Chem Biol Drug Des 2014; 84: 409419
Research Article
Synthesis and Biologic Evaluation of Substituted
5-methyl-2-phenyl-1H-pyrazol-3(2H)-one Derivatives as
Selective COX-2 Inhibitors: Molecular Docking Study
Pritam N. Dube1,*, Shweta S. Bule2, Santosh N.
Mokale1, Manoj R. Kumbhare2, Pravin R. Dighe2
and Yogesh V. Ushir2
1 ciated with inflammation and other pathologies, such as
Department of Pharmaceutical Chemistry, Y. B. Chavan
College of Pharmacy, Dr. Rafiq Zakaria Campus,
Aurangabad-431001, Maharashtra India
2
Department of Pharmaceutical Chemistry, S.M.B.T.
College of Pharmacy, Dhamangaon, Nashik-422403,
Maharashtra India
*Corresponding author: Pritam N. Dube,
pritamdube@gmail.com
The present study reported the synthesis and biologic
evaluation of new pyrazolone derivatives for COX-2
inhibitory activities and investigated in vivo for their
anti-inflammatory activities using carrageenan-induced
rat paw edema model as well as in vitro using HRBC
membrane stabilization and protein denaturation
method. Eight derivatives showed pronounced COX-2
inhibition, and 5a, 5d, and 5f exhibited the highest
COX-2 inhibition. The derivatives were further evalu-
ated for antioxidant activity wherein 5a and 5b showed
potent free radical-scavenging activity against DPPH,
nitric oxide, and hydrogen peroxide radicals. Molecular
docking study revealed the binding orientations of py-
razolone derivatives into the active sites of COX-2 and
thereby helps to design the potent inhibitors.
Key words: anti-inflammatory, antioxidants, molecular dock-
ing, pyrazolone
Received 3 December 2013, revised 14 February 2014 and
accepted for publication 12 March 2014
Cyclooxygenase is the key enzyme in the biosynthesis of
prostanoids, biologically active substances that are involved
in several physiologic processes but also in pathologic con-
ditions, such as inflammation (1). It is actually well known
that this enzyme exists under two forms: cyclooxygenase-1
(COX-1) and cyclooxygenase-2 (COX-2). COX-1 is a con-
stitutive enzyme and is responsible for the production of
cytoprotective prostaglandins in the gastrointestinal tract
(GI) and proaggregatory thromboxane in blood platelets.
However, COX-2 is an inducible enzyme which is induced
in response to the release of several proinflammatory medi-
2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12324
ators (24). Both enzymes are sensitive to inhibition by
conventional non-steroidal anti-inflammatory drugs (NSA-
IDs). Observations that COX-1 was involved in several
homeostatic processes, while COX-2 expression was asso-
cancer proliferation, have led to the development of COX-2
selective inhibitors to improve the therapeutic potency and
to reduce the classical side-effects associated with the use
of conventional NSAIDs (57).
Classical NSAIDs such as flurbiprofen (Figure 1A) non-
selectively inhibit both isoenzymes and cause gastric failure
like bleeding and ulcer. To prevent or decrease these side-
effects, a current strategy consists of designing selective
COX-2 inhibitors with an improved gastric safety profile.
The improved safety profile of COX-2 inhibitors may allow
the use of these new agents for long-term prophylactic use
in certain chronic diseases. This has led intense efforts in
search for potent and selective COX-2 inhibitors, which
could provide anti-inflammatory drugs with fewer risks.
Several classes of compounds having selective COX-2
inhibitory activity have been reported in the literature such
as SC-558 and celecoxib (Figure 1B and C, respectively).
It is interesting to note that most of the COX-2 inhibitors
bear a common structural feature, namely a central
heterocyclic five member ring (pyrazole in celecoxib/Celeb-
rex
, 3,4-dihydrofuran-2-one in rofecoxib/Vioxx
, or
1,2-oxazole in valdecoxib/Bextra
) and that several devel-
opments were based on that fact using, for example, tet-
rahydrofuran (5) or 1H-pyrrole (6); moreover, all these
described compounds are vicinal substituted. From these,
the pyrazole ring is considered a privileged structure and
an attractive scaffold for drug discovery.
A number of molecules based upon a monocyclic hetero-
