Synthesis, Characterization, Molecular Modeling, and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids as Potential Antimicrobial Agents

Article abstract of DOI:10.1002/jhet.3319

A novel 1,2,4-triazole-pyridine hybrid derivatives were synthesized by the reaction of nicotinohydrazide with carbon disulfide to yield potassium-3-pyridyl-dithiocarbazate (I). This was further cyclized with ammonia solution to yield 5-mercapto-substituted 1,2,4-triazole-pyridine hybrid (II). This was finally reacted with different substituted benzyl derivatives to produce 1,2,4-triazole-pyridine hybrid derivatives (III). The purity of the derivatives was confirmed by thin-layer chromatography and melting point. Structure of these derivatives was set up by determining its infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectroscopy. Further, the synthesized derivatives were evaluated for their in vitro antimicrobial activity against the three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Acinetbacter baumannii), three Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes, and Enterococcus faecalis), and two fungus (Aspergillus clavatus and Candida albicans). Minimal inhibitory concentration was also determined against same microorganism. Out of all synthesized derivatives, two derivatives, that is, 3-(5-(2-bromobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine and 3-(5-(2,4-dibromobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine showing more potent antibacterial activity. Docking studies were performed by using Argus lab, and all the derivatives exhibited good docking scores between ?10.5369 and ?11.8477?kcal/mol and were better as compared with standard drug methotrexate against a dihydrofolate reductase protein fragment from E.?coli and Lactobacillus (4DFR). Among all compounds, 4h has shown the maximum docking score and found in agreement to in vitro antimicrobial studies.

Full text of DOI:10.1002/jhet.3319

Month 2018
Synthesis, Characterization, Molecular Modeling, and Biological Evalua-
tion of 1,2,4-Triazole-pyridine Hybrids as Potential Antimicrobial Agents
a
J. Ahirwar,
D. Ahirwar,a S. Lanjhiyana,a A. K. Jha,b D. Dewangan,c
and H. Badwaikc
*
aSchool of Pharmacy, Chouksey Engineering College, Bilaspur, Chhattisgarh, India
bShri Shankaracharya Institute of Pharmaceutical Science, Bhilai, Chhattisgarh, India
cRungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India
*E-mail: jyoti_ah@yahoo.com
Received March 14, 2018
DOI 10.1002/jhet.3319
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A novel 1,2,4-triazole-pyridine hybrid derivatives were synthesized by the reaction of nicotinohydrazide
with carbon disulfide to yield potassium-3-pyridyl-dithiocarbazate (I). This was further cyclized with ammo-
nia solution to yield 5-mercapto-substituted 1,2,4-triazole-pyridine hybrid (II). This was finally reacted with
different substituted benzyl derivatives to produce 1,2,4-triazole-pyridine hybrid derivatives (III). The purity
of the derivatives was confirmed by thin-layer chromatography and melting point. Structure of these deriv-
atives was set up by determining its infrared spectroscopy, nuclear magnetic resonance spectroscopy, and
mass spectroscopy. Further, the synthesized derivatives were evaluated for their in vitro antimicrobial activ-
ity against the three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Acinetbacter
baumannii), three Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes, and Enterococ-
cus faecalis), and two fungus (Aspergillus clavatus and Candida albicans). Minimal inhibitory concentration
was also determined against same microorganism. Out of all synthesized derivatives, two derivatives, that is,
3-(5-(2-bromobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine and 3-(5-(2,4-dibromobenzylthio)-4H-1,2,4-tria-
zol-3-yl)pyridine showing more potent antibacterial activity.
Docking studies were performed by using Argus lab, and all the derivatives exhibited good docking
scores between À10.5369 and À11.8477 kcal/mol and were better as compared with standard drug metho-
trexate against a dihydrofolate reductase protein fragment from E. coli and Lactobacillus (4DFR). Among all
compounds, 4h has shown the maximum docking score and found in agreement to in vitro antimicrobial
studies.
J. Heterocyclic Chem., 00, 00 (2018).
© 2018 Wiley Periodicals, Inc.

J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Vol 000
INTRODUCTION
Among the broad range of heterocyclic that is being
explored for expansion of new components in the field of
medicinal chemistry, 1,2,4-triazole-pyridine hybrids and
their fused heterocyclic derivatives have acknowledged
significant consideration due to their synthetic and
effective biological importance [12–19]. The triazole is
an attractive bridge group, which could connect two
pharmacophores to produce novel bifunctional molecules,
while it is almost impossible to be hydrolyzed, oxidized,
or reduced.
Based on these literature data and the features described
previously, we have created a small library of 1,2,4-
triazole-pyridine hybrid derivatives as per Figure 1. All
the synthesized compounds were screened for
antimicrobial activity and compared with the commercial
antibiotic. The antimicrobial activity of derivatives was
assessed on three Gram-negative bacteria (Escherichia
coli, Pseudomonas aeruginosa, and Acinetbacter
baumannii), three Gram-positive bacteria (Staphylococcus
aureus, Streptococcus pyogenes, and Enterococcus
faecalis) by using plate hole diffusion method using
methotrixate as a standard drug, and two fungus
(Aspergillus clavatus and Candida albicans) by modified
microdilution method using fluconazole as a standard
drug. Dihydrofolate reductase (DHFR) inhibition has
long been identified as an important target for the
development of chemotherapeutic agents against bacterial
and parasitic infections. This made the DHFR as an ideal
target for rational and efficient drug design.
The emerging infectious diseases and the increasing
number of multidrug resistant microbial pathogens are the
main challenges behind the treatment of infectious
diseases. In spite of a wide range of antimicrobial drugs
with different mechanisms of action used to treat
microbial infections either alone or in combination and
also the existence many compounds in different phases of
clinical trials, microbial infections have been becoming a
worldwide problem. An increase in mortality might be
due to the emergence of antimicrobial resistance [1–3].
The toxic side effects and the increasing chances of
microbial resistance are the problems with clinically used
drugs that are often dose-limiting [4,5]. Moreover, the
long-term use of several drugs to treat microbial
infections may cause serious health problems, especially
in patients with impaired liver or kidney functions.
