Synthesis, characterization and anti-inflammatory activity evaluation of 1,2,4-triazole and its derivatives as a potential scaffold for the synthesis of drugs against prostaglandin-endoperoxide synthase

Article abstract of DOI:10.1080/07391102.2019.1711193

Substituted 1,2,4-triazole nucleus is common in several drugs used in a variety of clinical conditions including infections, hypoglycemia, hypertension and cancer. In this study, we synthesized 1,2,4-triazole and its 16 hydrazone derivatives (B1–B16), characterized them by IR, NMR and Mass spectroscopy, and evaluated their radical scavenging and anti-inflammatory activities in?vitro and in?vivo. Out of 16 derivatives, five (B1, B5, B6, B9, and B13) demonstrated a significant radical scavenging and anti-inflammatory activity in?vitro. B6, which possessed two electron-donating hydroxyl groups, was most active among all. Molecular docking and MD simulation of the complex of B6 with prostaglandin-endoperoxide synthase (PTGS) or cyclooxygenase (COX) showed that B6 occupied celecoxib binding site in COX with high affinity (the binding free energy of the complex with COX-1 was –10.5, and –11.2 kcal/mol with COX-2). Maximum anti-inflammatory activity was also shown by the B6 derivative in?vivo, in the rat model of carrageenan-induced inflammation. B6, along with four other derivatives (B1, B5, B9 and B13) exhibited 80–90% free radical scavenging activity. The IC₅₀ values of these compounds were ≥40 μM. Griess nitrite and dichloro-dihydro-fluorescein-diacetate assays suggested a significant inhibition of nitric oxide and reactive oxygen species, especially by B6 and B9. Taken together, out of 16 derivatives, B6 is reported to have highest anti-inflammatory and antioxidant activity at a low dose level, which may be attributed to its two electron-donating hydroxyls. B6 is proposed to be an important scaffold for the synthesis of new drugs against PTGS for use in a myriad of inflammatory and infectious diseases. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2019.1711193

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis, characterization and anti-inflammatory
activity evaluation of 1,2,4-triazole and its
derivatives as a potential scaffold for the synthesis
of drugs against prostaglandin-endoperoxide
synthase
Bushra Khan, Abdullah Naiyer, Fareeda Athar, Shakir Ali & Sonu Chand
Thakur
To cite this article: Bushra Khan, Abdullah Naiyer, Fareeda Athar, Shakir Ali & Sonu Chand
Thakur (2020): Synthesis, characterization and anti-inflammatory activity evaluation of
1,2,4-triazole and its derivatives as a potential scaffold for the synthesis of drugs against
prostaglandin-endoperoxide synthase, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2019.1711193
To link to this article: https://doi.org/10.1080/07391102.2019.1711193
View supplementary material
Accepted author version posted online: 03
Jan 2020.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tbsd20

Synthesis, characterization and anti-inflammatory activity evaluation of 1,2,4-triazole and
its derivatives as a potential scaffold for the synthesis of drugs against prostaglandin-
endoperoxide synthase
Bushra Khan1,#, Abdullah Naiyer1,#,Fareeda Athar1, Shakir Ali2, Sonu Chand Thakur1,*
1Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Delhi, India
2Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard, Delhi
3DBT BTISNet Bioinformatics infrastructure facility, BIF, Jamia Hamdard, Delhi, India
Running title: Synthesis and anti-inflammatory activity evaluation of 1,2,4-triazole derivatives
*To whom correspondence should be addressed: Sonu Chand Thakur, Ph.D., Centre for
Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central University), Jamia Nagar, New
Delhi110025, India. Tel. /Fax: +91- 011-26981717; E-mail: sonuchandthakur@gmail.com,
sthakur@jmi.ac.in
#Authors contributed equally
1

Abstract
Substituted 1,2,4-triazole nucleus is common in several drugs used in a variety of clinical
conditions including infections, hypoglycemia, hypertension and cancer. In this study, we
synthesized 1,2,4-triazole and its 16 hydrazone derivatives (B1-B16), characterized them by IR,
NMR and Mass spectroscopy, and evaluated their radical scavenging and anti-inflammatory
activities in vitro and in vivo. Out of 16 derivatives, five (B1, B5, B6, B9, and B13)
demonstrated a significant radical scavenging and anti-inflammatory activity in vitro. B6, which
possessed two electron-donating hydroxyl groups, was most active among all. Molecular
docking and MD simulation of the complex of B6 with prostaglandin-endoperoxide synthase
(PTGS) or cyclooxygenase (COX) showed that B6 occupied celecoxib binding site in COX with
high affinity (the binding free energy of the complex with COX-1 was -10.5, and -11.2 kcal/mol
with COX-2). Maximum anti-inflammatory activity was also shown by the B6 derivative in vivo,
in the rat model of carrageenan-induced inflammation. B6, along with four other derivatives (B1,
B5, B9 and B13) exhibited 80-90% free radical scavenging activity. The IC50 values of these
compounds were ≥40µM. Griess nitrite and dichloro-dihydro-fluorescein-diacetate assays
suggested a significant inhibition of nitric oxide and reactive oxygen species, especially by B6
and B9. Taken together, out of 16 derivatives, B6 is reported to have highest anti-inflammatory
and antioxidant activity at a low dose level, which may be attributed to its two electron-donating
hydroxyls. B6 is proposed to be an important scaffold for the synthesis of new drugs against
PTGS for use in a myriad of inflammatory and infectious diseases.
Supplementary material includes physical properties and spectral analysis data of synthesized
derivatives.
2

Keywords: 1,2,4-triazole; molecular docking; MD simulation; inflammation; cyclooxygenases
Abbreviations:
ACS: American Chemical Society; COX: Cyclooxygenase; DCFH-DA: Dichloro-dihydro-
fluorescein diacetate; DMSO-d6: Deuterated dimethyl sulfoxide; DPPH: 2,2-Diphenyl-1-
picrylhydrazyl; FTIR: Fourier transform infrared spectroscopy; LPS: Lipopolysaccharide; MD
simulation: Molecular dynamic simulation; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NO: Nitric oxide; NPT: constants, Number (N), Pressure (P), and
Temperature (T); NSAIDs: Non-steroidal anti-inflammatory drugs; NVT: constants, Number
(N), Volume (V), and Temperature (T); OD: Optical density; PTGS: Prostaglandin-
endoperoxide synthase; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TLC:
Thin layer chromatography; TMS: Tetramethyl silane.
Introduction
Triazoles are heterocyclic compounds consisting of a 5-membered ring along with two carbon
and three nitrogen atoms, which, on the basis of the position of nitrogen atom in the ring, can be
divided into three isomers: (i) 1,2,3‐ triazole (vicinal triazole); (ii) 1,2,4‐ triazole (asymmetrical;
asym‐ triazole); (iii) 1,3,4-triazole (symmetrical; sym‐ triazole) (Timur et al., 2018). Asym- or
1,2,4-Triazole is an interesting isomer of triazole which displays a number of pharmacologically
important properties including the antioxidant (Pokuri, Singla, Bhat, & Shenoy, 2014), anti-
inflammatory (Paprocka et al., 2015), anticancer (El-Sherief et al., 2018; Shahzad et al., 2019),
antiviral (Kucukguzel, Tatar, Kucukguzel, Rollas, & De Clercq, 2008), antifungal (Dheer, Singh,
& Shankar, 2017; Jin et al., 2018), antitubercular (S. Zhang et al., 2017), antibacterial (Eswaran,
3

