Antiproliferative diarylpyrazole derivatives as dual inhibitors of the ERK pathway and COX-2

Article abstract of DOI:10.1111/cbdd.12186

A series of 3,4-diarylpyrazole-1-carboxamide derivatives was designed and synthesized. A selected group of the target compounds was tested for in vitro antiproliferative activities over a panel of 60 cancer cell lines at the National Cancer Institute (NCI, Bethesda, MD, USA) at a single-dose concentration of 10 μm, and the four most active compounds 9a, 9l, 9n, and 10o were further tested in a five-dose testing mode to determine their IC₅₀ values over the 60 cell lines. In addition, a selected group of target compounds were tested for inhibitory effect over cyclooxygenase isozymes. Compounds 9a, 9l, 9n, and 10o were also tested for MEK and ERK kinase inhibitory activity using Western blot assay. Compound 10o was selective toward melanoma cell line subpanel, and its antiproliferative activity may be attributed to selective cyclooxygenase-2 inhibition and ERK pathway inhibition.

Full text of DOI:10.1111/cbdd.12186

Chem Biol Drug Des 2013; 82: 336–347
Research Article
Antiproliferative Diarylpyrazole Derivatives as Dual
Inhibitors of the ERK Pathway and COX-2
Mohammed I. El-Gamal1,2,3, Hong Seok Choi4,
USA according to the American Cancer Society report.a
More than 70% of all cancer deaths occurred in low- and
middle-income countries. Deaths from cancer worldwide
Kyung Ho Yoo5, Daejin Baek6
and Chang-Hyun Oh1,2,
*
are projected to exceed 13 million in 2030 according to
the World Health Organization report.b Despite the exten-
sive efforts and investment in research, the management
of human malignancies still constitutes a major challenge
for contemporary medicinal chemistry. There has been an
urgent need for development of more efficient anticancer
agents with minimal side-effects.
1Center for Biomaterials, Korea Institute of Science and
Technology, PO Box 131, Cheongryang, Seoul 130-650,
Korea
2Department of Biomolecular Science, University of
Science and Technology, 113 Gwahangno, Yuseong-gu,
Daejeon 305-333, Korea
3Department of Medicinal Chemistry, Faculty of Pharmacy,
University of Mansoura, Mansoura 35516, Egypt
4College of Pharmacy, Chosun University, Gwangju 501-
759, Korea
The RAS–RAF–MEK–ERK signaling pathway (ERK path-
way) plays an important role in tumorigenesis and cancer
progression. Abnormal activation of ERK pathway has
been linked to uncontrolled cell proliferation, increased cell
survival, and tumor progression (1). Sorafenib (Nexavarꢀ,
Figure 1), a diarylurea derivative, targets ERK pathway. It
inhibits basal phosphorylation of ERK in numerous cancer
cell lines in vitro, including melanoma cell lines (2). Sorafe-
nib is also a well-known inhibitor of B-RAF. Dysregulated
signaling through RAF kinase isoforms has been detected
in ~30% of human cancers (3). Constitutive B-RAF activity
can be caused by activating oncogenic mutations, such
as B-RAF V600E mutation (4). Much interest has been
focused on modulation of ERK pathway as a potential
strategy for anticancer agents.
5Chemical Kinomics Research Center, Korea Institute of
Science and Technology, PO Box 131, Cheongryang,
Seoul 130-650, Korea
6Department of Chemistry, Hanseo University, Seosan
356-706, Korea
*Corresponding author: Chang-Hyun Oh, choh@kist.re.kr
A series of 3,4-diarylpyrazole-1-carboxamide deriva-
tives was designed and synthesized. A selected group
of the target compounds was tested for in vitro anti-
proliferative activities over a panel of 60 cancer cell
lines at the National Cancer Institute (NCI, Bethesda,
MD, USA) at a single-dose concentration of 10 lM, and
the four most active compounds 9a, 9l, 9n, and 10o
were further tested in a five-dose testing mode to
determine their IC50 values over the 60 cell lines. In
addition, a selected group of target compounds were
tested for inhibitory effect over cyclooxygenase iso-
zymes. Compounds 9a, 9l, 9n, and 10o were also
tested for MEK and ERK kinase inhibitory activity using
Western blot assay. Compound 10o was selective
toward melanoma cell line subpanel, and its antiprolif-
erative activity may be attributed to selective cyclooxy-
genase-2 inhibition and ERK pathway inhibition.
