In silico binding affinity to cyclooxygenase-II and green synthesis of benzylpyrazolyl coumarin derivatives

Article abstract of DOI:10.1039/c5ra16643h

Here, we have reported for the first time, in silico binding affinity prediction of benzylpyrazolyl coumarin scaffolds to cyclooxygenase-II (COX-II) enzyme and a green synthetic route to access these scaffolds using a neutral ionic liquid, [pmIm]Br as the

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In-silico binding affinity to cyclooxygenase-II and green synthesis
of benzylpyrazolyl coumarin derivatives
Arijit Sahaa, Soumen Payraa, Sant Kumar Vermab, Mou Mandala, Suresh Tharejab and Subhash
Banerjeea,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Here, we have reported for the first time, in-silico binding affinity prediction of benzylpyrazolyl coumarin scaffolds to
cyclooxygenase-II (COX-II) enzyme and a green synthetic route to access these scaffolds using a neutral ionic liquid,
[pmIm]Br as the reusable catalyst and reaction media under metal-free conditions. Integrated scaffolds, benzylpyrazolyl
coumarins exhibited stronger binding affinity to COX-II inhibitor than it’s individual moieties, 3-benzyl coumarins and
pyrazolones. Moreover, 4-((4-hydroxy-2-oxo-2H-chromen-3-yl)(4-nitrophenyl)methyl)-5-methyl-2-phenyl-1H-pyrazol-
3(2H)-one (3c) has shown even better binding affinity than marketed anti-inflammatory drug, celecoxib.
www.rsc.org/
Design and development of integrated molecular scaffolds treatment of pain and inflammation, predominantly arthritis.4
for the biological applications has fascinated much attention as In this connection, design and development of NSAIDs are of
these moieties exhibit wide and improved bio-activities. In this much importance to overcome the general limitations of
connection, 3-benzyl substituted coumarins are of much present drugs such as GI bleeding, ulceration, perforation, and
interest as these moieties showed broad range of biological obstruction etc.5
activities and consequently these moieties were found in many
of the natural products namely as warfarin, phenprocumene
and coumatetralyl which were reported to show antibacterial,
anti-HIV,1a antiviral,1b anticoagulant,1c antioxidant1d and
anticancer activities1e (Fig. 1a). On the other hand, pyrazolones
have also exhibit broad spectrum of biological activities and
few of them for example, phenazone, propyphenazone,
ampyrone and edarvone are well known as antipyretic and
analgesic drugs (Fig. 1b).2 In addition, pyrazolones are also
recognized as antimicrobial, antifungal,3a antimycobacterial,3b
antibacterial,3c anti-inflammatory,3d,e antitumor,3f gastric
secretion stimulatory,3g antidepressant,3h antifilarial activities3i
and anti-tubercular activities.3j It is anticipated that the
integration of the 3-benzyl coumarin and pyrazolone moieties
leading to benzylpyrazolyl coumarin scaffolds (Fig. 1c) could be
fascinating and beneficial from the biological point of view.
However, interestingly, binding affinity and selectivity of
benzylpyrazolyl coumarin derivatives against any biological
target has not been investigated.
Over past decades, nonsteroidal anti-inflammatory drugs
(NSAIDs) are of enormous importance as therapeutics for the Fig. 1 Some bioactive individual (a) 3-benzyl substituted
coumarins,(b) pyrazolones and (c) model integrated scaffold.
The major pharmacological mechanism of action of NSAIDs
a.Department of Chemistry, Guru Ghasidas Vishwavidyalaya (A Central University),
Bilaspur-495009, Chhattisgarh, India.
is the enzymatic inhibition of cyclooxygenase (COX) mediated
b.School of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central
University), Bilaspur-495009, Chhattisgarh, India.