Adhikari, & Shetty, 2009; Y. L. Fan, 2018), antimicrobial (Eswaran et al., 2009; Fan, Shi, &
Bao, 2018), antitrypanosome (Papadopoulou et al., 2012), antiproliferative (Li et al., 2017),
antibiotic (Xiao et al., 2014), analgesic (Gajanan Khanage, Raju, Baban Mohite, & Bhanudas
Pandhare, 2013), anticonvulsant (Kapron et al., 2019), antidepressant (Kane, Dudley, Sorensen,
& Miller, 1988), CNS stimulant, sedative, antianxiety and antimigraine (Genc et al., 2016;
Kandile, Mohamed, & Ismaeel, 2017) properties. The wide range of biological activities of
1,2,4-triazole and its occurrence in common drugs including the anti-cancer (letrozole and
anastrozole), anti-viral (ribavirin), anti-fungal (fluconazole, voriconazole, and itraconazole), anti-
migraine (rizatriptan) and anti-anxiety (alprazolam) drugs has generated considerable amount of
interest in 1,2,4-triazole moiety as a biologically active nucleus with potential implications as a
scaffold for the synthesis of newer drugs.
Recent studies have demonstrated the effect of 1,2,4-triazole on lipophilicity, polarity and
hydrogen bonding capacity of the compound, which may enhance its biological activity and
improve physicochemical properties (Basri, Taha, & Ahmad, 2017; Gao, Wang, Xiao, & Huang,
2019; Kapron et al., 2019). Another interesting aspect of the 1,2,4-triazole nucleus is that it can
exert diverse weak non-covalent interactions (hydrogen bonds, hydrophobic interactions, van der
Waals interactions, coordination, ion-dipole, cation-π, π-π stacking etc.), which improve the
solubility and the ability of binding to biomolecular targets (H. Z. Zhang, Damu, Cai, & Zhou,
2014) (C. Gao et al., 2019; J. Zhang, Wang, Ba, & Xu, 2019). Modification of 1,2,4-triazole
may, therefore, be a useful approach in developing new triazole-based compounds with
improved medicinal properties, and at a low dose level, with least or no toxicity and improved
bioactivity. In literature, the nitrogen-containing heterocyclic compounds have been found useful
4

due to their broad range and significantly improved properties. Schiff bases of 1,2,4-triazoles are
important nitrogen-containing heterocyclic compounds with potential bioactivity (Jin et al.,
2018) and a Schiff base hydrazone nucleus might be a good candidate for designing and
synthesis of new 1,2,4-triazole derivatives with excellent biological activity in a facile and easy
synthesis (Kandile et al., 2017). In this study, we have designed, synthesized, and evaluated the
biological activity, particularly the anti-inflammatory and antioxidative properties of 1,2,4-
triazole and its derivatives, synthesized and characterized in our laboratory. The molecular
docking and molecular dynamics (MD) simulation, a computer simulation approach to analyze
the physical movements of atoms and molecules (which are allowed to interact for a fixed period
of time) to get a view of the dynamic "evolution" of a complex or system, were performed to
rationalize the potential of the chosen synthesized derivatives and elucidate its mechanism of
action in silico. Synthesized compounds showed an affinity to PTGS, prostaglandin-
endoperoxide synthase, a family of isozymes, commonly known as cyclooxygenase (COX) (EC
1.14. 99.1), which plays an important role in inflammation, cancer, and development. 1,2,4-
Triazole and its selected derivatives are suggested in this study to provide a scaffold for the
synthesis of new drugs in inflammation and other diseases where inhibition of PTGS may
constitute one of the mechanisms of pathogenesis.
Materials and Methods
Chemicals and instruments
ACS grade chemicals were used for the synthesis in this study. For biological activity
evaluation, chemicals/reagents were purchased from Sigma Aldrich and other standard
commercial sources. The melting point (C, uncorrected), wherever required, was recorded on a
5

Scitech melting point apparatus using capillary tubes closed at one end. For TLC, a pre-coated
aluminum sheet, silica gel 60 F254 (Merck) was used in a solvent system comprised of
chloroform/methanol and hexane/ethyl acetate, 9:1ml. Spots were visualized under a UV lamp.
Precursors, triazole, and substituted hydrazones were purified by silica gel column
chromatography (pore size 60Å, 200-400 mesh) and eluted with chloroform/methanol and
hexane/ethyl acetate. The elutants were concentrated in a Heidolph Laborota 4000 rotary
evaporator and re-crystallized using chloroform/methanol and hexane/ethyl acetate. FTIR spectra
were recorded in ATR mode in a Bruker Tensor 37 spectrophotometer. ʋmax was expressed as
cm-1. Mass spectra (ESI-MS) were recorded on an AB-Sciex 2000 instrument.1H NMR and 13C
NMR spectra were recorded on a Bruker AVANCE (300 MHz) spectrometer using DMSO-d6 as
solvent with TMS as an internal standard. Chemical shifts were measured in hertz (Hz) and ppm
on a δ scale. Splitting patterns were designated as follows: s, singlet; d, doublet; t, triplet; and m,
multiplet.
Synthesis of 1,2,4-triazole and its derivatives (B1-B16)
1-Formylthiosemicarbazide (a) and 2H-1,2,4-triazole-3-thiol (b) were synthesized as described
by Hu et al (Hu et al., 2013) with slight modifications. Ethyl 2-[5-(2-ethoxy-2-oxo-
ethyl)sulfanyl-1,2,4-triazol-1-yl]acetate (c) and 2-[5-(2-hydrazino-2-oxo-ethyl)sulfanyl-1,2,4-
triazol-1yl]acetohydrazide (d) were synthesized by the procedure described by Wani et al (Wani,
Bhat, Azam, & Athar, 2013). 1,2,4-Triazole derivatives were synthesized as follows: briefly,
0.01 mol of compound (d) was refluxed with 0.01 mol of appropriate aldehyde/ ketone and
glacial acetic acid (1ml) in ethanol for 6-24h. Progress of the reaction was monitored by TLC
(hexane: ethyl acetate, 9:1) / (chloroform: methanol, 9:1). The separated product was filtered and
6

washed with diethyl ether. Solvent was evaporated under reduced pressure to obtain the
derivative in good yield. The compound was crystallized from ethanol/methanol/chloroform and
air-dried.
Free radical scavenging and antioxidant activity evaluation
As shown in Workflow 1, all synthesized derivatives (B1-B16) were first evaluated for
antioxidant activity in vitro, followed by biological activity evaluation in cell culture and in vivo
studies on selected compounds in rat model of inflammation. Finally, molecular docking and
molecular dynamics (MD) simulation analyses of the docked complex was performed to
rationalize the potential of selected derivative (B6) found to have the maximum anti-
inflammatory effect in vivo and to elucidate its mechanism of action in silico against PTGS.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay
DPPH assay was performed by the protocol described by Manzocco et al (Manzocco, Anese, &
Nicoli, 1998). Briefly, each synthetic derivative (0-800 µg/ml) was mixed with a methanolic
solution followed by the addition of 150µl DPPH (1.3mg/ml). Optical density (OD) of the
resulting mixture was measured at 517nm and the percentage inhibition was calculated as
follows:
Percent inhibition= OD(Control) – OD(Sample)/ OD(Control)
Equation [1]
7