cyclic template have been investigated for their COX-2
inhibitory activity (810). However, the recent success of
pyrazole COX-2 inhibitors has highlighted the importance
of these heterocyclics in medicinal chemistry (8). Thus, our
main objective is to design novel heterocyclic compounds
as specific inhibitors of COX-2 in the hope that these mol-
ecules may be further explored as powerful and novel
non-ulcerogenic anti-inflammatory lead candidates. Thus,
our main objective is to design novel pyrazolones as spe-
cific inhibitors of COX-2 in the hope that these molecules
409
Dube et al.
may be further explored as powerful anti-inflammatory lead
candidates. In view of the rationale and in aiming to finding
new structure leads with potential anti-inflammatory activi-
ties and selective COX-2 inhibition (11,12), new series of
5-methyl-2-phenyl-1H-pyrazol-3(2H)-one derivatives (3,
5a5g) (Figure 1) have been synthesized and evaluated
the in vitro COX-1/COX-2 inhibition and in vivo and in vitro
assessment as anti-inflammatory activities.
Docking studies of this class of molecules have shown that
they accommodate entirely in the binding site of COX-2 in
positions very close to that of SC-558 in the crystal structure
of its complex with COX-2. So, we docked the most active
COX-2 inhibitors into COX-2 (1CX2) active site to predict
their binding modes, their binding affinities, and orientation
of these compounds at the active site of the COX-2 enzyme.
Materials and Methods
Chemistry
The melting points were determined by open capillary
method and were uncorrected. UV spectra were recorded
on Thermo scientific spectrum 2600 spectrophotometer.
The IR spectra were recorded on a Jasco FT-IR spectro-
photometer, model-4100 using KBr disk method. 1H and
13
C spectra were recorded on a Bruker AVANCE
400 MHz spectrometer at 25 C using tetramethylsilane as
an internal standard and DMSO-d6 as the solvent. The
chemical shifts are expressed in d ppm. Mass spectra
were recorded on a Varian Inc, 410 Prostar Binary LC with
500 MS IT PDA Detectors. Purity of the compounds was
checked on TLC plates using silica gel G as stationary
phase and iodine vapors as visualizing agent. For TLC
plate development, the benzenemethanol system at vari-
ous ratios was used as eluent. The% yield was reported
after the recrystallization. All chemicals used were of
410
Figure 1: Representative
examples of non-selective (A) and
selective COX-2 inhibitors (B and C)
and the designed pyrazolone
derivatives (3, 5a5g).
Research-Lab Fine Chem Industries, Mumbai. Solvents
and Chemicals were purified wherever required.
Step-I: Synthesis of 5-methyl-2-phenyl-1,2-
dihydro-3H-pyrazol-3-one (3)
Redistilled ethyl acetoacetate 50 g (49 mL, 0.384 mol) and
40 g (36.5 mL, 0.37 mol) of phenylhydrazine in a 250-mL
RBF were mixed together and refluxed for 3 h. The heavy
reddish syrup was allowed to cool for sometime, and then,
about 100 mL of ether was added, and the mixture was stir-
red vigorously. The syrup which was insoluble in ether got
solidified within 15 min. the solid was filtered at the pump
and washed thoroughly with ether to remove colored impu-
rities and crystallized from ethanol to give the corresponding
5-methyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one (13,14).
Colorless crystals; MP 126127 C; % yield: 80%; UV k
max: 302.
FT-IR (KBr) max/cm: 3132 (C-H aromatic); 2925 (C-H sat-
urated); 1728 (C=O lactam); 1620 (C=N); 1597, 1473 (C=C
aromatic); 1410, 1518 (N=O nitro group); 1340 (C-N);
1026 (C-O); 1455, 1378 (C-H); 1357 (N-N); 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.76.9 (m, 5H, Ar-H), 5.1
(s, 1H, Ar-CH), 2.3 (s, 3H, CH3), 1.9 (s, 1H, NH); 13C
NMR (400 MHz, DMSO-d6) d ppm: 172.8 (C=O of Pyrazo-
lone), 159.5 (C-N of pyrazolone), 139.6 (C-N of Ar),
129.3120.4 (5C, Ar), 91.7 (C4 of pyrazolone), 14.6 (CH3
of pyrazolone). MS: m/z = 175.0 [M+H].
Step-II: General method of synthesis of 5-methyl-
2-phenyl-1,2-dihydro-3H-pyrazol-3-one derivative
(5a5g)
To a solution of compound 5-methyl-2-phenyl-1,2-dihy-
dro-3H-pyrazol-3-one (0.01 mol) in ethanol (30 mL),
Chem Biol Drug Des 2014; 84: 409419

