An expedient synthesis of N-(1-(5-mercapto-4-((substituted benzylidene)amino)-4H-1,2,4-triazol-3-yl)-2-phenylethyl)benzamides as jack bean urease inhibitors and free radical scavengers: Kinetic mechanism and molecular docking studies

Article abstract of DOI:10.1111/cbdd.12998

In this study, some new azomethine-triazole hybrids 5a–5l derived from N-benzoyl-L-phenylalanine were synthesized and characterized. The synthesized compounds showed first-rate, urease inhibition, and compounds 5c and 5e were found to be most effective inhibitors with 0.0137?±?0.00082?μm and 0.0183?±?0.00068?μm, respectively (thiourea 15.151?±?1.27?μm). The kinetic mechanism of urease inhibition revealed the compounds 5c and 5e to be non-competitive inhibitors, whereas compounds 5d and 5j were found to be of mixed-type inhibitors. Docking studies also indicated better interaction patterns with urease enzyme. The results of enzyme inhibition, kinetic mechanism and molecular docking suggest that these compounds can serve as lead compounds in the design of more effective urease inhibitors.

Full text of DOI:10.1111/cbdd.12998

DR. AAMER SAEED (Orcid ID : 0000-0002-7112-9296)
Article type : Research Article
An Expedient Synthesis of N-(1-(5-mercapto-4-((substituted benzylidene)amino)-4H-
1,2,4-triazol-3-yl)-2-phenylethyl)benzamides as Jack bean Urease inhibitors and free
radical scavengers; kinetic mechanism and molecular docking studies.
a∗
* a
a
a
Aamer Saeed , Fayaz Ali Larik , Pervaiz Ali Channar , Haroon Mehfooz , Mohammad
a
b
b
b
Haseeb Ashraf , Qamar Abbas , Mubashir Hassan , Sung-Yum Seo ,
a
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
b
Department of Biological Sciences, College of Natural Sciences, Kongju National
University, 56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea
Abstract
In the present study some new azomethine-triazole hybrids 5a-5l derived from N-benzoyl-L-
phenylalanine were synthesized and characterized. The synthesized compounds showed first-
rate, urease inhibition and compounds 5c and 5e were found to be most effective inhibitors
with 0.0137±0.00082 µM and 0.0183±0.00068 µM respectively (thiourea 15.151±1.27 µM).
The kinetic mechanism of urease inhibition revealed the compounds 5c and 5e to be non-
∗
Email: aamersaeed@yahoo.com, fayazali@chem.qau.edu.pk Tel +92-51-9064-2128; Fax: +92-
51-9064-2241
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12998
This article is protected by copyright. All rights reserved.

competitive inhibitors, whereas compounds 5d and 5j were found to be of mixed type
inhibitors. Docking studies also indicated better interaction patterns with urease enzyme. The
results of enzyme inhibition, kinetic mechanism and molecular docking suggest that these
compounds can serve as lead compounds in the design of more effective urease inhibitors.
Keywords: Drug-design; Urease Inhbitors; antioxidant activity; triazolyl benzamides;
Kinetic Mechanism; Molecular docking
Introduction
In 1926, Sumner reported the crystal structure of urease enzyme. After 70 years, Karplus
proposed the correct structure of urease (1). Urease is pervasive in nature, it is found in soils,
microorganisms including bacteria, fungi, algae and in plants and invertebrates (2). Urease
(
urea amidohydrolase, EC 3.5.1.5) is nickel containing metalloenzyme belongs to the super
family of aminohydrolase and phosphotriestreases (3). Urease hydrolysis the urea into carbon
dioxide and ammonia, which results in the deficiency of nitrogen in the soil and toxicity of
excess ammonia makes soil infertile and as a result in the spoiling of food and crops. In
humans, it helps Helicobacter pylori to survive in the stomach and which can cause gastritis,
gastric, duodenal ulcers, and even gastric cancer (4). Urease is directly involved in the
formation of infectious stones and contributes to the development of urolithiasis,
pyelonephritis, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation (5,
6). Urease contain two nickel (II) cations which play pivotal role in the breaking down of
urea into ammonia and carbondioxide.
Ciurli et al. proposed mechanism related to urease hydrolysis by urease is still widely
recognized (7). The two nickel cations interact with hydroxyl ions, and the three molecules of
This article is protected by copyright. All rights reserved.

water immerse in the active site of enzyme. The hydrogen bonding plays a critical part in the
binding of urea with two nickel atoms in the active site of enzyme, because urea possesses
two hydrogen bonding sites, the carbonyl and the amino group. The binding affinity of urea is
not much stronger so the bridging of urea with metal ions is stabilized by amino acid residues
His a222, His a232 and Ala a366, found in the active site of the enzyme. The carbonyl of
urea is susceptible to the nucelophilic attack, conformational transitions favour the
nucleophilic attack of hydroxide ion on the electrophilic centre of urea in a tertahderal
fashion which after protonation collapses into ammonia gas and carbamate motif and
carbamate is cleaved rapidly into carbon dixode and second molecule of ammonia. This rapid
release of ammonia promptly enhances the basicity by increasing pH from 6.5 to 9.0 and this
paves the way for the survival of Helicobacter pylori which causes peptic and gastric ulcer
which may lead into cancer in the digestive system.
Chemical bio-drug designing relies on the innovative ideas and medicinal chemists have
come up with new ideas to deisgn the urease inhibitors by making use of small molecules
which can significantly play active part in inhibiting urease enzyme (8-15). The synthetic
derivatives are quite reliable as they can be chemically modified. Since, several classes of
organic compounds has been evaluated as urease inhbitors such as hydroxamic acid
derivatives are best known to inhibit urease activity. In addition, phosphoramidate based
derivatives showed great potential against urease. However, phosphoramidate are prone to
decomposition at lower pH value and fetal dysfunctioning in rats hampers the applicability of
hydroxamide compounds to be used in vivo. Metal containing Schiff bases have also been
investigated as urease inhibitors but they are toxic to human health. Following are the
structures of urease inhbitors bearing different classes of organic compounds (Figure 1).
This article is protected by copyright. All rights reserved.