production of pro-inflammatory prostaglandins and
thromboxanes. COX and Lipoxygenase (LOX) are the key
enzymes which catalyze the rate-limiting steps in the
biosynthesis of prostaglandins and leukotrienes from
arachidonic acid.6 The two different forms of the COX enzymes
†Electronic Supplementary Information (ESI) available: Plausible mechanism for
the synthesis of benzylpyrazolyl coumarins, Reusability of [pmIm]Br, Preparative
method for the synthesis of benzylpyrazolyl coumarin derivatives, Methods for the
Preparation of ionic liquids, Copies of 1H NMR, 13C NMR spectra and HPLC data of
benzylpyrazolyl coumarin derivatives. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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are COX-I (constitutive) and COX-II (inducible), respectively. imported into the workspace. During import process, intense
COX-I is expressed in many organs and is responsible for care should be taken that all the crystallographic water
homeostatic processes such as platelet aggregation, gastric molecules were removed from COX-II. Further, COX-II and all
protection, and renal function while COX-II enzyme induced in the ligands were subjected to molecules preparation. The
response to the release of several pro-inflammatory mediators secondary structure of COX-II with co-factors is presented in
leading to the inflammatory response and pain.7 However, Fig. 3. Five distinct cavities (See Fig.S1 in ESI-1) with different
simultaneous inhibition of COX-I results the gastrointestinal surface area and volume have been mapped within COX-II
toxicities and the mild bleeding diathesis.8 Whereas, COX-II (PDB Id.:4COX) using detect cavity module in MVD software.10
enzyme was found to be anti-inflammatory, responsible for
the selective inhibition but non-ulcerogenic in both the
carrageenin-induced inflammation and the adjuvant arthritis.9
Therefore, the research in the field of inflammation around
the world relies on the search of potent, selective and specific
inhibitors of the COX-II.
Computer aided drug design tools have been widely used
for the prediction of biological activity, toxicity, binding modes,
selectivity and screening of a library of synthetic compounds
against a particular biological target. It is evident from the
literature that the pyrazolones widely exhibits COX-II inhibitory
activity.3d,e Here, we have evaluated their binding affinity
against COX-II inhibitor enzyme of integrated benzylpyrazolyl
coumarin scaffolds (3a-g, Fig. 2) and compared with those of
individual moieties of pyrazolones (1a-b, Fig. 2) and 3-benzyl
coumarins (2a-e, Fig. 2).
Fig. 3 Secondary structure of COX-II enzyme showing its co-
factors and cavity 1
.
During this computational process, the maximum numbers of
cavities were fixed to 5, grid resolution 0.80 Å and probe size
1.2 Å; while the other parameters were kept as default. The
volume and surface area of these cavities are depicted in Table
1.
Table 1 Major cavities (1-5) detected in COX-II along with their
volume and surface area
Cavity No.
Volume (Å3)
Surface
Area (Å2)
3776.000
3566.080
3257.600
1539.840
807.680
1
2
3
4
5
1561.090
1426.940
1370.620
698.880
242.688
Fig. 2 Structure of individual pyrazolones (1a-b), 3-benzyl
coumarins (2a-e) and their integrated scaffold, benzylpyrazolyl
coumarins (3a-g).
Molecular docking technique is used in the present study
for the comparison of binding affinity and selectivity of parent The Cavity 1 (Fig. 5) has highest volume (1561.090Å3) along
scaffolds and their integrated complex against COX-II. Molegro with largest surface area (3776.000Å2). The key residues of
virtual docker (MVD 2013.6.0.0 evaluation version) was used cavity 1 included Ala 151, Arg 44, Arg 469, Asn 39, Asn 43, Asp
for the flexible docking study of parent scaffolds and their 125, Cys 36, Cys 47, Gln 42, Gln 46, Glu 46, Glu 465, Gly 45, Leu
union complexes with COX-II which is based on molecular 152, Lys 468, Lys 546, Pro 40, Pro 153, Ser 38, Ser 471, Tyr 130,
docking simulations such as intramolecular interaction energy Tyr 466 (Fig. 4). For performing docking the option docking
of the ligand (Eintra), ligand and macromolecular interaction wizard in docking window was selected and the docking
energy i.e. intermolecular interaction energy (Einter
) and algorithm was set to MolDock Simplex Evolution (MolDock SE)
docking scoring function (Escore). Initially, for performing with population size 50.RMSD thresholds were set 1.00 Å for