formaldehyde (0.02 mol) and corresponding substituents
like urea, p-chlorophenyl urea, thiourea, N-phenyl thiourea,
guanidine, sulfonamide, semicarbazide were added. The
reaction mixture was refluxed for 610 h. The solvent was
distilled off, and the residue was poured into ice water/
purified water. The precipitated solid was filtered off, dried,
and recrystallized from ethanol.
1-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)methyl]urea (5a). White powder; MP 235238 C; %
yield: 61.22%; UV k max: 313.
FT-IR (KBr) max/cm: 3351 (C-H aromatic); 2969 (C-H sat-
urated); 1630 (C=O lactam); 1620 (C=N); 1547 (C=C aro-
matic); 1495, 1398 (C-H); 1370 (C-N); 1359 (N-N); 1037
(C-O); 1H NMR (400 MHz, DMSO-d6) d ppm: 8.07.1 (m,
5H, Ar-H), 6.16.0 (s, 3H, NH), 3.7, 3.4 (m, 2H, CH2), 3.1
(s, 1H, CH), 1.4 (s, 3H, CH3), 13C NMR (400 MHz, DMSO-
d6) d ppm: 173.8 (C=O of pyrazolone), 163.2 (C=O of
urea), 156.1 (C-N), 132.6120.4 (Ar-C), 49.3 (C4 of
pyrazolone), 21.5 (CH3). MS: m/z = 246.20 [M] +.
1-(4-chlorophenyl)-3-[(3-methyl-5-oxo-1-phenyl-4,5-
dihydro-1H-pyrazol-4-yl) methyl]urea (5b). White
powder; MP 8082 C; % yield: 65.30%; UV k max: 328.
FT-IR (KBr) max/cm: 3328 (C-H aromatic); 2976 (C-H sat-
urated); 1704 (C=O lactam); 1633 (C=N); 1594, 1492 (C=C
aromatic); 1455, 1378 (C-H); 1400 (N=O nitro group);
1357 (N-N), 1340 (C-N); 1026 (C-O); 754 (C-Cl); 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.97.1 (m, 9H, Ar-H), 6.2,
6.1 (s, 2H, NH), 3.8, 3.4 (d, 2H, CH2), 3.1 (s, 1H, CH), 1.9
(s, 3H, CH3); 13C NMR (400 MHz, DMSO-d6) d ppm:
172.4 (C=O of pyrazolone), 168.1 (C=O of urea), 154.3
(C-N), 138.3120.2 (Ar-C), 46.9 (C4 of pyrazolone), 19.3
(-CH3). MS: m/z = 357.1 [M+H].
1-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)methyl]thiourea (5c). White powder; MP 98100 C;
% yield: 69.46%; UV k max: 357.
FT-IR (KBr) max/cm: 3300 (C-H aromatic); 2969 (C-H satu-
rated); 1711 (C=O lactam); 1703 (C=N); 1594, 1499 (C=C
aromatic); 1406 (N=O nitro group); 1455, 1378 (C-H); 1368
(N-N), 1336 (C-N); 1026 (C-O); 1H NMR (400 MHz, DMSO-
d6) d ppm: 8.6 (s, 2H, NH2), 7.87.2 (m, 5H, Ar-H), 3.7, 3.1
(d, 2H, CH2), 2.9 (s, 1H, CH), 2.7 (s, 1H, NH), 1.8 (s, 3H,
CH3); 13C NMR (400 MHz, DMSO-d6) d ppm: 182.1 (C=S),
171.4 (C=O of pyrazolone), 132.6120.9 (Ar-C), 42.1 (C5 of
pyrazolone), 21.7 (CH3). MS: m/z = 263.10 [M+H].
1-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)methyl]-3-phenylthiourea (5d). White powder; MP
150152 C; % yield: 85.58%; UV k max: 335.
FT-IR (KBr) max/cm: 3190 (C-H aromatic); 2925 (C-H sat-
urated); 1759 (C=O lactam); 1611 (C=N); 1611, 1527 (C=C
aromatic); 1453, 1385(C-H); 1369 (N-N); 1341 (C-N); 1062
Chem Biol Drug Des 2014; 84: 409419
Synthesis and Evaluation of COX-2 Inhibitors
(C-O); 1047 (C=S); 1H NMR (400 MHz, DMSO-d6) d ppm:
8.06.9 (m, 10H, Ar-H), 4.2 (s, 1H, NH), 4.1, 3.5 (d, 2H,
CH2), 2.9 (s, 1H, CH), 2.2 (s, 1H, NH), 1.9 (s, 3H, CH3);
13
C NMR (400 MHz, DMSO-d6) d ppm: 181.9 (C=S),
177.4 (C=O), 155.6 (C=N), 137.2121.7 (Ar-C), 20.1 (C5 of
pyrazolone). MS: m/z = 339.12 [M+H].
1-((3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)methyl)guanidine (5e). White powder; MP
185187 C; % yield: 79.18%; UV k max: 311.
FT-IR (KBr) max/cm: 3395 (C=NH); 3062 (C-H aromatic);
2973 (C-H saturated); 1712 (C=O lactam); 1625 (C=N);
1591, 1477 (C=C aromatic); 1458, 1372 (C-H); 1324 (N-
N); 1330 (C-N); 1027 (C-O); 1H NMR (400 MHz, DMSO-
d6) d ppm: 8.7 (s, 2H, NH2), 7.97.1 (m, 5H, Ar-H), 6.1 (s,
1H, NH), 3.7, 3.3 (d, 2H, CH2), 3.0 (s, 1H, CH), 2.0 (s, 1H,
NH), 1.6 (s, 3H, CH3); 13C NMR (400 MHz, DMSO-d6) d
ppm: 174.4 (C=O), 162.7 (C=N), 155.6 (C-N of pyrazo-
lone), 134.9120.2 (Ar-C), 19.3 (C5 of pyrazolone). MS: m/
z = 245.1 [M] +.
4-{[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
4-yl)methyl]amino} benzene sulfonamide (5f). White
powder; MP 220222 C; % yield: 63.60%; UV k max:
390.
FT-IR (KBr) max/cm: 2973 (C-H aromatic); 2900 (C-H sat-
urated); 1702 (C=O lactam); 1595 (C=N); 1501, 1489 (C=C
aromatic); 1020 (C-O); 1452, 1377 (C-H); 1321 (sulfon-
amide); 1321 (N-N); 1H NMR (400 MHz, DMSO-d6) d
ppm: 8.17.0 (m, 9H, Ar-H), 4.4 (s, 1H, NH), 3.8, 3.2 (s,
2H, CH2), 2.8 (s, 1H, CH), 2.1 (s, 2H, NH2), 1.9 (s, 3H,
CH3); 13C NMR (400 MHz, DMSO-d6) d ppm: 178.1
(C=O), 156.3 (C-N of pyrazolone), 151.4 (Ar-N), 132.7
116.2 (Ar-C), 22.5 (C5 of pyrazolone). MS: m/z = 359.12
[M+H].
N-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
4yl)methyl] hydrazine carboxamide (5g). Brown pow-
der; MP 188189 C; % yield: 59.74%; UV k max: 393.
FT-IR (KBr) max/cm: 3050 (C-H aromatic); 1668 (C=O lac-
tam); 1630 (C=N); 1592, 1499 (C=C aromatic); 1448, 1373
(C-H); 1354 (N-N); 1340 (C-N); 1021 (C-O); 1H NMR
(400 MHz, DMSO-d6) d ppm: 7.87.0 (m, 5H, Ar-H), 6.1,
6.0 (s, 2H, NH), 3.9, 3.4 (d, 2H, CH2), 3.1 (s, 1H, CH), 2.2
(s, 1H, NH), 1.8 (s, 3H, CH3); 13C NMR (400 MHz, DMSO-
d6) d ppm: 173.8 (C=O of pyrazolone), 160.2 (C=O), 156.3
(C-N of pyrazolone), 137.4121.7 (Ar-C), 20.5 (C5 of
pyrazolone). MS: m/z = 262.2 [M+H].
Biologic screening
In vitro cyclooxygenase (COX) inhibition assay
The ability of the test compounds listed in Table 2 to
inhibit ovine COX-1 and COX-2 (IC50 values, lM) was
411
Dube et al.
determined using an enzyme immunoassay (EIA) kit (cat-
alog number 560101, Cayman Chemical, Ann Arbor, MI,
USA). Cyclooxygenase catalyzes the first step in the bio-
