Modeling molecular interactions of propounded pyrazole based drug candidates against bacterial DNA gyrase: Validation by syntheses and biological studies

Article abstract of DOI:10.1016/j.molstruc.2019.05.125

A class of nitrogen-based heterocycles, pyrazoles and their derivatives, are cardinal agents in the field of pharmacology. Here, we have conducted in silico studies on newly designed pyrazole based drug molecules, thereby revealing their activities and in

Full text of DOI:10.1016/j.molstruc.2019.05.125

Accepted Manuscript
Modeling molecular interactions of propounded pyrazole based drug candidates
against bacterial DNA gyrase: Validation by syntheses and biological studies
Shubhangi, Nikhil Kumar, Rajendiran Kanagaraj, Kashmiri Lal, Avijit Kumar Paul
PII:
S0022-2860(19)30697-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.05.125
MOLSTR 26632
Reference:
To appear in: Journal of Molecular Structure
Received Date: 26 April 2019
Revised Date: 28 May 2019
Accepted Date: 28 May 2019
Please cite this article as: Shubhangi, N. Kumar, R. Kanagaraj, K. Lal, A.K. Paul, Modeling molecular
interactions of propounded pyrazole based drug candidates against bacterial DNA gyrase: Validation
by syntheses and biological studies, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.05.125.
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Graphical Abstract

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Modeling molecular interactions of propounded pyrazole based
drug candidates against bacterial DNA gyrase: Validation by
syntheses and biological studies
Shubhangi1†, Nikhil Kumar1†, Rajendiran Kanagaraj1, Kashmiri Lal2 and
Avijit Kumar Paul1*
1Department of Chemistry, National Institute of Technology Kurukshetra, Kurukshetra-136119,
Haryana, India.
2Department of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar-
125001, Haryana, India.
Keywords: Substituted pyrazoles; Docking studies; DNA gyrase; Antimicrobial
activity; DFT study.
†: Both the authors contributed equally
* Email id: apaul@nitkkr.ac.in
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Abstract
A class of nitrogen-based heterocycles, pyrazoles and their derivatives, are cardinal agents in the field of
pharmacology. Here, we have conducted in silico studies on newly designed pyrazole based drug molecules, thereby
revealing their activities and interaction behaviors when docked against S. aureus DNA gyrase upon comparison
with a standard drug ciprofloxacin and previously reported few compounds. The drug likeliness of the compounds
was analyzed using QikProp module of Maestro 11.5 (Schrödinger) through ADME (Absorption, Distribution,
Metabolism and Excretion) analysis. Among the predicted compounds, we have synthesized four pyrazole
derivatives; namely 5-(3, 5-dimethyl-1H-pyrazol-1-yl) 1,3-benzoic acid, 5-(3, 5-dimethyl-1H-pyrazol-1-yl) 1,3-
benzenedicarboxylic acid, 4-(4-methyl-1H-pyrazole-1-yl) benzoic acid and 1-(4-carboxyphenyl)-1H-pyrazole-4-
carboxylic acid. All the four compounds are characterized by 1H and 13C NMR, IR, UV, and mass
spectrophotometry. We have successfully tested the same compounds for anti-microbial activities involving testing
with standard bacterial as well as fungal strains. Anti-bacterial activity testing was performed against two Gram-
positive bacteria: Staphylococcus aureus, Bacillus subtilis and three Gram-negative bacteria: Escherichia coli,
Pseudomonas aeruginosa, Salmonella enterica. Anti-fungal activity was carried out against a well-known fungal
strain Candida albicans. The observed antimicrobial activities are in well agreement with the docking results. The
DFT study was also carried out to compare the conformational stability of the optimized structures and docked-pose
of the four molecules.
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1. Introduction
Biologically active heterocycles, namely oxadiazoles, triazoles, thiadiazoles or pyrazoles, have been widely
used for therapeutics [1–3]. The azole groups in various heterocycles have compelling hydrophobicity and
pharmacokinetic properties that impart transmembrane diffusion ability to the drug to reach the target [1]. Pyrazoles
are indispensable members of the azole class having two adjacent nitrogen and three carbon atoms with a five-
membered ring. The pyrazole scaffold can be better understood in scheme1 as shown below. The N-1 atom in the
structure is “pyrrole-like” because of its unshared electrons are conjugated with the aromatic system while, the N-2
atom is “pyridine-like” as the paired electrons are not compromised with resonance, similar to pyridine systems.
Due to such differences in the N-atoms, pyrazoles are reactive against both acids and bases [4].
Scheme 1: Pyrazole skeleton
Pyrazole, though bearing high biological activity, is rarely found in nature because of the difficulty faced
by living organisms to form N-N bond [5]. Synthesis of the first pyrazole derived compound was reported in 1883
which was later followed by its usage in therapeutics [2]. A few primitive examples of naturally occurring pyrazoles
were: one as an isomer of histidine, isolated from the juice of watermelon from Citrulus vulgaris [5] and the other as
3-Nonyl-1H-pyrazole, extracted from Houttuynia cordata, which is a common plant in tropical Asia [4].
