Design, synthesis, anti-inflammatory activity and molecular docking of potential novel antipyrine and pyrazolone analogs as cyclooxygenase enzyme (COX) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.01.043

As a part of a directed program for development of new active agents, novel heterocyclic derivatives with antipyrine and pyrazolone moieties -incorporated in- have been designed and synthesized. Starting with 4-arylidene-3-methyl-1-phenyl-5-pyrazolone derivative 2a,b novel Mannich bases derivatives have been synthesized and biologically evaluated for their anti-inflammatory activity. Furthermore, the activity of such compounds has been tested interestingly as COX-1 and COX-2 inhibitors. Structure elucidation of the synthesized compounds was attained by the use of elemental analysis, IR, ¹H NMR, ¹³C NMR, and Mass spectrometry techniques. Compounds 3b, 3d and 4b represent the high % inhibition values for both COX-1 and COX-2. On the other hand, compound 8 showed little selectivity against COX-2 while compound 10 showed good selectivity against COX-1 only. Structure activity relationship has been discussed and the results were confirmed by molecular docking calculations.

Full text of DOI:10.1016/j.bmcl.2018.01.043

Accepted Manuscript
Design, Synthesis, anti-inflammatory activity and Molecular docking of Poten-
tial novel antipyrine and pyrazolone Analogs as cyclooxygenase enzyme (COX)
Inhibitors.
Mardia T. El Sayed, Marwa A.M.Sh. El-Sharief, Eman S. Zarie, Nesrin M.
Morsy, Ahmed R. Elsheakh, Andrey Voronkov, Ghada S. Hassan
PII:
S0960-894X(18)30052-0
https://doi.org/10.1016/j.bmcl.2018.01.043
BMCL 25569
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 December 2017
11 January 2018
21 January 2018
Please cite this article as: El Sayed, M.T., El-Sharief, M.A.M., Zarie, E.S., Morsy, N.M., Elsheakh, A.R., Voronkov,
A., Hassan, G.S., Design, Synthesis, anti-inflammatory activity and Molecular docking of Potential novel antipyrine
and pyrazolone Analogs as cyclooxygenase enzyme (COX) Inhibitors., Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.01.043
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1
Design, Synthesis, anti-inflammatory activity and Molecular docking of Potential novel
antipyrine and pyrazolone Analogs as cyclooxygenase enzyme (COX) Inhibitors.
1
Mardia T. El Sayed 1,*, Marwa A.M.Sh. El-Sharief , Eman S. Zarie 2,3, Nesrin M. Morsy 4, Ahmed R.
Elsheakh 5,Andrey Voronkov 6, Ghada S. Hassan 7,*
1Deparment of Applied Organic Chemistry, National Research Centre, 12622-Dokki, Egypt.
2Department of Chemotherapy, National Research Centre, 12622-Dokki, Egypt
3Material science department, Aalto University,Kemistintie 1, 00076 Aalto, Finland
4Deparment of Organometallic and Organometalloid Chemistry, National Research Centre, 12622-Dokki, Egypt.
5Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt.
6Digital Bio Pharm Ltd., 145-157 St. John Street, London, EC1V 4PW, U.K.
7Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt.
Correspondence:
Dr. Mardia T. El Sayed, mardia.elsayed2016@gmail.com
Dr. Ghada S. Hassan, ghadak25@yahoo.com
Abstract
As a part of a directed program for development of new active agents, novel heterocyclic derivatives
with antipyrine and pyrazolone moieties -incorporated in- have been designed and synthesized.
Starting with 4-arylidene-3-methyl-1-phenyl-5-pyrazolone derivative 2 a,b novel Mannich bases
derivatives have been synthesized and biologically evaluated for their anti-inflammatory activity.
