An Organocatalyzed Efficient One-pot Synthesis, Biological Evaluation, and Molecular Docking Studies of 4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)

Article abstract of DOI:10.1002/jhet.2681

The synthesis of 4,4′-(arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ol) derivatives catalyzed by ?-proline is a simple, versatile, and efficient method. In vitro antioxidant, anti-inflammatory, and antimicrobial activities of compounds 4a, 4b, 4c, 4d

Full text of DOI:10.1002/jhet.2681

Month 2016
An Organocatalyzed Efficient One-pot Synthesis, Biological Evaluation,
and Molecular Docking Studies of 4,4′-(Arylmethylene)bis-(3-methyl-1-
phenyl-1H-pyrazol-5-ols)
Pravin S. Mahajan,a Mukesh D. Nikam,a Vijay Khedkar,b,c Prakash Jha, Pravin V. Badadhe, and Charansingh H. Gill
d
a
a
*
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431 004, India
bCombi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune 411008, India
cSchool of Health Sciences, Discipline of Pharmaceutical Sciences, University of KwaZulu-Natal, Westville, Durban
4000, South Africa
dSchool of Chemical Sciences, Central University of Gujarat, Sector-30, Gandhinagar 38200 Gujarat, India
*
E-mail: chgill16@gmail.com
Received October 9, 2015
DOI 10.1002/jhet.2681
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
The synthesis of 4,4′-(arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ol) derivatives catalyzed by
ʟ-proline is a simple, versatile, and efficient method. In vitro antioxidant, anti-inflammatory, and antimicro-
bial activities of compounds 4a–j were studied. Furthermore, to rationalize the observed anti-inflammatory
activity data, molecular docking study has been performed against COX-2 enzyme which revealed a good
binding affinity for these molecules and could provide an insight into the various bonded and non-bonded
interactions guiding their binding affinity.
J. Heterocyclic Chem., 000, 00 (2016).
INTRODUCTION
in clinic which is an ever-increasing public health haz-
ard. Because of constant change in bacteria, the search
and development of effective antimicrobial agents are
essential in the fight against infectious diseases. The
increase in prevalence of multiple drug resistance has
shown the development of synthetic antimicrobial, an-
tioxidative, and anti-inflammatory drugs.
In the past few decades, pyrazoles and their chemistry
have drawn considerable attention of the researchers owing
to their high therapeutic values. Some of the drugs, like
deracoxib, phenazone, and celecoxib, are non-steroidal
anti-inflammatory agents, sulphaphenazole is an antimicro-
bial agent, possessing pyrazole as basic moiety, and are
already active in the market. It is also well known that,
the hydroxyl groups are mainly responsible for the radical
scavenging or antioxidant activity. Ascorbic acid with its
four hydroxyl groups is a widely used as antioxidant agent
(Fig. 1). These findings created our attention on the
compounds in which both the pyrazole and hydroxyl
Pathophysiological conditions and aerobic metabo-
lism lead to oxidative stress because of imbalance be-
tween
oxidants
and
antioxidants
that
is
overproduction of free radicals [1,2]. High level of Re-
active Oxygen Species (ROS) can cause damage to cell
structure, nucleic acids, lipids, and proteins and associ-
ated with several human diseases like cancer, neurode-
generative disorder, atherosclerosis, and aging [3,4].
The infection process frequently induces inflammation
and during inflammatory processes free radicals are re-
leased from phagocytes [5]. It is also well known that
free radicals are involved in the biosynthesis of prosta-
glandins which are major mediators of inflammation.
Therefore the compounds with antioxidant activity are
supposed to have good anti-inflammatory activity. Re-
sistance of bacteria to the toxic effects of antimicrobial
agents develops fairly easily both in the laboratory and
© 2016 Wiley Periodicals, Inc.

P. S. Mahajan, M. D. Nikam, V. Khedkar, P. Jha, P. V. Badadhe, and C. H. Gill
Vol 000
groups are in one core. In this respect, 4,4′-(arylmethylene)
bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols) were prepared
by one pot multicomponent synthesis. They act as
antihypnotic [6], antipyretic [7], gastric secretion stimula-
tory [8], antidepressant [9], and antifilarial [10] agents.
Because of the presence of pyrazole and hydroxyl group,
these compounds have attracted the present work to study
the combination of antioxidant, anti-inflammatory, and an-
timicrobial activity (Fig. 1).
In general, synthesis of 4,4′-(arylmethylene)bis
(3-methyl-1-phenyl-1H-pyrazol-5-ols) is performed under
non conventional conditions such as ultrasound irradiation,
microwave, and electrolysis [11] and also in catalytic
conditions like acetic acid or piperidine [12], sodium
dodecyl sulfate [13], TEBA [14], silica bonded
S-sulphonic acid [15], PEG-400 at 110°C [16], CAN
[17], and NH4OAc [18]. However, most of these synthetic
protocols suffer from some demerits. Therefore, the
development of new synthetic route with significant
practical value still remains a challenge.
Recently, we have reported our work on pyrazole
containing compounds with their molecular docking [22].
In continuation of this work, and our continuous focus on
safe and potent synthesis of biologically active heterocy-
cles [23–26], herein we report a simple and facile multi-
component one-pot synthesis of 4,4′-(arylmethylene)bis
(3-methyl-1-phenyl-1H-pyrazol-5-ols) (Scheme 1), using
ʟ-proline as an organocatalyst with their potential antimi-
crobial, antioxidant, and anti-inflammatory activities. Al-
though the synthesized compounds are achiral, we have
used chiral ʟ-proline; this is because the aim was not only
to synthesize pyrazol-5-ols using organocatalyst but also to
study their biological potential and their molecular docking
to rationalize the observed anti-inflammatory activity data.
RESULTS AND DISCUSSION
Chemistry.
In the initial study, the reaction of
phenyl hydrazine (1) (2 equiv), ethyl acetoacetate (2)
The application of enantiomerically pure, “small”
organic molecules represents a promising alternative
catalytic concept in addition to other frequently used
syntheses based on metal containing catalysts [19]. In
this respect, ʟ-proline has drawn much interest in dif-
ferent organic reactions because of its rigid ring struc-
ture, experimental simplicity, ease of handling, cost
effectiveness, and excellent solubility in water and or-
ganic solvents [20]. In need of environmentally com-
patible solvents, ethanol and water are gaining
considerable significance for organic reactions. As a
result, the discovery of synthetic methodologies to fa-
cilitate the preparation of compound libraries using
MCRs without the use of hazardous solvents and cata-
lysts are the pivotal focal points in industry and acade-
mia [21].