synthesis of arachidonic acid (AA) to PGH2. PGF2a, pro-
duced from PGH2 by reduction with stannous chloride, is
measured by enzyme immunoassay (ACETM competitive
EIA). Stock solutions of test compounds were dissolved
in a minimum volume of DMSO. Briefly, to a series of
supplied reaction buffer solutions (960 lL, 0.1 M TrisHCl
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 concentra-
tions of test drug solutions (0.01, 0.1, 1, 10, 50, and
100 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 AA (100 lM) solution was added and the COX
reaction was stopped by the addition of 50 lL of 1M
HCl after 2 min. PGF2a produced from PGH2 by reduc-
tion with stannous chloride was measured by enzyme
immunoassay. This assay was based on the competition
between PGs and a PG-acetylcholinesterase conjugate
(PG tracer) for a limited amount of PG antiserum. The
amount of PG tracer that is able to bind to the PG anti-
serum is inversely proportional to the concentration of
PGs in the wells because the concentration of PG tracer
is held constant while the concentration of PGs varies.
This antibodyPG complex binds to a mouse anti-rabbit
monoclonal antibody that had been previously attached
to the well. The plate was washed to remove any
unbound reagents, and then, Ellmans reagent, which
contains the substrate to acetylcholine esterase, was
added to the well. The product of this enzymatic reaction
produces a distinct yellow color that absorbs at 406 nm.
The intensity of this color, determined spectrophotometri-
cally, is proportional to the amount of PG tracer bound
to the well, which is inversely proportional to the amount
of PGs present in the well during the incubation: Absor-
bance a [Bound PG Tracer] a1/PGs. Percent inhibition
was calculated by the comparison of compounds treated
to various control incubations. The concentration of the
test compounds causing 50% inhibition (IC50, lM) was
calculated from the concentrationinhibition response
curve (duplicate determinations).
In vivo anti-inflammatory activity
The test compounds were evaluated using in vivo rat car-
rageenan-induced foot paw edema model reported previ-
ously (15). Male Sprague Dawley rats (250 g) were fasted
with free access to water at least 16 h prior to experi-
ments. Edema was produced by injecting 0.2 mL of a
solution of 1% k-carrageenan in the hind paw. Paw vol-
ume was measured by water displacement with a plethys-
mometer (UGO BASILE) before, 1, and 2 h after
treatment. The compounds were administered intraperito-
neally with a 1 mL suspension of test compounds in vehi-
cle (0.5% methyl cellulose). The percentage was
calculated by the following equation: anti-inflammatory
412
activity (%) = (1 D/C) 100, where D represents the
difference in paw volume before and after compounds
were administered to the rats, and C stands for the differ-
ence of volume in the control groups.
In vitro anti-inflammatory activity
HRBC membrane stabilization. The test sample con-
sisted of stock erythrocyte (RBC) suspension 0.030 mL
mixed with 5 mL of hypotonic solution (154 mM NaCl in
10 mM sodium phosphate buffer at pH 7.4) containing
synthesized compound ranging from concentration 5,
10, 15, and 20 lg/mL (16). The control sample con-
sisted of 0.030 mL RBC suspension mixed with hypo-
tonic buffered solution alone. The standard drug Analgin
was treated similar to test at 5, 10, 15, and 20 lg/mL
concentrations. The experiment was carried out in tripli-
cate. The mixtures were incubated at 10 min at room
temperature, centrifuged for 10 min at 1077 9 g, and
absorbance of the supernatant was measured spectro-
photometrically at 540 nm. The percentage inhibition of
hemolysis or membrane stabilization was calculated by
following equation:
%Inhibition of hemolysis 100  A1  A2 =A1 ;
where A1 = absorbance of hypotonic buffered solution
alone, and A2 = absorbance of test/standard sample in
hypotonic solution.
Effect on protein denaturation. Test solution consist-
ing of 1 mL of different concentrations of synthesized
compounds ranging from 5, 10, 15, and 20 lg/mL or
standard Analgin 5, 10, 15, and 20 lg/mL was mixed with
1 mL of egg albumin solution (1 mM) and incubated at
27  1 C for 15 min (17,18). Denaturation was induced
by keeping the reaction mixture at 70 C in a water bath
for 10 min. After cooling, the turbidity was measured
spectrophotometrically at 660 nm. Percentage inhibition of
denaturation was calculated from control where no drug
was added. Each experiment was carried out in triplicate,
and the average was taken.
Antioxidant activity
Scavenging activity against 1,1-diphenyl-2-picryl
hydrazyl radical (DPPH). First, the antiradical activity
was measured as the scavenging activity of the stable
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. In its
radical form, DPPH has an absorption band at 516 nm
which disappears upon reduction by an antiradical com-
pound (19,20). The reaction mixture contained in 3 mL of
ethanol, 80 lM DPPH and test compounds at different
concentrations (10, 15, 20, 25 lg/mL). By the same pro-
cedure, standard was also prepared. After 30 min at room
temperature, the absorbance was recorded at 516 nm on
an UV spectrophotometer. All experiments were carried
out in triplicate. The percentage of remaining DPPH (DPPH
REM) was calculated as follows:
Chem Biol Drug Des 2014; 84: 409419

