Synthesis, characterization, in vitro biological and computational evaluation of 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones

Article abstract of DOI:10.1007/s13738-021-02156-5

Triazoles and their heterocyclic analogues are nitrogen-rich versatile pharmacophores easily synthesized and converted to a range of biologically relevant heterocycles. In this context, the present research reports the synthesis, characterization, in vitr

Full text of DOI:10.1007/s13738-021-02156-5

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-021-02156-5
ORIGINAL PAPER
Synthesis, characterization, in vitro
biological and computational evaluation
of 5‑benzyl‑4‑(benzylideneamino)‑2H‑1,2,4‑triazole‑3(4H)‑thiones
Hamid Aziz · Aamer Saeed · Ashfaq Ur Rehman2,3 · Farukh Jabeen4,5 · Bakht Nasir · Ashraf Ullah Khan ·
1
1
6
6
Ismat Ullah Khan7
Received: 8 June 2020 / Accepted: 2 January 2021
©
Iranian Chemical Society 2021
Abstract
Triazoles and their heterocyclic analogues are nitrogen-rich versatile pharmacophores easily synthesized and converted to
a range of biologically relevant heterocycles. In this context, the present research reports the synthesis, characterization,
in vitro antioxidant, cytotoxic and α-glucosidase inhibitory potential of 4-amino-5-benzyl-2H-1,2,4-triazol-3(4H)-thione (3)
and 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones 5(a–g). Consequently, the percent DPPH free radical
scavenging ability was found to be in the decreasing order of 5 g>5e>3>5d>5b>5c>5a>5f. The most potent deriva-
tives (5 g), (5e) and (3) showed signiꢀcant dose-dependent scavenging ability with IC values of 61.22, 74.06 and 94.87 µg/
5
0
ml, respectively. The antioxidant derivatives were screened in brine shrimp lethality as well as protein kinase inhibitory
assay to unveil their toxic nature. The percent mortality was found to be decreasing in the order of 5b>5f>3>5a>5 g>5
d>5e>5c at 200 µg/ml in brine shrimp lethality assay. Accordingly, three derivatives (5b), (3) and (5f) showed signiꢀcant
percent mortality with LC values of 25.88, 32.94 and 34.87 µg/ml, respectively. Similarly, in protein kinase inhibitory assay,
50
maximum inhibitory potential was observed for the derivatives (5 g), (3) and (5e) with MIC values of 50 µg/disc in each
case. Likewise, in α-glucosidase inhibition assay, the screened derivatives (3) and (5e) showed notable percent α-glucosidase
inhibition (66.78 and 55.15%) with IC value of 36.11 µg/ml and 60.33 µg/ml, respectively. Molecular docking studies
5
0
of the screened derivatives were performed in order to assess their binding potential and mechanism of their binding with
α-glucosidase, α-kinase and β-kinase enzymes. Docking simulation revealed that the molecules stabilize themselves inside
*
Aamer Saeed
aamersaeed@yahoo.com
1
2
Department of Chemistry, Quaid-I-Azam University
Islamabad, Islamabad 45320, Pakistan
Department of Biochemistry, Abdul Wali Khan
University, Mardan, Shankar Campus, Mardan 23200,
Khyber Pukhtoonkhwa, Pakistan
3
4
5
Department of Bioinformatics and Biostatistics, Shanghai
Jiao Tong University, 800 Dongchuan Road, Minhang
District, Shanghai 200240, People’s Republic of China
Department of Chemistry and Biochemistry, Laurentian
University, 935 Ramsey Lake Road, Sudbury, ON P3E 2C6,
Canada
Computation, Science, Research and Development
Organization, 1401, 2485 Hurontraio Street, Mississauga,
ON L5A2G6, Canada
6
7
Department of Pharmacy, Quaid-I-Azam University
Islamabad, Islamabad 45320, Pakistan
Institute of Chemical Sciences, Gomal University,
Dera Ismail Khan 29220, Khyber Pukhtoonkhwa, Pakistan
Vol.:(0
1
123456789)
3

Journal of the Iranian Chemical Society
the active site by establishing non-covalent interactions with critical residues. Noteworthy were the derivatives (3) and (5e)
which anchored themselves through various signiꢀcant electrostatic interactions with the critical residues.
Graphic abstract
Keywords Antioxidants · DPPH · Molecular docking · Protein kinase inhibitors · Triazoles
Introduction
hydrogen bond donor or accepter, moderate dipole character,
rigidity and stability which enhances their biological proꢀle
[24–26]. The 1,2,4-triazole core shows considerable toler-
ance to metabolic degradation and exhibits target speciꢀc-
ity and vast range of activities. The triazole core due to its
polar nature can enhance the solubility of ligands and aꢂord
improved pharmacodynamic and pharmacokinetic activities
[27]. Moreover, the presence of nitrogen atoms in a triazole
ring aids intercalation and impart chromophore a polarized
character resulting in an optimal DNA interaction [27–30].
Notable examples include ꢁuconazole, voriconazole and
posaconazole used as antimycotic, anti-anxiety and CNS
stimulants [31]. 1,2,4-Triazole-based drugs like vorozole,
letrozole and anastrozole serve as potent aromatase inhibi-
tors which prevent breast cancer [32]. Thus, 1,2,4-triazole
rings are inserted in a range of diverse and therapeutically
relevant heterocycles to turn them into eꢃcient drug candi-
dates. Based on the sale values, the 1,2,4-triazole-containing
antifungal drugs have made their position in the list of top
200 drugs [33, 34]. The antifungal drugs with 1,2,4-triazole
core include voriconazole, ravuconazole and itraconazole
[35, 36]. Consequently, some of the potent and commercially
available 1,2,4-triazole-based drugs are shown in Fig. 1.
Schiff bases are nitrogen analogues of aldehyde and
ketones containing imine/azomethine linkage in their
1
,2,4-Triazoles are nitrogen-rich ꢀve-membered aromatic
heterocycles with molecular formula C H N commonly
2
3
3,
known as bio-isostere of oxadiazoles [1]. Depending on the
relative positions of N-atoms in the ring, triazoles occur in
two isomeric forms: 1,2,4-triazoles or 1,2,3-triazoles. Both
these isomers of triazoles show tautomerism and are found
in two forms, i.e., 1,2,4-triazoles can occur as 1,2,4-(4H)-
triazoles and 1,2,4-(1H)- triazoles, whereas 1,2,3-triazoles
can occur as 1,2,3-(2H)-triazole and 1,2,3-(1H)-triazole.
1
,2,4-triazole moiety has gained considerable attention of
scientists due to its vast range of uses in pharmaceuticals,
material sciences, and agrochemicals [2]. For example, vari-
ously substituted 1,2,4-triazoles are described to have vast
range of biological activities like antimicrobial, antifungal
[
[
[
[
3], anti-inꢁammatory [4], antibacterial [5], hypoglycemic
6], anti-tubercular [7], antidiabetic [8], anticonvulsant
9], antidepressant [10], anti-malarial [11], anti-migraine
12], analgesic [13], arthritis [14], antiviral [15], antihy-
pertensive [16, 17], antileishmanial [18], antiplatelet [19]
potassium channel activators [20], and antioxidant [21].
Several 1,2,4-triazoles are commercialized as imperative
herbicides, fungicides [22] and growth regulators in plants
[
23]. 1,2,4-Triazoles possess unusual properties like strong
1
3