Cyclooxygenase-2 (COX-2) is an inducible enzyme involved
in the conversion of arachidonic acid to prostaglandins and
other eicosanoids. Cyclooxygenase-2 and its product, pros-
taglandin E2 (PGE2), play a crucial role in tumor microenvi-
ronment (5). Several reports have shown that PGE2 and
COX-2 have a wide range of effects including induction of
cellular proliferation, promotion of angiogenesis, promotion
of cancer cell resistance to apoptosis, stimulation of tumor
invasion, and suppression of immune responses (6,7).
Molecular pathology studies have revealed that COX-2 is
overexpressed in cancer and stroma cells during tumor pro-
gression, and anticancer chemoradiotherapies induce
expression of COX-2 in cancer cells (8). Cyclooxygenase-2
has been proven to be overexpressed in several types of
human cancer including melanomas (9–12), colon (13), non-
small cell lung (14), intestinal (15), colorectal (16), pancreatic
(17), cervical (18), breast (19), endometrial (20), laryngeal
(21), papillary thyroid (22), and gastric (23) cancers. Much
attention has been focused on COX-2 inhibitors as a benefi-
cial avenue for cancer chemotherapy (7,8). Several
Key words: 3,4-diarylpyrazole-1-carboxamide, anticancer,
cyclooxygenase-2, ERK pathway, pyrazole, vicinal diaryl het-
erocycle
Received 29 December 2012, revised 2 May 2013 and
accepted for publication 1 July 2013
Cancer is a major leading cause of death worldwide.
577 190 patients with cancer died, and more than 1.6
million new cancer cases were identified in 2012 only in
336
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12186

Dual Inhibitors of the ERK Pathway and COX-2
In the present investigation, 54 final compounds were
designed and synthesized in rational to the potent inhibi-
tors of ERK pathway A (25), B (26), and C (27) (Figure 2).
The target compounds 9a–r, 10a–r, 20a–i, and 21a–i
were designed by introduction of derivatives of the carb-
oxamide side chain of compound B into position 1 of the
pyrazole ring of compound A. The phenolic hydroxyl group
of A was retained in compounds 10a–r and 21a–i and
modified into methoxy group in compounds 9a–r and
20a–i to examine its effect on the activity. In addition, the
furan ring of the lead compound C was isosterically
replaced with pyrazole, and the carboxamide chain was
Figure 1: Structure of sorafenib.
experimental and clinical studies have established potent
anticancer activity of COX-2 inhibitors such as celecoxib.
Targeting both COX-2 and ERK pathway simultaneously
has been reported to produce synergistic antiproliferative
activity. ERK1/2 activation or phosphorylation was reported
to increase COX-2 protein expression in some cancer cells
treated with selective COX-2 inhibitor NS398. The combi-
nation of selective COX-2 inhibitor NS398 and MEK inhibi-
tor U0126 produced a synergistic antiproliferative effect at
cellular level. Both phosphorylated ERK1/2 and COX-2 pro-
tein expression were concentration dependently decreased
by combined treatment. Moreover, the combination of
selective COX-2 inhibitor and ERK1/2 inhibitor could
enhance apoptosis over that by individual treatment (24).
modified (Figure 2). The presence of
a vicinal diaryl
heterocyclic system, 3,4-diarylpyrazole scaffold, can con-
fer COX-2 inhibitory activity of the target compounds, sim-
ilar to celecoxib (Figure 3). We have previously reported
their antiproliferative activity against A375P human mela-
noma cell line (28–30). Herein, we report their in vitro anti-
proliferative activities against National Cancer Institute
(NCI)-60 cancer cell line panel of 9 different cancer types,
MEK/ERK kinase inhibitory activity, and their inhibitory
effect on cyclooxygenase isozymes: COX-2 and COX-1.
The target compounds were rationally designed to
Figure 2: Rational design of the
target 3,4-diarylpyrazole-1-
carboxamide derivatives.
Chem Biol Drug Des 2013; 82: 336–347
337

El-Gamal et al.
Figure 3: Structures of celecoxib
and the target compounds. The
vicinal diaryl heterocycle scaffold is
essential for cyclooxygenase-2
(COX-2) inhibition.
possess dual mechanisms of antiproliferative activity
through COX-2 inhibition and targeting RAS-RAF–MEK–
ERK pathway.
