Novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine derivatives embedded with benzimidazole moiety as potent antioxidants

Article abstract of DOI:10.1002/jccs.201900452

Fourteen novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine derivatives bearing benzimidazole moiety (7a-n) have been synthesized using the one-pot nitro reductive cyclization method. All the synthesized compounds were confirmed by ¹H nuclear magne

Full text of DOI:10.1002/jccs.201900452

Received: 23 October 2019
DOI: 10.1002/jccs.201900452
Revised: 13 February 2020
Accepted: 12 March 2020
A R T I C L E
Novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
derivatives embedded with benzimidazole moiety
as potent antioxidants
1
1
Reshma Sathyanarayana | Boja Poojary
|
2
2
Revanasiddappa B. Chandrashekarappa | Hemanth Kumar
|
2
Vijay K. Merugumolu
1
Department of Chemistry, Mangalore
Abstract
University, Mangalore, India
2
Fourteen novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine derivatives bearing
benzimidazole moiety (7a-n) have been synthesized using the one-pot nitro
reductive cyclization method. All the synthesized compounds were confirmed
Department of Pharmaceutical
Chemistry, NGSM Institute of
Pharmaceutical Sciences Nitte University,
Mangalore, India
1
1
13
by H nuclear magnetic resonance ( H NMR), C NMR, fourier-transform
infrared (FT-IR), mass spectrum, and elemental analyses. All the title com-
pounds were subjected to in vitro antioxidant activity. The free radical scav-
enging activity of the compounds was examined using DPPH, nitric oxide, and
superoxide radical scavenging methods. The results demonstrated that com-
Correspondence
Boja Poojary, Department of Chemistry,
Mangalore University, Mangalagangothri,
Mangalore 574199, Karnataka, India.
Email: bojapoojary@gmail.com
pound
-4-tolyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7c) was potent in scaveng-
ing both DPPH and nitric oxide radical with IC50 values of 13.57 and
3-(2-(3,4-dimethoxyphenyl)-1-propyl-1H-benzo[d]imidazol-5-yl)-
6
1
8.55 μg/ml when compared to the standard with IC50 values of 23.75 and
3.14 μg/ml, respectively, which was due to the presence of electron-donating
2
groups. The activity was found to decline when electron-donating groups were
replaced by electron-withdrawing groups. Moderate scavenging activity was
observed for the superoxide radical. Structure activity relationship and
physiochemical properties were studied for all the derivatives.
K E Y W O R D S
bioorganic chemistry, medicinal chemistry, organic chemistry
1
| INTRODUCTION
amyotrophic lateral sclerosis (death of neurons control-
[
2]
ling voluntary muscles), Alzheimer's disease (memory
[
3]
[4]
Free radicals can be defined as an atom or molecule with
unpaired electrons in the outer most shell. The presence
of excessive free radicals causes oxidative injury, which
leads to various neurodegenerative diseases such as
loss),
and schizophrenia (mental health illness).
The presence of excess radicals is also responsible for the
crosslinking of proteins, lipids, and DNA, which results
[
5]
in immune system decline and cancer. These oxidative
damages are facilitated by reactive oxygen species, such
[
1]
Parkinson's disease (a disorder that affects movement),
©
2020 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2020;1–16.
http://www.jccs.wiley-vch.de
1

2
SATHYANARAYANA ET AL.
as the hydroxyl radical (˙OH); peroxy radical (RCOO˙);
oxide radical scavenging activity, and superoxide anion
scavenging activity.
Due to poor pharmacokinetic properties, most of the
drug molecules fail to enter the market. For a molecule
to become a successful lead, it should obey Lipinski's rule
of five, which states that the drug should be easily
hydrogen peroxide (H O ); hypochlorous acid (HOCl);
2
2
−
the superoxide anion radical (O ˙ ); and reactive nitrogen
species such as peroxynitrite anion (ONOO ) and nitric
2
−
oxide (˙NO), which are generated in vivo during autoxi-
dation of polyunsaturated fatty acids, NADPH-dependent
oxidases, mitochondrial electron transfer chain, photoly-
absorbed, distributed to its target site, not easily metabo-
[6]
[31]
sis, and catecholamines. Antioxidants play a vital role
by preventing the initiation of oxidation by various mech-
anisms such as decomposing peroxide, binding or remov-
ing catalyzing metal ions, radical scavenging, and
lized, and excreted easily,
also known as absorption,
distribution, metabolism and elimination (ADME) prop-
erties. Nowadays, computer-based methods are gradually
becoming popular as it takes less time to determine the
ADME profile. Thus, the pharmacokinetic profile of syn-
thesized drugs has been calculated using the QikProp
program (Schrödinger) running in fast mode.
[
7]
limiting the propagation of the oxidative reaction.
Although many enzymatic antioxidants are known, it is
necessary to develop a novel drug that has a greater anti-
oxidant defense mechanism.
In wide search of novel antioxidants, it was found
that benzimidazole moiety has a broad spectrum of anti-
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
[8–11]
oxidant properties.
various pharmacological effects such as antileukemic,
anti-inflammatory,
Benzimidazole also demonstrates
[
12]
[13]
[14]
[15]
antiobesity,
and antimicrobial
analgesic,
[16]
[17]
antiproliferative,
activities. In
The synthesis of target compounds 7a-n is depicted in
addition, benzimidazoles exhibit similar structural prop-
erties with purine, which also hinders bacterial nucleic
Scheme 1. The procedure for the synthesis of hydrazide is
[
18]
followed according to literature,
where 4-chloro-
[18]
acids and proteins.
of novel benzimidazole-5-carbohydrazides using the
one-pot” nitro reductive cyclization method, which does
Vasantha et al. synthesized a series
3-nitrobenzoic acid as a starting material was treated
with a catalytic amount of concentrated H SO in ethanol
2
4
“
to yield ethyl-4-chloro-3-nitrobenzoate 1 (92%). Interme-
diate 1, upon treatment with n-propylamine as a nucleo-
phile and triethylamine as a base in dry THF at room
temperature, underwent a nucleophilic substitution reaction
to afford ethyl-3-nitro-4-(propylamino)benzoate 2 (82%).
Intermediate 2, under treatment with sodium dithionite and
substituted aldehyde, was allowed to undergo a one-pot
nitro reductive cyclization reaction to yield ethyl-2-phenyl-
1-propyl-1H-benzo[d]imidazole-5-carboxylate 3a and 3b in
89–90% yields. Esters 3a and 3b, upon treatment with
hydrazine hydrate, afforded 2-phenyl-1-propyl-1H-benzo[d]
imidazole-5-carbohydrazide 4a and 4b (80–81%). To the
not use reducing agents bearing metals such as Zn/HCl,
Sn/HCl, and Fe/HCl as these reagents are costly and
involve tedious workup. Instead, they have used one of
the cheapest available reducing agents, sodium
dithionite, which aids in reduction and cyclization.
Henceforth, this method is found to be more advanta-
[
19]
geous over the stepwise synthesis of benzimidazole.
Fused heterocycles have gained the attention of many
researchers in recent years as they are better pharmacologi-
cal agents. Heterocycles that have been derived from
1,2,4-triazoles have led to N-bridged heterocycles, which
play a vital role in pharmacological applications. Notice-
ably, triazolothiadiazines formed by the combination of
hydrazides (4a, b) in ethanol, KOH and CS were added
2
and refluxed for 8 hr to produce 5a and 5b in 80–81% yields,
which were then treated with hydrazine hydrate in ethanol
to yield 4-amino-5-(2-phenyl-1-propyl-1H-benzo[d]imidazol-
5-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6a and 6b in
50–55% yield. With these thione intermediates in hand,
substituted phenacyl bromide under refluxing ethanol could
achieve target compounds 7a-n in 75–90% yields.
1
,2,4-triazoles and 1,3,4-thiadiazines play a key role as
[20]
[21]
[22]
antiproliferative,
antimicrobial,
alkaline phosphatase inhibitors ; anti-inflammatory,
antihelmintic,
and PDE4 inhibitors.
antitubercular,
tubulin inhibitor,
[23]
acetyl- and butyryl-cholinesterases and
[24]
[25,26]
[27]
[28]
[29];
analgesic,
and molluscicidal agents
[
30]
1
Accounting for these factors, 14 novel [1,2,4]triazolo
The formation of compounds was confirmed by H
1
3
[
3,4-b][1,3,4]thiadiazine derivatives clubbed with benz-
NMR, C NMR, FT-IR, LCMS, and elemental ana-
lyses. The formation of intermediate 4-amino-5-(2-(3,
4-dimethoxyphenyl)-1-propyl-1H-benzo[d]imidazol-5-yl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (6a) was confirmed
imidazole in a single-molecular framework have been
designed and synthesized. All the structures were deter-
1
13
mined by H NMR, C NMR, FT-IR, mass, and elemen-
tal analyses. These derivatives have been subjected to
preliminary screening of antioxidant activity by three
methods such as DPPH radical scavenging activity, nitric
1
by H NMR spectrum, which showed a characteristic peak
at 5.83 ppm because of ─NH and peak at 13.6 ppm was
2
identified for N─H of the [1,2,4]-triazole ring. LCMS

SATHYANARAYANA ET AL.
3
SCHEME 1 The synthesis of target
compounds 7a-n. Reagents and conditions:
(
2 4
a) H SO , ethanol, 16 hr; (b) n-propylamine,
1
triethylamine, room temperature; (c) R -Ph-
ꢀ
CHO, sodium dithionite 90 C, 3 hr;
ꢀ
(
d) NH
2
NH
2
/EtOH, 90 C, 16 hr; (e) CS
2
/EtOH,
ꢀ
ꢀ
KOH 90 C, 8 hr; (f) NH
2
NH
2
/EtOH, 100 C,
2
ꢀ
8
hr; and (g) R -Ph-CH
2
COBr/EtOH 90 C, 4 hr
+
spectrum showed peak [M + 1] at 411.15 m/z, thus con-
firming its molecular mass. Taking 7f into consideration,
the propyl group. A further singlet at 4.55 ppm for S─CH2
confirmed the formation of [1,2,4]triazolo[3,4-b][1,3,4]
thiadiazine ring. The presence of a doublet at 7.29 ppm
(J = 8 Hz) was observed for H of 3,4-(MeO) C H due to
characteristic stretching frequencies of C═N and Ar─O N,
were observed at 1,604, 1,510 (N-O asymmetric stretch),
and 1,340 (N-O symmetric stretch) cm respectively. H
NMR spectrum confirmed the structure as the peaks at
2
5
2
6
4
−1
1
ortho coupling. Peaks at 7.48–7.50 ppm showed a doublet
for H of 3,4-(MeO) C H and H of benzimidazole with
6
2
6
4
7
5
.83 and 13.6 ppm attributed to ─NH and NH, respec-
coupling constant J = 8 Hz, which also represents ortho
coupling. The presence of a singlet was observed at
8.22 ppm for H of 3,4-(MeO) C H and H of benzimid-
2
tively, of the compound 6a disappeared in the compound
f. The two singlets were observed at 3.86 and 3.90 ppm
7
2
2
6
4
4
for ─MeO. The characteristic triplet at 0.83–0.87 ppm for
azole moiety. The chemical shift values at 8.25–8.28 and
C─Me, multiplet at 1.80–1.86 ppm for C─CH , and triplet
8.38–8.41 ppm showed two doublets attributed to H and
2
2
at 4.44–4.48 ppm for N─CH confirmed the presence of
H3 of 4-O N-phenyl, H6 of benzimidazole ring with
2
2