 
Control  Sample
% DPPH  100:
Control
For all derivatives, IC50, that is, the concentration of test
compound needed to reduce DPPH absorption by 50% at
516 nm was determined.
Assay of nitric oxide (NO)-scavenging activity. The
absorbance of the chromophore formed during diazotiza-
tion of nitrite with sulfanilamide and on subsequent cou-
pling with N-naphthylene diamine was read at 546 nm
(21,22).
The different concentrations (10,15, 20, 25 lg/mL) of
tested compound were taken in volume of 200 lL. Nitric
oxide production was initiated with the addition of 200 lL
of phosphate buffer (0.1 M, pH 7.4) and 800 lL sodium ni-
troprusside (10 ml), and the reaction mixture was incu-
bated at 25 C for 150 min. A control tube was also
proceeding in the same way except for test compound
ethanol was used. After incubation, 1.2 mL of Griess
reagent was added to the test-tubes, and they were kept
at RT for 30 min for color development. By the same pro-
cedure, standard was also prepared. The optical density
was read at 540 nm. All experiments were carried out in
triplicate.
A0  A1   100
% Inhibition of NO ;
A0
where A0 is the absorbance before reaction, and A1 is the
absorbance after reaction has taken place. The IC50 value
was derived from% Inhibition at different concentrations.
Assay of hydrogen peroxide-scavenging activity. The
H2O2-scavenging ability of the synthesized compounds
5ag was determined spectrophotometrically according to
the method of Ruch et al (23). Briefly, a solution of hydrogen
peroxide (2 mM) was prepared in 0.17 M phosphate buffer
(pH 7.4). Various concentrations of the samples (in ethanol)
were added to the reaction mixture containing 2 mM hydro-
gen peroxide. After 10 min incubation at room temperature,
the absorbance was read against a blank at 230 nm.
H2O2-scavenging activity (%) = [(A control A sample)/A
blank] 9 100, where A control is the absorbance of the
control reaction (containing all reagents except the test
compound), and A sample is the absorbance of the test
compound.
Molecular modeling
The compounds were modeled using BUILD application of
Maestro 9.3. The geometry was optimized by molecular
mechanics using IMPACT in a dynamic environment using
standard TIP4P water model. The energy minimization was
done using optimized potentials for liquid simulations 2005
(OPLS 2005) force field. Energy minimization was done
Chem Biol Drug Des 2014; 84: 409419
Synthesis and Evaluation of COX-2 Inhibitors
using Polak-Ribier conjugate gradient and Truncated New-
ton conjugate gradient algorithms. The convergence
threshold used was RMS gradient of 0.01. Conformational
models of the ligands were generated.
Docking
Docking of the ligands was carried out using extra preci-
sion (XP) method called grid-based ligand docking with
energetics (GLIDE) for flexible ligand docking. The ligands
were prepared for docking using LIGPREP from Schro-
dingers molecular modeling softwares. The co-ordinate
for COX-2 enzyme (PDB ID 1CX2) was taken from RCSB
Protein Data Bank and prepared for docking using protein
preparation wizard in Maestro v9.3.
The receptor grid generation for docking was prepared
using both the centroid of selected active site residues
and blind docking. The results of both the methods were
similar. The different conformations of the compounds
were docked flexibly, and 1000 poses per compound
were obtained. The poses, complexes, and the binding
affinities between the receptor and ligands were analyzed
using Schrodingers modeling software (24). All the molec-
ular modeling studies were performed on Schrodinger
Maestro, and their binding affinities with the receptor were
analyzed.
Result and Discussion
Chemistry
Synthesis of compounds (3)
The preparation of target 5-methyl-2-phenyl-1H-pyrazol-3
(2H)-one (3) is shown in Scheme 1. The cyclization of ethyl
acetoacetate with phenylhydrazine occurred in the first step.
Synthesis of compounds 5a5g
The addition of amine derivatives to compound 3 afforded
the corresponding 5-methyl-4-(methylamino)-2-phenyl-1H-
pyrazol-3(2H)-one derivatives (5a5g) as the major product
(Table 1). The amino alkylation of an acidic proton was
placed next to a carbonyl functional group by formalde-
hyde and amines. The reaction is known as Mannich reac-
tion. The structures of the isolated products 3, 5a5g