In vitro cyclooxygenase inhibition assay
The ability of the test compounds to inhibit bovine COX-1
and COX-2 was determined using an enzyme immunoas-
say (EIA; kit catalog number 560101; Cayman Chemical,
Ann Arbor, MI, USA) according to the manufacturer’s
instructions. Cyclooxygenase catalyzes the first step in the
biosynthesis of arachidonic acid to PGH2. PGF2a, pro-
duced from PGH2 by reduction with stannous chloride,
was measured by enzyme immunoassay (ACETM compet-
itive EIA). Stock solutions of test compounds were dis-
solved in a minimum volume of DMSO. Briefly, to a series
of supplied reaction buffer solutions (960 lL, 0.1 M Tris–
HCl, pH 8.0 containing 5 mM EDTA and 2 mM phenol) with
either COX-1 or COX-2 (10 lL) enzyme in the presence of
heme (10 lL) was added 10 lL of various concentrations
of test drug solutions (0.01, 0.1, 1, 10, and 50 lM in a final
volume of 1 mL). These solutions were incubated for a
period of 5 min at 37 °C after which 10 lL of arachidonic
acid (100 lM) solution was added and the COX reaction
was stopped by the addition of 50 lL of 1 M HCl after
2 min. PGF2a, produced from PGH2 by reduction with
stannous chloride, was measured by EIA. This assay is
based on the competition between PGs and a PG-acetyl-
cholinesterase conjugate (PG tracer) for a limited amount
of PG antiserum. The amount of PG tracer that is able to
bind to the PG antiserum is inversely proportional to the
concentration of PGs in the wells because the concentra-
tion of PG tracer is held constant while the concentration
of PGs varies. This antibody–PG complex bound to a
mouse anti-rabbit monoclonal antibody that was previously
attached to the well. The plate was washed to remove any
unbound reagents, and then, Ellman’s reagent, which con-
tains the substrate to acetylcholine esterase, was added
to the well. The product of this enzymatic reaction
produced a distinct yellow color that absorbs at 405 nm.
The intensity of this color, determined spectrophotometri-
cally, was proportional to the amount of PG tracer bound
to the well, which was inversely proportional to the amount
of PGs present in the well during the incubation: absor-
bance a [bound PG tracer] a1/PGs. Per cent inhibition was
calculated by the comparison of compound treated to vari-
ous control incubations. The concentration of the test
compound causing 50% inhibition (IC50, lM) was calcu-
lated from the concentration–inhibition response curve
(duplicate determinations).
Experimental
Synthesis of the target compounds
They were synthesized by the previously reported methods
(28–30).
NCI-60 cancer cell line screening
Screening against a panel of 60 cancer cell lines of nine
different cancer types was carried out at the NCI, Beth-
esda, MD, USA,c applying the standard protocol of the
NCI.d Sulforhodamine B colorimetric assay method was
utilized (31).
Kinase screening
Immunoblot analysis
For immunoblotting, A375P melanoma cells grown to
70–80% confluence were harvested in RIPA lysis buffer and
disrupted by sonication and centrifuged at 12 000 rpm for
15 min. The quantity of protein was determined with DC
protein assay kit (Bio-Rad Lab., Hercules, CA, USA). Protein
samples were subjected to SDS–PAGE and immunoblotted
with the appropriate primary antibody overnight at 4 °C. The
protein bands were visualized using chemiluminescence
detection kit (Amersham HRP Chemiluminescent Sub-
strates; Amersham Biosciences, Piscataway, NJ, USA) after
hybridization with the HRP-conjugated secondary antibody
from rabbits or mice. The LAS4000-mini (Fujifilm, Tokyo,
Japan) was used for chemiluminescence detection.
Compound treatment in A375P cells
To assess the effect of the target compounds on the RAF-
1/MEK/ERK signaling pathway, A375P cells were treated
with the tested compounds and sorafenib in a dose-
dependent way (1, 3, and 10 lM) for 24 h and immunob-
lotted with antibodies against phospho-MEK1/2, ERK1/2,
and b-actin, respectively.
