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Letters in Drug Design & Discovery, 2020, 17, 000-000 1

RESEARCH ARTICLE

Synthesis, Characterization, Antimicrobial, Anti-tubercular, Antioxidant

Activities and Docking Simulations of Derivatives of 2-(pyridin-3-yl)-1H-
benzo[d]imidazole and 1,3,4-Oxadiazole Analogy

Shipra Bhati1,#, Vijay Kumar2,*,#,Simranjeet Singh3,4,5,#, and Joginder Singh3,*

1
Department of Chemistry, The Oxford College of Engineering, Bangalore (Karnataka) 560068, India; 2Department of
Chemistry, Regional Ayurveda Research Institute for Drug Development, Gwalior (M.P.) 474009, India; 3Department of
Biotechnology, Lovely Professional University, Jalandhar (Punjab) 144111, India; 4Regional Advanced Water Testing
laboratory, Mohali (Punjab) 160059, India; 5Punjab Biotechnology Incubators, Mohali (Punjab) 160059, India

Abstract: Background: Antimicrobial Resistance (AMR) and Tuberculosis (TB) are global
concern. According to the WHO fact sheet on tuberculosis, in 2017, 10 million people fell ill with
TB, and 1.6 million including 230,000 children died from the disease. There is a critical need of
design and development of novel chemotherapeutic agents to combat the emergence and increasing
prevalence of resistant pathogens. In the present study, a new series of 1,3,4-oxadiazoles
incorporating benzimidazole and pyridine scaffolds in a single molecular framework has been
ARTICLE HISTORY reported.

Received: June 5, 2019
Revised: September 14, 2019
Accepted: November 20, 2019
DOI:
10.2174/1570180816666191122105313
Keywords: Antimicrobial, anti-tubercular, antioxidant, bioavailability, docking simulations.
Methods: The structures of the synthesized derivatives (4a to 4e) were assigned by IR, NMR and
mass spectral techniques. The hybrid compounds were evaluated for their antimicrobial,
antitubercular and antioxidant activities. In addition, docking simulations were performed to study
ligand-protein interactions and to determine the probable binding conformations.
Results: Molecule 4a has shown anti-tubular activities with MIC 1.6 µg/ml. As compared to
ascorbic acid activities (IC50 = 62.91 µg/ml), molecule 4e exhibited better antioxidant activities
(IC50 = 24.85 µg/ml). Also, molecule 4e has shown significant antimicrobial activities.
Conclusion: The synthesized derivatives from 4a to 4e have exhibited various medicinal activities
and could be emerged as lead compounds and further explored as potential therapeutic agents.

1. INTRODUCTION contagious disease caused by the bacterium Mycobacterium

Antimicrobial Resistance (AMR) and Tuberculosis (TB)
are global concern. The report from WHO's Global
Antimicrobial Resistance Surveillance System (GLASS),
2016-17 shows that Escherichia coli, Klebsiella pneumoniae,
Staphylococcus aureus, Streptococcus pneumoniae and
Salmonella species are the most commonly reported resistant
bacteria [1]. The report reveals the widespread occurrence of
antibiotic resistance. The emergence of antimicrobial
resistance in microorganisms is a natural phenomenon and to
overcome it, the understanding of its mechanism of action as
well multidisciplinary research is needed [2-5]. TB is a
*Address correspondence to these authors at the Department of Chemistry,
Regional Ayurveda Research Institute for Drug Development, Gwalior
(M.P.) 474009, India; E-mail: vijaychem99@gmail.com; Department of
Biotechnology, Lovely Professional University, Jalandhar (Punjab)-144111,
India; E-mail: simnav14@gmail.com
#
These authors contributed equally to this work.
tuberculosis. Multidrug-resistant TB (MDR-TB) remains a
public health crisis and a health security threat [6-8].
According to the WHO fact sheet on tuberculosis, in 2017,
10 million people fell ill with TB, and 1.6 million including
230,000 children died from the disease [9]. There is a critical
need for design and development of novel chemotherapeutic
agents to combat the emergence and increasing prevalence of
resistant pathogens.
Heterocyclic compounds are key components for the
development of chemotherapeutic agents. Pyridine is the
parent ring system of a large number of naturally occurring
products and important industrial, pharmaceutical and
agricultural chemicals [10]. Pyridine-based compounds have
been playing a crucial role as agrochemicals or pesticides
including fungicides, insecticides/ acaricides and herbicides
etc. [11]. Presently there are many drugs available in the
market like Pioglitazone HCl, Rosigitazone HCl, Isoniazid,
Ethionamide, Prothionamide, Nikethamide, etc., that have

