Molecular docking studies and synthesis of 3, 4-disubstituted triazoles as mycobacterium tuberculosis enoyl-acp reductase and CYP-51 inhibitors

Article abstract of DOI:10.22159/ijpps.2019v11i1.29428

Objective: To design, synthesize and in vitro antitubercular, antifungal and antioxidant evaluation of some novel mercapto 1, 2, 4–triazole derivatives. Methods: New derivatives were designed by using various software like ACD Lab chemsketch, molinspiration and autodock. Designed molecules are obeying Lipinski’s rule of five and having highest binding score was selected for the synthesis. The synthesized compounds were subjected to TLC, melting point determination, FTIR,¹H NMR,¹³C NMR and mass spectral analysis. The newly synthesized compounds were investigated for in vitro antitubercular evaluation by MABA method, antifungal evaluation by cup plate method and antioxidant evaluation by DPPH scavenging assay. Results: A virtual screening was carried out through docking designed compounds into the InhA and CYP-51 binding site to predict if these compounds have an analogous binding mode of the enoyl ACP reductase (InhA) and CYP-51 inhibitors. Three derivatives (4a1, 4a2 and 4a3) were selected for the synthesis with the help of in silico modeling. The selected derivatives were synthesized by a conventional method. All the synthesized compounds showed a characteristic peak in FT IR,¹H and¹³C NMR and mass spectroscopic studies. All the selected derivatives showed antitubercular, antifungal and antioxidant activity. Conclusion: The derivatives were synthesized adopting simple and laboratory friendly reaction conditions to give the target compounds in quantitative yields. Newer derivatives possess good antitubercular, antifungal and antioxidant activity.

Full text of DOI:10.22159/ijpps.2019v11i1.29428

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 11, Issue 1, 2019
Original Article
MOLECULAR DOCKING STUDIES AND SYNTHESIS OF 3, 4 - DISUBSTITUTED TRIAZOLES AS
MYCOBACTERIUM TUBERCULOSIS ENOYL-ACP REDUCTASE AND CYP-51 INHIBITORS
NEETHU DASAN, G. BABU, SHINY GEORGE*
Department of Pharmaceutical Chemistry, Devaki Amma Memorial College of Pharmacy, Malappuram, Kerala, India 673634
Email: georgeshiny28@gmail.com
Received: 31 Aug 2018 Revised and Accepted: 28 Nov 2018
ABSTRACT
Objective: To design, synthesize and in vitro antitubercular, antifungal and antioxidant evaluation of some novel mercapto 1, 2, 4–triazole derivatives.
Methods: New derivatives were designed by using various software like ACD Lab chemsketch, molinspiration and autodock. Designed molecules
are obeying Lipinski’s rule of five and having highest binding score was selected for the synthesis. The synthesized compounds were subjected to
1
TLC, melting point determination, FTIR, H NMR, 13C NMR and mass spectral analysis. The newly synthesized compounds were investigated for in
vitro antitubercular evaluation by MABA method, antifungal evaluation by cup plate method and antioxidant evaluation by DPPH scavenging assay.
Results: A virtual screening was carried out through docking designed compounds into the InhA and CYP-51 binding site to predict if these
compounds have an analogous binding mode of the enoyl ACP reductase (InhA) and CYP-51 inhibitors. Three derivatives (4a1, 4a2 and 4a3) were
selected for the synthesis with the help of in silico modeling. The selected derivatives were synthesized by a conventional method. All the
synthesized compounds showed a characteristic peak in FT IR, 1H and 13C NMR and mass spectroscopic studies. All the selected derivatives showed
antitubercular, antifungal and antioxidant activity.
Conclusion: The derivatives were synthesized adopting simple and laboratory friendly reaction conditions to give the target compounds in
quantitative yields. Newer derivatives possess good antitubercular, antifungal and antioxidant activity.
Keywords: Triazole, Docking, Antitubercular, Antifungal, Antioxidant
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2019v11i1.29428
INTRODUCTION
Mycobacterium tuberculosis enoyl-acyl carrier protein (ACP) reductase
(InhA) has been validated as a promising target for antitubercular
agents. InhA was identified as an NADH-dependent enoyl-ACP
In the past, most drugs have been discovered either by identifying
the active ingredient from traditional remedies or by serendipitous
discovery. A new approach has been identified to understand how
disease and infections are controlled at the molecular and
physiological level and to target specific entities based on this
knowledge [1]. Molecular docking is a method, which predicts the
preferred orientation of one molecule to a second when bound to
each other to form a stable complex. Knowledge of the preferred
orientation, in turn, may be used to predict the strength of
association or binding affinity between two molecules using scoring
functions which are used to predict the strength of the non-
covalent interaction (also referred to as binding affinity) between
two molecules after they have been docked [2]. Molecular docking
has become an increasingly important tool for drug discovery.