338
Chem Biol Drug Des 2013; 82: 336–347

Dual Inhibitors of the ERK Pathway and COX-2
tested at a single-dose concentration of 10 lM, and the
percentages of growth inhibition over the 60 tested cell lines
were determined. The mean inhibition percentages of all of
the tested compounds over the full panel of cell lines are
illustrated in Table 1. The results revealed that methoxy
derivatives 9j, 9l, 9n, and 20f showed higher mean%inhibi-
tion than the corresponding hydroxyl derivatives 10j, 10l,
10n, and 21f. In addition, the para-chloro compounds were
the lowest active compared with meta-chloro derivatives.
So, para-chloro group on the aryl ring at position 3 of the
pyrazole moiety is unfavorable for antiproliferative activity of
this series of compounds.
Results and Discussion
Synthesis
Synthesis of the target compounds 9a–r and 10a–r
The pathway for synthesis of compounds 9a–r and 10a–r
was reported and discussed in details (Scheme 1) (28,29).
Synthesis of the target compounds 20a–i and
21a–i
The pathway for synthesis of compounds 9a–r and 10a–r
was reported and discussed in details (Scheme 2) (30).
The effect of the terminal tertiary amine moiety on the activ-
ity was investigated. Compound 9a, with the highest mean
%inhibition, possessing dimethylamino moiety showed
higher activity than compounds 9c, 9d, 9f, and 9g with
morpholino, 2′,6′-dimethylmorpholino, N-acetylpiperazinyl,
and piperidinyl moieties, respectively. In case of 3-chloro-5-
methoxyphenyl derivatives 9j–r, morpholino compound 9l
demonstrated higher mean%inhibition compared with the
other analogs. But in cases of 3-chloro-5-hydroxyphenyl
and 4-chloro-3-methoxyphenyl analogs, diethyl amino,
N-methylpiperazinyl, and N-acetylpiperazinyl moieties were
more favorable for their activities (compounds 10k, 10n,
10o, 20b, 20e, and 20f).
Biological evaluation
Antiproliferative activities of
3,4-diarylpyrazole-carboxamide target compounds
against NCI-60 cell line panel
Structures of the target compounds were submitted to
NCI, Bethesda, MD, USA,c and the 29 compounds
shown in Table 1 were selected on the basis of degree of
structural variation and computer modeling techniques for
evaluation of their antineoplastic activities. The selected
compounds were subjected to in vitro anticancer assay
against tumor cells in a full panel of 60 cell lines taken
from nine different tissues (blood, lung, colon, CNS, skin,
ovary, kidney, prostate, and breast). The compounds were
Upon comparing the activities of methoxy and hydroxyl
analogs, it was found that the methoxy compounds 9c, 9j,
Cl
Cl
Cl
Cl
a
b
c
O
Cl
O
O
OH
OH
O
O
O
O
O
O
N
1a,b
2a,b
3a,b
4a,b
Cl
Cl
Cl
COOPh
O
O
e
f
d
N
N
N
N
N
COOPh
NH
+
N
N
N
7a,b
5a,b
6a,b
R
Cl
Cl
Cl
O
NH
R
R
g
O
O
HO
HN
O
HN
N
N
N
N
N
N
+
O
N
N
N
8a-r
9a-r
10a-r
Scheme 1: Reagents and conditions: (a) sodium methoxide, HMPA, 115–120 °C, 15 h; (b) acetyl chloride, MeOH, 0 °C then room
temperature, 15 h, 70% (for 3a), 65.1% (for 3b); (c) 4-picoline, LHMDS, THF, À25 °C then room temperature, overnight, 50% (for 4a),
40% (for 4b); (d) (i) DMF-DMA, room temperature, 18 h, (ii) hydrazine monohydrate, EtOH, room temperature, overnight, 74% (for 5a),
81% (for 5b); (e) phenyl chloroformate, TEA, THF, 0 °C, 2 h; (f) appropriate ethanamine, K2CO3, CH2Cl2, room temperature, 1 h, 16–80%
(for 9a–i), 40–70% (for 9j–r); (g) BF3.Me2S, CH2Cl2, room temperature, 48 h, 38–63% (for 10a–i), 41–63% (for 10j–r).
Chem Biol Drug Des 2013; 82: 336–347
339

El-Gamal et al.