Furthermore, the activity of such compounds has been tested interestingly as COX-1 and COX-2
inhibitors. Structure elucidation of the synthesized compounds was attained by the use of elemental
1
analysis, IR, H-NMR, 13C-NMR, and Mass spectrometry techniques. Compounds 3b, 3d and 4b
represent the high % inhibition values for both COX-1 and COX-2. On the other hand, compound 8
showed little selectivity against COX-2 while compound 10 showed good selectivity against COX-1
only. Structure activity relationship has been discussed and the results were confirmed by Molecular
docking calculations.
Keywords: Synthesis, antipyrine, pyrazolone derivatives, COX-1, COX-2, anti-inflammatory
activity, Molecular Modeling.
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Non-steroidal anti-inflammatory drugs (NSAIDs) belong to the most widely used therapeutics.
Through their anti-inflammatory, anti-pyretic and analgesic activities, they represent drugs of choice
in various inflammatory diseases such as: arthritis, rheumatisms as well as relieving the pains of
everyday life. They also represent a heterogeneous family of pharmacologically active compounds
used to alleviate acute and chronic inflammation, pain and fever. Their clinical efficacy is related to
their ability to inhibit both COX-1 and COX-2 isoforms of the enzyme cyclooxygenase (COX); it
also referred to prostaglandin H2 synthase since it catalyzes the conversion of arachidonic acid to
prostaglandin H2 (PGH2) that represents the main mediator for pain and inflammation [1,2]. The
constitutive COX-1 isoform is mainly responsible for the synthesis of prostaglandins which exert
cytoprotective effect on the gastrointestinal (GI) tract and control renal function in the kidneys,
whereas, the inducible COX-2 is selectively activated by pro-inflammatory stimuli and facilitates the
release of prostaglandins involved in the inflammatory process [3]. Consequently, their long-term
clinical employment is associated with significant side effects such as gastrointestinal lesions,
bleeding, and nephrotoxicity [4]. Since the introduction of antipyrine - the first pyrazolone derivative
used in pain management, inflammation and fever - into clinical use in 1884, great attention has been
focused on antipyrine and pyrazole derivatives as potent anti-inflammatory, analgesic and antipyretic
agents [5-7]. As a result of such researches, large number of pyrazoles derivatives have been
obtained and gained application on the clinical level. Since then, many pyrazolone compounds have
been obtained and introduced to the market as shown in (Fig. 1).They display activities such as
analgesic and antipyretic (propylphenazone, phenazone, metamizole) [8], anti-cancer (TELIN) [9],
anti-ischemic (edaravone) [10], and anti-anxiolytic [11]. Pyrazolones are gaining importance
especially in drug discovery programs towards cerebral ischaemia [12] and cardiovascular diseases
[13-14].
Figure 1: Structures of some biologically significant pyrazolones
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In view of the above-mentioned data, it was of interest to study the reactivity of some antipyrine and
pyrazolone derivatives aiming to discover new compounds with potential biological activity. The
scope and applicability of the use 4-arylidene-3-methyl-1-phenyl-5-pyrazolone derivative 2 a,b as a
unique precursor for the synthesis of novel Mannich bases with non-classical method were
performed. Some other Mannich bases have been synthesized by unique method in which antipyrine
moiety was incorporated. All prepared compounds were structurally confirmed by different
spectroscopic techniques; in addition they were investigated for their anti-inflammatory activities,
and evaluated for their COX-1 and COX-2 inhibition action. Structure activity relationship has been
discussed and the active candidates have been docked with the crystal structure of COX-1 and COX-
2 enzymes via Smina software (the fork of Autodock Vina).