(CoA) reductase specific for long-chain enoyl thioesters and is
a
member of the Type II fatty acid biosynthesis system, which elongates
acyl fatty acid precursors of mycolic acids which are components of
the mycobacterial cell wall [4, 5].
Azoles exert antifungal activity through inhibition of cytochrome
P450 14α-demethylase (CYP51), which is crucial in the process of
biosynthesis of ergosterol by a mechanism in which the heterocyclic
nitrogen atom (N-4 of 1,2,4-triazole) bind to the heme iron atom.
Selective inhibition of CYP 51 would cause depletion of ergosterol
and accumulation of lanosterol and other 14-methyl sterols
resulting in the growth inhibition of fungal cells [6]. When a
mercapto group attached to the heterocyclic rings enhance
fungicidal activity. It has been reported that structural properties of
triazoles, like moderate dipole character, hydrogen bonding
capability, rigidity and stability under in vivo conditions are the main
reasons for their superior pharmacological activities [7].
Mycobacterium tuberculosis (M. tuberculosis) is the fundamental
etiologic agent for human tuberculosis and is the biggest killer due
to bacterial infection in the world today. Tuberculosis accounts for
about 7% of all deaths in developing countries and 26% of avoidable
adult deaths [3]. Due to the reduced effectiveness of current drugs
resulting from the emergence of multidrug-resistant tuberculosis
(MDR TB) and co-infection with human immunodeficiency virus
(HIV), there is an urgent need to develop new natural or synthetic
antitubercular drugs. The major drawbacks with the therapeutic
agents for mycobacterial and fungal infections are prolonged
treatment regimen with a combination of drugs associated with
significant toxicity and the emergence of multi-drug resistant
bacteria and fungi causing morbidity and mortality in
immunocompromised hosts. The necessity for effective therapy has
stimulated research into the design and synthesis of novel
compounds which can treat both mycobacterial and fungal
infections. Design of compounds having good anti-tubercular activity
is gaining much importance in the field of tuberculosis research due
to the resurgence of antibiotic-resistant strains.
To date, the prevention of oxidative stress related diseases has been
tentatively achieved by the development of antioxidant compounds
that are able to scavenge reactive oxygen species and thus avoid
radical-induced oxidation damage. Excessive free radical attack can
damage DNA, proteins and lipids, resulting in diseases like cancer,
neurological degeneration and arthritis, as well as the process of aging
[8]. Therefore, considerable speculation has been directed towards the
identification of antioxidants for use in preventive medicine.
In recent years, triazole derivatives have acquired conspicuous
significance due to their wide spectrum of biological activities such as
antibacterial [9], antifungal [10], antiviral [11], anti-inflammatory [12,
13], anthelmintic [14], antitubercular [15], antioxidant [16],
antiplasmodial [17] and antileishmanial [18] activity. Nowadays, many
triazole containing drugs are available in the market such as fluconazole,
itraconazole, voriconazole, posoconazole anastrozole, estazolam,

George et al.
Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
ribavirin, triazolam [19], etc. The search and evaluation of 1,2,4-triazole
compounds and their derivatives with a specific pharmacological activity
is a demanding task in the drug discovery process. This study aims to
discover potentially active new 1, 2, 4-triazole derivatives through in
silico drug designing as InhA and cyp 51 inhibitor. Also, we present an
evaluation of the antioxidant effect of substituted triazole derivatives.
receptor with novel compounds were carried out by using AutoDock
4.2.1 and Auto Dock Tools (ADT) v 1.5.4 from the Scripps Research
Institute. Firstly, all bound waters, ligands and cofactors were
removed from the proteins. Then macromolecule was checked for
polar hydrogen. Partial atomic Kollman charges were assigned, and
then atomic solvation parameters were allotted. Torsion bonds of
the inhibitors were selected and defined. Secondly, the three-
dimensional grid box was created by AutoGrid algorithm to evaluate
the binding energy on the macromolecule coordinates. Grid maps
representing intact ligand in the actual docking target site were
calculated with AutoGrid. The three-dimensional grid box with a
grid map of size 80x80x80 of x, y and z-axis for different grid
parameters with a spacing of 0.300 A ° was created. Eventually,
cubic grids encompassed the binding site where the intact ligand
was embedded. Finally, resultant compounds were used to compute
molecular stimulation parameters like Lamarckian genetic algorithm
of population size 150, the mutation rate of 0.02 and crossover rate
of 0.8, these simulations were performed up to 2.5 million energy
and the evaluations were maximum at 27000 generations. Each
simulation was carried about 10 times which ultimately yielded 10
docked conformations. From this, the lowest energy conformations
were regarded as the best binding conformations.
