Synthesis and biological evaluation of N-substituted-3,5-diphenyl-2- pyrazoline derivatives as cyclooxygenase (COX-2) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.10.005

Eighteen new 1-N-substituted-3,5-diphenyl-2-pyrazoline derivatives have been synthesized and cyclooxygenase (COX-1 and COX-2) inhibitory activities have been evaluated. The results of these biological assays showed that all of new derivatives are not endowed with improved anti-inflammatory activity against COX-1, but some of them showed a good activity against COX-2. To evaluate the binding mode of the most significative compounds (2d, 2f, 2g and 2k) docking studies were carried out. These studies confirmed biological data, in fact these compounds were able to fit into the active site of COX-2.

Full text of DOI:10.1016/j.ejmech.2010.10.005

European Journal of Medicinal Chemistry 45 (2010) 6135e6138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of N-substituted-3,5-diphenyl-2-pyrazoline
derivatives as cyclooxygenase (COX-2) inhibitors
,
a
a
a
b
Rossella Fioravanti a , Adriana Bolasco , Fedele Manna , Francesca Rossi , Francisco Orallo ,
*
Francesco Ortuso c, Stefano Alcaro c, Roberto Cirilli d
a Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy
b Departamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur, E-15782 Santiago de
Compostela (La Coruña), Spain
c Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro “Magna Graecia”, “Complesso Ninì Barbieri”, 88021 Roccelletta di Borgia (CZ), Italy
d Istituto Superiore di Sanità, Dipartimento del Farmaco, Viale Regina Elena 299, I-00161 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen new 1-N-substituted-3,5-diphenyl-2-pyrazoline derivatives have been synthesized and cyclo-
oxygenase (COX-1 and COX-2) inhibitory activities have been evaluated. The results of these biological
assays showed that all of new derivatives are not endowed with improved anti-inflammatory activity
against COX-1, but some of them showed a good activity against COX-2. To evaluate the binding mode of
the most significative compounds (2d, 2f, 2g and 2k) docking studies were carried out. These studies
confirmed biological data, in fact these compounds were able to fit into the active site of COX-2.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 28 July 2010
Received in revised form
24 September 2010
Accepted 4 October 2010
Available online 25 October 2010
Keywords:
Pyrazoline
COX inhibitors
Molecular modeling
1. Introduction
inhibition of COX-1 causes unfavorable GI side effects [8]. Therefore,
development of novel compounds having anti-inflammatory
Cyclooxygenase (COX) or prostaglandin endoperoxide synthase
(PGHS) catalyzes the first step in the biosynthesis of the prosta-
glandins (PGs) from the substrate arachidonic acid (AA) [1]. COX
enzyme possesses two distinct catalytic activities: (1) cyclo-
oxygenase activity that catalyzes the oxidation of AA to produce
hydroperoxy endoperoxide (PGG2) and (2) peroxidase activity that
reduces the hydroperoxide PGG2 to the hydroxy endoperoxide
(PGH2). The PGH2 is transformed by a range of enzymic and
nonenzymic mechanisms into the primary prostanoids. In addition,
arachidonic acid is a substrate for a variety of additional oxidative
enzymes such as lipoxygenase, which generates biologically active
lipids: hydroperoxyeicosatetraenoic acid (HPETE), hydroxyeico-
satetraenoic acid (HETE), and leukotrienes (LTA4, LTB4, LTC4, and
LTE4). The cyclooxygenase (COX) enzymes, were identified as the
molecular targets of all nonsteroidal anti-inflammatory drugs
(NSAIDs) [2e4]. COX-1 is the constitutive isoform and is mainly
responsible for the synthesis of cytoprotective PGs in gastrointes-
tinal (GI) tract whereas COX-2 is inducible and plays a major role in
PG biosynthesis in inflammatory cells [5e7]. It is believed that the
activity with an improved safety profile is still a necessity. Litera-
ture survey revealed that many pyrazole derivatives [9] have found
their clinical application as NSAIDs. Among the highly marketed
COX-2 inhibitors that comprise the pyrazole nucleus, celecoxib is
the one which is treated as a safe anti-inflammatory and analgesic
agent. It is considered as a typical model of the diaryl heterocyclic
template that is known to selectively inhibit the COX-2 enzyme.
Some other examples of pyrazole derivatives as NSAIDs are mefo-
butazone, ramifenazone, famprofazone [10e13]. (Fig. 1).
But due to serious adverse effects (such as bone marrow
depression, water and salt retention and carcinogenesis), the use of
pyrazole derivatives is limited. This limitation has led to the inves-
tigation of new pyrazole derivatives with that more potent activity
and less toxicity.