Pyrazoles and its derivatives are an important class of heterocyclic compounds which have immense scope
in various biological activities and catalysis due to their ability to coordinate metal ions [6] and form complexes
with metals like Pt, Pd, Cu, Zn, Au, Co, Ni, Fe, Ga and Au [7]. Scientists across the globe have left no stone
unturned in devising the different biological applications of these compounds including anti-inflammatory,
antibacterial, analgesic, anti-cancer, anti-tumor, anti-glaucoma, cardio-vascular, anti-chagasic, anti-pyretic, anti-
spasmodic, anti-neoplastic, anti-diabetic, anti-oxidant, anti-viral, anti-leishmanial, anti-parasitic and anti-allergic [2-
19]. Certain classes of condensed pyrazoles such as pyrrolo pyrazoles, pyrazolo indoles, pyrazolo triazoles, pyrazolo
isoindoles, pyrazolo pyrazolones have exhibited profound biological activities such as kinase inhibitors,
topoisomerase I & II inhibitors, antitumorigenic, herbicides, pesticides and anti-bacterial agents [1,2,5,12]. Some
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pyrazole-based drugs have been successfully tested against cyclin kinase and are into clinical trials for the treatment
of human cancers [20]. The target for pyrazole as anticancer drug ranges from human telomerase reverse
transcriptase [16], tyrosine kinase, cyclin dependent kinase, Aurora A. kinase, Activen receptor like kinase 5,
Mitogen activated protein kinase to fibroblast growth factor, Apoptosis inducing factor, and tumor growth factor
[21]. Some of the commercially popular pyrazole based drugs are Celecoxib (gastrointestinally safe NSAID,
analgesic and COX-2 inhibitor), Tepoxalin (a NSAID to treat osteoarthritis), Deracoxib (to treat osteoarthritis in
dogs) [3], Antipyrine, Phenylbutazone, Tepoxalin, Sildenafil (to treat erectile dysfunction), Dipyrone (analgesic and
antipyretic) and Rimonabant (for obesity treatment).
Among all the biological activities, anti-inflammatory and anti-microbial activities are the most
investigated behaviors of the pyrazole ring structure [22]. Quinolone-based drugs like ciprofloxacin, which have
always been broad spectrum antibiotics, are now prone to resistance against the disease-causing microbes for whom
pyrazoles are a good alternative which act upon DNA gyrase. Tanitame et.al had also successfully synthesized
pyrazole-based drugs targeting DNA gyrase [23-26]. In continuation with our earlier work on triazole–substituted
drugs [27-28], we have now focused on new pyrazole-based systems. Designing of new organic drugs has revealed
the extensive applications of pyrazole-based compounds; therefore, we have performed initial computational
analysis post in silico designing of some pyrazole-based molecules. Use of computational method allows us to
validate and evaluate all aspects of drug discovery at this time. Researchers with proficiency in computational drug
designing have a benefit of proposing new candidate quickly and at a cheaper value than others. When the structure
of the target is predictable, then molecular docking is the best tool for the evaluation of possible proffered candidates
[29]. The theoretical observed results should always be compared with a commercially available drug, like common
antibiotic drug ciprofloxacin, to obtain a broad spectrum of comparative behavior.
Herein, we have designed 47 new pyrazole derivatives and performed their docking studies along with 15
reported analogs, keeping in mind the basic desirable features of a good pharmacophore: aromatic ring current,
hydrophobic-hydrophilic substituents, H-bond acceptors and donors and optimum molecular weight. All the
electronic interactions and drug likeliness behaviors have also been compared with commercially available
ciprofloxacin drug which is effective against bacterial infections including S. enterica, S. aureus, and E. coli [24].
The predicted activities have been validated by experimental antimicrobial studies after synthesizing four parent
structures. The detailed structural predictions, docking studies, and validation of the theoretically predicted
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behaviors have been described in the present work. DFT calculations of the synthesized compounds have also been
carried out to elaborately understand the difference in molecular conformations, post docking.
2. Experimental Section
2.1 Molecular docking studies
Antibiotic resistant gram positive bacteria such as S. aureus, S. pnemoniae, enterococci are causative
agents of nosocomial infections and hospital acquired infections worldwide [30]. The commercially available drugs
against S. aureus are Cephalothin, Amikacin, Gentamicin, Cefpirome, Chloramphenicol, Tetracycline, etc. [22],
which are on the verge of impotency due to the increasing drug resistance by the bacterium. Therefore, we have
concentrated our docking studies done on crystal structure of DNA gyrase of S. aureus.
