Structural basis of binding and justification for the urease inhibitory activity of acetamide hybrids of N-substituted 1,3,4-oxadiazoles and piperidines

Article abstract of DOI:10.1016/j.molstruc.2020.129141

In present, we have performed the Michaelis–Menten kinetics studies of urease inhibitors (6a–o), having basic skeleton of acetamide hybrids of N-substituted 1,3,4-oxadiazoles and piperidines. From the Lineweaver-Burk plot, Dixon plot and their secondary replots, it has been confirmed that all the compounds have inhibited the enzyme competitively with K_i values of in range from 3.11 ± 0.2 to 5.20 ± 0.7 μM. Compound 6a was found to have lowest K_i among the series, while compounds 6d, 6e, 6gand 6i were found subsequently the excellent K_i values after 6a. Molecular docking has supported their types of inhibitions and structure activity-relationship. Most frequently, the nitro group oxygen atoms were found in contact with nickel ions of the active site. Moreover, all the compounds were subjected to toxicity tests and were found nontoxic against human neutrophils and plants, respectively.

Full text of DOI:10.1016/j.molstruc.2020.129141

Journal of Molecular Structure 1223 (2021) 129141
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Structural basis of binding and justification for the urease inhibitory
activity of acetamide hybrids of N-substituted 1,3,4-oxadiazoles and
piperidines
Farman Ali Khana, Aziz Ur Rehmanb, Muhammad Athar Abbasib, Sahib Gul Afridia,
Asifullah Khana, Muhammad Arif Lodhia,∗, Ajmal Khanc,∗
a Department of Biochemistry, Abdul Wali Khan University, Mardan, Khyber Pakhtunkhwa-23200, Pakistan
b Department of Chemistry, Government College University, Lahore-54000, Pakistan
c Natural and Medical Sciences Research Center, University of Nizwa, Birkat-ul-Mouz 616, Nizwa, Sultanate of Oman, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
In present, we have performed the Michaelis–Menten kinetics studies of urease inhibitors (6a–o),
having basic skeleton of acetamide hybrids of N-substituted 1,3,4-oxadiazoles and piperidines. From
the Lineweaver-Burk plot, Dixon plot and their secondary replots, it has been confirmed that all the
Received 12 May 2020
Revised 3 August 2020
Accepted 22 August 2020
Available online 30 August 2020
compounds have inhibited the enzyme competitively with Ki values of in range from 3.11
0.2 to
5.20 0.7 μM. Compound 6a was found to have lowest Ki among the series, while compounds 6d, 6e, 6g
and 6i were found subsequently the excellent Ki values after 6a. Molecular docking has supported their
types of inhibitions and structure activity-relationship. Most frequently, the nitro group oxygen atoms
were found in contact with nickel ions of the active site. Moreover, all the compounds were subjected to
toxicity tests and were found nontoxic against human neutrophils and plants, respectively.
Key words:
Enzyme kinetics
Acetamide hybrids
Oxadiazole
Cytotoxicity
© 2020 Elsevier B.V. All rights reserved.
Phytotoxicity
1. Introduction
The urea is considered to be the most prominent source of ni-
trogen (46%) in the field of agriculture [14]. It is supplied to the
Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel dependent
metalloenzyme, which enhances the hydrolysis of urea into ammo-
nia and carbamate [1]. The hydrolysis of carbamate later produces
carbon dioxide and ammonia at physiological pH [2]. Such an ex-
cess production of ammonia makes the environment basic, which
favor the colonization of various bacterial species [3,4]. This rate
of urease hydrolysis is approximately 1014 times to that of uncat-
alyzed reaction [5].
soil in the form of dehydrated granules, prills, and fluids in combi-
nation with NH4NO3. Adverse action of urease released by the soil
microbes will loss a greater amount of urea (upto 30%) in the form
of ammonia to elevate the surrounding pH which will suppress the
growth of seeds and seedling germination as well as deprive plants
from their nitrogenous nutrient [15]. It also causes an imbalance
in the nitrogen cycle [7], eutrification which result in algae bloom
to reduce fish and animal population and cause addition of green-
house gasses in the environment [16].