Journal of the Iranian Chemical Society
Fig. 1 Some commercially
F
available drugs containing
CN
1
,2,4-triazole core skeleton
Cl
NC
N
N
CN
F
N
N
N
CN
N
OH
N
N
N
N
N
N
N
N
N
N
N
N
Vorozole
Letrozole
Anastrozole
Fluconazole
H
N
N
N
O
N
N
F
NH2
O
F
F
N
N
N
F
N
O
N
O
N
F
F
Cl
Cl
Cl
Rizatrplan
Flupoxam
Propiconazole
F
O
N
N
N
N
HO
HO
O
N
N
Cl
NH2
N
N
OH
OH
N
F
N
OH
N
N
N
OH
Ribavirin
Cl
N
Tebuconazole
Voriconazole
Metconazole
F
Cl
O
Cl
O
O
N
O
Si
N
N
N
N
N
N
N
Cl
O
N
N
N
N
F
N
N
Itraconazole
Flusilazole
Triadimefon
F
N
N
N
F
O
N
N
N
N
N
O
Cl
N
N
N
N
N
O
N
N
N
N
Posaconazole
Trapidil
Alprazolam
structures [37] and are frequently utilized for industrial
means and exhibit diverse biological activities [38]. They
are found in various natural, natural-derived and non-natural
heterocycles [39–41]. Their electrophilic carbon and nucleo-
philic nitrogen atoms interact nucleophiles and electrophiles
that lead to enzymes inhibition and DNA replication [42].
Azomethine linkages impart heterocycles potent biological
potential including anticancer, antimicrobial, antipyretic,
antiproliferative and antidiabetic [43]. Metal complexes
of Schiꢂ bases are very active catalysts in many biological
systems [44–47], dyes [48], polymers [49] and medicinal
industries [50]. Their uses in food packages, birth control
and as an oxygen sensor are also delineated. In this regard,
literature survey reveals that the pharmacokinetic proꢀle of
are reported as potent and eꢂective antimicrobials [52, 53],
antitumor [54], cytotoxic, growth regulating, antioxidant,
and antidiabetic agents [55]. Pillai et al. recently reported the
enhanced pharmacokinetic proꢀle of novel 4-amino-1,2,4-
triazole-based Schiff bases. Based upon the drug-like-
ness calculations and in vitro antidiabetic assay against
α-glucosidase, the screened 4-amino-1,2,4-triazole-based
Schiꢂ bases displayed signiꢀcant inhibitory potential in
terms of their IC values compared to the reference standard
5
0
acarbose [56]. Moreover, the 4-amino-1,2,4-triazole-based
Schiꢂ bases displayed strong antioxidant potential in DPPH
and total reducing power assays [56].
Keeping in view the synthetic utility and biologi-
cal potential of 1,2,4-triazoles, on the one hand, and the
enhancement of pharmaceutical potential of the Schiff
bases, on the other hand, the current research aims at the
1
,2,4-triazoles is improved by incorporating Schiꢂ bases in
their core skeleton [51]. 1,2,4-Triazoles-derived Schiꢂ bases
1
3