4
SATHYANARAYANA ET AL.
coupling constant J = 12 Hz, respectively. This was also
supported by C NMR spectrum, which showed charac-
in concentration, which is represented in Figure 1. The
results of the scavenging activity of the compounds 7a-n
and the standard (ascorbic acid) are expressed in IC50
and are presented in Table 2. From Table 2, it can be seen
that the compounds 7a-g showed good activity due to the
13
teristic peak at δ 154.7 assigned for S─C═N. LCMS
+
showed a peak [M + 1] at m/z 556.2, which matches its
molecular formula (Table 1, Appendix S1).
presence of 3,4-(MeO) when compared with 7h-n bear-
2
ing 2,3-Cl groups attached to the phenyl ring of benz-
2
2
.2 | Antioxidant activities and
imidazole. Among the compounds 7a-g, the best
scavenging activity was observed for the compound bear-
ing ─Me group, which is attached to the phenyl ring of
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (7c), which is bet-
ter than the standard ascorbic acid. The scavenging activ-
ity of the compounds 7g, 7f, 7a, and 7b decreased due to
the presence of electron-withdrawing groups such as F,
structure-active relationship analysis
2
.2.1 | DPPH radical scavenging activity
Benzimidazole derivatives donate labile electrons to
,1-diphenyl-2-picrylhydrazyl (DPPH) to form a diamagnetic
1
molecule. After accepting the electrons DPPH is converted
into a nonradical form (1,1-diphenyl-2-picrylhydrazine),
which can be identified spectrophotometrically by the
change in color from purple to yellow. The two well-known
mechanisms for antioxidants to scavenge DPPH is direct
hydrogen atom abstraction (Equation 1) and proton con-
NO , Cl, and Br in the order of 7g > 7f > 7a > 7b, respec-
2
tively; 7d showed moderate scavenging activity due to
the absence of a substituent group to the phenyl ring of
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine. Even though com-
pound 7e possessed electron-donating groups, it exhibited
lower scavenging activity compared to one of the com-
pounds bearing electron-withdrawing groups. The com-
pounds 7a-g showed better scavenging activity compared to
the standard ascorbic acid except for the compound 7d.
[32]
certed electron-transfer process (Equation 2).
ꢁ
DPPH + RXH ! DPPHH + RXꢁ
ð1Þ
The compounds (7h-n) possessing 2,3-(Cl) attached to
2
ꢁ
−
+
DPPH + RXH ! DPPH + RXHꢁ ! DPPHH + RXꢁ
ð2Þ
a phenyl ring of benzimidazole exhibited average to least
scavenging activity with IC50 values of 28.4–51.75 μg/ml
compared to standard. The compounds 7a-g were found to
interact with DPPH strongly may be due to the presence
of electron-donating groups attached to the phenyl ring of
benzimidazole moiety. The IC50 values for these com-
pounds were 13.57–24.95 μg/ml, while that of standard
was 23 μg/ml.
The radical scavenging activity was performed for the
synthesized compounds (7a-n) with varying concentra-
tions, namely, 10, 20, 30, 40, and 50 μg/ml. The results
indicated that scavenging activity increased with increase
1
2
ꢀ
TABLE 1 Physical data of 7a-n
Compound
R
R
Mol. formula
ClO
BrO
Mol. weight
544.14
588.09
524.20
510.18
540.19
555.17
528.17
596.00
552.05
536.08
548.10
532.10
518.08
563.07
M.p. ( C)
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
3,4-(MeO)
3,4-(MeO)
3,4-(MeO)
3,4-(MeO)
3,4-(MeO)
3,4-(MeO)
3,4-(MeO)
2
2
2
2
2
2
2
4-Cl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
28
28
29
28
29
28
28
26
26
26
27
27
26
26
H
H
H
H
H
H
H
H
H
H
H
H
H
H
25
25
28
26
28
25
25
19
19
19
22
22
20
19
N
N
N
N
N
N
N
N
N
N
N
N
N
N
6
6
6
6
6
7
6
6
6
6
6
6
6
7
2
S
213–214
218–220
180–181
224–225
195–196
238–240
235–236
203–204
194–195
198–200
178–179
218–219
130–131
213–214
4-Br
2
S
4-Me
4-H
O
O
2
2
S
S
4-MeO
3
FO S
4-O
2
N
4
O S
g
h
i
4-F
FO
2
S
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2
2
2
2
2
2
2
4-Br
4-Cl
4-F
BrCl
2
S
Cl
Cl
Cl
Cl
Cl
Cl
3
2
2
2
2
2
S
j
FS
OS
S
k
l
4-MeO
4-Me
4-H
m
n
S
4-O
2
N
2
O S

SATHYANARAYANA ET AL.
5
FIGURE 1 Graphical
representation of concentration
dependent percentage inhibition
DPPH radical scavenging activity
100
8
6
4
2
0
0
0
0
0
Compounds
1
0μg/ml
20μg/ml
30μg/ml
40μg/ml
50μg/ml
Nitric oxide scavenging activity
1
00
8
6
4
2
0
0
0
0
0
Compounds
1
0ꢀg/ml
20ꢀg/ml
30ꢀg/ml
40ꢀg/ml
50ꢀg/ml
Superoxide scavenging activity
1
00
8
6
4
2
0
0
0
0
0
Compounds
1
0ꢀg/ml
20ꢀg/ml
30ꢀg/ml
40ꢀg/ml
50ꢀg/ml
2.2.2 | Nitric oxide scavenging activity
with oxygen, leading to the reduction in the produc-
tion of nitrite ions.
Nitric oxide scavenging activity can be determined by
the Griess IlIosvoy method. The compound sodium
nitroprusside decomposes in aqueous solution produc-
The capacity to scavenge nitric oxide was determined
spectrophotometrically for the synthesized compound on
the basis of inhibition. The synthesized compound was
screened for five different concentrations, namely, 10, 20,
30, 40, and 50 μg/ml, using ascorbic acid as a standard.
Inhibition rate with respect to concentration is shown in
Figure 1. It is evident from the figure that, as the concen-
tration increases, the percentage of inhibition also
ꢁ
ing a nitric oxide radical (NO ) at physiological pH 7.2.
ꢁ
NO reacts with oxygen under aerobic conditions to
produce stable products nitrate and nitrite, the amount
of which can be estimated by Griess reagent. The
mechanism of the antioxidant scavenger is to compete

6
SATHYANARAYANA ET AL.
TABLE 2 The antioxidant activity of 7a-n
dissolved oxygen reduces nitro blue tetrazolium chloride
ꢁ
−
(NBT). The consumption of O2 by the antioxidant in the
Radical scavenging activity IC50 (μg/ml)
Compound DPPH Nitric oxide Super oxide
23.86 ± 7.25
reaction mixture can be determined spectroscopically.
Superoxide radical scavenging activity was also per-
formed in all the synthesized compounds 7a-n. The
obtained results are displayed in Table 2. Percentage inhi-
bition increased with increase in concentration (10, 20,
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
20.06 ± 1.07
24.95 ± 2.14
13.57 ± 1.3
25.7 ± 2.01
21.73 ± 1.65
19.67 ± 1.62
19.14 ± 2.4
28.4 ± 6.68
36.75 ± 9.32
51.45 ± 7.58
39.66 ± 6.53
39.83 ± 3.8
46.31 ± 7.71
40.88 ± 11.69
23.75 ± 4.36
30.65 ± 3.72
31.3 ± 4.24
30.4 ± 0.53
32.17 ± 4.26
34.52 ± 2.83
29.94 ± 1.8
29.98 ± 1.59
28.51 ± 2.62
29.74 ± 4.64
31.3 ± 0.94
35 ± 4.56
23.06 ± 1.16
18.55 ± 3.79
25.05 ± 2.17
20.86 ± 0.91
25.13 ± 3.99
24.21 ± 5.25
25.04 ± 4.39
38.73 ± 5.43
46.83 ± 4.17
35.26 ± 2.89
46.01 ± 6.88
37.93 ± 10.52
32.03 ± 5.64
23.14 ± 5.03
30, 40, and 50 μg/ml), which is shown in Figure 1. The
results showed that all the synthesized compounds were
least active compared to the standard.
g
h
i
2.2.4 | Structure-active relationship
analysis
j
The results highlighted that the presence of electron-
donating and electron-withdrawing groups in the mole-
cule implies a major role in the antioxidant activity. Inter-
estingly, the molecule with an electron-donating group
showed moderate to better activity, whereas the molecule
with electron-withdrawing group showed least activity
compared to standard. Of the 14 compounds synthesized,
k
l
30.87 ± 2.23
29.15 ± 5.4
29.71 ± 2.98
26.82 ± 2.65
m
n
a
SD
a
The superscript a corresponds to Ascorbic acid was used as a standard
compounds 7a-g had a 3,4-(MeO) group attached to the
2
antioxidant.
phenyl ring in the second position of benzimidazole mol-
Each value is expressed as mean ± SD of three replicates
ecule, whereas compounds 7h-n had a 2,3-Cl group. The
2
increased. The IC50 values of nitric oxide scavenging
activity of the synthesized compounds are given in
Table 2.
presence of an electron-donating group in compounds
7a-g were highly active for both DPPH and nitric oxide
scavenging assays compared to the standard. Further-
more, to check the variation in the activity of compounds
in 7a-g, the phenyl group attached to [1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine was substituted with both
electron-donating and electron-withdrawing groups. Fas-
The results of nitric oxide scavenging activity mim-
icked that of DPPH assay, and based on the results, we
can conclude that electron-donating groups plays a major
role in inhibiting nitric acid production. The screened
compounds showed that compounds 7c and 7e were very
effective in inhibiting nitric oxide production due to the
presence of an electron-donating group in the phenyl ring
of benzimidazole and [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine
moiety with IC50 values of 18.55 and 20.86 μg/ml, respec-
tively, which is considerably stronger than standard ascorbic
acid with IC50 values of 23.14 μg/ml. Compounds 7a and 7b
showed almost the same IC50 values as that of standard
ascorbic acid. A decrease in the scavenging activity was
observed for compounds 7h-n, which may be attributed to
the presence of electron-withdrawing groups in the phenyl
ring of benzimidazole.
2
cinatingly, when R was substituted with an electron-
donating group, activity was enhanced compared to when
2
R was an electron-withdrawing group. DPPH radical
scavenging activity is one of the quickest methods to
determine the antioxidant property of the compounds. Of
all synthesized compounds (7a-n), 7c showed high DPPH
1
scavenging activity, which demonstrated R = 3,4-(MeO)
2
2
2
and R = Me. When R was replaced with electron-
withdrawing groups, F, Cl, Br, and NO activity declined
2
when compared to 7c, but it was as potent as that of stan-
1
dard as it contained R = 3,4-(MeO) . However, compound
2
2
7e with R = MeO showed moderate activity when com-
pared to the compounds bearing R with electron-
withdrawing group, and the compound 7d with R = H
2
2
2
.2.3 | Superoxide radical scavenging
showed the least activity compared to that of standard.
However, in the case of nitric oxide radical scavenging
activity
2
activity, compound 7e with R = MeO was potent when
2
The superoxide radical scavenging activity can be estimated
compared to R as an electron-withdrawing group. For the
1
using the PMS–NADH–NBT system. The superoxide radical
compounds 7h-n, when R was 2,3-Cl , there was a drastic
2
ꢁ
−
(O2 ) produced in the reaction of N-methylphenazine
decrease in the DPPH and nitric oxide scavenging activity.
This explains the effect of substitutions for the radical
methosulphate (PMS) and NADH in the presence of