were established on the basis of their elemental and spec-
tral analyses.
Biologic activity
In vitro COX inhibition study
It was found that the structurally simple compounds, py-
razolone derivatives 3 and 5a5g (Figure 1), were obtained
as a potent and slightly COX-2-selective inhibitor. Accord-
413
Dube et al.
Step -1
H 2N
NH O O
a
+ H 3C OC 2 H5
1 2
Step-2
CH3
O NH
N b
+ R NH2
4 5a-5g
3 610 h.
ing to the above rationale, the compounds synthesized in
this work were evaluated for their ability to inhibit COX-1
and COX-2 using an ovine COX-1/COX-2 assay kit (Cata-
log No. 560101, Cayman Chemicals Inc.). IC50 (lM) was
determined and means of two determinations acquired,
and the deviation from the mean is <10% of the mean
value. The selectivity index (SI values) was defined as IC50
(COX-1)/IC50 (COX-2). The known COX inhibitor celecoxib
was used as positive control, and the IC50 values of celec-
oxib on COX-1 and COX-2 were determined to be >100
and 0.40 lM, respectively, indicating that celecoxib is a
selective COX-2 inhibitor [COX-2 (SI) > 250.0]. The results
showed that most of the compounds showed potent inhi-
bition against COX-2 (IC50  0.12.5 lM) compared to the
inhibition for COX-1 (IC50 > 100 lM) as listed in Table 2.
Nearly 8 of the compounds (5a, 5d, and 5f) were found to
be potent and selectively similar to celecoxib against COX-
2. Compound 5d was the most potent inhibitor in this ser-
ies with the activity (IC50 = 0.2 lM) twofold higher than cel-
ecoxib (IC50 = 0.4 lM). The effects of substituents
introduced into the pyrazolone moiety of compound 3
were revealed to be directional, being dependent on the
electronic nature of the substituents, that was introduction
of a phenylthiourea group at the 3-position (5d) enhanced
both COX-1- and COX-2-inhibiting activity with higher
potency for the latter, resulting in a COX-2-selective inhibi-
tor (SI = 400). Moreover, introduction of halogen such as
chloro (5b) resulted in lowering the COX-2-inhibiting activ-
ity, while introduction of an electron-donating sulfonyl
group (5f) showed just the opposite effects, resulting in a
COX-2-selective inhibitor (SI= >100). The activities of syn-
thesized analogs (5a5g) are shown in Table 2. Among
them, compounds 5a and 5f are a COX-2-selective inhibi-
tor (SI = >40.0). No clear-cut structureactivity relation-
ships could be deduced at this stage. Interestingly, benzyl
substituents on the N-pyrazolone ring play critical roles in
414
CH3
NH
O N
3
HN CH 3
R
O NH
N
Scheme 1: synthesis of designed
compounds. Reaction conditions:
(A) reflux for 3 h, (B) reflux for
the COX-inhibiting activity of the tested compounds. The
phenylurea analog (5d and 5f) showed the most potent
activity, being 1.25 and 2 times more active than celecoxib
in COX-1-and COX-2-inhibiting activity, respectively.
In vivo anti-inflammatory studies
All the synthesized compounds were tested for anti-inflam-
matory activities. The results of anti-inflammatory activities
against carrageenan-induced rat paw edema and ED50
(mg/kg) of all tested compounds are shown in Table 3. Of
the eight compounds, three compounds (5a, 5d, and 5f)
were found to possess potent anti-inflammatory activity
(8292% reduction in inflammation) and some of which
have shown higher inhibition in comparison with reference
drug diclofenac. In general, pyrazolones which were
substituted with chloro moiety at 3-phenyl ring (5b) and
unsubstituted urea (5c, 5g, and 5e) have shown lower
percentage of inhibition of edema when compared with
compounds containing urea-phenyl group at the same
position (5d and 5f). These findings may be attributed to
the importance of sulfonyl group and thiourea derivative in
COX receptor binding site.
In vitro Anti-inflammatory activity
It was found that the structurally simple compound, py-
razolone derivatives, was obtained as a potent and slightly
COX-2-selective inhibitor. According to the above ratio-
nale, the compounds synthesized in this work are evalu-
ated for their ability to inhibit COX-2 using hypotonic
solution hemolysis and protein denaturation methods
(Table 4).
Initially, the compounds were tested at 30 lM and their
effects on COX-2 inhibition. The study was further extended
Chem Biol Drug Des 2014; 84: 409419
 Synthesis and Evaluation of COX-2 Inhibitors