Carbocyclic chemistry has been overshadowed by heterocyclic chemistry, especially in the
last few decades. The N-substituted amino acids are key building blocks in the design and
syntheses of heterocyclic compounds (21). Diverse Cd(II) compounds based on N-benzoyl-L-
glutamic acid and N-donor ligands display photoluminescent properties (22). 1,2,4-Triazole
moiety represents the major pharmacophore, possessing a wide range of biological activities
(23-24). Similarly, hydrazone compounds possessing hydrazine moiety –NHN=CH-
constitute an important class of compounds for new drug development (25). These ligands
play an important role in the development of coordination chemistry. Synthesis of new
spirooxindole derivatives through 1,3-dipolar cycloaddition of azomethine ylides and their
antitubercular activity have been reported. In addition, the synthesized compounds serve as
an excellent ligands for metal complexes (26, 27). Moreover, our research group was
motivated by the biological properties of triazol systems, imine functionality coupled with
our synthetic and biological experiences in the field of heterocycles. Herein, the synthesis of
title hybrid compounds by obtained by condensation of N-(1-(4-amino-5-mercapto-4H-1,2,4-
triazol-3-yl)-2-phenylethyl)benzamide with variously substituted benzaldehydes is described.
1
1
. Experimental
.1 Methods and material
All the solvents used were dried and distilled according to standard procedures prior to use.
All reagents were purchased from Sigma Aldrich and used without further purification.
Melting point was determined on a Stuart SMP3 melting point apparatus. The NMR spectra
1
13
were recorded on a Bruker 300 ( H at 300 MHz and for C at 75.5 MHz) and chemical shifts
This article is protected by copyright. All rights reserved.

are reported in ppm versus tetramethylsilane or the residual solvent resonance used as an
internal reference standard. The reactions were monitored by thin layer chromatography
using Thin layer chromatography (TLC) was performed using aluminum sheets coated with
silica gel F254 (Merck) detection by means of UV light at 254 and 360ꢀnm.
1.2 Synthesis of N-benzoyl-L-phenylalanine (1)
8.5g, L-phenylalanine, 3g, sodium hydroxide, 25ml water, 30 ml ethanol and 6.5 g benzoyl
o
chloride were kept in a water bath at 100 C, for four hours, and the precipitated and
recovered N-benzyl-L-phenylalanine was washed with water, ether, and ethanol and
recrystallized from 40% acetic acid. The purified needle-shaped crystals weighed 5.7g (62%
o
yield) and melted at 234 C.
1
.3
Synthesis
of
N-(1-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (4):
A mixture of N-benzoyl-L-phenylalanine (0.1 mol) and thiosemicarbazide (0.1 mol) in dry
pyridine (20 ml) was added to a 50ꢀmL Erlenmeyer flask. The mixture was subjected to reflux
for the 30 minutes. After the completion of the reaction, the reaction mixture was poured into
water and pH was adjusted to afford compound 4 as crude solid which was recrystallized
from ethanol.
N-(1-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-2-phenylethyl)benzamide (4)
1
Yield = 79%, R
f
= 0.78 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.21 (s, -SH), 9.3 (s, =CH), 7.7 (m, 5H, Ar-H), 7.6(m, 5H, Ar-
This article is protected by copyright. All rights reserved.

2
H), 7.0 (s, -NH ), 5.9 (s, NH) 5.7 (dd, 1H, J= 7.2, 7.0, Hz), 3.4 (dd, 1H, J=6.3, 6.0, Hz ), 3.1
13
(
dd, 1H, J=6.1Hz) ), C NMR (75 MHz, DMSO-d6): δ (ppm):169, 160, 157, 152, 147, 144,
1
38, 134, 130, 127, 124, 58, 43 Anal.Calcd. for C17
.02 found: C, 59.30; H, 4.97; N, 19.26; S, 7.83.
17 5
H N OS: C, 60.29; H, 5.52; N, 20.64; S,
7
1.4 Synthesis of N-(1-(4-(substitued Benzylideneamino)-5-mercapto-4H-1,2,4-triazol-3-
yl)-2-phenylethyl)benzamides (5a-l).
A solution of 2 (1.0 mmol) in 25 mL absolute ethanol, suitably substituted aldehydes (3a-l
1
.0 mmol) along with 1-2 drops of acetic acid was added to a 50ꢀmL Erlenmeyer flask. The
mixture was refluxed. The progress of the reaction was monitored by TLC (Pet Ether: Ethyl
Acetate 7: 3) after regular intervals and completion the reaction mixture was added to water;
extracted with ethyl acetate (3×50ꢀmL). The combined organic layers were dried over
anhydrous magnesium sulfate, and filtered. The ethyl acetate was evaporated under reduced
pressure to give the crude substituted benzamides were filtered and recrystallized from
ethanol to give compounds 5a–l in good yields.
N-(1-(4-(Benzylideneamino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-phenylethyl)benzamide
(5a).
1
f
Yield = 69%, R = 0.68 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.21 (s, SH), 9.8 (s, NH), 9.3 (s, =CH), 7.9 (m, 5H, Ar-H),
This article is protected by copyright. All rights reserved.

7
3
1
7
.8(m, 5H, Ar-H),7.3 (m, 5H, Ar-H), 5.7 (dd, J= 6.4, 6.2 Hz,1H), 3.9 (dd, J= 5.5,5.8 Hz, 1H),
1
3
.7 (dd, J= 5.3 Hz, 5.1 Hz, 1H) C NMR (75.5 MHz, DMSO-d6): δ (ppm):171.3, 168.4,
64.5, 160.3, 152.5, 144.6, 141.2, 140.7, 138.7, 135.8, 132.4, 129.5, 125.2, 123.2, 121.3,
5.4, 58.3, 43.3 DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH
2
negative. Anal.Calcd. for C24
H, 4.87; N, 14.86; S, 8.03.
20 5
H N OS: C, 61.29; H, 4.52; N, 15.24; S, 7.02 found: C, 60.30;
N-(1-(5-Mercapto-4-((4-methylbenzylidene)amino)-4H-1,2,4-triazol-3-yl)-2-phenylethyl)
benzamide (5b).
1
Yield = 79%, R
f
= 0.61 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.11 (s, SH), 9.9 (s, NH), 9.5 (s, =CH), 7.8 (dd, J=7.6, 7.3 Hz,
H,Ar-H), 7.61 (dd, J=2.3, 2.1 Hz, 2H, Ar-H), 7.73 (m, 5H,Ar-H),7.31 (m, 5H, Ar-H), 5.74
dd, J= 6.4Hz, 6.2Hz, 1H), 3.93 (dd, J= 5.5,5.8Hz, 1H), 3.73 (dd, J= 5.2,4.9 Hz, 1H), 2.73
2
(
(
13
s,3H), C NMR (75.5 MHz, DMSO-d6): δ (ppm):173.5, 169.6, 166.6, 163.5, 155.6, 146.6,
1
1
42.5, 140.7, 137.6, 135.6, 132.4, 129.3, 125.2, 123.4, 121.3, 75.4, 58.5, 43.4, 28.3. DEPT-
35 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH3 positive and one CH
2
negative. Anal.Calcd. for C H N OS: C, 68.29; H, 5.52; N, 15.24; S, 7.02 found: C, 68.13;
25
23
5
H, 5.39; N, 15.16; S, 7.88.
N-(1-(5-Mercapto-4-((4-methoxybenzylidene)amino)-4H-1,2,4-triazol-3-yl)-2
phenylethyl) benzamide (5c)
This article is protected by copyright. All rights reserved.