The docking studies of the molecules were carried out using Glide5.0 module of Schrödinger 11.5 software
[31]. The ligands were docked against protein structure 2XCT taken from PDB database with a resolution 3.35 Å.
The synthesized compound was further derivatized by substituting groups on the benzene ring and the pyrazole.
These derivatives were also docked at the same site in the protein and the results were compared with compounds
synthesized by Allison et. al [32] as well as with the standard drug ciprofloxacin. Out of the computationally
designed compounds derived from 4 basic skeletons (N-1, N-2, R-1 and R-16), we have successfully synthesized the
four aforementioned compounds.
2.2 DFT studies
Quantum mechanical calculations were performed for four synthesized compounds using DFT method,
with the help of Gaussian 09 software, at Becke’s three-parameter hybrid functional and correlation functional of
Lee, Yang And Pan (B3LYP), with 6-311+G (d,p) basis set in gas phase [33-35]. The default optimization and SCF
procedures were followed in the program execution. All the geometrical parameters and optimized structures were
determined for the four parent pyrazole compounds [36-38]. Then, the pose obtained for these compounds after
docking, were used for “Single point calculations” using DFT. A comparative analysis of the geometrical data of
parent compounds in their global minima state (obtained by geometry optimization) with the energies of their post
docking poses (bioactive minima) was also done. All the results pertaining to DFT studies were visualized through
Gauss view 5.0 software [39].
2.3 Materials preparation and characterizations
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All the starting materials and solvents: 5-aminoisophthalic acid (Sigma Aldrich), 4-hydrazinobenzoic acid
(Sigma Aldrich), Sodium nitrite (CDH), Sodium sulphite (TCI), Phosphorus oxychloride (Sigma Aldrich),
Potassium permanganate (CDH), Potassium carbonate (CDH), Ethyl acetate (Avra), Potassium hydroxide (Avra),
Conc. HCl (CDH), 2,4-Pentanedione (TCI), Dimethylformamide (Avra), ethanol (Avra) and isopropanol (Avra)
were used as purchased without any further purification. The IR spectra were obtained with KBr pellets on a
1
Shimadzu FT-IR spectrometer. The mass spectra were collected with XEVO G2-XS QTOF mass spectrometer. H
and 13C NMR were recorded at room temperature (298 K) on Bruker Avance-III spectrometer operating at 400
MHz. All the compounds are also characterized by UV-visible spectra measured from UV-Visible
Spectrophotometer, Perkin Elmer (Fig. S8 in Supporting information).
Scheme 2
Synthesis of compound N-1: The compound N-1 was synthesized and characterized by earlier reported method
[40].
Scheme 3
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Synthesis of compound N-2: 5-aminoisophthalic acid (1g, 5.52 mmol) was suspended in ~6 mL water along with
the addition of 1.5 mL conc. HCl. The resultant mixture was cooled at 0-5oC with constant stirring for next 15 min.
Then, 20 mL aqueous solution of sodium nitrite (NaNO2, 0.36 g, 5.25 mmol) was added dropwise with controlled
internal temperature 0-5oC and stirred continuously for 1 h. In a separate beaker, sodium sulphite (Na2SO3, 1.75g,
13.9 mmol) was mixed with ~7 mL water and 2.8 mL conc. HCl. Then, the resulting sulphite mixture was added to
the reaction mixture and the solution was stirred for next 1 h at 0-5oC. After this, the reaction mixture was stirred at
60oC for 3-5 h. The internal temperature was brought down to room temperature, followed by filtration and washing
the product 4-5 times with distilled water. The washed product was air dried to obtain white colored 5-hydrazinyl-1,
3-benzene dicarboxylic acid with a yield of 95%. For the next step, 5-hydrazinyl-1, 3-benzene dicarboxylic acid
(0.546 g, 3 mmol) and 2,4-pentanedione (0.34 mL, 3 mmol) were dissolved in isopropanol (20 mL) and refluxed for
3-5 h. The reaction mixture was filtered by using vacuum pump and it was air-dried to obtain pale yellow colored 5-
(3, 5-dimethyl-1H-pyrazol-1-yl) 1,3-benzenedicarboxylic acid (N-2) compound with ~80% yield. The ligand
1
structure was confirmed by using IR spectroscopy, mass spectrometry, H NMR and 13C NMR (DMSO-d6) (see
Supporting Information Fig. S1, S2, S3 and S4, respectively).