Urease exists in fungi, algae, yeast, bacteria, plants, animals and
soil [6,7]. It plays a pivotal role in their growth by the degrada-
tion of urea and supply of nitrogen. Moreover, it participates in
the metabolism of nitrogen within cattle and many other animals
[8]. From medicinal point of view, the ureases from various bacte-
ria (H. pylori) adversely affect the host and cause various diseases
like gastric/peptic ulcer, gastric coma and stomach cancer [9–11].
In addition, it causes kidney stone formation, hepatic encephalopa-
thy, hepatic coma, pyelonephritis and urinary catheter blockage
[4,12,13].
During the last two decades, antibiotic combination of amoxi-
cillin and clarithromycin along with omeprazole was used for the
treatment of H. pylori eradication. However, in the recent years, the
prominent drug resistance of H. pylori especially to clarithromycin
along with increased side effects, these drugs has lost its medi-
cal importance [17,18]. Likewise the bismuth salt along with pro-
ton pump cell inhibitors and certain other antibiotics such as fluo-
roquinolones, aminopenicillins, and tetracyclines etc. efficacy have
also reduced towards the treatment of H. pylori infections [17,18].
The most efficient way to control urease complications is to dis-
cover a variety of novel and safe natural or synthetic urease in-
hibitors in order to improve human, animal and agriculture life
quality and to promote the fields of pharmaceutical, agricultural
∗
Corresponding authors.
E-mail address: ajmalkhan@unizwa.edu.om (A. Khan).
https://doi.org/10.1016/j.molstruc.2020.129141
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
Scheme 1. Synthetic route of 2-(5-(1-(4-nitrophenylsulfonyl) piperidin-4-yl)−1,3,4-oxadiazol2-ylthio)-N-(substituted) acetamide (6a–o).
and environmental sciences. Certain synthetic compounds like hy-
droxyurea, flurofamide and hydroxamic acid have shown potential
efficacy. However, some of these compounds were banned due to
their toxic or unstable nature; for example, acetohydroxamic acid
was found with teratogenic effect in rats [19]. Likewise, majority
of the recognized inhibitors were redundant due to their harmful
effects.
solutions at room temperature. Type X urease (Cat. No. U4002–
50 KU), urea (Cat. No. U5378), phenol (Cat. No. 16,017) and di-
potassium hydrogen phosphate (Cat. No. 16,788–57–1) were pur-
chased from Sigma-Aldrich, USA. NaOCl (Cat. No. 230,394 M) and
methanol (Cat. No. 20,864.320/0,823,601) were purchased from
BDH Lab. Supplies, UAE. Sodium hydroxide (Cat. No. S41298–
4
J) was purchased from Unichem, India. Thiourea (Cat. No.
The identification of new natural and synthetic urease in-
hibitors is promising due to the involvement of urease in agri-
culture, health, environmental and economical problems. We have
previously identified number of potent urease inhibitors which
might use as alternate to standard thiourea [20,21]. In the same
connection, we further try to discover effective inhibitors having
considerably high efficacy against urease without any side effects.
In the present work, we have resynthesized acetamide hybrids
of N-substituted 1,3,4-oxadiazoles and piperidines already identi-
fied urease inhibitors [22] and performed their detailed enzyme
kinetics, in order to know about their mechanism of inhibition.
Both oxadiazole and piperidines are active pharmacophores of
many therapeutics compounds, such as antibacterial [23,24], anti-
fungal [25,26], anticonvulsant, anti-enzymatic [27], and anticancer
[28] agents. The combined effect of oxadiazole and piperidine com-
pounds in one unit gave the best impetus for the search of highly
active biological and pharmacological agents.
1,079,791,000) was provided by Merck, Germany.