molecular docking, the 3D structures of COX-II, molecular cluster similar poses as well as for ignore similar poses (for
mechanics (MM2) and Hamiltonian approximation (AM1) multiple runs only) both. Number of independent runs were
optimized (both the optimizers were run till the root-mean- selected ten and each of these runs was returning to a single
square gradient value reaches a value smaller than 0.001 final solution i.e. pose. Only negative lowest-energy
kcal/mol Å) parent scaffolds and their union complexes were representative cluster was returned from each of them after
2 | J. Name., 2012, 00, 1-3
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completion of docking and the similar poses were removed
keeping the best scoring one. The clusters were ranked
through compare the conformation of the lowest binding
energy in each cluster. The first minimum binding free energy
pose was selected for the analysis of the docking results. Other
parameters such as binding radius, grid resolution and
maximum iterations parameters were set to 15 Å, 0.3 Å and
1,500 respectively.11
3e
-150.385
-92.549
-6.167
-2.294
-3.627
-0.238
-3.182
-3.627
-5.173
-2.680
-124.305
-80.324
-97.152
-124.316
-47.830
-97.152
-114.596
-126.570
1b
2e
-118.182
-154.521
-58.282
3f
1a
2e
-118.182
-152.536
-156.232
3g
Celecoxib
It is sighted from the Table 2 that the MolDock scores and
re-rank scores of all the integration complexes (3a-g) were
greater than that of their parent scaffolds. The compound, 3c
demonstrated highest MolDock score
(
-162.027
)
with
comparable re-rank score (-105.214) which are even greater
than celecoxib, a marketed selective COX-II inhibitor and
signifies its better affinity and selectivity towards COX-II (Table
2). The hydrogen bond and steric interactions of 3c with COX-II
is depicted in Fig.5 and Fig.6 respectively.
Fig. 4 Showing cavity 1 (green colour) of COX-II with amino acid
residues.
Further, MolDock score, Re-rank score and H-bond score,
obtained after docking simulation are the markers of the
affinity of the parent scaffolds and their integration complexes
with COX-II (Table 2). These scores permit us to recognize the
significant interactions take place between different
conformations of each ligand and the amino acid residues
present in active site of COX-II in order to get best minimum
energy conformation of each ligand.12
Table 2 Moldock score, re-rank score and H-Bond score of
parent ligands and their integration complexes
Ligand
MolDock Score H-Bond Score Re-rank
Score
1a
2a
3a
1b
2a
3b
1b
2b
3c
1b
2c
3d
1b
2d
-58.282
-3.182
0.000
-47.830
-92.001
-104.615
-80.324
-92.001
-118.887
-80.324
-106.286
-105.214
-80.324
-98.150
-113.761
-80.324
-101.978
-105.935
-134.877
-92.549
-6.372
-2.294
0.000
-105.935
-145.568
-92.549
-4.964
-2.294
-5.744
-3.399
-2.294
-2.076
-1.524
-2.294
-1.560
-124.023
-162.027
-92.549
-113.784
-154.438
-92.549
Fig. 5 Hydrogen bonds labeled in dash bonds as 1 and 2 (
steric ( ) interactions of 3c with COX-II.
A) and
B
-118.953
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Here, [pmIm]Br acts as both catalyst and reaction medium and
no other metal catalysts or organic solvents were used for this
synthesis.
Initially, we have chosen synthesis of 4-((4-hydroxy-2-oxo-2H-
chromen-3-yl)(phenyl)methyl)-5-methyl-3H-pyrazol-3-one via
a
four component reaction of benzaldehyde, ethyl
acetoacetate, hydrazine hydrate and 4-hydroxy coumarin as a
model reaction. When stoichometric amount of four
a
components mixed in [pmIm]Br (2 ml) at room temperature
for 2 h good yield (85 %) of product was obtained (entry 1,
Table 3). The same reaction was failed to produce desired
product even at elevated temperature (80 oC) without ionic
liquid under neat or in water (entry 2, Table 3).
Table 3 Optimization of reaction conditions
Fig. 6 H-bond interactions (dash bonds shown in yellow color,
labeled with 1 and 2) of compound 3c with in cavity 1 (red
colored amino acids: hydrophilic, blue colored amino acids:
hydrophobic) of COX-II.
The integrated molecular scaffolds possess diverse range of
functionalities (e.g. nitro, methoxy, keto, hydroxy, phenyl etc.)
for the desired H-bonding and electrostatic interactions with
the amino acid moieties of COX-II enzyme, which results
significant enhancement of binding affinity and selectivity of
integrated benzylpyrazolyl coumarin derivatives to COX-II
inhibitor compared to individual moieties.