Scheme 4
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Synthesis of compound R-16 & R-1: 4-hydrazinybenzoic acid (1.065g, 14 mmol) and propionaldehyde (0.33 mL,
14 mmol) were refluxed in 35 mL ethanol for 4h at 70°C, and then the mixture was poured into 50 g ice-water
mixture [41-43]. The yellow colored precipitate was collected with a yield of 0.94g (88%). In a separate beaker, the
Vilsmeier-Haack reagent was prepared by adding of 0.87 mL (45 mmol) POCl3 to 0.94 mL DMF at 0 °C in round-
bottom flask in ice-cold condition (0-5°C) under constant stirring. Appropriate amount of 4-(2-
propylidenehydrazinyl) benzoic acid, 0.6g (15 mmol) dissolved in 4 mL DMF were added to the Vilsmeier-Haack
reagent and stirred for further an hour. Then, the reaction mixture was kept on an oil bath at 70 ºC for 4h. After the
reaction, the mixture was poured into 20g of crushed ice under constant manual stirring. After neutralization with
K2CO3 solution, ethyl acetate (30 mL) was added and the organic phase was separated from the aqueous phase by
extraction with 60 mL AcOEt. The combined organic solutions were washed with water and brine, dried, and
concentrated. Yellow colour solid 4-(4-methyl-1H-pyrazole-1-yl) benzoic acid (R-16) was collected with moderate
yield. For the synthesis of 4-(4-methyl-1H-pyrazole-1-yl) benzoic acid (R-1), we took 4-(4-methyl-1H-pyrazole-1-
yl) benzoic acid (R-16) 0.1g in pyridine (0.78 mL) and added a hot solution of potassium permanganate (0.97g) in
H2O (5.2 mL) at 115 °C along with constant stirring for 2h. After an additional 5h heating, the deposited manganese
dioxide was filtered out and the product was washed with 0.3% aqueous KOH. The filtrate was collected and
concentrated. After acidification with conc. HCl bringing down the pH to 1, the precipitated1-(4-carboxyphenyl)-
1H-pyrazole-4-carboxylic acid (R-1) was filtered, washed with water, followed by acetone and dried. White colored
solid was collected and the yield was found to be 0.015g. The decomposition temperature of the product was 250°C,
and was soluble in DMSO. The ligand was confirmed by IR spectroscopy, mass spectrometry and 1H NMR (DMSO-
d6) (see Supporting Information Fig. S5, S6 and S7). The UV-Vis spectra of all the four synthesized ligands (N-1, N-
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2, R-1 & R-16) have also been collected and plotted against absorbance vs wavelength (see Supporting Information
Fig. S8).
2.4 Anti-microbial activity
Two methods have been adopted for the determination of anti-microbial activity of the synthesized ligands. In
anti-bacterial studies, the “Standard serial dilution” was adopted for determination of ligand potency against test
bacterial strains [27,44]. The gram-positive strains were S. aureus (MTCC 3160), B. subtilis (MTCC 441), and gram
negative strain were E. coli (MTCC 16521), P. aeruginosa (MTCC 424) and S. enterica (MTCC 3858). The potency
of ligand against bacteria was compared to the MIC value of the standard drug Ciprofloxacin. In this method, the
stock solution of 50µg/mL concentration was diluted to get a concentration of 25-1.56 µg/mL in different test tubes
containing 1 mL of double strength nutrient broth. Then, these test tubes individually inoculated with 100 mL
suspension of test microorganism in sterile saline. The test tubes were incubated undisturbed at 37 °C ± 1 for
duration of 24 hours. Dimethyl sulfoxide (DMSO) was kept as solvent control.
Secondly, the anti-fungal activity of the synthesized molecule was evaluated against the most common fungal
strains, C. albicans by the method of “Standard serial dilution” [28,45]. For C. albicans, freshly prepared fungal
medium was taken into account to evaluate the anti-fungal activity using the agar well diffusion method. In this
method, a plate of fungus was obtained from pure isolate and was allowed to grow at 37 °C for 24 h. 4 colonies from
the plate were transferred into normal saline (0.85%) under sterile environment and the density of the suspension
was adjusted to 106 cfu/mL (in accordance to 0.5 Mc Farland standard). This suspension was used as inoculum. For
performing the assay 100 µL of the inoculum was swabbed on agar plates consisting of 20 mL agar, to achieve
confluent fungal growth. The agar plates were dried and punched with sterile cork borer of 10 mm diameter to create
wells for loading 100 µL of test sample with concentration of 8.0 mg/mL in 20% Dimethyl sulfoxide as solvent.
The plates were incubated at 37 °C ±1 for 24 h, followed by evaluation of zone of growth inhibition with zone
reader (Hi Antibiotic zone scale). Clomitrazole was taken as positive control for the fungus. The experiment was
repeated for three times to minimize error. Dimethyl sulfoxide was taken as solvent control.