Journal of the Iranian Chemical Society
synthesis, characterization, biological evaluation and
molecular docking studies of 4-amino-5-benzyl-2H-1,2,4-
triazole-3(4H)-thione and their corresponding 5-benzyl-
was raised up to 90 ℃ for another half an hour. During the
course of the reaction, TLC was employed to check the reac-
tion’s progress using chloroform/methanol (70:30) solvent
system. Once the reaction was completed, the reaction mix-
ture was poured in ice cold water which resulted in the for-
mation of solid precipitates. The resulting precipitates were
ꢀltered and dried in an oven at 70 ℃ for two hrs to obtain
solid white precipitates of the product [56].
4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones. The
synthesis of 4-amino-5-benzyl-2H-1,2,4-triazol-3(4H)-one
was performed in a facile way using thiocarbohydrazide and
phenylacetic acid. Condensing 4-amino-5-benzyl-2H-1,2,4-
triazol-3(4H)-one with various aldehydes results in a series
of 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-
3
(4H)-thiones. The synthesized compounds were identiꢀed
4‑Amino‑5‑benzyl‑2H‑1,2,4‑triazole‑3(4H)‑thione (3)
and conꢀrmed using various spectroscopic techniques like
1
13
FT-IR, H-NMR, C-NMR and elemental analysis (CHNS)
and were screened in various in vitro biological assays.
White powder; (80%); R ; 0.54 (chloroform/methanol,
f
−
1
70:30); m.p 155 ℃; Mol. wt: 206.27; IR (neat, cm ) ν:
3
286.44, 3237.35 (NH ), 3148.91 (NH), 3084.74, 3033.33
2
2
3
(
sp CH), 2934.46 (sp CH), 1624.52 (C=N), 1567.18,
Experimental protocols
1494.95, 1451.50 (Ar–C=C), 1417.83 (C–N), 1364.01 (-CH
1
bending), 1295.68 (C=S); H NMR (DMSO-d 300 MHz) δ:
6,
Chemistry
13.54 (b, s, 1H, NH), 7.33 (d, 2H, J=6.90 Hz, Ar–H), 7.30
(
t, 2H, J=7.50 Hz, Ar–H), 7.25 (t, 1H, J=7.50 Hz, Ar–H),
1
3
The reactions were carried out under nitrogen atmosphere
using freshly dried distilled solvents obtained from sigma-
Aldrich. NMR spectra were recorded using Bruker 300 MHz
spectrometer at ambient temperature. The chemical shifts
values are reported relative to the residual solvent peak of
5.55 (s, 2H, C–H), 4.02 (b, s, 2H, NH ); C NMR (DMSO-
2
d , 75 MHz) δ: 166.47 (C=S), 151.83 (C=N), 135.96 (Ar),
6
129.31 (Ar), 128.94 (Ar), 127.25 (Ar), 30.61 (C–H); Anal.
Cald. For C H N S: C, 52.41; H, 4.89; N, 27.16; S, 15.55:
9
10
4
Found: C, 52.37; H, 4.94; N, 27.20; S, 15.50.
1
13
DMSO (δ 2.50 for H and δ 39.52 for C) or acetone (δ
1
13
2
.05 for H and δ 29.84, δ 206.26 for C). TMS was used as
Synthesis of 5‑benzyl‑4‑(benzylideneamino)‑2H‑1,2,
4‑triazole‑3(4H)‑thione 5(a–g)
1
an internal reference, and the HNMR data are reported as
the chemical shift value (δ), relative integral, multiplicity s
(
singlet), d (doublet), t (triplet), quartet (q), m (multiplets),
The synthesis of 5-benzyl-4-(benzylideneamino)-2H-1,2,4-
triazole-3(4H)-thiones 5(a–g) was performed as reported in
the literature. Brieꢁy, aldehydes (15 mmol) completely dis-
solved in (05 ml) dry distilled ethanol containing catalytic
amount of concentrated sulfuric acid was stirred at reꢁux
for half an hour. After half an hour, a solution of 4-amino-
5-benzyl-2H-1,2,4-triazole-3(4H)-thione (10 mmol) pre-
pared using dry distilled ethanol (10 ml) was sequentially
added to the acidic solution of aldehydes in ethanol. The
resulting reaction mixture was set for further 24 h at reꢁux.
During the course of the reaction, TLC was employed to
check the reaction’s progress using chloroform/methanol
(70:30) solvent system. Once the reaction was completed,
ethanol was removed under reduced pressure to obtain the
product in a solid form which was recrystallized from etha-
nol at room temperature [56].
b (broad), coupling constant (J, Hz), and the atom assign-
13
ment. C NMR data are reported as the chemical shift value
(
δ) and assignment of the atom. The FT-IR spectra were
−
1
measured in wave numbers (cm ) using Vertex 70 Bruker
apparatus. Elemental analysis (CHNS) was performed to
ꢀ
nd out the percentage of each element present in the syn-
thesized compound. Thin layer chromatography (TLC) on
2
6
.0 cm×5.0 cm aluminum sheets precoated with silica gel
0F254 with a layer thickness of 0.25 mm (Merck) was per-
formed to check the reaction’s progress. Ultraviolet ꢁuo-
rescence was used for the identiꢀcation of the compounds
while using TLC.
Synthesis of 4‑amino‑5‑benzyl‑2H‑1,2,4‑tria‑
zole‑3(4H)‑thione (3)
The 4-amino-5-benzyl-2H-1,2,4-triazole-3(4H)-thione (3)
was synthesized eꢃciently using a reported methodology
but with slight modifications. Briefly, equimolar ratios
5‑Benzyl‑4‑(propylideneamino)‑2H‑1,2,4‑tria‑
zole‑3(4H)‑thione (5a)
(
10 mmol) of phenyl acetic acid and thiocarbohydrazide
Brown powder; (70%); R ; 0.50 (chloroform/methanol, 70:30);
f
−
1
were mixed in a 100-ml round-bottom ꢁask. The reaction
mixture was allowed to stir at room temperature for half an
hour (hr) to ensure homogenous mixing of the reactants.
After half an hour, the temperature of the reaction mixture
m.p 165 ℃; Mol. wt: 246.33; IR (neat, cm ) ν: 3193.62
2
3
(NH), 3065.25 (sp CH), 2992.59, 2890.46 (sp CH), 1639.61
(HC=N), 1590.32, 1579.48, 1524.10 (Ar–C=C), 1439.57
1
(–CH bending), 1371.30 (-CH bending), 1280.52 (C=S); H
1
3