To search and synthesize of combinational
chemotherapeutic drugs with different mechanisms of
action and with low side effects constitute an important
part of the methods that aims to overcome the
antimicrobial resistance. Beside the development of
completely new agents possessing chemical characteristics
that clearly differ from those of existing ones, there is
another approach containing to combine two or more
pharmacophores into a single molecule [6–8]. Therefore,
more than one pharmacophores (with a different mode of
action) containing a hybrid molecule could be beneficial
for the treatment of microbial infections. These merged
pharmacophores may be addressing the active site of
different targets and offer the possibility to overcome drug
resistance. In addition, this approach can also reduce
unwanted side effects [9–11].
Several studies showed that triazole analogs can be used
as potential DHFR inhibitors [20,21]. It is a well-known
fact that DHFR is a key enzyme in the folate metabolism.
DHFR enzyme inhibition results in depletion of
intracellular reduced folates necessary for one-carbon
Figure 1. Scheme for synthesis of 1,2,4-triazole-pyridine hybrid derivatives.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Molecular Modeling and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids
transfer reactions which, in turn, are important for the
biosynthesis of thymidylate, purine nucleotides,
methionine, and many other compounds necessary for
RNA, DNA, and protein synthesis [22]. Molecular
docking study was performed to interpret the comparative
differences in the binding interactions of the synthesized
and commercial antibiotic at the molecular level as
inhibitors of DHFR as an antimicrobial agent.
Synthesis of potassium-3-pyridyl-dithiocarbazate (I) [29].
A solution of 8.4 g (0.15 M) of potassium hydroxide (CAS
1310-58-3), 200 mL of absolute ethanol (CAS 64-17-5),
and 13.7 g (0.10 M) of pyridyl-2-carbohydrazide (CAS
1452-63-7) was treated to the addition of 11.4 g
(0.15 M) of carbon disulfide (CAS 75-15-0). The
mixture was diluted with 150 mL of absolute ethanol
and agitated for 12–16 h. It was then diluted with
200 mL of dry ether and dried at 65°C. The salts,
prepared as described above were utilized in the next
step without further purification.
EXPERIMENTAL
Synthesis of 5-mercapto-3-pyridyl-1,2,4-triazole (II) [30].
A suspension of I (24 g, 0.096 M), 95% ammonia (CAS
7664-41-7) 20 mL (0.864 M), and water 40 mL was
refluxed with stirring for 3 to 4 h. The color of this
reaction mixture changed to yellow, hydrogen sulfide was
evolved, and a homogeneous solution resulted. A white
solid was precipitated by dilution with cold water
(100 mL) and acidify with concentrated HCL, filtered and
washed with cold water and recrystallized.
An open capillary method was adopted to determine the
melting points, and the purity of compounds was checked
by thin-layer chromatography (TLC) [23,24]. Fourier
transform infrared spectra (KBR, cmÀ1) were recorded on
1
Perkins Elmer Infrared-283 Fourier transform infrared; H
NMR (DMSO) and 13C NMR were on a Bruker
300 MHz spectrometer using tetramethylsilane as an
internal reference; and the mass spectrometry (MS) was
recorded on Aapi 3000 LC–MS.
Synthesis of 3-benzylthio-5-pyridin-3-yl,1,2,4-triazole
(III). A mixture of II (0.006 M), (0.69 g, 6 M) in dry
The synthesized compounds 3a–3n were examined for
their in vitro antimicrobial activity against the three
Gram-negative bacteria [E. coli (MTCC 443),
P. aeruginosa (MTCC424), and A. baumannii (MTCC
1425)], three Gram-positive bacteria [S. aureus (MTCC
96), S. pyogenes (MTCC 442), and E. faecalis
(MTCC 439)] by using plate hole diffusion method [25–27],
and two fungus [A. clavatus (MTCC 1323) and C. albicans
(MTCC 227)] by modified microdilution method [28]. Zone
of inhibition of all derivatives was determined and
compared with standard methotrexate and fluconazole,
which were used as reference drug. The minimal inhibitory
concentration study was performed by microdilution
susceptibility method [22].
N,N-dimethylformamide (CAS 68-12-2) (3 mL) was
added to a solution of sodium (0.14 g, 6 M) in dry
methanol (CAS 67-56-1) (2 mL). After 10 min of stirring
at room temperature, benzyl halide (6 M) was added. The
resultant suspension was stirred with CaCl2 guard tube at
room temperature 1–23 h. The completion of the reaction
was confirmed by TLC and then resultant solution was
poured in crushed ice.
Molecular docking study.
Molecular docking study
was performed using Argus Lab 4.0 docking software to
find the mode of corresponding interactions of the test
compound with the target. Protein structure of DHFR
from E. Coli and Lactobacillus (4DFR) was obtained
Table 1
Docking parameters used in Argus Lab 4.0.1.
Compound ID(s)
Constant term
vdW coefficient
H-bond coefficient neutral-neutral
Rotors coefficient
Hydrophobic coefficient
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
2.783
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
À0.00096
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
0.38
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
À0.1
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
0.0373
Grid parameters: spacing 0.375 Å and grid size 80X Å, 80Y Å, and 80Z Å.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Vol 000
from the Protein Data Bank. The three-dimensional
structure of the investigated molecules in their natural
form was constructed using Chem Draw Ultra 10.0. In
the Argus Lab 4.0 program, lowest energy conformer of
each of the new analogs was docked into DHFR binding
domain. In the present study, protein–ligand interaction
was determined by using Pymol 1.3 software.
techniques like combustion analysis, TLC, IR, NMR,
and MS. Finally, these compounds were screened for
in vitro antibacterial and antifungal activity.
The synthetic way used to produce different 1,2,4-
triazole derivatives is outlined in Figure 1. Total 14
different 1,2,4-triazole derivatives were prepared
by treating 5-mercapto-3-pyridyl-1,2,4-triazole with
differently substituted benzyl halides. Docking parameters
used in Argus lab 4.0.1 were mentioned in Table 1.
Properties of ligands on the basis of their molecular
structure were mentioned in Table 2. Chemical properties
of ligands were mentioned in Table 3. Docking results of
ligands and standard drug against DHFR (4DFR) were
mentioned in Table 4. Chemical structure, melting
point, and other physical data were mentioned in Table 5.
