Novel pyrazoles and pyrazolo[1,2-a]pyridazines as selective COX-2 inhibitors; Ultrasound-assisted synthesis, biological evaluation, and DFT calculations

Article abstract of DOI:10.1016/j.bmcl.2017.04.020

COX-2 is an inducible enzyme mediating inflammatory responses. Selective targeting of COX-2 is useful for developing anti-inflammatory agents devoid of ulcerogenic activity. Herein, we report the design and synthesis of a series of pyrazoles and pyrazolo[1,2-a]pyridazines with selective COX-2 inhibitory activity and in vivo anti-inflammatory effect. Both series were accessed through acid-catalyzed ultrasound-assisted reactions. The most active compounds in this study are two novel molecules, 11 and 16, showing promising selectivity and decent IC₅₀ of 16.2 and 20.1?nM, respectively. These compounds were also docked into the crystal structure of COX-2 enzyme (PDB ID: 3LN1) to understand their mode of binding. Finally, Mulliken charges and electrostatic surface potential were calculated for both compound 11 and celecoxib using DFT method to get insights into the molecular determinants of activity of this compound. These results could lead to the development of novel COX-2 inhibitors with improved selectivity.

Full text of DOI:10.1016/j.bmcl.2017.04.020

Accepted Manuscript
Novel Pyrazoles and Pyrazolo[1,2-a]pyridazines as Selective COX-2 Inhibitors;
Ultrasound-assisted Synthesis, Biological Evaluation, and DFT Calculations
Nagat Ghareb, Hosam A. Elshihawy, Mohamed M. Abdel-Daim, Mohamed A.
Helal
PII:
S0960-894X(17)30383-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.020
BMCL 24873
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 January 2017
4 April 2017
6 April 2017
Please cite this article as: Ghareb, N., Elshihawy, H.A., Abdel-Daim, M.M., Helal, M.A., Novel Pyrazoles and
Pyrazolo[1,2-a]pyridazines as Selective COX-2 Inhibitors; Ultrasound-assisted Synthesis, Biological Evaluation,
and DFT Calculations, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.04.020
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Novel Pyrazoles and Pyrazolo[1,2-a]pyridazines as Selective COX-2 Inhibitors;
Ultrasound-assisted Synthesis, Biological Evaluation, and DFT Calculations
Nagat Ghareba, Hosam A. Elshihawya, Mohamed M. Abdel-Daimb, and Mohamed A. Helalc*
aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.
bPharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt.
cMedicinal Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.
* Corresponding author:
Mohamed A. Helal
Email: mohamed_hilal@pharm.suez.edu.eg
Tel: 002-01201122213
Fax: 002-064-3230741
Keywords: Pyrazole; pyrazolo[1,2-a]pyridazine; COX-2; anti-inflammatory; Ultrasound-
assisted reactions; DFT calculations
Abstract
COX-2 is an inducible enzyme mediating inflammatory responses. Selective targeting of COX-2
is useful for developing anti-inflammatory agents devoid of ulcerogenic activity. Herein, we
report the design and synthesis of a series of Pyrazoles and pyrazolo[1,2-a]pyridazines with
selective COX-2 inhibitory activity and in-vivo anti-inflammatory effect. Both series were
accessed through acid-catalyzed ultrasound-assisted reactions. The most active compounds in
this study are two novel molecules, 11 and 16, showing promising selectivity and decent IC50 of
16.2 and 20.1 nM, respectively. These compounds were also docked into the crystal structure of
COX-2 enzyme (PDB ID: 3LN1) to understand their mode of binding. Finally, Mulliken charges
and electrostatic surface potential were calculated for both compound 11 and celecoxib using
DFT method to get insights into the molecular determinants of activity of this compound. These
results could lead to the development of novel COX-2 inhibitors with improved selectivity.
______________________________________________________________________________
Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most widely used
classes of medicinal agents for the treatment of pain, inflammation, and fever associated with
some diseases. NSAIDs are generally indicated for the symptomatic relief of a variety of
conditions including rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, acute gout,
dysmenorrhoea, headache, and migraine.1 They produce their pharmacological effects by the
common ability to inhibit cyclooxygenase (COX), a key enzyme that catalyzes the conversion of
arachidonic acid to prostaglandin H2 (PGH2), the immediate precursor to prostaglandins,
thromboxane A2 and prostacyclin.2 Sir John Vane proved in 1971 that aspirin and other NSAIDs
1