Total reduction capability (TRC) assay
TRC was measured by the method described by Oyaizu (Oyaizu, 1986). Briefly, 2.5 ml of a
1% (w/v) solution of potassium ferricyanide hexacyanoferrate, K3Fe(CN)6 was mixed with 1ml
of the test compound in 2.5ml of 0.2 M phosphate buffer (pH 6.6). The mixture was incubated
for 20 minutes at 50C in an incubator followed by the addition of 2.5ml of 10% trichloroacetic
acid, 2.5ml distilled water and 0.5ml ferric chloride (0.1%, w/v). The absorbance of the reaction
mixture was measured at 700nm against blank.
Nitric oxide radical scavenging assay
Nitric oxide (NO) radical scavenging activity was measured by the method described by
Temraz and El-Tantawy (Temraz & El-Tantawy, 2008). Briefly, the test compound
(concentration range, 10-800 µg/ml) was mixed with 20 mM sodium nitroprusside (2 ml) and the
mixture was incubated at room temperature for 15 minutes followed by the addition of 1ml
Griess reagent (1%sulphanilamide, w/v, in 2% orthophosphoric acid, v/v, and 0.1% naphthyl
ethylene diaminedihydrochloride, w/v). The mixture was vortexed and incubated in dark for 10
minutes and the development of pink color, an indication of the nitric oxide radical scavenging
capability of the test compound, was measured at 540 nm against blank. Percent inhibition in
activity was calculated as in Equation [1].
Cell culture studies
Murine blood macrophage cells, RAW 264.7 and J774.1A, purchased from the National Centre
for Cell Science (NCCS), Pune were used to measure cell viability, nitrite and ROS. For all
purposes, cells were maintained in 75 cm2 culture flask containing DMEM supplemented with
10% FBS and 1% antibiotic solution in a humidified atmosphere at 5% CO2 and 37 C in a
8

water-jacketed CO2 incubator (Thermo Fischer Scientific) (Routray & Ali, 2019). Cells were
passaged 4-5 times.
Cytotoxicity
Test compounds were evaluated for their effect on cell viability or cytotoxicity as per the
published protocols (Teng et al., 2012) . Briefly, cells (1105) plated in a 96-well plate and
treated with the test compound (5-160 µM) were incubated in a CO2 incubator at 37 C (5%
CO2) for 48 h. After the incubation, the medium was discarded and an equal volume of serum
free medium and MTT (20 µL) was added into each well and the plate was further incubated at
37 C for 3 h. Thereafter, 150µl DMSO was added to dissolve the formed formazan crystals. The
plate was wrapped in foil and shaken on an orbital shaker for 15 minutes before taking the
absorbance of the solution in a microplate reader at 570nm. The analysis was performed in
triplicate (n=3). Data were expressed as Mean ± SD. IC50 was determined by the concentration-
cell viability curve. The cell viability was calculated as follows:
Cell viability = (Absorbance of the sample/ Absorbance of control)  100
Equation [2]
Griess Nitrite assay
Nitrite in the culture medium was measured by the Griess colorimetric method (Routray & Ali,
2019). Briefly, cells (1105cells/ml) were plated in a 96-well plate and treated with the test
compounds (B1, B5, B6, B9, and B13) and indomethacin as standard drug at a dose level of 40
µM, IC50. Cells were stimulated with the LPS (Escherichia coli O111:B4, lg/mL) followed by
9

48h incubation at 37C in a CO2 incubator. The medium containing the cells (100µl) was mixed
with an equimolar amount of Griess reagent and the absorbance of the reaction mixture was
taken at 540nm in a microplate reader using a sodium nitrite solution to draw the standard
calibration curve. For statistical analysis, one-way ANOVA was used followed by Dunnett’s
multiple comparison tests.
Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay
Production of ROS by the LPS-stimulated macrophage was analyzed by the DCFH-DA assay
(Pan et al., 2014). Briefly, cells were treated with 40, 80 or 160µM of the test compound in
culture medium incubated at 37C in a CO2 incubator for 48 h, followed by the stimulation with
LPS for 30 minutes. DCFH-DA (10µM) was used to detect the ROS level as per the published
procedure and the fluorescence was measured in a Jasco spectrofluorometer (FP-6200) after
excitation at 485 nm, and an emission wavelength of 500-550nm, respectively.
Computational analysis: molecular docking and MD simulation
The binding modes of selected synthetic derivatives (B1, B5, B6, B9 and B13) with PTGS
(COX) were understood by the molecular docking and MD simulation analysis of each complex.
Chemical structures of COX-1 (PDB ID: 3KK6) and COX-2 (PDB ID: 5KIR) were obtained
from the protein databank, PDB (www.rcsb.org/pdb) and converted to three-dimensional
structures (3D) in ChemBio3D Ultra 12.0 and saved (Shinkafi et al., 2019). Docking simulations
were studied by the Lamarckian genetic algorithm method (Morris et al., 1998). Standard
docking procedure was employed for rigid protein and flexible ligand, whose torsion angles were
identified for 10 independent runs per ligand. Docking grids were generated through bound co-
10

crystallized ligand. A grid of 28, 28, and 32 points in x, y, and z directions was built with a grid
spacing of 0.375 Å . Distance-dependent function of the dielectric constant was used for
calculating the energy maps. Default settings were used for other parameters. Docking
parameters were validated by re-docking of co-crystal ligand at the catalytically active site of
COX. At the end of the docking procedure, analysis of best pose interaction was done through
The PyMOL Molecular Graphics System, Maestro10.5 visualizer, and Chimera 1.11.
Two independent sets of MD simulations were run to understand the binding affinities and
binding modes of the compounds of interest – one set was performed with COX-1 (without
celecoxib and co-crystal celecoxib) and the docked complex system (celecoxib docked and
compound B6 docked), while another set was performed for COX-2 and docked complex system
(celecoxib docked and B6 docked) using Gromacs v5.1.2. The X-ray crystal structure of COX-1
and COX-2 was recruited from PDB. Before performing the simulation, the wild type of each
target was solvated in an octahedral TIP3P water box and relaxed at neutral pH for 500 ps. The
last frame of the trajectory obtained after the relaxation of wild type targets in water, was used.
Through steepest descent method, the entire system was minimized for 10,000 steps, followed by
minimization of another 10,000 steps by conjugate gradient method at 10 K to 277.15 K, through
Langevin thermostat in canonical (NVT) ensemble (Pastor, Brooks, & Szabo, 1988; Uberuaga,
Anghel, & Voter, 2004). Then, 100 ps simulation was performed under constant temperature and
pressure under the isothermal-isobaric (NPT) ensemble. The whole protein and solvent system
was minimized for 5,000 steps and heated from 10 K to 300 K for 100 ps in the NVT ensemble.
The production run for each protein was carried throughout 40 ns through the NPT ensemble. All
simulations conducted in this study involved a time step of 2 fs. Root mean square deviation
11

(RMSD), angles, and root mean square fluctuation (RMSF) were determined through Gromacs in
built tools, g_rms, g_sgangle, and g_rmsf, respectively.
Animal studies
Animals. The anti-inflammatory activity of selected test compounds was demonstrated in rat
against carrageenan-induced paw edema. Briefly, adult Wistar strain of rat, weighing 120-180 g,
was used for the study. Animals were acclimatized for one week before starting the experiment.
All animals were kept in an environmentally controlled room maintained at 30-70% relative
humidity, 18-26 ºC room temperature and a 12 h light/ dark cycle, and at least 10 room air
changes per hour. Animals were housed in polypropylene rat cages in a group containing not
more than six rats (n=6). Animals had free access to laboratory chow and water ad libitum. The
study was approved by the Institutional Animal Ethics Committee, and all experiments were
performed as per the guidelines laid down by the CPCSEA, the Committee for the Purpose of
Control and Supervision of Experiments on Animals (Registration No. 173-CPCSEA).
Treatment protocol and dose levels. Rats were randomly divided into 7 different groups (6 rats
in each group). Inflammation was induced in hind paw of each rat by injecting a suspension of
0.1 ml of 1% carrageenan in normal saline in the sub planter region as described by Winter et al.,
1962 (Winter, Risley, & Nuss, 1962). The test compound, suspended in tween 80 (1%, w/v), was
administered orally 1 h prior to the injection of carrageenan at a dose level of 5, 10, or 20 mg/kg
body weight orally. Indomethacin was used as standard drug at a dose level of 5 mg/kg body
weight by the same route. Normal control group of animals did not receive any treatment. The
group of animals injected with carrageenan alone served as negative control to study the
12