Data of combustion analysis was mentioned in Table 6.
RESULTS AND DISCUSSION
In the present study, for the first time, we have reported the
antimicrobial evaluation of synthesized 3-(5-(substituted-
benzylthio)-4H-1,2,4-triazol-3-yl)pyridine derivatives. The
synthesized 1,2,4-triazole derivatives were subjected to
confirmation of physical and analytical parameters by using
Table 2
Properties of ligands on the basis of their molecular structure.
Molecular parametersa
Compound
ID(s)
Stretch
0.7730
Bend
Stretch-bend
Torsion
Non-1,4 VDW 1,4 VDW Dipole/dipole
Total energy (kcal/mol)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
12.5043
12.8240
12.4803
12.8141
12.8492
12.5493
12.5204
13.0036
13.0722
12.5706
12.4597
12.4649
12.5149
12.5290
À0.0414
À0.0112
À0.0430
À0.0289
À0.0198
À0.0317
À0.0331
À0.0088
0.0111
À0.0136
À0.0551
À0.0559
À0.0590
À0.0637
À9.4392
À11.0387
À9.4399
À9.1133
À9.1127
À9.4394
À9.4401
À9.4293
À9.4316
À9.4381
À9.4390
À9.4394
À9.4402
À9.4390
À2.2321
À2.3892
À2.2626
À1.7173
À1.8841
À2.2782
À2.3236
À1.6553
À1.8956
À2.6184
À2.1162
À2.1201
À1.9861
À2.0087
13.8876
15.1217
13.9150
14.1282
14.6221
14.0425
14.0762
14.3367
15.0143
14.7617
13.3324
13.3347
13.4803
13.3673
À2.8071
À0.2819
À2.8233
À3.0422
À2.3606
À2.8039
À2.8186
À2.9906
À2.3989
À2.2393
À2.8105
À2.8284
À3.1684
À2.3683
12.6451
15.1273
12.6056
13.9479
15.0306
12.8199
12.7695
14.2167
15.3740
13.8581
12.1247
12.1099
12.1288
12.8094
0.9027
0.7791
0.9073
0.9365
0.7813
0.7882
0.9604
1.0024
0.8353
0.7534
0.7541
0.7872
0.7927
aData generated using ChemBioDraw 13 software after MM2 energy minimization.
Table 3
Chemical properties of ligands.
Compound ID(s)
Molecular formula
Molecular mass (g/mol)
CLogP values*
CMR
Gibbs energy: [kJ/mol]
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
C14H11ClN4S
C14H10Cl2N4S
C14H11ClN4S
C14H11ClN4S
C14H10Cl2N4S
C14H11BrN4S
C14H11BrN4S
C14H11BrN4S
C14H10Br2N4S
C14H10Br2N4S
C14H11FN4S
C14H11FN4S
C14H11FN4S
C14H10F2N4S
302
337
302
302
337
347
347
347
426
426
286
286
286
304
2.88
3.47
2.88
2.88
3.47
3.03
3.03
3.03
3.89
3.89
2.31
2.31
2.31
2.45
8.26
8.76
8.26
8.26
8.76
8.55
8.55
8.55
9.33
9.33
7.89
7.89
7.89
7.80
782.77
761.21
782.77
782.77
761.21
809.02
809.02
809.02
813.71
813.71
599.89
599.89
599.89
395.45
*Data generated using ChemBioDraw 13 software.
Journal of Heterocyclic Chemistry
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Month 2018
Molecular Modeling and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids
Table 4
Docking results of ligands and standard drug against DHFR (4DFR).
Compound
ID(s)
Binding energy No. of hydrogen Bond length of H-bond with
(kcal/mol)
bonds
H-bonds in Å
receptor residue Enzyme’s binding site residue
3a
À11.016
1
2.695
211 ARG
188 ALA (ALPHA HELIX), 211 ARG
(BETA STRAND), 216 ARG (COIL), 209 ILE
(ALPHA HELIX), 253 ILE (COIL), 187 LEU
(ALPHA HELIX), 213 LEU (BETA STRAND),
197 LYS (ALPHA HELIX), 201 MET
(BETA STRAND), 190 PHE (ALPHA HELIX),
205 THR (ALPHA HELIX)
3b
À11.2976
À11.8369
À11.6663
2
1
2
2.633
300 HIS
185 ALA (ALPHA HELIX), 188 ALA
(ALPHA HELIX), 298 GLU (BETA STRAND),
300 HIS (BETA STRAND), 314 ILE (BETA
STRAND), 319 LYS (BETA STRAND), 296
PHE (BETA STRAND), 312 PHE (BETA
STRAND), 189 TRP (ALPHA HELIX), 310
TYR (BETA STRAND)
2.825
2.467
300 HIS
181 TRP
3c
165 ALA (BETA STAND), 166 ALA (BETA
STAND), 186 ASP (ALPHA HELIX), 164 ILE
(BETA STRAND), 173 ILE (COIL), 209 ILE
(ALPHA HELIX), 253 ILE (COIL), 187 LEU
(ALPHA HELIX), 213 LEU (BETA STRAND),
179 MET (COIL), 190 PHE (ALPHA HELIX),
205 THR (ALPHA HELIX), 181 TRP (COIL),
259 TYR (ALPHA HELIX)
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 57 ARG (BETA STRAND), 15
GLY (COIL), 95 GLY (COIL), 96 GLY (COIL),
97 GLY (COIL), 14 ILE (COIL), 50 ILE
(ALPHA HELIX), 94 ILE (COIL), 28 LEU
(ALPHA HELIX), 54 LEU (BETA STRAND),
31 PHE (ALPHA HELIX), 46 THR
3d
2.989
94 ILE
(ALPHA HELIX), 100 TYR (ALPHA HELIX)
2.999
2.553
46 THR
211 ARG
3e
À11.3277
2
188 ALA (ALPHA HELIX), 211 ARG
(BETA STRAND), 216 ARG (COIL), 209 ILE
(ALPHA HELIX), 253 ILE
(COIL), 187 LEU (ALPHA HELIX), 213 LEU
(BETA STRAND), 197 LYS (ALPHA HELIX),
201 MET (BETA STRAND), 190 PHE (ALPHA