Table 1: Physical characterization of synthesized compounds

CH3 HN CH3
R
O NH NH
N O N

3 5a-5g

Compound code R Molecular formula Molecular weight Rf valuea

3 C10H10N2O 174.19 0.50

5a O C12H14N4O2 246.10 0.68
H 2N

5b Cl C18H17ClN4O2 356.80 0.31

O

N
H

5c S C12H14N4OS 262.33 0.55

H2N

5d S C18H18N4OS 338.43 0.67

N
H

5e NH C12H15N5O 245.28 0.42

H2 N

5f O C17H18N4O3S 358.41 0.63

S NH2
O

5g O C12H15N5O2 261.28 0.72

H 2N
N
H

a
eluent used for TLC plates Benzene/Methanol (4:1).

Table 2: In vitro COX-1/COX-2 enzyme inhibition assay of the Table 3: In vivo anti-inflammatory activity against carrageenan-
designed compounds induced rat paw edema and ED50 (mg/kg) of the designed
compounds
IC50 (lM)a
% Inhibition of edemaa
Compound no COX-1 COX-2 SIb
Compound no After 1 h After 2 h ED50b(mg/kg)
3 >100 35.00 >2.86
5a >100 2.30 >43.48 3 69 77 137
5b >100 >100 5a 76 82 118
5c >100 85.00 >1.17 5b 51 75 185
5d 80.00 0.20 400.0 5c 33 59 193
5e >100 50.70 >1.97 5d 83 91 102
5f >100 1.00 >100.0 5e 48 69 162
5g >100 >100 5f 88 92 109
Celecoxib >100 0.40 >250.0 5g 27 38
Diclofenac 77 83 121
a
IC50 value is the compound concentration required to produce
50% inhibition of COX-1 or COX-2 for means of two determina- a
The carrageenan-induced rat paw edema assay was carried out
tions using an ovine COX-1/COX-2 assay kit (catalog no. 560101, using six animals (male Sprague Dawley rats)/group following IP of
Cayman Chemicals Inc.), and deviation from the mean is <10% of the test compound. The results are expressed as means  SEM
the mean value. (n = 46) following a 100 mg/kg IP of the test compounds.
b
Selectivity index = (COX-1 IC50/COX-2 IC50). b
ED50 was the effective dose calculated after 2 h.

Chem Biol Drug Des 2014; 84: 409419 415

Dube et al.

Table 4: In vitro anti-inflammatory activity evaluated by hypotonic highly relevant to the design of selective COX-2 inhibitors.
solution hemolysis and protein denaturation methods This COX-2 2-pocket arises due to a conformational
change at Tyr355, that is, attributed to the presence of
Compound % Protein
code Hemolysis IC50 denaturation IC50
Ile523 in COX-1 relative to Val523 having a smaller side
chain in COX-2. The level of COX-2 inhibition and anti-
3 61.43 6.76 78.00 1.75 inflammatory activities of synthesized compounds prompted
5a 60.02 36.36 77.04 4.74 us to perform molecular docking studies to understand the
5b 55.64 8.31 72.53 18.49 ligandprotein interactions in detail. The pdb entry (1CX2)
5c 56.59 15.98 32.78 71.95
was selected as a dataset to compare the binding of syn-
5d 66.27 0.61 45.62 40.17
thesized molecules with that of pyrazole derivative celecoxib
5e 63.95 22.50 36.34 53.68
5f 61.43 3.15 46.44 38.51 which is a well-established COX-2 antagonist. Docking of
5g 59.78 12.39 82.92 15.41 compounds was done with COX-2, and the results not only
Celecoxib 61.57 8.5 85.65 17.90 validated our docking protocol but also established the fol-
lowing findings.
IC50 values are reported as an average of three determinations.
Access of ligands to the pocket of COX-2 is controlled by
Leu352, Gln192, and Arg513. Interaction of Leu352 with the
to examine the concentrationactivity responses at different bound drug is a requirement for time-dependent inhibition of
concentrations to determine the IC50 values for COX-2. COX-2. With few exceptions, all the ligands showed similar
Four compounds (3, 5a, 5d, and 5f) showed better COX-2 orientation in the COX-2 active site, and the complex formed
inhibitory activity than celecoxib with IC50 values in the was stabilized by formation of classical and non-classical
range of 0.618.5 lM. Compounds 3 and 5d (3-phenylthio- hydrogen bonds (Figure 2). The compound 5d was found to
urea pyrazolone derivative) exhibited the highest COX-2 dock into the active site of COX-2 with interaction energy of
inhibition (IC50 0.61 lM) in protein denaturation model fol- 12.94 kcal/mol. It formed hydrogen bonds with Ser530
lowed by 5d (IC50 0.61 lM) and 5f (IC50 3.15 lM) in percent-
age hemolysis model (Table 4). The COX-2 inhibitory effect
Table 5: In vitro antioxidant activity of pyrazolone derivatives
of 5d was >10-fold as compared to celecoxib (IC50 8.5 lM).
Introduction of either urea or guanidine results in decrease % Inhibition  SDa
in COX-2 inhibitory activity. The substitution of pyrazolone Sr. Compound
with distal aryl ring results in reasonable COX-2 inhibition no. code DPPH NO H2O2
profile. The increased activity was also observed in thiourea
1 3 62.01  0.11 40.41  0.70 64.13  1.56
derivative than urea and guanidine. 2 5a 27.77  0.56 13.46  0.58 18.56  0.56
3 5b 29.14  0.56 11.54  1.56 22.13  0.06
4 5c 39.43  0.56 18.55  2.56 23.67  1.58
In vitro Antioxidant activity 5 5d 60.67  2.54 30.28  0.51 62.43  0.66
It has been reported that in inflammatory disorders, exces- 6 5e 53.76  0.54 29.97  0.22 59.67  2.54
sive free radical generation takes place and several NSA- 7 5f 55.24  2.56 23.97  0.22 25.77  0.76
8 5g 51.67  1.56 14.23  0.66 32.54  0.06
IDs and pyrazolone compounds with anti-inflammatory
9 Standard 22.13  0.06 18.56  0.56 15.65  0.33
activity are reported to act as radical scavengers (25).
Therefore, antioxidant property of the synthesized pyrazol- a
Average of three determinations.
ones was evaluated using DPPH, nitric oxide, and hydro-
gen peroxide radical-scavenging in vitro assays (26,27).
The compounds were tested at different concentrations,
Table 6: IC50 value of synthesized compounds and standard
and it was observed that all compounds have shown ascorbic acid
antioxidant property (Tables 5 and 6). The free OH group
on substituted distal aryl ring is responsible for antioxidant IC 50  SDa
activity as it is proved by testing compounds 5a, 5b, and Sr. Compound
5c that showed good activity. no. code DPPH NO H2O2