3. Results and discussion
3.1 Docking studies
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A series of compounds were designed (Table S1, supporting information) and analyzed using latest version
of Glide 5.0 module of Schrodinger 11.5 software. We have designed four classes of compounds (N-1, N-2, R-1 &
R-16) and have taken one class of reported compounds (A series) for the computational study. The designed
molecules belonging to the S-series are also a derivative of the N-series. In N-1, a carboxylic acid group is present at
para-position in phenyl ring with respect to the pyrazole ring and two methyl substituents are present at 3- and 5-
positions in pyrazole ring. In N-2, there are two carboxylic acid groups at 3- and 5- positions on the benzene ring
with respect to the pyrazole ring and two methyl substituents on the pyrazole ring similar to N-1. In case of R-1,
there is one carboxylic group is present at para position similar to N-1 and a carboxylic group at the 4 position on
the pyrazole ring. In R-16, the structure is quite similar to R-1 where the carboxylic acid group on the pyrazole is
replaced by a lipophilic methyl group. All compounds were docked against DNA gyrase of S. aureus adopted from
PDB file 2XCT. Here, 3.35Å structure complex satisfies our interest since it gives the complete tetrameric structure
for DNA gyrase (A2B2) of S. aureus consisting of four active sites and 692 amino acids with a bound antibacterial
drug molecule ciprofloxacin (Fig. S9 in Supporting Information). The DNA strands present in the crystal structure
of DNA gyrase make it more versed in letting us to understand the intercalation of drug moiety in the active site of
the protein and its interactions with components of DNA structure. Docking studies were performed on the four
parent compounds (N-1, N-2, R-1, R-16) and certain derivatives of these compounds were designed to study the
increased ligand-protein interactions along with any other existing electronic interactions (see Supporting
information Fig. S10). The various substitutions of hydrophobic and hydrophilic groups in the aromatic rings can
play a major role for the biological activity.
The compounds, N-1, N-2, R-1 and R-16 when evaluated on the basis of various interaction parameters
through docking, show comparable results with the standard drug ciprofloxacin (Table 1). The major advantage of
these molecules is the low molecular weight along with the retention of desirable properties like extensive hydrogen
bonding (value is 1.2 in case of R-1 which is significantly high compared to ciprofloxacin). There is an H-bonding
interaction of O-atom of one of the carboxylic acid groups of all the four ligands with the polar uncharged amino
acid residue of Ser 1084 of the B-chain. The compounds containing carboxylic groups (as enlisted in Table S1)
show similar hydrogen bonding interactions with DNA component. In case of R-1, there is additional hydrogen-
bonding due to an extra carboxylic group substituted on the pyrazole ring, with Arg 458 residue of the B-chain. The
pyrazole rings of the ligands show π-π stacking interactions with the nitrogenous bases of the DNA (DA H:13
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fragment of DNA), denoted by the green arrow lines as shown in Fig. 1. Here, Mn+2 ion is present in protein pocket
(a part of G-chain) which is mandatorily forming salt bridges with carbonyl and carboxyl oxygens of R-1 and
carboxyl oxygens of N-1, N-2 and R-16. The metal coordination bond length is nearly 2.06 Å in case of N-1, R-1
and R-16 whereas it is 1.97 Å in case of N-2 (Fig. 2). This metal-coordination bond length is highly reduced in
present compounds when compared to the standard drug (2.32 Å and 2.19 Å in case of ciprofloxacin), as shown in
Fig. 3.
In order to improvise these interactions, the pyrazole structure was modified by changing the functional
groups at various positions either in phenyl ring or in the pyrazole ring. The derivatization of the compounds was
done computationally, followed by their docking at the same binding sites and evaluated their interaction scores in
contrast to the standard drug ciprofloxacin. The results are compiled in Table S1 (see Supporting Information). If we
try to get an insight of the various interaction scores obtained, we can observe that the compounds of the A-series
have a comparatively poor M.W (Molecular weight) score than the rest which depend on the size of ligands. The
interaction diagrams were analyzed for the compounds with highest H-bonding scores, like compounds R-1, R8,
R22, S5, S6 and S7. Compounds exhibiting highest interactions at the docking site of DNA gyrase along with their
respective scores are specified in Table 2. All the compounds have two interactions in common that are between the
O-atom of the substituted carboxylic group and the Ser 1084 residue along with the salt bridging with Mn+2.
Compounds S7 and S6 show extensive H-bonding of their hydroxyl oxygen with Gly 458 and Asp 437 residue as
well as oxygen of the substituted carboxyl group with Ser 1084. R8 shows a similar H-bonding interaction as R-1. In
case of R22, S5 and S7 another contributor to the H-bonding interaction is the solvent exposure of the substituted F-
atom. Compounds A8-A15 (excluding A13) though have good interaction scores in various parameters, the resultant
score become low in the overall Glide score due to heavy penalty scores which is discussed later. The highest
interactions were observed in case of S7 whereas the compound A14 showed the lowest favorable interactions.
When we carefully examine the interaction parameters of S7, A14 and standard drug ciprofloxacin, we
observed pi-pi interactions between aromatic rings of the compounds and with the nitrogenous bases present in the
DNA residues (DA H:13 DNA fragment) surrounding the molecules (as denoted by gray color, indicated as
unspecified residues). The metal coordination bond and salt bridge between Mn+2 atom are present in the protein
active site pocket which are mainly observed due to the presence of polar carboxylate groups of the compounds. The
metal bond length is almost comparable in case of S7 and ciprofloxacin ( i.e. 2.08 Å in S7 and 2.19 Å in the drug).