HELIX), 214 PRO (BETA STRAND), 205 THR
(ALPHA HELIX)
2.995
2.814
211 ARG
7 ALA
3f
À10.6901
À11.1236
À11.8477
1
0
2
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 27 ASP (ALPHA HELIX), 5 ILE
(BETA STRAND), 50 ILE (ALPHA HELIX),
94 ILE (COIL), 28 LEU (ALPHA HELIX), 54
LEU (BETA STRAND), 20 MET (COIL), 31
PHE (ALPHA HELIX), 46 THR (ALPHA
HELIX), 22 TRP (COIL), 100 TYR
(ALPHA HELIX), 182 ASN (COIL)
3g
3h
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 57 ARG (BETA STRAND), 15 GLY
(COIL), 95 GLY (COIL), 96 GLY (COIL), 97
GLY (COIL), 14 ILE (COIL), 50 ILE (ALPHA
HELIX), 94 ILE (COIL), 28 LEU (ALPHA
HELIX), 54 LEU (BETA STRAND), 31 PHE
(ALPHA HELIX), 46 THR (ALPHA HELIX), 100
TYR (ALPHA HELIX)
2.93
94 ILE
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 57 ARG (BETA STRAND), 15 GLY
(Continues)
Journal of Heterocyclic Chemistry
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J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Vol 000
Table 4
(Continued)
Compound
ID(s)
Binding energy No. of hydrogen Bond length of H-bond with
(kcal/mol)
bonds
H-bonds in Å
receptor residue Enzyme’s binding site residue
(COIL), 95 GLY (COIL), 96 GLY (COIL), 97
GLY (COIL), 14 ILE (COIL), 50 ILE (ALPHA
HELIX), 94 ILE (COIL), 28 LEU
(ALPHA HELIX), 54 LEU (BETA STRAND), 20
MET (COIL), 31 PHE (ALPHA HELIX), 46 THR
(ALPHA HELIX), 100 TYR (ALPHA HELIX)
2.997
2.929
46 THR
100 TYR
3i
À11.251
1
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 27 ASP (ALPHA HELIX), 5 ILE
(BETA STRAND), 50 ILE (ALPHA HELIX),
94 ILE (COIL), 8 LEU (BETA STRAND), 28
LEU (ALPHA HELIX), 54 LEU (BETA
STRAND), 20 MET (COIL), 31 PHE (ALPHA
HELIX), 46 THR (ALPHA HELIX), 22 TRP
(COIL), 100 TYR (ALPHA HELIX), 13 VAL
(COIL)
3j
3k
3l
À10.7919
À10.6243
À11.0509
3
2
2
2.486
216 ARG
211 ARG (BETA STRAND), 216 ARG (COIL),
209 ILE (ALPHA HELIX), 253 ILE (COIL), 187
LEU (ALPHA HELIX), 213 LUC (BETA
STRAND), 191 LYS (ALPHA HELIX), 201
MET (BETA STRAND), 190 PHE (ALPHA
HELIX), 214 PRO (BETA STRAND), 205 THR
(ALPHA HELIX)
2.562
2.159
2.734
216 ARG
216 ARG
166 ALA
165 ALA (BETA STAND), 166 ALA (BETA
STAND), 174 GLA (COIL), 164 I;LE (BETA
STRAND), 173 ILE (COIL), 209 ILE (ALPHA
HELIX), 253 ILE (COIL), 187 ILU (ALPHA
HELIX), 213 LEU (BETA STRAND), 175
MET (COIL), 179 MET (COIL), 190 PHE
(ALPHA HELIX), 205 THR (ALPHA HELIX),
181 TRP (COIL), 259 TYR (ALPHA HELIX)
2.895
2.982
166 ALA
100 TYR
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 57 ARG (BETA STRAND), 15 GLY
(COIL), 95 GLY (COIL), 96 GLY (COIL), 97
GLY (COIL), 5 ILE (BETA STRAND),
14 ILE (COIL), 50 ILE (ALPHA HELIX), 94 ILE
(COIL), 28 LEU (ALPHA HELIX), 54 LEU
(BETA STRAND), 31 PHE (ALPHA HELIX), 46
THR (ALPHA HELIX), 100 TYR
(ALPHA HELIX)
2.993
2.999
46 THR
46 THR
3m
À10.7385
1
6 ALA (BETA STRAND), 7 ALA (BETA
STRAND), 57 ARG (BETA STRAND), 15 GLY
(COIL), 95 GLY (COIL), 96 GLY (COIL), 97
GLY (COIL), 5 ILE (BETA STRAND),
14 ILE (COIL), 50 ILE (ALPHA HELIX), 94 ILE
(COIL), 28 LEU (ALPHA HELIX), 54 LEU
(BETA STRAND), 31 PHE (ALPHA HELIX), 46
THR (ALPHA HELIX), 100 TYR
(ALPHA HELIX)
3n
À10.5369
1
2.622
211 ARG
188 ALA (ALPHA HELIX), 211 ARG (BETA
STRAND), 216 ARG (COIL), 209 ILE (ALPHA
HELIX), 253 ILE (COIL), 187 LEU (ALPHA
HELIX), 213 LEU (BETA STRAND), 191 LYS
(ALPHA HELIX), 201 MET (BETA STRAND),
190 PHE (ALPHA HELIX), 214 PRO
(BETA STRAND), 205 THR (ALPHA HELIX)
(Continues)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Molecular Modeling and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids
Table 4
(Continued)
Compound
ID(s)
Binding energy No. of hydrogen Bond length of H-bond with
(kcal/mol)
bonds
H-bonds in Å
receptor residue Enzyme’s binding site residue
Mithotrixate À10.0287
4
1.847
52 ARG
7 ALA (BETA STRAND), 52 ARG (BETA
STRAND), 57 ARG (BETA STRAND), 23 ASN
(COIL), 27 ASP (ALPHA HELIX), 50 ILE
(ALPHA HELIX), 94 ILE (COIL), 24 LEU
(COIL), 28 LEU (ALPHA HELIX), 54
LEU (BETA STRAND), 32 LYS (ALPHA
HELIX), 31 PHE (ALPHA HELIX), 46 THR
(ALPHA HELIX), 22 TRP (COIL), 182 ASN
(COIL)
2.339
2.679
2.8539
52 ARG
182 ASN
22 TRP
Grid parameters (Argus Lab.): spacing 0.375 Å and grid size 80X Å, 80Y Å, and 80Z Å.