OMe
SH
N
N
N
HN
O
N
1
f
Yield = 83%, R = 0.7 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.22 (s, SH), 9.91 (s, NH), 9.5 (s, =CH), 7.83 (dd, J=6.5, 6.2
Hz, 2H, Ar-H), 7.64 (dd, J=4.2, 4.0 Hz, 2H, Ar-H), 7.75 (m, 5H, Ar-H),7.3 (m,5H,Ar-H), 5.7
(dd, J= 5.5, 5.8Hz 1H), 3.94 (dd, J= 5.5, 5.8Hz, 1H), 3.75 (dd, J= 5.2, 4.9 Hz ,1H), 3.54
13
(
s,3H), C NMR (75.5 MHz, DMSO-d6): δ(ppm):173.6, 169.6, 166.5, 163.5, 155.6, 146.7,
1
1
42.5, 140.6, 137.8, 135.7, 132.5, 129.4 ,125.6, 123.5, 121.4, 75.5, 58.6, 52.4, 43.5. DEPT-
35 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH positive and one CH
S: C, 68.29; H, 5.52; N, 15.26; S, 7.02 found: C, 68.15;
3
2
negative. Anal.Calcd. for C25
H, 5.41; N, 15.16; S, 6.89.
23 5 2
H N O
N-(1-(5-Mercapto-4-((3-nitrobenzylidene)amino)-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5d)
1
f
Yield = 64%, R = 0.73 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 13.89 (s, SH), 9.91 (s, NH), 9.40 (s, =CH), 8.33 (dd, J=3.7,3.5
Hz, 1H, Ar-H), 8.12 (dd,J=4.2, 4.0 Hz, 1H, Ar-H), 8.01 (dd, J=5.1, 4.9 Hz, 1H, Ar-H), 7.92
This article is protected by copyright. All rights reserved.

(dd,J= 2.2,2.1 Hz,1H,Ar-H), 7.83 (m, 5H, Ar-H),7.3 (m, 5H, Ar-H), 5.73 (dd, J= 6.4, 6.2Hz,
1
3
1
H), 3.93 (dd, J =5.5,5.8Hz, 1H), 3.73 (dd, J=5.2 ,4.9 Hz, 1H) C NMR (75.5 MHz, DMSO-
d6): δ (ppm):176.4, 167.5, 163.6, 161.4, 154.6, 145.5, 142.6, 140.5, 138.5, 135.4, 132.5,
29.3, 125.3, 123.4, 121.4, 75.5, 58.6, 43.3 DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10
1
CH positive, one CH2 negative. Anal.Calcd. for C24
S, 7.21 found: C, 64.18; H, 4.44; N, 15.06; S, 7.13
20 6 3
H N O S: C, 64.29; H, 4.52; N, 15.14;
N-(1-(5-Mercapto-4-((4-nitrobenzylidene)amino)-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5e)
1
Yield = 73%, R = 0.73 (Pet Ether: Ethyl Acetate 7: 3). H NMR
f
(300 MHz, DMSO-d6): δ (ppm): 13.77 (s, SH), 9.81 (s, NH), 9.60 (s, =CH), 8.3 (dd,
J=6.5,6.3 Hz ,2H,Ar-H), 8.1(dd, J=4.2, 4.0 Hz, 2H, Ar-H), 7.8(m, 5H, Ar-H),7.3 (m, 5H, Ar-
13
H), 5.7 (dd, J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5, 5.8 Hz,1H), 3.7 (dd, J=5.2, 4.9 Hz, 1H) C
NMR (75.5 MHz, DMSO-d6): δ (ppm):177.4, 166.3, 161.2, 158.4, 154.6, 145.7, 142.5,
1
40.5, 138.6, 135.7, 132.4, 129.5, 125.4, 123.5, 118.4, 70.5, 54.6, 37.4; DEPT-135 (75.5
MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH2 negative. Anal.Calcd. for
S: C, 59.29; H, 4.62; N, 15.13; S, 7.14 found: C, 59.19; H, 4.53; N, 15.02; S,
24 20 6 3
C H N O
7.03.
N-(1-(4-((4-Fluorobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5f)
This article is protected by copyright. All rights reserved.

1
Yield = 73%, R
MHz, DMSO-d6): δ (ppm): 14.34 (s, SH), 9.91 (s, NH), 9.70 (s, =CH), 8.1 (dd, J=5.5,5.3 Hz,
H, Ar-H), 7.9(dd,J=3.5,3.3 Hz, 2H, Ar-H), 7.6(m, 5H, Ar-H),7.4 (m, 5H, Ar-H), 5.9 (dd,
f
= 0.73 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
2
13
J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5,5.8Hz, 1H), 3.8 (dd, J=5.2 ,4.9 Hz, 1H) C NMR (75.5
MHz, DMSO-d6): δ (ppm):177.3, 166.4, 161.5, 158.4, 154.6, 145.7, 142.5, 140.5, 138.4,
1
35.4, 132.5, 129.4, 126.5, 124.5, 120.4, 71.3, 50.4, 34.5; DEPT-135 (75.5 MHz, DMSO-d6):
δ (ppm): 10 CH positive, one CH negative. Anal.Calcd. for C24 OFS: C, 61.29; H, 4.32;
N, 14.54; S, 7.02 found: C, 61.18; H, 4.17; N, 14.46; S, 6.90.
2
20 6
H N
N-(1-(4-((3-Bromobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5g)
1
Yield = 64%, R
MHz, DMSO-d6): δ (ppm): 13.84 (s, SH), 9.94 (s, NH), 9.47 (s, =CH), 8.2 (dd, J=3.5, 3.3Hz,
H, Ar-H), 8.1(dd, J=2.9,2.7 Hz, 1H, Ar-H), 8.0 (dd, J=6.4, 6.1 Hz,1H, Ar-H), 7.9 (dd,
f
= 0.73 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
1
J=4.9, 4.7 Hz, 1H, Ar-H), 7.8(m, 5H, Ar-H),7.3 (m, 5H, Ar-H), 5.9 (dd, J= 6.4, 6.2Hz ,1H),
This article is protected by copyright. All rights reserved.

1
3
3
.9(dd, J=5.5,5.8Hz, 1H), 3.6 (dd, J=5.2 ,4.9 Hz, 1H) C NMR (75.5 MHz, DMSO-d6): δ
ppm):176.3, 167.4, 163.5, 161.5, 154.6, 145.6, 142.7, 140.7, 138.6, 135.6, 132.4, 129.3,
25.5, 123.6, 121.4, 75.6, 58.7, 43.5; DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH
positive, one CH negative. Anal.Calcd. for C24 OBrS: C, 61.29; H, 4.42; N, 15.11; S,
.11 found: C, 61.17; H, 4.33; N, 15.06; S, 7.03; [α]
(
1
2
20 6
H N
2
5
o
7
D
= +18.09
N-(1-(4-((4-Bromobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5h)
1
Yield = 79%, R
MHz, DMSO-d6): δ (ppm): 13.88 (s, SH), 9.71 (s, NH), 9.63 (s, =CH), 8.1 (dd, J=6.7,6.4 Hz,
H, Ar-H), 8.0(dd, J=2.9,2.7 Hz, 2H, Ar-H), 7.8(m, 5H, Ar-H),7.3 (m, 5H, Ar-H), 5.7 (dd,
f
= 0.53 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
2
1
3
J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5,5.8Hz, 1H), 3.7 (dd, J=5.2 ,4.9 Hz ,1H) C-NMR (75.5
MHz, DMSO-d6): δ (ppm):176.4, 167.5, 163.5, 156.6, 152.4, 145.6, 142.5, 140.3, 138.5,
135.6, 132.3, 129.4, 127.3, 124.5, 119.6, 70.5, 50.4, 34.3; DEPT-135 (75.5 MHz, DMSO-
d6): δ (ppm): 10 CH positive, one CH negative. Anal.Calcd. for C H N OBrS: C, 61.29; H,
2
24 20
6
4.52; N, 14.24; S, 7.32 found: C, 60.30; H, 4.97; N, 13.36; S, 7.93 %.
N-(1-(4-((2-Chlorobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5i)
This article is protected by copyright. All rights reserved.