MATERIALS AND METHODS
Lead optimization
Lead optimization was done through in-silico Lipinski filter.
Molinspiration server was used for this purpose [20]. The structure
drawn in the JME molecule editor was subjected to analysis of
Lipinski rule of five.
Selection of target protein
The 3D crystal structure of InhA receptor (entry code: 4U0J) [21]
and CYP 51 receptor/fungal protein (entry code: 1EA1) [22] used for
docking was recovered from the Brookhaven Protein Data Bank
(http://www. rcsb. org/pdb/home). Reference compounds such as
pyrazinamide and fluconazole are directly downloaded from
DrugBank 2.5 database [23, 24], and they are potentially competitive
inhibitor against target proteins.
Chemistry
Active site prediction
Chemicals used were purchased from Merck and Hi-Media, Mumbai,
India. Vanillic acid (4-hydroxy-3-methoxy benzoic acid), carbon-
disulfide, potassium hydroxide, hydrazine hydrate, ethanol, and
anhydrous ether was purchased from Merck. DMSO and aromatic
amines (4-nitroaniline, 2-nitroaniline, 4-bromo aniline) were
purchased from Hi-Media, India. All the chemicals were used
without further purification. Melting points were determined in
open capillary tube and were uncorrected. Purity of the synthesized
compounds was routinely checked by thin layer chromatography on
silica gel G with the solvent system–benzene: methanol (8:2) using
iodine vapour for detection. IR spectra of the compounds were
recorded using KBr pellets in the range of 4000-500 cm-1 on a Jasco
FTIR spectrophotometer in Devaki Amma Memorial College of
Pharmacy, Malappuram, India. 1H and 13C NMR spectra were
recorded in CDCl3 on Bruker ultra shield DPX 400 spectrometer
using tetramethylsilane as an internal standard (chemical shifts in δ,
ppm) and mass spectra on LC-MSD Trap-SL 2010 A-Shimadzu
spectrometer at Interdisciplinary School of Indian System of
Medicine (ISISM), SRM University, Chennai, India.
A prediction of the active site and ligand binding sites of 3D protein
structure was done by using Computed Atlas of Surface Topography
of proteins (CASTp) which is a web server that provides online
services for locating, delineating and measuring geometric and
topological properties of protein structures [25].
Preparation of ligand files
Ligand files for the molecular docking studies were prepared in
Chem Draw Ultra software, Cambridge Soft Corporation, USA.
Version-12.0, 1997-2010. It is a Chem Tech tool used for the
drawing of ligand molecules. Compound structures were drawn in
ChemDraw software and converted to pdb format (. pdb file) with
the standard settings and further used for docking studies.
Docking study
Molecular docking studies performed with various targets like
enoyl-ACP reductase and cytochrome P450 14α-demethylase
NH2
O
OH
O
O
O
NH
C2H5
NH2NH2.H2O
C2H5OH
H2SO4
CH3
CH3
O
CH3
O
O
OH
4-hydroxy-3-methoxybenzoic acid
OH
OH
ethyl 4-hydroxy-3-methoxybenzoate
4-hydroxy-3-methox
2a
ybenzohydrazide
1a
CS2 /KOH
SK
O
NH
NH
O
S
N
SH
ArNH2
N
CH3
N
Ar-
OH
potassium 2-(4-hydroxy-3-methoxybenzoyl)hydrazinecarbodithioate
3a
HO
O
H3C
4 - [4 -(substituted)-5 -sulfanyl - 4H 1,2,4triazole -3 -yl] - 2 -methoxyphenol
4a 1 -3
Fig. 1: Schematic presentation of synthesis of 4a1-4a3
86

George et al.
Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
Synthesis of ester (1a)
fluorescent, oxidized form becomes pink and fluorescent upon
reduction. Growth was therefore determined by a visual color
change. The derivatives were considered active (have inhibitory
activity) for the well of the plate with unchanged color or the blue,
non-fluorescent, oxidized form and if the color of the agent or
resazurin is changed to pink (fluorescent) the derivative is inactive
or the microorganism is considered resistant strain to the derivative
[27].
A mixture of 0.01 mol substituted acid, 10 ml of absolute ethanol and
2 ml of concentrated sulphuric acid was refluxed for 4 h. The
product was recrystallized from ethanol.
Synthesis of acid hydrazide (2a)
A mixture of 0.01 mol ester and 0.2 mol hydrazine hydrate was
refluxed in 50 ml of 95% ethanol for 2 h. The resultant mixture was
acidified with concentrated hydrochloric acid. The solid mass thus
separated out was filtered, dried and purified by recrystallization
from ethanol.