Table 5
Characterization data of synthesized 1,2,4-triazole derivatives.
Melting
point
(°C)
Molecular
formula
Molecular
weight
Retention
factor
% yield
(w/w)
λ max
(nm)
Compounds
R1
4-Cl
Appearance
White solid
Solubility
Ethanol
Chemical name
3a
C14H11ClN4S
C14H10Cl2N4S
C14H11ClN4S
C14H11ClN4S
C14H10Cl2N4S
C14H11BrN4S
C14H11BrN4S
C14H11BrN4S
C14H10Br2N4S
C14H10Br2N4S
C14H11FN4S
C14H11FN4S
C14H11FN4S
C14H10F2N4S
302
337
302
302
337
347
347
347
426
426
286
286
286
304
207
250
207
207
250
537
237
237
310
310
178
178
178
311
0.72
0.86
0.73
0.72
0.85
0.89
0.90
0.90
0.96
0.95
0.63
0.63
0.63
0.78
73.73
66.82
70
345
350
345
346
352
342
341
342
361
362
340
340
340
354
3-(5-(4-chlorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3b
3c
3d
3e
3f
3,4-(Cl)2
3-Cl
Creamy
DMF
3-(5-(3,4-dichlorobenzylthio)-
4H-1,2,4-triazol-3-yl)pyridine
3-(5-(3-chlorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3-(5-(2-chlorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3-(5-(2,4-dichlorobenzylthio)-
4H-1,2,4-triazol-3-yl)pyridine
3-(5-(4-bromobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
white solid
Light brown
solid
Ethanol
Ethanol
Ethanol
Water
2-Cl
Brown solid
73
2,4-(Cl)2
4-Br
Yellow solid
White solid
Brown solid
Yellow solid
Dark brown
66.19
62.19
70.39
59.90
63.73
76.89
87.25
78.94
75.90
89.35
3g
3h
3i
3-Br
Water
3-(5-(3-bromobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3-(5-(2-bromobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3-(5-(2,4-dibromobenzylthio)-
4H-1,2,4-triazol-3-yl)pyridine
3-(5-(3,5-dibromobenzylthio)-
4H-1,2,4-triazol-3-yl)pyridine
3-(5-(4-fluorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
2-Br
DMF
2,4-(Br)2
3,5-(Br)2
4-F
Ethanol
Ethanol
Ethanol
DMF
3j
Pale yellow
solid
Yellow solid
3k
3l
3-F
Red-orange
solid
Yellow solid
3-(5-(3-fluorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3m
3n
2-F
Ethanol
Ethanol
3-(5-(2-fluorobenzylthio)-4H-
1,2,4-triazol-3-yl)pyridine
3-(5-(2,4-difluorobenzylthio)-
4H-1,2,4-triazol-3-yl)pyridine
2,4-(F)2
Yellow
brown solid
Formation of different 3-(5-(substituted-benzylthio)-4H-
1,2,4-triazol-3-yl)pyridine derivatives was established by
recording there IR, 1H NMR, and MS.
1630.41 cmÀ1 is due to C=C str, a band at 735.83 cmÀ1
is due to C─Cl str, and band at 657.11 cmÀ1 is due to
C─S str. Strong absorption at around 3078.85 and
around 1620.47 cmÀ1 was present in all final derivatives,
which was confirmed for aromatic C─H and C═C
bonds, respectively. The presence of specific functional
groups in final synthesized derivatives was confirmed by
1H NMR data. The 1H NMR spectrum of triazole
derivatives 3-(5-(4-chlorobenzylthio)-4H-1,2,4-triazol-3-
Infrared spectrum of one 1,2,4-triazole derivative 3-(5-(4-
chlorobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine
showed
strong C═N stretching (str) band at 1595.76 cmÀ1 and
C─N absorption band at 1252.41 cmÀ1 that indicate ring
closure of 1,2,4-triazole ring. An absorption band at
2978.85 cmÀ1 is due to aromatic (Ar) C-H str, band at
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Vol 000
Table 6
Combustion analysis of synthesized 1,2,4-triazole derivatives.
Combustion analysis
Compounds
Theoretical value
Observed values
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
C (55.53%) H (3.66%) Cl (11.7%) N (18.50) S (10.59%)
C (49.86%) H (2.99%) Cl (21.03%) N (16.61%) S (9.51%)
C (55.53%) H (3.66%) Cl (11.7%) N (18.50) S (10.59%)
C (55.53%) H (3.66%) Cl (11.7%) N (18.50) S (10.59%)
C (49.86%) H (2.99%) Cl (21.03%) N (16.61%) S (9.51%)
C (48.43%) H (3.19%) Cl (23.01%) N (16.14%) S (9.23%)
C (48.43%) H (3.19%) Cl (23.01%) N (16.14%) S (9.23%)
C (48.43%) H (3.19%) Cl (23.01%) N (16.14%) S (9.23%)
C (39.46%) H (2.37%) Br (37.50%) N (13.15%) S (7.52%)
C (39.46%) H (2.37%) Br (37.50%) N (13.15%) S (7.52%)
C (58.73%) H (3.87%) F (6.64%) N (19.57%) S (11.20%)
C (58.73%) H (3.87%) F (6.64%) N (19.57%) S (11.20%)
C (58.73%) H (3.87%) F (6.64%) N (19.57%) S (11.20%)
C (55.25%) H (3.31%) F (12.49%) N (18.41%) S (10.54%)
C (54.53%) H (3.60%) Cl (11.5%) N (18.21) S (10.49%)
C (49.26%) H (2.91%) Cl (20.82%) N (16.27%) S (9.41%)
C (54.55%) H (3.60%) Cl (11.38%) N (18.10) S (10.41%)
C (55.01%) H (3.59%) Cl (11.38%) N (18.36) S (10.39%)
C (49.06%) H (2.94%) Cl (21.0%) N (16.41%) S (9.41%)
C (48.02%) H (3.16%) Cl (22.91%) N (16.04%) S (9.20%)
C (48.02%) H (3.16%) Cl (22.91%) N (16.04%) S (9.20%)
C (48.02%) H (3.16%) Cl (22.91%) N (16.04%) S (9.20%)
C (39.26%) H (2.32%) Br (37.40%) N (13.11%) S (7.50%)
C (39.26%) H (2.32%) Br (37.40%) N (13.11%) S (7.50%)
C (57.79%) H (3.85%) F (6.60%) N (19.47%) S (11.12%)
C (57.79%) H (3.85%) F (6.60%) N (19.47%) S (11.12%)
C (57.79%) H (3.85%) F (6.60%) N (19.47%) S (11.12%)
C (54.35%) H (3.30%) F (12.45%) N (18.35%) S (10.50%)
Table 7
Spectral data of synthesized 1,2,4-triazole derivatives.