OH
O
O
O
Cl
Cl
Cl
Cl
a
c
b
d
OH
O
O
O
11
12
13
14
O
O
O
Cl
Cl
Cl
O
COOPh
Cl
N
N
N
e
f
N
COOPh
N
NH
+
O
N
N
N
N
18
15
16
17
R
O
O
OH
O
Cl
Cl
Cl
NH
R
R
HN
O
N
HN
N
N
h
g
N
N
N
+
O
N
N
N
20a-i
19a-i
21a-i
Scheme 2: Reagents and conditions: (a) (CH3)2SO4, K2CO3, acetone, reflux, 1 h; (b) KMnO4, C5H5N, H2O, 50 °C, 24 h then room
temperature, 13 h, 90%; (c) acetyl chloride, MeOH, 0 °C then room temperature, 15 h, 85%; (d) 4-picoline, LHMDS, THF, À25 °C then
room temperature, overnight, 45%; (e) (i) DMF-DMA, room temperature, 18 h, (ii) hydrazine monohydrate, EtOH, room temperature,
overnight, 81%; (f) phenyl chloroformate, TEA, THF, 0 °C, 2 h; (g) appropriate ethanamine, K2CO3, CH2Cl2, room temperature, 1 h, 44–
80% (for 20a–i); (h) BF3.Me2S, CH2Cl2, room temperature, 48 h, 40–61%.
9l, 9n, and 20f were more active than the corresponding
hydroxyl analogs 10c, 10j, 10l, 10n, and 21f. So, it can
be concluded that the bulkier and more hydrophobic
methoxy group on the phenyl ring at position 3 of the
pyrazole ring is more favorable for antiproliferative activity
of this series of 3,4-diarylpyrazole-1-carboxamide compounds.
MDA-MB-435 melanoma cell line and EKVX non-small cell
lung cancer (NSCLC) cell line, respectively.
MEK/ERK kinase inhibitory activity of compounds
9a, 9l, 9n, and 10o
To study the mechanism of action of 3,4-diarylpyrazole-
1-carboxamide derivatives, the four compounds 9a, 9l, 9n,
and 10o, selected for five-dose testing mode over NCI-60
cancer cell line panel, were taken as representative
examples of these series of compounds to be tested for
their MEK and ERK kinase inhibitory activity. The MEK/ERK-
containing A375P cell lysate was treated with three different
concentrations of each of the test compounds (1, 3, and
10 lM), and their inhibitory activities were compared with that
of sorafenib. The results showed that the tested compounds
and sorafenib significantly suppressed phosphorylation of
MEK1/2 and ERK1/2 in a dose-dependent manner (Fig-
ure 4). Quantification of the results was performed by mea-
suring density of each spot at each concentration, and the
inhibition percentages are provided in Table 4. The ERK
and MEK activities were inhibited by 87.5% and 80.6%,
respectively, with 10 lM of compound 10o possessing
hydroxyl group on the chlorophenyl ring and N-acetylpiper-
azinyl terminal moiety. Moreover, the inhibition percentages
expressed by compound 10o over both ERK and MEK kin-
ases at 1 and 3 lM were higher than those demonstrated by
sorafenib at the same concentrations. Compound 10o
showed selectivity toward melanoma subpanel more than
Compounds 9a, 9l, 9n, and 10o were further tested in a
five-dose testing mode to determine their IC50 values over
the 60 cancer cell lines. The mean IC50 values of these
four compounds over the nine cancer types are shown in
Table 2.
As shown in Table 2, most of the compounds exhibited high
potency (in micromolar scale) over all the nine cancer types.
Compounds 9a and 9l showed broad-spectrum antiprolifera-
tive activities over all the nine cancer types with mean IC50
<10.5 lM. Compound 9n was more active against CNS and
renal cancer cell lines. While the phenolic compound 10o pos-
sessing N-acetylpiperazinyl terminal moiety was more selective
for melanoma. Its selectivity index was 1.72 compared
with the second most susceptible cancer type, leukemia.