Journal of the Iranian Chemical Society
NMR (CD COCD -d 300 MHz) δ: 11.28 (b, s, 1H, NH),
4‑(4‑Hydroxy‑3‑methoxylbenzylideneamino)‑5‑benzyl‑2H‑
1,2,4‑triazole‑3(4H)‑thione (5d)
3
3
6,
7
.53 (s, 1H, HC=N), 7.17 (t, 2H, J=8.10 Hz, Ar–H), 7.10
(
t, 1H, J=8.10 Hz, Ar–H), 7.09 (d, 2H, J=8.10 Hz, Ar–H),
4
3
.35 (s, 2H, C–H), 1.43 (q, 2H, J=7.20 Hz, C–H), 0.93 (t,
Red powder; (75%); R ; 0.42 (chloroform/methanol, 70:30);
f
13
−1
H, J=7.20 Hz, C–H); C NMR (CD COCD -d 75 MHz)
m.p 190 ℃; Mol. wt: 340.40; IR (neat, cm ) ν: 3294.44
3
3
6,
2
3
δ: 168.0 (C=S), 161.0 (C=N), 156.24 (HC=N), 137.53 (Ar),
(OH), 3183.34 (NH), 3029.87 (sp CH), 2981.17 (sp CH),
1647.20 (HC=N), 1597.42, 1576.55, 1547.78, 1502.39
1
29.13 (Ar), 128.73 (Ar), 125.83 (Ar), 30.92 (C–H), 21.83
1
(
C–H), 10.43 (C–H); Anal. Cald. For C H N S: C, 58.51; H,
(Ar–C=C), 1336.17 (-CH bending), 1243.46 (C=S); H
12 14 4
5
.73; N, 22.74; S, 13.02: Found: C, 58.43; H, 5.75; N, 22.70;
NMR (CD COCD -d 300 MHz) δ: 11.28 (b, s, 1H, NH),
3
3
6,
S, 13.72.
8.32 (s, 1H, HC=N), 7.32 (t, 2H, J=8.0 Hz, Ar–H), 7.08 (t,
1
H, J=8.0 Hz, Ar–H), 7.07 (d, 2H, J=8.10 Hz, Ar–H), 7.06
4‑(4‑Chlorobenzylideneamino)‑5‑benzyl‑2H‑1,2,4‑tria‑
(s, 1H, Ar–H), 7.05 (d, 1H, J=8.02 Hz, Ar–H), 6.75 (d, 1H,
J=8.02 Hz, Ar–H), 4.80 (b, s, 1H, OH), 4.27 (s, 2H, C–H),
zol‑3(4H)‑thione (5b)
1
3
3
.72 (s, 3H, O–C–H); C NMR (CD COCD -d 75 MHz)
3 3 6,
Yellow powder; (75%); R ; 0.45 (chloroform/methanol, 70:30);
δ: 186.30 (C=S), 155.30 (C=N), 151.30 (Ar), 148.30
(Ar), 143.33 (HC=N), 137.53 (Ar), 129.93 (Ar), 128.73
(Ar), 127.73 (Ar), 125.73 (Ar), 122.63 (Ar), 117.63 (Ar),
114.23 (Ar), 56.13 (O–C), 38.63 (C–H); Anal. Cald. For
C H N O S: C, 59.98; H, 4.74; N, 16.46; S, 9.42: Found:
f
−
1
m.p 212 ℃; Mol. wt: 328.82; IR (neat, cm ) ν: 3237.70
2
3
(
(
(
NH), 3084.76, 3032.18 (sp CH), 2933.95 (sp CH), 1624.70
HC=N), 1566.86, 1493.37, 1417.81 (Ar–C=C), 1453.16
1
-CH bending), 1360.63 (-CH bending), 1294.05 (C=S); H
1
7
16
4
2
NMR (CD COCD -d 300 MHz) δ: 11.28 (b, s, 1H, NH),
C, 59.92; H, 4.77; N, 16.45; S, 9.42.
3
3
6,
8
.13 (s, 1H, HC=N), 7.65 (d, 2H, J=8.10 Hz, Ar–H), 7.35 (d,
2
H, J=8.10 Hz, Ar–H), 7.17 (t, 2H, J=8.0 Hz, Ar–H), 7.09
5‑Benzyl‑4‑(decylideneamino)‑2H‑1,2,4‑tria‑
zole‑3(4H)‑thione (5e)
(
t, 1H, J=8.0 Hz, Ar–H), 7.03 (d, 2H, J=8.10 Hz, Ar–H),
1
3
4
1
1
.26 (s, 2H, C–H); C NMR (CD COCD -d 75 MHz) δ:
3 3 6,
56.62 (C=S), 151.70 (C=N), 149.80 (HC=N), 138.98 (Ar),
35.54 (Ar), 129.43 (Ar), 128.98 (Ar), 128.59 (Ar), 126.98
Brown powder; (70%); R ; 0.57 (chloroform/methanol,
f
−
1
70:30); m.p 145 ℃; Mol. wt: 344.52; IR (neat, cm ) ν:
2
3
(
Ar), 124.10 (Ar), 30.53 (C–H); Anal. Cald. For C H ClN S:
3274.13 (NH), 2982.54 (sp CH), 2958.42, 2859.80 (sp
16
13
4
C, 58.44; H, 3.98; N, 17.04; S, 9.75: Found: C, 58.30; H, 3.92;
CH), 1643.61 (NH Bending), 1629.25 (HC=N), 1595.02,
1549.60, 1508.74 (Ar–C=C), 1445.60 (-CH bend-
N, 17.14; S, 9.79.
1
ing), 1365.58 (-CH bending), 1249.18 (C=S); H NMR
4‑(4‑Nitrobenzylideneamino)‑5‑benzyl‑2H‑1,2,4‑tria‑
(CD COCD -d 300 MHz) δ: 11.28 (b, s, 1H, NH), 7.53
3
3
6,
zole‑3(4H)‑thione (5c)
(s, 1H, HC=N), 7.17 (t, 2H, J=8.0 Hz, Ar–H), 7.10 (t, 1H,
J = 8.0 Hz, Ar–H), 7.09 (d, 2H, J = 8.02 Hz, Ar–H), 4.23
(s, 2H, C–H), 2.16 (t, 2H, J=7.50 Hz, C–H), 1.87 (q, 2H,
J=7.20 Hz, C–H), 1.28–1.49 (m, 12H, C–H), 0.94 (t, 3H,
Yellow powder; (86%); R ; 0.32 (chloroform/methanol,
f
−
1
7
3
0:30); m.p 135 ℃; Mol. wt: 339.37; IR (neat, cm ) ν:
2
3
13
383.39 (NH), 3091.16, 3050.63 (sp CH), 2941.86 (sp
J = 7.20 Hz, C–H); C NMR (CD COCD -d 75 MHz)
3
3
6,
CH), 1651.33 (HC=N), 1597.31, 1574.28, 1514.57, 1455.67
δ: 186.30 (C=S), 158.33 (C=N), 155.33 (HC=N), 137.53
(Ar),129.13 (Ar), 128.73 (Ar), 125.83 (Ar), 38.63 (C–H),
31.93 (C–H), 29.73 (C–H), 29.53 (C–H), 29.47 (C–H),
29.43 (C–H), 26.73 (C–H), 26.13 (C–H), 22.83 (C–H), 14.23
(C–H); Anal. Cald. For C H N S: C, 66.24; H, 8.19; N,
(
Ar–C=C), 1344.37 (-CH bending), 1266.70 (-CH bend-
1
ing), 1216.20 (C=S); H NMR (CD COCD -d 300 MHz)
3
3
6,
δ: 12.80 (b, s, 1H, NH), 10.81 (s, 1H, HC=N), 8.40 (d, 2H,
J=8.70 Hz, Ar–H), 8.19 (d, 2H, J=9.0 Hz, Ar–H), 7.40 (d,
1
9
28
4
2
H, J=7.2 Hz, Ar–H), 7.32 (t, 2H, J=7.50 Hz, Ar–H) 7.25
16.26; S, 9.31: Found: C, 66.15; H, 8.18; N, 16.36; S, 9.31.
1
3
(
t, 1H, J=7.20 Hz, Ar–H), 4.29 (s, 2H, C–H); C NMR
(
CD COCD -d 75 MHz) δ: 156.64 (C=S), 151.78 (C=N),
4‑(3,4,5‑Trimethoxybenzylideneamino)‑5‑benzyl‑2H‑1,2,4
‑triazole‑3(4H)‑thione (5f)
3
3
6,
1
1
49.84 (HC=N), 138.95 (Ar), 135.34 (Ar), 129.39 (Ar),
28.96 (Ar), 128.58 (Ar), 126.99 (Ar), 124.08 (Ar), 30.89
(
C–H); Anal. Cald. For C H N O S: C, 56.63; H, 3.86; N,
White powder; (73%); R ; 0.39 (chloroform/methanol, 70:30);
16
13
5
2
f
−
1
2
0.64; S, 9.45: Found: C, 56.50; H, 3.93; N, 20.70; S, 9.39.
m.p 201 ℃; Mol. wt: 384.46; IR (neat, cm ) ν: 3280.85
2
3
(
NH), 3236.14 (sp CH), 2995.58, 2839.31 (sp CH), 1609.74
HC=N), 1576.32, 1520.40, 1504.89, 1465.71 (Ar–C=C),
(
1
361.42 (-CH bending), 1280.62 (-CH bending), 1248.76
1
(
C=S); H NMR (CD COCD -d 300 MHz) δ: 11.28 (b, s,
3
3
6,
1
H, NH), 8.13 (s, 1H, HC=N), 7.17 (t, 2H, J=8.0 Hz, Ar–H),
1
3