suppression of inflammation by the drug. The paw volume was measured at 1, 3 and 5h post-
carrageenan injection with a Vernier caliper. Change in paw volume or percent inhibition by the
test drug was calculated as follows: Percent inhibition = (Vc-Vt) /Vc100, where Vc is mean
increase in paw volume by the carrageenan injection and Vt, the mean increase in paw volume in
treatment group. Potency of the test compound relative to indomethacin was calculated as
follows: Potency = Percent inhibition obtained after the treatment with test compound / Percent
inhibition after treatment with standard drug.
Statistical analysis
Graph Pad Prism version 6.0 was used for all statistical analysis. Each value represents
MeanSD of analysis in triplicate, or n=6, for studies on animals. Statistical difference between
standard and test compounds was determined using a two-tailed Student’s t-test. Differences
were considered significant at p≤0.05 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Results
Synthesis and structural determination
1,2,4-Triazole and its 16 derivatives were synthesized as per the newly designed synthetic
route outlined in Scheme1, using thiosemicarbazide (Sigma Aldrich) as starting material without
further purification.
13

The reaction of thiosemicarbazide with formic acid resulted in the synthesis of 1-
formylthiosemicarbazide, the intermediate which reacts with 10% KOH and form 2H-1,2,4-
triazole-3-thiol (due to the removal of water molecule). Esterification of 2H-1,2,4-triazole-3-
thiol with ethylchloroacetate forms ethyl 2-[5-(2-ethoxy-2-oxo-ethyl)sulfanyl-1,2,4-triazol-1-
yl]acetate which when reacted with hydrazine hydrate forms 2-[5-(2-hydrazino-2-oxo-
ethyl)sulfanyl-1,2,4-triazol-1yl]acetohydrazide. Finally, the reaction of hydrazide with different
aldehydes and ketones yielded different derivatives (B1-B16). Various substituted R groups are
depicted in Table 1. The yield was good and the compounds could be easily recrystallized using
chloroform and methanol.
The structures of all newly synthesized compounds were confirmed by the FTIR, 1H NMR, 13C
NMR, and ESI-MS. The physical properties and the results of the spectral analysis data of all
derivatives are shown in supplementary files, Supplementary material, S1 and S2,
respectively. The presence of sharp bands in IR spectra around 650-750 cm-1 and 1040-1095 cm-
1
signify the peaks corresponding to C-S and C-N. Simultaneously appearance of the peaks of
C=O, C=N and NH in 1600-1675 cm-1, 3100-3200 cm-1 supports the synthesis of different
1
derivatives, B1-B16. In H NMR spectra, the singlet for N=C-H of the triazole ring, S-CH2 and
N-CH2 at 7.43-9.75, 3.27-3.98 and 4.05-5.03 ppm was achieved. Also, the singlet for –NH of
hydrazone was achieved from 8.60-11.99 ppm, followed by multiplets of aromatic protons at
6.71-8.16 ppm for B1-B16. The carbon NMR also confirmed the formation of the desired
derivatives, B1-B16, with signals at 40.33-53.22 ppm for CH2, carbon bonded to –S and –N
atoms. The clear signal at 153.52-173.54 ppm appeared for the C=O group of the synthesized
hydrazone. Singlet indicating the formation of the C=N bond appeared at 161.21-170.04 ppm.
14

The appearance of the desired characteristic peaks confirmed the formation of respective
compounds. In the Mass spectra (ESI-MS), the appearance of the molecular ion peaks at desired
intensities, corresponding to the molecular weight of the compounds (B1-B16), further
confirmed the formation of derivatives.
Free radical scavenging activity and TRC
As per the flow chart Workflow 1, synthesized derivatives were first tested for efficacy in
vitro. All 16 derivatives had considerable antioxidant activity, with maximum DPPH radical
scavenging activity exhibited by B13 (95.45 ± 0.64%) and an almost similar activity by B6
(95.31 ± 0.12%), followed by B9 (91.67 ± 1.20%), B1 (88.69 ± 1.05%) and B5 (80.98 ± 3.40%),
when compared with the positive control L-ascorbic acid (L-AA) (95.36 ± 1.24%) at 800µg/ml
(Fig. 1(A)(B)). B1, B6, B9 and B13, which possess electron-donating (-OCH3,-OH and -CH3)
groups as a substituent, displayed better free radical scavenging activity than B5 (which has an
electron-withdrawing nitro (-NO2) group as a substituent). The radical scavenging activity of
these compounds was comparable to the standard substance, L-AA. IC50 of B1, B5, B6, B9 and
B13 were 82.88 ± 1.732, 179.69 ± 1.05, 38.79 ± 1.88, 43.30 ± 1.79, and 55.66 ± 0.53 µg/ml
(p<0.001), respectively, when compared with L-AA (35.58 ± 1.52 µg/ml) (Table 2). The total
reduction capability among all compounds was best for B1, B5, B6, B9, and B13. B6 TRC,
expressed as absorbance at 700 nm, increased from 0.176 ± 0.017 at 10µg/ml to 2.639 ± 0.170 at
800µg/ml (Fig. 2). TRC of B1, B5, B9 and B13 at 800µg/ml was in the following order: B9
(2.205)> B13 (1.924) >B1 (1.079) >B5 (0.697). In comparison, TRC of L-AA was 2.602. B5
reported the lowest activity among all derivatives, while B6 showed the highest activity in a
15

concentration-dependent manner, reflecting the potential of B6 to reduce the transition state of
iron.
Nitric oxide radical scavenging activity
Results of the nitric oxide radical scavenging activity of 1,2,4-triazole derivatives are shown in
Fig. 3(A). A remarkable inhibitory effect was observed with B1, B5, B6, B9 and B13 in a
concentration-dependent manner in the following order: B6 (92.55 ± 1.18%), B9 (88.65 ±
2.22%), B13 (88.03 ± 1.94%), B5 (83.49 ± 1.72%) and B1 (79.75 ± 2.46%), when compared
with L-AA (94.31 ± 0.99%) at 800µg/ml, as shown in Fig. 3(B). IC50 values for B1, B5, B6, B9
and B13 were in the following order, 57.29 ± 1.73, 60.49 ± 1.72, 32.40 ± 0.62, 36.33 ± 1.71,
37.39 ± 1.94 µg/ml (p<0.001), respectively. IC50 of the standard was 43.46 ± 1.3 µg/ml (Table
2).
Synthesis and secretion of free radicals by the stimulated macrophages and cell viability
As shown in Fig. 4(A)(B), IC50 of B1, B5, B6, B9 and B13 was ≥40µM (Table 3). There was a
significant reduction (70-80%) in NO production by the LPS-stimulated macrophages by B1, B5,
B6, B9 and B13 in comparison to indomethacin, as shown in Fig. 5(A)(B) and Table 4. The
extent of inhibition was more in case of B6 and B9. The intracellular antioxidant activity, as
measured by the DCFH-DA assay, was also high particularly in the presence of B6 and B9, as
reported in Fig. 6(A)(B) and Fig. 7(A)(B), at 40, 80 and 160µM.
16