1
Yield = 81%, R
MHz, DMSO-d6): δ (ppm): 13.99 (s, SH), 9.92 (s, NH), 9.52 (s, =CH), 8.2 (dd, J=4.4,4.2 Hz,
H, Ar-H), 8.1(dd, J=3.8,3.6Hz, 1H, Ar-H), 8.0 (dd, J=4.8,4.7Hz, 1H,Ar-H), 7.9 (dd, J=2.8,2.6
Hz, 1H,Ar-H), 7.7(m, 5H, Ar-H),7.3 (m, 5H, Ar-H), 5.8 (dd, J= 6.4, 6.2Hz ,1H), 3.9(dd,
f
= 0.43 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
1
13
J=5.5,5.8Hz, 1H), 3.6 (dd, J=5.2,4.9 Hz, 1H) C NMR(75.5 MHz,DMSO-d6):δ (ppm):171.4,
1
67.5, 163.6, 161.5, 154.6, 145.6, 142.5, 140.5, 138.4, 135.6, 132.4, 129.5, 125.6, 123.4, 121.5,
3.5, 53.5, 40.4; DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH
7
2
negative. Anal.Calcd. for C24
H, 4.40; N, 14.16; S, 6.95 %
20 6
H N OClS: C, 62.29; H, 4.52; N, 14.24; S, 7.02 found: C, 62.14;
N-(1-(4-((2,3-Dichlorobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5j)
1
Yield = 85%, R
f
= 0.53 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.10 (s, SH), 9.82 (s, NH), 9.63 (s, =CH), 8.2 (dd, J=6.5,6.3Hz,
H, Ar-H), 8.1(dd, J=4.6,4.3Hz, 1H, Ar-H), 8.0 (dd, J=2.9, 2.7Hz, 1H,Ar-H), 7.7(m, 5H, Ar-
1
H),7.3 (m, 5H, Ar-H), 5.8 (dd, J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5,5.8Hz, 1H), 3.6 (dd, J=5.2
This article is protected by copyright. All rights reserved.

13
,
4.9 Hz, 1H) C NMR (75.5 MHz, DMSO-d6): δ (ppm):171.3, 167.4, 163.5, 161.4, 154.5,
1
45.6, 142.6, 140.5, 138.6, 135.7, 132.8, 129.5, 125.6, 123.5, 121.4, 73.6, 53.4, 40.5; DEPT-
35 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH negative. Anal.Calcd. for
OCl S: C, 61.29; H, 4.52; N, 14.24; S, 7.02 found: C, 61.12; H, 4.49; N, 14.16; S,
1
2
C
24
H
20
N
6
2
6.93%
N-(1-(4-((4-fluoro-3-nitrobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5k)
1
Yield = 86%, R = 0.73 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
f
MHz, DMSO-d6): δ (ppm): 14.15 (s, SH), 9.78 (s, NH), 9.63 (s, =CH), 8.2 (dd, J=7.2,6.9Hz,
1H, Ar-H), 8.1(dd, J=2.8, 2.6Hz, 1H, Ar-H), 8.0 (dd, J=3.2, 3.0 Hz, 1H, Ar-H), 7.7(m, 5H,
Ar-H),7.3 (m, 5H, Ar-H), 5.8 (dd, J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5,5.8Hz, 1H), 3.6 (dd,
1
3
J=5.2 ,4.9 Hz ,1H) C NMR (75.5 MHz, DMSO-d6): δ (ppm):173.6, 168.2, 164.5, 161.3,
54.6, 145.7, 142.3, 140.5, 138.6, 135.6, 132.4, 129.5, 125.6, 122.4, 121.3, 75.5, 55.6, 38.6;
DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH negative. Anal.Calcd.
OCl S: C, 61.29; H, 4.52; N, 14.24; S, 7.44 found: C, 61.01; H, 4.47; N, 14.06;
1
2
for C24
H
20
N
6
2
S, 7.33%
This article is protected by copyright. All rights reserved.

N-(1-(4-((3-chloro-2-fluorobenzylidene)amino)-5-mercapto-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamide (5l)
1
Yield = 89%, R
f
= 0.63 (Pet Ether: Ethyl Acetate 7: 3). H NMR (300
MHz, DMSO-d6): δ (ppm): 14.23 (s, SH), 9.79 (s, NH), 9.73 (s, =CH), 8.2 (dd, J=6.5, 6.3
Hz, 1H, Ar-H), 8.1(dd, J=4.7,4.5 Hz, 1H, Ar-H), 8.0 (dd, J=2.9,2.7 Hz, 1H, Ar-H), 7.7(m,
5H, Ar-H),7.3 (m, 5H, Ar-H), 5.8 (dd, J= 6.4, 6.2Hz, 1H), 3.9(dd, J=5.5,5.8Hz, 1H), 3.6 (dd,
1
3
J=5.2 ,4.9 Hz ,1H) C NMR (75.5 MHz, DMSO-d6): δ (ppm):171.6, 167.5, 163.4, 161.3,
54.3, 145.3, 142.4, 140.4, 138.4, 135.6, 132.4, 129.5, 125.6, 123.4, 121.5, 73.5, 53.4, 40.5;
DEPT-135 (75.5 MHz, DMSO-d6): δ (ppm): 10 CH positive, one CH negative. Anal.Calcd.
1
2
for C24
H
20
N
6
OCl
2
S: C, 62.29; H, 4.52; N, 14.24; S, 7.02 found: C, 61.30; H, 4.97; N, 13.26;
25
o
S, 8.03 [α]
D
= +18.09
2
. Enzyme inhibition assay
.1 Material and Methods
2
2.4 Urease inhibition assay
The Jack bean urease activity was determined by measuring amount of ammonia produced
with indophenols method described by Weatherburn (28). The reaction mixtures, comprising
2
0 µL of enzyme (Jack bean urease, 5 U/mL) and 20 μL of compounds in 50 μL buffer (100
mM urea, 0.01 M K HPO , 1 mM EDTA and 0.01 M LiCl, pH 8.2), were incubated for 30
min at 37 ºC in 96-well plate. Briefly, 50 μL each of phenol reagents (1%, w/v phenol and
.005%, w/v sodium nitroprusside) and 50 μL of alkali reagent (0.5%, w/v NaOH and 0.1%
2
4
0
This article is protected by copyright. All rights reserved.