Journal of the Iranian Chemical Society
7
.10 (t, 1H, J=8.0 Hz, Ar–H), 7.09 (d, 2H, J=8.02 Hz, Ar–H),
.66 (s, 2H, Ar–H), 4.43 (s, 2H, C–H), 3.76 (s, 9H, O–C–H);
C NMR (CD COCD -d 75 MHz) δ: 186.93 (C=S), 155.30
Table 1 Structures
of
the
synthesized
5-benzyl-
4
-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones
derivatives
6
13
5(a–g)
3
3
6,
(
(
C=N), 150.93 (Ar), 143.30 (HC=N), 141.30 (Ar), 137.30
S. No. Compound
R
S. No. Compound R
Ar), 129.13 (Ar), 128.73 (Ar), 128.13 (Ar), 125.83 (Ar),
1
2
5a
5b
C H
5
6
5e
5f
CH (CH )
2
5
3
2 8
106.73 (Ar), 56.65 (O–C), 56.23 (O–C), 38.63 (C–H); Anal.
4-Cl
3,4,5-tri-
Cald. For C H N O S: C, 59.36; H, 5.24; N, 14.57; S, 8.34:
19
20
4
3
OCH3
Found: C, 59.46; H, 5.20; N, 14.51; S, 8.38.
3
4
5c
5d
4-NO2
4-OH,
7
5 g
…
C H
3
7
…
…
5‑Benzyl‑4‑(butylideneamino)‑2H‑1,2,4‑tria‑
3-OCH
3
zole‑3(4H)‑thione (5g)
Brown powder; (72%); R ; 0.53 (chloroform/methanol, 70:30);
Brine shrimp lethality assay
f
−
1
m.p 260 ℃; Mol. wt: 260.35; IR (neat, cm ) ν: 3197.12
2
3
(
(
(
NH), 3092.64, 2963.94 (sp CH), 2864.26 (sp CH), 1638.42
Brine shrimp lethality assay is a simple, high throughput
screening test used to determine the cytotoxicity of the
compounds based on their ability to kill a simple zoo-
logical organism-brine shrimp (Artemia salina). The
synthesized derivatives (3, 5(a–g)) were tested in a 24-h
lethality assay performed in a 96-well plate against Arte-
mia salina (Ocean star, USA) [58]. The eggs of Artemia
salina were incubated for a hatching period of 24–48 h
under light and warmth (30–32 °C) in simulated seawater
(38 g/l supplemented with 6 mg/l dried yeast) in a spe-
cially designed two-compartment plastic tray. Using Pas-
teur pipette, mature phototropic nauplii were harvested and
transferred to each well of plate. The corresponding vol-
ume of each derivative containing≤1% DMSO in seawater
at ꢀnal concentrations of 200, 100, 50, 25 , and 12.50 μg/
ml was transferred to the wells containing seawater and
shrimp larvae. The ꢀnal volume in each well was kept
300 μl. Positive and negative control wells included serial
concentrations (50, 25, 12.5, 6.25, and 3.125 ug/ml) of
doxorubicin and 1% DMSO in seawater, respectively. After
24-h incubation, live shrimps were counted, and the per-
centage of deaths was determined. TableCurve 2D v5.01
software was used to calculate median lethal concentration
HC=N), 1586.18, 1549.23, 1454.63 (Ar–C=C), 1419.52
1
-CH bending), 1376.10 (-CH bending), 1274.30 (C=S); H
NMR (CD COCD -d 300 MHz) δ: 11.31 (b, s, 1H, NH),
3
3
6,
7
.53 (s, 1H, HC=N), 7.17 (t, 2H, J=8.0 Hz, Ar–H), 7.10 (t,
1
H, J=8.0 Hz, Ar–H), 7.09 (d, 2H, J=8.02 Hz, Ar–H), 4.36
(
s, 2H, C–H), 2.21 (t, 2H, J=7.50 Hz, C–H), 1.64 (sixtet,
13
J=7.50 Hz, C–H), 0.97 (t, 3H, J=7.50 Hz, C–H); C NMR
(
CD COCD -d 75 MHz) δ: 186.30 (C=S), 158.33 (C=N),
3 3 6,
1
55.30 (HC=N), 137.53 (Ar), 129.13 (Ar), 128.73 (Ar),
25.83 (Ar), 38.63 (C–H), 28.93 (C–H), 19.43 (C–H), 14.02
1
(
C–H); Anal. Cald. For C H N S: C, 59.97; H, 6.19; N,
1
3
16
4
2
1.52; S, 12.32: Found: C, 59.80; H, 6.30; N, 21.58; S, 12.32.
In vitro biological evaluation
Biological evaluation was performed through numer-
ous in vitro assays including antioxidant, cytotoxic and
α-glucosidase to assess the biological potential of the synthe-
sized derivatives (3, 5(a–g)). All the in vitro assays were per-
formed following some standard literature protocols.
DPPH free radical scavenging assay (FRSA)
(
LC ). The assay was done in triplicate.
50
DPPH (2,2-diphenyl-1-picrylhydrazyl) is a red color free radi-
cal having an absorbtion at a wavelength of 517 nm and turns
yellow upon its elimination. FRSA of the synthesized deriva-
tives (3, 5(a–g)) was assessed from a reported methodology
Protein kinase inhibition assay
[
57]. Ascorbic acid served as a positive control and DMSO
Kinases are an important class of enzymes that phospho-
rylate their substrate proteins. The assay was performed
in triplicates by observing hyphae formation in puriꢀed
isolates of Streptomyces 85E strain [59]. The bacterial
served as a negative control. The FRS potential in terms of %
inhibition is given in Table 1 and was determined from Eq. (1)
provided.
(
Absorbance of the Control − Absorbance of the Sample)
%
Inhibition =
× 100
Absorbance of the Control
1
3