(2 equiv), and benzaldehyde (1 equiv) was carried out
using ʟ-proline as
a catalyst in water at reflux
condition, but the reaction proceeded to completion
with low yield and more time (Table 1, entry 1). To
improve the yield and reduce the reaction time
different solvents like non-polar (THF, 1,4-dioxane)
and polar-aprotic (acetonitrile, DMF), polar-protic
(methanol, ethanol) were used but best result was
obtained in ethanol (Table 1, entries 2–7).
With these optimistic results in hand, further investi-
gation was carried out for the catalytic evaluation of ʟ-
proline for the optimum reaction conditions. It was
found that the amount of catalyst plays a vital role.
An increase in the amount of ʟ-proline from 5 to 20
mol % led to an increase in the yield (Table 1, entries
7–10). The use of 25 mol% of ʟ-proline in ethanol did
Figure 1. Structures of pyrazole and hydroxy group containing drugs and the studied molecules.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
Scheme 1. Synthesis of 4,4′-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ols).
not improve the yield (Table 1, entry 11). However, in
the absence of ʟ-proline, no product was obtained (Ta-
ble 1, entry 12). This proves that a carboxylic acid
group with secondary nitrogen may be essential for
better catalytic activity. This is because ʟ-proline can
play a role of acid or a base or both simultaneously,
as a nucleophile and its ability to form iminium inter-
mediates upon reaction with carbonyl compounds [27].
Thus the optimum conditions required the use of 20
mol %, ʟ-proline in ethanol solvent at reflux.
In order to generalize the reaction a range of aromatic
and hetero-aromatic aldehydes was subjected to react
with ethyl acetoacetate and phenyl hydrazine in the pres-
ence of 20 mol % of ʟ-proline and ethanol as solvent
(Table 2). It was found that both aromatic and
heteroaromatic aldehydes reacted equally smoothly to af-
ford 4,4′-(arylmethylene)bis(3-methyl-1-phenyl-pyrazol-
5-ols). Interestingly, it is observed that the reaction
conditions are best suited for heterocyclic rings, which
gave excellent yield.
were displayed in the range of δ 6.73–7.74, and broad
singlet was observed at δ 13.97 ppm corresponding to
―OH group. Similarly, the 13C NMR showed reso-
nances at δ 12.1 (methyl group), δ 28.4 (―CH) and
also δ 121.5–154.4 confirms the presence of aromatic
carbon.
Based on the above observation and in accordance
with reports from the literature [28,29], a plausible
mechanism was proposed for the current reaction
(Fig. 2). The initial nucleophilic reaction of ʟ-proline
on the ethyl acetoacetate 2 followed by dehydration af-
fords intermediate 5, which on reaction with phenyl
hydrazine 1 and subsequently removal of ʟ-proline
gives the desired 3-methyl-1-phenyl-1H-pyrazol-5-ol
6. In the next step, nucleophilic addition of 6 on inter-
mediate 7 takes place to give arylidene 8. Finally, the
second molecule of 3-methyl-1-phenyl-1H-pyrazol-5-
ol adds in the Michael addition way to give
4,4′-(arylmethylene)bis(3-methyl-1-phenyl-pyrazol-5-
ols) 4a–j.
The structure of all compounds were compared and
confirmed by IR, 1H, 13C NMR, and HRMS spectral
techniques [11,13]. HRMS of compound 4g displayed
molecular ion peak at 443.1533 in consistent with its
molecular formula, C25H22N4O2S. The IR spectrum
showed ―OH stretching at 3420 cmꢀ1. The 1H NMR
In vitro antioxidant activity.
The antioxidant
potential of 4,4′-(arylmethylene)bis(3-methyl-1-phenyl-
1H-pyrazol-5-ol) derivatives (4a–j) was evaluated
using DPPH (1,1-diphenyl-2-picryl-hydrazil) radical
scavenging method. Radical scavenging activity of all
tested compounds was indicated by the interaction of
compounds with the stable free radical DPPH. As
shown in Table 3, in radical scavenging activity,
standard drug BHT showed EC50 at 8.25 μg/mL.
Among the tested compounds 4a–j, compound 4g with
EC50 (7.69 0.59 μg/mL) has shown the most potent
scavenging activity, and compound 4b with EC50
(19.03 1.16 μg/mL) showed good scavenging activity.
On the other hand compounds 4c, 4d, 4e, and 4i
showed moderate activity.
spectrum of 4g exhibited singlet at
δ 2.36 ppm
(―CH3) and δ 5.11 ppm (―CH). Aromatic protons
Table 1
Screening of solvent and catalyst for the synthesis of 4aa.
Catalyst
(mol %)
Time
(min)
Yieldb
(%)
Entry
Solvent
Water
THF
1,4-
1
2
3
ʟ-proline(20)
ʟ-proline(20)
ʟ-proline(20)
95
90
95
35
40
28
In vitro anti-inflammatory activity. Anti-inflammatory
agents play
a
vital role in the inhibition of
Dioxane
Acetonitrile
DMF
Methanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
cyclooxygenase enzymes that are responsible for the
conversion of arachidonic acid to prostaglandins. Because
human red blood cell (HRBC) membranes are similar to
these lysosomal membrane components [30], the
prevention of hypotonicity induced HRBC membrane
lysis was evaluated in estimating the anti-inflammatory
property. Compounds 4a–j were screened for in vitro
anti-inflammatory activity (Table 3). The standard drug
Diclofenac sodium showed EC50 at 11.70 μg/mL.
Compound 4g showed potent anti-inflammatory activity
with EC50 of 10.87 0.65 whereas, compound 4c showed
4
5
6
7
8
9
10
11
12
ʟ-proline(20)
ʟ-proline(20)
ʟ-proline(20)
ʟ-proline(20)
ʟ-proline(5)
ʟ-proline(10)
ʟ-proline(15)
ʟ-proline(25)
—
85
80
80
65
80
75
75
70
—
55
48
62
88
55
62
68
86
No
product
aReaction conditions: benzaldehyde (1 mmol), Ethyl acetoacetate
(2 mmol), Phenyl hydrazine (2 mmol), EtOH (10 mL).
bYields based on crystallization from ethanol.