Sodium hypochlorite NaOCl) were added to each well. The absorbance at 625 nm was
measured after 10 min, using a microplate reader (OPTI Max, Tunable). All reactions were
performed in triplicate. The urease inhibition activities were calculated according to the
following formula:
Urease inhibition activity (%) = (ODcontrol – ODsample × 100) / ODcontrol
Where ODcontrol and ODsample represents the optical densities in the absence and presence of
sample, respectively. Thiourea was used as the standard inhibitor for urease.
2
.5 Free radical scavenging assay
Radical scavenging activity was determined by modifying already reported method by 2, 2-
diphenyl-1-picrylhydrazyl (DPPH) assay (29,30). The assay solution consisted of 100 µL of
DPPH (150 µM), 20 µL of increasing concentration of test compounds and the volume was
adjusted to 200 µL in each well with DMSO. The reaction mixture was then incubated for 30
minutes at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor.
The assay measurements were carried out by using a micro plate reader (OPTI Max, Tunable)
at 517 nm. The reaction rates were compared and the percent inhibition caused by the
presence of tested inhibitors was calculated. Each concentration was analyzed in three
independent experiments run in triplicate.
2
.6 Kinetic mechanism study
Kinetic analysis was carried out to determine the mode of inhibition. Four inhibitors (5c, 5d,
e and 5j) were selected on the basis of most potent IC values. Kinetics were carried out by
5
5
0
varying the concentration of urea in the presence of different concentrations of compound 5c
0.0, 0.0055, 0.011, 0.022 and 0.088 µM), compound 5d (0.00, 0.01, 0.02, 0.04 and 0.08
µM), compound 5e (0.00, 0.0018, 0.036 and 0.072 µM) and compound 5j (0.00, 0.0045,
(
0
6
.009 and 0.018 µM). In detail the urea concentration was changed from 100, 50, 25, 12.5,
.25, and 3.125 mM for urease kinetics studies and remaining procedure was same for all
kinetic studies as described in the urease inhibition assay protocol. Maximal initial velocities
were determined from the initial linear portion of absorbances up to 10 minutes after the
addition of enzyme at per minute’s interval. The inhibition type on the enzyme was assayed
by Lineweaver-Burk plot of inverse of velocities (1/V) versus the inverse of substrate
-1
concentration 1/ [S] mM . The EI dissociation constant Ki was determined by the secondary
plot of 1/V versus inhibitor concentration and ESI dissociation constant Kiʹ was calculated by
intercept versus inhibitors concentrations. Urease activity was determined by measuring
This article is protected by copyright. All rights reserved.

ammonia production using the indophenol method as reported previously [36]. The results
change in absorbance per min) were processed by using SoftMaxPro.
(
Computational Methodology
Jack bean urease structure from PDB
The Jack bean urease structure was retrieved from Protein Data Bank (PDB) (www.rcsb.org)
with PDBID 4H9M. The selected crystal structure of urease was minimized by employing
UCSF Chimera 1.10.1 tool (31). Furthermore, the stereo-chemical properties of urease
structure and Ramachandran plot and values were generated by Molprobity server (32) and
Protparam (33). The Discovery Studio 4.1 Client tool was employed to generate the
hydrophobicity graph of target protein (34). The protein architecture and statistical
percentage values of receptor proteins helices, beta-sheets, coils and turn were predicted from
online server VADAR 1.8 (35).
Molecular docking
The synthesized chemical structures (5a-l) were electronically sketched in ACD/ChemSketch
tool and minimized by UCSF Chimera 1.10.1 tool. PyRx docking tool (36) was employed to
perform molecular docking experiment. The grid box center values of (center_X=10.48,
center_Y=-55.22 and center_ Z=-26.48) and size values were adjusted as (X=66.60, Y=60.08,
and Z=56.84). The default exhaustiveness value = 8 was used to maximize the binding
conformational analysis. All the synthesized ligands were docked separately against urease.
The predicted docked complexes were evaluated on the basis of lowest binding energy
(
Kcal/mol) values and structure activity relationship (SAR) analyses. The three dimensional
3D) graphical depictions of all the docked complexes were accomplished by Discovery
(
Studio (2.1.0).
Results and discussion
The synthetic strategy adopted for the synthesis of the triazole-azomethine scaffolds is
depicted in the scheme-1. Initially amide was formed by using benzoyl chloride which was
further treated with a key component for heterocyclic compounds, that is thiocarbazide,
This article is protected by copyright. All rights reserved.

which afforded 1,2,4-triazole, and in last step Schiff bases were synthesized by making use of
free amino group present in 4, aromatic substituted aldehydes were used to obtain desired
products N-(1-(5-mercapto-4-((substituted benzylidene) amino)-4H-1,2,4-triazol-3-yl)-2-
phenylethyl)benzamides (5a-l).
Scheme-1 Synthesis of N-(1-(5-mercapto-4-((substituted benzylidene) amino)-4H-1,2,4-
triazol-3-yl)-2-phenylethyl)benzamides (5a-l)
The starting material N-benzoylphenylalanine was conveniently synthesized from the
commercial L-phenylalanine with benzoyl chloride in dry pyridine and the melting point was
[1, 17]
compared with the literature value
. In the next step, the cyclization reaction of 3 with
thiocarbazide in dry pyridine afforded the N-(1-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-
2-phenylethyl)benzamide (4) as a key precursor to heterocylic derivatives with retention of
1
configuration. H NMR showed the most deshielded singlet at δ 14.21 for -SH proton, and
1
3
characteristic singlet for CH at δ 9.3, besides signals for aromatic and amino protons. In C-
This article is protected by copyright. All rights reserved.

NMR the signal for amidic carbonyl appeared at δ 171.3 ppm and for ipso carbons at δ 168,
64, 160 ppm.
1
The hydrazone derivatives (5a-l) were obtained by acid catalyzed condensation of the free 4-
amino available in compound (4) with a variety of suitably substituted benzaldehydes. The
1
13
synthesized compounds were confirmed by using H, C and DEPT, NMR spectroscopy.
1
13
The characteristic imine singlets were observed at δ 9-10 region in H NMR. In the C NMR
spectra, the imine carbon appeared around δ 165 ppm, region characteristic of Schiff bases,
confirming the linkage of aromatic aldehydes to the triazole moiety. The amide carbonyl
appeared around δ 170 ppm, the ipso carbons of aromatic rings were deshielded and appeared
at high ppm. Moreover, DEPT-135 and DEPT-90 NMR provided further evidence for the
carbon skeleton, all the ten CH carbons appeared positive, while the single CH
negative in DEPT-135, and no quaternary carbon was found.
2
appeared
Free radical scavenging assay
Newly synthesized N-(1-(5-mercapto-4-((substituted benzylidene) amino)-4H-1, 2, 4-triazol-
-yl)-2-phenylethyl) benzamides compounds were screened for their possible radical
3
scavenging potency. Compounds 5d, 5i and 5l showed excellent radical scavenging potency
in comparison to reference drug vitamin c other nine compounds did not show significant
radical scavenging potency even at high concentration (100µg/mL). (Table.1.). All the
compounds showed great potency against urease enzyme and the newly synthesized
derivatives showed better activity compared to standard (Table 2).
Structural and physiochemical evaluation of Jack bean urease
Jack bean urease is a class of hydrolase protein which consist of four domains having
different numbers of amino acids. A couple of nickel atoms are also present in domain four of
targeted protein which involves in the downstream signaling pathways. The VADAR analysis
showed the protein architecture having 27% helices, 31% β sheets and 41% coils in the target
protein. Moreover, Ramachandran plots indicated that 97.5% of residues were present in
This article is protected by copyright. All rights reserved.