exert their action by preventing the synthesis of prostaglandins.3, 4 In 1997, Needleman and
Isakson proposed two isoenzymes of cyclooxygenase; “House Keeping” enzyme, COX-1, which
is responsible for a basic level of PGs for the maintenance of physiological homeostasis, such as
gastrointestinal integrity, and an “Inducible” enzyme, COX-2, which is activated by different
stimuli mediating inflammatory reactions. Therefore, the design of novel NSAIDs with selective
targeting of COX-2 over COX-1 is useful for the treatment of inflammatory disorders with
reduced gastrointestinal toxicities when compared with the traditional non-selective NSAIDs.5, 6
Generally, reported COX-2 selective inhibitors can be classified into three major classes;
8
diarylheterocycles including celecoxib and roficoxib,7,
sulfonanilide derivatives such as
11-13
nimesulide,9, 10 and compounds obtained via modification of traditional NSAIDs (Fig. 1).
Over the past few years, several attempts have been made to exploit traditional NSAIDs as
starting points for the design of novel COX-2 selective inhibitors, including aspirin, meloxicam,
meclofenamic acid, ketoprofen, and indomethacin.8, 11, 13-15
Salicylamide is a non-prescription drug with analgesic and antipyretic properties comparable
to those of aspirin. It can be considered as the simplest anti-inflammatory agent and its
derivatives were reported for their ability to ease aches, pains and reduce fever since ancient
times.16 In this study, we designed novel COX-2 inhibitors, as hybrid molecules, by merging
salicylamide with the pyrazole ring of celecoxib to obtain a molecular architecture similar to that
of the diarylheterocycles COX-2 inhibitors. We report herein the design and synthesis of two
series of compounds, pyrazole and pyrazolo[1,2-a]pyridazine derivatives, and the biological
evaluation of their analgesic and anti-inflammatory activities using enzyme assay as well as
common rodent pain and inflammatory models (Fig. 1). The most active compounds were also
docked into the crystal structure of COX-2 to gain insights into the binding mode of this series of
inhibitors. We used ultrasound-assisted reactions for the synthesis of both pyrazole and
pyrazolo[1,2-a]pyridazine derivatives. This utilizes acoustic activation that abolishes the
attractive forces of molecules in the solution phase.17 This technique allowed us to get access to a
diverse library of pyrazoles in a one-step reaction under mild conditions.
2

Figure 1: Selected reported COX-2 inhibitors and, below, the hybrid-molecules designed in the current study.
Substructures in green represent the structural elements derived from salicylamide.
Following the FDA approval of celecoxin in 1998, several pyrazole-bsed selective COX-2
inhibitors have been reported (compounds A-D, Fig. 2). The majority of these compounds were,
similar to celecoxib, diarylpyrazoles, with the central pyrazole ring carrying a trifluoromethyl
moiety or an isostere of it such as methyl or methoxy groups.18-22 Representative examples of
these ligands are given in Figure 2. Most of them showed decent inhibitory activities for COX-2.
However, selectivity and/or pharmacokinetic profiles hampered the introduction of real
competitors to celecoxin into the market. In addition, few pyrazolopyridazines were reported in
only two studies in 2004 and 2012 (compounds E-G, Fig. 2).23, 24 To the best of our knowledge,
the pyrazolo[1,2-a]pyridazine scaffold has not yet been explored for COX-2 inhibition.
As mentioned above, several research groups have made use of the traditional NSAIDs as a
starting point for the design of novel molecules with selective COX-2 inhibitory activity. For
example, Hu and co-workers utilized indole ring as a scaffold for the design of new selective
COX-2 inhibitors.25 This study led to the discovery of a series of sulfonylphenylindole
derivatives with higher COX-2 inhibitory activity than celecoxib by replacing the central
pyrazole ring of the latter with indole. Few years later, Wey and coworkers reported a series of
3