2.2. General procedure for the synthesis of target compounds (6a–p)
Equimolar of 4-nitrobenzene sulfonyl chloride (a) and
ethyl isonipecotate (b) at 0.390 mol were react in the pres-
ence of sodium carbonate in aqueous medium to get ethyl
1-(4-nitrophenylsulfonyl)piperidin-4-carboxylate (1) at room
temperature. The compound (1) (0.015 mol) was reacted
with hydrazinium hydroxide (5 mL) in methanol to get 1-(4-
nitrophenylsulfonyl)piperidine-4-carbohydrazide (2). The 5-(1-
(4-nitrophenylsulfonyl)piperidin-4-yl)−1,3,4-oxadiazol-2-thiol (3)
was synthesized by adding equimolar of KOH (0.0304 mol) and
compound 2 (0.0304 mol) in 10 mL of ethanol followed by the
addition of CS2 (0.0608 mol) and refluxed for 6 h. Aralkyl/aryl
amines (4a–o; 0.02 mol) were stirred with 2-bromoacetyl bromide
(0.02 mol) in distilled water for 2 h to get N-(substituted)−2-
bromoacetamides (5a–o). The targeted series of 2-(5-(1-(4-
nitrophenylsulfonyl)piperidin-4-yl)−1,3,4-oxadiazol-2-ylthio)-N-
(substituted) acetamide (6a–o) were obtained in the presence LiH
and 10 mL of N,N-dimethyl form amide as reaction medium 24 h
(Scheme 1).
Therefore, due to the existence biological applications of 1,3,4-
oxadiazoles and piperidine, we hereby report the synthesis, detail
kinetics studies against urease and safety profile of hybrids N-
(substituted)−2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)−1,3,4-
oxadiazol-2ylthio)acetamide having both 1,3,4-oxadiazoles and
piperidine in one unit.
2.3. Urease inhibition assay (Indophenol’s method)
2. Materials and methods
Initially, the reaction mixture consists of 25 μL (one unit per
well) of Jack bean urease solution and 5.0 μL of a test compound
(0.5 mM in solvent methanol) and was incubated at 30 °C for
15 min in 96-well plates. Later 55 μL of potassium phosphate
2.1. General
All the reagents and chemicals used were of high analytical
level. Deionized H2O was utilized for the preparation of various

F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
3
buffer (4 mM, pH 6.8) containing urea (100 mM) was added in
each well and plate was incubated at 30 °C for additional 15 min.
Activity of urease was determined by the measurement of the
amount of NH3 formed during urease catalysis as mentioned by
Weatherburn [29]. After that, each well of the plate was inoculated
with 45 μL of phenol reagent (1% w/v phenol + 0.005% w/vNa2-
nitroprusside) and 70 μL of alkali reagents (0.5% w/v sodium
hydroxide+ 0.1% sodium hypochlorite). Finally, after 50 min the in-
crease in absorbance was measured at 630 nm by the use of mi-
croplate readers (Molecular Devices., CA, USA). The entire reactions
were performed (in triplicate) till to achieve a final volume 200 μL.
Analysis of the results was carried out by the SoftMax Pro6.3 soft-
ware (Molecular Device, USA). Thiourea was used as standard by
applying this method. The percentage (%) inhibition was measured
by using the formula;
which is important for structure-based drug designing. These re-
sults were then correlated with the experimental values. Molecular
Operating Environment (MOE 2009–2010, Quebec, Canada) docking
software was used to perform such studies (MOE. 2010). Ligands as
well as the receptor protein (urease) preparation are the two basic
steps prior to perform the docking.
(і) Ligands preparation
The 3D structures of all the ligands were sketched through
ChemDraw Ultra 12.0 (Cambridge Soft-2001, Cambridge, USA)
(Cambridge Soft. 2001) and saved in the mol format. The com-
pounds scaffolds were 3D protonated by Protonate 3D Option
in MOE software. The energies of identified ligands were mini-
mized by applying the default parameters of energy minimization
algorithm already adjusted in MOE (gradients: 0.05, force field:
MMFF94x). The Database of compounds series was created in mdb
file format.
%age inhibition = 100-(ODtest well/ODcontrol) × 100.