Journal of Heterocyclic Chemistry
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P. S. Mahajan, M. D. Nikam, V. Khedkar, P. Jha, P. V. Badadhe, and C. H. Gill
Vol 000
Table 2
Physical data of 4,4′-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ols) 4a–ja.
S. no.
R
Time (min)
Yieldb (%)
M.p. (lit. m.p.)/°C
4a
4b
4c
4d
4e
4f
4g
4h
4i
C6H5
3-Cl-C6H4
2-Cl-C6H4
4-OCH3-C6H4
4-N(CH3)2-C6H4
4-NO2-C6H4
2-Thiophene
2-Furyl
65
60
70
80
65
60
50
55
55
60
88
89
86
84
86
90
95
94
92
93
170–172 (170–172 [11])
152–154 (150–151 [11])
237–239 (237–239 [11])
198–200 (199–201 [13])
180–182
230–232 (230–232 [11])
190–192 (190–192 [11])
188–191 (189–191 [11])
170–173
2-Imidazolyl
2-Indolyl
4j
158–162
aReaction conditions: Aldehyde (1 mmol), Ethyl acetoacetate (2 mmol), Phenyl hydrazine (2 mmol), EtOH (10 mL).
bYields based on crystallization from ethanol.
Figure 2. Plausible mechanism for the formation of 4a–j.
good activity with EC50 of 12.25 2.03 μg/mL compared
to DFS. Compounds 4d, 4i, and 4j displayed moderate
activity.
In vitro antimicrobial activity.
compounds were evaluated for in vitro antimicrobial
activity against four different bacterial strains and two
All the synthesized
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4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
Table 3
In vitro antioxidant and anti-inflammatory activity of compounds 4a–j.
Antioxidant activity
Anti-inflammatory activity
EC50 SD (μg/mL)
Compound
Glide score
Glide energy
EC50 SD (μg/mL)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
BHT
DFS
32.13 2.03
19.03 1.16
23.25 1.03
28.01 1.19
25.45 1.37
56.11 1.92
7.69 0.59
63.79 1.16
23.03 1.45
39.19 1.66
8.25 0.33
—
50.00 2.13
44.67 1.36
12.25 2.03
27.60 1.04
39.83 0.94
48.13 0.38
10.87 0.65
55.94 0.54
29.67 2.13
30.23 2.18
—
ꢀ6.661
ꢀ7.00
ꢀ8.00
ꢀ7.92
ꢀ7.14
ꢀ6. 912
ꢀ8.91
ꢀ6.417
ꢀ7.652
ꢀ7.440
—
ꢀ25.320
ꢀ28.595
ꢀ52.058
ꢀ40.669
ꢀ31.595
ꢀ28.920
ꢀ55.593
ꢀ23.585
ꢀ37.032
ꢀ34.595
—
11.70 0.98
ꢀ8.07
ꢀ54.592
Values are expressed as mean standard deviation (n = 3), BHT (butylated hydroxy toluene), DFS—diclofenac sodium.
fungal strains. The results for antimicrobial activity are
summarized in Table 4. In between the tested strains,
compound 4g which contains thiophene ring exhibited
potent antimicrobial activity against Gram-negative
bacterial strains Escherichia coli, and Micrococcus
luteus and antifungal strain Aspergillus niger compared
to the used standard. Compound 4e bearing ―N(CH3)
Candida albicans. Other compounds showed moderate
antibacterial and antifungal activity.
Molecular docking.
Molecular docking study was
performed to gain an insight into the mode of binding
and the associated molecular interactions that govern
the binding of the title compounds 4,4′-(arylmethylene)
bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
with
the
group on the phenyl ring was found to exhibit
excellent activity against E. coli strain. Compound 4c
with ―Cl substitution at position 2 of phenyl ring
target enzyme COX-2. While doing so, the structures
of the title compounds (4a–j) as well as the enzyme
were kept flexible to explore the conformational space
within the active site(s) and the best docked complex
obtained from it has been analyzed. The molecular
docking protocol adopted in the current study was
validated by extracting the native ligand diclofenac
from its binding site and re-docking it which produced
2
exhibited
good
activity
against
Pseudomonas
aeruginosa and both fungal strains. Compound 4d
possessing methoxy at position 4 of phenyl ring
exhibited good activity against bacterial strains E. coli
and Staphylococcus aureus and antifungal strain
Table 4
In vitro antimicrobial activity of compounds 4a–j.
Antibacterial activitya
Antifungal activitya
Gram negative
Gram positive
S. aureus
Compound
E. coli
P. aeruginosa
M. luteus
C. albicans
A. niger
4a
4b
4c
4d
4e
4f
4g
4h
08 0.11
08 0.23
09 0.22
18 0.21
20 0.20
07 0.52
22 0.21
10 0.29
16 0.11
14 0.27
20 0.11
19 0.09
NA
06 0.07
06 0.15
15 0.06
13 0.20
07 0.22
12 0.18
14 0.20
12 0.23
13 0.13
12 0.11
18 0.12
20 0.08
NA
08 0.28
11 0.21
08 0.25
17 0.08
08 0.10
11 0.22
13 0.11
14 0.33
13 0.22
11 0.08
19 0.10
21 0.14
NA
09 0.18
08 0.08
14 0.27
14 0.18
11 0.21
09 0.50
16 0.06
13 0.13
12 0.16
09 0.10
22 0.13
20 0.17
NA
13 0.21
14 0.17
16 0.20
18 0.12
13 0.16
11 0.25
15 0.13
10 0.29
14 0.12
10 0.32
NA
12 0.23
14 0.13
16 0.08
09 0.42
12 0.21
11 0.12
18 0.15
08 0.23
11 0.21
09 0.47
NA
4i
4j
Ciprofloxacin
Streptomycin
Fluconazole
NA
20 0.12
NA
16 0.14
aZone of inhibition in mm (mean three replicate standard deviation), NA—not applicable.