Antifungal activity
All the synthesized compounds (200 µg/ml) in DMSO solvent were
screened for in vitro antifungal activity by agar well diffusion
method [26]. The activity was evaluated against pathogenic fungal
strains such as Candida albicans (ATCC. 4563), Aspergillus niger
(ATCC. 20611) using Sabouraud’s dextrose agar medium. The fungal
strains were procured from the Department of Microbiology, Devaki
Amma Memoria College of Pharmacy. The results were compared
with standard fluconazole (25 µg/ml). The plates were incubated for
48 h at 27 °C in an incubator. Plates were read only if the lawn of
growth was confluent or nearly confluent. The diameter of the
inhibition zone was measured to the nearest whole millimeter by
holding the measuring device. The test was carried out in triplicate.
Synthesis of the potassium salt of dithiocarbazinate (3a)
Potassium hydroxide (0.01 mol) was dissolved in absolute ethanol
(75 ml). To the above solution, acid hydrazide (0.01 mol) was added
with stirring and cooling in ice. To this, carbon disulphide (10 ml)
was added in small portions. The reaction mixture was refluxed for
4-6 h. To the resulting solution, anhydrous ether (250 ml) was
added and precipitated potassium dithiocarbazinate was collected
by filtration, washed with diethyl ether and dried. The
dithiocarbamates were obtained in quantitative yield. As most of the
potassium salt of dithiocarbazinates were moisture sensitive, they
were employed directly for the preparation of aminomercapto
triazoles without further purification.
Antioxidant activity
In vitro antioxidant activity was carried out by 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay method [28]. Assay
was carried out using UV spectrophotometer at 517 nm. To 2 ml
solutions of synthesized compounds (50, 100,150 and 200 μg/ml), 2
ml DPPH solutions (400 μmol) in ethanol were added into the test
tube. The solution was incubated at 37 °C for 30 min and the
absorbance of each solution was measured at 517 nm against a
reagent blank solution. Ascorbic acid was used as reference
antioxidant. Experimental values summarized for DPPH radical
scavenging assays are expressed as the mean±standard error of
mean (SEM). The percent free radical scavenging activity was
calculated by the formula given by
Synthesis of 5-mercapto-1, 2, 4-triazole derivatives (4a1-4a3)
A suspension of potassium salt dithiocarbazinate (0.01 mol),
hydrazine hydrate (2 ml), water (80 ml) and a primary aromatic
amine (0.01 mol) was refluxed for 3h. The colour of the reaction
mixture changed to green, hydrogen sulphide was evolved and a
homogenous solution resulted. A white solid was precipitated by
dilution with cold water. The product was filtered, washed with cold
water and recrystallised from ethanol [26].
Antitubercular activity
Microplate Alamar Blue Assay (MABA) method was used for the
study. Prior to the bioassay, a standard stock solution with the
concentration of 32 μg/ml was prepared and stored to be used for
the positive control. Control wells without tested derivatives and
sterility controls were assayed simultaneously. Pyrazinamide was
prepared from the stock solution just prior to inoculation time to the
concentration of 5 μg/ml in the total volume of 200 μl. The growth
inhibition result was explained by MABA using 1 % resazurin. The
reagent allows the detection of microbial growth in microtiter plates
without the use of a spectrophotometer. The susceptibility test
conducted by the MABA was using 96 well microtitre plates to
evaluate the susceptibility of H37Rv MTB reference strain to the
derivative. The inhibitory concentration of all derivative was
evaluated with concentrations of 250 mg/ml, 500 mg/ml and 1000
mg/ml in the total volume of 200 μl. The measured derivatives were
mixed with the bacterial suspension and the diluent media (7H9) in
the well. The alamar blue oxidation-reduction dye is a general
indicator of cellular growth and/or viability; the blue, non-
Percentage scavenging
Control absorbance − Testabsorbance
=
× 100
Control absorbance
The experiment was done in triplicate. As opposed to increasing
concentration of specimens decline of absorbance is an indication
that destroyed DPPH radical. Antioxidant activity results are
expressed as IC50 value (μg/ml) that reduces by half the effective
concentration of DPPH radicals and was calculated by interpolation
from the linear regression analysis.
RESULTS AND DISCUSSION
In silico molecular analysis of different triazole derivatives was done.
All these compounds obeyed “Lipinski rule of five”. These analogues
were taken for computing molecular descriptors and which have the
best docking score was taken for synthesis. The results are shown in
table 1.