SATHYANARAYANA ET AL.
7
TABLE 3 Predicted Lipinski's parameter for 7a-n
a
b
Compounds
Molecular weight
≤500
Donor HB
Acceptor HB
log Po/w
#rotor
Rule of 5
Acceptable range
≤5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
≤10
6.0
6.0
6.0
6.0
6.75
7.0
6.0
4.5
4.5
4.5
5.25
4.5
4.5
5.5
(−2.0 to 6.5)
7.08
≤10
4
<5
2
2
2
2
2
3
2
2
2
2
2
2
2
2
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
544.14
588.09
7.18
4
524.02
6.19
4
510.18
6.59
4
540.19
6.65
5
555.17
5.83
5
g
h
i
528.17
6.83
4
596.00
7.83
2
552.05
7.77
2
j
536.08
7.50
2
k
l
548.1
7.33
3
532.10
7.63
2
m
n
518.08
7.26
2
563.07
6.55
3
a
Predicted octanol/water partition coefficient log P.
b
Predicted rotatable bonds.
scavenging activity of the synthesized compound. From
the superoxide radical scavenging activity, no inference
can be drawn as all the compounds showed moderate
activity.
compounds, 85% have PSA <80 Å, indicating that these
molecules have good intestinal absorption. Few factors
such as flexibility, shape, lipophilicity, and molecular size
are considered when determining liphophilicity. Molecu-
lar flexibility, in turn, depends on number of rotatable
bonds. The recommended range is <10 rotatable bonds.
All the synthesized derivatives have <5 rotatable bonds.
Drug-likeness parameter, indicated by #stars, determines
the number of properties that fall outside the 95% range
of similar values of known drugs. The molecular descrip-
tors calculated by Qikprop, such as MW, dipole, SASA,
IP, EA, PISA, FOSA, WPSA, FISA, PSA, volume, #rotor,
donorHB, accptHB, glob, QPpolrz, QPlogPw, QPlogPC16,
QPlogPoct, QPlogPo/w, QPLogKhsa, logS, QPlogBB, and
#metabo, are used to determine #star parameter. All the
synthesized compounds have #star <5, indicating these
molecules have properties similar to the known drugs.
The blood/brain coefficients (log B/B) parameter deter-
mines the access of a drug to the central nervous system
(CNS). Drugs that are overly polar fail to enter CNS. The
computed value for CNS active is +2 and −2 inactive.
The compounds 7f and 7n are CNS inactive. Madin-
Darby canine kidney (MDCK) is a good imitator of the
blood–brain barrier (BBB). This additional information
helps in predicting BBB penetration. QPPCaCO predicts
apparent Caco-2 cell permeability in nm/sec. Caco-2 cells
are a model for the gut–blood barrier. QikProp predic-
tions are made for nonactive transport. LogKHSA deter-
mines the binding efficiency of drug to human serum
albumin. The lesser the binding, the more efficiently
2.3 | Physiochemical properties
According to Lipinski's rule of five, the molecule should
have molecular weight ≤500, partition coefficient (log P)
≤
5, and a number of hydrogen bond donor and hydrogen
bond acceptor ≤5 and ≤10, respectively. Polar surface
area (PSA) ≤140 Å and molecular flexibility are also con-
sidered additional criteria to determine the oral bioavail-
[33]
ability.
In addition, percentage human oral absorption,
#
#
stars, CNS, QPPMDCK, QPHERG, QPlogKhsa, QlogKp,
metab, and QPPCaCO parameters have also been studied
and summarized in Tables 3 and 4.
Absorption of the drug candidate depends on perme-
ability and solubility, which in turn determines the bio-
availibity. All the synthesized compounds 7a-n have
good percentage human oral absorption >80%. As these
compounds have high percentage human oral absorption,
a violation of Lipinski's rule of five will not affect
[34]
bioavailabity.
The most potent compound, 7c, showed
excellent percentage human oral absorption (100%). PSA
is defined as the ability of a molecule to form hydrogen
bonds. This information correlates with intestinal absorp-
.
[35]
tion of drugs in humans
PSA >140 Å of a molecule is
considered to be poorly absorbed. Of the synthesized

8
SATHYANARAYANA ET AL.

SATHYANARAYANA ET AL.
9
the drug can diffuse. Compounds 7h-m fall outside the
recommended value, indicating it might bind strongly to
human serum albumin. Qikprop also estimates the num-
ber of possible metabolic reactions, which helps to pre-
dict whether the drug can easily reach the target site. It is
denoted by #metab, and 1–8 is the recommended range.
All the derivatives fall within the 2–5 range. LogKp deter-
mines the dermal penetration of the drug. The estimated
value for compounds 7a-n indicates that the drug can be
administrated dermally. The calculated IC50 of HERG K
(45.8 mmol) and triethylamine (45.8 mmol) were added
at room temperature to the above mixture and stirred
overnight at the same temperature. Completion of reac-
tion was monitored by thin-layer chromatography.
Excess solvent was removed under high vacuum, and the
residue left was added to ice cold water. Precipitate
formed was filtered, dried, and recrystallized from etha-
nol to yield intermediate 2 as a yellow solid. Yield:
9.5 g (82%).
[36]
+
determines the toxicity of cardiac drugs.
The rec-
ommended value is >−5. A value below −5 is considered
to be cardiac toxic. All molecules showed values above
3.3 | General procedure for the synthesis
of intermediate 2-phenyl-1-propyl-1H-
benzo[d]imidazole-5-carboxylate (3a, b)
−
5, which implies that these molecules are nontoxic.
To a stirred solution of intermediate 2 (5 g, 19.8 mmol) in
dry dimethyl sulfoxide solvent (50 ml), appropriate alde-
hyde (3.4 g, 19.8 mmol) and sodium dithionite (6.8 g,
3
| EXPERIMENTAL
ꢀ
The reagents used for the synthesis of this series were of
analytical grade and were purchased from S. D. Fine,
Sigma-Aldrich, and Spectrochem, which were used
directly without further purification. The melting point
was determined by the open capillary method and was
uncorrected. Bruker Avance III, 400 MHz was used to
record H NMR and C NMR spectra. The chemical shift
values were stated in parts per million (ppm) and coupling
constants in Hertz (Hz). TMS is used as internal standard.
FT (ATR)-IR absorption spectra were recorded on Thermo
Nicolet, Avatar 370 in the range of 4,000–400 cm . Mass
spectra were recorded on Waters, synapt G2 high-detection
mass spectrometry and are uncorrected. Thin-layer chro-
matography on a silica-coated aluminum sheet was used
to check the completion of reaction and the purity.
Ethylacetate and hexane was used as the mobile phase.
39.6 mmol) were added and heated to 90 C with stirring
for 3 hr. After completion of the reaction, reaction mass
was diluted with EtOAc (250 ml) and given water washes
(2 × 100 ml). The organic layer was separated, dried over
sodium sulfate, and evaporated under high vacuum. The
solid formed was recrystallized from ethanol to afford
intermediate 3a, b as off-white solid. Yield: 6.5 g (89%).
1
13
3.4 | General procedure for the synthesis
of 2-phenyl-1-propyl-1H-benzo[d]imidazole-
5-carbohydrazide (4a, b)
−1
Hydrazine hydrate 98% (20 ml) was added to the solution of
intermediate 3a-b (6.5 g, 17.6 mmol) in ethanol and heated to
reflux for 16 hr. Excess solvent was evaporated under high
vacuum. The obtained intermediate 4a was directly taken to
the next step, whereas for intermediate 4b, the reaction mix-
ture was added to ice cold water. The precipitate formed was
filtered, dried, and recrystallized using ethanol.
3
3
.1 | Synthesis of ethyl-4-chloro-
-nitrobenzoate (1)
A catalytic amount of concentrated H SO was added to a
2
4
solution of 4-chloro-3-nitrobenzoic acid (10 g, 49.6 mmol)
in ethanol (100 ml) and heated to reflux for 16 hr. After
completion of reaction, excess solvent was removed under
high vacuum, and the residue was poured into ice water.
The precipitate formed was filtered and dried to afford
intermediate 1 as white solid. Yield: 10.5 g (92%).
3.4.1 | 2-(3,4-Dimethoxyphenyl)-1-propyl-
1H-benzo[d]imidazole-
5-carbohydrazide (4a)
1
Brown gum; yield: 5.0 g (81%); H NMR (400 MHz,
DMSO-d ): δ = 1.08–1.11 (t, 3H, J = 12 Hz, Me),
6
2
.12–2.15 (m, 2H, C─CH ), 3.70 (s, 3H, MeO), 3.86 (s, 3H,
2
MeO), 4.42–4.46 (m, 2H, N─CH ), 4.51 (s, 2H, NH ),
2
2
3
4
.2 | Synthesis of ethyl-3-nitro-
-(propylamino)benzoate (2)
6.94–6.96 (m, 1H, H of 3,4-(MeO) C H ), 7.14–7.28 (m,
5
2
6
4
2H, H of 3,4-(MeO) C H and H of benzimidazole),
2 2 6 4 6
7
.34–7.35 (d, 1H, J = 4 Hz, H6 of 3,4-(MeO) C H ),
2
6
4
Ethyl-4-chloro-3-nitrobenzoate (10.5 g, 45.8 mmol) was
taken in dry tetrahydrofuran solvent. Propylamine
7.76–7.78 (d, 1H, J = 8 Hz, H of benzimidazole), 7.93 (s,
1H, H4 of benzimidazole), 9.75 (s, 1H, NH). MS:
7