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2 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0
pyridine ring in their structure which is responsible for
biological activities. Grombein and co-workers had
presented 1-Phenylsulfinyl-3-(pyridin-3-yl)naphthalen-2-ols
derivatives as potent aldosterone synthase inhibitors [12].
Abdel-Megeed and co-workers synthesized and evaluated
antimicrobial activity of a series of diphenyl-1-(arylamino)-
1-(pyridin-3-yl) ethylphosphonates derivatives and found
that the compounds showed high antimicrobial activity
against selected bacterial and fungal strains at low
concentrations (10-100 µg/mL). Also, these compounds
showed significant cytotoxic anticancer activities against
liver carcinoma cell line (HepG2) and human breast
adenocarcinoma cell line (MCF7) [13].
Benzimidazole derivatives have been extensively studied
in the field of medicinal chemistry due to their versatile and
eminent biological profile. A large number of activities such
as antimicrobial [14-16], anti-mycobacterial [17], analgesic
[18], anticonvulsant [19], antitumor [20], antioxidant and
anti-inflammatory [21], anti HIV activity [22], antiviral [23],
antimalarial [24] and anti-hypertension activity [25] etc. had
been displayed by compounds adjoining this heterocyclic
system. The introduction of benzimidazole derivatives in
triphenylamine-based organic dyes can improve their
photovoltaic performance because of the extension of the π-
conjugation structures in dye-sensitized solar cells [26].
Polybenzimidazole based nanocomposite membranes have
also been reported as promising electrolyte used for high
temperature proton exchange membrane fuel cells [27].
Lijun Tang and co-workers designed and synthesized
benzimidazole-based fluorescent chemo sensor for Cu2+ and
sulfide anion detection in water [28].
Oxadiazole rings are commonly employed as bioisosteric
replacement for carbonyl-containing groups such as esters,
amides, carbamates and hydroxamic esters and are
comparatively more stable in biological media and hence
they are frequently used motif in structure of drug candidates
[29]. 1,3,4-oxadiazoles are nitrogen oxygen containing
heterocyclic compounds that are known for numerous thera-
peutic effects and are recognized as important phar-maco-
phore in drug discovery. 1,3,4-oxadiazole derivatives have
occupied prominent place in medicinal chemistry and are
known for their diverse range of biological activities include-
ing antimicrobial [30, 31], anti-mycobacterial [32] anti-
inflammatory and analgesic [33], anticancer [34, 35], anti-
allergic [36], antioxidant [37] insecticidal [38], anticonv-
ulsant [39] etc.
In the view of the facts mentioned above and in
continuation of our research work, in the present report, the
synthesis and characterization of some novel heterocyclic
compounds containing three potential heterocyclic ring
systems namely pyridine, benzimidazole and 1,3,4-
oxadiazole has been carried out. The ring of synthesized
molecules having phenyl ring substituted with electron
releasing groups (-OH, OCH3) and electron withdrawing
group (-C6H5,-Cl,-NO2) in a single framework. The
synthesized molecules were evaluated for their
antimicrobial, anti-tubercular and antioxidant potential.
Further, molecular docking studies were also carried out to
understand the binding interactions of ligands and selected
protein targets.
Bhati et al.
2. EXPERIMENTAL
2.1. Chemicals and Reagents
All reagents were purchased from SD fine chemicals and
were used without further purification. Melting points were
determined in open capillary using the melting point
apparatus and are uncorrected. The Proton Magnetic
Resonance (1H-NMR) spectra were recorded on a Bruker
300 MHz instrument in DMSO using tetramethylsilane as an
internal standard. The Infrared spectra of the compounds
were recorded in KBr on a Perkin-Elmer FTIR Spectrometer.
Mass spectra were recorded on JEOL SX 102/DA-6000 mass
spectrometer data system using argon/Xenon (6 KV, 10 MA)
as FAB gas. To monitor the reactions and to confirm the
purity of the compounds, Thin Layer Chromatography
(TLC) was performed and iodine/UV Chamber was used for
visualization of spots.
2.1.1. Synthesis of 2-(pyridin-3-yl)-1H-benzimidazole (1)
Equimolar amount of o-phenylenediamine and pyridine-
3-carboxylic acid were refluxed in 4 N HCl for 5 h on a
water bath. The completion of the reactions was monitored
by TLC. After completion of the reaction, the solution was
poured onto crushed ice, ammonia solution was added
dropwise to neutralize and the resulting solid was filtered,
washed with cold water, dried and recrystallized with
ethanol. Yield 90%, m.p.255°C, 253-257°C (lit. m.p. [5]) IR