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Compound S7 shows very strong H-bonding interactions with Ser 1084 and Gly 459 residue of the protein. Such
kind of strong H-bond interactions are even not observed in the ciprofloxacin molecule, though it shows single such
interaction with one of the DNA fragments DA H:13. The active site of the protein is flanked with five various
amino acids: Arg 458 (B-chain) which is positively charged amino acid, Asp 437 and Glu 435, 477 (B-chain) which
are negatively charged amino acids, Phe 1123 which is a hydrophobic amino acid, Gly 1082, 459(B-chain) which
are nonpolar charged amino acids and Ser 1084 which is a polar uncharged amino acid. The compounds
ciprofloxacin and S7 fit into the protein pocket to maximize their interactions with the favorable amino acid as
observed in the Fig. 3. The occupancy of ligand S7 in protein pocket can be better understood using Fig. 5 and the
interactions of ligand inside the protein pocket can be visualized from Fig. S12, S13 and Table S2 (see Supporting
Information). The interaction diagram of compound A14 shows the certain familiar interactions like S7 compound,
but it lags in overall score. As, the Cl- ions are exposed to solvent which could have easily contributed in H-bond
formation if present in the inner cavity. Its orientation does not suit the residues flanking it, giving it a high penalty
score of 4 (refer Table S1 in Supporting information). If we observe the docking of all compounds at the active site
of the protein, we can easily conclude that Ser 1084, Gly 1082 along with the DNA fragments are actively taking
part for interactions with the drug molecules.
The compounds which exhibited the highest interactions with protein are given in Table 2. Most important
parameter for measuring activity is the G-Score which signifies the total glide score that sums up all interaction
values and deducting the penalties, lipophilicity (sum of the hydrophobic grid potential), H-bonding interactions,
electrostatic interactions (including the electrostatic rewards), molecular weight (rewards for ligands with low
molecular weights). The G-Score and related interaction values for all other docked molecules have been supplied in
Table S1 (Supporting information). Interestingly, few compounds have been shown higher values of H-bonding
interactions compared to standard drug ciprofloxacin. The G-score of the drug can be inferred from the high
electrostatic interactions and the hydrophobicity as well. Here, compound S6 has electrostatic interaction score of -
4.7 which is more than ciprofloxacin but it lags in the hydrophobicity thus deteriorating its overall G-Score. Low
molecular weight is another advantage of our molecules over the standard drug which is obvious by the specified
low M.W Score. Since, the protein exists as a tetramer with four chains, there are four binding sites of the drug in
each of these chains. All of the parent compounds along with their derivatives were docked at these sites and a
peculiar difference was observed in the G-score for each compound, which can draw us towards the conclusion that
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even though the sites seem similar superficially but there might be difference in the interactions of the residues with
these compounds. The above observations can be well explained by further computational and experimental studies
discussed below.
3.2 Penalty Analysis
The Glide 5.0 module offers a feature of highlighting penalty score for any ligand, at various grounds,
during the process of docking. This analysis is important aspect of visualizing the faulty positioning of the
functional groups in ligands with an attempt to rectify and improvise the interactions. A high penalty was charged
for compounds A8 to A15 (excluding A13) on the basis of position of various protein residues and their ill
interactions with the ligand functional groups. In case of A14, there is high penalty due to burial of charged group
on the protein (referring to negatively charged amino acids like Glu 477 and Asp 437) by the ligand pertaining to
minimum H-bond interactions made to the charged group. Also, there is a hydrophobic group of the ligand against
donor groups of protein in a protein region which normally would give a favorable phobic packaging score. No such
penalty was charged for compounds belonging to N and R series due to the appropriate positioning of the various
lipophilic and hydrophilic moieties.
3.3 ADME analysis
The well-known ADME analysis i.e., Absorption (A), distribution (D), metabolism (M) and excretion (E)
of any organic molecule is an important analysis which states about the disposition of any pharmaceutical compound
inside an organism and therefore, influences the pharmacological activity of it [46]. In our work, this study was
performed using Qikprop (ligand based ADME analysis) module of Maestro 11.5, which provides ranges for
comparing particular molecule’s properties with those of 95% of known drugs. The ADME analysis of all
compounds in the data set was done. The parent compounds N-1, N-2, R-1 and R-1-6 show good drug like
properties and no violations were observed in accordance to Lipinski’s rule of five (as shown in Table 3). Other
important properties of any pharmacologically active molecule, playing crucial role as drug candidate, are also taken
into consideration like molecular weight, QPlogPw (which predicts water and gas partition coefficients), QPPCaco
(which predicts permeability of a molecule for the gut-blood barrier through passive transport), percentage human
oral absorption, globularity and the number of H-bonds that would be donated by the solute molecule to the water
molecules in the aqueous solution (donor HB). The results of ADME analysis propound the theoretical drug-like
behavior of these compounds which are very promising and can be considered beneficial for further laboratory
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analysis of them. The top 20 candidates obtained after the interaction studies of molecules data set post docking
were also subjected to ADME analysis (Table S3 in supporting information). They also exhibited similar behavior in
comparison to the standard drug Ciprofloxacin, though some were showing improved donor HB ability with lesser
molecular weights which were inferred previously in other sections as well. Moreover, the percentage of oral
absorption for few molecules was another striking result which was enhanced in our molecules than the standard
drug.