favored regions which shows the precision of phi (φ) and psi (ψ) angles among the
coordinates of jack bean urease. The Ramachandran graph is mentioned in the supplementary
data (Fig S1).
The physiochemical properties of jack bean urease such as molecular weight (Mw) was
calculated by aggregation of average isotopic masses of residues. Whereas, the theoretical pI
was computed using pK values of amino acids of target protein (38). Literature study
revealed the computational predicted pI range values (4.31 to 11.78) of proteins (39). The
predicted pI value 6.05 is comparable with the standard values which showed the accuracy of
targeted protein. The predicted value of aliphatic and instability indexes were also presented
the constancy and relative volume engaged by aliphatic side chains of targeted protein (Table
3
). The GRAVY value is the sum of hydropathic values of all amino acids present in the
protein (40), however, more negative value indicates it’s more hydrophilic and less
hydrophobic behavior (38).
Molecular docking analysis
Binding energy evaluation of synthesized compounds
To predict the best fitted conformational position of synthesized compounds (5a-l) were
docked against target protein. The generated docked complexes were examined on the basis
of minimum energy values (Kcal/mol) and bonding interaction pattern. Docking results
justified that compound 5j exhibited a good binding energy value (-9.30 kcal/mol) compared
to others compounds. The binding energy value depicts the best conformational position
within the active region of target protein. The other compounds such as 5c-5e also presents a
significance energy values (-9.10, -8.70, and -8.80 Kcal/mol), respectively against receptor
molecule. Although, the basic nucleus of all the synthesized compounds were similar,
therefore most of ligands possess good efficient energy values and have no big energy
fluctuations. The comparative binding energy analysis reveals that 5j was most active
compound and confined a good compatible position within binding pocket. Our
computational analysis also showed a good association with in-vitro study (Figure. 2).
<<Insert Figure 2>>
Binding pocket analysis of urease docked complexes
The docked complexes were further analyzed on the basis of binding interactions. Docking
analysis showed that all compounds were confined in the active binding region of receptor
This article is protected by copyright. All rights reserved.

molecule with different conformational poses (Figure. 3). However, the best conformational
energy valued docked complex (5j) is mentioned in figure 3. Whereas the other docking
complexes (5c-5e) which also show comparable results are depicted in (Figures. 4, 5, 6).
<<Insert Figure 3>>
Structure activity relationship (SAR) analysis
In detail, the structure activity relationship (SAR) study showed that single active hydrogen
bond was observed in 5j docked complex. The amino group of benzene ring is directly
interacts with Ala636 by hydrogen interaction having bonding distance 2.40 Å. Whereas, 4c
docking complex two hydrogen bonds were observed. The oxygen and methoxy functional
groups of 4c forms hydrogen bonds against Arg439 and Glu493 with bonding distances 3.00
and 2.64 Å, respectively. The 4d and 4e are also involves in hydrogen interactions against
targeted protein residues. The sulfur moiety of 4d form double hydrogen bonds against
Arg439 with bonding distance 3.16 and 3.21 Å. The amino group of 4e makes hydrogen
bonds with Arg439 and Asp494 with bond length 3.18 and 2.80Å, respectively. Literature
data also ensured the importance of these residues in bonding with other urease inhibitors
which strengthen our docking results. The comparative binding energy and SAR analysis
showed the significance of 5e compound and may consider as potent inhibitors by targeting
jack bean urease.
Kinetic mechanism
Presently four most potent compounds were studied for their mode of inhibition 5c, 5d, 5e,
and 5j against urease. The potential of these compounds to inhibit the free enzyme and
enzyme-substrate complex was determined in terms of EI and ESI constants respectively. The
kinetic studies of the enzyme by the Lineweaver–Burk plot of 1/V versus 1/[S] in the
presence of different inhibitors concentrations gave a series of straight lines as shown in
Figure 8-10(a). The results of compound 5c, and 5e gives a series of straight lines, all of
which intersected at the same point on the x-axis Figure. 8 and 9 (a). The analysis showed
that Vmax decreased to a new value while that of Km remains the same as a result of increase
in the concentrations of compound 5c and 5e. This behavior indicated that the compound 5c
and 5e inhibits urease non-competitively to form enzyme inhibitor (EI) complex. The
secondary plot of the slope against concentration of 5c and 5e showed EI dissociation
constant (Ki) Figure. 8 and 9 (b). The results of Figure 8 and 10 (a) showed that compounds
5d, and 5j intersected within the second quadrant. The analysis showed that Vmax decreased
with increasing Km in the presence of increasing concentrations of compounds 5d, and 5j,
This article is protected by copyright. All rights reserved.

respectively. This behavior of derivatives 5d, and 5j indicated that it inhibits urease by two
different pathways; competitively forming enzyme inhibitor (EI) complex and interrupting
enzyme-substrate-inhibitor (ESI) complex in a noncompetitive manner. The secondary plots
of slope versus concentration of compounds 4d, and 4j showed EI dissociation constants Ki
Figure. 8 and 10 (b), while ESI dissociation constants Ki′ were shown by secondary plots of
intercept versus concentration of compounds 5d, and 5j, Figure. 8 and 10 (c). A lower value
of Ki than Ki′ pointed out stronger binding between enzyme and compounds 5d, and 5j
which suggested preferred competitive over noncompetitive manners (Table 4). The results of
kinetic constants and inhibition constants are presented in Table 4.
CONCLUSIONS
Facile synthesis of library of title triazolyl benzamides in excellent yields (82-92%) was
achieved. The protocol is clean and convenient with easier work-up, shorter reaction time,
and higher yields as the important features. The synthesized N-(1-(5-mercapto-4-((substituted
benzylidene) amino)-4H-1, 2, 4-triazol-3-yl)-2-phenylethyl) benzamides were evaluated for
jack bean urease enzyme inhibition and antioxidant activity to validate their role as potent
enzyme inhibitors. The kinetic mechanism was explored for urease inhibition and it was
found that compounds 5c and 5e appeared non-competitive inhibitors while compound 5d
and 5j found to be mixed type of inhibitors. Antioxidant activity of compounds was
performed and all compounds showed better free radical scavenging potential and compound
5j was found to be most potent with 14.590±0.137 compared to Vitamin C standard
9
5.379±0.862. Docking studies were performed to delineate the binding affinity of the
synthesized compounds and compound 5j exhibited a good binding energy value (-9.30
kcal/mol) compared to others compounds. After the analyses of kinetic mechanism and
molecular docking, it can be predicted that designed compounds can serve as potent
inhibitors in chemical bio drug designing against urease enzyme.
This article is protected by copyright. All rights reserved.

Conflict of Interest
The authors declare no any conflict of interest.
Legends of figures
Figure 1: Some urease inhibitors designed and synthesized by various research groups (16-
20).
This research aims to provide these heterocyclic N-(1-(5-mercapto-4-((substituted
benzylidene) amino)-4H-1,2, 4-triazol-3-yl)-2-phenylethyl)benzamides, as important Jack
bean urease inhibitors.
Figure 2. The graphical depiction of docking energy values.
Figure. 3. Binding pocket of urease with binding position of all synthesized compounds
Figure 4. Docking complex of 5j. The ligand structure is mentioned in green color, while the
functional groups such as amino, sulfur and oxygen are highlighted in light blue, yellow and
red colors, respectively. The protein interacted amino acids are mentioned in purple color in
stick format. Two nickel atoms are also present. The black dotted lines represent the
hydrogen binding and bond distances measured in angstrom (Å).
Figure 5. Docking complex of 5c. The ligand structure is mentioned in green color, while
the functional groups such as amino, sulfur and oxygen are highlighted in light blue, yellow
and red colors, respectively. The protein interacted amino acids are mentioned in purple color
in stick format. Two nickel atoms are also present. The black dotted lines represent the
hydrogen binding and bond distances measured in angstrom (Å).
Figure 6. Docking complex of 5d. The ligand structure is mentioned in green color, while
the functional groups such as amino, sulfur and oxygen are highlighted in light blue, yellow
and red colors, respectively. The protein interacted amino acids are mentioned in purple color
in stick format. Two nickel atoms are also present. The black dotted lines represent the
hydrogen binding and bond distances measured in angstrom (Å).
This article is protected by copyright. All rights reserved.

Figure 7. Docking complex of 5e. The ligand structure is mentioned in green color, while
the functional groups such as amino, sulfur and oxygen are highlighted in light blue, yellow
and red colors, respectively. The protein interacted amino acids are mentioned in purple color
in stick format. Two nickel atoms are also present. The black dotted lines represent the
hydrogen binding and bond distances measured in angstrom (Å).
Figure 8. Lineweaver–Burk plots for inhibition of urease in the presence of Compound 5c.
(
a) Concentrations of 5c were 0.0, 0.0055, 0.011, 0.022 and 0.088 µM. Substrate urea
concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 mM. (b) The insets represent the plot
of the slope.
Figure 9. Lineweaver–Burk plots for inhibition of urease in the presence of Compound 5d.
(
a) Concentrations of 5d were 0.0, 0.01, 0.02, 0.04 and 0.08 µM. Substrate urea
concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 mM. (b) The insets represent the plot
of the slope (c) the vertical intercepts versus inhibitor 4c concentrations to determine
inhibition constants.
Figure.10. Lineweaver–Burk plots for inhibition of urease in the presence of Compound 5e.
(a) Concentrations of 5e were 0.0, 0.0018, 0.036 and 0.072 µM. Substrate urea concentrations
were 100, 50, 25, 12.5, 6.25, and 3.125 mM. (b) The insets represent the plot of the slope.
Figure.11. Lineweaver–Burk plots for inhibition of urease in the presence of Compound 5j.
(
a) Concentrations of 5j were 0.0, 0.0045, 0.009, and 0.018 µM. Substrate urea
concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 mM. (b) The insets represent the plot
of the slope (c) the vertical intercepts versus inhibitor 5j concentrations to determine
inhibition constants.
This article is protected by copyright. All rights reserved.

References
1
2
3
. P.A. Karplus, M.A. Pearson, R.P. Hausinger, Acc. Chem. Res. 1997, 30, 330–337
. B. Krajewska, J. Mol. Catal. B: Enzym. 2009, 59, 9–21.
. H.L. Mobley, R.P. Hausinger, Microbiol. Rev. 1989, 53, 85–108.
4
2
. A. Agrawal, A. Gupta, M. Chandra, S. Koowar, Indian J. Gastroenterol. 2011, 30,
9–32.
5
6
7
. B. Zambelli, F. Musiani, S. Benini, S. Ciurli, Acc. Chem. Res. 2011, 44, 520–530
. H. Beraldo, D. Gambinob, Mini Rev. Med. Chem. 2004, 4, 31–39.
. B. Zambelli, F. Musiani, S. Benini, S. Ciurli, Acc. Chem. Res. 2011, 44, 520–530
8. L. Holm, C. Sander, Proteins: Struct. Funct. Genet. 1997, 28, 72–82.
9. S. Benini, W.R. Rypniewski, K.S. Wilson, S. Miletti, S. Ciurli, S. Mangani,
Structure 1999, 7, 205–216.
1
2
1
0. S. Benini, W.R. Rypniewski, K.S. Wilson, S. Mangani, S. Ciurli, J. Am. Chem. Soc.
004, 126, 3714–3715.
1. Z. Amtul, Atta-ur-Rahman, R.A. Siddiqui, M.I. Choudhary, Curr. Med. Chem. 2002, 9
1323–1348.
1
4
1
2. J.G. Kusters, A.H.M.v. Vliet, E.J. Kuipers, Clin. Microbiol. Rev. 2006, 19, 449–
90.
3. P. Kosikowska, Ł. Berlicki, Expert Opin. Ther. Pat. 2011, 21, 945–957 (and
1
4. K.M. Khan, F. Rahim, A. Khan, M. Shabeer, S. Hussain, W. Rehman, M. Taha, M.
Khan, S. Perveen, M.I. Choudhary, Bioorg. Med. Chem. 2014, 22, 4119–4123.
5. K.M. Khan, F. Naz, M. Taha, A. Khan, S. Perveen, M.I. Choudhary, W. Voelter,
1
Eur. J. Med. Chem. 2014, 74, 314–323.
This article is protected by copyright. All rights reserved.

1
6. Z.-P. Xiao, T.-W. Ma, W.-C. Fu, X.-C. Peng, A.-H. Zhang, H.-L. Zhu, Eur. J. Med.
Chem. 2010, 45, 5064–5070.
7. Z.-P. Xiao, Z.-Y. Peng, J.-J. Dong, J. He, H. Ouyang, Y.-T. Feng, C.-L. Lu, W.-Q. Lin,
J.-X. Wang, Y.-P. Xiang, H.-L. Zhu, Eur. J. Med. Chem. 2013, 63, 685–695.
1
1
5
8.T. Akhtar, S. Hameed, K.M. Khan, M.I. Choudhary, Med. Chem. 2008, 4, 539–
43.
1
9. M.A.S. Aslam, S.-U. Mahmood, M. Shahid, A. Saeed, J. Iqbal, Eur. J. Med. Chem.
2
2
2
2
011, 46, 5473–5479.
0. a) Saeed, A., Zaib, S., Pervez, A., Mumtaz, A., Shahid, M. Iqbal, J., Med Chem Res
013, 22, 3662. b) Saeed, A., Khan, M.S., Rafique, H., Shahid, M. Iqbal, J., Bioorg Chem,
014, 52, 7. c) Saeed, A., Imran, A., Channar, P.A., Shahid, M., Mahmood, W. Iqbal, J.,
Chem Bio Drug Design, 2015, 85, 230. d) Saeed, A., Mahmood, S.U., Rafiq, M., Ashraf, Z.,
Jabeen, F. Seo, S.Y., Chem. Bio. Drug. Design, 2016, 87, 443.
21. 17.Singh, M., and N. Raghav." Intl. J. Pharm. Pharma Sci 2011, 34, 32.
2
2
2
2. M.M. Heravi, V. F.Vavsari, Advances in Heterocyclic Chemistry, 2015, 114, 145
3. N. Ma, W. Guo, H. Song, H.Yu, J. Solid State Chem., 2016, 233, 389
4.a) Colanceska-Ragenovic, K.; Dimova, V.; Kakurinov, V.; Molnar, D. G.; Buzarovska, A.
Molecules 2001, 6, 824. b) Saeed A., P.A. Channar, Q. Iqbal, J. Mahar, Chin. Chem.
Lett.,2016, 27, 40. c) Larik, F.A., Saeed, A., Channar, P.A., Ismail, H., Dilshad, E., Mirza,
B., Bangladesh J. of Pharmacol, 2016, 11, 902. D) Saeed, A., Zaman, S., Jamil, M., Mirza,
B., Turk. J. Chem, 2008, 32, 592. e) Saeed, A., Larik, F.A., Channar, P.A., Res. Chem
Intermed, 2016, 1-9. f) Saeed A., S.A. Ejaz, A. Khurshid, S. Hassan, M. al-Rashida, M.
This article is protected by copyright. All rights reserved.

Latif, J. Lecka, J. S´evigny J. Iqbal, RSC Advances, 2015, 5, 86439. (g) Saeed, A., Channar,
P.A., Larik, F.A., Flörke, U., Synlett, 2016, 27, 1374.
2
5. Kraunsoe, J.A., Claridge, T.D., Lowe, G., Biochem, 1996, 35, 9096.
6. Kim, S.J., Sancheti, S.A., Sancheti, S.S., Um, B.H., Yu, S.M., Seo, S.Y., Acta Pol
Pharm, 2010, 67, 50.
2
2
5
7. Ashraf, Z, Muhammad, R, Sung,Y.S, Mustafeez M.B. Bioorg. Med. Chem 2015, 23,
880.
2
8. Weatherburn, M.W., Phenol-hypochlorite reaction for determination of
ammonia. Analytical chemistry, 1967, 39, 974.
2
9. Reddy, C. V. K., Sreeramulu, D., & Raghunath, M., Food Research International, 2010,
43, 285–288.
30. Zhou, J.T., Li, C.L., Tan, L.H., Xu, Y.F., Liu, Y.H., Mo, Z.Z., Dou, Y.X., Su, R., Su,
Z.R., Huang, P. and Xie, J.H., 2017.. PloS one, 2017, 12, e0168944.
3
1. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C.,
Ferrin, T.E. J Comput Chem 2004, 25, 1612.
2. Chen, V.B., Arendall, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J.,
3
Murray, L.W., Richardson, J.S. and Richardson, D.C. MolProbity: all-atom structure
validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 2010, 66,
2
3
1.
3. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch
A.; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press 2005,
571-607
3
4. Studio D, Discovery. "version 2.1." Accelrys: San Diego, CA (2008).
This article is protected by copyright. All rights reserved.

3
5. Willard, L., Ranjan, A., Zhang, H., Monzavi, H., Boyko, R.F., Sykes, B.D., Wishart, D.S
VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids
Res 2003, 31, 3319.
3
6. Dallakyan S, Olson A.J., Small-molecule library screening by docking with PyRx.
Methods Mol Biol 2015, 1263, 250.
7. Prerana B. Jadhav, Akshay R.Yadav and Megha G. Gore. Concept of drug likeness in
pharmaceutical research. Int J Pharm Bio Sci 2015, 6, 154.
3
3
3
4
8. H. E. Selick, A. P. Beresford and M. H. Tarbit, Drug discovery today, 2002, 7, 116.
9. P. Bonnet, Eur J Med Chem, 2012, 54, 689.
0. Abdul, H, Khalid M.K., Syeda, T.Z., Ramasa, A., Zahid, S., Syeda M.B., Muhammad, Y.,
Mazhar, H., Antonio de la, Vega de, L.,, Norbert ,F., Jürgen, B., Hazoor, A., Muhammad,
N.T., Jamshed, I., Bioorg Chem. 2015; 61, 7.
This article is protected by copyright. All rights reserved.

List of tables
Table. 1. Inhibition % of newly synthesized compounds against free radical scavenging
_
_
__________________________________________________________________________
__
Compound
100µg/mL
50 µg/mL
25 µg/mL
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
27.425±0.822
22.629±0.768
45.040±1.531
92.422±1.772
38.761±2.162
17.105±0.153
41.195±2.051
37.932±1.678
91.972±2.761
14.590±0.137
19.355±0.258
89.747±1.230
95.379±0.862
7.511±0.105
1.236± 0.009
28.016±0.460
45.758±0.115
13.466±0.269
3.221± 0.043
24.314±0.386
28.663±0.473
58.125±1.436
15.153±0.206
13.089±0.161
78.595±1.831
96.088±1.927
6.452±0.280
1.236±0.039
16.813±0.572
26.850±1.074
13.278±0.531
1.678±0.012
20.170±0.606
16.168±0.646
29.245±0.782
12.781±0.140
7.828±0.125
36.341±1.36
96.311±3.852
g
h
i
j
k
l
Vitamin C
All compound concentrations (100, 50 and 25 µg/mL) were used against Free radical
scavenging. SEM = Standard error of the mean; values are expressed in mean ± SEM.
This article is protected by copyright. All rights reserved.

Table.2. The inhibitory effects of synthesized (4a-4l) on Jack bean Urease
Compound
Urease inhibition activity
IC50 ± SEM (µM)
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
0.0962±0.00189
0.0856±0.00368
0.0137±0.00082
0.0202±0.00073
0.0183±0.00068
0.0551±0.00156
0.750±0.0226
g
h
i
0.070±0.0011
0.0191±0.00057
0.00866±0.00018
0.113±0.0044
j
k
l
0.044±0.00148
15.151± 1.27
Thiourea
SEM = Standard error of the mean; values are expressed
in mean ± SEM.
This article is protected by copyright. All rights reserved.

Table 3. Physiochemical properties of Urease by ProtParam.
Sr. No. Parameters
Values
90747.7 Da
6.05
1
2
3
4
5
6
Molecular weight (MW)
Theoretical pI
Extinction coefficient* (assuming all Cys residues are reduced) 53290
Aliphatic index
90.48
31.75
-0.152
Instability index
Gran average of hydropathicity (GRAVY)
-1
-1
*
Extinction Coefficient units M cm at 280 nm
This article is protected by copyright. All rights reserved.