Table 1: Analysis of lipinski rule of five
Compound code
Ar
Log p
H donor (nON)
H acceptor
(nOHNH)
No. of rotatable bonds
(nrotab)
Mol. Wt
No.
of violation
4a1
C6H4NO2 2.86
C6H4NO2 3.02
8
8
5
4
1
1
1
1
4
4
3
3
344.34
344.34
378.24
362.54
0
0
0
0
4a2
4a3
Fluconazole
C6H4Br
-
3.71
3.86
Molecular modeling technique was used to explore, predict and
understand the protein/enzyme interactions with designed 1, 2,
4-triazole library; and also to visualize the probable binding.
Analogues were docked with various receptors using Autodock
4.2.1 and the binding energy obtained are shown in table 2.
Docking studies of targeted compounds with CYP51 protein
showed good binding interactions and formed various
hydrophobic interactions with active site residues. The ligand
4a3 exhibited docking score of 9.73, with the main
hydrophobic interactions with the surrounding residues
ALA191A, GLY192A, ILE194A and PRO193A, strongly
contributed to the stabilization with 1EA1. The hydrogen
bonding interaction of hydroxyl group protons of 4a3 were with
the residues SER94A and LYS166A.
a
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George et al.
Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
Table 2: Docking result with proposed derivatives
4U0J
Compound
code
1EA1
Binding energy (K.
Inhibition
constant
1.1µM
No. Of H
Binding energy (K.
Inhibition
constant
1.05 µM
4.06 µM
1.34 µM
-
No. Of H
Cal/Mol)
-8.64
-8.75
-9.73
-8.31
-
bonds
Cal/Mol)
-8.47
-8.39
-8.55
-
bonds
4a1
4
3
4
3
-
4
4
5
-
4a2
3.68 µM
693.55 nM
258.35 nM
-
4a3
Fluconazole
Pyrazinamide
-8.1
2.96 µm
3
Binding interactions of derivatives with 1EA1 and 4U0J
Fig. 2: Interaction of 1EA1 with 4a3
Fig. 3: Interaction of 1EA1 with 4a1
Fig. 4: Interaction of 4U0J with 4a3
Fig. 5: Interaction of 4U0J with 4a2
Analogues designed by in-silico studies were selected for wet lab
synthesis based on the good binding energy and availability. Three
analogues were synthesized by a conventional method. The
reaction sequence leading to the formation of titled compounds is
shown on the scheme. First step in synthesis was esterification of
carboxylic acids with alcohols which is a kind of nucleophilic acyl
substitution. In the second step, an efficient and general process,
involving preforming activated ester reacts with hydrazine, for the
preparation of hydrazides. This process gives the desired
hydrazides in excellent yield and purity under mild conditions
[29]. The main synthetic route to dithiocarbamates is based on the
interaction between the corresponding amine and CS2 in the
presence of a strong base. It was found that upon decreasing of the
protoning ability of the solvent, the rate of dithiocarbamate
formation increases. Finally, potassium salt of dithio carbazinates
was react with corresponding primary aromatic amines like 4-
bromo aniline, 4-nitroaniline and 2-nitroaniline, the ring cyclises
to form 1,2,4-triazole derivatives (4a1-4a3). All the compounds
were obtained in moderate to good yields. The physicochemical
data of synthesized derivatives is given in table 3.
Table 3: Physicochemical data of newly synthesized compounds
Compound
Physical appearance
Yellowish brown
Yellowish orange
Yellowish orange
Brown
Melting point in °C
58
Rf value
0.84
% yield
88
1a
2a
198
246
132
64
0.87
88
3a
0.76
84
4a1
4a2
4a3
0.80
78
Red
0.79
81
Pale yellow
86
0.82
67
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George et al.
Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
The structures of all the newly synthesized compounds were
confirmed by their IR, 1H and 13C NMR and mass spectral studies. All
the newly synthesized compounds exhibited satisfactory spectral
data consistent with their molecular structures.
136.04, 130.83, 125.80, 119.62, 115.86, 40.47, 40.30, 40.14, 39.97,
39.80, 39.64, 39.47, MS m/z (%):111.05, 132.95, 301.25 (base peak),
344.34 (molecular ion peak).
4a3: Molecular formula: C15H12BrN3O2S, IR υ (cm-1) (KBr) 2610 (S-
H-stretching), 510 (Ar-Br-stretching), 3517 (NH-stretching),1HNMR
(CDCl3) (ppm): δ 5.20 (S, 1H, OH), 6.56-7.4 (D, 7H,Aromatic H), 3.53
(S, 3H, CH3), 2.5 (S, 1H, C-SH), 13C NMR (CDCl3) ppm: δ 148.40,
131.82, 116.37, 106.83, 56.69, 40.50, 40.34, 40.17, 40.00, 39.67,
39.50, 19.06, MS m/z (%): 111.20, 206.10 (base peak), 378.24
(molecular ion peak).