nitric oxide-donating COX-2 selective inhibitors, obtained through modifications of the acidic
moiety of indomethacin (Fig. 1).13 Inspired by the previous observations, we decided to build a
hybrid scaffold through the combination of the structural elements of salicylamide and celecoxib
(Fig. 1) and to introduce diversity at the heterocyclic ring. We were encouraged by the synthetic
accessibility of pyrazole derivatives via acid catalyzed cyclization of hydrazine derivatives.26
Figure 2: Selected examples of reported pyrazoles and pyrazolopyridazines COX-2 inhibitors.
The crystal structure of the inhibitor-bound COX-2, PDB ID: 3LN1, shows that the catalytic
binding site is a long hydrophobic channel.27, 28 At the entrance of the binding site, Arg106 forms
a salt bridge with the carboxylic acid end of the traditional NSAIDs. However, this interaction is
not essential for COX-2 inhibition. On the other hand, many Cox-2 inhibitors were noticed to
form an H bond with Tyr341 at the entrance of the active site. It is worth noting that in COX-1,
Ile523 blocks the diarylheterocycle-type selective inhibitors from entering the catalytic site.
However, a side-pocket is created in COX-2 because of the shorter side chain of Val509. This
results in a 17% increase in size next to the main catalytic binding site which becomes accessible
for the diarylheterocycle inhibitors. This side pocket, lined by His75, Glu178, and Arg499, is
referred to as the “selectivity binding site” as traditional NSAIDs do not use it, while the
sulfonamide group of celecoxib fills it forming a strong H bond with Arg499 (Fig. 3a).28, 29 In
the designed compounds, we merged the salicylamide moiety with a substituted pyrazole ring to
obtain a heterocyclic core with two aryl arms, an arrangement similar to that of celecoxib. In
4

addition, we have investigated the effect of fusing one of the aryl groups with the pyrazole ring
to form a pyrazolo[1,2-a]pyridazine nucleus. Moreover, various hydrogen bonding groups, such
as amino, hydroxyl, and carbonyl, were installed on the aryl rings in order to form hydrogen
bonding interactions with the polar selectivity pocket of COX-2 catalytic site or the critical
Tyr341 at the entrance.
Retrosynthetic analysis of our first series of compounds showed that the substituted pyrazoles
(3 and 4) and the salicylamide derivatives (5-8) could be obtained via the reaction of 2-
hydroxybenzohydrazide (2) with β-diketones and Dihydro-2,5-furandiones, respectively. The
hydrazide key intermediate 2-hydroxybenzohydrazide (2) was prepared by the reaction of methyl
2-hydroxybenzoate (1) with excess hydrazine hydrate.30 The hydrazide 2 was cyclized with β-
diketones such as pentane-2,4-dione or ethyl 3-oxobutanoate by applying sonication or heat on
equimolar amounts of the reactants in presence of acetic acid to afford the novel pyrazole
derivatives 3 and 4 in high yields.31 Interestingly, sonication gave us better yields under mild
conditions with comparable reaction times (discussed later). Similarly, hydrazide 2 was
condensed with a series of Dihydro-2,5-furandione derivatives in acidic medium to give the
salicylamide derivatives 5-8.32 In addition, 2-Hydroxybenzohydrazide 2 was condensed with
chalcone 9 in ethanol, also under sonication, to give the dihydropyrazole derivative 10. The
chalcone 9 could be easily accessed through the reaction of acetanilide and salicyladehyde in
basic medium.33 The target pyrazole 11 was then obtained after oxidation of 10 with bromine
water. (Scheme 1).
5