Journal of Heterocyclic Chemistry
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P. S. Mahajan, M. D. Nikam, V. Khedkar, P. Jha, P. V. Badadhe, and C. H. Gill
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Vol 000
a
(ꢀ1.908 kcal/mol), Leu352 (ꢀ2.633 kcal/mol), Val116
(ꢀ1.897 kcal/mol), Val89 (ꢀ1.288 kcal/mol), and Pro86
(ꢀ0.164 kcal/mol) residues through the phenyl rings;
Glu524(ꢀ3.083 kcal/mol), Val523 (ꢀ4.558 kcal/mol),
Phe518 (ꢀ1.450 kcal/mol) through the pyrazole ring and
with residues Val349 (ꢀ2.600 kcal/mol), Gln350
(ꢀ1.311 kcal/mol), Leu534 (ꢀ1.379kcal/mol), and
Leu531 (ꢀ1.054 kcal/mol) through the thiophene ring
stabilizing the complex of 4g with COX-2. Furthermore,
significant electrostatic interactions were observed with
Ala527 (ꢀ1.205 kcal/mol) Ser353 (ꢀ1.162 kcal/mol), and
Arg120 (ꢀ3.731kcal/mol) through the pyrazole ring while
with Tyr355 (ꢀ1.106) through the phenyl rings
contributed to the enhanced binding affinity of 4g. This
enhanced binding affinity can also be attributed to the three
prominent pi-pi stacking interactions observed between the
phenyl rings of 4g and Tyr385 (1.854Å), Trp387
(2.384 Å), and Tyr355 (2.399Å) residues lining the active
site of COX-2.
The binding mode of the second most active compound
4c revealed a similar topology as observed for 4g (Fig. 5).
Furthermore, the per residue ligand interaction energy
distribution showed that this compound as well is anchored
to the active site of COX-2 through a chain of favorable
van der Waals and electrostatic interactions along with a
pi–pi stacking interaction. The compound binds with an
overall binding energy of ꢀ52.058kcal/mol and a docking
score of ꢀ8.00 which was slightly less than compound 4g.
The compound was observed to be stabilized within the
active site through a series of favorable van der Waals
interactions with Ser530 (ꢀ1.190), Gly526 (ꢀ1.407),
Met522 (ꢀ1.514) Leu359 (ꢀ1.284), Leu384 (ꢀ1.011),
Phe381 (ꢀ1.799), Leu352 (ꢀ2.509), Val116 (ꢀ1.575),
Val89 (ꢀ1.700), and Pro86 (ꢀ1.000) residues through
the phenyl rings. Similarly the pyrazole rings also exhib-
ited significant van der Waals interactions with Glu524
(ꢀ2.314), Val523 (ꢀ3.567), and Phe518 (ꢀ1.640) while
the ortho-chloro phenyl ring interacted with Val349
(ꢀ2.228), Gln350 (ꢀ1.201), Leu534 (ꢀ0.979), and
Leu531 (ꢀ0.941) residues in the active site. The electro-
static interactions observed with Ala527 (ꢀ1.194377),
Ser353 (ꢀ1.19639), and Arg120 (ꢀ3.034) further contrib-
uted to the binding affinity. While most of the per-residue
interactions were conserved among 4g and 4c, the pi–pi
stacking interactions observed between 4g and Tyr385
(1.854 Å) and Trp387 (2.384Å) residues were found to
be missing for 4c (Fig. 6). However, it did display a close
pi–pi stacking interaction with Tyr355 (2.766Å). In
addition, to relatively lower values of per-residue interac-
tions and the missing pi–pi stacking interactions in case
of 4c, the distance between the ligand and the interacting
residues was also found to be higher for 4c compared to
4g (Supporting Information) which could be also the
reason for lower binding affinity of 4c over 4g.
between the experimental conformation and that
predicted from docking (Fig. 3). This signifies the
reliability of the docking protocol to predict the
receptor bound conformation for the investigated
compounds.
Molecular docking study revealed that all the pyrazol-
5-ols were successfully localized into the active site of
COX-2 in positions very close to that of the native ligand
diclofenac with varying degree of affinities, and their
complexation was stabilized by formation of several
bonded and non-bonded interactions. A very significant
correlation was observed between theoretical predictions
and the experimentally observed anti-inflammatory activity
(expressed as EC50) with the active compounds showing
higher docking scores while those with relatively low ac-
tivity were also predicted to have lower scores (Table 3).
Their binding energies were also found to be negative
ranging from ꢀ55.593kcal/mol to ꢀ23.585 kcal/mol
indicating that these compounds could serve as a pertinent
starting point for the rational design of drugs targeting
COX-2.
A detailed per-residue interaction analysis
between the enzyme and the pyrazol-5-ols derivatives
was carried on each of the docked complex through which
we can speculate regarding the detailed binding patterns in
the cavity and also provide an explanation for the factors
governing the binding affinity towards the enzyme. For
the sake of brevity the per-residue interactions analysis only
can be elucidated for the most active compounds 4g and 4c.
The lowest energy docked conformation of the most ac-
tive compound 4g (Fig. 4) revealed that the compound
binds with an overall binding energy of ꢀ55.593 kcal/mol
(diclofenac: ꢀ54.592 kcal/mol) making intimate contact
with the residues lining the active site of COX-2 through
significant bonded and non-bonded interactions. An
extensive network of favorable van der Waals interactions
were observed with Ser530 (ꢀ1.413kcal/mol), Gly526
(ꢀ2.528 kcal/mol), Met522 (ꢀ1.948kcal/mol), Leu359
(ꢀ1.539 kcal/mol), Leu384 (ꢀ1.508 kcal/mol), Phe381
Figure 3. Validation of docking protocol: overlay of X-ray conformation
(green carbon) of Diclofenac over its best docked conformation (orange
carbon) into the active site of COX-2. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
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Month 2016
4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
Figure 4. The binding mode of 4g into the active site of COX-2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
phenyl-1H-pyrazol-5-ol) derivatives exhibited a good %
ABS ranging from 66.93 to 82.74%. Furthermore, only
compounds 4b, 4c, and 4j violated Lipinski’s rule of five
(miLog P ≤ 5) [34,35]. Other compounds did not violate
Lipinski’s rule of five, thus showing good drug like
properties. A molecule likely to be developed as an orally
active drug candidate should show no more than one
violation of the above shown criteria. All the compounds
except 4b, 4c, and 4j followed the criteria for orally
active drug, and therefore, these compounds may have a
good potential for eventual development as oral agents.