Entry
Ionic Liquid
[pmIm]Br
Time (h)
Yield (%)a
1
2
2
4
85
-
No ionic liquid
or H2O/80oC
[AcMIm]Cl
[AcMIm]Clb
[pmIm]Brc
3
4
5
2
2
2
35
69
74
Thus, the computer aided modeling data successfully
demonstrated that the integrated scaffolds (3a-g) selectively
and specifically binds with the amino acid residues present in
the cavity of COX-II inhibitor to a greater extent compared to
the individual moieties (1a-b and 2a-e). Motivated by these
results, we have tried to establish a green synthetic route to
access these scaffolds (3a-g) under neutral and metal-free
reaction conditions. The literature reports indicate that only
three methods are available for the synthesis of benzyl
pyrazolyl coumarin scaffolds12a-c (Fig. 1c). These methods have
utilized either glacial acetic acid,13a or lanthanide13b/alkaline
earth13c metal as catalyst. Thus, development of a metal-free
green synthetic protocol for the synthesis of scaffold, 3 is
highly desirable.
Reaction conditions: ethyl acetoacetate (1 mmol), phenyl
hydrazine (1 mmol), benzaldehyde (1 mmol), 4-
hydroxycoumarin (1 mmol), ionic liquid (2 ml), room
temperature, continuous stirring. Isolated yield. Heating at 70
oC, c1 ml of [pmIm]Br was used.
a
b
Using acidic ionic liquid, [AcmIm]Cl only 35% product was
isolated at room temperature (entry 3, Table3). However, the
yield has increased to 69% at 70oC (entry 4, Table 3). Thus, the
reaction using neutral ionic liquid, [pmIm]Br at room
temperature was considered as optimized conditions for the
synthesis of benzylpyrazolyl coumarin derivatives.
Next, we have explored the scope of this protocol for the
generation of a library of benzylpyrazolyl coumarin derivatives.
The results were listed in Table 4. A variety of aryl aldehydes
containing different substituent (e.g. –NO2, –Cl, –OMe, –Me
etc.) participated in this multicomponent reaction to provide
the excellent yields (85-90%) of products within practical time
period (2 h) and electronic effect of the substituent present in
aromatic aldehyde was not observed.
Over decades, ionic liquids (ILs) were reported to act as
green alternative reaction media14 and efficient catalysts15 in
organic synthesis. Here, we have observed remarkable
catalytic activity of a neutral IL, pentyl methyl imidazolium
bromide, [pmIm]Br rfor the one-pot four component synthesis
of benzylpyrazolyl coumarin derivatives at room temperature
(Scheme 1).
However, slight lower yields of the products were observed
in reactions when phenyl hydrazine is used in place of
hydrazine. This could be due to the delocalization of lone pair
of one nitrogen atom to the phenyl ring. After the reactions
the compounds were separated from ionic liquids and
recrystalized from hot ethanol and characterized primarily by
checking their melting points and compared these values with
the reported values. Further, the structure and purity of
Scheme 1 Synthesis of benzylpyrazolylcoumarin derivatives
using [pmIm]Br at room temperature.
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compounds were re-confirmed by 1H NMR and 13C NMR condensation reaction respectively. Subsequently, the two
studies (ESI-6) as well as high performance liquid intermediates react to each other via Michael addition
chromatography (HPLC) study (see ESI-7).
followed by cyclization and tautomerization to afford the
benzylpyrazolyl coumarin derivatives. A plausible mechanistic
pathway has been presented in ESI-4.
Table 4 Synthesis of benzylpyrazolyl cumarins using [pmIm]Br
Table 5 Comparative results of catalysts for the synthesis of
R
benzylpyrazolyl coumarin derivatives
OH
O
CHO
R
OH
O
O
O
O
[pmIm]Br (2 ml)
R.T
R1-NHNH2
OEt
N-R1
NH
O
Entry Catalyst
Reaction Time
condition
Yield
(%)
Ref.
O
(3a-g)
1
2
3
[bmIm]Br Neat/RT/ 2-2.5 h
25 oC
85-90 This
work
Entry (3a-g)
Time Yield
(h)
Melting Point (oC)
(%)a
Observed
Reportedref
Gl. AcOH
H2O/
30-55
min.