(KBr) cm-1: 1545 (C=N), 3444(N–H); 1H-NMR (DMSO) δ
ppm: 4.962 (s, 1H, NH) 7.081-7.923 (m, 4H, Ar-H),
8.14(ddd, 2H, pyridine-H), 8.77(ddd, 1H, pyridine-H),
9.11(t, 1H, pyridine-H); EI-MS: 196.08 [M+H]+; Anal calcd
for C12H9N3: C, 73.83; H,4.65; N, 21.52; Found: C, 73.85; H,
4.64; N, 21.53.
2.1.2. Synthesis of Ethyl-[2-(pyridin-3-yl)-1H-benzim-
idazol-1-yl] Acetate (2)
2-(pyridin-3-yl)-1H-benzimidazole (0.01 mol), ethyl chloro-
acetate (0.02 mol), anhydrous potassium carbonate (2 g)
were refluxed in 50 ml of dry acetone for 24 h. After
completion of the reaction the inorganic solid was filtered
off, the filtrate was concentrated under reduced pressure. On
cooling a solid separated out and washed with petroleum
ether and distilled water successively. The solid thus
obtained was recrystallized from ethanol. Yield 50%, m.p.
88–92 ˚C, IR (KBr) cm-1: 3583.6 (C-H str), 1632(-C=N),
1150 (C-N), 1739.57 (C=O ester), 1460 (-N-CH2); 1H-NMR
(DMSO) δ ppm: 1.213 (t, 3H, Ester- CH3), 4.188 (q, 2H,
Ester-CH2), 5.215 (s, 2H, -N-CH2), 7.516-7.550 (m, 4H, Ar-
H), 8.269 (ddd, 2H, pyridine-H), 8.781 (ddd,1H, pyridine-
H), 9.074 (t , 1H, pyridine-H) ; EI-MS: 281 (M+); Anal calcd
for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94; O, 11.37;
Found: C, 68.30; H, 5.40; N, 14.96; O, 11.36.
2.1.3. Synthesis of 2-[2-(pyridin-3-yl)-1H-benzimidazol-1-
yl] Acetohydrazide (3)
Ethyl-[2-(pyridin-3-yl)-1H-benzimidazol-1-yl] acetate
(0.01 mol) was added to a round bottom flask which
contained 20 ml of ethanol. The reaction mixture was
refluxed for 1h at 80˚C then hydrazine hydrate (99%, 0.06
mol) was added dropwise and the mixture was refluxed
Synthesis, Medicinal and Docking Simulations of Derivatives
further for 10h. After completion of the reaction, the mixture
was cooled and kept in the refrigerator overnight. The solid
so obtained was filtered, washed with cold water and
recrystallized from methanol. Yield 70%; m.p.178–180 ˚C,
IR (KBr) cm-1: 3068.42 (-CONH), 3044 (CH-Ar), 2847.16
(C-H str), 1680.35 (C = O, amide), 1645 (-C = N), 1461 (-N-
CH2); 1H-NMR(DMSO) δ ppm: 2.54 (s, 2H, NH2), 5.37 (s,
2H, -N-CH2), 7.091-7.949 (m, 4H, Ar-H), 8.167 (s, 1H,
CONH), 8.254(ddd, 2H, pyridine-H), 8.635 (ddd, 1H,
pyridine-H), 9.131 (t, 1H, pyridine-H); EI-MS: 267 (M+);
Anal calcd for C14H13N5O:C, 62.91; H, 4.90; N, 26.20; O,
5.99; Found: C, 62.93; H,4.93; N, 26.22; O, 5.98.
2.1.4. General Procedure for the Synthesis of Compounds
(4a-4e)
An equimolar amount of 2-[2-(pyridin-3-yl)-1H-
benzimidazol-1-yl] acetohydrazide (3) and suitable aromatic
acid were refluxed on a sand bath in the presence of POCl3
(10 ml) for 8-10 h. After completion of the reaction, the
mixture was cooled at room temperature and poured onto
crushed ice. On basification with sodium bicarbonate (10%),
a solid mass was separated out which was filtered, dried and
recrystallized by ethanol to give pure product.
2.1.4.1. Synthesis of 1-[(5-phenyl-1,3,4-oxadiazol-2-yl)
methyl]-2-(pyridin-3-yl)-1H-benzimidazole (4a)
Yield 70%; m.p. 215˚C, IR (KBr) cm-1: 3583.91 (CH
arom), 1630.31(C=N), 1262.27 (C-O-C asymm), 1097.34
(C–O–Csymm), 1022.94 (N–N), 925.92, 837.12 (CH); 1H-
NMR (DMSO) δ ppm: 5.52 (s, 2H, N-CH2), 7.235-7.948 (m,
9H, Ar-H), 8.229 (ddd , 2H, pyridine-H), 8.634 (ddd , 1H,
pyridine-H), 9.132 (t , 1H, pyridine-H); EI-MS: 353 (M+);
Anal calcd for C21H15N5O: C, 71.38; H, 4.28; N, 19.82; O,
4.53; Found: C, 71.39; H, 4.30; N, 19.80; O, 4.56.
2.1.4.2. Synthesis of 1-{[5-(4-chlorophenyl)-1,3,4-
oxadiazol-2-yl]methyl}-2-(pyridin-3-yl)-1H-benzimidazole
(4b)
Yield 50%; m.p. 245°C, IR (KBr) cm-1: 3580.07 (CH
arom), 1680.08 (C=N), 1282.74 (C-O-C asymm), 1090.3 (C–
O–Csymm), 1013.28 (N–N), 926.23, 849.54 (CH), 759.15
(C-Cl) ; 1H-NMR (DMSO) δ ppm: 5.62 (s, 2H, N-CH2),
7.091-7.972 (m, 8H, Ar-H), 8.227 (ddd , 2H, pyridine-H),
8.635 (ddd , 1H, pyridine-H), 9.134 (t , 1H, pyridine-H); EI-
MS: 387 (M+); Anal calcd for C21H14N5OCl: C, 65.04; H,
3.64; N, 18.06; O, 4.13; Found: C, 65.02; H, 3.65;N, 18.03;
O, 4.15.
2.1.4.3. Synthesis of 1-{[5-(4-hydroxy phenyl)-1,3,4-
oxadiazol-2-yl]methyl}-2-(pyridin-3-yl-1H-benzimidazole
(4c)
Yield 40%; m.p. 240°C, IR (KBr) cm-1: 3654.25 (OH),
3573.07 (CH arom), 1672.75 (C = N), 1272.77 (C-O-C
asymm), 1082.17 (C–O–Csymm), 1012.94 (N–N), 925.92,
837.12 (CH); 1H-NMR (DMSO) δ ppm: 5.54 (s, 2H, N-
CH2), 7.093-7.948 (m, 8H, Ar-H), 8.225 (ddd, 2H, pyridine-
H), 8.634 (ddd , 1H, pyridine-H), 9.132 (t , 1H, pyridine-H);
EI-MS: 369 (M+); Anal calcd for C21H15N5O2: C, 68.28; H,
4.09; N, 18.96; O, 8.66; Found: C, 68.26; H, 4.08; N, 18.97;
O, 8.68.
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 3
2.1.4.4. Synthesis of 1-{[5-(4-methoxy phenyl)-1,3,4-