Drug likeness results have shown that most of the
Figure 5. Overlay of best docked conformation of 4g (green carbon) on
compounds are having good model score, and hence,
these molecules showed good drug-like properties.
4c (orange carbon). [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
Structure–activity relationship (SAR).
The results of
the anti-inflammatory, antioxidant, and antimicrobial
screening confirmed some specific and remarkable facts
about the SAR of synthesized molecules. The structural
variations in molecules also show the variation and
dependence of the activity profile of molecules. The
important highlights of SARs are:
Effect of the substituent on phenyl ring. The presence of
phenyl ring attached to (4a) without any substitution at
ortho, meta, and para position does not make the
molecule specifically active towards biological activity.
A similar network of interaction was observed for the
other pyrazol-5-ols as well but decreasing gradually
with their observed anti-inflammatory activity. Overall,
the molecular docking results obtained for the pyrazol-
5-ol derivatives were found to be in agreement with
their observed anti-inflammatory activity. The per-
residue ligand–receptor interaction analysis revealed that
the primary driving force for the mechanical
interlocking of these ligands with COX-2 was their ste-
ric complementarity with the target as is evidenced from
the relatively higher contribution of van der Waals in-
teraction over other components contributing to the
binding scores. The binding pattern predicted by
docking complemented with a detailed per residue inter-
action analysis can aid in site specific modification in
scaffold to facilitate the rational design of anti-
inflammatory leads targeting COX-2 enzyme.
Effect of chloro group.
The chloro group and its
position to phenyl ring have an essential role in
determined the antioxidant and anti-inflammatory
activity of molecules. Substitution of the chloro group
at meta position on the phenyl ring (4b) causes the
molecule specifically active towards DPPH radical
scavenging activity. While substitution of chloro group
at ortho position (4c) makes the molecule active
against anti-inflammatory screening. This clearly
indicates that the change in position of the chloro
group from meta to ortho on the phenyl ring is
favorable regarding the development of broad spectrum
molecule active against antioxidant and anti-
inflammatory activity.
In silico absorption, distribution, metabolism, excretion
(ADME), and drug likeness analysis.
A computational
study of synthesized compounds 4a–4j was performed
for prediction of ADME properties using Molinspiration
online property calculation toolkit [31] and drug likeness
using molsoft [32], and the values are presented in Table
5. Absorption (% ABS) was calculated by:
ABS =109 ꢀ(0.345 × TPSA) [33]. It is observed that, all
the synthesized 4,4′-(arylmethylene)bis(3-methyl-1-
%
Effect of the substituent at 4-position on phenyl ring. The
substitution of electron donating methoxy group (4d)
Journal of Heterocyclic Chemistry
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P. S. Mahajan, M. D. Nikam, V. Khedkar, P. Jha, P. V. Badadhe, and C. H. Gill
Vol 000
Figure 6. The binding mode of 4c into the active site of COX-2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and dimethylamino group (4e) and electron
withdrawing nitro group (4f) at para position on the
phenyl ring clearly shows that, electron donating
group (active against E. coli and C. albicans) is
better than electron withdrawing group (the nitro
group which makes molecule inactive against
microbial strains), for the development broad
spectrum antimicrobial molecule.
Effect of heterocyclic aromatic ring. In compound 4g
the replacement of phenyl ring by thiophene ring gives
the most potent and broad spectrum molecule of the
series. From the obtained data compound 4g showed
more potency for antioxidant activity with EC50 7.69
0.59 μg/mL and against bacterial strain E. coli and
fungal strain A. niger with maximum zone of
inhibition. This indicates that such a modification
helps the molecule to make more potent against
oxidants and also the microbium E. coli and A. niger.
On the other hand, introduction of furan (4h), pyrazole
(4i), and indole (4j) resulted in the formation of less
active molecule, and these heterocyclic rings are not
suitable to develop antioxidant, anti-inflammatory, and
antimicrobial compounds.
EXPERIMENTAL
All chemicals and reagents used in current study were
of analytical grade. The melting points of all synthe-
sized compounds were determined in open capillary
tubes and were uncorrected. Reactions were monitored,
and purity of the products was checked by thin layer
chromatography (TLC). IR spectra were recorded on
1
Jasco FT-IR-4100, Japan, in KBr disc. H NMR spectra
were recorded on a Bruker Advance-II 300-MHz spec-
trometer in DMSO-d6 (δ in ppm relative to
tetramethylsilane). 13C NMR spectra were recorded on
a
Bruker Advance II 75-MHz spectrometer in
DMSO-d6. Mass spectra were recorded on a Q-Tof
Micromass spectrometer (Water) by the electro-spray
method (ES). In oxidant and anti-inflammatory activity
UV–visible spectrophotometer-Jasco model V-630 was
used to calculate absorbance.
General procedure for the synthesis of 4,4′-
(arylmethylene)-bis(3-methyl-1-phenyl-1H-pyrazol-5-ols)
(4a–j). A mixture of phenyl hydrazine 1 (2 mmol),
ethyl acetoacetate 2 (2 mmol), and ʟ-proline (20 mol
%) in ethanol (10 mL) was refluxed for 30 min;
Table 5
Pharmacokinetic properties important for good oral bioavailability of compounds 4a–j.
Compound
MW
% ABS
Volume (A3)
TPSA (A2)
NROTB
HBA
HBD
Log P
Lipinski’s violations
Rule
4a
4b
4c
4d
4e
4f
4g
4h
4i
<500
436.51
—
—
—
—
05
05
05
06
06
06
05
05
05
05
<10
06
06
06
07
07
09
06
07
08
07
<5
02
02
02
02
02
02
02
02
03
03
≤5
≤1
00
01
01
00
00
00
00
00
00
01
82.74
82.74
82.74
79.55
81.62
66.93
82.74
78.21
72.84
77.29
398.08
411.62
411.62
423.63
443.99
421.41
388.79
379.65
378.91
427.06
76.10
76.10
76.10
85.34
79.34
121.93
76.10
89.24
104.79
91.89
4.65
5.30
5.28
4.70
4.75
4.61
4.55
3.90
2.74
5.11
470.96
470.96
466.54
479.58
481.51
442.54
426.47
426.48
475.55
4j
% ABS—percentage of absorption; MW—molecular weight; TPSA—topological polar surface area; NROTB—number of rotatable bonds; Log P—
octanol–water partition coefficient; HBA—number of hydrogen bond acceptors; HBD—number of hydrogen bond donors.