10
SATHYANARAYANA ET AL.
+
calculated for C H N O is 354.17; found [M + 1]
calc. For C H N O S: C, 60.59; H, 5.08; N, 14.13%.
19
22
4
3
20 20 4 3
3
6
55.10 m/z; Anal.calc. For C H N O : C, 64.39; H,
.26; N, 15.81%. Found: C, 64.30; H, 6.24; N, 15.78%.
Found: C, 60.50; H, 4.99; N, 14.10%.
19
22 4 3
3
.5.2 | 5-(2-(2,3-Dichlorophenyl)-
3
1
5
.4.2 | 2-(2,3-Dichlorophenyl)-1-propyl-
H-benzo[d]imidazole-
-carbohydrazide (4b)
1-propyl-1H-benzo[d]imidazol-5-yl)-
1,3,4-oxadiazole-2(3H)-thione (5b)
ꢀ
Off-white solid; yield: 4.5 g (80%); melting point ( C):
ꢀ
1
Off-white solid; yield: 5.0 g (80%); melting point ( C):
224–225 (EtOH);
H NMR (400 MHz, DMSO-d6):
1
1
90–192 (EtOH);
H
NMR (400 MHz, DMSO-d6):
δ = 0.76–0.79 (t, 3H, J = 12 Hz, Me), 1.70–1.75 (m, 2H,
C─CH ), 4.31–4.35 (m, 2H, N─CH ), 7.13–7.16 (m, 1H,
δ = 1.06–1.09 (t, 3H, J = 12 Hz, Me), 2.13–2.16 (m, 2H,
C─CH ), 4.50–4.55 (m, 4H, N─CH and NH ), 7.19–7.21
2
2
H of 2,3-(Cl) C H ), 7.38–7.42 (m, 1H, H of 2,3-(Cl) C H ),
2
2
2
4
2
6
4
5
2 6 4
(
m, 1H, H of 2,3-(Cl) C H ), 7.39–7.43 (m, 1H, H of
7.58–7.61 (m, 2H, H and H of benzimidazole), 7.68–7.70
4
2
6
4
5
4
6
2
7
7
8
1
,3-(Cl) C H ), 7.47–7.50 (d, 1H, H of benzimidazole),
(d, 1H, J = 8 Hz, H of benzimidazole), 7.79–7.81 (d, 1H,
2
6
4
7
7
.66–7.69 (d, 1H, J = 12 Hz, H6 of 2,3-(Cl) C H ),
J = 8 Hz, H6 of 2,3-(Cl) C H ). MS: calculated for
2
6
4
2 6 4
+
+
.73–7.76 (d, 1H, J = 12 Hz, H6 of benzimidazole),
C H C N OS is 404.03; found [M + 1] 405.00, [M + 3]
18 14 l2 4
.08–8.09 (1H, J = 4 Hz, H of benzimidazole), 9.80 (s,
407.00 m/z; Anal.calc. For C H C N OS: C, 53.34; H,
18 14 l2 4
3.48; N, 13.82%. Found: C, 53.25; H, 3.40; N, 13.80%.
4
H, NH). MS: calculated for C H Cl N O is 362.07;
17
+
16
2 4
+
found [M + 1] 363.05, [M + 3] 365.01 m/z; Anal.calc.
For C H Cl N O: C, 56.21; H, 4.44; N, 15.42%.
17
16
2 4
Found: C, 56.19; H, 4.40; N, 15.40%.
3.6 | General procedure for the synthesis
of 4-amino-5-(2-phenyl-1-propyl-1H-benzo
[
d]imidazol-5-yl)-2,4-dihydro-3H-
3.5 | General procedure for the synthesis
1,2,4-triazole-3-thione (6a, b)
of 5-(2-phenyl-1-propyl-1H-benzo[d]
imidazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione
To a solution of intermediate 5a-b (4.5 g, 11.35 mmol)
in ethanol (40 ml), 98% hydrazine hydrate (10 ml) was
added and heated to reflux for 5 hr. Excess solvent
was evaporated. The reaction mixture was diluted
with EtOAc (250 ml) and given water washes
(2 × 100 ml). The organic layer was separated from
aqueous layer, dried over sodium sulfate, and evapo-
rated under high vacuum to yield intermediate 6a, b
as off-white solid.
(5a, b)
To a solution of intermediate 4a, b (5 g, 14.11 mmol) in eth-
anol (50 ml), potassium hydroxide (1.1 g, 21.1 mmol) was
added and stirred well to form a clear solution. Carbon
disulfide (1.8 ml, 28.2 mmol) was added and heated to reflux
for 8 hr. The reaction mixture was then added to ice cold
water and acidified using 10% dil.HCl to pH = 6. The pre-
cipitate formed was filtered, dried, and recrystallized from
ethanol to yield intermediate 5a, b as an off-white solid.
3
5
.6.1 | 4-Amino-
-(2-(3,4-dimethoxyphenyl)-1-propyl-1H-
3.5.1 | 5-(2-(3,4-Dimethoxyphenyl)-
1-propyl-1H-benzo[d]imidazol-5-yl)-
1,3,4-oxadiazole-2(3H)-thione (5a)
benzo[d]imidazol-5-yl)-2H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (6a)
ꢀ
Off-white solid; yield: 2.3 g (50%); melting point ( C):
ꢀ
1
Off-white solid; yield: 4.5 g (81)%; melting point ( C):
260–261 (EtOH); H NMR (400 MHz, DMSO-d6):
1
250–251 (EtOH);
H
NMR (400 MHz, DMSO-d6):
δ = 0.75–0.76 (t, 3H, J = 4 Hz, Me), 1.69–1.75 (m, 2H,
δ = 0.75–0.78 (t, 3H, J = 12 Hz, Me), 1.69–1.74 (m, 2H,
C─CH ), 3.83 (s, 3H, MeO), 3.85 (s, 3H, MeO),
2
C─CH ), 3.83 (s, 3H, MeO), 3.85 (s, 3H, MeO), 4.30–4.34
4.30–4.33 (m, 2H, N─CH ), 5.83 (s, 2H, ─NH ),
2
2
2
(
m, 2H, N─CH ), 7.14–7.16 (m, 1H, H of 3,4-(MeO) C H ),
7.13–7.16 (d, 1H, J = 12 Hz H of 3,4-(MeO) C H ),
2
5
2
6
4
5
2
6
4
7
.33–7.34 (m, 2H, H of 3,4-(MeO) C H and H of benz-
7.33–7.35 (d, 2H, J = 8 Hz, H of 3,4-(MeO) C H and
2
2
6
4
6
2
2
6
4
imidazole), 7.78–7.80 (d, 1H,
J
=
8
Hz, H6 of
H of benzimidazole), 7.76–7.78 (d, 1H, J = 8 Hz, H
6
6
3
,4-(MeO) C H ), 7.86–7.88 (d, 1H, J = 8 Hz, H of benz-
imidazole), 8.12 (s, 1H, H of benzimidazole). MS: calculated
for C H N O S is 396.1; found [M + 1] 397.1 m/z; Anal.
of 3,4-(MeO) C H ), 7.84–7.87 (d, 1H, J = 12 Hz, H of
2
6
4
7
2
6
4
7
benzimidazole), 8.42 (s, 1H, H4 of benzimidazole),
4
+
13.6 (s, 1H, N-H) ppm. MS: calculated for
20 20 4 3