oxadiazol-2-yl]methyl}-2-(pyridin-3-yl-1H-benzimidazole
(4d)
Yield 30%; m.p. 225°C, IR (KBr) cm-1: 3583.06 (CH
arom), 1682.75 (C=N), 1260.77 (C-O-C asymm), 1105.17
(C–O–Csymm), 1022.94 (N–N), 925.92, 837.12 (CH) ; 1H-
NMR (DMSO) δ ppm: 3.82 (s, 3H, OCH3), 5.52 (s, 2H, N-
CH2), 7.090-7.942 (m, 8H, Ar-H), 8.224 (ddd , 2H, pyridine-
H), 8.635 (ddd , 1H, pyridine-H), 9.130 (t , 1H, pyridine-H);
EI-MS: 383 (M+); Anal calcd for C22H17N5O2: C, 68.92; H,
4.47; N, 18.27; O, 8.35; Found: C, 68.94; H, 4.46; N, 18.28;
O, 8.33.
2.1.4.5. Synthesis of 1-{[5-(3-nitro phenyl)-1,3,4-oxadiazol-
2-yl]methyl}-2-(pyridin-3-yl-1H-benzimidazole (4e)
Yield 30%; m.p. 230°C , IR (KBr) cm-1: 3588.06 (CH
arom), 1692.05 (C=N ), 1260.77 (C-O-C asymm), 1064.12
(C-O-Csymm), 1517.98 (N=O asymm), 1317.38 (N=O
symm), 1025.12 (N–N), 940.67, 742.12 (CH); 1H-NMR
(DMSO) δ ppm: 5.58 (s, 2H, N-CH2), 7.092-7.947 (m, 8H,
Ar-H), 8.225 (ddd , 2H, pyridine-H), 8.638 (ddd , 1H,
pyridine-H), 9.134 (t , 1H, pyridine-H); EI-MS: 398 (M+);
Anal calcd for C21H14N6O3: C, 63.31; H, 3.54; N, 21.1; O,
12.05; Found: C, 63.33; H, 3.52; N, 21.0; O, 12.06.
2.2. Prediction of Molecular Properties Descriptors
Prediction of molecular properties descriptors is
significant in the discovery and development of potential
therapeutics. In the present study, Molinspiration program
[40] was used for calculating molecular properties, such as
milogP (partition coefficient), TPSA (topological polar
surface area), MW (molecular weight), nON ( number of
hydrogen bond acceptors), nOHNH ( number of hydrogen
bond donors), Nrotb (number of rotatable bonds), Nvio
(Number of violations), Natoms (range of atoms) and
Volume. The bioactivity scores of GPCR ligand, ion channel
modulator, nuclear receptor ligand, kinase inhibitor, protease
inhibitor and enzyme inhibitor were also calculated using
molinspiration server.
2.3. Antimicrobial Activity
The synthesized compounds 4a-4e were screened in vitro
for their antibacterial activity against four bacterial strains
i.e. Pseudomonas aeruginosa (MTCC 4673), Staphylococcus
aureus (MTCC 7443), Salmonella enterica (MTCC 164),
Streptococcus mutans (MTCC 497) and one fungal strain A
parasiticus (MTCC 646) by following standard literature
reported procedure of disk-diffusion method [41-45]. The
cultures used in this experiment were ordered from MTCC
Chandigarh (India). The media used for this purpose are
nutrient agar media and potato dextrose media. 28 grams of
nutrient agar and 39.0 grams of Potato dextrose agar was
added to the 1 liter of double distilled water separately, it
was mixed thoroughly and pH was adjusted at 7.5 ± 0.2. The
solution was heated to dissolve the ingredients completely
after which the media was autoclaved at 121°C for 45
minutes at 15lbs pressure. After autoclaving, 15-20 ml of
that media was poured into petri dish for studying
antimicrobial activities. Whatman no. 1 filter paper disks
4 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0
were sterilized by autoclaving at 160°C for 1 h. Then the
sterile disks were impregnated with the test compounds of
different concentrations (0 ppm, 250 ppm, 500 ppm and
1000 ppm). Cultures having 105 CFU/mL were used against
each concentration level. DMSO was used as solvent control
and as 0 ppm. The impregnated disks were placed on the
medium suitably spaced apart and the plates were incubated
at 37°C for 24 h for bacterial strains and 48 h for fungal
species. Streptomycin and ketoconazole were used as a
standard drug for antibacterial and anti fungal activity,
respectively. After incubation, the degree of inhibition was
observed by the formation of zones. The diameters of the
zones were measured in mm.
2.4. Antitubercular Activity
The antitubercular activity of compounds 4a-4e was
assessed against Mycobacteria tuberculosis (Vaccine strain,
H37 RV strain, ATCC-27294) using Microplate Alamar
Blue Assay (MABA) [46,47]. This methodology is non-
toxic, uses a thermally stable reagent and shows good
correlation with proportional and BACTEC radiometric
method. 200 µl of sterile deionzed water was added to all
outer perimeter wells of sterile 96 wells plate to minimize
evaporation of medium in the test wells during incubation.
The 96 wells plate received 100 µl of the Middlebrook 7H9
broth and serial dilution of compounds was made directly on
plate. The final drug concentrations tested were 100 to 0.2
µg/ml. Plates were covered and sealed with parafilm and
incubated at 37ºC for five days. After this time, 25µl of
freshly prepared 1:1 mixture of Almar Blue reagent and 10%
tween 80 was added to the plate and incubated for 24 hrs.
Pyrazinamide, Ciprofloxacin and Streptomycin were used as