Motivated by the aforesaid findings, and pursuing our studies on
pyrazole moiety [14e17], we were designed to synthesize a new
series of 1-N-substituted-3,5-diphenyl-2-pyrazoline derivatives
and tested them as COXs inhibitors.
Compared with Celecoxib, new pyrazoline derivatives were
synthesized by replacing pyrazole moiety, with a dihydropyrazole
nucleus, linked in C3 and C5 with phenyl group. In particular we
have introduced 4-methansulfonyl phenyl, in C3 position, which
* Corresponding author. Tel.: þ39 6 49693259; fax: þ39 6 49913975.
E-mail address: rossella.fioravanti@uniroma1.it (R. Fioravanti).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.005

6136
R. Fioravanti et al. / European Journal of Medicinal Chemistry 45 (2010) 6135e6138
CHO
R
H3CO2S
H3CO2S
+
(i)
CH3
F3C
H
N
R
H3C
H3C
N
O
O
1a-i
N
N
N
H3C
O
N
CH3
N
H3C
O
N
(ii) or (iii)
SO2NH2
H3CO2S
celecoxib
ramifenazone
famprofazone
R
N
N
F3C
Br
S
O
O
X
2a-r
N
N
Br
F
Scheme 1. Reagent and conditions: (i) Ba(OH)2∙8H2O, EtOH 96%, 25e30 ꢀC, 8 h; (ii)
NH2NH2, CH3COOH, EtOH dry, 78 ꢀC, 24 h; (iii) NH2NHCSNH2, NaOH, EtOH dry, 78 ꢀC,
24 h (for assignment of aer see Table 1).
SO2NH2
SC-558
SO2CH3
DuP-697
SO2CH3
rofecoxib
as COX-1 inhibitors. N-acetyl derivatives (2aei), were found to be
more potent, than corresponding N-thiocarbamoyl derivatives
(2jer).
Fig. 1. Chemical structures of some known coxib inhibitors.
Derivatives 2d, 2f and 2g are the most potent compounds of
N-acetyl series, whereas, among N-thiocarbamoyl derivatives the
most active compound is 2k. For the most active compounds we
have carried out HPLC enantioseparations (Supplementary Data) to
investigate which enantiomer was the best inhibitor. The biological
results showed a slight difference of activity between the two
isomers with respect to each other and with respect to the racemic
mixture. In order to deeply investigate the recognition of our most
potent and COX-2 selective pyrazoline derivatives, docking simu-
lations were carried out onto both enantiomers of compounds 2d,
2f, 2g and 2k. The Protein Data Bank (PDB) [18] was searched for
COX-1 and COX-2 experimental models. Human enzyme isoforms
were not found and, consequently, the crystallographic structures
1Q4G, [19] from Ovis aries, and 6COX, [20] from Mus musculus, were
selected for mimicking the cyclooxygenase 1 and 2 respectively. The
seems important in interaction with the secondary pocket of COX-2
active site. (Fig. 2).
2. Chemistry
The synthesis of the target compounds is shown in Scheme 1.
The intermediate chalcones 1aei, were obtained by direct Clai-
seneSchmidt condensation between different aromatic aldehyde
and 4-methyl sulfonyl acetophenone in a basic medium in ethanol
96%. A solution of appropriate 40-methyl sulfonyl chalcone 1aei in
acetic acid and hydrazine hydrate or thiosemicarbazide in 50 mL of
ethanol and sodium hydroxide was refluxed for 8 h, to obtain the
new derivatives 2aer (see Supplementary Data).
3. Biochemistry
Table 1
The potential effects of the test drugs on total hCOX activity
(bisdioxygenase and peroxidase reactions) were investigated by
measuring their effects on the oxidation of N,N,N0,N0-tetramethyl-p-
phenylenediamine (TMPD) to N,N,N0,N0-tetramethyl-p-phenylen-
ediimine, using AA as common substrate for both hCOX-1 and
hCOX-2, microsomal COX-2 prepared from insect cells (Sf 21 cells)
infected with recombinant baculovirus containing cDNA inserts for
hCOX-2 (SigmaeAldrich Química S.A., Alcobendas, Spain) and COX-
1 from human platelet microsomes (obtained as described in the
above paragraph since, unlike hCOX-2, hCOX-1 is not available
commercially).
IC50 values for the inhibitory effects of test drugs (new compounds and reference
inhibitors) on the enzymatic activity of recombinant human COX-2 expressed in Sf
21 cells.