SATHYANARAYANA ET AL.
11
+
C H N O S is 410.15; found [M + 1] 411.15 m/z;
H of 3,4-(MeO) C H ), 7.85–7.92 (m, 2H, H and H
2
0
22
6
2
6
2
6
4
2
6
Anal.calc. For C H N O S: C, 58.52; H, 5.40; N,
of 4-Cl-C H ), 8.19 (s, 2H, H of 3,4-(MeO) C H and
2
0
22
6
2
6 4 2 2 6 4
2
0.47%. Found: C, 58.50; H, 5.38; N, 20.45%.
H4 of benzimidazole), 8.35 (s, 1H, H of benzimid-
7
1
3
azole) ppm; C NMR (100 MHz): δ = 156.8, 152.5,
48.5, 148.0, 147.7, 132.5, 132.1, 131.4, 131.1,
1
3
.6.2 | 4-Amino-
-(2-(2,3-dichlorophenyl)-1-propyl-1H-benzo
129.4,128.8, 125.7, 123.7, 112.3, 55.88 (MeO), 55.80
5
(MeO), 46.8 (N─CH ─CH ─CH ), 23.7 (CH ─S), 22.3
2
2
3
2
[
d]imidazol-5-yl)-2,4-dihydro-3H-
,2,4-triazole-3-thione (6b)
(N─CH ─CH ─CH ), 10.5 (N─CH ─CH ─CH ); MS:
2 2 3 2 2 3
1
calculated for C H N ClO S is 544.1; found [M + 1]
28 25 6 2
+
5
45.0, [M + 3] 547.1 m/z; Anal.calc. For C H N ClO S: C,
28 25 6 2
ꢀ
Off-white solid; yield: 2.5 g (55%); melting point ( C):
61.70; H, 4.62; N, 15.42%. Found: C, 61.69; H,
4.61; N, 15.87%.
1
2
48–250 (EtOH);
H NMR (400 MHz, DMSO-d6):
δ = 0.76–0.79 (t, 3H, J = 12 Hz, Me), 1.70–1.75 (m, 2H,
C─CH ), 4.31–4.35 (m, 2H, N─CH ), 5.85 (s, 2H, ─NH ),
2
2
2
7
.13–7.16 (m, 1H, H of 2,3-(Cl) C H ), 7.38–7.42 (m, 1H,
3.7.2 | 6-(4-Bromophenyl)-
4
2
6
4
H of 2,3-(Cl) C H ), 7.58–7.61 (m, 2H, H and H of
3-(2-(3,4-dimethoxyphenyl)-1-propyl-1H-
benzo[d]imidazol-5-yl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (7b)
5
2
6
4
4
6
benzimidazole), 7.68–7.70 (d, 1H, H of benzimidazole),
7
7
.79–7.81 (d, 1H, J = 8 Hz, H of 2,3-(Cl) C H ), 12.9 (s,
6
2
6
4
1
H, N-H) ppm MS: calculated for C H C N S is 418.05;
18
16 l2 6
+
+
ꢀ
found [M + 1] 419.00, [M + 3] 421.00 m/z; Anal.calc.
Off-white solid; yield: 260 mg (90%); melting point ( C):
For C H C N OS: C, 51.56; H, 3.85; N, 20.04%.
Found: C, 51.50; H, 3.80; N, 20.00%.
218–220 (EtOH); FT-IR (ATR, υmax/cm): 2,970, 2,939,
18
14 l2 4
(C─H), 1,604, 1,510 (C═N), 1,267, 1,228 (C─O), 1,020
1
(
C─S), 650 (C─Br); H NMR (400 MHz, DMSO-d6):
δ = 0.83–0.87 (t, 3H, J = 16 Hz, Me), 1.78–1.84 (m, 2H,
3
.7 | General procedure for the synthesis
C─CH ), 3.87 (s, 3H, MeO), 3.89 (s, 3H, MeO), 4.43–4.45
2
of target compounds 7a-n
(m, 4H, N─CH and S─CH ), 7.28–7.30 (d, 1H, J = 8 Hz
2
2
H of 3,4-(MeO) C H ), 7.49–7.58 (m, 4H, H and H
5
2
6
4
3
5
To a solution of intermediate 6a, b (0.2 g, 4.8 mmol) in
of 4-Br-C H , H6 of benzimidazole and H6 of
6 4
ethanol (5 ml), appropriate phenacyl bromide (0.11 g,
3,4-(MeO) C H ), 7.95–8.05 (m, 2H, H and H of 4-Br-
6 4 2 2 6 4 4
2
6
4
2
6
4
.8 mmol) was added and heated to reflux for 4 hr. Reac-
C H ), 8.16 (s, 2H, H of 3,4-(MeO) C H and H of
tion mass was added to ice cold water. The precipitate
formed was filtered and dried. For the final compounds
benzimidazole), 8.30 (s, 1H, H of benzimidazole) ppm;
7
C NMR (100 MHz): δ = 155.7, 153.8, 150.8, 149.9,
1
3
7
a-g, purification was carried out by recrystallization
149.2, 148.3, 131.3, 130.8, 128.9, 126.2, 123.1, 122.0,
116.4, 115.1, 112.5, 111.7, 110.0, 55.90 (MeO), 55.70
(MeO), 46.70 (N─CH ─CH ─CH ), 23.5 (CH ─S), 22.1
from ethanol, whereas for compounds 7h-n, purification
was performed by column chromatography. The com-
pounds were eluted in 5% MeOH: DCM system.
Yield: 75–90%.
2
2
3
2
(N─CH ─CH ─CH ), 10.5 (N─CH ─CH ─CH ); MS:
2
2
3
2
2
3
+
calculated for C H N BrO S is 590.09; found [M + 1]
2
8
25
+
6
2
5
91.08,
[M + 3]
593.03 m/z;
Anal.calc.
For
C H N BrO S: C, 57.05; H, 4.27; N, 14.26%. Found: C,
28
25
6
2
3
3
.7.1 | 6-(4-Chlorophenyl)-
-(2-(3,4-dimethoxyphenyl)-1-propyl-1H-
57.03; H, 4.25; N, 14.25%.
benzo[d]imidazol-5-yl)-7H-[1,2,4]triazolo
3,4-b][1,3,4]thiadiazine (7a)
[
3.7.3 | 3-(2-(3,4-Dimethoxyphenyl)-
1
-propyl-1H-benzo[d]imidazol-5-yl)-
ꢀ
Off-white solid; yield: 240 mg (90%); melting point ( C):
6-4-tolyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
2
13–214 (EtOH); FT-IR (ATR, υmax/cm): 3003, 2,970
thiadiazine (7c)
(
(
C─H), 1,605, 1,503 (C═N), 1,220, 1,265 (C─O), 1,022
C─S), 817 (C─Cl); H NMR (400 MHz, DMSO-d6):
1
ꢀ
Off-white solid; yield: 220 mg (88%); melting point ( C):
180–181 (EtOH); FT-IR (ATR, υmax/cm): 2,975, 2,950,
δ = 0.81–0.85 (t, 3H, J = 16 Hz, Me), 1.78–1.84 (m, 2H,
C─CH ), 3.86 (s, 3H, MeO), 3.89 (s, 3H, MeO),
(C─H), 1,598, 1,515 (C═N), 1,256, 1,230 (C─O), 1,040
2
1
4
.43–4.45 (m, 4H, N─CH2 and S─CH ), 7.26–7.28
(C─S); H NMR (400 MHz, DMSO-d ): δ = 0.80–0.84 (t,
2
6
(
d, 1H, J = 8 Hz, H of 3,4-(MeO) C H ), 7.50–7.61 (m,
3H, J = 16 Hz, Me), 1.75–1.81 (m, 2H, C─CH ), 1.93 (s,
3H, Me), 3.85 (s, 3H, MeO), 3.84 (s, 3H, MeO), 4.40–4.42
5
2
6
4
2
4
H, H and H of 4-Cl-C H , H of benzimidazole and
3
5
6
4
6