1 3 14.75  0.51 22.11  1.20 20.45  2.14

2 5a 14.01  0.14 15.50  1.34 15.64  2.41
Molecular docking analysis 3 5b 17.00  0.41 20.15  0.11 22.50  0.15
The docking study may offer more insight into the under- 4 5c 26.72  0.32 25.13  1.10 23.70  1.36
standing of the proteinligand interactions and the structural 5 5d 25.75  0.01 36.36  0.32 38.00  0.45
features of the active site. The compounds were modeled 6 5e 36.34  1.54 37.00  0.18 25.70  0.10
7 5f 21.95  1.07 40.00  0.23 32.34  0.72
using BUILD application of Schrodinger Maestro 9.3, and
8 5g 21.91  1.09 38.00  0.02 25.77  1.37
the binding affinities of the synthesized compounds with the 9 Standard 6.50  0.06 8.90  1.44 7.25  2.87
receptor were analyzed. The COX-2 binding site possesses
a
an additional 2-pocket, that is, absent in COX-1, which is Average of three determinations.

416 Chem Biol Drug Des 2014; 84: 409419


A
C C
Figure 2: 2D ligand interaction diagrams of (A) celecoxib, (B)
compound 5d, and (C) compound 5f.
(Figure 2b) into the active site of COX-2 and bonding dis-
tance between NH of 5d and OH of Ser530 2.58
A (HO)
and pp stacking interaction between urea-substituted phe-
nyl ring with Trp387 and Tyr385 was observed. Similarly,
compound 5f forms two hydrogen bonds with Gln192 and
Ser353 as similar to that of celecoxib.
The urea-substituted phenyl ring of 5d was surrounded by
the active site amino acid residues Tyr385, Gly526, Phe381,
Leu384, Met522, and Trp387 (Figure 2B). The compound
Chem Biol Drug Des 2014; 84: 409419
Synthesis and Evaluation of COX-2 Inhibitors
A
Figure 3: Overlap of binding pose of celecoxib (purple color) with
(A) compound 3 (red), (B) compound 5a (green), and (C)
compound 5f (orange). Hydrogen bonds are shown by yellow
dotted line.
5d could dock successfully into the COX-2 active site as well.
Our docking study showed that compounds 5b, 5d, and 5f
were oriented so that their 4-substituted pyrazolone fragment
and phenyl moiety fill this adjunct pocket.
The complex generated by docking studies of 3, 5a, and 5f
with COX-2 and superimposition with the structure of the
417
Dube et al.
selective inhibitor, celecoxib, co-crystallized with COX-2,
illustrated in Figure 3, shows that compound 5f can bind in
the active site of this enzyme in approximately similar fashion
as the pyrazolic prototype (celecoxib). Comparison of the
interactions performed by SC-558 in the crystal and the
docked structure of 5f with COX-2 (Figure 3C) shows that
the sulfonamide groups of 5f hydrogen bonded to the amino
acid residue Ser353, similarly to the sulfonyl moiety of the
pharmacophoric sulfonamide group pertaining to SC-558
and celecoxib.
Conclusion
In conclusion, eight pyrazolone derivatives were synthesized
and showed more pronounced COX-2 inhibition than natu-
rally occurring chromone, stellatin. The SAR study revealed
that a pyrazolone skeleton with C3 methyl group, C=O at 5-
position and urea substitution on 4-position is an essential
feature for COX inhibition. Four compounds (3, 5a, 5d, and
5f) exhibited greater anti-inflammatory activity than stan-
dard, and two compounds (5a and 5b) showed the potent
radical-scavenging activity as an antioxidant agent. Molecu-
lar docking study further helped in supporting the observed
COX-2 selectivity. The findings of the study inferred that the
dual functioning of 5a as a COX inhibitor and antioxidant
agent renders it as a lead molecule for further development
of new anti-inflammatory agents.
Acknowledgments
The authors are thankful to Head of the Department, Phar-
maceutical Chemistry; A.S. Dhake, Principal, S.M.B.T Col-
lege of Pharmacy, Nashik, for providing necessary facilities
to carry out this research work. This work was supported
by the Department of Science and Technology of India
[Project File No. SR/FT/LS-132/2012 and Sanction Diary
No. SERB/F/4302/2012-13].
Conflict of Interest
No.
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