Journal of the Iranian Chemical Society
lawn was allowed to grow by spreading spores (myce-
lia fragments) of refreshed culture of Streptomyces on
sterile plates containing minimal ISP4 medium. About
complex crystallographic structure unavailability. Subse-
quently, we observed a hydrophobic cleft (also revealed in
the experimental study for kinases), where all the predicted
site spheres appeared, and ꢀnally, we selected the large size
pocket for MD purposes; the following approached already
successfully applied as described in our previous study [62]
was utilized. Due to the unavailability of the crystallographic
structure of the α-glucosidase enzyme, we have used the
homology model described in our previous study [63] for
docking purposes. All the structure was subjected to MOE
for preparation.
5
μl of each of the synthesized derivative (20 mg/ml of
DMSO) was loaded onto sterile 6 mm ꢀlter paper discs.
The impregnated paper discs with a ꢀnal concentration
of 100 μg/disc were applied directly on the surface of the
plates seeded with Streptomyces 85E. DMSO served as a
negative control, and surfactin infused discs served as a
positive control. The plates were then incubated at 30 °C
for 72 h (time required for hyphae formation in Strepto-
myces 85E), and the results were interpreted as bald zone
of inhibition around samples and controls infused discs.
Further, the protonation was done using default param-
eters of the structure preparation module of MOE. Next, all
the structure was subjected to energy minimization to get
minimal energy conformation. Finally, reꢀned structures
were used for docking study using the default parameters
of MOE; Placement: Triangle Matcher, rescoring 1: Lon-
don dG, Reꢀnement: Forceꢀeld, Rescoring 2: GBVI/WSA.
Before running the docking protocol, a total of ꢀve confor-
mations for the ligand were selected. The top-ranked con-
formations based on docking score (S) and protein–ligand
interaction (PLI) proꢀle were selected for analysis.
α‑glucosidase inhibition assay
α-Glucosidase is a glucosidase enzyme that is present in
the brush border of the small intestine and cleaves α (1→4)
bond of starch. The α-glucosidase assay was performed
using a literature reported methodology [60]. The assay
involves addition of 25 μl of 20 mM substrate solution,
6
9 µl of 50 mM phosphate buꢂer, and 5 µl of test sample
from 4 mg/ml DMSO stock solution. Subsequently, 1 µl of
enzyme solution was added into the designated wells of 96
well plate. The mixture was incubated at 37 °C for half an
hour. Following incubation, 100 µl of 0.5 mM sodium bicar-
bonate solution was added to stop the reaction and absorb-
ance was estimated at 405 nm using microplate reader. Per-
cent enzyme inhibition was calculated using Eq. (1):
Statistical analysis
The data obtained from this research work are presented in
mean± SD terms. One-way analysis of variance was carried
out to ꢀnd out variability among groups by Statistix 8.1.
The diꢂerent graphs were obtained using GraphPad Prism
(
Absorbance of the Control − Absorbance of the Sample)
%
Inhibition =
× 100
Absorbance of the Control
Negative control was employed in the assay using the
same procedure just by replacing the test derivative with
5. Tukey’s multiple comparison tests were used to calculate
signiꢀcant diꢂerences among groups. The statistical signiꢀ-
cance was set at P<0.05.
5
µl DMSO. Acarbose was used as a positive control, and
the assay was run in triplicate.
Molecular docking study
Results and discussion
Molecular docking (MD) analysis was performed using
molecular operating environment (MOE) [61] to explore
the binding mode of the screened derivatives (3, 5(a–g))
against kinases (i.e., α-IKK and β-IKK) and α-glucosidase
enzymes. First, the 3D coordinates for the screened deriva-
tives were generated using the MOE-builder module and
were protonated, and energy minimized using the default
parameters of the MOE (gradient: 0.05, Force Field:
MMFF94X). The crystal structure for α-IKK and β-IKK
was retrieved from the protein databank (PDB code; 3BRT
Synthetic work
The synthesis of 4-amino-5-benzyl-2H-1,2,4-triazole-3(4H)-
thione (3) and 5-benzyl-4-(benzylideneamino)-2H-1,2,4-
triazole-3(4H)-thiones 5(a–g) was performed as depicted
clearly in Scheme 1. The ꢀrst step involves the synthesis
of an intermediate 4-amino-5-benzyl-2H-1,2,4-triazole-
3(4H)-thione from phenylacetic acid and thiocarbo-
hydrazide. The synthetic work is performed by fusing
phenylacetic acid and thiocarbohydrazide at high temper-
atures. The reaction completes in a shorter period with-
out any side products. In the second step, the synthesized
&
3BRV). The active site for all the structures was predicted
using a site-ꢀnder module implemented in MOE due to the
1
3

Journal of the Iranian Chemical Society
S
O
H N
2
NH2
N
H
(
N
H
N
NH
N
Fusion
0°C
N
R
H 4(a-g)
NH
S
1)
+
9
N
C H OH/H SO /Reflux
2
5
2
4
N
S
H N
2
O
(
3)
R
HO
5(a-g)
(2)
Scheme 1 Synthetic scheme adopted for the synthesis of 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones 5(a–g)
4
-amino-5-benzyl-2H-1,2,4-triazole-3(4H)-thione was
In vitro biological evaluations
transformed into 5-benzyl-4-(benzylideneamino)-2H-1,2,4-
triazole-3(4H)-thiones 5(a–g) using substituted aldehydes
in dry distilled ethanol. This reaction involves the use of
a catalytic amount of concentrated sulfuric acid. Once
the synthetic work was performed, the next step involved
was to conꢀrm the structures of the synthesized 5-benzyl-
Biological screening of the synthesized 5-benzyl-
4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones (3,
5(a–g)) was performed through various in vitro biologi-
cal assays like DPPH free radical scavenging (anti-oxidant
assay), brine shrimp lethality, protein kinase inhibition
(cytotoxicity assay) and α-glucosidase (antidiabetic assay).
The in vitro assays were performed by preparing solutions
of the synthesized derivatives (3, 5(a–g)) in DMSO (4 mg/
ml, 20 mg/ml) following some well-established standard
protocols from the literature. Then serial dilutions of the
prepared solutions were performed to prepare their solutions
with diꢂerent concentrations.
4
-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones
1
13
5
(a–g) using spectral data (FT-IR, H and C NMR) and
elemental analysis (CHNS).
Spectroscopic characterizations
Structural assignments of the synthesized 4-amino-5-ben-
zyl-2H-1,2,4-triazole-3(4H)-thione (3) and 5-benzyl-
4
-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones
(a–g) were established from elemental (CHNS) and
DPPH free radical scavenging assay
5
1
13
spectroscopic analysis (FT-IR, H and C NMR). The
elemental analysis provided % results for the elements C,
H, N and S closely aligned with the structural formula of
the synthesized 5-benzyl-4-(benzylideneamino)-2H-1,2,4-
triazole-3(4H)-thiones 5(a–g). FT-IR spectrum of
In vitro DPPH free radical scavenging assay was performed
for the synthesized derivatives (3, 5(a–g)) with reference to
the control and was related with the standard antioxidant.
DPPH is a stable free radical owing to its delocalized elec-
tron that gives an absorption band at 517 nm.
4
-amino-5-benzyl-2H-1,2,4-triazole-3(4H)-thione (3)
shows broader absorption bands at 3286.44, 3237.35,
−
1
3
148.91 cm representing NH and NH protons. These
2
NH and NH protons are evident as broader signals from
2
Table 2 DPPH free radical scavenging potential of the screened
derivatives (3, 5(a–g))
1
the H NMR spectrum at their respective position of 13.54
and 4.02 ppm, respectively. FT-IR spectra of the synthe-
DPPH free radical scavenging potential
sized 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-
S. No.
Compound
% scavenging (200 µg/ml)
IC50 (µg/ml)
3
(4H)-thiones 5(a–g) lack peaks for the NH group protons
2
−1
at 3286.44, 3237.35 cm , and sharp singlet is observed
for the azomethine (HC=N) linkages in the range of
d
1
2
3
4
5
6
7
8
3
82.89± 0.67
43.0± 0.42
94.87
–-
165.32
–-
132.42
74.06
–-
a
5a
5b
5c
5d
5e
5f
−
1
1
651.33–1609.74 cm . The azomethine linkages are also
b
a
63.28± 0.56
45.27± 0.21
75.12± 0.74
84.08± 0.27
42.99± 0.63
88.76± 0.77
1
evident from H NMR spectra of the synthesized 5-ben-
zyl-4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thione
c
5
(a–g) as sharp singlets in the range of 10.81–7.53 ppm. The
de
a
coupling pattern of the para-substituted phenyl ring protons
5c) display double doublets at 8.40 and 8.19 ppm [55].
(
e
5 g
61.22
1
3