(іі) Preparation of receptor protein
The 3D structure of urease (4UBP) having resolution of 1.55 A,
˚
2.4. Determination of kinetic parameters
was retrieved from Protein Data Bank (PDB). H2O molecules from
the receptor protein were extracted. Like ligands, Protonate 3D op-
tion was applied for the protonation of urease 3D structure. Simi-
larly, the energy of urease protein was also minimized by applying
the default parameters of energy minimization algorithm with gra-
dients of 0.05 and force field Amber99, and saved in pdb file for-
mat. Later on, docking of all ligands was carried out in the binding
pocket of urease by following the default parameters of MOE-Dock
Program. In order to increase the accuracy of protocol, re-docking
was repeated [31]. After complete docking of all the ligands, the
most excellent 2D as well as 3D interaction images were chosen to
elucidate their specific types of interactions and bond lengths re-
spectively. All 3-D figures were made by discovery studio visualizer
software [32].
The IC50 inhibitory potential of compounds is the concentration
of the test compounds that blocked the urea (substrate) hydrolysis
values represent by 50% was measured by monitoring the effects of
their different concentrations (from high to low) in the assay. Such
calculation was carried out by using the EZ-Fit Enzyme Kinetics
Program (Perrella Scientific Inc., Amherst, USA). The ES represents
the complex of Jack bean urease and urea, while P stand for the
product obtained as a result of reaction. The Lineweaver-Burk plot
was then applied to identify the type of inhibition, while dissocia-
tion and inhibition constants (Ki) values were calculated by apply-
ing the Dixon plots and their secondary replots [30]. Non-linear re-
gression equation was applied to find out the Km, Ki, and Vmax val-
ues. At first, the values of 1/Vmaxapp were calculated at each inter-
section points of lines of every inhibitor concentration on y-axis of
the Lineweaver-Burk plot and then replotted against various con-
centrations of inhibitor. In the subsequent proceeding, the slope of
each line on the Lineweaver-Burk plot obtained as a result of in-
hibitor concentration was plotted against various concentrations of
inhibitor. Competitive inhibition can be classically represented as;
2.7. Cytotoxicity evaluation
The cytotoxicity of various potential urease inhibitors were
tested against neutrophil cell lines. The following steps were used
for the evaluation of cytotoxicity.
(і) Isolation of human neutrophils
Heparinized fresh venous blood was taken from the young and
healthy volunteers in a city blood collection center. Isolation of
neutrophils was carried out through Siddiqui et al., 1995 method
[33]. Accordingly, mixing of whole blood was made with Ficoll-
Paque (Pharmacia Biotech Amersham, Uppsala). When sedimenta-
tion occurred, the unnecessary red blood cells (RBCs) were layered
in a buffy coat way on a 3.0 mL cushion of Ficoll. Centrifugation
was then carried out for 30 min at a rate of 1500 rpm. Super-
natant was discarded, while pellets were collected. Furthermore, it
was mixed with 0.83% of hypotonic ammonium chloride solution
in order to lyse the RBCs. Again, the solution was centrifuged and
Modified Hank’s Solution (MHS) was used for the washing of col-
lected neutrophils. Later on, resuspension of neutrophil cells was
performed in the same solution at the rate of 1 × 107 cells per
mL.
Scheme 2. Classical representation of competitive inhibition.
2.5. Statistical analysis
Each experiment was performed in triplicate and results were
expressed as a mean of three. SoftMax Pro 6.3 Software (Molecular
Devices, CA, USA) was used for the analysis of these results. The
graph plotting was carried out through GraFit program. By using
the same program; we have obtained the values of correlation co-
efficients, intercepts, slopes, and their standard errors from linear
regression analysis. The relationship for each and every line in all
the graphs was more than 0.99.