In vivo anti-inflammatory activity
B1, B6, B9 and B13, which showed good radical scavenging activity in vitro, demonstrated a
good anti-inflammatory activity in vivo at a dose level of 5, 10, and 20 mg/kg body weight when
administered orally in rats (Fig. 8, Table 5). B6 exhibited the most significant anti-inflammatory
activity (64.44% inhibition and a potency of 0.92 at 20mg/kg body weight after 5h of inducing
inflammation). The effect was comparable to the standard drug indomethacin (potency, 1.00)
(Table 5). The percentage inhibition of edema for B1, B9 and B13 was 58.4, 61.21 and 62.27%,
respectively, after 5 h. The order of the anti-inflammatory activity demonstrated by B1, B6, B9
and B13 was as follows: B6 > B13 > B9 > B1.
Molecular docking and MD simulation
Molecular docking studies were carried out to rationalize the potential of chosen synthesized
compounds as anti-inflammatory agents by predicting their binding affinities, binding mode, and
proper orientation with PTGS or COX against co-crystal ligand celecoxib. The compounds B1,
B5, B6, B9, and B13, which exhibited good anti-inflammatory activity, were docked in the
catalytic binding pocket of COX-1 (PDB ID: 3KK6) and COX-2 receptor (PDB ID: 5KIR) with
reference to celecoxib as a co-crystal ligand. The binding free energies of these five derivatives
complexed with COX-1 and COX-2 are shown in Table 6.
The binding of celecoxib with catalytic ligand-binding pocket of COX-1 is considered to be a
complex interaction via Gln 192, Leu 352, Phe 518 and Ile 517 hydrogen bonding network
resulting in tight binding. Celecoxib also formed important hydrophobic interaction with Val
116, Val 349, Tyr 355, Ile 359, Leu 384, Trp 387, Met 522, Ile 523, Ala 527 and Leu 531(Table
6, Fig. 9(C)(D)). B6 was found to bind into the same ligand-binding pocket of COX-1 as
17

celecoxib (interaction energy, 10.5kcal/mol). Three H-bonding interactions between para-
hydroxyl of B6 with Arg 120, Leu 352, and Phe 518 of COX-1 active sites were observed (Fig.
9(A)(B)). B6 also exhibited the hydrophobic interaction with Phe 205, Phe 209, Val 228, Val
344, Tyr 348, Val 349, Tyr 355, Leu 359, Ile 377, Phe 381, Tyr 385, Trp 387, Ile 517, Met 522,
Ile 523, Ala527, Leu 531 and Leu 534 (Fig. 9(B)). It superimposed with the co-crystal ligand
celecoxib which displayed the same interaction in the catalytic domain of COX-1. The aromatic
moiety attached to the triazole ring of B6 was superimposed on the aromatic moiety with the
sulfonamide functional group of celecoxib (Fig.11(A)). B6 (para-hydroxyl substituted) showed
similar docking results as celecoxib and exhibited excellent docking with COX-1.B1, B5, B6, B9
and B13 also docked in the active site of COX-2 (PDB ID: 5KIR). Celecoxib binds through a
network of hydrogen bonding with Gln 192, Leu 352, Ser 353, Arg 513 and Phe 518, and (also)
forms hydrophobic interaction with Val 116, Val 349, Tyr 355, Ile 359, Leu 384, Trp 387, Ala
516, Ile 517, Met 522, Val 523, Ala 527 and Leu 531(Fig. 10(C)(D)). B6 exhibited similar
interactions in the catalytic ligand binding site of COX-2. The interaction energy was
11.2kcal/mol. B6 showed four hydrogen bonding interactions between para-hydroxyl (of B6)
with Gln 192, Leu 352, Asn 375, and Ile 517 in COX-2 active site. It had hydrophobic
interaction with Phe 205, Phe 209, Val 228, Val 344, Tyr 348, Val 349, Tyr 355, Ile 377, Phe
381, Tyr 385, Trp 387, Ala 516, Phe 518, Met 522, Ile 523, Ala 527, Phe 529, and Leu 534(Fig.
10(A)(B)). In order to see the positioning of B6 in the catalytic domain of COX-2,
superimpositions were performed with the co-crystal ligand celecoxib which displayed
almost similar interaction in the catalytic domain of COX-2 (Fig. 11(B)). To sum up, results of
the in-silico analyse ssuggested anti-inflammatory activity via PTGS.
18

For a better understanding of the binding affinity and binding mode, the complex docked with
the compound showing highest docking score, B6, was subjected to MD simulation for 40 ns in
an aqueous environment at 300K. An analysis of RMSD, RMSF, Rg and SASA suggested
structural stability of the ligand (B6)-PTGS complex. The examination of all C-α and backbone
RMSD suggested conformational stability of COX-1and COX-2 in an aqueous environment. The
trajectory obtained for COX-1 (3KK6_Without celecoxib (black)) achieved equilibrium after 19
ns (Fig. 12(A)(B)) and a stable conformation of COX-1 can be seen up to 36 ns with minor
fluctuation. Thereafter, the trajectory showed a sharp transition for a few nanoseconds and
dropped to attain equilibrium at 40 ns. The trajectory obtained for protein-celecoxib co-crystal
structure (blue) showed equilibrium achieved at 9 ns followed by fluctuation of RMSD in an
average of 0.3 nm toward 22 ns. A further increase of RMSD started after 22 ns and reached upto
2.7 nm at 35 ns, followed by a short decrease (toward 2.2 nm) at 40 ns with small fluctuations.
RMSD of docked celecoxib (red) showed a sharp increase up to 2.2 nm at 13 ns followed by the
equilibrium of the structure up to 40 ns with an average fluctuation of 0.8 nm. On the other hand,
B6 docked COX-1 (green) showed a slow increase at 6 ns, and the structure maintained an
equilibrium toward 40 ns with an average fluctuation of 0.5 nm. The change in RMSD with
respect to COX-1 without celecoxib suggest a tight binding (of celecoxib, as well as B6) in the
active region. The ligand was stabilized by hydrogen and hydrophobic interactions with
surrounding residues during the simulation period. B6 stabilized the complex more quickly than
celecoxib. COX-2 (black) showed a sharp increase in RMSD in first 12.5 ns, followed by
equilibrium at ~25.5 ns. Thereafter, trajectory showed a small increase up to 32.5 ns followed by
a small increase up to 39 ns; afterward, trajectory attained equilibrium at 40 ns. Celecoxib
docked structure of 5KIR (red) and B6 docked 5KIR (green) achieved equilibrium after 11 ns
19

and thereafter, the complexes fluctuated with an average RMSD of 0.6 nm and 0.7 nm
throughout the simulation run. In short, data suggest large conformational changes when
celecoxib or B6 bound to COX-2 (Fig. 13(A)(B)). The variation in conformation due to ligand
contributes to the stabilization of COX-2 upon binding.
The Radius of gyration (Rg), which is the weighted root mean square average of the distance of
all scattering atoms from the center of mass of the protein and which provides information of the
overall structural compactness of protein, is important to understand the conformational changes
and dynamic stability of the complexes in an aqueous environment. As shown in Fig.12(C)(D),
the secondary and tertiary structures of COX-1 (and its complexes with the ligand) remained
stable during the simulation run (40ns). Average Rg for the B6 complex remained low (~2.3
nm), whereas docked celecoxib complex showed a slight increase to 2.325nm. A comparative
analysis of the results suggested very less increase in compactness of the protein structures both
in co-crystal and B6 complexes, as compared with native COX-1 (removed celecoxib from co-
crystal). Rg analysis (COX-2 and COX-2–ligand complex) (Fig. 13(C)(D)) showed a change in
Rg of the docked complex (COX-2-B6) from 2.4 nm to 2.325 nm at 3 ns, followed by
equilibrium up to 40 ns with only minor fluctuation. In case of COX-2-celecoxib, Rg started
from 2.4 nm and reached to 2.315 nm at 5 ns. After that, the complex maintained equilibrium up
to 25 ns and thereafter, a sudden decrease was observed up to 2.26 nm at 27 ns followed by
equilibrium toward 40 ns with small fluctuations. The residual fluctuation in docking complexes
was around 0.1-0.3 nm and was lower than the native (removed celecoxib from co-crystal)(Fig.
12(E)). Moreover, the active site protein residues interacting with docked ligands showed a little
fluctuation and stabilized in their favorable conformations. RMSF values further illustrated that
20