3.4 DFT studies
The optimized geometries, total energies and dipole moments of the parent molecules were obtained from
the DFT calculations. The observed optimized geometries of the four parent compounds have been depicted in Table
4. The optimized structures are found without any imaginary frequency and hence, all are characterized in their
minimum energy states on the potential energy surface. The single point calculations were performed by importing
the docked pose structures. Since there were no alterations done in the imported pose, hence, the molecules depicted
are in ionized state with the removal of polar hydrogens as observed in their docked pose. A similar set of
calculations were also carried out after converting the imported poses into unionized states by addition of their polar
hydrogens but there were very tiny differences in the energies as well as in the torsional angles. We have tabulated
all the results obtained from the optimized structures and the post docked structures to observe the changes in their
conformations and energies (Table 5). The values mentioned in column ‘a’ correspond to the optimized structure of
the molecules whereas the values in column ‘b’ correspond to the values pertaining to the single point calculation
studies performed with post docking molecules. The torsional angles mentioned in both the columns correspond to
the similar angles, though the nomenclature may change for single point calculations of the post docked molecules
due to the ionized structures after removal of polar hydrogen.
On comparison of the data obtained after geometry optimization and single point energy calculations for
the same molecule, it is observed that, there are certain conformational changes observed in the molecule upon
docking. These changes can be explained by the hypothesis that the structure obtained on geometry optimization is
the ‘global minima’ whereas the one obtained after docking is the best suited pose resulted from the ligand
interaction with the surrounding entities of protein pocket, which can be referred as the ‘bioactive minima’ [47]. On
the comparison of the two molecules N1 and N2, their optimized structures are found to be completely overlapping
with each other but the molecules are posing differently in the protein pocket (Fig. 7a). This can be attributed to the
14

ACCEPTED MANUSCRIPT
mandatory interaction of the residue Gly 459 with –COOH group of substituted phenyl ring in case of N2 which can
be better understood from Fig 7b. Such conformational variation results to a significant change in the torsional
angles of the molecule, in order to remain in the vicinity of the interacting residues as mentioned in the Table 5. A
correlation between total energies of the molecule and their respective interaction scores can be established as well.
The lower energies were obtained in case of N2 (-24,852.08 e.V) and R1(-22,712.083 e.V), which are in the
agreement with their G-scores obtained after docking. The lowest energy in the compound N2 is observed due to the
presence of two –COOH groups playing a pivotal role in the better interaction behavior. Moreover, there is a
constant trend of increase in dipole moment after docking in the each molecule.
3.5 Anti-microbial studies of synthesized compounds
To verify the veracity of our computational results, four parent compounds were successfully synthesized
namely: N-1 [5-(3,5-dimethyl-1H-pyrazol-1-yl)1,3-benzoic acid], N-2 [5-(3,5-dimethyl-1H-pyrazol-1-yl)1,3-
benzenedicarboxylic acid], R-1[1-(4-carboxyphenyl)-1H-pyrazole-4-carboxylic acid] and R-16 [4-(4-methyl-1H-
pyrazole-1-yl)benzoic acid] as depicted in Table 6.
The anti-bacterial properties were concluded by the minimum inhibitory concentration (MIC) value of the
synthesized molecule against different bacterial strains (as shown in Table 6). The anti-bacterial testing of the
synthesized compounds revealed that the compounds were active against almost all strains of common bacteria (as
Table 6). Compound N-2 is showing higher activity against B. subtilis and P. aeruginosa with a MIC value of 12.5
which is better than earlier reported compound N-1, though it was not tested for anti-bacterial activities before.
Compound R-1 was found to be active against S. enterica and moderately active against all other bacterial strains.
The MIC values for our standard drug ciprofloxacin against E. coli, B. subtilis, S. aureus, P. aeruginosa and S.
enterica are 0.08, 0.06, 0.6, 0.15 and 0.06 µg/mL, respectively.