2-7
80-94 13a
Reflux
EtOH-
H2O/RT/
25 oC
1
2.0
85
229-231
231-23313a
ZrO2 NPs
93-97 13b
68-91 13c
min.
(3a)
4
Ca(OTf)2/ H2O/
Bu4NPF6
100 oC
3-5 h
2.5
85
232-234
232-23413a
2
(3b)
3
2.0
2.0
2.5
2.5
2.0
87
88
86
85
90
248-249
228-230
223-225
204-206
200-201
248-25013a
227-22913a
222-22413a
205-20713a
202-20413a
Conclusions
In conclusion, we have demonstrated enhanced target
specific molecular binding affinities of integrated molecular
scaffolds, benzylpyrazolyl coumarins with COX-IIinhibitor.
Moreover, It is noteworthy that the 4-((4-hydroxy-2-oxo-2H-
chromen-3-yl)(4-nitrophenyl)methyl)-5-methyl-2-phenyl-1H-
pyrazol-3(2H)-one (3c) moiety has exhibited even better
affinity in terms of H-bonds score and re-rank score than
marketed anti-inflammatory drug, celecoxib. Additionally, here
we have developed a green and synthetic methodology to
access benzylpyrazolyl coumarin moieties via a facile one-pot
multicomponent reaction using a neutral ionic liquid at room
temperature. Here, ionic liquids act as both catalyst and
reaction medium and of course performed the reactions under
neutral conditions. Finally, these scaffolds may further be used
for the development of potential and COX-II specific anti-
inflammatory agents.
(3c)
4
(3d)
5
(3e)
6
(3f)
7
(3g)
Reaction condition: ethyl acetoacetate (1mmol), hydrazine
hydrate/phenyl hydrazine (1mmol), 4-hydroxycoumarin
(1mmol), aryl aldehyde (1 mmol), [pmIm]Br (2ml), room
temperature with continuous stirring. a isolated yield.
Acknowledgment
We are pleased to acknowledge the funding agencies
Department of Science and Technology, New Delhi, Govt. of
India (NO.SB/FT/CS-023/2012). AS and SP thank to Guru
Ghasidas Vishwavidyalaya and SKV thanks ICMR for their
fellowship. We are also thankful to Dr. Rene Thomsen for
providing permission to use MVD software, Prof. B. C. Ranu
and Dr. K. V. S. Ranganath and their groups for helping in NMR
and HPLC studied.
In order to compare the results of synthesis of
benzylpyrazolyl coumarin using [pmIm]Br with those of
reported protocols,
a comparative study of catalytic
performance is presented in Table 5. This study clearly
indicates that the present protocol better alternative to those
of existing as present protocol avoided metal catalysts.
The synthesis of benzylpyrazolyl coumarins proceeds through
the catalytic cycle involving simultaneous synthesis of two
intermediates, pyrazolone (I) and 3-arylidenechroman-2,4-
dione (II) from general condensation and Knoevenagel
Notes and references
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DOI: 10.1039/C5RA16643H
Graphical Abstract
In-silico binding affinity to cyclooxygenase-II and green synthesis of
benzylpyrazolyl coumarin derivatives
Arijit Sahaa, Soumen Payraa, Sant Kumar Vermab, Mou Mandal, Suresh Tharejab and Subhash
Banerjeea,*
Here, we have reported for the first time, in-silico binding affinity prediction of benzylpyrazolyl coumarin scaffolds to
cyclooxygenase-II (COX-II) enzyme and a green synthetic route to access these scaffolds using a neutral ionic liquid,
[pmIm]Br as the reusable catalyst and reaction media under metal-free conditions. Integrated scaffolds, benzylpyrazolyl
coumarins exhibited stronger binding affinity to COX-II inhibitor than it’s individual moieties, 3-benzyl coumarins and
pyrazolones. Moreover, 4-((4-hydroxy-2-oxo-2H-chromen-3-yl)(4-nitrophenyl)methyl)-5-methyl-2-phenyl-1H-pyrazol-
3(2H)-one (3c) has shown even better binding affinity than marketed anti-inflammatory drug, celecoxib.