Comp
R
X
COX-2 IC50
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
H
4Cl
4F
4CH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
**
21.70 ꢁ 1.86
m
m
m
m
m
m
M
M
M
M
M
M
19.63 ꢁ 1.62
6.77 ꢁ 0.48
4CF3
19.83 ꢁ 1.76
4OCH3
2OCH2Ph
3OCH2Ph
4OCH2Ph
H
4Cl
4F
4CH3
4CF3
4 OCH3
2OCH2Ph
3OCH2Ph
4OCH2Ph
7.61 ꢁ 0.58
3.20 ꢁ 0.24
**
**
50.31 ꢁ 4.06
m
m
M
M
4. Results and discussion
9.35 ꢁ 0.76
**
The in vitro activity of derivatives 2aer, is reported in Table 1; we
depicted only COX-2 activity, because all derivatives were inactive
31.85 ꢁ 2.57
m
M
*
*
*
*
*
R = H;
2q
2r
H3CO2S
4-Cl
4-F;
Indometacin
Diclofenac
Nimesulide
DuP-697
35.20 ꢁ 1.41
23.62 ꢁ 1.97
m
m
M
M
mM
4-CH3;
4-OCH3;
2-OCH2C6H4;
3-OCH2C6H4;
4-OCH2C6H4.
231.40 ꢁ 19.84
N
N
X
126.32 ꢁ 7.41 nM
R
All IC50 values shown in this table are the mean ꢁ S.E.M. from five experiments. IC50
X = COCH3; CSNH2
values obtained against COX-2, as determined by ANOVA/Dunnett’s. * Inactive at
25
mM (highest concentration tested). ** Inactive at 100 mM (highest concentration
Fig. 2. 1-N-substituted-3,5-diphenyl-2-pyrazoline derivatives.
tested).

R. Fioravanti et al. / European Journal of Medicinal Chemistry 45 (2010) 6135e6138
6137
first PDB model reported the complex between the COX-1 and the a-
methyl-4-biphenylacetic acid and was characterized by the highest
resolution (2.0 Å) available for cyclooxygenase structures into the
PDB at the search time. The 6COX was selected taking into account
its co-crystallized ligand, the SC-588, which represents a COX-2
selective inhibitor with some structural similarities with respect to
our compounds. After a preliminary pre-treatment (Supplementary
Data) the above indicated PDB models were considered the targets
of our docking simulations carried out using the AutoDockeVina
software [21]. Theoretical results were submitted to both visual and
energy analysis. Interestingly within the COX-1 all our compounds
were not able to fit into the known active site preferring the
recognition of other areas, i.e. that related to the iron protopor-
phyrin IX (HEME) cofactor (Supplementary Data). Such an obser-
vation was not a surprise, in fact, the original PDB model 1Q4G
reported a glycerol molecules interacting to the cofactor iron ion,
and, recently [22], and we have described the HEME recognition of
anti-inflammatory oleuropein semi-synthetic derivatives. The
cofactor could be considered as an accessory binding site modu-
lating the COX activity but we are not confident about its relevance
into the anti-inflammatory properties of our compounds. We can
suggest that molecules with a large chemical structure could be not
able to fit into the know COX-1 binding site and, consequently, their
target recognition, in computational simulation, moves toward
more accessible sites. Actually, in order to verify the steric hindrance
role into the COX-1 recognition, we have performed, first of all, the
docking of the co-crystallized ligand and, then, of known non-
selective COX inhibitors, such as flurbiprofen, nimesulide and
diclofenac. In all these cases the resulting configurations have
highlighted the recognition of both classic binding sites and HEME
Fig. 3. Best pose of 2g into the COX-2 binding site. The ligand is depicted in polytube
model, interacting residues are labelled and displayed in wireframe, HEME cofactor is
in green carbon coloured spacefill. Yellow dotted line and cyan arrow indicate inter-
molecular hydrogen bond and hydrogen-pi interaction respectively. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article).
both the default AutoDockeVina scoring function and the MolInE
method [23,24] highlighting a good qualitative agreement with
respect to the experimental inhibition IC50 (Table 2).
The graphical analysis of the most stable COX-2 complexes
revealed similar configuration for all docked pyrazoline derivatives.
In all cases our compounds showed their 2-aryl-R substituent
directed to the inner cyclooxygenase side while the 4-methyl-
sulfonylphenyl moiety was located into the same pocket occupied
by the SC-588 sulfonamide group into the 6COX original PDB model
(Fig. 3 and Supplementary Data).
Such an additional binding cleft is known to be exclusive for the
COX-2 and its recognition is on the basis of the coxibs selectivity.
The number of recognized residues was quite similar among our
derivatives ranging from 19 amminoacids to 24. Excluding (S)-2d
and both enantiomers of 2k, our pyrazoline derivatives demon-
strated hydrogen-pi interaction to Ser530. A deeper analysis high-
lighted that only the (R) - and the (S) - enantiomers, of the most
potent inhibitor 2g, were able to produce hydrogen bonds to
Arg513 and Phe518 respectively. Moreover the phenoxy-group of
2g was well accommodated into an aromatic cage delimited by
Phe518, Trp387, Tyr385 and Phe384. We addressed to those
evidences the rational of the higher COX-2 inhibition of 2g with
respect to the other analogues.
In particular derivatives 2d, 2f, 2g and 2k, were the most
interesting compounds, with IC50 values in range micromolar.
Because these compounds have a carbon chiral we separated them
in two enantiomers and then re-tested; then we completed the
experimental part, using molecular modeling studies. No signifi-
cant differences between both enantiomers and racemic inhibition
activities were confirmed by the molecular modeling studies too.
moiety, in particular the complex with the
a-methyl-4-biphenyl-
acetic acid have reproduced the PDB model 1Q4G geometries with
the most stable ligand orientation and, at higher interaction ener-
gies directly in contacts with the cofactor. With the aim to stronger
validate our computational procedure, we have docked to COX-1
two known COX-2 selective inhibitors such as the SC-588 and the
DuP-697. The results of these last compounds were quite similar to
that indicated for our derivatives, actually both have interacted to
the HEME and were not able to fit into the classic binding site. On the
base of the above reported evidences, and taking into account the
experimental COX-1 IC50 values of our pyrazoline derivatives, we
focused our computational study onto their COX-2 interaction only.
The COX-2 docking experiments were validated including in our
simulations the selective inhibitors SC-588 and DuP-697. Such
molecules allowed us to verify both the theoretical complexes
geometries and the adopted scoring functions. Into the COX-2 the
best scored configurations of all compounds have fitted into the
known classic binding site, i.e. the cleft occupied by SC-588 into
the PDB original model (Supplementary Data). In order to evaluate
the binding modes of all docked compounds we have considered
Table 2
Comparison among experimental IC50 (mM) and theoretical binding energies (kcal/
mol). Best and average values were computed by AutoDockeVina software, DDG was
calculated applying the MolInE method to the AutoDockeVina interaction energies.
Compounds 2d, 2f, 2g and 2k results were obtained averaging the values of the
corresponding enantiomers.
5. Conclusion
Compound
Binding energies (kcal/mol)
IC50 (mM)
Best
Average
DDG
Molecularmodeling studies confirm biologicaldata, in factall our
compounds were not able to fit into the known active of COX-1,
while, into the COX-2 the best scored configurations of all
compounds have fitted into the known classic binding site. Our
studies have underlined the importance of 4-methylsulfonylphenyl
in C3 position on pyrazoline, but, very interesting results obtained
DuP-697
2d
2f
2g
2k
ꢂ7.70
ꢂ6.65
ꢂ2.20
ꢂ1.13
ꢂ2.37
ꢂ0.03
ꢂ7.07
ꢂ2.22
ꢂ1.38
ꢂ2.56
ꢂ0.15
0.13
6.77
7.61
3.20
9.35
ꢂ2.35S
ꢂ1.65S
ꢂ3.55S
ꢂ0.45R
R, S
energy value related to (R) - and (S)- enantiomers respectively.

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R. Fioravanti et al. / European Journal of Medicinal Chemistry 45 (2010) 6135e6138
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coxib series we have inserted a methylene group among two aryl
groups, to realize if the activity maintained. This shift of phenyl
group produced derivatives several selective COX-2 inhibitors, but
less active than references. Also we have investigated the impor-
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Acknowledgments
This work was supported by Grants from MURST (Italy), in
particular by the young research project PRIN 2007JERJPC. Minis-
terio de Sanidad Consumo (Spain; FISS PIO61537) and Conselleria
de Innovaciòn e Industria de la Xunta de Galicia (Spain; INCI-
TE07PXI203039ES, INCITE08E1R203054ES and 08CSA019203PR).
Appendix. Supplementary data
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P. Turini, F. Ortuso, S. Alcaro, M.C. Cardia, Synthesis, molecular modeling
studies and selective inhibitory activity against MAO of N1-propanoyl-3,5-
diphenyl-4,5-dihydro-(1H)-pyrazole derivatives, Eur. J. Med. Chem. 43 (10)
(2008) 2262e2267.
Supplementary data associated with this article can be found in
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