12
SATHYANARAYANA ET AL.
(
m, 4H, N─CH and S─CH ), 7.29–7.31 (d, 1H, J = 8 Hz
MeO), 3.90 (s, 3H, MeO), 4.41–4.43 (m, 4H, N─CH and
2
2
2
H of 3,4-(MeO) C H ), 7.51–7.60 (m, 4H, H and H
of 4-Me-C H , C6 of benzimidazole and H6 of
3
C H ), 8.16 (s, 2H, H of 3,4-(MeO) C H and H of benz-
imidazole), 8.30 (s, 1H, H of benzimidazole) ppm;
NMR (100 MHz): δ = 153.3, 151.5, 150.8, 149.9, 149.2,
S─CH2), 7.30–7.32 (d, 1H,
J
= 8 Hz H5 of
5
2
6
4
3
5
3,4-(MeO) C H ), 7.39–7.48 (m, 4H, H and H of 4-MeO-
6
4
2
6
4
3
5
,4-(MeO) C H ), 7.90–8.00 (m, 2H, H and H of 4-Me-
C H , H of benzimidazole and H of 3,4-(MeO) C H ),
2
6
4
2
6
6 4 6 6 2 6 4
7.89–7.99 (m, 2H, H and H of 4-MeO-C H ), 8.15 (s, 2H,
6
4
2
2
6
4
4
2
6
6
4
13
C
H of 3,4-(MeO) C H and H of benzimidazole), 8.28
7
2 2 6 4 4
1
3
(s, 1H, H7 of benzimidazole) ppm;
C NMR
1
1
48.3, 139.8, 134.3, 132.6, 129.3, 126.8, 126.2, 125.2, 116.0,
12.7, 111.0, 55.60 (MeO), 54.9 (MeO), 49.6
(100 MHz): δ = 158.3, 153.2, 151.5, 149.3, 149.0, 148.3,
138.4, 135.2, 131.3, 130.1, 126.4, 126.0, 125.1, 115.9,
112.5, 110.9, 56.8 (MeO), 56.2 (MeO), 55.5 (Ar─OMe),
50.6 (N─CH ─CH ─CH ), 29.7 (CH ─S), 20.9
(N─CH ─CH ─CH ), 29.7 (CH ─S), 21.3 (Ar─Me), 20.9
2 2 3 2
(N─CH ─CH ─CH ), 11.5 (N─CH ─CH ─CH ); MS: cal-
2
2
3
2
2
3
2
2
3
2
+
culated for C H N O S is 524.2; found [M + 1]
5
5
(N─CH ─CH ─CH ), 11.5 (N─CH ─CH ─CH ); MS:
29
28
6
2
2 2 3 2 2 3
+
25.1 m/z; Anal.calc. For C H N O S: C, 66.39; H,
calculated for C H N O S is 524.2; found [M + 1]
29 28 6 2
29
28
6
2
.38; N, 16.02%. Found: C, 66.38; H, 5.36; N, 16.03%.
525.1 m/z; Anal.calc. For C H N O S: C, 64.43; H,
29 28 6 3
5
.22; N, 15.54%. Found: C, 64.42; H, 5.21; N, 15.53%.
3
1
6
.7.4 | 3-(2-(3,4-Dimethoxyphenyl)-
-propyl-1H-benzo[d]imidazol-5-yl)-
-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
3.7.6 | 3-(2-(3,4-Dimethoxyphenyl)-
1-propyl-1H-benzo[d]imidazol-5-yl)-
6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b]
thiadiazine (7d)
[
1,3,4]thiadiazine (7f)
ꢀ
Off-white solid; yield: 210 mg (85%); melting point ( C):
ꢀ
2
24–225 (EtOH); FT-IR (ATR, υmax/cm): 3,001, 2,969
Pale yellow; yield: 220 mg (81%); melting point ( C):
238–240 (EtOH); FT-IR (ATR, υmax/cm): 2,970, 2,939
(
(
3
C─H), 1,604, 1,510 (C═N), 1,267, 1,228 (C─O), 1,020
C─S); H NMR (400 MHz, DMSO-d ): δ = 0.86–0.90 (t,
1
(C─H), 1,604 (C═N), 1,510, 1,340 (Ar─O N), 1,267, 1,228
(C─O), 1,020 (C─S); H NMR (400 MHz, DMSO-d6):
6
2
1
H, J = 16 Hz, Me), 1.80–1.86 (m, 2H, CH ), 3.87 (s, 3H,
2
MeO), 3.90 (s, 3H, MeO), 4.44–4.48 (t, 2H, N─CH ), 4.55
δ = 0.83–0.87 (t, 3H, J = 16 Hz, Me), 1.80–1.86 (m, 2H,
2
(
3
s, 2H, S─CH ), 7.27–7.29 (d, 1H, J = 8 Hz H5 of
CH ), 3.86 (s, 3H, MeO), 3.90 (s, 3H, MeO), 4.44–4.48
2
2
,4-(MeO) C H ), 7.39–7.47 (m, 5H, H H and H of
(t, 2H, N─CH ─), 4.55 (s, 2H, S─CH ) 7.28–7.30 (d, 1H,
2
6
4
3,
4
5
2
2
C H , H of benzimidazole and H of 3,4-(MeO) C H ),
J = 8 Hz, H5 of 3,4-(MeO) C H ), 7.48–7.50 (d, 2H,
6
5
6
6
2
6
4
2
6
4
7
3
.83–7.94 (m, 2H, H and H of C H ), 8.15 (s, 2H, H of
J = 8 Hz, H of 3,4-(MeO) C H and H of benzimidazole),
2
6
6
5
2
6 2 6 4 7
,4-(MeO) C H and H of benzimidazole), 8.30 (s, 1H,
8.22 (s, 2H, H of 3,4-(MeO) C H and H of benzimid-
2 2 6 4 4
2
6
4
4
13
H7 of benzimidazole) ppm;
δ = 156.0, 150.6, 149.2, 149.2, 148.9, 147.5, 133.0, 132.7,
1
5
C NMR (100 MHz):
azole), 8.25–8.28 (d, 2H, J = 12 Hz, H and H of 4-O N-
2
6
2
C H ), 8.38–8.41 (d, 3H, J = 12 Hz, H , H of 4-O N-C H
6
4
3
5
2
6
4
13
31.3, 130.9, 127.2, 124.1, 122.5, 115.2, 110.8, 54.0 (MeO),
and H of benzimidazole) ppm; C NMR (100 MHz):
6
3.9 (MeO) 46.5 (N─CH ─CH ─CH ), 30.1 (CH ─S), 21.3
δ = 154.7, 152.0, 151.9, 151.2, 149.1, 148.9, 142.7, 139.3,
134.0, 129.0, 124.8, 124.0, 123.2, 114.6, 113.5, 112.7, 112.0,
55.86 (MeO), 55.83 (MeO), 46.8 (N─CH ─CH ─CH ),
2
2
3
2
(N─CH ─CH ─CH ), 11.0 (N─CH ─CH ─CH ); MS: cal-
2 2 3 2 2 3
+
culated for C H N O S is 510.18; found [M + 1]
28
26
6
2
2
2
3
5
5
11.15 m/z; Anal.calc. For C H N O S: C, 65.86; H,
.13; N, 16.46%. Found: C, 65.85; H, 5.12; N, 16.43%.
23.0 (CH ─S), 22.1 (N─CH ─CH ─CH ), 10.7
28
26
6
2
2 2 2 3
(N─CH ─CH ─CH ); MS: calculated for C H N FO S
2 2 3 28 25 7 4
+
is 555.6; found [M + 1] 556.2 m/z; Anal.calc. For
C H N FO S: C, 60.53; H, 4.54; N, 17.65%. Found: C,
2
8
25
7
4
3
1
6
.7.5 | 3-(2-(3,4-Dimethoxyphenyl)-
-propyl-1H-benzo[d]imidazol-5-yl)-
-(4-methoxyphenyl)-7H-[1,2,4]triazolo
60.50; H, 4.53; N, 17.63%.
[3,4-b][1,3,4]thiadiazine (7e)
3.7.7 | 3-(2-(3,4-Dimethoxyphenyl)-
1
-propyl-1H-benzo[d]imidazol-5-yl)-
ꢀ
Off-white solid; yield: 230 mg (88%); melting point ( C):
95–196 (EtOH); FT-IR (ATR, υmax/cm): 3,002, 2,986
C─H), 1,598, 1,515 (C═N), 1,256, 1,230, 1,228 (C─O),
6-(4-fluorophenyl)-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazine (7g)
1
(
1
ꢀ
1
,040 (C─S);
H
NMR (400 MHz, DMSO-d6):
Off-white solid; yield: 200 mg (80%); melting point ( C):
δ = 0.81–0.85 (t, 3H, J = 16 Hz, Me), 1.75–1.81 (m, 2H,
235–236 (EtOH); FT-IR (ATR, υmax/cm): 3,003, 2,970
(C─H), 1,602, 1,508 (C═N), 1,224, 1,265 (C─O), 1,022
C─CH ), 1.93 (s, 3H, Me), 3.85 (s, 3H, MeO), 3.84 (s, 3H,
2

SATHYANARAYANA ET AL.
13
1
(
C─S), 1,141 (C─F); H NMR (400 MHz, DMSO-d6):
132.7, 132.5, 132.1, 131.4, 131.1, 129.4,128.8, 125.7, 123.7,
δ = 0.82–0.86 (t, 3H, J = 16 Hz, Me), 1.79–1.85 (m, 2H,
112.3, 46.0 (N─CH ─CH ─CH ), 22.7 (CH ─S), 22.3
2
2
3
2
C─CH ), 3.86 (s, 3H, MeO), 3.89 (s, 3H, MeO), 4.45–4.47
(N─CH ─CH ─CH ), 10.8 (N─CH ─CH ─CH ); MS: calcu-
2
2 2 3 2 2 3
+
(
m, 4H, N─CH and S─CH ), 7.27–7.29 (d, 1H, J = 8 Hz
lated for C H N Cl S is 552.08; found [M + 1] 553.1, [M
26 19 6 3
2
2
+
+
H of 3,4-(MeO) C H ), 7.41–7.49 (m, 4H, H and H of
4
+ 5]
557.0, [M + 7]
559.0 m/z; Anal.calc. For
5
2
6
4
3
5
-F-C H , H6 of Benzimidazole and H6 of
C H N Cl S: C, 56.38; H, 3.46; N, 15.17%. Found: C,
6 4
26 19
6
3
3
,4-(MeO) C H ), 8.08–8.09 (m, 2H, H and H of 4-F-
56.35; H, 3.45; N, 15.15%.
2
6
4
2
6
C H ), 8.20 (s, 2H, H of 3,4-(MeO) C H and H of Benz-
imidazole), 8.37 (s, 1H, H of benzimidazole) ppm;
6
4
2
2
6
4
4
13
C
7
NMR (100 MHz): δ = 163.0, 155.4, 152.0, 151.9, 148.9,
3.7.10 | 3-(2-(2,3-Dichlorophenyl)-
1-propyl-1H-benzo[d]imidazol-5-yl)-
6-(4-fluorophenyl)-7H-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazine (7j)
1
1
4
42.8, 130.3, 130.2, 129.9, 124.7, 123.2, 123.0, 116.3,
16.1, 113.5, 112.7, 112.0, 55.85 (MeO), 55.83 (MeO),
6.8 (N─CH ─CH ─CH ), 22.8 (CH ─S), 22.1 (N─CH ─
2
2
3
2
2
CH ─CH ), 10.7 (N─CH ─CH ─CH ); MS: calculated for
2
3
2
2
3
+
C H N FO S is 528.1; found [M + 1] 529.2 m/z; Anal.
Light brown solid; yield: 200 mg (77%); melting point
28
25
6
2
ꢀ
calc. For C H N FO S: C, 63.62; H, 4.77; N, 15.90%.
Found: C, 63.60; H, 4.75; N, 15.89%.
( C): 198–200; FT-IR (ATR, υ /cm): 1,582 (C═N),
28
25
6
2
max
1
1,008 (C─S), 1,139 (C─F), 773, 803 (C─Cl); H NMR
(
400 MHz, DMSO-d ): δ = 0.70–0.73 (t, 3H, J = 12 Hz,
6
Me), 1.62–1.67 (m, 2H, C─CH ), 4.10 (s, 2H, N─CH ),
2
2
3
3
.7.8 | 6-(4-Bromophenyl)-
-(2-(2,3-dichlorophenyl)-1-propyl-1H-benzo
4.43 (s, 2H, S─CH ), 7.63–7.79 (m, 4H, Ar─H),
2
13
7.96–8.11 (m, 5H, Ar─H), 8.38 (s, 1H, Ar─H) ppm;
C
[
[
d]imidazol-5-yl)-7H-[1,2,4]triazolo[3,4-b]
1,3,4]thiadiazine (7h)
NMR (100 MHz): δ = 160.3, 153.6, 145.4, 134.1, 133.5,
132.8, 132.5, 131.2, 130.1, 129.0,128.0, 126.7, 127.4, 113.3,
46.5
(N─CH ─CH ─CH ),
22.9
(CH ─S),
22.0
2
2
3
2
ꢀ
Off-white solid; yield: 240 mg (83%); melting point ( C):
03–204; FT-IR (ATR, υmax/cm): 1,585 (C═N), 1,004
(N─CH ─CH ─CH ), 10.6 (N─CH ─CH ─CH ); MS: cal-
2
2
3
2
2
3
+
2
culated for C H N Cl FS is 536.08; found [M + 1]
26 19 6 2
537.05, [M + 3] 539.08, [M + 5] 541.06 m/z; Anal.calc.
1
+
+
(
C─S), 705, 723, 804 (C─Cl), 559 (C─Br); H NMR
(
1
2
400 MHz, DMSO-d ): δ = 0.71–0.75 (t, 3H, J = 16 Hz, Me),
For C H N Cl FS: C, 58.10; H, 3.56; N, 15.64%.
Found: C, 58.05; H, 3.54; N, 15.60%.
6
26 19
6
2
.64–1.70 (m, 2H, C─CH ), 4.13–4.16 (s, 2H, N─CH ), 4.46 (s,
2
2
H, S─CH ), 7.61–7.75 (m, 1H, Ar─H), 7.75–7.81 (m, 3H,
2
Ar─H), 7.94–7.97 (m, 3H, Ar─H), 8.01–8.09 (m, 2H, Ar─H),
13
8
1
1
.37 (s, 1H, Ar─H) ppm; C NMR (100 MHz): δ = 152.3,
49.8, 142.5, 132.5, 131.9, 131.1, 131.0, 130.0, 129.7129.2,
28.5, 124.5, 123.2, 112.3, 46.2 (N─CH ─CH ─CH ), 22.5
3.7.11 | 3-(2-(2,3-Dichlorophenyl)-
1-propyl-1H-benzo[d]imidazol-5-yl)-
6-(4-methoxyphenyl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine (7k)
2
2
3
(CH ─S), 22.2 (N─CH ─CH ─CH ), 10.3 (N─CH ─CH ─
2 2 2 3 2 2
CH ); MS: calculated for C H N BrCl S is 596.00; found
3
26 19
6
2
+
+
+
[M + 1] 597.0, [M + 3] 599.0, [M + 7] 603.0 m/z; Anal.
Light brown solid; yield: 200 mg (80%); melting point
ꢀ
calc. For C H N BrCl S: C, 52.19; H, 3.20; N, 14.05%.
Found: C, 52.15; H, 3.17; N, 14.03%.
( C): 178–179; FT-IR (ATR, υ /cm): 2,888 (C─H), 1,583
26
19
6
2
max
1
(C═N), 1,225 (C─O), 1,008 (C─S), 825, 775 (C─Cl); H
NMR (400 MHz, DMSO-d ): δ = 0.71–0.75 (t, 3H,
6
J = 16 Hz, Me), 1.65–1.71 (m, 2H, C─CH ), 3.90 (s, 3H,
2
3
.7.9 | 6-(4-Chlorophenyl)-
-(2-(2,3-dichlorophenyl)-1-propyl-1H-benzo
MeO), 4.17–4.20 (t, 2H, N─CH ), 4.50 (s, 2H, S─CH ),
2
2
3
7.60–7.77 (m, 1H, Ar─H), 7.76–7.83 (m, 3H, Ar─H),
7.93–7.95 (m, 3H, Ar─H), 8.00–8.09 (m, 2H, Ar─H), 8.34
[
[
d]imidazol-5-yl)-7H-[1,2,4]triazolo[3,4-b]
1,3,4]thiadiazine (7i)
13
(s, 1H, Ar─H) ppm; C NMR (100 MHz): δ = 160.0,
52.6, 145.0, 134.1, 133.1, 132.8, 132.5, 131.2, 130.1,
1
ꢀ
Off-white solid; yield: 190 mg (72%); melting point ( C):
129.0,127.8, 126.7, 127.4, 113.3, 55.1 (Ar─MeO), 46.3
(N─CH ─CH ─CH ), 22.9 (CH ─S), 22.2 (N─CH ─
1
94–195; FT-IR (ATR, υ /cm): 1,587 (C═N), 1,006 (C─S),
max
1
2
2
3
2
2
773, 804, 846 (C─Cl); H NMR (400 MHz, DMSO-d6):
CH ─CH ), 10.6 (N─CH ─CH ─CH ); MS: calculated for
2 3 2 2 3
+
δ = 0.71–0.74 (t, 3H, J = 12 Hz, Me), 1.64–1.69 (m, 2H,
C─CH ), 4.13 (s, 2H, N─CH ), 4.46 (s, 2H, S─CH ), 7.60–7.76
C H N Cl OS is 548.1; found [M + 1]
549.0,
2
7
22
6
2
+
+
[M + 3] 551.1, [M + 5] 553.0 m/z; Anal.calc. For
2
2
2
(
m, 4H, Ar─H), 7.94–8.09 (m, 5H, Ar─H), 8.36 (s, 1H, Ar─H)
C H N Cl OS: C, 59.02; H, 4.04; N, 15.29%.
2
7
22
6
2
13
ppm; C NMR (100 MHz): δ = 155.3, 151.8, 142.5, 133.0,
Found: C, 59.01; H, 4.02; N, 15.25%.