The synthesis of the target compounds was illustrated in schemes 1-3. The starting compounds 4-
arylidene-3-methyl-1-phenyl-5-pyrazolone derivatives (2 a,b) were synthesized according to the
reported procedures of Rajeev Verma and co-workers [15], through the reaction of 3-methyl-1-
phenyl-5-pyrazolone 1 with aromatic aldehydes via Knoevenagel condensation reaction. It depends
on the activity of the methylene group at position-4 of that reacted with the selected aldehydes in the
presence of mixture of piperidine and glacial acetic acid to form derivatives 2a and 2b as colored
products. The introduction of the aromatic aldehydes was confirmed by NMR techniques in the
appearance of the peaks of two additional methyl group at 2.11 and 3.38 in case of 2a and the
appearance of the characteristic peak of (OCH2O) peak at 6.21 in case of 2b derivative.The two
arylidene derivatives 2a and 2b were utilized directly in a non-classical Mannich reaction by the use
of different primary and secondary amines. Reaction with secondary amines formed Mannich bases
3a-d and 5 a,b using piperidine, morpholine, 1-phenylpiperazine, and piperazine, whereas the
Mannich bases of type 4 a-c were produced by the use of primary amines. In both cases, the reaction
was performed by refluxing the ethanolic solutions of 4-arylidene-3-methyl-1-phenyl-5-pyrazolone
derivatives 2a or 2b with the requested primary or secondary amines derivatives for about 4 h over a
water bath. The progress of the reaction was monitored by TLC until the reaction finished then
worked up to get the target compounds as illustrated in the experimental section. The structure of
these Mannich products has been confirmed by their analytical and spectral data. For 3 a-d
derivatives, the structures of the final compounds were confirmed by the appearance of the
heterocyclic amine protons that appeared in the region 1.47-2.50 and 2.13-2.99, and by the
appearance of additional phenyl group in case of compound 3d of the phenyl piperazine moiety. In
case of compounds 4 a-c the appearance of additional 2 methyl groups at 0.89-2.11 and N-CH3 at
2.23 – 3.15 confirmed the structures of compounds 4a and 4c, while compound 4b is characterized
by the presence of the NH peak at 9.42 (Scheme 1). Compounds 5 a,b structures were confirmed by
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the presence of characteristic peaks of CH2 at 2.33, 2.50, 2.68, 2.88, and 5.89 in addition to the
additional peak of NCH3 at 3.32 in compound 5a. The synthesis of compounds 6 a,b was performed
through the reaction of 2 a,b with either 2-phenyl indole or nitromethane at 80oC followed by
crystallization of the obtained compounds from acetone (Scheme 2). The appearance of the
additional aromatic peaks of compound 6a and the additional CH2NO2 at 3.39 in compound 6b
confirmed the structure of the final compounds. The classical approach for synthesis of novel
antipyrine Mannich bases 8 and 9 was performed directly by using equivalent amounts of antipyrine,
formaldehyde and 2-(piperazin-1-yl)ethan-1-ol in ethanol solution by refluxing the mixture at about
80 oC to afford Mannich base 8. By the same procedure 1,3-di(piperidin-4-yl)propane has been used
with two equivalents of antipyrine and formaldehyde to give Mannich base 9 (Scheme 3). Both
structures 8 and 9 have been proved by analytical and spectral data. The appearance of the peaks of
CH2OH at 3.68 and 10.95 confirmed the structure of compound 8, while the appearance of the
multiplet peaks of the two piperidine moieties confirmed the structure of compound 9.Compound 10
was synthesized through incorporation of more than one antipyrine moiety in one structure to test the
effect of increasing number of antipyrine units on the potency of the target compounds as anti-
inflammatory agents. The reaction was performed by the usual method of condensation through the
use of four moles of antipyrin with one mole of glutaraldehyde 25 % solution (Scheme 3). The
appearance of the 3 peaks of the methylene group of the bridge between the two antipyrine groups in
addition to the duplication of the number of peaks representing the antipyrine moieties confirmed the
formation of the final product 10.
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The anti-inflammatory activity was performed to determine the percentage inhibition of edema for
each tested compound after 4 h of induction of inflammation using in vivo rat carrageenan-induced
foot paw edema model reported previously [16, 17], the reference drug Celecoxib was used as a
positive control in the study, however, a significant decrease in the activity of all compounds was
noticed after 6 h (Table 1). According to the provided results, compound 3d exhibited the highest I%
when compared with the other target compounds with value of 98%, in the same series compound 3b
produced 80% inhibition giving indication that this series represented the most potent one among the
synthesized drugs either in vitro or in vivo screening. On the other hand, compounds 4b and 9
exhibited I% value of 89.2% and 85.4 % respectively producing the next high level values for the
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6
anti-inflammatory screening. Some of the compounds produced moderate inhibition % such as 6b
and 10 and the rest of the compounds were considered inactive with % inhibition < 50 %.