1H NMR δ (ppm)
13C NMR (DMSO-d6,
100 MHz) δ (ppm)
Compounds
IR (KBr cmÀ1
)
(DMSO-d6)
MS
3a
2978.85 (Ar─C─H str), 1630.41 (Ar─C═C str),
1154.87 (Ar─C─C str), 1595.76 (C═Nstr),
1252.41 (─C─N-str), 657.11 (─C─S str), 735.
83 (C─Cl str)
3088.25 (Ar─C─H str), 1610.41 (Ar─C═C str),
1173.78 (Ar─C─C str), 1542.56 (C═Nstr),
1200.41 (─C─N-str), 647.12 (─C─S str),
717.33 (C─Cl str)
6.84–7.19 (m 4H, Ar─H), 4.25
(s 2H, ─CH2), 7.40–8.90
(m 4H, pyridine ring)
160.7, 154.2, 141.1,
140.0, 138.1, 135.1,
133.0, 130.7, 129.2,
127.9, 122.0, 36.2
156.2, 154.3, 150.2,
146.0, 140.2, 136.1,
135.4, 133.8, 131.3,
128.0, 125.3, 122.0,
37.9
159.8, 156.2, 150.1,
147.0, 141.9, 135.3,
134.3, 132.0, 131.2,
129.6, 126.3, 124.9,
122.0, 37.3
161.8, 159.5, 149.6,
145.0, 138.1, 135.3,
134.1, 131.0, 129.2,
129.9, 127.6, 126.2,
124.9, 33.1
159.2, 156.9, 149.5,
148.2, 134.1, 133.8,
132.0, 131.2, 130.2,
130.9, 126.0, 123.0,
32.1
162.8, 159.5, 150.1,
147.0, 139.7, 135.1,
133.0, 131.6, 129.0,
125.0, 123.5, 36.2
159.8, 156.5, 149.1,
144.0, 140.9, 136.1,
134.0, 132.2, 131.7,
130.1, 127.8, 125.0,
122.1, 34.5
161.3, 158.3, 152.1,
148.0, 138.2, 137.1,
134.0, 131.7, 129.0,
128.4, 126.8, 123.0,
121.7, 32.4
301+
3b
3c
3d
3e
6.88–7.29 (m 4H, Ar─H), 4.19
(s 2H, ─CH2), 7.44–8.86
(m 4H, pyridine ring)
336+
301+
301+
336+
3108.64 (Ar─C─H str), 1684.40 (Ar─C═C str),
1112.08 (Ar─C─C str), 1521.24 (C═Nstr),
1221.56 (─C─N─str), 612.41.11 (─C─S str),
712.98 (C─Cl str)
6.94–7.46 (m 4H, Ar─H), 4.21
(s 2H, ─CH2), 7.74–8.78
(m 4H, pyridine ring)
2968.46 (Ar─C─H str), 1598.35.47 (Ar─C═C str),
1175.47 (Ar─C─C str), 1500.36 (C═Nstr), 1285.47
(─C─N-str), 611.81 (─C─S str), 765.79
(C─Cl str)
7.02–7.27 (m 4H, Ar─H), 4.20
(s 2H, ─CH2), 7.63–8.63
(m 4H, pyridine ring)
2912.56 (Ar─C─H str), 1623.56 (Ar─C═C str),
1121.67 (Ar─C─C str), 1521.12 (C═Nstr), 1213.87
(─C─N-str), 641.78 (─C─S str), 735.13 (C─Cl str)
6.64–7.10 (m 3H, Ar─H), 4.12
(s 1H, ─CH2), 7.30–8.86
(m 4H, pyridine ring)
3f
2890.45 (Ar─C─H str), 1611.76 (Ar─C═C str),
1108.45 (Ar─C─C str), 1541.10 (C═Nstr), 1286.45
(─C─N-str), 698.34 (─C─S str), 812.12 (C─Br str)
6.94–7.30 (m 4H, Ar─H), 4.15
(s 2H, ─CH2), 7.60–8.86
(m 4H, pyridine ring)
346+
346+
3g
3134.78 (Ar─C─H str), 1652.89 (Ar─C═C str),
1146.89 (Ar─C─C str), 1511.21 (C═Nstr), 1264.76
(─C─N-str), 698.98 (─C─S str), 842.45 (C─Br str)
7.10–7.30 (m 4H, Ar─H), 4.19
(s 2H, ─CH2), 7.44–8.84
(m 4H, pyridine ring)
3h
3i
3078.45 (Ar─C─H str), 1662.67 (Ar─C═C str),
1109.78 (Ar─C─C str), 1500.90 (C═Nstr), 1210.43
(─C─N-str), 690.56 (─C─S str), 832.56 (C─Br str)
6.96–7.31 (m 4H, Ar─H), 4.19
(s 2H, ─CH2), 7.44–8.81
(m 4H, pyridine ring)
346+
425+
2910.56 (Ar─C─H str), 1623.56 (Ar─C═C str),
1101.67 (Ar─C─C str), 1561.12 (C═Nstr), 1210.87 (─C─N-str),
691.78 (─C─S str), 842.12 (C─Br str)
6.86–7.41 (m 3H, Ar─H), 4.19
(s 2H, ─CH2), 7.42–8.88
(m 4H, pyridine ring)
160.8, 156.5, 151.1,
149.0, 139.8, 138.2,
136.1, 135.0, 131.2,
130.2, 125.9, 124.0,
122.7, 31.4
(Continues)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Molecular Modeling and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids
Table 7
(Continued)
1H NMR δ (ppm)
(DMSO-d6)
13C NMR (DMSO-d6,
100 MHz) δ (ppm)
Compounds
IR (KBr cmÀ1
)
MS
3j
2922.12 (Ar─C─H str), 1615.45 (Ar─C═C str), 1135.60
(Ar─C─C str), 1515.10 (C═Nstr), 1235.10 (─C─N-str), 690.80
(─C─S str), 820.90 (C─Br str)
7.10–7.41 (m 3H, Ar─H), 4.18
(s 2H, ─CH2), 7.43–8.87