Journal of the Iranian Chemical Society
This assay is based on the fact that the DPPH free radi-
cals are being quenched by accepting electrons or hydrogen
radicals from the donor antioxidants which augments the
antioxidant capability of the screened derivatives. The var-
ied results of the FRSA are presented in terms of their %
inhibition as well as IC (µg/ml) values in Table 2. Accord-
Brine shrimp lethality assay The cytotoxic screening of
the synthesized derivatives (3, 5(a–g)) was performed
against brine shrimp larvae to reveal their lethality proꢀle.
For brine shrimp lethality assay, the initial concentration
employed was 200 µg/ml. Among the screened derivatives,
those having noteworthy results were further screened at
low concentrations (100, 50, 25, 12.5 ug/ml), and their LC
5
0
ingly, the antioxidant assay was evaluated initially at 200 µg/
ml and the screened derivatives showing signiꢀcant activ-
ity were further screened at lower concentrations (100, 50,
5
0
values were determined. Accordingly, the screened deriva-
tives (5b), (3) and (5f) showed signiꢀcant percent mortal-
2
5, and 12.5 ug/ml) in order to determine their IC val-
ity rate at 200 µg/ml with LC values of 25.88, 32.94, and
5
0
50
ues. The highest % free radical inhibition was shown by the
derivatives (5 g), (5e) and (3) with more than 80% of DPPH
free radical inhibition. The % DPPH free radical inhibitory
potential of the remaining derivatives decreased in the order
of 5d>5b>5c>5a>5f. In terms of their IC values, the
34.87 µg/ml, respectively, as shown in Table 3. Their LC
5
0
values were found to be well below 50 μg/ml and hence cat-
egorized as highly cytotoxic. Similarly, the screened deriva-
tives (5 g) and (5a) caused moderate to high cytotoxicity
at 200 µg/ml with LC values of 150.37 and 54.45 µg/ml,
5
0
50
screened derivatives (5 g), (5e) and (3) showed signiꢀcant
respectively. These derivatives were considered as moder-
free radical scavenging potential with IC values of 61.22,
ately cytotoxic in nature (LC value≥50 but≤200 μg/ml).
5
0
50
7
4.06, and 94.87 µg/ml, respectively. Moreover, marked free
The remaining derivatives (5d), (5e) and (5c) showed very
low mortality rate and were considered as weakly cytotoxic
in nature under the experimental conditions [58].
radical scavenging potential was shown by the derivatives
(
5d) and (5b) with IC values of 132.42 and 165.32 µg/ml,
5
0
respectively, well above 100.0 µg/ml. However, the remain-
ing derivatives (5c) and (5a) as well as (5f) did not show any
prominent radical scavenging potential [57].
Protein kinase inhibition assay Protein kinase inhibitory
potential of the synthesized derivatives (3, 5(a–g)) was
determined at an initial concentration of 100 µg/disc using
Streptomyces 85E strain. The results of the zones of inhibi-
tion are presented in Table 3. Among the screened deriva-
tives, those derivatives showing more than 12-mm bald
zone were considered to have noteworthy kinase inhibitory
action, and their lower concentrations were tested. Conse-
quently, in comparison with the reference standard surfactin
with bald zone of 25.50± 1.0 mm, the maximum inhibitory
potential was observed for the derivative (5e) with clear
and bald zones of 10± 0.58 and 15± 0.58 mm, respectively.
Values are represented as mean± SD of analysis run in
a−e
triplicate, means with diꢂerent superscript letters ( ) in a
column specify signiꢀcant diꢂerence at P<0.05. IC values
50
were calculated using TableCurve 2D v4 software.
In vitro cytotoxicity assays
The cytotoxicity proꢀle of the synthesized derivatives (3,
(a–g)) was assessed from brine shrimp lethality and protein
kinase inhibitory assays.
5
Table 3 The cytotoxicity assays of the screened 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-3(4H)-thiones (3, 5(a–g))
S. No.
Compound
Brine shrimp
Protein kinase inhibition
Diameter at 100 µg/disc
%
Mortality 200 µg/ LC50 µg/ml
MIC µg/disc
50
ml
Clear zone
Bald zone
a
a
14± 1.0b
…
1
2
3
4
5
6
7
8
9
3
5a
5b
5c
5d
5e
5f
5 g
100± 5.7
80± 5.7
32.94
54.45
25.88
–
–
–
34.87
150.37
5.65
–
8± 0.58
…
7± 0.58
…
…
10± 0.58
…
7± 1.0
b
c
100± 5.7
12± 0.58
100
…
…
50
…
50
–
d
d
10± 5.7
40± 5.7
20± 5.7
8± 1.0
9± 1.0
c
d
d
b
15± 0.58
…
a
a
100± 5.7
b
b
60± 5.7
14± 1.0
Doxorubicin
Surfactin
DMSO
100± 0.0
–
–
–
–
–
–
1
0
1
25.50± 1.0a
–
–
–
1
–
1
3