Journal of Heterocyclic Chemistry
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Month 2016
4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
further aromatic aldehyde 3 (1 mmol) was added to the
reaction mixture and reflux continued till completion
as shown in Table 1. Progress of the reaction was
monitored by TLC. After completion of reaction, the
residue was filtered and recrystallized from ethanol:
chloroform (7:3) to obtain the pure desired products
4a–j. All the compounds were characterized by
spectroscopic techniques (FT-IR, 1H NMR, 13C
NMR, and HRMS).
157.3; HRMS (ESI+) m/z: (M + H)+ calcd for
C28H26N4O3: 467.2081; found: 467.2070.
4-((4-(Dimethylamino)phenyl)(5-hydroxy-3-methyl-1-phenyl-
1H-pyrazol-4-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4e). Compound 4e was obtained as orange solid; ir
(KBr): 3426, 3045, 1589, 1571, 1480, 1371, 1305, 1284,
1
1130, 1065, 1010, 875, 755, 710cmꢀ1; H nmr (300MHz,
DMSO-d6): δ 2.24 (s, 6H, CH3), 3.12 (s, 6H, NCH3), 5.20
(s, 1H, CH), 7.21 (t, 2H, ArH), 7.38–7.43 (m, 6H, Ar―H),
7.49 (d, 2H, Ar―H), 7.83 (d, 4H, Ar―H), 13.90 (brs, 2H,
OH); 13C nmr (75 MHz, DMSO-d6): δ 12.5, 34.9, 55.2,
121.6, 124.4, 125.4, 128.8, 130.8, 133.3, 137.3, 140.1,
142.2, 146.1, 155.1; HRMS (ESI+) m/z: (M + H)+
calcd. for C29H29N5O2: 480.2397; found: 480.2392.
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
(4-nitrophenyl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4f). Compound 4f was obtained as white solid. ir (KBr):
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
(phenyl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol (4a).
Compound 4a was obtained as pale yellow solid; ir
(KBr): 3430 (OH), 2920 (CH), 1615, 1493, 1410, 1290,
1
1140, 1020, 913, 854, 789, 754, 730, 690cmꢀ1; H nmr
(300MHz, DMSO-d6): δ 2.32 (s, 6H, CH3), 4.96 (s, 1H, CH),
7.12 (m, 1H), 7.23–7.29 (m, 6H, Ar―H), 7.40 (t, J = 8.0 Hz,
4H, Ar―H), 7.87 (d, J = 7.6 Hz, 4H, Ar―H), 13.96
(brs, 2H, OH); 13C nmr (75 MHz, DMSO-d6): δ 12.1,
33.5, 121.3, 126.4, 126.5, 127.7, 128.7, 129.5, 130.0,
137.4, 142.5, 146.8; HRMS (ESI+) m/z: (M + H)+
calcd. for C27H24N4O2: 437.1975; found: 437.1969.
4-((3-Chlorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4b). Compound 4b was obtained as white solid; ir
(KBr): 3549 (OH), 3072, 2975, 2918 (CH), 1741 (C¼O),
1594 (C¼C), 1500, 1415, 1323, 1297, 1187, 889,
3390, 3080, 2905, 1585, 1470, 1400, 1333, 734, 669cmꢀ1
;
1H nmr (300 MHz, DMSO-d6): δ 2.35 (s, 6H, CH3), 5.26
(s, 1H, CH), 7.21–7.27 (m, 2H, Ar―H), 7.38–7.42 (t,
4H, Ar―H), 7.49–7.55 (d, 2H, Ar―H), 7.64–7.66 (d,
4H, Ar―H), 8.10–8.16 (d, 2H, Ar―H), 13.81 (brs, 2H,
OH); 13C nmr (75 MHz, DMSO-d6): δ 12.5, 34.4, 121.9,
124.6, 127.0, 129.9, 130.2, 133.5, 136.6, 143.3, 147.2,
147.5, 151.6; HRMS (ESI+) m/z: (M+H)+ calcd. for
C27H23N5O4: 482.1822; found: 482.1820.
1
756cmꢀ1; H nmr (300MHz, DMSO-d6): δ 2.30 (s, 6H,
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
(thiophen-2-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4g). Compound 4g was obtained as white solid; ir
(KBr): 3420, 2980, 2920, 1600, 1490, 1415, 1294, 769,
2CH3), 4.97 (s, 1H, CH), 7.21–7.69 (m, 14H, ArH),
12.60 (s, 1H, OH), 13.90 (s, 1H, OH); 13C nmr (75MHz,
DMSO-d6): δ 14.4 (CH3), 39.8 (CH), 104.3, 121.4, 125.0,
126.2, 128.0, 128.2, 129.0, 129.6, 130.9, 140.0, 144.0,
147.9, 148.3,; HRMS (ESI+) m/z: (M + H)+ calcd.
for C27H23ClN4O2: 471. 9579; found: 471. 9783.
4-((2-Chlorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4c). Compound 4c was obtained as white solid; ir (KBr):
3450, 3070, 2925, 1610, 1560, 1505, 1441, 1360, 1299,
1
692cmꢀ1; H nmr (300MHz, DMSO-d6): δ 2.36 (s, 6H,
CH3), 5.11 (s, 1H, CH), 6.73–6.75 (m, 1H, Ar―H),
6.89–6.91 (m, 1H, Ar―H), 7.23–7.31 (m, 3H, Ar―H),
7.40 (t, 4H, Ar―H), 7.74 (d, 4H, Ar―H), 13.97 (brs,
2H, OH); 13C nmr (75 MHz, DMSO-d6): δ 12.1, 28.4,
121.5, 125.0, 125.1, 126.2, 128.9, 131.9, 134.1, 147.7,
154.2; HRMS (ESI+) m/z: (M + H)+ calcd. for
C25H22N4O2S: 443.1541; found: 443.1533.
844, 757, 690cmꢀ1 1H nmr (300MHz, DMSO-d6): δ
;
2.24 (s, 6H, CH3), 5.08 (s, 1H, CH), 7.27–7.29 (m, 2H,
Ar―H), 7.41–7.43 (m, 8H), 7.75–7.77 (m, 4H), 13.92
(brs, 2H, OH); 13C nmr (75 MHz, DMSO-d6): δ 11.6,
32.4, 120.6, 123.6, 126.9, 128.0, 128.9, 129.4, 130.3,
135.9, 137.3, 139.4, 140.6, 141.1; HRMS (ESI+) m/z: (M
+H)+ calcd. for C27H23ClN4O2: 471.1586; found: 471.1579.
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
(4-methoxyphenyl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
4-((Furan-2-yl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol (4h).
Compound 4h was obtained as white solid; ir (KBr):
3420, 2980, 2920, 1600, 1490, 1415, 1294, 769,
1
692 cmꢀ1; H nmr (300MHz, DMSO-d6): δ 2.36 (s, 6H,
CH3), 5.11 (s, 1H, CH), 6.73–6.75 (m, 1H, Ar―H),
6.89–6.91 (m, 1H, Ar―H), 7.23–7.31 (m, 3H, Ar―H),
7.40 (t, 4H, Ar―H), 7.74 (d, 4H, Ar―H), 13.97 (brs,
2H, OH); 13C nmr (75 MHz, DMSO-d6): δ 12.1, 28.4,
121.5, 125.0, 125.5, 126.2, 128.9, 131.9, 134.1, 147.7;
HRMS (ESI+) m/z: (M + H)+ calcd. for C25H22N4O3:
427.1770; found: 427.1773.
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)
(1H-imidazol-2-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol
(4i). Compound 4i was obtained as white solid; ir (KBr):
3440, 3110 (NH), 2995, 2921, 1605, 1515, 1420,
(4d).
Compound 4d was obtained as white solid; ir
(KBr): 3431, 2930, 2827, 1607, 1508, 1410, 1290, 1150,
1004, 913, 854, 789, 754, 731, 688cmꢀ1 1H nmr
;
(300 MHz, DMSO-d6): δ 2.34 (s, 6H, CH3), 3.70 (s, 3H,
OCH3), 5.01 (s, 1H, CH), 7.25–7.29 (m, 4H), 7.38–7.46
(m, 6H), 7.71–7.73 (d, J = 8.0 Hz, 4H), 13.86 (brs, 2H,
OH); 13C nmr (75 MHz, DMSO-d6): δ 11.3, 31.7, 55.0,
114.8, 119.2, 126.5, 128.2, 129.3, 130.4, 134.9, 140.1,
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P. S. Mahajan, M. D. Nikam, V. Khedkar, P. Jha, P. V. Badadhe, and C. H. Gill
Vol 000
1
1300 cmꢀ1; H nmr (300 MHz, DMSO-d6): δ 2.34 (s, 6H,
incubated at 37°C for 30min and centrifuged for 30min at
3000rpm. The hemoglobin content of the supernatant
solution was estimated at 560 nm spectrophotometrically.
Each experiment was performed in triplicate and distilled
water as control in this study. Where the blood control
represents 100% lysis or zero percent stability, the
percentage of HRBC heamolysis calculated by formula,
CH3), 5.14 (s, 1H, CH), 6.65–6.76 (dd, 2H, pyrazole
ring-H), 6.85–7.71 (m, 10H, Ar―H), 10.80 (brs, 1H,
NH), 14.01 (brs, 2H, OH); 13C nmr (75 MHz, DMSO-d6):
δ 12.6, 38.4, 121.8, 125.1, 126.3, 128.2, 128.7, 128.6,
129.8, 132.5, 133.7, 140.5, 151.9; HRMS (ESI+) m/z: (M
+H)+ calcd. for C24H22N6O2: 427.1879; found: 427.1877.
4-((5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(1H-
indol-3-yl)methyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol (4j).
Compound 4j was obtained as white solid; ir (KBr): 3430,
% Heamolysis ¼ ðO: D: of Control- O: D: of Test
sample = O: D: of ControlÞ X 100
3322, 2923, 1604, 1499, 1408, 1282, 756cmꢀ1; H nmr
1
The concentration of a compound where
50% of its maximal effect is observed
ðEC50Þ using graph pad prism was measured:
(300 MHz, DMSO-d6): δ 2.36 (s, 6H, CH3), 5.14 (s, 1H,
CH), 6.61–6.63 (m, 2H), 6.82–7.71(m, 13H, Ar―H),
9.62 (brs, 1H, NH) 13.93 (brs, 2H, OH); 13C nmr
(75 MHz, DMSO-d6): δ 12.1, 31.6, 104.5, 117.9, 119.2,
120.1, 122.0, 123.7, 126.5, 128.0, 129.1, 136.3, 137.6,
138.1, 146.5, 147.8, 148.5; HRMS (ESI+) m/z: (M+ H)+
calcd. for C29H25N5O2: 476.2084; found: 476.2076.
General procedure for antimicrobial activity.
The
standard strains were procured from the NCIM, Pune,
India. All the synthesized compounds were screened for
in vitro antibacterial and antifungal activity (Table 4).
The determination of antibacterial and antifungal activity
was done by using disc diffusion method at 100 μg/mL
concentration [41,42]. Ciprofloxacin and streptomycin
were used as a standard drug for the comparison of
antibacterial activity and Fluconazole for antifungal
activity. Dimethyl sulfoxide (DMSO) was used as solvent
control. Inoculated Mueller Hinton agar for bacteria
culture and sabouraud dextrose agar for fungal culture
was preferred. Sterile filter paper disks (6 mm diameter)
were moistened with the test compound solution in
DMSO of specific concentration 100 μg/disc and were
carefully placed on the agar culture plates that had
been previously inoculated separately with the
microorganisms. The plates were incubated at 37°C, and
the results were recorded for antibacterial activity after
14h, and for antifungal activity after 30h. Each
experiment was carried out in triplicate, and the average
diameter of inhibition zones of bacterial or fungal growth
around the disks in mm were recorded by Himedia
antibiotic zone scale [43].
General procedure for antioxidant activity.
DPPH
(1,1-diphenyl-2-picryl-hydrazil) radical scavenging method
was used to evaluate the antioxidant potential of
4,4′-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol)
derivatives (4a–j) [36–38]. The reaction was performed in
2mL of solution containing 0.2 mM freshly prepared DPPH
in methanol and different concentration of synthesized
compounds (10–100μg/mL). After incubation at room
temperature for 30min, the absorbance at 517 nm was
measured spectrophotometrically [39]. Each experiment was
performed in triplicate, and the activity was compared with
standard BHT. DPPH radical scavenging capacity was
measured by using formula,
Scavenging activity % ¼ Acontrol– Atest=Acontrolꢁ 100
Acontrol ¼ Absorbance of the control; Atest
¼ Absorbance of the test compounds:
The concentration required to obtain a 50% antioxidant
effect (EC50) was calculated using graph pad prism and is
compared with the standard.
General procedure for anti-inflammatory activity. The
HRBC membrane stabilization method has been used to
study the anti-inflammatory activity and Diclofenac
sodium as a standard drug [40]. Blood are collected from
healthy volunteers. Fresh whole human blood was
collected, and it was mixed with equal volumes of
sterilized Alsever’s solution (Dextrose 2%, Sodium
citrate 0.8%, Citric acid 0.05%, Sodium chloride 0.42%,
and Distilled water 100mL). This blood solution was
centrifuged for 10 min at 3000rpm and then washed three
times with equal volume of normal saline. The volume of
the blood is measured and reconstituted as 10% v/v
suspension with normal saline. The reaction mixture consists
of 1.0 mL of test sample of different concentrations in
normal saline and 0.5mL of 10% HRBC suspension, 1mL
of 0.2 M phosphate buffer, 1-mL hypo saline were
Molecular docking.
Molecular docking study was
performed by using the Glide (grid based ligand docking
with energy) program incorporated in the Schrödinger
molecular modeling suite (Schrödinger, LLC, New York,
NY, 2015) [44–46] to study the binding affinity of the
title compounds 4,4′-(arylmethylene)bis-(3-methyl-1-
phenyl-1H-pyrazol-5-ols) to the active sites of
cyclooxygenase-2 enzyme in an effort to increase the
understanding of the action of these compounds as anti-
inflammatory agents. The 3D crystal structures of
cyclooxygenase-2 (COX-2) in complex with its inhibitor
Diclofenac were retrieved from the Protein Data Bank
(www.rcsb.org; PDB ID: 1PXX) and were subjected to
the Protein Preparation Wizard workflow implemented in
the Schrödinger package. It was preprocessed by deleting
the crystallographically observed water molecules
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
4,4′-(Arylmethylene)bis-(3-methyl-1-phenyl-1H-pyrazol-5-ols)
(because no water molecule was observed to be
conserved), addition of hydrogen atoms corresponding to
the pH 7.0 considering the appropriate ionization states
for the acidic and basic amino acid residues, assigning
the bond order, identifying overlaps, creating zero order
bonds to metals, and creating disulfide bonds. Following
the assignment of appropriate charge and protonation
states, the enzyme structure was subjected to energy
minimization (RMSD of the atom displacement for
terminating the minimization 0.3 Å) using the OPLS-
2005 force field to relieve the steric clashes among the
residues because of addition of hydrogen atoms. The
active site of COX-2 was defined for docking using the
Receptor Grid Generation panel in Glide. The receptor
grid was generated by defining a 14 × 14 ×14 Å box that
was centered on the geometric centroid of the COX-2
structure which was sufficiently big to explore a large
region of the enzyme structure. On the other hand, 3D
structures of the title compounds were sketched using the
build panel in Maestro followed by optimization using
LigPrep module which performs addition of hydrogens,
adjusting realistic bond lengths and bond angles,
correction of chiralities and ionization states, generation
of tautomers, and ring conformations. Thereafter these
structures were also subjected to energy minimization
setting a RMSD cutoff of 0.001 Å, and the resulting
structures were then used for docking. The Glide
algorithm carries out a systematic search for favorable
interactions between one or more ligands and a receptor,
which is usually a protein through a complete search of
the conformational, orientation, and positional space of
the docked ligand. The Extra-precision (XP) scoring
function incorporated in Glide was used to rank the
resulting docked poses and to measure the binding
affinity of the ligands. The output files were visualized
for analyzing the interactions using the Maestro’s Pose
Viewer utility.
4,4′-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-
ols). Further biological evaluation to describe the mode of
action as well as study of animal models to evaluate the full
potential of 4,4′-(arylmethylene)bis(3-methyl-1-phenyl-
pyrazol-5-ols) is warranted. Furthermore, analysis of the
ADME parameters for synthesized compounds showed
good drug like properties and can be developed as oral
drug candidate. Thus, suggesting that compounds from
present series 4a, 4d, 4e, 4f, 4g, 4h, and 4i can be further
optimized and developed as a lead molecule. The theoreti-
cal predictions from molecular docking studies could
establish a link between their binding affinity and the
observed anti-inflammatory activity providing insights into
specific bonded and non-bonded interactions guiding the
activity. In conclusion, the combined results from the
in vitro and in silico studies provide a strong platform for
new structure-based design efforts towards management
of inflammation.
Acknowledgments. We gratefully acknowledge the University
Grants Commission, New Delhi for Providing Junior Research
Fellowship and The Head, Department of Chemistry, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad,
Maharashtra for Providing Laboratory Facilities. We are
thankful to the Haffkine research institute, Mumbai for
providing biological activities. We are also thankful to
Schrodinger Inc. for providing the evaluation license of
Schrödinger molecular modeling suite, and especially Vinod
Devarji for delivering the valuable support.
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CONCLUSION
The combination of in vitro antioxidant, anti-
inflammatory,
and
antimicrobial
screening
of
4,4′-(arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-
ols) was studied for the first time. Compound 4g emerged
as the most interesting compound in this series exhibiting
excellent DPPH radical scavenging activity and found to
be more potent than the standard drug BHT used. The
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time, no chromatographic separation, higher yield, and
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