(m 4H, pyridine ring)
158.2, 156.5, 149.7,
14890, 143.1, 135.1,
132.2, 131.0, 130.5,
125.3, 124.0, 35.4
162.3, 158.2, 157.1,
150.1, 148.7, 138.3,
136.1, 134.0, 128.4,
124.8, 118.5, 38.0
162.2, 159.8, 157.8,
149.6, 148.0, 143.3,
137.1, 133.0, 131.4,
126.0, 125.4, 115.8,
111.9, 39.2
161.3, 159.8, 155.5,
149.1, 147.0, 138.1,
135.0, 130.4, 128.8,
127.1, 122.4, 115.9,
33.0
162.2, 161.6, 159.8,
154.5, 149.1, 144.0,
134.1, 132.0, 131.0,
125.0, 123.7, 110.1,
134.7, 29.4
425+
3k
3l
3113.20 (Ar─C─H str), 1590.45 (Ar─C═C str), 1130.60
(Ar─C─C str), 1492.10 (C═Nstr), 1230.10 (─C─N-str), 703.89
(─C─S str), 1370.90 (C─F str)
6.98–7.25 (m 4H, Ar─H), 4.19
(s 2H, ─CH2), 7.44–8.84
(m 4H, pyridine ring)
285+
285+
3087.89 (Ar─C─H str), 1657.89 (Ar─C═C str), 1147.78
(Ar─C─C str), 1547.89 (C═Nstr), 1290.56 (─C─N-str), 725.78
(─C─S str), 1400.89 (C─F str)
7.10–7.30 (m 4H, Ar─H), 4.22
(s 2H, ─CH2), 7.50–8.90
(m 4H, pyridine ring)
3m
3n
3089.56 (Ar─C─H str), 1634.56 (Ar─C═C str), 1111.89
(Ar─C─C str), 1543.87 (C═Nstr), 1233.89 (─C─N-str), 612.87
(─C─S str), 1321.89 (C─F str)
7.08–7.28 (m 4H, Ar─H), 4.17
(s 2H, ─CH2), 7.48–8.82
(m 4H, pyridine ring)
285+
303+
3099.90 (Ar─C─H str), 1555.12 (Ar─C═C str), 1172.98
(Ar─C─C str), 1496.89 (C═Nstr), 1298.67 (─C─N-str), 695.90
(─C─S str), 1380.90 (C─F str)
6.76–7.31 (m 3H, Ar─H), 4.15
(s 2H, ─CH2), 7.60–8.62
(m 4H, pyridine ring)
yl)pyridine shown in the region 6.84–7.19 is due to four
aromatic proton, 7.40–8.90 is due to four pyridine proton,
and 4.25 is due to two methylene proton. The presence of
another group like ─CH3 synthesized derivatives was
confirmed by shift value 3.67. The mass spectra of
triazole derivative 3-(5-(4-chlorobenzylthio)-4H-1,2,4-
triazol-3-yl)pyridine showed a molecular ion peak at
m/z 301+, which is in conformity with the molecular
formula C14H11ClN4S. In the same way, spectral data of
remaining derivatives are given in Table 7.
Table 8
Antimicrobial activity of synthesized 1,2,4-triazole derivatives using plate hole diffusion method (10 mg/mL).
Zone of inhibition (mm)
Antibacterial activity
Gram-negative bacteria
Gram-positive bacteria
Antifungal activity
Aspergillus Candida
Escherichia Pseudomonas Acinetbacter Staphylococcus Streptococcus Enterococcus
Compounds
coli
11
aeruginosa
baumannii
aureus
pyogenes
faecalis
clavatus
albicans
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
11
10
12
11
08
12
11
13
12
11
10
11
11
11
–
8
6
9
10
5.5
–
5.5
8
9
7
8
–
6
5.5
–
12
–
10
10
08
11
11
12
10
12
13
12
10
11
11
09
–
09
09
08
12
10
13
11
13
12
11
09
11
12
09
–
–
–
7
5.5
5.5
8
6
8
6
7
8
5.5
6
–
–
–
–
–
6
–
–
–
8
–
–
5.5
6
–
–
–
–
11
7
5.5
–
11
11
12
12
12
11
13
13
12
11
12
11
08
–
–
5.5
–
–
8
–
–
–
6
–
–
–
12
3m
3n
DMSO
Mithotrixate
Fluconazole
14
–
13
–
13
–
14
–
10
–
#Diameter of zone of inhibition expressed in mm.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Further, we have screened the antibacterial activity of
Vol 000
newly synthesized 1,2,4-triazole derivatives. The tested
derivatives exhibited 5.5–13 mm zone of inhibition in
10 mg/mL concentration against Gram-negative bacteria
and Gram-positive bacteria, whereas standard drug
methotrexate showed 10–14 mm zone of inhibition. The
synthesized compounds were shown very weak
antifungal activity. (Table 8).
The minimal inhibitory concentration study was
performed on synthesized compounds. The results were
shown in Table 9.