Scheme 1: (a) Hydrazine hydrate, EtOH, reflux, 6 h; (b) EtOH, ACOH, sonication or reflux, 1 h; (c) EtOH, NaOH,
rt, 16 h; (d) EtOH, ACOH, sonication or reflux, 4 h; (e) Br2, H2O, rt, 2 h.
After the completion of series one, we turned our attention to our second series, the
pyrazolo[1,2-a]pyridazine derivatives (13-16). This heterocyclic scaffold could be accessed via
the condensation of hydrazine with tetrabromo-o-xylene.34 Then, the products could be
condensed with the appropriate diketone in acidic medium. However, due to the low yield of this
multi-step procedure, we opted to use the recently reported ultrasound-assisted, one-pot, three-
component method. The target compounds (13-16) were obtained via one-pot reaction of
hydrazide, salicyladehyde, and ethyl cyanoacetate in ethanol under ultrasonic irradiation
(Scheme 2).35 The use of ultrasound irradiation gave significantly improved yield and
straightforward purification. It has been reported that ultrasound could effectively enhance
chemical reactivity by as much as a million-fold.36 It catalyzes the reaction by exciting the
molecular vibrational, rotational, and translational modes. Solid reactants usually undergo
extensive degradation by the energy released from the bubbles created by cavitation leading to a
great increase of their surface area as well as the observed rate of reaction.37
Scheme 2: (a) Et3N, EtOH, sonication, 50 °C, 1 h.
The aim of this study is to design novel and selective COX-2 inhibitors with in-vivo activity.
Therefore, before performing the enzyme assay, we screened the prepared compounds for
analgesic and anti-inflammatory activities using mild animal models, namely, tail-immersion test
6

and hind paw oedema, respectively.38 Compounds 4, 7, 11, 13, 14, and 16 showed acceptable
analgesic and anti-inflammatory activities comparable to the standard diclofenac sodium (Tables
S1 and S2, Supporting Information). To validate the mode of action and the selectivity of these
compounds, in-vitro assay for COX-1 and COX-2 enzymes inhibition was conducted. The most
active compounds were screened using enzyme-immuno assay (EIA) kit (Cayman Chemical
Company, Ann Arbor, MI 48108, USA).39 IC50 values for the most active compounds were
determined using celecoxib as the reference drug. Furthermore, selectivity index for COX-
1/COX-2 inhibition was calculated for each compound. From the screening results, compounds
11 and 16 showed the best inhibitory activity as well as selectivity for COX-2 with IC50 of 16.2
nM and 20.1 nM, respectively. The IC50 values for COX-1 and COX-2 inhibition are listed in
Table 1. It is obvious that simply merging salicylamide with pyrazole gave a compound that is
too small to fill the relatively large catalytic site of COX-2 (compound 4). Merging salicylamide
with phthalimide ring in compound 7 led to a slight improvement in COX-2 inhibitory activity.
The best activity and selectivity was noticed in compound 11 which possesses a very similar
special arrangement to celecoxib with an additional aryl group. The pyrazolo[1,2-a]pyridazines
series showed a gradual improvement of activity with increasing the molecular size and/or
rigidity. Compound 16 is the most active in this series with more than a 3-fold selectivity for
COX-2, perhaps, due to formation of additional hydrophobic and hydrogen bonding interactions
with the enzyme (discussed later). Interestingly, compound 15, which is very similar to 16, lacks
the COX-2 inhibitory activity. This might be attributed to the pyridine moiety of compound 16
with a nitrogen atom that can act as an H bond acceptor.
Table 1: In-vitro enzyme assay against COX-1 and COX-2 with selectivity ratios.*
COX-1
IC50 (nM)
COX-2
IC50 (nM)
Selectivity
(COX-1/COX-2)
Compound
125
NA
4.8
NA
Celecoxib
26.0
--
3
4
82.1
NA
151.4
NA
0.54
--
5
NA
NA
--
6
92.2
NA
130.4
NA
0.71
--
7
8
11
13
70.4
88.1
16.2
181.0
4.35
0.47
7

