
c Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
d Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
e Department of Beauty Science, Hanseo University, Seosan 31962, Republic of Korea
f Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
g Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 02792, Republic of Korea
h Department of Nuclear Medicine, Molecular Imaging & Therapeutic Medicine Research Center, Jeonbuk National University Medical School and Hospital, 20 Geonji-ro,
Deokjin-gu, Jeonju 54907, Republic of Korea
i Research Institute of Clinical Medicine of Jeonbuk National University-Biomedical Research Institute of Jeonbuk National University Hospital, 20 Geonji-ro, Deokjin-gu,
Jeonju 54907, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Amide
In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as
inhibitors of nitric oxide (NO) and prostaglandin E2 (PGE2) release induced by lipopolysaccharide (LPS) in
murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their
cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the in-
hibitory effects against NO and PGE2 production were not caused by non-specific cytotoxicity. Compounds 1h
and 2f were the most active PGE2 inhibitors with IC50 values of 2.94 μM and 4.21 μM, respectively. Western
blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein ex-
pression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-
induced murine RAW 264.7 macrophages.
Antiinflammatory
COX-2
iNOS
NO
PGE2
Triarylpyrazole
Urea
The inflammatory response is a part of the immune system of higher
organisms protecting them from injury and infections.1 Its activity is to
surround and remove both the injurious agent and damaged tissue
components allowing the body to start the healing process. Although
inflammation is a beneficial mechanism of defense in some cases, it can
lead to allergy, autoimmune diseases, and rejection of organs. Chronic
inflammatory conditions have been linked to several diseases including
cardiovascular diseases,2 cancer,3 inflammatory bowel syndrome,4 ar-
thritis,5 pulmonary disease, Alzheimer’s disease,6 etc.
situations. The inducible nitric oxide synthase (iNOS) secretes NO as an
inflammatory mediator resulting in vasodilation at the location of in-
flammation in chronic inflammation, thus causing edema.10 So inhibi-
tion of NO production through inhibition of iNOS enzyme activity and/
or protein expression can be a useful approach for treatment of in-
flammation. Moreover, cyclooxygenase-2 (COX-2) converts the arachi-
donic acid into another inflammatory mediator, PGE2.11 Similarly, in-
hibition of PGE2 production via inhibition of COX-2 protein expression
and/or enzyme activity is another potential mechanism of inflamma-
tion therapy.
Nitric oxide (NO) plays a crucial role in the development of in-
flammation despite of acting as an anti-inflammatory agent under
normal physiological conditions.7–9 On the contrary, it could be con-
sidered as a pro-inflammatory mediator that could induce localized
inflammation because of increased secretion in abnormal
Several triarylpyrazole derivatives have been recently reported as
antiinflammatory agents.12–15 In the present work, we report the in-
hibitory effects of a series of substituted pyrazole derivatives on LPS-
induced NO and PGE2 productions. They possess structural similarity to
⁎ Corresponding authors at: Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-
701, Republic of Korea (Kyung-Tae Lee) & Department of Beauty Science, Hanseo University, Seosan 31962, Republic of Korea (Daejin Baek).
E-mail addresses: ktlee@khu.ac.kr (K.-T. Lee), djbaek@hanseo.ac.kr (D. Baek).
https://doi.org/10.1016/j.bmcl.2019.126884
Received 21 October 2019; Received in revised form 27 November 2019; Accepted 2 December 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MahmoudM.GamalEl-Din,etal.,Bioorganic&MedicinalChemistryLetters,
https://doi.org/10.1016/j.bmcl.2019.126884

M.M. Gamal El-Din, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
tether attached to the pyridyl ring on activity. The most promising
compounds were further considered at cellular and molecular levels for
more understanding of their redundancy mechanisms of action.
Compounds 1a-h, 2a-f, and 3a-h were synthesized performing the
route illustrated in Scheme 1. 3-methoxybenzoic acid (4) was refluxed
in methanol with catalytic amount of sulfuric acid to give methyl
benzoate ester 5. The resulted ester was reacted with 2-bromo-4-me-
thylpyridine using the strong base, lithium bis(trimethylsilyl)amide
(LiHMDS) to get compound 6.17 The intermediate ketide compound 6
was then treated with dimethylformamide dimethylacetal (DMF-DMA),
followed by phenylhydrazine to yield the cyclized 1,3,4-triarylpyrazole
7 possessing bromopyridyl moiety. Compound 7 was refluxed with
ethylenediamine or 1,3-propylenediamine to get compounds 8a and 8b,
respectively. The primary NH2 group of 8a or 8b was reacted with the
appropriate acid chloride and using triethylamine as a base to give the
corresponding amide compounds 1a-h. Compounds 2a-f with urea
linker were prepared through reacting compound 8a or 8b with the
appropriate aryl isocyanate derivatives to afford the target aryl urea
compounds 2a-f. Regarding hydroxyl derivatives 3a-h, they were syn-
thesized by treating compounds 1a-h with BBr3 for demethylation of
the methoxy group affording the corresponding compounds.18 The in
vitro anticancer activity of the target compounds were previously re-
ported.18 The final target compounds are illustrated in Table 1.