The IC50 values of compounds 9a, 9l, 9n, and 10o tested
in five-dose mode over the most sensitive cell line of each
subpanel are summarized in Table 3. From these
data, we find that most of the IC50 values are in micromo-
lar scale. Of special interest, the IC50 values of
compounds 9l and 9n were in submicromolar range over
340
Chem Biol Drug Des 2013; 82: 336–347

Dual Inhibitors of the ERK Pathway and COX-2
Table 1: Mean inhibition percentages observed with 3,4-diarylpyrazole-1-carboxamide target compounds in single-dose (10 lM)
60-cancer cell line screening
R2
R1
HN
O
N
N
N
Compound no. R1
R2
Mean%inhibitiona Compound no. R1
R2
Mean% inhibitiona
25.42
CH3
CH3
CH3
CH3
9a
3-Cl, 4-OMe
62.22
10k
3-Cl, 5-OH
N
N
9c
9d
3-Cl, 4-OMe
3-Cl, 4-OMe
7.02
4.85
10l
3-Cl, 5-OH
3-Cl, 5-OH
5.20
N
N
O
N
N
N
O
CH3
O
10n
22.39
CH3
CH3
O
O
CH3
9f
3-Cl, 4-OMe
26.49
10o
3-Cl, 5-OH
25.51
N
N
N
N
CH3
9g
9j
3-Cl, 4-OMe
3-Cl, 5-OMe
2.85
10r
3-Cl, 5-OH
11.77
14.88
N
N
N
N
CH3
CH3
CH3
CH3
16.48
20b
4-Cl, 3-OMe
CH3
9k
3-Cl, 5-OMe
25.33
20c
4-Cl, 3-OMe
À2.32
N
O
N
CH3
9l
3-Cl, 5-OMe
3-Cl, 5-OMe
37.03
29.42
20e
20f
4-Cl, 3-OMe
4-Cl, 3-OMe
28.18
18.83
N
N
O
N
N
N
N
CH3
O
9n
N
CH3
CH3
9p
9r
3-Cl, 5-OMe
3-Cl, 5-OMe
3-Cl, 4-OH
À2.16
7.94
20g
21c
21d
4-Cl, 3-OMe
4-Cl, 3-OH
4-Cl, 3-OH
11.94
À3.62
À2.97
N
N
N
N
O
CH3
10c
À7.29
N
O
N
O
CH3
O
10g
3-Cl, 4-OH
2.32
21f
4-Cl, 3-OH
4-Cl, 3-OH
À9.59
N
N
N
N
CH3
10i
10j
3-Cl, 4-OH
3-Cl, 5-OH
À4.09
21g
17.31
N
N
CH3
CH3
10.92
aMean%inhibition represents the mean inhibition percentages over the 60 cell lines. The inhibition percentages are calculated by subtracting
the growth percentages from 100.
Chem Biol Drug Des 2013; 82: 336–347
341

El-Gamal et al.
Table 2: Mean IC50 values (lM) of compounds 9a, 9l, 9n, and
10o over in vitro subpanel cancer cell linesa
Table 4: Inhibition percentages expressed by compounds 9a, 9l,
9n, 10o, and sorafenib over ERK and MEK kinases in Western
blot analysis at different concentrationsa
No. of cell
lines in each
subpanel
Compound no.
% Inhibition
9a
9l
9n
10o
ERK
1
MEK
1
Subpanel cancer cell line
Concentration (lM)
3
10
3
10
Leukemia
Non-small cell
lung cancer
6
9
6.47
6.13
5.06 29.7
9.96 14.07 15.47
7.31
Compound no.
9a
–
–
–
29.3
15.4
19.4
2.0
28.6
62.9
50.2
70.6
55.5
64.8
87.5
94.9
–
19.3
30.0
27.4
67.0
7.5
52.2
82.0
63.1
80.6
80.7
Colon Cancer
CNS Cancer
Melanoma
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
7
6
9
7
8
2
6
4.52
3.25
5.90
4.42 16.61
4.67 7.12 12.77
6.79 11.22 4.25
8.77
9l
9n
35.9
12.5
3.6
–
10o
Sorafenib
6.28 10.29 13.12 19.16
4.35
5.78
5.13
5.37
5.93 11.41
6.35 15.90 18.95
5.76 13.12 12.88
aThe inhibition percentages were calculated by densitometry of
results shown in Figure 4.
aMean IC50 values were calculated by dividing the summation of
IC50 values of the compound over cell lines of the same cancer
type by the number of cell lines in the subpanel.
Much interest has been given to vicinal diaryl heterocycles as
selective COX-2 inhibitors. So, a selected group (29 final
compounds) was tested for COX-2 inhibitory activity at
molecular level. The best nine compounds in terms of
potency against COX-2 were further tested for COX-1 inhibi-
tion to determine their selectivity toward COX-2 than COX-1.