(іі) Assay procedure for neutrophil based cytotoxicity
Isolated human neutrophils (1 × 107cells per mL) were treated
first with compounds under trial for thirty minutes and then with
0.25 mM WST-1(Dojindo Laboratories Kumamoto, Japan) on shak-
ing water bath at 37 °C [34]. Change in absorbance was calculated
after incubation for 3 h, at 450 nm using 96-well plate reader
(Spectra-MAX-340, Molecular Devices, CA, USA). Here the OD rep-
resents the mean of 5 investigational replicates. Percent (%) viabil-
ity of cells was measured as:
2.6. Molecular docking
%age viability of cells = {(OD test compound × 100/OD control)
-100}–100
Molecular docking was performed for prediction the orienta-
tion of protein active site residues and potential inhibitors binding,

4
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
2.8. Phytotoxicity evaluation
The urease inhibitors are used in agriculture to reduce the pH
of soil and to control the loss of urea. Therefore, the identified
inhibitors were checked for their phytotoxic effects according to
the modified assay of McLaughlin et al., 1991 [35]. The identi-
fied inhibitors with their three different concentrations (i.e. 10, 100
and 1000 μg/mL in CH3OH) were dissolved in sterilized E-medium.
Later on, inhibitors having fixed concentrations (made by the di-
lution of stock solution) were taken in sterilized conical flasks and
allowed their evaporation for overnight. In each of the flask, 20 mL
of sterilized E-medium and 10 Lemna aequinocitalis Welv plants
were inoculated, each having a rosette of 3 fronds. In some of
flasks CH3OH and phosporoamide (commercially available urease
inhibitors) were added which act as negative and positive control
respectively. Such treatments were performed in triplicates and the
flasks were kept in Fisons Fi-Totron 600H growth cabinet at 30 °C
for a whole week having the environmental conditions (light in-
tensity: 9000 lux, relative humidity: 56
10 rh) at a short day
span of 12 hrs. Growth rate of Lemna aequinocitalis Welv was mea-
sured by counting the number of fronds per dose present in flasks
containing inhibitors, while the inhibition of growth was calculated
with the reference to negative control.
3. Results and discussion
3.1. Chemistry
In the current study, we have synthesized
a series of N-
(substituted)−2-(5-(1-(4-nitrophenylsulfonyl)piperidin-4-yl)−1,3,4-
oxadiazol-2-ylthio)acetamide having a wide range biological ap-
plications [23–28]. The synthesis of targeted compounds was
initiated by the synthesis of sulfonamide through 4-nitrobenzene
sulfonyl chloride and piperidine termed as compound 1, which
was further modified into hydrazide the key product of this series.
Hydrazide was converted into 1,3,4-oxadiazole and finally by the
reaction 1,3,4-oxadiazole and a variety of acetamides to synthesize
(6a–o) (Table 1). All the synthesized compounds were character-
ized by spectroscopic techniques like IR, EI-MS, 1H NMR and 13C
NMR. The detail spectroscopic data were published in our previous
article [22].
3.2. Biology
X-ray crystallography based resolved 3D structure identified
two nickel ions in the active site of urease, which are coordinated
by the nitrogen of imidazole ring, carboxylate group and water
molecule. The architecture of the active site is almost similar in
all ureases obtained from different origins and adopts tetrahedral
geometry between two Ni atoms due to the presence of four H2O
molecules that were observed in native Bacillus pasteurii (BP) and
Klebsiella aerogenes (KA) ureases [36]. To understand about accu-
rate mechanism concerning urease activity is basically the initia-
tives towards the effective drugs design in order to treat the ure-
ase related problems. It is essential to know clearly about the co-
ordination mechanism of inhibitors with the active site of urease
in order to understand about its inhibitory potential. Nearly from
100 years ago, the scientists are trying to know about the exact
mechanism of action of urease but still it has been remained un-
der investigation [37]. Several scientists have described about their
mechanism of action, but the initial mechanism of urease was pro-
posed by Zerner and his coworkers, who suggested that one nickel
Fig. 1. Steady state inhibition of urease by acetamide hybrid compound No. 6o, A is
the Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of four
fixed Jack bean urease concentrations in absence (ꢀ) and presence of 12.5 μM (ꢁ),
25.0 μM (●), 50 μM (◦) of compound No. 6o. B is the Dixon plot of reciprocal of
the initial velocities versus various concentrations of compound 15 at fixed urease
concentrations, (ꢀ) 50 μM, (ꢁ) 25.0 μM, (●)12.5 μM and (◦) 6.2 μM. C is the, 1/
Kmapp versus various concentrations of compound No. 6o.

F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
5
Table 1
Chemical structures of different acetamide hybrid synthetic derivatives 6a-o.