standard drugs. A blue color in the well was interpreted as no
bacterial growth, and pink color was scored as growth. The
MIC was defined as the lowest drug concentration that
prevented the color change from blue to pink.
2.5. Antioxidant Activity
The synthesized compounds 4a-4e were evaluated for
their antioxidant activity by using DPPH (2,2-diphenyl-1-
picrylhydrazyl), free radical scavenging method according to
the reported method with slight modifications [48, 49].
Briefly, 1 mg/ml solutions of compounds and ascorbic acid
were prepared by dissolving them into DMSO. 25, 50, 75
and 100 µL of each was added separately to 10.0 mL amber
color volumetric flasks containing 2.0 ml of 0.01mM DPPH
(prepared in ethanol). The final volume was made up to 3.0
ml and after 30 minutes the absorbance was checked at 517
nm by using UV-visible spectrophotometer. Pure DPPH
solution (0.01mM) was used as a control and ethanol was as
a blank. The decrease in absorbance equates the DPPH
radical scavenging capacity. The above process was repeated
three times for ascorbic acid (positive control) and
compounds. The radical scavenging ability was calculated
according to the formula: Radical scavenging activity = (A0-
AT/ A0) × 100; where, A0 is the absorbance of pure DPPH
solution (0.01mM), and AT is the absorbance of (DPPH)
and compounds. The IC50 was defined as half maximal
inhibitory concentration of the compounds that inhibit
DPPH radical.
Bhati et al.
2.6. Docking Simulations
The molecular docking simulations were performed to
analyze drug-target interactions of the synthesized
compounds using the Autodock 4.2 software [50]. The
chemical structures of the compounds were drawn and their
3D structures were optimized in the ACD Labs ChemSketch
12.0 software. Docking study of antimicrobial activity was
carried out against three different protein targets i.e.
Isocitrate lyase (P. aeruginosa, PDB ID: 6g1o),
Dihydrofolate reductase (S. aureus, PDB ID: 5isp) and
Noranthrone synthase (A. parasiticus, PDB ID: 5kbz).
Methionine aminopeptidase (M. Tubeculosis, PDB ID: 5yxf)
and tyrosine-protein kinase (Homosapiens, PDB ID: 6il3)
were selected for in-silico docking simulations of
antitubercular and antioxidant activity respectively of the
synthesized compounds. The X-ray crystallographic
structure of the target proteins was retrieved from the protein
data bank [51]. Prior to the simulations all bound ligands,
cofactors, and water molecules were removed from the
proteins. Gasteiger charges were computed, and the Auto
Dock atom types were defined using Auto Dock version 4.2.
The three-dimensional grid boxes were created, the spacing
between grid points was 0.375 angstroms, and the grid maps
representing the intact ligand in the actual docking target site
were calculated with Auto Grid algorithm. Finally, Auto
Dock was used to calculate the binding free energy of a
given inhibitor conformation in the macromolecular
structure. Lamarckian Genetic Algorithm (LGA) parameters
were set to default settings, which include 150 runs, 150
conformational possibilities, 50 populations and 2,50,0000
energy evaluations. During the docking process, a maximum
of 10 conformers was considered for each compound. The
results were evaluated based on the binding compatibility i.e.
binding energy in kcal/mol and inhibition constant. The
resultant protein-ligand complex structures were explored
using Pymol program [52].
3. RESULTS AND DISCUSSION
3.1. Chemistry
The synthesis of the compounds (4a-4e) was carried out
as given in Scheme 1. The IR, NMR and Mass spectrums of
all the molecules including 1 to 3 are given under
supplementary data. The IR spectra of compound (1)
displayed absorption bands at 1545 and 3444 cm-1,
corresponding to C = N and -NH group respectively. The 1H
NMR spectra of compound (1) showed signals at δ 4.962 and
δ 7.08 to 9.11 corresponding to NH and CH (ring) protons
respectively. The IR spectra of compound (2) displayed
absorption bands at 1150, 1460, 1632 and 1739.57 cm-1,
corresponding to C-N, N-CH2, C = N and C = O group
respectively. The NMR spectra of compound (2) showed
signals at δ 1.213 and δ 4.188 corresponding to CH3 and CH2
protons of ester, respectively. The IR spectra of compound
(3) displayed characteristic absorption band at 1680.35 cm-1
due to C = O, amide group, whereas the absorption band at
3068.42 cm-1 confirming the presence of -CONH group. The
1
H NMR spectra of compound (3) showed signals at δ 2.54,
δ 5.37 and δ 8.167 corresponding to NH2, -N-CH2 and
CONH protons, respectively. The structure assigned to the
Synthesis, Medicinal and Docking Simulations of Derivatives Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 5