Table (1): The inhibition of edema (I%), percentage of inhibition of COX-1 and COX-2 of the
synthesized compounds.
Selectivity Index (SI)
(COX-2/COX-1)
IC50 µM
(COX-1)
> 5.0
22.2
1.33
1.57
> 5.0
1.6
> 5.0
1.74
2.52
1.16
> 5.0
> 5.0
1.25
> 5.0
1.23
1.46
15.0
Comp.
2a
2b
3a
3b
3c
3d
4a
4b
4c
5a
5b
6a
6b
8
% inhibition (I%)
%I COX-1a
%I COX-2a
49.1 ± 2.7
20.3 ± 0.9
40.5 ± 1.0
80.0 ± 6.9
33.8 ± 3.1
98.0 ± 4.2
39.4 ± 2.1
89.2 ± 3.5
48.3 ± 1.1
49.5 ± 5.4
21.1 ± 7.5
35.9 ± 2.3
68.0 ± 0.8
48.2 ± 2.2
85.4 ± 1.5
75.0 ± 0.7
89.0 ± 1.3
87.02
75.96
16.69
73.65
80.55
52.14
82.35
59.44
81.96
69.24
75.74
51.58
54.34
71.83
77.19
68.76
39.13
61.0
0.87
25.01
77.98
88.92
58.30
84.41
58.80
82.97
84.23
87.36
42.91
69.13
88.20
73.40
88.53
81.34
2.0
0.67
0.95
0.91
0.90
0.98
1.01
0.99
0.82
0.87
1.20
0.79
0.82
1.05
0.78
0.48
0.03
9
10
Celecoxib
a Values represents means of two determinations acquired using COX-2/COX-1 assay kits.
Percentage of inhibition of COX-1 and COX-2 of test compounds at concentration of 5.0 µM were
illustrated in (Table 1). In general, compounds 3b, 3d and 4b represented the high % inhibition
values for both COX-1 and COX-2 showing the non-selectivity of the tested compounds. To lesser
extent, compounds 4c, 6b and 9 had less % inhibition than the former compounds against both
enzymes with increased activity against COX-1. On the other hand, it was also noticed that
compounds 2a and 5a had slight selectivity against COX-1. Furthermore, it was found that
compound 8 had little selectivity against COX-2 while compound 10 showed good selectivity against
COX-1 only. The selectivity index of the tested compounds is illustrated in Table 1. The IC50 of the
synthesized compounds was determined using Cayman colorimetric COX (ovine) inhibitor screening
assay kit and the calculations were performed as per the kit guidelines. Compounds 5a, 9, 8, 3a, 3b
and 3d had the lowest IC50 values among the tested compounds with values of 1.16, 1.23, 1.25, 1.33,
1.57 and 1.60 µM respectively. The rest of the compounds had higher IC50 values with decrease in
the activity.
Docking study started with the selection of the X-ray structures. Those of COX-1 and COX-2 were
taken from PDB database www.rcsb.org (PDB code 3KK6 and 4 COX). Docking of the compounds
3b, 3d, 4d, 9 and 10 was performed via Smina software (the fork of AutodockVina) [18]. In order
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7
to confirm the adequacy of the docking procedure, docking of co-crystallized ligands was carried out
into COX-1 and COX-2 complexes. As a result, one can talk about the sound reproducibility of the
position of a known ligand with Smina. RMSD from the co-cristallized ligand for the position of the
atoms was less than 1 Å. RMSD calculations were carried by the means of PyMOL program [19].
Estimations of binding energy for each compound and target were shown in the Table 2. The
position of celecoxib (co-crystallized ligand) in the binding site of COX-1 was shown in Figure 2.