6
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
Table 2
Inhibitory potency data of 6a-o synthetic compounds against Jack bean urease.
Enzyme
Compounds
Ki (μM) SEM
Km (mM)
Km (mM) app
Vmax (μmol / min)−1
Vmaxapp
Type of Inhibition
Urease
6a
6b
6c
6d
6e
6f
3.11
5.20
3.14
5.15
3.24
3.28
3.23
3.16
3.17
3.25
4.11
4.14
3.12
3.32
11.2
0.2
0.7
1.1
0.5
0.3
0.4
0.6
0.5
1.1
0.1
0.7
0.5
1.1
0.7
0.3
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
8.4
8.0
8.5
6.4
8.6
9.7
8.5
9.3
9.9
9.7
7.3
9.1
9.4
5.9
8.0
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
104
104
106
105
107
102
104
103
104
104
102
105
103
107
107
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
6 g
6h
6i
6j
6k
6l
6m
6n
6o
Ki (dissociation constant or inhibition constant) was determined from nonlinear regression analysis by Dixon plot and secondary.
Table 3
Table 4
Viability results of human neutrophils (1 × 107 cells per mL)
Results of LemnaWelv Phytotoxicity assay of 6a-o acetamide
hybrids.
against 6a-o acetamide hybrids.
Compounds
Conc. μg / mL
Viability [%]
Compounds
Concentration of compounds (μg/ml)
6a
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
76.45
92.86
86.41
78.35
68.12
99.94
100
4.0
5.2
4.0
3.5
3.1
4.0
1000
80.0
69.6
87.55
100
100
10
6b
6a
58.3
17.5
6c
6b
43.56
78.54
85.67
42.3
23.5
6d
6c
27.8
6e
6d
35.32
23.2
6f
6e
64.5
100
6 g
1.0
5.2
4.4
6f
100
29.32
12.34
14.00
43.56
28.12
23.6
6h
95.41
71.9
6 g
61.08
70.6
100
37.65
23.99
62.04
74.05
77.02
17.0
6i
6h
6j
69.05
63.14
79.39
81.35
73.39
68.32
4.9
2.2
2.6
4.2
3.9
1.2
6i
6k
6j
89.7
83.6
32.5
87.58
79.2
100
6l
6k
6m
6l
7.7
6n
6m
48.53
60.36
36.43
70.3
24.18
24.01
22.71
19.0
6o
6n
Acetohydroxamic acid
88.04 5.0
6o
Phosphoroamide
81.5
Mean
SD of three experiments.
activates the H2O molecules while the other Ni activates the sub-
strate urea [38].
derstand not only about the suitable configuations of the ligands
obtained after adjustment in the active pocket of enzyme, but also
supported us about the type of inhibition as well as about the
strength of inhibition after measuring the bond lengths of inter-
acted inhibitors. After docking repeatition for several turns, we
have selected the images with best interactions and less dock-
ing scores for 3D configuration analysis. When measured their
bondlengths, the most feasible images were selected for each com-
pound. It was observed that all the bioassay identified inhibitors
were supported strongly by the docking as they were mostly inter-
acted with the Ni ions of the active site with more interactions at
very short bond distances. Furthermore, it was also identified that
terminal nitro group oxygen atoms of the main skeleton of ligands
were in front line interactions with the Ni ions and have proved
as potent inhibitors against urease. Some of the interaction images
are listed below.
All the synthesized acetamide hybrids (6a–o) have inhibited
the urease very effectively in a concentration dependent ways. We
have determined their Ki values, in order to know about their
strength of inhibition. Three different methods were applied for
the determination of Ki values. The whole series of the compounds
have inhibited the enzyme very strongly, showing low Ki values of
3.11
0.2 to 11.2
0.3 μM. To know about the possible mode of
inhibition of enzyme and binding moieties of the inhibitors with
enzyme, we have determined their type of inhibition. In case of
all the inhibitors, the Km values were increased, while Vmax values
were remained fixed, which indicates competitive nature of inhi-
bition. It has also been confirmed from the Lineweaver-Burk plots,
Dixon plots and their secondary replots, that urease was inhibited
at their active site as is clear from the Fig. 1 (A, B & C). In addition
to this, Km, Ki, Vmax, Vmaxapp and Kmapp values as well as the type
of inhibition of each compound are illustrated in Table 2.
The proposed structure activity relationship (SAR) showed that
the activity of these compounds may be due the sulfonamide and
nitro moieties, while substitution at R group have not significantly
influence their activities, as all compounds have almost same Ki
values (table 2).
From the docking results, it has come to know that compound
6a has made four interactions in the active site of urease enzyme.
The first acidic interaction was made by the ligand through its sul-
fonyl oxygen with NH hydrogen of imidazole moiety of His323
˚
showing bond length of 2.42 A. The second metallic interaction
was made between terminal nitro group oxygen of ligand and Ni
˚
Molecular docking was performed to confirm the accurate
protein-ligand interactions geometries and also evaluated their
predictive structure activity relationship (SAR). From this, we un-
ion of the receptor at a bond distance of 2.60 A. The remaining two
acidic as well as arene-cation interactions were established among
sulfonyl oxygen, nitrophenyl ring and Arg339 amino acid residue

F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
7
Fig. 2. The docked conformations of compound 6a in 2D (A) and 3D (B) in the active site of urease. Compound is depicted in purple color thick stick, while protein is shown
in ribbon with active site residues in thin stick.
˚
From the Fig. 4 (A & B), it is clear that compound 6e has
blocked very strongly the active pocket of urease by making five
interactions. The NH proton of the imidazole ring of His323 and
sulfonyl oxygen has made a acidic interaction with a bond length
showing bond lengths of 2.70 and 2.74 A, respectively as indicated
in Fig. 2 (A & B).
Similarly, compound 6d has inhibited the enzyme very strongly
by making four interactions. The first hydrogen bond interaction
existed between sulfonyl oxygen and NH group hydrogen of imida-
˚
of 2.09 A. Couples of the terminal oxygen atoms of the nitro
˚
group and Ni ions have established metallic interactions with bond
zole ring of His323 showing bond distance of 1.98 A. The next two
˚
˚
lengths of 2.47 A and 2.81 A, respectively. His324 by itself has
made an arene-cation as well as arene-arene interactions with the
metallic interactions were found between two Ni ions of the urease
and a couple of terminal nitro group oxygen atoms of the ligand
˚
˚
oxadiazole ring (bond length: 3.06 A) and terminal phenyl moi-
indicating bond lengths of 2.59 and 2.85 A, respectively. Somewhat
˚
ety (average bond length: 4.52 A), respectively. It has come to
interaction exhibited by the oxadiazole ring of compound with im-
˚
know from the Fig. 5 (A & B) that compound 6g have inhibited
idazole moiety of His324 at a distance of 4.50 A, which is illus-
the enzyme by showing three interactions. The first hydrogen bond
trated through Fig. 3 (A & B).

8
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
Fig. 3. The docked conformations of compound 6d in 2D (A) and 3D (B) in the active site of urease. Compound is depicted in purple color thick stick, while protein is shown
in ribbon with active site residues in thin stick.

F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
9
Fig. 4. The docked conformations of compound 6e in 2D (A) and 3D (B) in the active site of urease. Compound is depicted in purple color thick stick, while protein is shown
in ribbon with active site residues in thin stick.

10
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
Fig. 5. The docked conformations of compound 6 g in 2D (A) and 3D (B) in the active site of urease. Compound is depicted in purple color thick stick, while protein is
shown in ribbon with active site residues in thin stick.
formed between sulfonyl oxygen and proton of NH group in imi-
fore, it is must to know about the toxic nature of newly identified
inhibiting compounds. In this regard, we have subjected all these
synthesized compounds to human neutrophils by applying an al-
ready discussed standard assay and by using the positive control
of most widely used urease inhibitor of acetohydroxamic acid. It
has been known from the results that all 6a–o inhibitors of the
series are non toxic against human
˚
dazole ring of His323 (bond length: 1.83 A). In the same sequence,
another metallic interaction has been found between Ni1 ion of
the urease and nitro group oxygen of the ligand in the range of
˚
2.56 A. The NH2 proton of Arg339 and benzyl moiety of com-
pound also made an interaction of arene-cation with a bond length
˚
of 2.85 A.