Out of all synthesized derivatives, two derivatives, that is,
3-(5-(2-bromobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine and
3-(5-(2,4-dibromobenzylthio)-4H-1,2,4-triazol-3-yl)pyridine
showing more potent antibacterial activity (Fig. 2). Triazole
nucleus containing compounds has revealed nice-looking
pharmacological activity. It may note that the 3-position
and 5-position of triazole nucleus are extremely important
sites for molecular modification, which can play a
dominant role in determining the pharmacological activities
of triazole derivatives. Therefore, we have attempted to
develop the derivatives of triazole nucleus at 3-position and
5-position with a view to evaluating further the potency of
the molecules. In the present study, as discussed earlier, we
have modified the 1,2,4-triazole derivatives linked
substituted benzyl groups via thio linkage. The resulting
compounds produced 80–90% efficacy as compared with
standard against Gram-negative, Gram-positive bacteria.
Results revealed that novel 1,2,4-triazole-linked with
Figure 2. Comparative antibacterial evaluation of compound 3f, 3h, and
3i with respect to methotrexate. [Color figure can be viewed at
wileyonlinelibrary.com]
substituted benzyl groups via thio linkage showed significant
antibacterial activities as compared with standard drug
methotrexate.
DOCKING STUDY
To rationalize the potency of the molecules as an
antimicrobial agent, these compounds were docked
against DHFR from E. coli and Lactobacillus (4DFR)
obtained from protein data bank (RCSB). The binding
Table 9
Antimicrobial activity of the synthesized compounds expressed as MIC (mg/mL).
MIC (mg/mL)
Antibacterial activity
Gram-negative bacteria
Gram-positive bacteria
Antifungal activity
Aspergillus Candida
Escherichia Pseudomonas Acinetbacter Staphylococcus Streptococcus Enterococcus
Compounds
coli
1.57
1.78
1.83
1.29
1.1
1.21
1.74
0.98
0.99
1.23
1.86
1.17
2.1
aeruginosa
baumannii
aureus
pyogenes
faecalis
clavatus
albicans
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
2.03
2.01
1.73
1.97
3.89
1.33
1.98
0.91
1.21
1.98
2.12
2
1.98
2.01
–
0.82
NT
3.97
5.96
3.02
2.86
6.96
13.03
6.09
3.84
3.10
3.08
3.8
15.3
3.9
6.1
–
0.95
NT
2.15
2.8
4
2.1
2.1
1.15
2.75
1.2
1
1.3
2.2
1.9
1.95
2.9
–
4
15
11
7.5
9.2
23.4
12.5
24.5
8.9
12.8
11.5
3.8
24.5
23.6
23.2
8.6
3.9
4.75
1.1
2.1
0.95
1.75
0.98
1.2
1.9
3.5
2.15
1.25
3.2
–
16.7
6.9
7.1
8.5
3.75
9
3.9
8.5
3.8
3.2
9.5
8.6
12
23.5
22.1
6.9
21.0
12.1
23.0
4.2
24.1
22.5
8.2
8.4
3m
3n
DMSO
Mithotrixate
Fluconazole
23.1
22.8
–
NT
1.10
3.01
–
0.8
22.5
–
NT
–
1.25
NT
0.96
NT
0.8
NT
NT
1.20
MIC, minimal inhibitory concentration; NT, not tested.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Molecular Modeling and Biological Evaluation of 1,2,4-Triazole-pyridine Hybrids
energy of drug-receptor complex and standard drug is
shown in Table 4.
synthesized compounds are responsible for the form
hydrogen bond with the different amino acid of a receptor
(Table 6). Out of 16 amino acid residue of DHFR that is
responsible for the formation of bonds with methotrexate,
8 to 10 were found to be similar in compounds 3d, 3f,
3g, 3h, 3i, 3l, and 3m. The synthesized compounds
active site alignment showed a pattern resemblance to the
binding pocket alignment of methotrexate (Fig. 3).
According to docking study results, the difference in
biological activity of tested compounds despite their
similar chemical structure could be explained on the basis
of its alignment with receptor binding pocket as well as
interaction with particular amino acid residue. The
All synthesized molecules interacted with the active
sites of DHFR enzyme (4DFR) through various bonds
like hydrogen, van der Waals, p-cation, p-anion, p-alkyl,
p-donor hydrogen, p-sulfur, carbon-hydrogen bond, and
p-sigma. As shown in Table 6, binding energies of the
molecules against the active sites indicated a higher
affinity of the molecules to bind to the protein leading to
the inhibition. The binding energies of the molecules
ranging from À10.5369 to À11.8477 kcal/mol and were
better as compared with standard drug methotrexate. The
nitrogen atoms of the pyridine and triazole ring of
Figure 3. Interaction of compound 3f and methotrexate with the binding domain of the dihydrofolate reductase enzyme (4DFR). [Color figure can be
viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

J. Ahirwar, D. Ahirwar, S. Lanjhiyana, A. K. Jha, D. Dewangan, and H. Badwaik
Vol 000
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A new series of different 3-(5-(substituted-benzylthio)-
4H-1,2,4-triazol-3-yl)pyridine derivatives were prepared
by treating 5-mercapto-3-pyridyl-1,2,4-triazole with
differently substituted benzyl halides, by a simple,
suitable, and well-organized synthetic route. Physical and
analytical parameters of the newly synthesized 1,2,4-
triazole derivatives were confirmed by TLC, IR, NMR,
and MS. Subsequently, in antimicrobial screening, the
compounds showed antimicrobial activity. In antimicrobial
screening, 3-(5-(2-bromobenzylthio)-4H-1,2,4-triazol-3-yl)
pyridine and 3-(5-(2,4-dibromobenzylthio)-4H-1,2,4-
triazol-3-yl)pyridine showing more potent antibacterial
activity as compare with other derivatives. The results of
docking studies also conform the mechanism of
antimicrobial action, that is, by inhibition of DHFR
enzyme. Thus, we concluded that the data of the present
study may pave a way for the development of novel
antibacterial agents with good efficacy and lesser adverse
effects. Moreover, results of docking study enforce us for
anticancer evaluation of synthesized molecules in future
due to its higher binding affinity to DHFR than
methotrexate.
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Acknowledgments. The authors thank to the Management of
School of Pharmacy, Chouksey Engineering College, Bilaspur,
for providing necessary infrastructural facilities, and RGPV,
Bhopal, for providing immense help in the spectral analysis.
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SUPPORTING INFORMATION
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Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet