Convergent synthesis, free radical scavenging, Lineweaver-Burk plot exploration, hemolysis and in silico study of novel indole-phenyltriazole hybrid bearing acetamides as potent urease inhibitors

Article abstract of DOI:10.1002/jhet.4006

In the current paper, through a convergent multi-step approach, a library of novel indole-phenyltriazole hybrids containing an amide moiety (9a-k) was synthesized. The structural verification of all synthesized molecules was accomplished by CHN and spectral analyses data. These synthesized bi-heterocyclic derivatives (9a-k) were evaluated for their anti-ulcer potential by inhibitory action against Jack bean urease enzyme and subsequently their structure-activity relationship was perceived. Moreover, these compounds were inspected for cytotoxic profile by hemolytic activity and it was professed that nearly all the synthesized compounds showed low cytotoxicity. In addition, free radical scavenging activity and kinetic analysis were also carried out for these compounds to understand their mode of inhibition. So, it was summated that these derivatives might lead to further research gateways for obtaining better and safe anti-ulcer agents.

Full text of DOI:10.1002/jhet.4006

Received: 29 February 2020
DOI: 10.1002/jhet.4006
Revised: 10 April 2020
Accepted: 16 April 2020
A R T I C L E
Convergent synthesis, free radical scavenging, Lineweaver-
Burk plot exploration, hemolysis and in silico study of novel
indole-phenyltriazole hybrid bearing acetamides as potent
urease inhibitors
1
1
1
Wajiha Khan | Muhammad A. Abbasi
| Aziz-ur Rehman
|
1
1
2
3
Sabahat Z. Siddiqui | Majid Nazir | Syed A. Ali Shah | Hussain Raza
|
4
5
3
Mubashir Hassan | Muhammad Shahid | Sung Y. Seo
1
Department of Chemistry, Government
Abstract
College University, Lahore, Pakistan
2
In the current paper, through a convergent multi-step approach, a library of
novel indole-phenyltriazole hybrids containing an amide moiety (9a-k) was
synthesized. The structural verification of all synthesized molecules was
accomplished by CHN and spectral analyses data. These synthesized bi-
heterocyclic derivatives (9a-k) were evaluated for their anti-ulcer potential by
inhibitory action against Jack bean urease enzyme and subsequently their
structure-activity relationship was perceived. Moreover, these compounds were
inspected for cytotoxic profile by hemolytic activity and it was professed that
nearly all the synthesized compounds showed low cytotoxicity. In addition,
free radical scavenging activity and kinetic analysis were also carried out for
these compounds to understand their mode of inhibition. So, it was summated
that these derivatives might lead to further research gateways for obtaining
better and safe anti-ulcer agents.
Faculty of Pharmacy and Atta-ur-
Rahman Institute for Natural Products
Discovery (AuRIns), University
Technology MARA, Shah Alam, Malaysia
3
Department of Biological Sciences,
College of Natural Sciences, Kongju
National University, Gongju, South Korea
4
Institute of Molecular Biology and
Biotechnology, The University of Lahore,
Lahore, Pakistan
5
Department of Biochemistry, University
of Agriculture, Faisalabad, Pakistan
Correspondence
Muhammad A. Abbasi, Department of
Chemistry, Government College
University, Lahore 54000, Pakistan.
Email: atrabbasi@yahoo.com;
abbasi@gcu.edu.pk
1
| INTRODUCTION
the hydrolysis of urea up to 1014 times compared to
uncatalyzed reaction. Basically, this enzyme works by cat-
[7]
Urea has exclusive role in the history of organic and medici-
nal chemistry. It was the first organic compound that was
synthesized from an inorganic compound by Friedrich
Wohler in 1928. It is an exogenous product of amino acids
and proteins dissimilation, human body excretes about
alyzing the hydrolysis of urea into NH and carbamate
3
(which yields second molecule of NH on decomposition).
3
Release of these two NH molecules causes considerable
3
[1]
increase in pH which imparts negative effects on human
[8]
body and agriculture. One of the most common urease
associated gram-negative bacterial specie is Helicobacter
pylori which is capable of living in acidic environment like
stomach (pH 1-2) and is the cause of a chief public health
2
0 to 30 g of urea in human urine in a single day. Urea is
also used as important fertilizer in agriculture. Ureases
urea aminohydrolase E.C.3.5.1.5) are the members of phos-
(
[9]
photriesterases and aminohydrolases family which exhibit
active metals in the active sites of these enzymes. Ureases
problem. Its infections stimulate stomach inflammation
and enhance the possibility of emergent gastric and duode-
+2
[2–6]
[10–13]
contain two Ni ions in their active sites
that catalyze
nal ulcers, gastric lymphoma, gastric adenocarcinoma,
J Heterocyclic Chem. 2020;1–14.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC
1

2
KHAN ET AL.
[
14,15]
stomach cancer and peptic ulcer.
Around 50% of
is a safe natural compound present in many edibles in large
quantities. One of the remarkable characteristic of Vitamin
C is that its activities are pH dependent. It is unstable at neu-
worldwide population is septic to H. pylori because this spe-
cie can survive in stomach all through the life of infected
people without any indication of disease. This high occur-
rence of H. pylori in humans points to the development of
bacterial immunity against human host immunity. A broad
range of urease enzymes have been isolated from fungi,
[20]
tral pH and degrades at pH 6 to 7. Other urease inhibitors
[
21]
[22]
used are benzoquinones,
hydroxyurea,
barbituric and
[18]
[23]
thiobarbituric acid, and triazoles. Thus, the novel and
safe treatment of gastric ulcers with lesser side effects is an
attractive field for pharmaceutical researchers.
[16]
algae, bacteria and plants
and all the urease enzymes
persue the similar catalysis pattern. This is due to the analo-
gous sequence of amino acids and the active sites of enzyme
containing two nickel ions, showing common ancestry.
Therefore, urease inhibitors have been considered as target
Indole is a naturally occurring heterocyclic molecule
found in indole alkaloids, marine natural products and
fungal metabolites. Structurally, it is a benzopyrrole ring
which is composed of benzene and pyrrole rings fused
with each other at positions 2- and 3- of pyrrole ring.
Here C-2 and C-3 bonds often react like alkenes. The
N-substitution reactions are always favored by ionic salts
[17]
for novel antiulcer drugs.
Keeping in view the associa-
tion of ureases in several pathological conditions, the explo-
ration of safe and potent urease enzyme inhibitors has
become a significant topic of research in pharmaceutical
+
+
+
(Li , Na , K , etc.) while softer counter ions prefer C-3
substitution. This helps in binding of indole with differ-
[18]
industry.
Imidazole, phosphorodiamidates, hydroxamic
[
24]
acid derivatives and many other urease inhibitors have been
examined in past decades, but majority of these compounds
are unstable or too toxic to be allowed for their use in living
organisms. Therefore, more research is needed for the syn-
thesis of new urease inhibitors with predicting levels of
ent compounds.
Indole and its derivatives containing
thiazole and pyrazole moieties are of great importance as
[
25,26]
[27]
antioxidant,
agents.
anti-cancer,
and anti-proliferative
[
28,29]
Moreover, indole derivatives are also found
to have numerous biological activities like analgesic,
anti-inflammatory, anti-malarial, anti-convulsant, anti-
[19]
activities.
[
24]
[30–32]
[33]
Ascorbic acid (Vitamin C) is a urease inhibitor, an anti-
oxidant and also a free radical scavenger. High doses of anti-
microbials can enhance the risk of side effects but ascorbate
ulcer,
anti-depressant,
and anti-hypertensive.
Similarly, 1,2,4-triazole nuclei and their derivatives have
also been accounted for their wide range of
FIGURE 1 Rationale of current study

KHAN ET AL.
3
pharmacological and medicinal properties like anti-
inflammatory, anti-tumor, anti-tubercular, anti-convul-
excess of water was added in workup followed by solvent
extraction. The purpose of adding base here was to neutral-
ize the unreacted amount of conc. H SO and acid (1). The
[8]
sant, antimicrobial, and anti-cancer activities.
2
4
[
34–42]
From the already reported literature,
the bio-
resulting ester, ethyl 4-(1H-indol-3-yl)butanoate (2) moved
to organic layer and remaining salts of acids partitioned to
aqueous layer in solvent extraction. Ester 2 obtained was a
brownish liquid in appearance. Next step was the conver-
sion of this ester into respective hydrazide 3, which was
done by refluxing it with hydrazine hydrate in methanol
for 14 hours. The hydrazide, 4-(1H-indol-3-yl)but-
anohydrazide (3) was recovered as light brown colored
solid. The third step involved the cyclization into 5-[3-(1H-
indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5) by
treating hydrazide 3 with phenyl isothiocyanate (4). The
triazole (5) was having SH group at C-3 position, which
was a lone nucleophilic site for attachment with electro-
philes. In a sidewise set of reactions, different electrophiles,
activity of triazole and indole cores encouraged us to
produce some hybrid compounds having both indole
and 1,2,4-triazole moieties altogether (Figure 1). Based
on our preceding efforts, in this current research, the
designed bi-heterocyclic molecules were examined for
inhibitory potential against urease enzyme in addition
to assess the kinetic mechanism. Moreover, their
molecular docking study was also done to find the
interactions of synthesized compounds with the target
enzyme.
2
| RESULTS AND DISCUSSION
8a-k, were synthesized by reacting substituted/
The convergent synthesis of targeted bi-heterocyclic
hybrids converged with an amide moiety was carried out
in ample yields through a number of steps. In first step, the
acid (1) was esterified by using EtOH and conc. H SO (cat-
alytic amount). Here, EtOH was utilized both as reactant
and as solvent to avoid reversible reaction. Weak base with
unsubstituted anilines (6a-k) with 2-bromoethanoyl bro-
mide (7). In the final step, the nucleophilic thiol (5) was
amalgamated with different acetamides (8a-k) using LiH
as catalyst in polar aprotic medium, resulting in the forma-
tion of designed derivatives, 9a-k, as illustrated in Scheme 1
and Table 1.
2
4
O
O
O
(CH2)3
C
(
CH )
2 3
C
(CH )
2 3
C
OH
OEt
N2H3
C
A
B
N
N
N
Ph N
C
S
H
H
H
4
1
2
3
O
C
N
N
7
Br
(CH2)3
SH
Br
CH2
R1
N
H
N
+
CH2 Br
R1
E
C
O
D
NH2
N
H
R2
5
8a-k
R2
6a-k
N1
"
N
2""
R1
5
"
3"
CH2 1"" NH
H C
3""'
2
S
C
1
'
N
1""'
2
'
CH2
H C
2
9a-k
O
3
'
1"'
R2
""'
9
5
4
5"'
3
1
3"'
6
8
N
H
SCHEME 1 Outline for the synthesis of 9a-k. Reagents and conditions: (A) EtOH/H
·H O/14 hours reflux. (C) MeOH/4/16 hours reflux/dissolved precipitate in 10% NaOH followed by filtration and acidification of filtrate
in cold state. (D) Aq. Na CO solution/pH 9-10/vigorous shaking for 20-30 minutes at RT. (E) DMF/LiH/60-70 hours stirring
2 4
SO /8 hours reflux. (B) MeOH/
N
2
H
4
2
2
3

4
KHAN ET AL.
TABLE 1
Compound
Different groups ( R
1
and
R
2
) in Scheme 1
activity due to presence of relatively bulky but polar eth-
oxy group at para-position in the aromatic ring
R
1
R
H
H
2
(
Figure 3).
While discussing dimethl substituted compounds,
6
6
6
6
6
6
6
6
6
6
6
a, 8a, 9a
b, 8b, 9b
c, 8c, 9c
d, 8d, 9d
e, 8e, 9e
f, 8f, 9f
H
2-CH
H
3
compound 9i (IC50 = 0.029 ± 0.028 μM) was the most
potent compound of this series with one methyl at meta
and other at para position of the ring. However, when
these dimethyl substituents were repositioned at different
ortho, meta and para positions on the ring, a slight
change was observed in inhibitory activities of those
4-C
2
H
5
H
4-OC
2
H
5
2-CH
2-CH
2-CH
2-CH
3-CH
3-CH
3
3
3
3
3
3
3-CH
4-CH
5-CH
6-CH
4-CH
5-CH
6-CH
3
3
3
3
3
3
3
g, 8g, 9g
h, 8h, 9h
i, 8i, 9i
compounds as 9e (IC50 = 0.667 ± 0.099 μM), 9f (IC50
=
0
.158 ± 0.215 μM), 9g (IC = 0.246 ± 0.113 μM), 9h (IC
50
50
=
0.224 ± 0.072 μM) and 9j (IC50 = 0.378 ± 0.224 μM)
j, 8j, 9j
(Figures 4 and 5).
Compound 9k (IC50 = 0.497 ± 0.049 μM) with one
ethyl group and one methyl group at two ortho positions on
the ring also showed efficient inhibitory effect due to pres-
ence of two electron donating groups (Figure 6). The activ-
ity of this molecule was also closely analogous to those of
some other di-substituted molecules like 9g, 9h, and 9j.
k, 8k, 9k
2 5
2-C H
2.1 | Urease inhibitory activity
Urease enzyme was used to test the inhibitory potential
of the synthesized compounds (9a-k) and it was evident
from the resultant IC50 (μM) values that all the synthe-
sized compounds were having remarkable inhibitory
potential against urease enzyme relative to that of thio-
urea (standard) (Table 2). The IC50 values of compounds
2.3 | Hemolytic activity
To determine the cytotoxicity of the synthesized derivatives,
9a-k, these were subjected to hemolytic assay. Percentage
hemolysis (%) results are given in Table 2. According to the
results, almost all the derivatives of this series showed mild
cytotoxicity towards RBC plasma membrane. Compound
9d (23.52%) showed maximum membrane cytotoxicity
while 9f (1.63%) gave minimum membrane toxicity as com-
pared to the standard Triton-X (89%). Rest of the com-
pounds depicted relatively low cytotoxicity values of 9a
9
±
a-k were lower than the standard thiourea (IC50 = 4.721
1.374 μM). Keeping in view the inhibitory effects of
these compounds (9a-k) with different alkyl (R) groups
on them, a confined structure-activity relationship (SAR)
was streamlined.
2.2 | Structure-activity relationship
(4.93%), 9b (4.41%), 9c (3.54%), 9e (3.24%), 9g (4.45%), 9h
Compound 9a (IC50 = 1.448 ± 0.992 μM) with unsubstituted
phenyl group depicted remarkable inhibiton compara-
tively to that of thiourea (IC50 = 4.721 ± 1.374 μM).
Compound 9b (IC50 = 2.272 ± 0.997 μM) is the only sin-
gle methyl-substituted compound in the series. It showed
higher IC50 value as compared to unsubstituted 9a,
owing to the positive inductive effect of electron-
donating methyl group substituted at ortho position on
the ring, however the enzyme inhibitory effect of 9b was
lower than the standard used (Figure 2).
(2.45%), 9i (5.31%), 9j (2.48%), and 9k (3.02%).
2.4 | Kinetic analysis
Based on the IC50 results of synthesized compounds, it
was pertinent to select the most potent compound 9i to
find its inhibition type and constant on Jack bean ure-
ase. The inhibition potential of this compound was
checked in terms of enzyme substrate inhibitor and
enzyme inhibitor constants. A number of straight lines
graphs were obtained showing kinetic studies of urease
enzyme (Lineweaver Burk 1/[S] vs 1/V) (Figure 7A).
According to results, 9i intersected in the second
quadrant, Km (constant) remained same while in Vmax
(reaction velocity) a decrease was noticed with increasing
dose of 9i, showing non-competitive behavior of the said
A significant increase in the inhibition (IC50
=
0
.139 ± 0.006 μM) was observed in compound 9c, when
an ethyl group was present at para position of the aro-
matic ring. It can be explained by the fact that ethyl
group is more electron donating as compared to methyl
group (in 9b), therefore the molecule with an ethyl group
will interact with urease enzyme more competently
than that having a methyl group. Compound 9d
compound. K (EI dissociation constant) is shown in sec-
i
(IC50 = 0.594 ± 0.743 μM) also showed potent inhibitory
ond plot of slope vs 9i concentration (Table 3).

KHAN ET AL.
5
TABLE 2
Free radical scavenging, urease inhibition activity and hemolytic activity of manufactured derivatives (9a-k).
Mean ± SEM, n = 3
Free radical
scavenging
(100 μg/mL)
Urease activity,
%
Hemolysis (%)
(mean ± SEM)
Compound
R
IC50 (μM) ± SEM
9
a
H
N
15.765 ± 0.854
1.448 ± 0.992
4.93 ± 0.04
CH2
C
O
9
b
CH3
3.163 ± 0.144
2.272 ± 0.997
4.41 ± 0.035
H
N
CH2
CH2
C
O
9
9
c
H
N
8.467 ± 0.691
4.567 ± 0.874
0.139 ± 0.006
0.594 ± 0.743
3.54 ± 0.023
23.52 ± 0.08
C
O
CH3
CH2
d
H
N
CH2
C
O
CH3
CH2
O
9
9
e
f
CH3
CH3
3.602 ± 0.541
6.665 ± 1.257
0.667 ± 0.099
0.158 ± 0.215
3.24 ± 0.021
1.63 ± 0.11
H
N
CH2
CH2
CH3
C
O
H
N
C
O
CH3
9
g
CH3
6.787 ± 2.015
0.246 ± 0.113
4.45 ± 0.03
H
N
CH2
C
O
CH3
CH3
9
9
h
5.135 ± 0.988
28.374 ± 3.335
0.224 ± 0.072
0.029 ± 0.028
2.45 ± 0.02
5.31 ± 0.04
H
N
CH2
CH2
C
O
H C
3
i
H
N
CH3
CH3
C
O
(
Continues)

6
KHAN ET AL.
TABLE 2 (Continued)
Free radical
scavenging
Urease activity,
%
Hemolysis (%)
Compound
R
(100 μg/mL)
IC50 (μM) ± SEM
(mean ± SEM)
9
j
H
N
4.142 ± 0.886
0.378 ± 0.224
2.48 ± 0.019
CH2
CH3
C
O
CH3
CH3
9
k
2.623 ± 0.774
0.497 ± 0.049
3.02 ± 0.02
H C
2
H
N
CH2
C
O
H C
3
Standards
Vitamin C:
5.562 ± 7.40
Thiourea:
4.721 ± 1.374
Triton X: 89.00 ± 0.67;
PBS hemolysis:
9
(
%) = 2.93 ± 0.018
N N
N
N N
N
H
N
FIGURE 2 Struture-activity
relationship of compounds 9a
and 9b
H
N
C
O
C
O
S
S
H C
3
N
H
N
H
9a (IC50 =1.448 ± 0.992 µM)
9b (IC50 = 2.272 ± 0.997 µM)
N
N
H
N
N
FIGURE 3 Struture-activity
relationship of compounds 9c
and 9d
N
H
N
C
O
C
O
N
S
N
S
CH3
CH3
O
N
H
N
9c (IC50 = 0.139 ± 0.006 µM)
H
9d (IC50 = 0.594 ± 0.743 µM)
2
.5 | Free radical scavenging
(IC50 = 8.467 ± 0.691 μg/mL), 9d (IC50 = 4.567 ±
.874 μg/mL), 9e (IC50 = 3.602 ± 0.541 μg/mL), 9f (IC50
= 6.665 ± 1.257 μg/mL), 9g (IC50 = 6.787 ± 2.015 μg/
0
Synthesized bi-heterocyclic amides (9a-k) were assessed
for 2,2-diphenyl-1-picrylhydrazyl free radical scavenging
potential. Some derivatives did not show considerable
scavenging potential even in increased concentrations
mL), 9h (IC50 = 5.135 ± 0.988 μg/mL), and 9j (IC50
4.142 ± 0.886 μg/mL).
=
(
100 μg/mL) (Table 2). Maximum radical scavenging
potential was showed by compound 9i (IC50 = 28.374
3.335 μg/mL) while minimum scavenging potential
was showed by compound 9k (IC50 = 2.623 ± 0.774 μg/
mL) against standard ascorbic acid (Vitamin C) (IC50
5.562 ± 7.40 μg/mL). However, low values of scavenging
potential was seen for compound 9a (IC50 = 15.765
0.854 μg/mL), 9b (IC50 = 3.163 ± 0.144 μg/mL), 9c
2.6 | Binding energy estimation of
synthesized derivatives
±
=
All the synthesized compounds, 9a-k, were docked to
check their conformational arrangements, in relation to
their bonding interaction pattern and minimum energy
value (kcal/mol). Results showed that all the
9
±

KHAN ET AL.
7
H C
3
H C
3
N
N
H
CH3
N
N
H
N
N
C
O
C
O
S
S
N
N
.
CH3
N
N
H
9e (IC50 = 0.667 ± 0.099 µM)
H
9f (IC50 = 0.158 ± 0.215 µM)
H C
3
H C
3
N
N
H
N
N
H
N
N
C
O
C
O
S
N
N
S
H C
3
CH3
N
N
9g (IC50 = 0.246 ± 0.113 µM)
9h (IC50 = 0.224 ± 0.072 µM)
H
H
FIGURE 4 Struture-activity relationship of compounds 9e, 9f, 9g, and 9h
N N
N
H
N
CH3
CH3
N N
H
N
CH3
C
O
S
N
S
C
O
CH3
N
H
N
H
9j (IC50 = 0.378 ± 0.224 µM).
9
i (IC50 = 0.029 ± 0.028 µM)
FIGURE 5 Struture-activity relationship of compounds 9i and 9j
ΔGbinding = ΔGGauss + ΔGrepulsion + ΔGhbond
ΔGhydrophobic + ΔGtors
CH3
+
N
N
H
N
where ΔGGauss is the scattering of two Gaussian func-
tions, ΔGrepulsion is the square of distance (if closer than
entrance value), ΔGhbond is the used metal ions interac-
tions, ΔGhydrophobic is the ramp function, and ΔGtors is
the relative to the no. of rotatable bonds.
C
O
S
N
H C
3
N
H
9k (IC50 = 0.497 ± 0.049 µM)
2
.7 | Binding pocket evaluation of urease
FIGURE 6 Struture-activity relationship of compound 9k
docked compounds
Based on in vitro and in silico results, 9i complex was fur-
ther assessed for binding interactions. Although all the
compounds showed different conformational arrangements
with active site of binding receptor but most excellent
compounds presented significant values of binding
energy as compared to standard values (Figure 8). Fol-
lowing equation was used to evaluate energy values:

8
KHAN ET AL.
FIGURE 7 Lineweaver-Burk plots for urease inhibition using 9i. (A) Concentrations of 9i were 0.00, 0.029, 0.059, and 0.0118 μM. (B)
The intersect shows the plot of slope or the vertical against 9i concentrations
TABLE 3
Jack bean urease with some different
Kinetic parameters of
Dose (μM)
V
max (ΔA/Min)
K
m
(mM)
Inhibition type
i
K (μM)
0
0
0
0
.00
0.00286
3.333
3.333
3.333
3.333
Non-competitive
—
concentrations of 9i
.029
.059
.118
0.0004512
0.000169
0.000123
0.012
—
—
–
12
FIGURE 8 Binding energy of
derivatives 9a-k
-
10.4
-
9.6
–10
-9.4
-
9
-9.2
-9.2
-9.2
-
8.7
-8.6
-8.7
-
8
–
8
6
4
2
–
–
–
0
9
a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
Docked Complexes
energy value of docked compound 9i is given in Figure 9.
Rest of docked compounds is mentioned in
Figures S23-S32. In SAR analysis, three hydrogen and two
hydrophobic connections were studied in 9i docked com-
pounds. Amino group of carbonyl pentene ring forms H-
interactions with Ala636 containing bond distances 2.84
and 3.19 Å. Moreover, another amino group of carbonyl
interacts to Gln635 having bond length 2.71 Å. The met-
hoxy benzene form couple of hydrophobic interactions
against Pro434 and Ile411 having bond distances 4.89 and
4.95 Å, respectively. Already reported literature on signifi-
cance of these residues supported our docked results.
[43–47]
3 | EXPERIMENTAL
We purchased all the chemicals from Alfa Aesar and
Sigma Aldrich (Germany). Solvents bought from local

KHAN ET AL.
9
0
00
000
000
dealers were of analytical grades. Open capillary tube
method was implemented to find the melting points on
Griffin and George apparatus. TLC (mobile phase: EtOAc
and n-hexane in 30:70 ratio) was used to detect initial
purity of synthesized compounds at 254 nm. KBr pellet
method and Jasco-320-A spectrometer were used to
δ/ppm): 10.75 (1H, s, NH-1), 7.52 (3H, m, H-3 , H-4
&
0
00
000
H-5 ), 7.36 (3H, dist.d, J = 8.8 Hz, H-2 , H-6 & H-7),
7.04 (1H, br.t, J = 9.1 Hz, H-6), 6.97 (1H, s, H-2), 6.93
(1H, br.t, J = 8.9 Hz, H-5), 2.64 (2H, t, J = 8.7 Hz, CH -
3 ), 2.47 (2H, t, J = 8.8 Hz, CH -1 ), 1.84 (2H, quintet,
J = 8.7 Hz, CH -2 ). C-NMR (150 MHz, DMSO-d ,
δ/ppm): 168.10 (C-3 ), 152.63 (C-5 ), 136.75 (C-8), 134.26
000 000 000 000 000
(C-1 ), 129.82 (C-3 , C-4 & C-5 ), 128.65 (C-2 & C-
6 ), 127.48 (C-9), 122.69 (C-2), 121.30 (C-6), 118.63 (C-7),
118.58 (C-5), 113.87 (C-3), 111.75 (C-4), 26.48 (C-2 ),
2
0
0
2
0
13
2
6
1
13
00 00
record IR peaks. H-NMR (600 MHz) and C-NMR
150 MHz) signals were recorded in DMSO-d6 using
(
0
00
Bruker spectrometers.
Ethyl 4-(1H-indol-3-yl) butanoate (2). Carboxylic
acid (1, 0.2 mol) was dissolved in EtOH (70 mL) in
0
0
0
25.45 (C-1 ), 24.28 (C-3 ).
5
00 mL round bottom flask and conc. H SO (20 mL)
2-Bromo-N-(un/substituted-phenyl)acetamides
(8a-k). Various un/substituted anilines (6a-k) were
reacted with 2-bromoacetyl bromide (7) in equimolar
amount (0.001 mol) and manual shaking in sodium car-
bonate 10% solution. After 20 to 30 minutes precipitates
will start forming, filtration followed by washing using
cold water will result in separation of desired electro-
2
4
was added in catalytic amount. It was refluxed for around
hours under the supervision of TLC to check maximum
reaction completion. Ten percent aq. Na CO (40 mL)
8
2
3
was added in workup for neutralization of reaction mix-
ture followed by solvent extraction with CH Cl
3
(50 mL × 3). Ester (2) formed was recovered from organic
[34,48]
[48]
solvent layer as reddish brown liquid. Yield: 98%.
philes (8a-k).
4
-(1H-indol-3-yl)butanohydrazide (3). Ester (2,
2-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-
1,2,4-triazol-3-yl}sulfanyl)-N(un/substituted-phenyl)
acetamides (9a-k). Triazole (5, 0.2 g), DMF (5 mL) and
pinch of catalyst LiH were stirred for half hour in 250 mL
RB (room temperature). Equimolar concentration of dif-
ferent electrophiles (8a-k) was mixed in reaction mixture
with 60 to 70 hours stirring. TLC was used for monitoring
of reaction completion. After that reaction contents were
poured in 100 mL chilled water and precipitates of deriv-
atives (9a-k) were filtered or extracted by solvent
according to the physical state of product formed.
0
(
1
3
.15 mol) was dissolved in 60 mL CH OH and N H ·H O
25 mL, 80%) in round bottom flask (500 mL). After
4 hours stirring at room temperature, acid hydrazide
was formed. Workup was done by filtration of precipi-
tate followed by washing with cold n-hexane and air dry-
3
2
4
2
[34,48]
ing. Yield: 80%.
-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-tri-
5
azole-3-thiol (5). Hydrazide (3, 0.13 mol) was mixed
with KOH (0.13 mol) and EtOH (30 mL) in flask (500 mL
RB), after that phenyl isothiocyanate (4) was added.
Refluxing the reaction mixture for 16 hours resulted in
the formation of uncyclized product, which was further
cyclized by adding excess of chilled water with dilute HCl
to adjust pH at 5 to 6. The resulting precipitate was
washed after filtration and air dried to get final cyclized
2-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,
2,4-triazol-3-yl}sulfanyl)-N-phenyl acetamide (9a).
Light brown sticky liquid; Yield: 72%; Mol. Formula:
−
1
C H N SO; Mol. Weight: 467 g/mol; IR (KBr, cm ): υ
27
25 5
3310 (N H str.), 2955 (C H aromatic str.), 1654 (C O
str.), 1595 (C C aromatic str.), 1525, 1481, 1445 (str. for
1
product 5. Yield: 70%; H-NMR (600 MHz, DMSO-d6,
FIGURE 9 Binding pockets of urease with active binding position of 9i docked compound

10
KHAN ET AL.
triazole), 1151 (C O C str.), 1100 (C N str.), 685 (C S
3298 (N H str.), 2961 (C H aromatic str.), 1653 (C O
str.), 1581 (C C aromatic str.), 1527, 1483, 1441 (str. for
triazole), 1157 (C O C str.), 1121 (C N str.), 689 (C S
1
str.); H-NMR (600 MHz, DMSO-d , δ/ppm): 10.71 (1H, s,
6
0
00
NH-1), 10.34 (1H, s, CONH), 7.57-7.53 (5H, m, H-3 , H-
0
00
000
00000
00000
1
4
7
7
, H-5 , H-2
& H-6 ), 7.41 (2H, br.dd, J = 1.2,
str.); H-NMR (600 MHz, DMSO-d , δ/ppm): 10.74 (1H, s,
6
0
00
000
000
.6 Hz, H-2 & H-6 ), 7.36 (1H, br.d, J = 7.8 Hz, H-7),
.32-7.29 (3H, m, H-4,H-3 & H-5 ), 7.07-7.02 (1H, m,
H-6 & H-4 ), 6.97 (1H, dist.d, J = 2.04 Hz, H-2), 6.92
1H, br.dt, J = 0.7, 7.8 Hz, H-5), 4.09 (2H, s, CH -2 ),
NH-1), 10.29 (1H, s, CONH), 7.54-7.51 (3H, m, H-3 ,
00000
00000
000
000
00000
H-4 & H-5 ), 7.48 (2H, br.d, J = 7.5 Hz, H-2 &
0
0000
00000
000
000
H-6 ), 7.41 (2H, dist.d, J = 6.9 Hz, H-2 & H-6 ), 7.38
(1H, br.d, J = 7.5 Hz, H-7), 7.31 (1H, br.d, J = 7.9 Hz, H-
0
000
(
2
0
00000
00000
2
.64 (2H, t, J = 7.4 Hz, CH -3 ), 2.59 (2H, t, J = 7.5 Hz,
4), 7.14 (2H, br.d, J = 7.6 Hz, H-3 & H-5 ), 7.04 (1H,
2
0
0
13
CH -1 ), 1.89 (2H, quintet, J = 7.3 Hz, CH -2 ). C-NMR
br.t, J = 7.5 Hz, H-6), 6.97 (1H, s, H-2), 6.93 (1H, br.t, J =
2
2
0000
0000
(
150 MHz, DMSO-d , δ/ppm): 165.56 (C-1 ), 155.57 (C-
7.4 Hz, H-5), 4.10 (2H, s, CH -2 ), 2.64 (2H, t, J = 7.1
6
2
00
00
00000
0 0
Hz, CH -3 ), 2.60 (2H, t, J = 7.2 Hz, CH -1 ), 2.54 (2H, q,
2 2
00000
J = 7.3 Hz, CH -CH -4 ), 1.14 (3H, t, J = 7.2 Hz,
3 2
CH CH -4 ), 1.90-1.86 (2H, m, CH -2 ).
5
), 149.38 (C-3 ), 138.72 (C-1 ), 136.21 (C-8), 132.96
0
00
000
000
000
(
C-1 ), 129.93 (C-4 ), 129.90 (C-3 & C-5 ), 128.75 (C-
00000
00000
000
000
00000
0
13
3
& C-5 ), 127.14 (C-2 & C-6 ), 126.99 (C-9), 123.48
C-NMR
3
2
2
0
0000
00000
0000
(
C-4 ), 122.17 (C-2), 120.77 (C-6), 119.07 (C-2 & C-
(150 MHz, DMSO-d , δ/ppm): 165.32 (C-1 ), 155.56 (C-
6
00000
00 00 00000 00000
6
4
), 118.18 (C-7), 118.06 (C-5), 113.50 (C-3), 111.26 (C-
5 ), 149.43 (C-3 ), 138.91 (C-1 ), 136.43 (C-4 ), 136.24
0
000
0
0
0
000 000 000
(C-8), 132.97 (C-1 ), 129.88 (C-4 ), 129.84 (C-3 & C-
), 36.96 (C-2 ), 26.93 (C-2 ), 24.27 (C-1 ), 23.91 (C-3 );
0
00
00000 00000 000 000
& C-5 ), 127.13 (C-2 & C-6 ),
Anal. Calc. for C H N SO (467.18): C, 69.35; H, 5.39; N,
5 ), 127.91 (C-3
127.01 (C-9), 122.17 (C-2), 120.78 (C-6), 119.18 (C-2
27
25 5
00000
1
4
4.98. Found: C, 69.31; H, 5.36; N, 14.91. EI-MS m/z:
67, 334, 324, 190, 158, 143, 130, 120, 91, 78.
&
00000
C-6 ), 118.19 (C-7), 118.06 (C-5), 113.52 (C-3), 111.26
0000 00000 0
(C-4), 36.97 (C-2 ), 27.56 (CH CH -4 ), 26.95 (C-2 ),
3 2
2
-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,
0 0 00000
24.29 (C-1 ), 23.93 (C-3 ), 15.62 (CH CH -4 ); Anal.
3 2
2,4-triazol-3-yl}sulfanyl)-N-(2 methylphenyl) acet-
amide (9b). Light brown sticky liquid; Yield: 91%; Mol.
Calc. for C H N SO (495.21): C, 70.27; H, 5.90; N,
29 29 5
Formula: C H N SO; Mol. Weight: 481 g/mol; IR (KBr,
cm ): υ 3286 (N H str.), 2945 (C H aromatic str.), 1652
14.13. Found: C, 70.21; H, 5.86; N, 14.08. EI-MS (m/z):
495, 352, 334, 190, 158, 143, 130, 120, 91, 78.
28
27 5
−
1
(
(
6
1
C O str.), 1599 (C C aromatic str.), 1517, 1478, 1441
N-(4-Ethoxyphenyl)-2-({5-[3-(1H-indol-3-yl)pro-
pyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9d). Brown sticky liquid; Yield: 82%; Mol.
Formula: C H N SO ; Mol. Weight: 511 g/mol; IR
str. for triazole), 1159 (C O C str.), 1115 (C N str.),
1
89 (C S str.); H-NMR (600 MHz, DMSO-d , δ/ppm):
6
0.72 (1H, s, NH-1), 9.71 (1H, s, CONH), 7.55-7.54 (1H,
29
29
5
2
0
00
000
000
−1
m, H-3 , H-4 & H-5 ), 7.46 (1H, br.d, J = 7.7 Hz, H-
6
(KBr, cm ): υ 3292 (N H str.), 2949 (C H aromatic
str.), 1666 (C O str.), 1599 (C C aromatic str.), 1528,
1487, 1449 (str. for triazole), 1159 (C O C str.), 1109
00000
000
000
), 7.41 (2H, dist.d, J = 5.9 Hz, H-2 & H-6 ), 7.37
(1H, br.d, J = 7.7 Hz, H-7), 7.31 (1H, br.d, J = 7.8 Hz, H-
00000
1
4
), 7.19 (1H, br.d, J = 7.5 Hz, H-3 ), 7.14 (1H, br.t,
(C N str.), 688 (C S str.); H-NMR (600 MHz, DMSO-
0
0000
00000
J = 7.4 Hz, H-5 ), 7.08-7.03 (2H, m, H-6, & H-4 ), 6.97
1H, s, H-2), 6.93 (1H, br.t, J = 7.2 Hz, H-5), 4.12 (2H, s,
d , δ/ppm): 10.72 (1H, s, NH-1), 10.18 (1H, s, CONH),
6
000 000 000
(
7.57-7.51 (3H, m, H-3 , H-4 & H-5 ), 7.45 (2H, br.d,
0
000
0
00000 00000
J = 8.8 Hz, H-2 & H-6 ), 7.41 (2H, dist.d, J = 6.4 Hz,
CH -2 ), 2.65 (2H, t, J = 7.2 Hz, CH -3 ), 2.60 (2H, t, J =
7
J = 7.1 Hz, CH -2 ). C-NMR (150 MHz, DMSO-d ,
δ/ppm): 165.83 (C-1 ), 155.61 (C-5 ), 149.52 (C-3 ),
1
2
5
1
1
3
2
2
0
00000
000
000
.2 Hz, CH -1 ), 2.19 (3H, s, CH -2 ), 1.88 (2H, quintet,
H-2 & H-6 ), 7.36 (1H, br.d, J = 7.8 Hz, H-7), 7.30 (1H,
br.d, J = 8.0 Hz, H-4), 7.04 (1H, br.t, J = 7.5 Hz, H-6),
6.97 (1H, s, H-2), 6.92 (1H, br.t, J = 7.4 Hz, H-5), 6.86
2
3
0
13
2
6
0000
00
00
0
0000
000
00000 00000
(2H, br.d, J = 8.8 Hz, H-3 & H-5 ), 4.06 (2H, s, CH -
2
36.24 (C-8), 135.98 (C-1 ), 132.97 (C-1 ), 131.13 (C-
0
0
0000
00
00000
000
000
0000 00000
2 ), 3.96 (2H, q, J = 6.9 Hz, CH CH O-4 ), 2.64 (2H, t,
3 2
), 130.28 (C-3 ), 129.91 (C-4 ), 129.86 (C-3 & C-
), 127.14 (C-2 & C-6 ), 127.01 (C-9), 125.94 (C-6 ),
25.14 (C-4 ), 124.24 (C-5 ), 122.19 (C-2), 120.78 (C-6),
18.19 (C-7), 118.06 (C-5), 113.51 (C-3), 111.26 (C-4),
6.43 (C-2 ), 26.98 (C-2 ), 24.29 (C-1 ), 23.92 (C-3 ), 17.72
000
000
00000
0 0
J = 7.3 Hz, CH -3 ), 2.59 (2H, t, J = 7.3 Hz, CH -1 ), 1.88
2 2
0
0000
00000
0
(2H, quintet, J = 7.3 Hz, CH -2 ), 1.29 (3H, t, J = 6.9 Hz,
2
0
0000 13
CH CH O-4 ). C-NMR (150 MHz, DMSO-d , δ/ppm):
3
2
6
0
000
0
0
0
0000 00000 00
164.99 (C-1 ), 155.53 (C-4 ), 154.60 (C-5 ), 149.41 (C-
0
0000
00 000 00000
3 ), 136.23 (C-8), 132.99 (C-1 ), 131.79 (C-1 ), 129.88
(CH -2 ); Anal. Calc. for C H N SO (481.19): C,
3
28 27 5
000 000 000 000 000
(C-4 ), 129.84 (C-3 & C-5 ), 127.16 (C-2 & C-6 ),
6
1
1
9.83; H, 5.65; N, 14.54. Found: C, 69.79; H, 5.60; N,
4.48. EI-MS (m/z): 481, 338, 334, 190, 158, 143,
30, 120, 91, 78.
N-(4-Ethylphenyl)-2-({5-[3-(1H-indol-3-yl)propyl]-
-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acetamide
00000
127.00 (C-9), 122.18 (C-2), 120.77 (C-6), 120.61 (C-2
C-6 ), 118.19 (C-7), 118.05 (C-5), 114.39 (C-3
&
0
0000
00000
& C-
00000
0
0000
5
), 113.51 (C-3), 111.25 (C-4), 63.04 (CH CH O-4 ),
3
2
0000 0 0 0
4
(
36.91 (C-2 ), 26.95 (C-2 ), 24.30 (C-1 ), 23.93 (C-3 ), 14.63
00000
(CH CH O-4 ); Anal. Calc. for C H N SO
3 2 29 29 5 2
9c). Dark brown sticky liquid; Yield: 86%; Mol. Formula:
−1
C H N SO; Mol. Weight: 495 g/mol; IR (KBr, cm ): υ
(511.20): C, 68.08; H, 5.71; N, 13.69. Found: C, 68.05; H,
29
29 5

KHAN ET AL.
11
0
0000 00000
), 13.84 (CH -2 ); Anal. Calc. for C H N SO
3 29 29 5
5
1
.66; N, 13.66. EI-MS (m/z): 511, 369, 368, 353, 334, 190,
58, 143, 130, 91, 78.
4
(495.21): C, 70.27; H, 5.90; N, 14.13. Found: C, 70.23; H,
5.85; N, 14.07. EI-MS (m/z): 495, 352, 334, 190, 158, 143,
130, 120, 91, 77.
N-(2,5-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9g). Brown sticky liquid; Yield: 93%; Mol. For-
mula: C H N SO; Mol. Weight: 495 g/mol; IR (KBr,
N-(2,3-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9e). Brown sticky liquid; Yield: 78%; Mol. For-
mula: C H N SO; Mol. Weight: 495 g/mol; IR (KBr,
29
29 5
−
1
cm ): υ 3296 (N H str.), 2958 (C H aromatic str.), 1652
(C O str.), 1591 (C C aromatic str.), 1531, 1489, 1444
29
29 5
−1
(str. for triazole), 1155 (C O C str.), 1122 (C N str.),
cm ): υ 3317 (N H str.), 2955 (C H aromatic str.), 1654
(C O str.), 1599 (C C aromatic str.), 1535, 1484, 1449
1
6
87 (C S str.); H-NMR (600 MHz, DMSO-d , δ/ppm):
6
1
0.75 (1H, s, NH-1), 9.66 (1H, s, CONH), 7.56-7.55 (3H,
(str. for triazole), 1155 (C O C str.), 1103 (C N str.),
0
00
000
000
1
m, H-3 , H-4 & H-5 ), 7.42 (2H, dist.d, J = 5.8 Hz, H-
681 (C S str.); H-NMR (600 MHz, DMSO-d , δ/ppm):
6
0
00
000
2
& H-6 ), 7.38 (1H, br.d, J = 7.7 Hz, H-7), 7.32 (2H, br.
10.74 (1H, s, NH-1), 9.68 (1H, s, CONH), 7.56-7.53 (3H,
00000
000
000
000
000
000
t, J = 8.28 Hz, H-4 & H-6 ), 7.05 (1H, br.t, J = 7.4 Hz,
H-6), 6.99 (1H, s, H-2), 6.96-6.93 (3H, m, H-5,H-4 & H-
m, H-3 , H-4 & H-5 ), 7.44-7.41 (2H, m, H-2 & H-6 ),
00000
7.37 (1H, br.d, J = 7.8 Hz, H-7), 7.31 (1H, br.d, J = 8.1 Hz,
0
0
0000
0000
00000
5
3
2
2
1
3
1
1
1
1
2
1
7
1
1
), 4.11 (2H, s, CH -2 ), 2.67 (2H, t, J = 7.2 Hz, CH -
H-4), 7.29 (1H, s, H-6 ), 7.07-7.03 (2H, m, H-6 & H-
2
2
00000
0
00000
), 2.61 (2H, t, J = 7.3 Hz, CH -1 ), 2.23 (3H, s, CH -3 ),
.16 (3H, s, CH -2 ), 1.90 (2H, quintet, J = 7.3 Hz, CH2-
3
), 7.00 (1H, dist.d, J = 1.86 Hz, H-2), 6.89-6.86 (1H, m,
2
3
0
0000
00000
H-4 ), 6.93 (1H, br.t, J = 7.6 Hz, H-5), 4.12 (2H, s, H-
3
0
0
0
13
0000 0
2 ), 2.75 (2H, t, J = 7.5 Hz, CH -3 ), 2.61 (2H, t, J = 7.4
2
). C-NMR (150 MHz, DMSO-d , δ/ppm): 166.24 (C-
6
000
0000
00
00
0 00000 00000
Hz, CH -1 ), 2.22 (3H, s, CH -5 ), 2.15 (3H, s, CH -2 ),
2 3 3
), 156.10 (C-5 ), 150.03 (C-3 ), 136.76 (C-8), 134.75 (C-
00000
000
00000
0
1.91 (2H, s, CH -2 ). C-NMR (150 MHz, DMSO-d ,
2 6
13
), 133.90 (C-1 ), 133.49 (C-1 ), 131.65 (C-2 ),
0
0000
000
000
000
0000 00 00
δ/ppm): 165.77 (C-1 ), 155.60 (C-5 ), 149.57 (C-3 ),
31.24 (C-4 ), 130.39 (C-4 ), 130.34 (C-3 & C-5 ),
27.58 (C-2 & C-6 ), 127.53 (C-9), 126.86 (C-5 ),
24.84 (C-6 ), 122.68 (C-2), 121.27 (C-6), 118.69 (C-7),
18.56 (C-5), 114.04 (C-3), 111.77 (C-4), 36.95 (C-2 ),
7.49 (C-2 ), 24.80 (C-1 ), 24.42 (C-3 ), 20.91 (CH -3 ),
8.13 (CH -2 ); Anal. Calc. for C H N SO (495.21): C,
0
00
000
00000
00000 00000
136.25 (C-8), 135.77 (C-1 ), 134.95 (C-5 ), 132.97 (C-
0
0000
000 00000 000 000 000
1 ), 130.08 (C-3 ), 129.90 (C-4 ), 129.86 (C-3 & 5 ),
0
000
00000 000 000
128.99 (C-2 ), 127.14 (C-2 & 6 ), 127.03 (C-9), 125.61
0
0
0
00000
00000
00000
(C-4 ), 125.61 (C-6 ), 122.19 (C-2), 120.78 (C-6),
118.20 (C-7), 118.06 (C-5), 113.52 (C-3), 111.28 (C-4),
36.44 (C-2 ), 26.99 (C-2 ), 24.29 (C-1 ), 23.93 (C-3 ),
3
0
0000
3
29 29 5
0000 0 0 0
0.27; H, 5.90; N, 14.13. Found: C, 70.22; H, 5.85; N,
4.08. EI-MS (m/z): 495, 352, 334, 190, 158, 143,
30, 120, 91, 78.
0
0000
00000
20.57 (CH -5 ), 14.04 (CH -2 ); Anal. Calc. for
3
3
C H N SO (495.21): C, 70.27; H, 5.90; N, 14.13.
2
9
29
5
N-(2,4-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
Found: C, 70.25; H, 5.86; N, 14.10. EI-MS (m/z):
495, 352, 334, 190, 158, 143, 130, 120, 91, 77.
N-(2,6-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9h). White colored amorphous powder; Yield:
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9f). Brown sticky liquid; Yield: 71%; Mol. For-
mula: C H N SO; Mol. Weight: 495 g/mol; IR (KBr,
2
9
29 5
−
1
cm ): υ 3274 (N H str.), 2953 (C H aromatic str.), 1703
ꢀ
(
(
6
1
C O str.), 1590 (C C aromatic str.), 1531, 1489, 1452
79%; Melting Point 93 C; Mol. Formula: C H N SO;
29
29 5
−
1
str. for triazole), 1159 (C O C str.), 1100 (C N str.),
Mol. Weight: 495 g/mol; IR (KBr, cm ): υ 3286 (N H
str.), 2957 (C H aromatic str.), 1656 (C O str.), 1599
(C C aromatic str.), 1527, 1481, 1441 (str. for triazole),
1141 (C–O–C str.), 1101 (C N str.), 677 (C S str.); H-
NMR (600 MHz, DMSO-d , δ/ppm): 10.72 (1H, s, NH-1),
1
66 (C S str.); H-NMR (600 MHz, DMSO-d , δ/ppm):
6
0.74 (1H, s, NH-1), 9.77 (1H, s, CONH), 7.55 (3H, br.s,
0
00
000
000
000
000
1
H-3 , H-4 & H-5 ), 7.42 (2H, br.s, H-2 & H-6 ), 7.37
1H, dist.d, J = 5.3 Hz, H-7), 7.31 (1H, dist.d, J = 5.8 Hz,
(
6
00000
00000
000 000
H-4), 7.16 (1H, br.s, H-6 ), 7.03 (2H, br.s, H-6 & H-3 ),
.99 (1H, s, H-2), 6.98 (1H, s, H-5 ), 6.93 (1H, s, H-5),
.11 (2H, s, CH -2 ), 2.65 (2H, s, CH -3 ), 2.60 (2H, s,
9.62 (1H, s, CONH), 7.56-7.55 (3H, m, H-3 , H-4 & H-
5 ), 7.42 (2H, m, H-2 & H-6 ), 7.37 (1H, br.d,
00000
000
000
000
6
4
0
000
0
J = 7.8 Hz, H-7), 7.31 (1H, br.d, J = 7.8 Hz, H-4),
2
2
0
00000
00000
00000
00000
00000
CH -1 ), 2.22 (3H, s, CH -4 ), 2.05 (3H, s, CH -7 ),
7.03-7.02 (4H, m, H-6,H-3 , H-4 & H-5 ), 6.97 (1H,
2
3
3
0
13
1
.89 (2H, s, CH -2 ). C-NMR (150 MHz, DMSO-d ,
δ/ppm): 165.82 (C-1 ), 155.56 (C-5 ), 149.49 (C-3 ),
36.92 (C-1 ), 136.24 (C-8), 135.72 (C-2 ), 132.99 (C-
), 130.74 (C-4 ), 129.89 (C-4 ), 129.85 (C-3 & 5 ),
27.15 (C-2 & 6 ), 127.01 (C-9), 126.93 (C-3 ), 125.15
C-5 ), 122.96 (C-6 ), 122.18 (C-2), 120.77 (C-6), 118.19
s, H-2), 6.93 (1H, br.t, J = 7.1 Hz, H-5), 4.12 (2H, s, CH -
2
6
2
0000
00
00
0000 0
2 ), 2.65 (2H, t, J = 7.2 Hz, CH -3 ), 2.60 (2H, t, J = 7.2
2
0
0000
00000
0 00000 00000
Hz, CH -1 ), 2.09 (6H, s, CH -2 & CH -6 ), 1.87 (2H,
2 3 3
1
1
1
(
(
0
00
00000
000
000
000
000
0
quintet, J = 7.2 Hz, CH -2 ). C-NMR (150 MHz, DMSO-
2
13
0
00
00000
0000 00 00
d , δ/ppm): 165.34 (C-1 ), 155.49 (C-5 ), 149.38 (C-3 ),
6
00000
00000
00000 00000 00000
& C-6 ), 134.64 (C-1 ),
133.01 (C-1 ), 129.86 (C-4 ), 129.82 (C-3 & C-5 ),
00000 00000 000 0000
127.59 (C-3 & C-5 ), 127.13 (C-2 & C-6 ), 127.00
136.23 (C-8), 135.13 (C-2
000 000 000 000
C-7), 118.05 (C-5), 113.51 (C-3), 111.27 (C-4), 36.39 (C-
0000
0
0
0
2
), 26.97 (C-2 ), 24.30 (C-1 ), 23.93 (C-3 ), 20.09 (CH -
3

12
KHAN ET AL.
0
0000
0 00000 00000
Hz, CH -1 ), 2.21 (6H, s, CH -3 & CH -5 ), 1.88 (2H,
2 3 3
(
(
C-9), 126.50 (C-4 ), 122.18 (C-2), 120.78 (C-6), 118.19
0
quintet, J = 7.4 Hz, CH -2 ). C-NMR (150 MHz, DMSO-
2
13
C-7), 118.05 (C-5), 113.51 (C-3), 111.26 (C-4), 35.84 (C-
0
0
000
0000
0
00
0
0000 00 00
d , δ/ppm): 165.40 (C-1 ), 155.53 (C-5 ), 149.40 (C-3 ),
6
2
2
7
1
1
), 27.00 (C-2 ), 24.28 (C-1 ), 23.91 (C-3 ), 17.95 (CH -
& CH -6 ); Anal. Calc. for C H N SO (495.21): C,
0.27; H, 5.90; N, 14.13. Found: C, 70.23; H, 5.85; N,
4.09. EI-MS (m/z): 495, 352, 334, 190, 158, 143,
30, 120, 91, 77.
3
00000
00000
00000 00000
& C-5 ), 136.23 (C-8),
138.59 (C-1 ), 137.70 (C-3
3
29 29 5
000 000 000 000
132.98 (C-1 ), 129.88 (C-4 ), 129.84 (C-3 & C-5 ), 127.14
000
000
00000
(C-2 & C-6 ), 127.00 (C-9), 125.00 (C-4 ), 122.18 (C-2),
00000
120.77 (C-6), 118.19 (C-7), 118.05 (C-5), 116.85 (C-2 & C-
00000 0000 0
N-(3,4-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
6 ), 113.51 (C-3), 111.25 (C-4), 36.96 (C-2 ), 26.96 (C-2 ),
0
0
00000 00000
& CH -5 );
3
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9i). Brown colored sticky liquid; Yield: 69%; Mol.
Formula: C H N SO; Mol. Weight: 495 g/mol; IR (KBr,
24.29 (C-1 ), 23.93 (C-3 ), 21.04 (CH -3
3
Anal. Calc. for C H N SO (495.21): C, 70.27; H, 5.90; N,
29 29 5
14.13. Found: C, 70.23; H, 5.85; N, 14.07. EI-MS (m/z):
495, 352, 334, 190, 158, 143, 130, 120, 91, 77.
29
29 5
−
1
cm ): υ 3293 (N H str.), 2966 (C H aromatic str.), 1642
(
(
6
1
C O str.), 1598 (C C aromatic str.), 1529, 1488, 1447
N-(2-Ethyl-6-methylphenyl)-2-({5-[3-(1H-indol-
3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)
acetamide (9k). Light brown colored sticky liquid;
Yield: 76%; Mol. Formula: C H N SO; Mol. Weight:
509 g/mol; IR (KBr, cm ): υ 3250 (N H str.), 2956
(C H aromatic str.), 1684 (C O str.), 1591 (C C aro-
str. for triazole), 1153 (C O C str.), 1126 (C N str.),
1
87 (C S str.); H-NMR (600 MHz, DMSO-d , δ/ppm):
6
0.72 (1H, s, NH-1), 10.19 (1H, s, CONH), 7.55-7.51 (3H,
30
31 5
0
00
000
000
−1
m, H-3 , H-4 & H-5 ), 7.41 (2H, br.d, J = 7.6 Hz, H-
2
H-2 ), 7.31 (1H, br.d, J = 8.0 Hz, H-4), 7.27 (1H, br.d,
J = 7.9 Hz, H-6 ), 7.04 (2H, dist.t, J = 7.6 Hz, H-6 & H-
5
4
2
2
2
1
000
000
& H-6 ), 7.37 (1H, br.d, J = 7.6 Hz, H-7), 7.33 (1H, s,
00000
matic str.), 1508, 1471, 1433 (str. for triazole), 1157
0
0000
1
(C O C str.), 1126 (C
N str.), 696 (C S str.); H-
0
0000
), 6.99 (1H, s, H-2), 6.92 (1H, br.t, J = 7.5 Hz, H-5),
NMR (600 MHz, DMSO-d , δ/ppm): 10.72 (1H, s, NH-1),
6
0
000
0
000 000
9.61 (1H, s, CONH), 7.56-7.55 (3H, m, H-3 , H-4 & H-
.07 (2H, s, CH -2 ), 2.66 (2H, t, J = 7.3 Hz, CH -3 ),
.59 (2H, t, J = 7.4 Hz, CH -1 ), 2.17 (3H, s, CH -4 ),
2
2
0
00000
000
000
000
5 ), 7.42-7.41 (2H, m, H-2 & H-6 ), 7.37 (1H, br.d,
2
3
0
0000
.15 (3H, s, CH -3 ), 1.89 (2H, quintet, J = 7.3 Hz, CH2-
J = 7.9 Hz, H-7), 7.31 (1H, br.d, J = 7.5 Hz, H-4), 7.11
3
0
0
13
00000
). C-NMR (150 MHz, DMSO-d , δ/ppm): 165.21 (C-
), 155.53 (C-5 ), 149.42 (C-3 ), 136.48 (C-3 ), 136.31
(1H, dist.d, J = 7.7 Hz, H-4 ), 7.05-7.02 (3H, m, H-6,H-
6
000
00
00
00000
00000
00000
3
& H-5 ), 6.97 (1H, s, H-2), 6.93 (1H, br.t, J = 7.4
0
0000
000
00000
0000
(C-1 ), 136.23 (C-8), 132.99 (C-1 ), 131.21 (C-4 ),
Hz, H-5), 4.12 (2H, s, CH -2 ), 2.65 (2H, t, J = 7.1 Hz,
2
0
00
00
000
000
00000
0 0
CH -3 ), 2.60 (2H, t, J = 6.7 Hz, CH -1 ), 2.46 (2H, q, J =
2 2
1
1
29.84 (C-4 ), 129.80 (C-3 & C-5 ), 129.56 (C-5 ),
0
000
00000 00000
7.3 Hz, CH CH -2 ), 2.07 (3H, s, CH -6 ), 1.88 (2H,
3 2 3
27.15 (C-2 & C-6 ), 127.00 (C-9), 122.18 (C-2), 120.77
0
0000
0
quintet, J = 7.2 Hz, CH -2 ) 1.03 (3H, t, J = 7.3 Hz,
2
(C-6), 120.29 (C-2 ), 118.19 (C-7), 118.05 (C-5), 116.63
0
0000
0000
00000
CH CH -2 ). C-NMR (150 MHz, DMSO-d , δ/ppm):
3 2 6
13
(C-6 ), 113.51 (C-3), 111.26 (C-4), 36.96 (C-2 ), 26.95
0
0
0
00000
0000 00 00
(C-2 ), 24.29 (C-1 ), 23.93 (C-3 ), 19.56 (CH -3 ), 18.74
165.76 (C-1 ), 155.47 (C-5 ), 149.41 (C-3 ), 141.04 (C-
3
0
0000
00000 00000 00000
), 136.23 (C-8), 135.58 (C-6 ), 134.01 (C-2 ), 133.00
(CH -4 ); Anal. Calc. for C H N SO (495.21): C,
1
3
29 29 5
000 000 000 000
7
1
1
0.27; H, 5.90; N, 14.13. Found: C, 70.25; H, 5.87; N,
4.10. EI-MS (m/z): 495, 352, 334, 190, 158, 143,
30, 120, 91, 77.
(C-1 ), 129.86 (C-4 ), 129.81 (C-3 & C-5 ), 127.59 (C-
0
0000
000 000 00000
), 127.11 (C-2 & C-6 ), 126.87 (C-3 ), 127.00 (C-9),
4
0
0000
125.94 (C-5 ), 122.17 (C-2), 120.77 (C-6), 118.18 (C-7),
0000
N-(3,5-Dimethylphenyl)-2-({5-[3-(1H-indol-3-yl)
118.04 (C-5), 113.51 (C-3), 111.26 (C-4), 35.79 (C-2 ),
0 0 00000
27.00 (C-2 ), 24.28 (C-1 ), 24.17 (CH CH -2 ), 23.91 (C-
3 2
propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)acet-
amide (9j). Red colored amorphous powder; Yield: 84%;
Melting Point 91 C; Mol. Formula: C H N SO; Mol.
0
00000
00000
3 ), 17.96 (CH CH -2 ), 14.58 (CH -6 ); Anal. Calc. for
3
2
3
ꢀ
C H N SO (509.22): C, 70.70; H, 6.13; N, 13.74.
30 31 5
Found: C, 70.63; H, 6.08; N, 13.68. EI-MS (m/z): 509, 366,
334, 190, 158, 143, 130, 120, 91, 78.
In vitro urease inhibition assay. Jack bean urease
activity was done by determining the quantity of NH3
29
29
5
−1
Weight: 495 g/mol; IR (KBr, cm ): υ 3282 (N H str.),
945 (C H aromatic str.), 1664 (C O str.), 1599 (C C
aromatic str.), 1535, 1473, 1456 (str. for triazole), 1155
2
1
(C O C str.), 1116 (C N str.), 689 (C S str.); H-NMR
(
(
7
600 MHz, DMSO-d , δ/ppm): 10.72 (1H, s, NH-1), 10.17
formed with indo-phenols method as described in
6
000
000
000
[49–51]
1H, s, CONH), 7.56-7.52 (3H, m, H-3 , H-4 & H-5 ),
literature.
Equimolar concentrations (20 μL) of
000
000
.40 (2H, dist.d, J = 7.7 Hz, H-2 & H-6 ), 7.36 (1H, br.d,
compound and urease enzyme with KH PO buffer
2 4
J = 7.8 Hz, H-7), 7.31 (1H, br.d, J = 8.0 Hz, H-4), 7.17
(pH 8.2) were left in 96 well plate for half hour incuba-
tion at 37 C. Equal amount (50 μL) of phenol and base
0
0000
00000
ꢀ
(2H, br.s, H-2 & H-6 ), 7.04 (1H, br.t, J = 7.8 Hz, H-
6
), 6.97 (1H, dist.d, J = 1.4 Hz, H-2), 6.92 (1H, br.t,
were poured into each well. A microplate reader
(SpectraMax ABS) was used to calculate the absorbance
(at 625 nm) after 10 minutes, all the readings were taken
00000
J = 7.4 Hz, H-5), 6.69 (1H, br.s, H-4 ), 4.07 (2H, s, CH2-
0
000
0
), 2.65 (2H, t, J = 7.4 Hz, CH -3 ), 2.59 (2H, t, J = 7.4
2
2

KHAN ET AL.
13
in triplicate. Inhibition activity was calculated by follow-
ing formula:
helped in three-dimensional graphical interpretations of
the entire docked compounds.
Urease inhibition activity ð%Þ
À
Á
−
=
OD
OD
× 100 =ODcontrol,
4 | CONCLUSION
control
sample
where ODcontrol is the optical density in the absence of sample
and ODsample is the optical density in the presence of sample.
Standard enzyme inhibitor used for urease is thiourea.
Hemolytic assay. Bovine blood samples were taken
for the formation of RBCs suspension following already
Aimed synthesis of indole-triazole hybrids bearing
N-substituted acetamides (9a-k) was accomplished in
excellent yields. All the molecules exposed an efficient
potential against urease enzyme and depicted lower IC50
values relative to standard used. Moreover, all the com-
pounds were attributed with mild cytotoxicity and suit-
able free radical scavenging potential. Consequently, it
was concluded that these chemical entities might be com-
plemented as valuable additions to the already existing
anti-ulcer therapeutic agents.
[52,53]
reported method.
Solution (10 mg/mL, 20 μL) of
synthesized compound was incubated with 180 μL of red
blood cells suspension at room temperature. Triton 100-X
and PBS was used as positive and negative control,
respectively. Percentage of hemolysis was calculated by
following formula:
Statistical analysis. Microsoft Excel 2010 was used
to perform statistical analysis. The results are displayed
as mean ± SEM with CL 96%.
%
of hemolysis
Absorbance of sample−Absorbance of negative control
=
× 100:
Absorbance of positive control
ACKNOWLEDGMENT
We highly acknowledge, Higher Education Commission
(HEC), Pakistan, for their kind support in the providing
the financial support and spectroscopic facilities.
Kinetic assay. Mode of inhibition was followed
through kinetic analysis. For this purpose, 9i derivative
with best IC50 value was selected. Kinetic study was per-
formed by changing the amount of urea with varying con-
centrations of selected compound. Procedure is same for all
ORCID
Muhammad A. Abbasi https://orcid.org/0000-0003-
3439-9286
[54]
kinetic activities as discussed in urease inhibition assay.
Free radical scavenging assay. Previously reported
method was utilized after a slight change, to measure the
radical scavenging potential of synthesized com-
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