14
SATHYANARAYANA ET AL.
3
1
6
.7.12 | 3-(2-(2,3-Dichlorophenyl)-
-propyl-1H-benzo[d]imidazol-5-yl)-
-4-tolyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
S─CH ), 7.59–7.75 (m, 4H, Ar─H), 7.93–8.08 (m, 5H, Ar─H),
2
13
8.32 (s, 1H, Ar─H) ppm; C NMR (100 MHz): δ = 154.5,
152.2, 151.5, 151.2, 133.9, 132.7, 132.5, 132.0, 131.4, 131.1,
129.7, 127.8, 125.7, 123.7, 112.0, 46.2 (N─CH ─CH ─CH ),
thiadiazine (7l)
2
2
3
22.9
(CH ─S),
22.0
(N─CH ─CH ─CH ),
10.5
2
2
2
3
ꢀ
Off-white solid; yield: 220 mg (88%); melting point ( C):
18–219; FT-IR (ATR, υmax/cm): 1,583 (C═N), 1,005
(N─CH ─CH ─CH ); MS: calculated for C H N Cl O S is
2 2 3 26 19 7 2 2
+
+
+
2
563.07; found [M + 1] 564.04, [M + 3] 566.07, [M + 5]
568.1 m/z; Anal.calc. For C H N Cl O S: C, 55.32; H,
1
(C─S), 811, 728 (C─Cl); H NMR (400 MHz, DMSO-d ):
6
26 19
7
2 2
δ = 0.72–0.76 (t, 3H, J = 16 Hz, Me), 1.67–1.73 (m, 2H,
C─CH ), 1.90 (s, 3H, Me), 4.16–4.19 (t, 2H, N─CH ),
3.39; N, 17.37%. Found: C, 55.30; H, 3.35; N, 17.35%.
2
2
4
.48 (s, 2H, S─CH ), 7.63–7.77 (m, 1H, Ar─H), 7.79–7.83
2
(
m, 3H, Ar─H), 7.95–7.98 (m, 3H, Ar─H), 8.03–8.11 (m, 2H,
3.8 | Biological screening
13
Ar─H), 8.39 (s, 1H, Ar─H) ppm; C NMR (100 MHz):
δ = 152.3, 150.8, 141.5, 133.0, 132.7, 132.0, 131.9, 131.4,
3.8.1 | DPPH free radical scavenging
activity
1
31.1, 129.0,128.6, 125.7, 122.7, 112.3, 46.0 (N─CH ─
2
CH ─CH ), 22.7 (CH ─S), 22.3 (Ar─Me), 21.3 (N─CH ─
2
3
2
2
CH ─CH ), 10.3 (N─CH ─CH ─CH ); MS: calculated for
The free radical scavenging activity of the title compounds
was measured by using the 2, 2-diphenyl-1-picrylhydrazyl
(DPPH) method. A total solution of 0.2 mM of DPPH in
methanol was prepared, and 100 μl of this solution was
added to various concentrations of title compounds at con-
centrations of 10, 20, 30, 40, and 50 μg/ml. After 30 min,
absorbance was measured at 517 nm, and the scavenging
activity was calculated as a percentage of radical reduc-
2
3
2
2
3
+
+
C H N Cl S is 532.1; found [M + 1] 533.0, [M + 3] 535.1,
27
22
6
+
2
[
M + 5] 537.1 m/z; Anal.calc. For C H N Cl S: C, 60.79; H,
27 22 6 2
4.16; N, 15.75%. Found: C, 60.75; H, 4.17; N, 15.73%.
3.7.13 | 3-(2-(2,3-Dichlorophenyl)-
1-propyl-1H-benzo[d]imidazol-5-yl)-
6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]
[37]
tion. Ascorbic acid was used as the reference material.
thiadiazine (7m)
DPPH radical scavenging activity ð%Þ
ꢀ
Pale yellow; yield: 220 mg (88%); melting point ( C): 130–131;
FT-IR (ATR, υmax/cm): 1,570 (C═N), 1,011 (C─S), 845, 780
= ½ðAcontrol – ATestÞ=Acontrol ꢂ × 100
1
(
C─Cl); H NMR (400 MHz, DMSO-d ): δ = 0.72–0.76 (t, 3H,
6
J = 16 Hz, Me), 1.66–1.72 (m, 2H, C─CH ), 4.18–4.21 (t, 2H,
3.8.2 | Nitric oxide radical scavenging
2
N─CH ), 4.52 (s, 2H, S─CH ), 7.62–7.79 (m, 2H, Ar─H),
activity
2
2
7
(
(
1
.78–7.85 (m, 3H, Ar─H), 7.95–7.98 (m, 3H, Ar─H), 8.03–8.09
13
m, 2H, Ar─H), 8.35 (s, 1H, Ar─H) ppm; C NMR
100 MHz): δ = 151.3, 149.7, 143.2, 136.2, 133.7, 132.7, 132.6,
30.9, 128.9, 128.7, 127.2, 124.4, 123.0, 116.2, 110.8, 46.0
N─CH ─CH ─CH ), 25.5 (CH ─S), 22.3 (N─CH ─CH ─
Nitric oxide (NO) radical are generated from sodium
nitroprusside solution at physiological pH. Sodium
nitroprusside (1 ml of 10 mM) was mixed with 1 ml of test
compounds at different concentrations (10–50 μg/ml) in
phosphate buffer (pH 7.4). The mixture was incubated at
(
2
2
3
2
2
2
CH3), 10.8 (N─CH ─CH ─CH ); MS: calculated for
2
2
3
+
+
ꢀ
C H N Cl S is 518.08; found [M + 1] 519.07, [M + 3]
25 C for 150 min. To 1 ml of the incubated solution, 1 ml
2
6
20
6
2
+
5
21.05, [M + 5] 523.08 m/z; Anal.calc. For C H N Cl S: C,
of Griess's reagent (1% sulphanilamide, 2% o-phosphoric
acid, and 0.1% naphthyl ethylene diamine dihydrochloride)
was added. Absorbance was read at 546 nm. Ascorbic acid
26
20
6
2
60.12; H, 3.88; N, 16.18%. Found: C, 60.10; H, 3.87; N, 16.17%.
[38]
was used as standard drug material.
3
1
6
.7.14 | 3-(2-(2,3-Dichlorophenyl)-
-propyl-1H-benzo[d]imidazol-5-yl)-
-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-b]
Nitric oxide radical scavenging activity ð%Þ
=
½ðA
– A Þ=A
ꢂ × 100
control
Test
control
[1,3,4]thiadiazine (7n)
ꢀ
Pale yellow; yield: 210 mg (78%); melting point ( C): 213–214;
3.8.3 | Superoxide anion scavenging
activity
FT-IR (ATR, υmax/cm): 2,958 (C─H), 1,575 (C═N), 1,007
C─S), 1,510, 1,340 (Ar─O N), 770, 815 (C─Cl); H NMR
1
(
2
(
1
400 MHz, DMSO-d ): δ = 0.72–0.75 (t, 3H, J = 12 Hz, Me),
About 1 ml of nitroblue tetrazolium (NBT) solution
(156 μM NBT in 100 mM phosphate buffer, pH 7.4), 1 ml
6
.59–1.64 (m, 2H, C─CH ), 4.11 (s, 2H, N─CH ), 4.45 (s, 2H,
2
2