N N N N
Dry acetone, K2CO3

ClCH2COOC2H5 N
N
H

1 2
O

NH2NH2.H20

C2H5OH

N N N
N POCl3
RCOOH

N N

4 3
O
O
R = C6H5 (4a), 4-ClC6H4 (4b),
4-OHC6H4 (4c), 4-OCH3C6H4 (4d),3-NO2C6H4(4e)
R N HN
NH2
N

Scheme 1. Synthetic route of compounds from 1 to 4 including 4a to 4e.

Table 1. Prediction of molecular properties descriptors of the titled compounds (4a-4e).

Compound miLogP TPSA MW (mol/g) n-ON n-OH or n-NH Nrotb Nvio Volume Natom

4a 3.56 69.64 353.38 6 0 4 0 309.37 27

4b 4.24 69.64 387.83 6 0 4 0 322.91 28

4c 3.08 89.87 369.38 7 1 4 0 317.39 28

4d 3.62 78.88 383.41 7 0 5 0 334.92 29

4e 3.49 115.47 398.38 9 0 5 0 332.71 30

X -5.35 336.45 581.58 19 16 9 3 497.25 40

Y 3.77 69.08 531.44 8 0 7 1 452.47 36

Z -1.40 107.22 176.12 6 4 2 0 139.71 12

Here, X = Streptomycin, Y = Ketoconazole, Z = Ascorbic acid, logarithm of compound partition coefficient between n-octanol and water (miLogP), topological polar surface area
(TPSA), percentage of absorption (% ABS), molecular weight (MW), number of hydrogen bond acceptors (n-ON), number of hydrogen bond donors (nOH/NH), number of rotatable
bonds (Nrotb), Number of violations (Nvio).

compounds 4a-4e were supported by IR spectrum showing
characteristics absorption bands at 3573-3588, 1630-1692,
1260-1282, 1105-1097 and 1012-1025 cm-1, corresponding
to the aromatic CH, C = N, C-O-C asymmetrical, C-O-C
symmetrical and -N-N groups, respectively. The 1H NMR
spectrum of compound 4a-4e showed signals range at δ
5.52-5.62 and 7.090-9.134 ppm corresponding to the –N-
CH2 and aromatic CH protons, respectively. All the other
aliphatic and aromatic protons were observed within the
expected regions. Mass spectra also supported the structures
of the synthesized compounds.
3.2. Molecular Properties Descriptors
The synthesized compounds (4a-4e) were evaluated
against Lipinski’s rule of five by calculating molecular
descriptors using Molinspiration and results are presented in
Table 1 [53]. According to Lipinski’s rule of five, molecules
that have miLogP<5, molecular weight <500, the number of
hydrogen bond acceptors <10 and the number of hydrogen
bond donors<5, possess good oral bioavailability. The
compounds were in accordance with Lipinski’s rule of five
showing good drug-likeness and bioactivity score for drug
targets with no violations. The bioactivity scores of the
compound 4a-4e were also predicted by calculating the
activity score of GPCR ligand, ion channel modulator,
nuclear receptor legend, kinase inhibitor, protease inhibitor
and enzyme inhibitor by Molinspiration and were presented
in Table 2. A molecule having bioactivity score more than
zero, it is presumed to be active, while values -0.50 to 0.00
are expected to be moderately active and if the score is less
than -0.50, it is considered to be inactive [40,41]. Against
GPCR ligand compound 4c was found to be active with
bioactivity score 0.01. The compounds were moderately
active against ion channel modulator, nuclear receptor ligand
6 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 Bhati et al.

Fig. (1). Binding modes of compound 4b with protein targets 6g1o (a), 5isp (b), 5kbz (c), 5yxf (d) and 6il3 (e) respectively visualized using
Pymol software. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Table 2. Prediction of bioactivity score of the titled compounds (4a-4e).

Compound GPCR ICM KI NRL PI EI

4a -0.03 -0.26 0.08 -0.36 -0.12 0.03

4b -0.03 -0.26 0.06 -0.37 -0.16 -0.01

4c 0.01 -0.21 0.11 -0.23 -0.11 0.07

4d -0.07 -0.31 0.04 -0.35 -0.17 -0.02

4e -0.16 -0.29 -0.03 -0.42 -0.24 -0.07

X 0.09 -0.16 -0.17 -0.18 0.65 0.38

Y 0.26 -0.14 -0.17 -0.32 -0.21 0.15

Z -0.53 -0.24 -1.09 -1.01 -0.81 0.20

Here, X = Streptomycin, Y = Ketoconazole, Z = Ascorbic acid, Ligand (GPCR, GPCR), Ion channel modulator (ICM), Kinase inhibitor (KI), Nuclear receptor ligand (NRL), Protease
inhibitor (PI), Enzyme inhibitor (EI).
Synthesis, Medicinal and Docking Simulations of Derivatives Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 7

Fig. (2). Binding modes of compound 4e with protein targets 6g1o (a), 5isp (b), 5kbz (c), 5yxf (d) and 6il3 (e) respectively visualized using
Pymol software. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Table 3. Antimicrobial activities of the synthesized compounds (4a-4e) with Minimum inhibitory concentration MIC mg/l
(MBC/MFC) values.

Conc. Inhibition zone (mm)a

Strain
(mg/l) 4a 4b 4c 4d 4e X Y

50 7 6 6 5 13 11 NA
P. aeruginosa
250 10 8 8 7 18 12 NA
(MTCC 4673)b
500 18 16 14 8 35 34 NA

50 6 ND ND ND 6 8 NA
S. aureus
250 8 6 ND 7 9 16 NA
(MTCC 7443)b
500 12 11 6 12 12 28 NA

50 ND ND ND 6 7 8 NA
S. enterica
250 ND ND ND 8 10 11 NA
(MTCC 164)b
500 ND 7 6 28 15 30 NA

50 ND ND ND ND 7 9 NA
S. mutans
250 ND ND ND ND 8 12 NA
(MTCC 497)b
500 11 ND ND 7 18 26 NA
(Table 3) Contd…
8 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 Bhati et al.