Before conducting the screening tests of NO and PGE2 production
inhibition, it was crucial to detect compounds’ cytotoxicity to de-
termine a safe and non-toxic concentration of each compound. After
confirmation that 10 µM concentration of each of the target compounds
is not cytotoxic against murine RAW 264.7 macrophages, the target
compounds were tested at this single-dose concentration for inhibitory
effect of various tested compounds on LPS-induced NO and PGE2 pro-
duction in murine RAW 264.7 macrophages. The results are summar-
ized in Table 1.
Fig. 1. Chemical structures of the lead compound I,16 celecoxib, and the final
target compounds.
the marketed pyrazole anti-inflammatory drug, celecoxib (Fig. 1). In
addition, we have reported the in vitro anti-inflammatory effects of
compound I that contains 1,3,4-triarylpyrazole scaffold (Fig. 1).16
Herein, we decided to test the target compound 1a-h, 2a-f, and 3a-h as
inhibitors of NO and PGE2 productions, and investigated the effect of
It is noticeable that the targeted compounds are more active as
Scheme 1. Reagents and conditions: a) methanol,
Conc. sulfuric acid, overnight, reflux; b) 2-bromo-4-
pyridine, lithium bis(trimethylsilyl)amide, tetra-
hydrofuran, 6 h, −78 °C to rt; c) dimethylforma-
mide dimethylacetal,
3 h, 80 °C; d) PhNHNH2,
ethanol, overnight, rt,; e) ethylenediamine or 1,3-
propylenediamine, reflux,8h; f) appropriate acid
chloride, Et3N, dichloromethane, overnight, 0 °C; g)
boron tribromide, dichloromethane, 3 h, −78 °C to
rt; h) appropriate isocyanate, tetrahydrofuran,
overnight, rt.
2

M.M. Gamal El-Din, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
Chemical structures of the final compounds 1a-h, 2a-f, and 3a-h, their effect on cell viability, and inhibitory effects on LPS-induced NO and PGE2 production in
murine RAW 264.7 macrophages.
Compound No.
R1
R2
n
% cell viability
NO % inhibition
PGE2 production
1a
1b
1c
1d
1e
1f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
C6H5
1
1
1
1
2
2
2
2
1
1
1
2
2
2
1
1
1
1
2
2
2
2
131.5
136.1
124.7
116.5
117.8
132.9
115.3
112.1
142.6
165.1
129.4
143.4
123.6
120.7
104.8
127.4
135.3
124.3
145.1
120.3
123.2
126.5
12.8
15.4
19.1
12.3
18.7
18.6
28.8
48.8
12.2
26.1
22.0
22.2
31.3
38.6
12.2
20.4
18.6
34.5
2.1
0.53
0.89
0.72
0.53
0.64
0.44
0.43
0.58
0.34
0.39
0.45
0.56
0.65
0.34
0.41
0.37
0.31
0.35
36.2
47.9
58.1
59.6
6.1
0.54
0.65
0.75
0.73
4-F-C6H4
3-CF3-C6H4
4-OCH3-C6H4
C6H5
0.33
4-F-C6H4
28.9
73.8
75.4
53.9
69.9
76.1
56.3
ND
0.83
0.89
0.74
0.29
0.43
0.39
0.12
1g
1h
2a
2b
2c
2d
2e
2f
3-CF3-C6H4
4-OCH3-C6H4
C6H5-NH
3-CF3-C6H4-NH
3,4-Cl2-C6H4-NH
C6H5-NH
3-CF3-C6H4-NH
3,4-Cl2-C6H4-NH
C6H5
77.1
20.9
−1.9
12.2
−56.4
12.6
63.1
11.6
31.1
0.47
0.36
0.26
0.24
3a
3b
3c
3d
3e
3f
H
4-F- C6H4
3-CF3-C6H4
4-OH-C6H4
C6H5
H
H
0.23
H
0.43
0.31
0.35
0.43
0.89
H
4-F-C6H4
32.6
ND
0.56
0.39
3g
3h
H
3-CF3-C6H4
4-OH-C6H4
H
39.7
a Values represent means
S.D. of three independent experiments. Cells were pretreated with tested compounds (10 μM) over 1 h followed by LPS (1 μg/mL) over
24 h for stimulation. Both PGE2 and NO production were quantified in the culture media applying an enzyme immunoassay (EIA) kit and the Griess assay. Added
below data or explain contents. L-NIL (40 μM –31.5
used for PGE2 production.