This part of the work was done among our attempts to inves-
tigate the possible mechanism(s) of antiproliferative activities
of our target compounds. The results are illustrated in Table 5
(IC50, lM).
Table 3: IC50 values (lM) of the tested compounds 9a, 9l, 9n,
and 10o over the most sensitive cell line of each subpanel
Compound no.
9a
9l
9n
10o
Cancer cell line
HL-60(TB)a
EKVXb
5.30
2.23
2.17
1.68
1.15
1.73
2.19
4.62
1.95
3.22
5.57
3.98
1.73
0.60
7.61
2.67
4.24
2.00
25.60
0.97
3.32
1.75
10.40
2.94
2.41
19.50
10.50
7.79
24.60
3.28
13.50
2.70
12.20
3.67
HT29c
The results showed that methoxy derivatives 9l and 9q
were more potent than the corresponding hydroxyl deriva-
tives 10l and 10q. Compound 9l also showed higher
mean%inhibition over the NCI-60 cancer cell line panel
than its hydroxyl analog 10l. This may be attributed to
differences in steric and/or electronic effects between
methoxy and hydroxyl groups. So, the methoxy group on
the chlorophenyl ring may enhance the affinity of the ligand
to COX-2 enzyme and hence the potency.
SNB-75d
MDA-MB-435e
OVCAR-8f
RXF-393g
PC-3h
13.60
4.59
MDA-MB-468i
aLeukemia cell line.
bNon-small cell lung cancer cell line.
cColon cancer cell line.
dCNS cancer cell line.
Compound 9q with terminal 2′-methylpiperidinyl moiety
showed higher potency than 9p with piperidinyl ring. This
result together with the higher potency of methoxy deriva-
tives compared with the corresponding hydroxyl deriva-
tives suggests that the bulkier the compound, the more
appropriate will be the drug–receptor interaction and
hence the higher the COX-2 inhibitory activity.
eMelanoma cell line.
fOvarian cancer cell line.
gRenal cancer cell line.
hProstate cancer cell line.
iBreast cancer cell line.
the other cancer types (Table 2). In addition, compounds 9l
and 9n were more active against MEK kinase than sorafenib
at 1 and 3 lM concentration. Upon investigating the results
of compounds 9a, 9l, and 10o over the most sensitive cell
line of each cancer subpanel (Table 3), we found that MDA-
MB-435 melanoma cell line was the most susceptible for
both compounds. These compounds may inhibit melanoma
cell proliferation through inhibition of ERK pathway.
The effect of substituents on the phenyl ring at position 3
of the pyrazole ring and their positions was investigated.
Compounds 9n and 9q with 3-chloro-5-methoxyphenyl
ring were more potent than 20e and 20h possess-
ing 4-chloro-3-methoxyphenyl moiety. Similarly, compound
9l with 3-chloro-5-methoxyphenyl demonstrated higher
potency against COX-2 enzyme and higher mean%inhibi-
tion over NCI-60 cancer cell line panel than 9c with
3-chloro-4-methoxyphenyl ring. 3-Chloro-5-hydroxyphenyl
derivatives 10m and 10q were more potent than 10h with
3-chloro-4-hydroxyphenyl and 21d and 21h containing
In vitro cyclooxygenase inhibition assay
The target 3,4-diarylpyrazole-1-carboxamide derivatives pos-
sess a vicinal diaryl heterocyclic scaffold, 3,4-diarylpyrazole.
342
Chem Biol Drug Des 2013; 82: 336–347

Dual Inhibitors of the ERK Pathway and COX-2
Figure 4: Inhibition of MEK and
ERK kinase activities by
compounds 9a, 9l, 9n, 10o, and
sorafenib.
4-chloro-3-hydroxyphenyl moiety. However, 3-chloro-4-
methoxyphenyl derivatives 9a and 9g exerted higher
potency than 9p, 20a, and 20g. So, the lowest potency
was encountered with 4-chloro-3-methoxyphenyl and
4-chloro-3-hydroxyphenyl rings. It can be concluded that
para-chloro group on this aryl ring at position 3 of the pyr-
azole ring is unfavorable for COX-2 inhibitory effect of this
series of compounds.
melanoma cells, was better than the corresponding mor-
pholino derivative 10l.