N-terminal part of COX-1 native and complex showed more fluctuations than C-terminal part. A
comparative analysis of COX-2 and COX-2-B6 complex demonstrated fluctuations in 0.1-0.3
nm range at the catalytic site of COX-2 (Fig. 13(E)). On the other hand, little fluctuation was
detected at the same catalytic site of COX-2 with celecoxib, as well as COX-2-B6 complex.
Apart from this, we observed a high level of mobility in some parts of COX-2, as well as COX-
2-ligand complexes, but these were not considered as our focus was to study the dynamic
behavior at the catalytic site. SASA, the solvent-accessible surface area, of COX-1 and the
docked complex (Fig. 12(F)) showed an average area of 309 nm2 and 311 nm2 with the native
removed celecoxib from co-crystal and native protein-celecoxib co-crystal structure of COX-1.
Docked complexes COX-1-celecoxib and COX-1-B6 were exposed to solvent environment with
an average area of 314 and 310 nm2. It can be concluded that the docked complex (B6) showed
either similar SASA or lower to native (without celecoxib). The lower value of SASA obtained
from the complex suggested that B6 on binding to COX-1 active site may lead to a tight packing
of hydrophobic residues aligned at the core of the active site. SASA of COX-2 and docked
complex (Fig. 13(F)) showed an average area of 327 nm2; the docked complexes (COX-2-
celecoxib and COX-2-B6) had an average area of 308 nm2 and 305 nm2, respectively. A lower
SASA for COX-2-celecoxib and COX-2-B6, when compared to COX-2, clearly suggested that
these compounds, when interacted with COX-2 active site, may lead to a tight packing of
hydrophobic residues aligned at the core of the active site.
Discussion
The heterocyclic compounds are commonly used in designing and development of newer
classes of medicinally important structural moieties with improved biological and
pharmacological properties such as the antimicrobial (Eswaran et al., 2009; Fan et al., 2018),
21

antibacterial (Eswaran et al., 2009; Y. L. Fan, 2018), anti-inflammatory (Paprocka et al., 2015),
analgesic (Gajanan Khanage et al., 2013), anticonvulsant (Kapron et al., 2019), and anticancer
properties (El-Sherief et al., 2018; Shahzad et al., 2019), to name a few. Triazole is an important
heterocyclic moiety possessing a five-membered ring along with two carbon and three nitrogen
atoms. Triazoleand its derivatives are known for their diverse and a wide range of biological
activities. Many studies have shown that the biological activities of 1,2,4-triazole and its
derivatives are due to the existence of the1,2,4-triazole nucleus (Q. Zhang et al., 2007). Some of
the compounds with triazole ring are reported as potent anticancer CA-4 analogues (Mur Blanch
et al., 2012). The presence of S-linkers in the structure improves some drug-like parameters such
as increasing water solubility, decreasing lipophilicity, and offering good hydrogen bond
acceptors (Bogolubsky et al., 2015). 1,2,4-Triazole along with Schiff base hydrazone ligands are
known to display excellent pharmacological activities, including the ability to inhibit oxidative
stress (Pokuri et al., 2014) and inflammation (Paprocka et al., 2015), which have been reported to
play an important role in the pathogenesis of many diseases, ranging from infection to cancer. In
this study, we have synthesized a new series of hydrazone derivatives of 1,2,4-triazole (Scheme
1). All synthesized derivatives were obtained in good yield and had sharp melting points. The
1
newly synthesized moieties were characterized by FTIR, H NMR, 13C NMR, and ESI-MS.
From proton (1H) NMR spectra, the singlet for N=C-H of the triazole ring, S-CH2, and N-CH2 at
7.43-9.75 ppm, 3.27-3.98 ppm and 4.05-5.03 ppm was achieved. Also, the singlet for –NH of
hydrazone was achieved from 8.60-11.99 ppm, followed by multiplets of aromatic protons at
6.71-8.16 ppm for B1-B16. The appearance of desired peaks at respective intensities confirmed
the formation of derivatives. All 16 derivatives (B1-B16) when evaluated in vitro for antioxidant
and anti-inflammatory activity by the DPPH assay, TRC and NO assays showed significant
22

activities when compared with known antioxidant and anti-inflammatory substance, L-ascorbic
acid. B6, which has electron-donating–OH as ring substituent easily stabilized the DPPH free
radical, displaying antioxidant activity, and enhanced the reductive ability by conversion of
ferricyanide complex Fe3+ to ferrous form. B6 also showed a significant nitric oxide radical
scavenging activity with an IC50of32.40 ± 0.62 µg/ml. Excessive and rapid production of both
reactive oxygen and reactive nitrogen species occurs during various inflammation-related
diseases and its mitigation has been found to be beneficial in a number of pathological conditions
(Afonso, Champy, Mitrovic, Collin, & Lomri, 2007). We also studied the cytotoxicity of B6 and
found it to be much less cytotoxic in MTT assay in RAW 264.7 and J774.1A cells. The
compound significantly suppressed the nitric oxide radical production by the LPS-primed
macrophages. The effect was comparable to indomethacin, a standard drug used in this study.
The level of ROS is also reported to be significantly reduced in this study by B6. In vivo
evaluation of the activity of B6 in rat model of carrageenan-induced inflammation (rat paw
edema inhibition by the drug) confirmed the anti-inflammatory action of the drug. B6 caused a
significant reduction in the size of edema.
In order to rationalize the potential of B6 as a novel synthetic drug with appreciable anti-
inflammatory activity, binding mode of the selected derivative, B6, and its proper orientation in
the active site of a key enzyme involved in inflammation, PTGS or COX was studied, against co-
crystal ligand celecoxib. The docking analysis suggested that the most active derivative
identified in this study, B6, nicely binds to COX-1(G(Binding)= -10.5 kcal/mol) and COX-2
(G(Binding)= -11.2 kcal/mol). MD simulation of B6 and the standard revealed stability of the
complex with both COX-1 and COX-2. The interaction was consistent with the essential active
23

site residues during the period of simulation. The activity of B6 may be attributed to the presence
of phenyl ring with two hydroxyl groups as substituents. This study implicates B6 as an
important scaffold for the synthesis of new medicinal agents in inflammatory conditions. A
graphical representation depicting the beneficial effects of B6 is shown in Fig. 14.
Conclusions
This study reports the synthesis, characterization and free radical scavenging and anti-
inflammatory activity of 1,2,4-triazole and its hydrazone derivatives. The structure of the
1
synthesized compounds was confirmed by IR, H NMR, 13C NMR and MS. Out of 16
derivatives, five (B1, B5, B6, B9, B13) (IC50≥40 µM) demonstrated 80-90% free radical
scavenging and antioxidant activity at low dose level; in particular, B6 and B9 significantly
ameliorated the reactive oxygen species and nitrite production by the LPS-stimulated
macrophages in culture. B6 demonstrated a significant anti-inflammatory effect in animal model
of inflammation. Computational studies suggested stability of the B6-PTGS / COX complex, and
its binding free energies, against bound co-crystal ligand celecoxib—the interaction was similar
to celecoxib, and the resulting complex was strong and stable. The presence of phenyl ring with
two hydroxyl groups as substituents in B6 is suggested to explain a better B6 efficacy,
implicating B6 as an important scaffold for the synthesis of new compounds targeted against
PTGS in inflammation and infections.
Acknowledgements
BK acknowledges UGC for Maulana Azad National Fellowship. BK expresses her gratitude to
Professor Faizan Ahmad, for his unconditional support, advice and guidance. AN acknowledges
24