74.4
NA
90.1
NA
0.83
--
14
15
16
66.7
20.1
3.32
* Enzyme-immuno assay, NA: Not Active
The two most active compounds 11 and 16 were docked into the crystal structure of the
celecoxib-bound COX-2 enzyme (PDB ID: 3LN1).40 Molecular docking investigations were
performed using Glide software within Schrodinger Molecular Modeling package and the
standard parameters.41 The designed inhibitors were docked into the same binding site of the
reference drug celecoxib (fig. 3). As mentioned earlier, the COX-2 catalytic site is lined with
polar residues at the entry with His75, Glu178, and Arg499 forming the “selectivity pocket”.
Deeper, there is a wider hydrophobic pocket, formed by Leu338, Leu345, Val509, which
accommodates the central aromatic part of celecoxib. The bottom of the catalytic site is mostly
aromatic with Tyr371 and Trp373 making contacts with most co-crystallized NSAIDs.
Redocking of celecoxib resulted in a docking pose very similar to the crystallized ligand
with RMSD value of 1.51 Å and a docking score of -9.12 KJ/mol. Also, the characteristic H
bond with Arg499 in the selectivity pocket was maintained (fig. 3a). The synthesized ligands 11
and 16 achieved lower docking scores of -8.02 KJ/mol and -6.10 KJ/mol, with binding poses
very similar to that of celecoxib and RMSD values of 1.9Å and 2.2 Å, respectively. Figure 3b
shows the alignment of these compounds with celecoxib in the active site of COX-2. Compound
11 docked vertically inside the pocket to avoid steric clashes and, as expected, formed a strong
hydrogen bonding interaction with the critical TYR341 using its NH group (fig. 3c). The aniline
moiety at position 3 of the pyrazole nucleus docked smoothly deep inside the selectivity pocket
avoiding implausible clashes with polar amino acids populated inside this domain. The lower
salicylamide moiety was found to be flanked by Leu345 and Leu517 making preferable
hydrophobic interactions without forming H bonds with Arg106. Meanwhile, the third arm of
8

this compound, the phenolic group at position 5 of the pyrazole, showed contacts with Tyr371 at
the bottom of the catalytic site. Compound 16 docked in a similar manner to compound 11
showing interactions with the three aforementioned pockets. However, its ethyl ester group
pointed downwards forming a hydrogen bond with Arg106. This could rationalize, at least in
part, the reduced selectivity of this compound as most NSAIDs that form polar interactions with
Arg106 usually suffer from low selectivity for COX-2.42, 43 Also, the rigid tricyclic ring system
of 16 approached the selectivity pocket, unfortunately, without forming any π-stacking
interactions or H bonds with His75 or Arg499. Again, its phenyl moiety made contacts with
Tyr371 and Trp373 deep inside the pocket. Generally speaking, by comparing the prepared
compounds with celecoxib, it was noticed that the presence of H bonding groups on the para
position of one of the aryl arms or on the central pyrazole ring (such as the NH group of the
aniline moiety of 11) could enhance the COX-2 inhibitory activity. This is mediated by
interaction with residues of the selectivity pocket or Tyr341, respectively. These observations
showed that salicylamide-pyrazole hybrids could offer a good starting point for the design of
novel COX-2 inhibitors if substituted with aryl groups of suitable size and H bonding groups in
the appropriate positions.
9

Figure 3: (a) Docked pose of celecoxib in the COX-2 crystal structure (PDB: 3LN1), the compound adopts a Y-
shaped conformation and forms an H bond with the Arg499. (b) Alignment of the docked poses of the most active
compounds 11 (cyan) and 16 (magenta) on the crystallized ligand celecoxib (green). (c) Proposed binding mode of
11 in COX-2 crystal structure forming an H bond with Tyr341. (d) Proposed binding mode of 16 in COX-2 crystal
structure showing an H bond with Arg106. Protein is shown as a grey cartoon with the important binding site
residues displayed as sticks.
To further investigate and verify the mode of binding of the most active compound in this
study, compound 11, we decided to examine the hydrophobic/aromatic interactions as they
represent the major interaction forces in case of both celecoxib and this compound. Visual
examination of both complexes reveals the absence of any aromatic (π/π) interactions. On the
other hand, many hydrophobic contacts with Val509, Ala513, Tyr341, and Leu345 could be
10