0.38 inhibition %) was used as positive controls for NO and NS-398 (10 nM-59.5
0.96 inhibition %) was
Table 2
and 2f. The inhibitory effects of compounds 1h and 2f on PGE2 pro-
duction are illustrated in Fig. 2. Compounds 1g, 1h, 2b, 2c, 2f, and 3f
are more potent than the lead compound I (Fig. 1).16 The lead com-
pound I possesses unsubstituted pyridyl ring, so the tether attached to
the pyridyl ring in compounds 1g, 1h, 2b, 2c, 2f, and 3f could con-
tribute to stronger potency.
IC50 values of the most active compounds as inhibitors of
PGE2 production in LPS-induced murine RAW 264.7
macrophages.
Compound No.
IC50 (µM)
1g
1h
2b
2c
2f
5.62
2.94
5.11
4.51
4.21
7.06
0.94
Furthermore, these most promising compounds 1h and 2f were se-
lected for further investigation of their molecular mechanism of action.
They were tested for inhibitory effects on COX-2 protein expressions in
LPS-induced murine RAW 264.7 macrophages by western blotting
(Fig. 3). Compounds 1h and 2f showed dose-dependent inhibition of
the COX-2 expression, however, compound 1h was more active. So, the
PGE2 inhibition could occur as a result of inhibition of COX-2 protein
expression in a dose-dependent manner.
0.66
1.21
0.89
0.75
1.38
3f
inhibitors of PGE2 production rather than NO production. The most
active compounds were 1g, 1h, 2c, and 2f. All of them possess 3-
methoxyphenylpyrazole ring. The methoxy derivatives 1g and 1h are
more active than the corresponding hydroxyl analogues 3g and 3h.
Moreover, the propylene linker is generally more favorable for activity
than ethylene. In case of urea derivatives, terminal 3,4-di-
chlorophenylurea moiety is the best for activity. The most active urea
derivatives, 2c and 2f possess that moiety.
Compound 1h, the most potent as PGE2 production inhibitor, was
further tested for its effect on COX-2 mRNA expression in LPS-induced
murine RAW 264.7 macrophages (Fig. 4). Compound 1h strongly in-
hibited the COX-2 mRNA expression in dose-dependent manner. Thus
PGE2 production inhibition could be a result of inhibition of both COX-
2 mRNA expression as well as COX-2 protein expression.
In conclusion, our target triarylpyrazole derivatives were tested as
inhibitors of LPS-induced NO and PGE2 productions in murine RAW
264.7 macrophages. We could get several compounds act as potent
inhibitors of PGE2 production rather than inhibitors of NO release.
Among them, we obtained two promising PGE2 production inhibitors,
Based on the preliminary results illustrated in Table 1, the six
compounds that exerted more than 60% inhibition of PGE2 production
(compounds 1g, 1h, 2b, 2c, 2f, and 3f) were tested in 4-dose mode to
measure their IC50 values (Table 2). The most potent compounds are 1h
3

M.M. Gamal El-Din, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 2. Effects of compounds 1h (Fig. 2A) and 2f (Fig. 2B) on PGE2 production in LPS-induced murine RAW264.7 macrophages following treatment with compound
1h or 2f (3.13–25 μM) for 1 h, cells were stimulated using LPS (1 μg/ml) over 24 h. The amount of PGE2 in the cell supernatant was estimated using EIA kit. NS-398
was used as a positive control. #P < 0.05 vs. the control group;*** P < 0.001 vs. LPS-stimulated cells.
Fig. 3. Inhibitory effects of compounds 1h
and 2f on LPS-induced COX-2 protein ex-
pression in murine RAW 264.7 macro-
phages. The cellular lysates were prepared
from cells pretreated with/without com-
pound 1h or 2f over 1 h and then stimulated
with LPS (1 µg/mL) over 24 h. Total cellular
proteins were resolved by SDS-PAGE,
transferred to PVDF membranes, and de-
tected with specific COX-2 antibodies using
β-actin as an internal standard.
Acknowledgments
This work was funded by 2019 research program of Hanseo
University in Korea. We would like to thank Kyung Hee University in
Korea for its support to this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.126884.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4