the Indian Council of Medical Research (ICMR) for Senior Research Fellowship. SA
acknowledges the DBT Bioinformatics infrastructure facility, BIF, Jamia Hamdard under
BTISNet.
Conflict of Interest
The authors declare no conflict of interest.
References
Afonso, V., Champy, R., Mitrovic, D., Collin, P., & Lomri, A. (2007). Reactive oxygen species
and superoxide dismutases: role in joint diseases. Joint Bone Spine, 74(4), 324-329. doi:
S1297-319X(07)00159-5 [pii] 10.1016/j.jbspin.2007.02.002
Basri, A. M., Taha, H., & Ahmad, N. (2017). A Review on the Pharmacological Activities and
Phytochemicals of Alpinia officinarum (Galangal) Extracts Derived from Bioassay-
Guided Fractionation and Isolation. Pharmacogn Rev, 11(21), 43-56. doi:
10.4103/phrev.phrev_55_16 PRev-11-43 [pii]
Bogolubsky, A. V., Moroz, Y. S., Mykhailiuk, P. K., Ostapchuk, E. N., Rudnichenko, A. V.,
Dmytriv, Y. V., . . . Tolmachev, A. (2015). One-Pot Parallel Synthesis of Alkyl Sulfides,
Sulfoxides,
and
Sulfones.
ACS
Comb
Sci,
17(6),
348-354.
doi:
10.1021/acscombsci.5b00024
Dheer, D., Singh, V., & Shankar, R. (2017). Medicinal attributes of 1,2,3-triazoles: Current
developments. Bioorg Chem, 71, 30-54. doi: S0045-2068(16)30332-7 [pii]
10.1016/j.bioorg.2017.01.010
El-Sherief, H. A. M., Youssif, B. G. M., Abbas Bukhari, S. N., Abdelazeem, A. H., Abdel-Aziz,
M., & Abdel-Rahman, H. M. (2018). Synthesis, anticancer activity and molecular
modeling studies of 1,2,4-triazole derivatives as EGFR inhibitors. Eur J Med Chem, 156,
774-789. doi: S0223-5234(18)30580-4 [pii] 10.1016/j.ejmech.2018.07.024
Eswaran, S., Adhikari, A. V., & Shetty, N. S. (2009). Synthesis and antimicrobial activities of
novel quinoline derivatives carrying 1,2,4-triazole moiety. Eur J Med Chem, 44(11),
4637-4647. doi: 10.1016/j.ejmech.2009.06.031 S0223-5234(09)00360-2 [pii]
Fan, Z., Shi, J., & Bao, X. (2018). Synthesis and antimicrobial evaluation of novel 1,2,4-triazole
thioether derivatives bearing a quinazoline moiety. Mol Divers, 22(3), 657-667. doi:
10.1007/s11030-018-9821-8 10.1007/s11030-018-9821-8 [pii]
Gajanan Khanage, S., Raju, A., Baban Mohite, P., & Bhanudas Pandhare, R. (2013). Analgesic
activity of some 1,2,4-triazole heterocycles clubbed with pyrazole, tetrazole, isoxazole
and pyrimidine. Adv Pharm Bull, 3(1), 13-18. doi: 10.5681/apb.2013.003
Gao, C., Chang, L., Xu, Z., Yan, X. F., Ding, C., Zhao, F., . . . Feng, L. S. (2019). Recent
advances of tetrazole derivatives as potential anti-tubercular and anti-malarial agents. Eur
25

J
Med
Chem,
163,
404-412.
doi:
S0223-5234(18)31038-9
[pii]
10.1016/j.ejmech.2018.12.001
Gao, F., Wang, T., Xiao, J., & Huang, G. (2019). Antibacterial activity study of 1,2,4-triazole
derivatives. Eur J Med Chem, 173, 274-281. doi: S0223-5234(19)30358-7 [pii]
10.1016/j.ejmech.2019.04.043
Genc, M., Karagoz Genc, Z., Tekin, S., Sandal, S., Sirajuddin, M., Hadda Taibi, B., & Sekerci,
M. (2016). Design, Synthesis, in vitro Antiproliferative Activity, Binding Modeling of
1,2,4,-Triazoles as New Anti-Breast Cancer Agents. Acta Chim Slov, 63(4), 726-737. doi:
Genc-2014-4 [pii] 10.17344/acsi.2016.2428
Hu, J., Wang, Y., Wei, X., Wu, X., Chen, G., Cao, G., . . . Li, X. (2013). Synthesis and biological
evaluation of novel thiazolidinone derivatives as potential anti-inflammatory agents. Eur
J Med Chem, 64, 292-301. doi: 10.1016/j.ejmech.2013.04.010 S0223-5234(13)00242-0
[pii]
Jin, R. Y., Zeng, C. Y., Liang, X. H., Sun, X. H., Liu, Y. F., Wang, Y. Y., & Zhou, S. (2018).
Design, synthesis, biological activities and DFT calculation of novel 1,2,4-triazole Schiff
base derivatives. Bioorg Chem, 80, 253-260. doi: S0045-2068(18)30272-4 [pii]
10.1016/j.bioorg.2018.06.030
Kandile, N. G., Mohamed, M. I., & Ismaeel, H. M. (2017). Synthesis of new Schiff bases bearing
1,2,4-triazole, thiazolidine and chloroazetidine moieties and their pharmacological
evaluation.
J
Enzyme
Inhib
Med
Chem,
32(1),
119-129.
doi:
10.1080/14756366.2016.1238365
Kane, J. M., Dudley, M. W., Sorensen, S. M., & Miller, F. P. (1988). 2,4-Dihydro-3H-1,2,4-
triazole-3-thiones as potential antidepressant agents. J Med Chem, 31(6), 1253-1258. doi:
10.1021/jm00401a031
Kapron, B., Luszczki, J. J., Plazinska, A., Siwek, A., Karcz, T., Grybos, A., . . . Plech, T. (2019).
Development of the 1,2,4-triazole-based anticonvulsant drug candidates acting on the
voltage-gated sodium channels. Insights from in-vivo, in-vitro, and in-silico studies. Eur
J Pharm Sci, 129, 42-57. doi: S0928-0987(18)30553-0 [pii] 10.1016/j.ejps.2018.12.018
Kucukguzel, I., Tatar, E., Kucukguzel, S. G., Rollas, S., & De Clercq, E. (2008). Synthesis of
some novel thiourea derivatives obtained from 5-[(4-aminophenoxy)methyl]-4-alkyl/aryl-
2,4-dihydro-3H-1,2,4-triazole-3-thiones and evaluation as antiviral/anti-HIV and anti-
tuberculosis agents. Eur J Med Chem, 43(2), 381-392. doi: S0223-5234(07)00192-4 [pii]
10.1016/j.ejmech.2007.04.010
Li, Y. H., Zhang, B., Yang, H. K., Li, Q., Diao, P. C., You, W. W., & Zhao, P. L. (2017).
Design, synthesis, and biological evaluation of novel alkylsulfanyl-1,2,4-triazoles as cis-
restricted combretastatin A-4 analogues. Eur J Med Chem, 125, 1098-1106. doi: S0223-
5234(16)30913-8 [pii] 10.1016/j.ejmech.2016.10.051
Manzocco, L., Anese, M., & Nicoli, M. C. (1998). Antioxidant Properties of Tea Extracts as
Affected by Processing. LWT - Food Science and Technology, 31(7-8), 694–698
26