Sulfonamide group formed two hydrogen bonds with side chains of GLN192 and SER516.
Trifluoromethyl group of celecoxib also formed hydrophobic interactions with LEU359 and
VAL116. Docking of test compounds showed that they can occupy the same binding site as a co-
crystallized ligand. Compounds 3b, 3d and 4b shared pretty similar binding mode with celecoxib.
They all had hydrophobic interactions with side chains of LEU359, LEU352 and LEU531.
Compound 3b – the most energetically favorable according to scoring function additionally formed
hydrogen bond with hydroxyl group of SER530. Figure 3 - showed its interactions within the
binding site of COX-1. Compounds 9 and 10 due to their size had different binding mode. They
formed hydrophobic interactions with LEU531, VAL116, LEU384, THR94, ILE89. Compound 10
also formed hydrogen bond with ARG120. Figure 4 (in the supplementary section) showed the
binding mode of compound 10. The position of indomethacin (co-crystallized ligand) in the binding
site of COX-2 was shown on the Figure 5 (in the supplementary section). Carboxyl group formed
hydrogen bond with side chains of ARG120, methoxy group participated in the formation of a
hydrogen bond with TYR355; carbonyl group also had a possibility to interact with SER530 via
hydrogen bond. Indomethacin interacted with binding site via hydrophobic interactions with
LEU384, LEU352, LEU531, TYR385. Compounds 3b, 3d and 4b as in the previous case showed
almost the same binding mode: hydrophobic interactions with PHE381, TYR355, LEU531, LEU359,
compound 4b formed hydrogen bond with TYR385. Binding mode of compound 4b was shown on
the Figure 6 (in the supplementary section). Compounds 9 and 10 partially repeated the mode of
binding of the remaining compounds as they formed hydrophobic interactions with TYR355,
LEU531, and LEU359. However, in addition, hydrophobic interactions with LEU123, LEU112 were
also possible for them. Compound 9 can from hydrogen bond with SER119. Binding mode of
compound 9 was shown on the Figure 7 (in the supplementary section).
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Table 2.Values of binding affinities estimated via«Vina» scoring functions.
Compound COX-1 (kcal/mol)
COX-2 (kcal/mol)
-9.0
-9.3
3b
3d
4b
9
-7.3
-6.8
-8.4
-10.8
-11.5
-10.5
-28.9
-24.0
10
Figure 2: Illustrated the position of celecoxib (Co-crystallized ligand) in the binding site of COX-1.
Figure 3: Shows compound 3b interactions within the binding site of COX-1.
To construct a structure-activity relationship for the synthesized compounds, the GI % inhibition of
both COX-1 and COX-2 activity was observed and related with the corresponding structures. It was
noticed that the presence of the 3-benzo[d][1,3]dioxole ring system and the N-containing
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heterocyclic amine attached to the methyl group at 4-position of the antipyrine structure is very
essential for activity as in case of compounds 3b and 3d that had the greatest GI% values
respectively among the synthesized compounds. This criteria was proved by replacement of the
piperazine moiety with morpholine as in case of 3c or replacement both of the for-mentioned groups
with two antipyrine moieties as in case of compound 4a, it was found that such changes resulted in
dramatic decrease in the activity by one third of the original one. Similar effect was achieved by the
introduction of a larger group such as indolyl moiety as in compound 6a that leaded to a decrease in
the activity against both enzymes. In addition, the removal of the secondary amine moiety as in case
of 2b resulted in abolishing the inhibition activity against both enzymes. By taking a closer look to
the activity against COX-2, the presence of four moieties of antipyrine linked by aliphatic chain
dramatically decreased the activity against COX-2 enzyme while the activity against COX-1 is
preserved as in case of compound 10. Furthermore, changing the aliphatic chain with a piperazine
moiety as in compound 5a or two piperidine rings as in compound 9 regained the activity against
both enzymes. The in-vivo results obtained were consistent with in-vitro ones as shown in Table 1.