noticed. Interestingly, the orientation of several amino acids within the active site suggests a
CH/π interaction, a weak molecular attraction force occurring between a CH group and an
electron-rich π-system. This attractive force is generally weak and difficult to distinguish from
the non-directional van der Waals interaction. We assume that the existence of several CH/π
interactions could play a critical role in the binding of celecoxib that has been overlooked in the
previous studies. To examine the presence of this type of interaction quantitatively, we used the
CHPi module as implemented in the Biostation Viewer software. As expected, both molecules
showed an extensive network of CH/π interactions (Fig. 4). The right phenyl arm of celecoxib is
sandwiched between Ser339 and Val509, while the other phenyl ring and pyrazole forms
contacts with Ala513 and Val335, respectively. Compound 11 shows similar CH/π bonds with
Val509 on the right side, while the lower benzoyl group exhibits more interactions of this type
with Val335 and Ala513, analogous to celecoxib. These results give more emphasis on the
importance of the aforementioned CH/π contacts for ligand binding in the hydrophobic pocket of
COX2 enzyme.
11

Figure 4: CH/π interactions as calculated using the CHPi module as implemented in the Biostation Viewer software
for celecoxib (a) and compound 11 (b). Both compounds are shown as yellow balls and sticks, protein side chains
are displayed as green sticks, and the predicted interactions are illustrated as red lines.
To visualize the charge distribution on both celecoxib and Compound 11 and to better
estimate the contribution of polar interactions in their binding, we ran an energy calculation in
Guassian 09 software using Density Functional Theory (DFT) method on the conformation of
crystallized celecoxib as well as the docked conformation of compound 11.44, 45 The B3LYP
functional was employed with 6-311++G (d, p) basis set to calculate the Mulliken charge
distribution for all the atoms as well as the Electrostatic Surface Potential (ESP) for both
molecules.46 Figure 5 shows the distribution of Mulliken atomic charges. It is obvious that both
12

compounds possess relatively negative phenyl moieties which indicates a high electron density
responsible for the CH/π interactions. Interestingly, the calculated atomic charge of the celecoxib
sulfonamide oxygen interacting with Arg499 was found to be ‒ 0.541 (Fig. S1, Supporting
Information). This suggests an ionic bond nature of this interaction rather than a traditional H
bond and highlights the importance of interaction with the residues of the selectivity pocket for
celecoxib binding. The ESP of both compounds (Fig. 5) reveals a very similar shape and size
with mostly hydrophobic three arms. The only striking difference is the strong negative charge
localized on the apex of the right arm of celecoxib. This highly negative surface represents the
sulfonamide group of celecoxib which interacts with the polar residues of the selectivity pocket,
especially Arg499. This may rationalize, to some extent, the higher inhibitory activity of
celecoxib compared to compound 11. It is worth noting that, for ease of comparison, we placed
the compounds in figure 5 in a similar orientation to their docked poses in figure 3.
Figure 5: Mulliken charges (left) as calculated using DFT method with B3LYP functional and 6-311++G (d, p)
basis set (color-coded from red to green). Electrostatic Surface Potential (ESP) calculated using the same
methodology and mapped onto both compounds (color-coded from red to blue).
13

With the extensive use of anti-inflammatory medications, there is still a need for the
development of novel COX-2 selective inhibitors with improved pharmacokinetics and safety
margin. In this study, we reported the design and synthesis of some salicylamide-pyrazole hybrid
molecules; two of them with promising selectivity. Docking studies and DFT calculations
suggests that these molecules possess a suitable size and complementarity to the COX-2 active
site. According to our results, the ideal selective COX-2 inhibitor should have three aryl arms to
form the required hydrophobic or CH/π interactions with the hydrophobic residues in the active
site such as Ala513 and Val509. In addition, the molecule should possess two polar regions; one
in the center to interact with Tyr341 and the other at the upper aryl arm to fill the selectivity
pocket interacting with Arg499 in a similar manner to celecoxib. Further studies are undergoing
in our laboratory to improve the activity of our lead compounds 11 and 16 according to these
proposed guidelines and to fine-tune their selectivity towards COX-2 enzyme.
Acknowledgments
The authors would like to thank Dr. Mohamed M. Said, professor of organic chemistry,
Faculty of Pharmacy, Suez Canal University, for his support and insightful comments during
compounds synthesis as well as his help reviewing the manuscript.
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