Morris, G. M., Goodsell, D. S., Halliday, D. S., Huey, R., Hart, W. E., Belew, R. K., & Olson, A.
J. (1998). Automated Docking Using a Lamarckian. Genetic. J Comput Chem 19, 1639-
1662
Mur Blanch, N., Chabot, G. G., Quentin, L., Scherman, D., Bourg, S., & Dauzonne, D. (2012).
In vitro and in vivo biological evaluation of new 4,5-disubstituted 1,2,3-triazoles as cis-
constrained analogs of combretastatin A4. Eur J Med Chem, 54, 22-32. doi:
10.1016/j.ejmech.2012.04.017 S0223-5234(12)00269-3 [pii]
Oyaizu, M. (1986). Studies on products of browning reaction--antioxidative activities of products
of browning reaction prepared from glucosamine. J. Nutrit, 44, 307–315
Pan, X., Cao, X., Li, N., Xu, Y., Wu, Q., Bai, J., . . . Lan, L. (2014). Forsythin inhibits
lipopolysaccharide-induced inflammation by suppressing JAK-STAT and p38 MAPK
signalings and ROS production. Inflamm Res, 63(7), 597-608. doi: 10.1007/s00011-014-
0731-7
Papadopoulou, M. V., Bloomer, W. D., Rosenzweig, H. S., Chatelain, E., Kaiser, M., Wilkinson,
S. R., . . . Ioset, J. R. (2012). Novel 3-nitro-1H-1,2,4-triazole-based amides and
sulfonamides as potential antitrypanosomal agents. J Med Chem, 55(11), 5554-5565. doi:
10.1021/jm300508n
Paprocka, R., Wiese, M., Eljaszewicz, A., Helmin-Basa, A., Gzella, A., Modzelewska-
Banachiewicz, B., & Michalkiewicz, J. (2015). Synthesis and anti-inflammatory activity
of new 1,2,4-triazole derivatives. Bioorg Med Chem Lett, 25(13), 2664-2667. doi:
10.1016/j.bmcl.2015.04.079 S0960-894X(15)00411-4 [pii]
Pastor, R. W., Brooks, B. R., & Szabo, A. (1988). An analysis of the accuracy of Langevin and
molecular dynamics algorithm Mol. Phys, 65(6), 1409–1419
Pokuri, S., Singla, R. K., Bhat, V. G., & Shenoy, G. G. (2014). Insights on the antioxidant
potential of 1, 2, 4-triazoles: synthesis, screening & QSAR studies. Curr Drug Metab,
15(4), 389-397. doi: CDM-EPUB-62195 [pii]
Routray, I., & Ali, S. (2019). Boron inhibits apoptosis in hyperapoptosis condition: Acts by
stabilizing the mitochondrial membrane and inhibiting matrix remodeling. Biochim
Biophys Acta Gen Subj, 1863(1), 144-152. doi: S0304-4165(18)30320-9 [pii]
10.1016/j.bbagen.2018.10.007
Shahzad, S. A., Yar, M., Khan, Z. A., Shahzadi, L., Naqvi, S. A. R., Mahmood, A., . . . Bajda,
M. (2019). Identification of 1,2,4-triazoles as new thymidine phosphorylase inhibitors:
Future anti-tumor drugs. Bioorg Chem, 85, 209-220. doi: S0045-2068(18)30954-4 [pii]
10.1016/j.bioorg.2019.01.005
Shinkafi, T. S., Kaushik, A., Mahmood, A., Tiwari, A. K., Alam, M. M., Akhter, M., . . . Ali, S.
(2019). Computational prediction and experimental validation of the activator function of
C2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone on pancreatic and hepatic
hexokinase. J Biomol Struct Dyn, 1-12. doi: 10.1080/07391102.2019.1650829
Temraz, A., & El-Tantawy, W. H. (2008). Characterization of antioxidant activity of extract
from Artemisia vulgaris. Pak J Pharm Sci, 21(4), 321-326
Teng, P., Liu, H. L., Zhang, L., Feng, L. L., Huai, Y., Deng, Z. S., . . . Li, J. X. (2012). Synthesis
and biological evaluation of novel sinomenine derivatives as anti-inflammatory agents.
27

Eur J Med Chem, 50, 63-74. doi: 10.1016/j.ejmech.2012.01.036 S0223-5234(12)00051-7
[pii]
Timur, I., Kocyigit, U. M., Dastan, T., Sandal, S., Ceribasi, A. O., Taslimi, P., . . . Ciftci, M.
(2018). In vitro cytotoxic and in vivo antitumoral activities of some aminomethyl
derivatives
of
2,4-dihydro-3H-1,2,4-triazole-3-thiones-Evaluation
of
their
acetylcholinesterase and carbonic anhydrase enzymes inhibition profiles. J Biochem Mol
Toxicol, e22239. doi: 10.1002/jbt.22239
Uberuaga, B. P., Anghel, M., & Voter, A. F. (2004). Synchronization of trajectories in canonical
molecular-dynamics simulations: observation, explanation, and exploitation. J Chem
Phys, 120(14), 6363-6374. doi: 10.1063/1.1667473
Wani, M. Y., Bhat, A. R., Azam, A., & Athar, F. (2013). Nitroimidazolyl hydrazones are better
amoebicides than their cyclized 1,3,4-oxadiazoline analogues: In vitro studies and
Lipophilic efficiency analysis. Eur
J
Med Chem, 64, 190-199. doi:
10.1016/j.ejmech.2013.03.034 S0223-5234(13)00194-3 [pii]
Winter, C. A., Risley, E. A., & Nuss, G. W. (1962). Carrageenin-induced edema in hind paw of
the rat as an assay for antiiflammatory drugs. Proc Soc Exp Biol Med, 111, 544-547
Xiao, Z. P., Wang, X. D., Wang, P. F., Zhou, Y., Zhang, J. W., Zhang, L., . . . Zhu, H. L. (2014).
Design, synthesis, and evaluation of novel fluoroquinolone-flavonoid hybrids as potent
antibiotics against drug-resistant microorganisms. Eur J Med Chem, 80, 92-100. doi:
10.1016/j.ejmech.2014.04.037 S0223-5234(14)00358-4 [pii]
Y. L. Fan, X. K., M. Li. . J. (2018). Coumarin-triazole hybrids and their biological activities.
Heterocyclic Chem. , 55, 791-802
Zhang, H. Z., Damu, G. L. V., Cai, G. X., & Zhou, C. H. (2014). Current developments in the
syntheses of 1,2,4-triazole compounds. Current Organic Chemistry, 18, 359-406
Zhang, J., Wang, S., Ba, Y., & Xu, Z. (2019). 1,2,4-Triazole-quinoline/quinolone hybrids as
potential anti-bacterial agents. Eur J Med Chem, 174, 1-8. doi: S0223-5234(19)30348-4
[pii] 10.1016/j.ejmech.2019.04.033
Zhang, Q., Peng, Y., Wang, X. I., Keenan, S. M., Arora, S., & Welsh, W. J. (2007). Highly
potent triazole-based tubulin polymerization inhibitors. J Med Chem, 50(4), 749-754. doi:
10.1021/jm061142s
Zhang, S., Xu, Z., Gao, C., Ren, Q. C., Chang, L., Lv, Z. S., & Feng, L. S. (2017). Triazole
derivatives and their anti-tubercular activity. Eur J Med Chem, 138, 501-513. doi: S0223-
5234(17)30505-6 [pii] 10.1016/j.ejmech.2017.06.051
28

Workflow 1: Flow chart depicting stepwise analysis of synthetic derivatives to shortlist the
compound with maximum anti-inflammatory activity and elucidation of its mechanisms of
action.
29