SATHYANARAYANA ET AL.
15
NADH solution (468 μM in 100 m M phosphate buffer,
pH 7.4), and 0.1 ml of sample solution in water were
mixed. The reaction was started by adding 100 μl of
phenazinemethosulphate (PMS) solution (60 μM PMS in
ACKNOWLEDGMENTS
The authors are thankful to Mangalore University for
providing facility; IISC Bangalore for providing spectral
data; and the Department of Biochemistry, Mangalore
University for computational studies.
1
00 m M phosphate buffer, pH 7.4) to the mixture. The
ꢀ
reaction mixture was incubated at 25 C for 5 min, and the
absorbance was recorded at 560 nm, measured against
blank. Ascorbic acid was used as standard drug material.
Superoxide radical scavenging activity (%)
CONFLICT OF INTERESTS
The authors declare no conflicts of interest.
[39]
=
[
(Acontrol – ATest)/Acontrol] × 100
ORCID
Boja Poojary https://orcid.org/0000-0001-6725-3289
3.9 | Physiochemical properties
REFERENCES
[1] E. R. Whittemore, D. T. Loo, J. A. Watt, C. W. Cotmans, Neu-
roscience 1995, 67, 921.
The physiochemical parameters were simulated at
Mangalore University, India on an Acer Veriton i5
[
2] C. W. Olanow, Ann. Neurol. 1992, 32, S2.
[
40]
[3] C. W. Olanow, Trends Neurosci. 1993, 16, 439.
Workstation using QikProp
017-1, Schrödinger LLC, New York, NY, 2017, running
in fast mode.
program Maestro Suite
[
[
4] S. J. Richardson, Ann. N. Y. Acad. Sci. 1993, 695, 73.
5] R. D. Reddy, J. K. Yao, Prostaglandins Leukot. Essent. Fatty
Acids 1996, 55, 33.
2
[6] G. Ayhan-Kllcigll, C. Kus, T. Coban, B. Can-Eke, S. Ozbey,
M. Iscan, J. Enzyme Inhib. Med. Chem. 2005, 20, 503.
4
| CONCLUSIONS
[7] W. R. Markesbery, T. J. Montine, M. A. Lovell, Neurodegener.
Dis. 2001, 21. https://doi.org/10.1007/978-1-59259-106-0_2
[
8] C. Kus, G. Ayhan-Kilcigil, S. Ozbey, F. B. Kyanak, M. Kaya,
T. Coban, B. Can-Eke, Bioorg. Med. Chem. 2008, 16, 4294.
9] G. Avhan-Kilcigil, C. Kus, E. D. Ozdamar, B. Can-Eke,
M. Iscan, Arch. Pharm. Chem. Life Sci. 2007, 340, 607.
In this study, a novel series of [1,2,4]triazolo[3,4-b]
[
1,3,4]thiadiazine derivatives embedded with benzimid-
[
azole moiety has been synthesized via one-pot nitro
reductive cyclization protocol. The results of DPPH and
nitric oxide radical scavenging showed better activity
when compared to standard, whereas the superoxide
result was relatively low as the mechanism of these
methods are different. Among the synthesized deriva-
tives, 7c exhibited excellent scavenging activity. The
scavenging activity of the compounds bearing electron-
donating groups was relatively higher compared to the
electron-withdrawing groups. As there is no active
hydrogen to participate in the scavenging process, the
plausible scavenging activity of our molecules con-
taining electron-donating group may be due to con-
certed electron transfer process. The reaction sequence
to inhibit superoxide radical is given below.
[
[
[
[
[
10] H. Bektas, C. Albay, B. B. Sokmen, S. Avdin, E. Mentese,
G. Aydin, D. Sen, J. Heterocycl. Chem. 2018, 55, 2400.
11] Z. Ates-Alagoz, C. Kus, T. Coban, J. Enzym. Inhib. Med. Chem.
2005, 20, 325.
12] N. T. Gowda, C. V. Kavitha, K. K. Chiruvella, O. Joy,
K. S. Rangappa, S. C. Raghavan, Bioorg. Med. Chem. 2009, 19, 4594.
13] R. Paramashivappa, P. P. Kumar, P. S. Rao, A. S. Rao, Bioorg.
Med. Chem. 2003, 13, 657.
14] H. J. Kwak, Y. M. Pyun, J. Y. Kim, H. S. Pagire, K. Y. Kim,
K. R. Kim, S. D. Rhee, W. H. Jung, J. S. Song, M. A. Bae,
H. D. Lee, J. H. Ahn, Bioorg. Med. Chem. 2013, 23, 4713.
[15] K. C. Achar, K. M. Hosamani, H. R. Seetharamareddy, Eur.
J. Med. Chem. 2010, 45, 2048.
[
16] L. Garuti, M. Roberti, D. Pizzirani, A. Pession, E. Leoncini,
V. Cenci, S. Hrelia, Il Farmaco 2004, 59, 663.
ꢁ
ꢁ
−
ꢁ
+
[17] V. Klimesova, J. Koci, M. Pour, J. Stachel, K. Waisser,
O2 + HO + H ! H O + O
2
ꢁ−
2
2
2
J. Kaustova, Eur. J. Med. Chem. 2002, 37, 409.
−
+
O2 + O + 2H ! H O + O
2
2
2
2
[
18] Y. Ozkay, Y. Tunali, H. Karaca, I. I s¸ lkdag, Eur. J. Med. Chem.
010, 45, 3293.
19] K. Vasantha, G. Basavarajaswamy, M. V. Rai, P. Boja,
V. R. Pai, N. Shruthi, M. Bhat, Bioorg. Med. Chem. 2015, 25,
1420.
Hence, possibly because of the absence of active
hydrogen atom, moderate activity was observed in the
case of superoxide radical scavenging activity. The exact
mechanism for the scavenging activity needs to be inves-
tigated. Compound 7c obeyed most of the ADME param-
eters. In our study, the preliminary antioxidant tests and
physiochemical property results are encouraging, which
demonstrated the importance of substitution of the func-
tional group for potential applications in the pharmaceu-
tical field.
2
[
[
[
[
20] B. Zhang, Y. H. Li, Y. Liu, Y. R. Chen, E. S. Pan, W. W. You,
P. L. Zhao, Eur. J. Med. Chem. 2015, 103, 335.
21] A. K. Gadad, M. N. Noolvi, R. V. Karpoormath, Bioorg. Med.
Chem. 2004, 12, 5651.
22] Q. Xu, K. Bao, M. Sun, J. Xu, Y. Wang, H. Tian, D. Zuo,
Q. Guan, Y. Wu, W. Zhang, Sci. Rep. 2017, 7, 1197.

16
SATHYANARAYANA ET AL.
[
[
[
23] V. Sumangala, B. Poojary, N. Chidananda, T. Arulmoli,
[35] D. E. Clark, J. Pharm. Sci. 1999, 88, 807.
S. Shenoy, Eur. J. Org. Chem. 2012, 54, 59.
[36] A. M. Aronov, Drug Discov. Today 2005, 10, 149.
[37] S. Jamuna, S. Paulsamy, K. Karthika, J. Appl. Pharm. Sci.
2012, 02, 149.
24] I. Khan, S. Zaib, A. Ibrar, N. H. Rama, J. Simpson, J. Iqbal,
Eur. J. Org. Chem. 2014, 78, 167.
[
[
[
38] S. K. Mondal, G. Chatraborty, M. Gupta, U. K. Mazumder,
Ind. J. Exp. Biol. 2004, 44, 39.
39] I. Gulcin, M. Oktay, I. K. O. Vioglu, A. Ali,
J. Ethanopharmacol. 2002, 79, 325.
25] D. H. Boschelli, D. T. Connor, C. R. Kostlan, J. B. Kramer,
M. D. Mullican, Sircar, U.S. Patent and Trademark Office,
Washington, DC 1992 U.S. Patent No. 5,102,897.
26] P. Karegoudar, D. J. Prasad, M. Ashok, M. Mahalinga,
B. Poojary, B. S. Holla, Eur. J. Org. Chem. 2008, 43, 808.
27] P. Puthiyapurayil, B. Poojary, C. Chikkanna, S. K. Buridipad,
Eur. J. Med. Chem. 2012, 57, 407.
28] P. Puthiyapurayil, B. Poojary, S. K. Buridipad, J. Heterocycl.
Chem. 2014, 51, E55.
29] M. F. El Shehry, A. A. Abu-Hashem, E. M. El-Telbani, J. Med.
Chem. 2010, 45, 1906.
30] A. P. Skoumbourdis, C. A. LeClair, E. Stefan, A. G. Turjanski,
W. Maguire, S. A. Titus, R. Huanga, D. S. Auld, J. Inglese,
C. P. Austin, S. W. Michnick, M. Xia, C. Thomas, Bioorg. Med.
Chem 2009, 19, 3686.
31] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv.
Drug Deliv. Rev. 2001, 46, 3.
32] G. Litwinienko, K. U. Ingold, J. Org. Chem. 2003, 68, 3433.
33] D. F. Veber, S. R. Johnson, H. Y. Cheng, B. R. Smith,
K. W. Ward, K. D. Kapple, J. Med. Chem. 2002, 45, 2615.
34] M. A. A. El-Sayed, N. I. Abdel, A. M. Abdel-Aziz,
A. S. El-Azab, K. E. H. ElTahir, Bioorg. Med. Chem. 2012,
[
[
[
[
[
40] Schrödinger. QikProp 3.4 User Manual. LLC, New York, NY
2011.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Sathyanarayana R,
Poojary B, Chandrashekarappa RB, Kumar H,
Merugumolu VK. Novel [1,2,4]triazolo[3,4-b][1,3,4]
thiadiazine derivatives embedded with
[
[
[
benzimidazole moiety as potent antioxidants.
J Chin Chem Soc. 2020;1–16. https://doi.org/10.
[
1002/jccs.201900452
2
0, 3306.