4a1: Molecualr formula: C15H12N4O4S, IR υ (cm-1) (KBr) 3470 (NH-
1
stretching), 1700 (NO2-stretching), 2610 (S-H-stretching), H NMR
(CDCl3) (ppm): δ 5.01 (S, 1H, OH), 3.514 (S, 1H, C-SH), 6.61-6.68 (M,
5H, C6H5), 2.507 (S, 3H, CH3), 7.94-7.96 (D, 2H, CH2), 13C NMR
(CDCl3) ppm: δ 156.08, 136.23, 126.79, 112.85, 56.58, 40.41, 40.25,
40.17, 40.08, 39.91, 39.75, 39.58, 39.41, 18.92, MS m/z (%):132.95,
176.95, 193.05, 239.10 (base peak), 344.34 (molecular ion peak).
Antitubercular evaluation
4a2: Molecular formula: C15H12N4O4S, IR υ (cm-1) (KBr) 3470 (NH-
Antitubercular study was performed by MABA method using
Mycobacterium tuberculosis H37Rv strain (ATCC 27294). In table 4
showed the antitubercular effect of synthesized derivatives.
1
stretching), 1700 (NO2-stretching), 2610 (S-H-stretching), H NMR
(CDCl3) (ppm): δ 2.51 (S, 1H, C-SH), 3.42 (S, 3H, CH3) 5.01 (M, 1H, C-
OH), 6.6-7.95 (M, 7H, Aromatic H), 13C NMR (CDCl3) ppm: δ 146.65,
Table 4: Antitubercular screening of synthesized derivatives
MIC in µg/ml
Test drugs
4a1
25
25
12.5
3.125
4a2
4a3
Pyrazinamide
Fig. 6: Mycobacterial sensitivity to synthesized derivatives
Among the compounds evaluated 4-[4-(4-chlorophenyl)-5-sulfanyl-
4H-1,2,4-triazol-3-yl]-2-methoxyphenol (4a3) showed good binding
affinity with Mycobacterial InhA, having minimum inhibitory
concentration found to be 12.5µg/ml compared with pyrazinamide
as standard drug. The antitubercular data revealed that compounds
having an electron withdrawing group like bromo attached to
triazole nucleus may prove a template for antitubercular activity for
further development.
was determined using digital zone reader, and the datas are given in
table 5. Azoles perform their antifungal action in two steps:
inhibition of ergosterol synthesis, a major component of the fungal
membrane and second step involves the blocking of P450-
dependent enzyme i.e., lanosterol 14-α-demethylase (CYP 51). The
3D model of CYP 51 obtained on the basis MTCYP51 (M. Tuberclosis
14-α-demethylase) showed that triazole ring coordinated to the
heme iron of CYP51, a key step in antifungal activity of triazole. Lack
of
ergosterol
and
accumulation
of
14-α-demethylase
Antifungal evaluation
disfunctionalize the fluidity of several enzyme located in the
membrane which results in inhibition of fungal growth and
replication of its DNA [30].
The antifungal evaluation was done using 2 microorganisms i.e.
Candida albicans and Aspergillus niger. Growth of inhibition of wells
Table 5: Zone of inhibition of 4a1-4a3 in C. albicans and A. niger
Diameter of zone of inhibition (mm) in
Compound
C. albicans
10.53±0.251
11.86±0.305
16.63±0.028
17.20±0.264
0
A. niger
4a1
8.25±0.031
10.85±0.041
15.69±0.015
17.86±0.638
0
4a2
4a3
Fluconazole (25µg/ml)
DMSO
*Values are given mean±SD (n=3)
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Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
Antioxidant evaluation
Many of them were shown to possess antioxidant properties and
inhibit radicals by the effect of electron donating groups or
supplying hydrogen atom. Based on the IC50 values of standard
ascorbic acid and synthetic derivatives as showed in table 6, it is
indicated that 4a3 shows better DPPH scavenging activity.
Antioxidant evaluation was carried out by DPPH assay using
ascorbic acid as a standard. The compounds 4a1-4a3 exhibited
significant scavenging activity ranging from 40 to 88% and the data
are expressed in mean±SEM in table 6.
Table 6: Antioxidant evaluation of synthesized derivatives
Compound
Ascorbic acid
Concentration (µg/ml)
12.5
25
50
Percentage inhibition
18.23±0.483
36.99±0.745
52.48±0.485
75.01±0.530
40.05±0.028
47.41±0.005
56.43±0.005
62.89±0.010
45.37±0.0152
47.29±0.0115
53.17±0.0208
75.01±0.530
54.21±0.0208
70.31±0.0057
80.44±0.0057
88.9±0.0100
IC50 (µg/ml)
54.11
100
50
100
150
200
50
100
150
200
50
100
150
200
4a1
4a2
4a3
114.12
122.46
22.32
*Values are given mean±SEM (n=3).