The compound 3d represented the most active compound among the synthesized one with %
inhibition of the edema of 98% value, followed by 4b and finally compound 9 with (I%) values of
89.2% and 85.4% respectively. These findings proved that the presence of the 3-
benzo[d][1,3]dioxole ring system and the N-containing heterocyclic amine attached to the methyl
group at 4-position of the antipyrine structure is very essential for activity. In addition the IC50 of
these compounds was the lowest values among the tested compounds.
Conclusion
A novel series of compounds that incorporate the antipyrine and pyrazole moieties in was designed
and synthesized according to the reported procedures in the literature. The structures of the
synthesized compounds were confirmed using different spectroscopic techniques. The anti-
inflammatory activity of the final products was performed using the carrageenan-induced foot paw
edema model, in addition the enzymatic inhibition activity of either COX-1 or COX-2 enzymes was
evaluated and the results showed that compounds 3b, 3d, 4b, and 9 represented the most active
compounds among the final ones. Molecular modeling technique has been used to confirm the results
obtained the in vitro and the in vivo results obtained. The results gained can be used for future
development of more active agents.
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Acknowledgment
This work was financially supported by the National Research Centre and Faculty of pharmacy,
Mansoura University, Egypt. The NMR and Mass Spec service were carried out in the Material
science department, Aalto University, Finland. The Digital Bio Pharm LTD Company (London, UK)
and IVO and AA acknowledge the support of the US National Institutes of health (R25GM067122).
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Captions
Figure 1: Structures of some biologically significant pyrazolones.
Figure 2: Illustrated the position of celecoxib (Co-crystallized ligand) in the binding site of COX-1.
Figure 3: Shows compound 3b interactions within the binding site of COX-1.
Figure 4: Shows the binding mode of compound 10.
Figure 5: Indicate the position of indomethacin in the binding site of COX-2.
Figure 6: Shows binding mode of compound 4b.
Figure 7: Shows the binding mode of compound 9.
Scheme 1: Synthesis of compounds 2 a,b, 3 a-d, 4 a-c
Scheme 2: Synthesis of compounds 5 a,b, 6 a,b
Scheme 3: Synthesis of compounds 8-10
Table 1: The inhibition of edema (I%), percentage of inhibition of COX-1 and COX-2 of the
synthesized compounds.
Table 2: Values of binding affinities estimated via«Vina» scoring functions.
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Figure 4: Shows the binding mode of compound 10.
Figure 5: Indicate the position of indomethacin (co-crystallized ligand) in the binding site of COX-2.
Figure 6: Shows binding mode of compound 4b.
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Figure 7: Shows the binding mode of compound 9.
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Research Highlights
 Synthesis of Antipyrine and pyrazolone analogs was performed.
 Anti-inflammatory activity of the synthesized compounds was performed.
 Compounds 3b, 3d and 4b represented the high % inhibition values for both COX-1 and COX-2
 Compound 8 showed little selectivity against COX-2 while compound 10 showed good
selectivity against COX-1 only.
 Structure activity relationship has been discussed and the results were confirmed by Molecular
docking calculations.
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Design, Synthesis, Anti-inflammatory activities and Molecular docking of Potential novel
antipyrine and pyrazolone Analogs as cyclooxygenase enzyme (COX) Inhibitors.
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Mardia T. El Sayed1,*, Marwa A.M.Sh. El-Sharief , Eman S. Zarie2,3, Nesrin M. Morsy4, Ahmed R.
Elsheakh5, Andrey Voronkov6, Ghada S. Hassan7,*
1Deparment of Applied Organic Chemistry, National Research Centre, 12622-Dokki, Egypt.
2Department of Chemotherapy, National Research Centre, 12622-Dokki, Egypt
3Material science department, Aalto University,Kemistintie 1, 00076 Aalto, Finland
4Deparment of Organometallic and Organometalloid Chemistry, National Research Centre, 12622-Dokki, Egypt.
5Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt.
6Digital Bio Pharm Ltd., 145-157 St. John Street, London, EC1V 4PW, U.K.
7Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt.
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