Conc. Inhibition zone (mm)a

Strain
(mg/l) 4a 4b 4c 4d 4e X Y

50 4 6 ND ND 7 NA 10
A. parasiticus
250 6 11 6 6 9 NA 15
(MTCC 646)f
500 11 18 8 10 12 NA 28

Minimum Inhibitory Concentration MIC (MBC/MFC) mg/l

Strain 4a 4b 4c 4d 4e X Y

P. aeruginosa 25 50 50 12.5
100 (>200) 12.5 NA
(MTCC 4673)b (100) (>200) (>200) (50)

S. aureus 50 100 25
b
---- 100 (>200) 25 NA
(MTCC 7443) (>100) (>200) (50)

S. enterica 25
b
---- ---- ---- ---- 25 NA
(MTCC 164) (50)

S. mutans 25
---- ---- ---- 50 (>200) 25 NA
(MTCC 497)b (50)

A. parasiticus 50 50 100 25
100 (>200) NA 12.5
(MTCC 646)f (200) (200) (>200) (50)
Here X (standard) = Streptomycin, Y (standard) = Streptomycin, a = mean of triplicates, b = bacterial strain, f = fungal strain, ND = not detected, NA = not applicable.

and protease inhibitor. Compounds 4a, 4b, 4c and 4d were
active against kinase inhibitor with the bioactivity scores
ranging from 0.04 to 0.11. Compounds 4a (0.03) and 4c
(0.07) were found to be active against enzyme inhibitor
while other compounds were moderately active with
bioactivity score between -0.01 to -0.07 (Figs. 1 and 2). The
result suggested that most of the compounds were
moderately active with all drug targets and none of the
compound was found to be inactive.
3.3. Antimicrobial Activity
The synthesized compounds (4a-4e) were screened for
their in-vitro antibacterial activity against two gram negative
bacterial strains i.e. Pseudomonas aeruginosa, Salmonella
enterica and two gram positive bacterial strain i.e.
Staphylococcus aureus, Streptococcus mutans by taking
Streptomycin as standard drug while in vitro antifungal
activity was carried out against a fungal strain Aspergillus
parasiticus by taking Ketoconazole as standard drug at
concentration 50, 250 and 500 ppm by disk diffusion
method. The results of the zone of inhibition measured for
antibacterial and fungal activity are shown in Table 3 and
Supplementary data. The majority of the compounds was
found to be active against selected bacterial and fungal
strains. A maximum degree of inhibition was observed at
500 ppm concentration. The molecule 4e has shown better
anti-bacterial activities and least by 4d. Compound 4e has
shown prominent activities against all bacterial strains,
whereas compound 4a was active against two bacterial
strains viz. P. aeruginosa and S. aureus. All the compounds
have shown significant activity against strain P. aeruginosa
with an order 4e >4a >4b >4d. The results of antifungal
activity revealed that compound 4b was found to be
moderately active against Aspergillus parasiticus while
compounds 4a, 4c and 4e were active. All the compounds have
shown antifungal activities with an order 4b >4e >4a >4c.
The Minimum Inhibitory Concentration (MIC) values of
all compounds were evaluated and reported under Table 3.
The MIC values were ranged from 12.5 to 200 mg/l. As
compared to the standards, molecules 4a and 4e have shown
prominent antimicrobial activities over ranged from 12.5 to
25 mg/l (Table 3). The results of antimicrobial screening
revealed that the compounds showed significant activity in
comparison to standard drugs. Compound 4e emerged as a
potent antimicrobial derivative in the series against selected
bacterial and fungal strains.
3.4. Antitubercular Activity
The synthesized compounds 4a-4e were screened for
their in vitro antitubercular activity against Mycobacteria
tuberculosis at a concentration ranging from 0.8-100 µg/ml
(Table 4 and Supplementary data) by using Micro-plate
Alamar Blue Assay (MABA). Pyrazinamide, Ciprofloxacin
and Streptomycin were used as reference drugs. The
compounds showed significant activity compared to the
standard drugs. The Minimum Inhibitory Concentration
(MIC) was determined as the lowest drug concentration
which prevented the color change from blue to pink. The
results indicated that compound 4a having phenyl ring at 5th
position of oxadiazole ring was most effective against M.
tuberculosis with MIC value 1.6 µg/ ml in the series. It was
noted that compounds 4b, 4d and 4e exhibited moderate
anti-TB activity having MIC of 12.5 µg/ ml.
3.5. Antioxidant Activity
The in vitro antioxidant activity of the compound 4a-4e
was determined spectrophotometrically by DPPH free
Synthesis, Medicinal and Docking Simulations of Derivatives Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0 9

Table 4. Anti-tubercular activity of the synthesized compounds (4a-4e).

Concentration (µg/ml)
Compound MIC µg/ml
100 50 25 12.5 6.25 3.12 1.6 0.8

4a S S S S S S S R 1.60

4b S S S S R R R R 12.5

4c S S S R R R R R 25.0

4d S S S S R R R R 12.5

4e S S S S R R R R 12.5

K S S S S S S R R 3.12

L S S S S S S R R 3.12

M S S S S S R R R 6.25
Here, S – Sensitive R- Resistant, K = Pyrazinamide, L = Ciprofloxacin, M = Streptomycin.

Table 5. Antioxidant activity of the synthesized compounds (4a-4e).

% inhibition at Different Concentrations (µg/ml)

Compound IC50 (µg/ml)
25 50 75 100

4a 32.87 45.28 50.57 57.24 73.83

4b 28.27 41.83 46.89 55.86 81.88

4c 39.54 47.81 56.09 64.59 56.63

4d 3.21 5.51 9.19 11.49 436.11

4e 49.88 55.63 62.06 66.66 24.85

Ascorbic Acid 32.87 40.68 55.40 70.34 62.91

Table 6. Docking simulations for antimicrobial activity of the synthesized compounds (4a-4e).