The anti-fungal activity was also measured for the best performing candidate N-2 (in the anti-bacterial
testing) along with compound N-1. Although the compound N-2 showed promising anti-bacterial activity, the anti-
fungal activity was better in case of N-1. The diffusion diameter was found to be 3 mm for compound N-2, whereas
it was 10 mm for compound N-1 as shown in Fig. 8. Hence, compound N-2 can be a better anti-bacterial candidate
but N1 is better anti-fungal drug.
4. Conclusions
15

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Pyrazoles are an evolving class of azoles as antimicrobial agents due to the increasing resistance of
pathogenic microbes to multiple drugs including ciprofloxacin. Therefore, an efficient strategy has been employed
to design noble pyrazole based drug molecules, keeping in mind the basic pre-requisites of any good
pharmacophore: aromaticity, optimum molecular weight, hydrophobic and hydrophilic substituents along with H-
bond accepting and donating groups. On the basis of the computational analysis, a noble bunch of pyrazole based
compounds have been propounded that could be good antimicrobial agents. Four parent skeletons have also been
synthesized and studied anti-microbial testing against various gram-positive, gram-negative bacteria and a fungal
species. Computational biological activity was well matched with preliminary experimental results. Structural
optimizations of synthesized compounds have also been carried out to validate the conformational arrangement of
the docked structures. The synthesis of other derivatives from these four compounds is under process. The current
research on pyrazole based molecules can be a useful aid in the development of newer pyrazole based drug
candidates for various laboratories and pharmaceutical industries in future.
Acknowledgements
Authors acknowledge the financial support from the Department of Science and Technology through INSPIRE
Award (No. IFA14-CH-144) & Science and Engineering Research Board, India in the form of Start-Up Research
Grant (No.SB/FT/CS-098/2014). AKP and NK are also thankful to UGC-DAE-CSR for support in various aspects.
Conflicts of interest
There are no conflicts to declare.
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Table 1: Important docking parameters and corresponding scores for the four synthesized compounds N-1, N-2, R-
1, R-16 and standard drug ciprofloxacin.
Compounds
Structure
N-1
N-2
R-1
R-16
S.D.
G-Score
-7.2
-3.0
-7.5
-7.61
-2.8
-7.02
-3.2
-11.3
Electrostatic
interactions
-2.8
-4.58
H-bonding
-0.4
-3.4
-0.3
-3.8
-1.2
-3.1
-0.5
-2.8
-0.55
-4.27
Lipophilic
interactions
Low M.W
-0.5
-0.5
-0.5
-0.5
-0.4
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Table 2: Compounds with higher interactions at the docking site of DNA gyrase in S. aureus.
S.
Compound
Name
Code
G-
Electrostatic H-
Lipophilic
Low
No.
Score
interactions
-4.4
Bonding
M.W
interactions
1.
2.
3.
S7
-10
-1.5
-1.6
-0.9
-3.8
-0.3
S6
-9.6
-4.7
-2.5
-0.5
A13
-10.28 -3.76
-4.06
-0.06
Where R=
4.
R11
-8.65
-2.94
-0.83
-4.39
-0.49
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5.
R12
-8.43
-2.95
-0.61
-4.42
-0.44
6.
R18
-8.38
-2.96
-0.51
-4.41
-0.5
7.
A6
-8.33
-3.41
0.0
-4.79
-0.13
Where R=
8.
R21
-8.3
-3.12
-0.97
-3.72
-0.5
H3C
N
N
H2N
HOOC
COOH
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9.
R24
-8.22
-3.16
-0.9
-3.91
-0.26
10.
R15
-8.21
-2.8
-0.84
-4.07
-0.5
11.
R25
-8.2
-2.86
-0.7
-4.42
-0.22
12.
R23
-8.15
-2.91
-1.17
-3.66
-0.41
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13.
R6
-8.15
-2.93
-0.76
-3.97
-0.49
14.
S11
-9.1
-4.0
0.0
-3.6
-0.5
15.
A3
-8.12
-3.4
-0.63
-3.98
-0.11
Where R=
16.
A2
-8.1
-3.63
-1.61
-3.73
-0.13
WhereR=
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17.
A4
-8.07
-3.4
-0.64
-3.87
-0.17
Where R=
18.
R26
-8.24
-3.28
-0.91
-3.64
-0.41
19.
R22
-8.12
-3.4
-1.26
-3.15
-0.5
20.
S.D*
-11.3
-4.58
-0.55
-4.27
-0.4
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Table 3: Various parameters obtained from ADME analysis of the four synthesized compounds N-1, N-2, R-1 and
R-16.
S. No.
Molecules
M.W
QPlog
Pw
QPPCaco
%Human oral
Absorption
81.445
Rule of
Five
0
Globin Donor HB
1.
2.
3.
4.
N-1
N-2
R-1
216.239
260.239
233.195
202.212
7.01
174.032
4.691
0.870
0.857
0.876
0.878
1
2
2
1
10.616
11.234
7.284
50.103
44.549
79.728
0
0
0
3.545
R-16
156.838
28