CONCLUSION
tubercular
Targets 2007;8:399-411.
and
anti-malarial
agents.
Curr
Drug
An attempt was made to explore the antitubercular potential of
triazole scaffold in drug development. In this study, new
mercapto1,2,4-triazole derivatives were designed and synthesized
from potassium salt dithiocarbazinate and aromatic amines. The
6. Zhongjun Guan, Xiaoyun Chai, Shichong Yu, Honggang Hu,
Yuanying Jiang, Qingguo Meng, et al. Synthesis, molecular
docking and biological evaluation of novel triazole derivatives
as antifungal agents. Chem Biol Drug Des 2010;76:496–504.
7. Stefania Felicia Barbuceanu, Diana Carolina Ilies, Gabriel
Saramet, Valentina Uivarosi, Constantin Draghici, Valeria
Radulescu. Synthesis and antioxidant activity evaluation of new
compounds from hydrazinecarbothioamide and 1, 2, 4-triazole
class containing diaryl sulfone and 2,4-difluoro-phenyl
moieties. Int J Mol Sci 2014;15:10908-25.
8. Ahmet Cetin, Ibrahim Halil Gecibesler. Evaluation as
antioxidant agents of 1,2,4-triazole derivatives: essential
functional group effects. J Appl Pharm Sci 2015;5:120-6.
9. Nareshvarma Seelam, Shrivastava SP, Prasanthi S, Supriya
Gupta. Synthesis and in vitro study of some fused 1, 2, 4-
triazole derivatives as antimycobacterial agents. J Saudi Chem
Soc 2016;20:411–8.
data obtained from H NMR, 13C NMR and mass spectra confirmed
1
the proposed structures. The products were obtained in good yield.
The synthesized compounds have been screened for in vitro
antifungal activity and showed good to moderate antifungal activity.
Results of in vitro antifungal, antitubercular activity, and molecular
docking study revealed that the synthesized compounds have
potential antifungal, antitubercular activity and can be further
optimized and developed as a lead compound. 1, 2, 4-triazole
derivative compounds were shown to have significant antioxidant
properties as measured by DPPH scavenging assay.
ACKNOWLEDGMENT
The authors are thankful to SRM University for providing necessary
facilities to carry out the NMR and mass spectral analysis and Leads
Clinical Research, Bengaluru, India for providing antitubercular data.
10. Sheng CQ, Zhang WN, Ji HT, Zhang M, Song YL, Xu H, et al.
Structure-based optimization of azole antifungal agents by
CoMFA, CoMSIA and molecular docking.
2006;49:2512–25.
J Med Chem
AUTHORS CONTRIBUTIONS
11. Wang Z, Wu B, Kuhen KL, Bursulaya B, Nguyen TN, Nguyen DG,
et al. Synthesis and biological evaluations of sulfanyl triazoles
as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.
Bioorg Med Chem Lett 2006;16:4174–7.
12. Navidpour L, Shafaroodi H, Abdi K, Amini M, Ghahremani MH,
Dehpour AR, et al. Design, synthesis, and biological evaluation
of substituted 3-alkylthio-4,5-diaryl-4H-1, 2, 4-triazoles as
selective COX-2 inhibitors. Bioorg Med Chem 2006;14:2507–
17.
13. Prabhu Jalihal C, Vaibhav Rajoriya, Varsha Kashaw. Design,
synthesis, and evaluation of new derivative of 1,2,4-triazoles
for antimicrobial and anti-inflammatory activity. Int J Curr
Pharm Res 2018;10:29-35.
14. Jeetendra Kumar Gupta, Pradeep Mishra. Antimicrobial and
anthelmintic activities of some newly synthesized triazoles.
Asian J Pharm Clin Res 2017;10:139-45.
All the authors have contributed equally.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest.
REFERENCES
1. John Warren. Drug discovery: lessons from evolution. Br J Clin
Pharmacol 2011;71:497–503.
2. Lengauer T, Rarey M. Computational methods for biomolecular
docking. Curr Opin Struct Biol 1996;6:402–6.
3. Dolin, Gerald Mandell L, John Bennett E, Raphael Mandell.
Douglas and bennett's principles and practice of infectious
diseases. 7th
ed.
Philadelphia
PA:
Churchill
Livingstone/Elsevier; 2010.
4. Ling LL, Xian J, Ali S, Geng B, Fan J, Mills DM, et al. Identification
and characterization of inhibitors of bacterial enoyl-acyl carrier
protein reductase. Antimicrob Agents Chemother 2004;
48:1541-7.