After docking, compound 6i has given four interactions with
Due to hyper activity, urease is also involved in a number of
agricultural as well as neutrophil cells and can be proved as lead
series of drugs for the urea related complications (Table 3) en-
vironmental problems. To control such complications, urease in-
hibitors were used. It has been known from the previous litera-
ture that most of the urease inhibitors have shown severe side ef-
fects against plants. To confirm such effects, we have checked all
these compounds (6a–o) through standard LemnaWelv phytotoxic-
ity assay. The results proved that mostly they were non phytotoxic
against plants, except of the compound 6l which is slightly phyto-
toxic against plants as indicated in Table 4.
a variety of amino acids of protein. Ala366 has made back bone
donor interaction with NH hydrogen of the ligand (bond length:
˚
2.06 A), while His222 through its NH hydrogen of imidazole ring
has given hydrogen bond with carbonyl oxygen (bond length:
˚
2.36 A). In addition, Lys169 and His324 have made arene-cation
interactions with nitrophenyl and oxadiazole rings of the ligand in
˚
the range of 3.65 and 4.60 A respectively (Fig. 6 A & B).
From the previous studies, we have understood that most of the
identified urease inhibitors were toxic against animals, specifically
for the human beings and were banned after some time. There-

F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
11
Fig. 6. The docked conformations of compound 6i in 2D (A) and 3D (B) in the active site of urease. Compound is depicted in purple color thick stick, while protein is shown
in ribbon with active site residues in thin stick.
4. Conclusion
non phytotoxic against plants. Shortly, it can be concluded that
these compounds can serve lead drug candidates against urease re-
lated issues.
Kinetics studies have confirmed their competitive type of in-
hibition. In silico studies have also shown close agreement with
their competitive type of inhibition, as most of the compounds
have interacted well with an individual or both the Ni ions of the
active site, mostly through their nitro group oxygen atoms and
rarely through their other functional groups. Moreover, they en-
compassed low cytotoxic effects against human neutrophils and
Declaration of Competing Interest
The authors declare that they have no conflicts of interest con-
cerning the publication of this paper.

12
F.A. Khan, A.U. Rehman and M.A. Abbasi et al. / Journal of Molecular Structure 1223 (2021) 129141
Acknowledgement
[19] K.M. Khan, A. Wadood, M. Ali, Z. Ul-Haq, M.A. Lodhi, M. Khan, S. Perveen,
M.I. Choudhary, Identification of potent urease inhibitors via ligand-and struc-
ture-based virtual screening and in vitro assays, J. Mol. Graph. Model. 28 (2010)
792–798.
The authors are thankful to Higher Education Commission
(HEC) of Pakistan for generous support.
[20] S. Shamim, K.M. Khan, U. Salar, F. Ali, M.A. Lodhi, M. Taha, F.A. Khan, S. Ashraf,
Z. Ul-Haq, M. Ali, 5-Acetyl-6-methyl-4-aryl-3, 4-dihydropyrimidin-2 (1H)-ones:
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Supplementary materials
[21] M.A. Lodhi, S. Shams, M.I. Choudhary, A. Lodhi, Z. Ul-Haq, S. Jalil, S.A. Nawaz,
K.M. Khan, S. Iqbal, A.-u. Rahman, Structural basis of binding and rationale for
the potent urease inhibitory activity of biscoumarins, Biomed Res. Int. 2014
(2014).
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129141.
[22] A.-u. Rehman, J. Iqbal, M.A. Abbasi, S.Z. Siddiqui, H. Khalid, S. Jhaumeer Laulloo,
N. Akhtar Virk, S. Rasool, S.A.A. Shah, Compounds with 1, 3, 4-oxadiazole and
azinane appendages to evaluate enzymes inhibition applications supported by
docking and BSA binding, Cogent Chem. 4 (2018) 1441597.
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