Compounds 9a, 9g, 9l, 9n, 9q, 10m, 10o, 10q, and 21b
possessing the most promising results against COX-2
together with celecoxib were further tested for COX-1
inhibition. All the tested compounds did not show any
inhibitory activity against COX-1 up to 50 lM concentration.
All the nine compounds showed highly promising selectivity
indices toward COX-2 than COX-1. Of special interest,
compounds 9a, 9q, 10o, and 10q showed selectivity indi-
ces more than 106 comparable to that of celecoxib.
The effect of terminal tertiary amine on biological activities
was also investigated. The results were parallel with data
obtained over NCI-60 cell line panel. Compound 9a with
dimethylamino terminal moiety demonstrated higher potency
against COX-2 and higher mean%inhibition over NCI-60 cell
line panel than the corresponding analogs 9c, 9d, 9g, and
9i possessing morpholino, 2′,6′-dimethylmorpholino, pipe-
ridinyl, and pyrrolidinyl moieties, respectively. Compounds
9l and 9n possessing morpholino and N-methylpiperazinyl
moieties, respectively, showed higher potency against
COX-2 enzyme and higher mean%inhibition values over
NCI-60 cell line panel than compounds 9k and 9p with
diethylamino and piperidinyl moieties, respectively. On the
other hand, compound 10o with the highest potency
against COX-2 and ERK kinase, and with selectivity toward
Among all the target compounds, compound 10o possessing
3-chloro-5-hydroxyphenyl and N-acetylpiperazinylmoieties
demonstrated the best results. It was equipotent to celecoxib
against COX-2. It showed almost the same selectivity index
as celecoxib.
Conclusions
A series of 3,4-diarylpyrazole-1-carboxamide derivatives
has been designed and synthesized based on our
Chem Biol Drug Des 2013; 82: 336–347
343

El-Gamal et al.
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Dual Inhibitors of the ERK Pathway and COX-2
Chem Biol Drug Des 2013; 82: 336–347
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El-Gamal et al.
previous literature studies and as a continuation of our
ongoing anticancer development program. Compounds 9a
and 9l showed broad-spectrum cytotoxicity over all the
nine types of cancers tested at the NCI. Compound 9n
was selective for CNS and renal cancer cell line subpan-
els, while compound 10o demonstrated selectivity toward
melanoma. Compounds 9n and 10o are promising leads
for future development of potent and selective antiprolifer-
ative agents. The IC50 values of compounds 9l and 9n
were in submicromolar scale, 0.60 lM and 0.97 lM over
MDA-MB-435 melanoma and EKVX NSCLC cell lines,
respectively.
4. Brose M.S., Volpe P., Feldman M., Kumar M., Rishi I.,
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Upon testing the inhibitory effect of the target compounds
over COX-2 and COX-1, compound 10o possessing
3-chloro-5-hydroxyphenyl and N-acetylpiperazinyl moieties
showed equal potency and selectivity toward COX-2 enzyme
as celecoxib. Compounds 9a, 9l, 9n, and 10o inhibited
MEK and ERK kinases in a dose-dependent manner.
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Its selectivity toward melanoma subpanel and COX-2
enzyme together with its inhibitory activity toward MEK
and ERK kinases make compound 10o a promising lead
for future development of potential anticancer agents.
Compound 10o might exert its antiproliferative activity
against melanoma due to dual inhibition of ERK pathway
and COX-2.
Acknowledgments
This work was supported by Korea Institute of Science
and Technology (KIST), KIST Project (2E22360). We would
like to thank the National Cancer Institute (NCI), Bethesda,
Maryland, USA, for performing the anticancer testing of
the target compounds over 60 cancer cell lines.
Conflict of Interest
14. Huang M., Stolina M., Sharma S., Mao J.T., Zhu L.,
Miller P.W., Wollman J., Herschman H., Dubinett S.M.
(1998) Non-small cell lung cancer cyclooxygenase-
2-dependent regulation of cytokin balance in lympho-
cytes and macrophages: up-regulation of interleukin
10 and down-regulation of interleukin 12 production.
Cancer Res;58:1208–1216.
15. Williams C.S., Shattuk-Brandts R.L., Dubois R.N.
(1999) The role of COX-2 in intestinal cancer. Expert
Opin Investig Drugs;8:1–12.
The authors have declared no conflict of interest.
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