Journal of the Iranian Chemical Society
Similarly, the derivatives (3) and (5 g) displayed the next
higher kinase inhibitory potential with clear and bald zones
of 08± 0.58 and 14± 1.0 mm for the derivative (3) and
inhibitory potential was found to decreasing in the order of
5f>5d>5a>5c>5b>5 g [60].
0
7± 1.0 and 14± 1.0 mm for the derivative (5 g), respec-
Molecular docking study
tively. However, the MIC values obtained for these three
derivatives were found to be 50 µg/disc. The remaining
derivatives displayed lower kinase inhibitory potential with
bald zones much lower than the reference standard surfactin
α‑IKK and β‑IKK
The MD results revealed that all the screened derivatives
(3, 5(a–g)) showed the best-ꢀt well pattern of binding in the
predicted active site for both α- and β-kinase enzyme by site
ꢀnder module implemented in MOE.
[
59].
Values presented are the mean± SD (n=3) values analy-
sis run in triplicate. Results of standards are given in respec-
tive columns. Means with diꢂerent superscripts (a–d) rep-
resent signiꢀcantly diꢂerent values at p<0.05. LC values
In the case of α-IKK, we have noticed that all the deriva-
tives (3, 5(a–g)) possess diꢂerent substituted groups, i.e.,
electron-donating groups (EDG’s) or electron-withdrawing
groups (EWG’s) as shown in the ribbon representation in
Fig. 3a. In the case of the most potent derivatives (5e and 3),
the highest in vitro activity and several favorable interactions
with critical residues which might have a role in the enhance-
ment of the activity of the corresponding enzyme from the
MD perspective were observed. The potent derivatives (5e
and 3) shared similar protein–ligand interaction (PLI) pro-
ꢀle, but the only diꢂerences found were in their prospective
docking score (DS), which was 5.4 for (5e) (Fig. 3b) and 4.5
for (3) (Fig. 3c). In the case of (5e), the R101 participated
in arene–cation interaction with the 5-ring, and the Q98
adopted side-chain donor interaction with the sulfur, which
is a typical interaction in both compounds.
5
0
were calculated using TableCurve 2D v4 software.
In vitro α‑glucosidase assay α-glucosidase inhibition assay
was employed to determine the antidiabetic potential of the
synthesized derivatives (3, 5(a-g)) at an initial concentra-
tion of 100 μg/ml. Results of the α-glucosidase inhibition
assay are given in Fig. 2 as mean± SEM of the % inhibition
of α-glucosidase. Among the screened derivatives, (3) and
(
5e) showed notable glucosidase inhibitory potential (66.78
and 55.15%). The inhibitory potential was determined in
terms of their IC value. The IC value was found to be
5
0
50
3
6.11 µg/ml and 60.33 µg/ml in case of (3) and (5e). The
remaining derivatives were found to be less potent with %
α-glucosidase inhibitory potential lower than 50%; hence,
there IC values cannot be determined. However, their
5
0
Fig. 2 α-Glucosidase inhibi-
tory potential of the screened
derivatives (3, 5(a–g)): Results
are expressed as mean± SD
of percent inhibition of the
α-glucosidase enzyme. Acar-
bose was used as a positive
control, and its IC value was
50
found to be 4.2 µg/ml. DMSO
was used as a negative control
with enzyme inhibition noted.
IC values were calculated
50
using TableCurve 2D v4
software
1
3

Journal of the Iranian Chemical Society
Fig. 3 The protein–ligand
interaction (PLI) proꢀles against
kinase (α-IKK and β-IKK)
and α-glucosidase enzyme. (a)
indicates the α-IKK enzyme
surface, (b, c) Represent the PLI
proꢀle for the derivatives (5e
and 3). (d) shows the surface
of the β-IKK enzyme. (e, f)
Represent the PLI proꢀle for
the derivatives (5e) and (3). (g)
shows the surface representation
of the α-glucosidase enzyme.
(
h, i) Represent the PLI proꢀle
for the derivatives (3) and (5e).
Double-sided arrows represent
the arene–arene interaction
In the case of β-IKK, the potent derivatives (5e and 3)
shared diꢂerent PLI proꢀle with key residues, but the only
similarity found was, in their interacting mode; the sulfur
α‑glucosidase
The molecular docking results for both the derivatives (3
and 5e) against the α-glucosidase enzymes revealed that
the derivative (3) showed the best inhibitory potential based
on the PLI proꢀle and the docking score. The active site
was mostly occupied by polar and acidic residues and some
hydrophobic residues supporting the bridge for maintaining
the interaction (Fig. 3g). In the case of the potent deriva-
tives (3), the PLI proꢀle revealed that the derivative mostly
adopted favorable interactions with polar (H111, Y71,
H348) and acidic (D349 and D214) residues, and only one
essential residue (R212) was found, as shown in Fig. 3h.
Both the derivative shared a typical interaction moiety (sul-
fur), as was observed in kinase activity. In the case of the
second most potent derivative (5e), we have noticed that
only one acidic (E304) and basic (R312) and one additional
hydrophobic (F300) residue adopted interaction with the
derivative (Fig. 3i). The derivative (3) high potency might
be due to the attached EDG (NH2), which has a substantial
activation over others.
(
S) atom of the 4-methyl-2H-1,2,4-triazole-3(4H)-thione
moiety participated in interaction with diꢂerent residues
Fig. 3d–f). For example, the experimental results indicate
(
the importance of hydrophobic residue F734, which plays
an essential role in interaction with other proteins. In our
case, we observed that the derivative (5e) adopted hydro-
gen bond interaction with the 5-ring structure of the ligand.
Also, we observed that the nearby residues to this critical
residue F734 also participated in the interaction. Due to the
triggering of these important residues, it might be allosteri-
cally raised the inhibitory potential of this compound. The
nearby residue at a distance ~ 2-3 angstrom includes the
polar uncharged side-chain residue, i.e., Q732. Both the
residues shared a similar interaction, as the only diꢂerence
found in their side-chain moiety. Generally, the excellent
potential of the (5e) against the beta-kinase might be due
to the triggering potential of the critical important residue
F734, which might trigger other residues to participate in
the interaction. Also, the binding pose of 5e is similar to a
cage-like structure, where both the hydrophobic and polar
uncharged side-chain residues rescue the compound from
dissociation.
1
3

Journal of the Iranian Chemical Society
1
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This study reports the synthesis and characterization of
the 5-benzyl-4-(benzylideneamino)-2H-1,2,4-triazole-
1
1
5. A.K. Jordao, P.P. Afonso, V.F. Ferreira, M.C. De Souza, M.C.
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3
(4H)-thiones (3, 5(a–g)). The synthesized derivatives
were screened in various in vitro assays. As evident from
the results, some of the screened derivative displayed
dose-dependent response in the tested in vitro assays. The
derivatives (5 h), (5f), and (5a) showed signiꢀcant free radi-
cal scavenging potential with IC values of 61.22, 74.06,
1
1
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5
0
and 94.87 µg/ml, respectively. The derivatives (5c), (5a),
and (5 g) showed signiꢀcant percent mortality at 200 µg/
ml with LC values of 25.88, 32.94, and 24.87 µg/ml,
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5
0
respectively. In case of protein kinase inhibitory assay,
the maximum inhibitory potential was observed in case
of (5f) and (5a) with MIC that was observed to be 50 µg/
disc in both cases. Moreover, the derivatives (5a) and (5f)
showed notable enzyme inhibitory action with IC values
2
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5
0
of 36.11and 60.33 µg/ml, respectively. Thus, based on the
results, it can be concluded that the screened 4-amino-
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-benzyl-2H-1,2,4-triazole-3(4H)-thione and their 5-ben-
2
2
2
2
2
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possess better inhibitory potential for the α-glucosidase and
Kinases.
Acknowledgements The author Mr. Hamid Aziz gratefully acknowl-
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edges the ꢀnancial support of the Higher Education Commission
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