PDB ID: 6g1o PDB ID: 5isp PDB ID: 5kbz

Compound Binding Energy Ki Inhibition Binding Energy Ki Inhibition Binding Energy Ki Inhibition
(Kcal/mol) Constant (µM) (Kcal/mol) Constant (nM) (Kcal/mol) Constant (nM)

4a -6.77 10.82 -10.75 13.21 -8.24 915.68

4b -6.97 7.74 -11.06 7.77 -9.95 50.62

4c -6.81 10.21 -10.17 35.02 -9.28 156.8

4d -6.25 26.31 -10.57 18.02 -9.28 158.19

4e -7.68 2.34 -11.03 8.23 -9.42 124.37

Streptomycin -4.38 614.68 -3.8 1.63mM - -

Ketoconazole - - - - -9.41 126.09

10 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 0
Table 7. Docking simulations for anti-tubercular and antioxidant activity of the synthesized compounds (4a-4e).
PDB ID: 5yxf
Compound
Binding Energy (Kcal/mol) Ki Inhibition Constant (nM)
4a -8.35 763.36
4b -9.01 247.29
4c -8.25 897.5
4d -8.61 489.23
4e -9.66 83.15
Streptomycin -6.84 9.67 µM
Ascorbic acid - -
radical scavenging method at a concentration ranging from
25 µL to 100 µL and the results were summarized in Table 5.
Ascorbic acid was used as a positive control. The reduction
capacity of DPPH radicals was determined by the decrease in
its absorbance at 517
 nm, which is induced by antioxidants. bearing chloro group at phenyl ring emerged as prominent
The compounds showed moderate scavenging activity on
DPPH compared to the standard. It was observed that
antioxidant potential of the compounds increased with an
increase in concentration. Compound 4e having nitro group
substitution at phenyl ring on oxadiazole ring appeared to be the
most active derivative with IC50 value of 24.85 µg/ml in the
series followed by compound 4c with IC50 value of 56.63 µg/ml.
3.6. Docking Simulations
AutoDock 4.2, the docking program was employed to
estimate the binding affinity of ligand-receptor complex in
the receptor binding site. The receptor was kept rigid, while
ligands were set flexible to rotate and explore the most
probable binding poses. For docking study, the antimicrobial
activity of all the synthesized derivatives 4a-4e were docked
with three protein target namely Isocitrate lyase (P.
aeruginosa, PDB ID: 6g1o), Dihydrofolate reductase (S.
aureus, PDB ID: 5isp) and Noranthrone synthase (A.
parasiticus, PDB ID: 5kbz) and results are presented in
Table 7. Docking studies revealed that the synthesized
compounds 4a-4e showed good binding energy towards
selected protein targets compared to the standard drugs.
Compound 4e and 4b showed minimum binding energy in
the series against selected protein targets and hence can be
considered as potential antimicrobial agents. Docking
simulation for antitubercular activity and antioxidant activity
was carried out against protein target Methionine
aminopeptidase (M. Tubeculosis, PDB ID: 5yxf) and
tyrosine-protein kinase (Homosapiens, PDB ID: 6il3),
respectively and results are presented in Table 7. The results
suggested that compound 4e (-9.66 Kcal/ mol) and 4b (-9.01
Kcal/ mol) were the best ligands in the series in terms of
binding energy and inhibition constant against protein target
5yx. Docking results of antioxidant activity revealed that
compound 4a (-10.4 Kcal/mol) showed a strong affinity with
protein target 6il3 followed by compound 4e (-10.28
Kcal/mol) in the series. From the in silico docking
simulations, it is quite evident that all the synthesized
compounds 4a-4e had great potential as antimicrobial,
Bhati et al.
PDB ID: 6il3
Binding Energy (Kcal/mol) Ki Inhibition Constant (nM)
-10.4 24.0
-9.77 69.13
-9.9 55.8
-9.79 66.43
-10.28 29.27
- -
-4.88 263.77 µM
antitubercular and antioxidant agents. Figs. 1 and 2 depict
the binding mode of the best conformer of compounds 4b
and 4e against the selected protein targets for docking
studies. Compound 4e bearing nitro group and compound 4b
ligands in the series against selected protein targets.
CONCLUSION
In the present study a series of 1-[(5-phenyl/substituted
phenyl-1, 3, 4-oxadiazol-2-yl) methyl]-2-(pyridin-3-yl)-1H-
benzimidazole derivatives (4a-4e) were synthesized by
multistep reaction scheme. The structures of the synthesized
compounds were confirmed by IR, 1HNMR and mass
spectral techniques. The synthetic compounds were assessed
for their in vitro antimicrobial, antitubercular and antioxidant
potential and showed significant activity. The derivatives
were in accordance with Lipinski’s rule of five and showed
good bioavailability [53]. Docking simulations were
performed to gain an insight into the binding modes of the
synthesized derivatives and good binding affinity was
recorded against selected protein targets. A good correlation
was observed between the experimental results and
theoretical predictions. Among the synthesized compou-
nds,1-{[5-(3-nitro phenyl)-1,3,4-oxadiazol-2-yl]methyl}-2-
(pyridin-3-yl-1H-benzimidazole (4e) and 1-{[5-(4-chlorop-
henyl)-1,3,4-oxadiazol-2-yl]methyl}-2-(pyridin-3-yl)-1H-
benzimidazole (4b) emerged as promising compound in the
series and can be considered for further in vivo validation.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
Not applicable.
FUNDING
None.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
Synthesis, Medicinal and Docking Simulations of Derivatives
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s
web site along with the published article.
ACKNOWLEDGEMENTS
The authors are thankful to the Head, SAIF
(Sophisticated Analytical Instrument Facility)/ CDRI
(Central Drug Research Institute), Lucknow for FTIR, 1H
NMR and mass spectral analysis. The authors also
acknowledge Dr. Kishore G. Bhat, MD, Microbiology,
Maratha Mandal's Central Research Laboratory, Belgaum for
antitubercular activity and Head, Department of
Biotechnology, Lovely Professional University, Jalandhar
for antioxidant activity.
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