5. Oliveira JS, Vasconcelos IB, Moreira IS, Santos DS, Basso LA.
Enoyl reductases as targets for the development of anti-
15. Yasodakrishna Sajja, Sowmya Vanguru, Hanmanth Reddy
Vulupala, Rajashaker Bantu, Perumal Yogeswari, Dharmarajan
Sriram, et al. Design, synthesis and in vitro anti-tuberculosis
activity of benzo [6, 7] cyclohepta [1,2-b]pyridine-1,2,3-triazole
derivatives. Bioorg Med Chem Lett 2017;27:5119-21.
90

George et al.
Int J Pharm Pharm Sci, Vol 11, Issue 1, 85-91
16. Arif Mermer, Neslihan Demirbas, Yakup Sirin, Harun Uslu,
mutants of isoniazid-resistant Mycobacterium tuberculosis
enoyl reductase (InhA) in complex with NADH: toward the
understanding of NADH-InhA different affinities. Biophys J
2005;89:876-84.
Zeynep Ozdemir, Ahmet Demirbas. Conventional and
microwave prompted synthesis, antioxidant, anticholinesterase
activity screening and molecular docking studies of new
quinolone triazole hybrids. Bioorg Chem 2018;78:236-48.
17. Bakunov SA, Bakunova SM, Wenzler T, Ghebru M, Werbovetz
KA, Brun R. Synthesis and antiprotozoal activity of cationic 1,4-
diphenyl-1H-1,2,3-triazoles. J Med Chem 2010;53:254-72.
18. Guimaraes TT, Pinto FR, Lanza JS, Melo MN, Monte-Neto RL,
Melo IMM. Potent naphthoquinones against antimony sensitive
and resistant Leishmania parasites: synthesis of novel α-and
nor-α-lapachone based 1,2,3-triazoles by copper catalyzed
azide-alkyne cycloaddition. Eur J Med Chem 2013;63:523-30.
19. Zhou CH, Wang Y. Recent researches in triazole compounds as
medicinal drugs. Curr Med Chem 2012;19:239-80.
24. Bellamine A, Lepesheva GI, Waterman MR. Fluconazole binding
and sterol demethylation in three CYP 51 isoforms indicate
defferences in active site topology. J Lipid Res 2004;45:2000-7.
25. Tian W, Chen C, Lei X, Zhao J, Liang J. CASTp 3.0: computed
atlas of surface topography of proteins. Nucleic Acids
Res 2018;2:W363-7.
26. Shashikanth Pattan, Priyanka Gadhave, Vishal Tambe.
Synthesis and evaluation of some novel 1,2,4-triazole
derivatives for antimicrobial, antitubercular and anti-
inflammatory activities. Indian J Chem 2012;51B:297-301.
27. Holla BS, Mahalinga M, Karthikeyan MS, Akberali PM, Shetty
NS. Synthesis of some novel pyrazolo[3,4-d]pyrimidine
derivatives as potential antimicrobial agents. Bioorg Med Chem
2006;14:2040-7.
28. Sethupandian Geetha, Kokkaiah Irulandi, Palanichamy
Mehalingam. Evaluation of antioxidant and free radical
scavenging activities of different solvent extracts of leaves of
piper umbellatum. Asian J Pharm Clin Res 2017;10:274-6.
29. Xini Zhang, Michael Breslav, Jeffrey Grimm, Kailin Guan. A new
procedure for preparation of carboxylic acid hydrazides. J Org
Chem 2002;67:9471–4.
30. Hitchcock CA, Dickinson K, Brown SB, Evans EGV, Adams DJ.
Interaction of azole antifungal antibiotics with cytochrome P-
450-dependent 14 alpha-sterol demethylase purified from
Candida albicans. Biochem J 1990;266:475-80.
20. Rucha Wadapurkar M, Shilpa MD, Anil Kumar Katti S,
Sulochana MB. In silico drug design for Staphylococcus aureus
and development of host-pathogen interaction network.
Informatics Medicine Unlocked 2018;10:58–70.
21. He X, Alian A, Stroud RM, Ortiz de Montellano PR. Pyrrolidine
carboxamides as a novel class of inhibitors of enoyl acyl carrier
protein reductase from Mycobacterium tuberculosis. J Med
Chem 2006;19:6308-23.
22. Podust LM, Poulos TL, Waterman MR. Crystal structure of
cytochrome P450 14alpha-sterol demethylase (CYP51) from
Mycobacterium tuberculosis in complex with azole inhibitors.
Proc Natl Acad Sci USA 2001;13:3068-73.
23. Schroeder EK, Basso LA, Santos DS, de Souza ON. Molecular
dynamics simulation studies of the wild-type, 121V and I16T
91