Benzenesulfonohydrazides inhibiting urease: Design, synthesis, their in vitro and in silico studies

Article abstract of DOI:10.1016/j.molstruc.2020.128740

Keeping in view the therapeutic importance of ureases due to its involvement in different pathological conditions, its inhibition was investigated by newly synthesized benzenesulfonohydrazides. Elemental analysis, IR, ¹H NMR and ¹³C

Full text of DOI:10.1016/j.molstruc.2020.128740

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Benzenesulfonohydrazides inhibiting urease: Design, synthesis, their in vitro and in
silico studies
Mahmood Ahmed, Muhammad Imran, Muhammad Muddassar, Riaz Hussain,
Muhammad Usman Khan, Saghir Ahmad, Muhammad Yasir Mehboob, Saira Ashfaq
PII:
S0022-2860(20)31065-6
DOI:
https://doi.org/10.1016/j.molstruc.2020.128740
MOLSTR 128740
Reference:
To appear in: Journal of Molecular Structure
Received Date: 25 February 2020
Revised Date: 9 June 2020
Accepted Date: 21 June 2020
Please cite this article as: M. Ahmed, M. Imran, M. Muddassar, R. Hussain, M.U. Khan, S. Ahmad,
M.Y. Mehboob, S. Ashfaq, Benzenesulfonohydrazides inhibiting urease: Design, synthesis, their
in vitro and in silico studies, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.128740.
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Benzenesulfonohydrazides inhibiting urease: Design, synthesis, their in vitro and in silico studies
1
*
2
3
4
Mahmood Ahmed , Muhammad Imran , Muhammad Muddassar , Riaz Hussain , Muhammad
4
,5
6
4
2
Usman Khan , Saghir Ahmad , Muhammad Yasir Mehboob , Saira Ashfaq
1
Renacon Pharma Limited, Lahore 54600, Pakistan
2
School of Life Sciences, FC College (A Chartered University) Lahore- Pakistan
Department of Biosciences, COMSATS University, Park Road, Islamabad- Pakistan
Department of Chemistry, University of Okara, Okara-56300, Pakistan
3
4
5
6
Department of Applied Chemistry, Government College University, Faisalabad-38000, Pakistan
Institute of Chemistry, University of the Punjab. Lahore 54590, Pakistan
Running title: Benzenesulfonohydrazide as urease inhibitors
To whom correspondence should be addressed
Dr. Mahmood Ahmed
mahmoodresearchscholar@gmail.com
1

Abstract
Keeping in view the therapeutic importance of ureases due to its involvement in different pathological
conditions, its inhibition was investigated by newly synthesized benzenesulfonohydrazides. Elemental
1
13
analysis, IR, H NMR and C NMR spectral studies were performed to elucidate the structure of
benzenesulfonohydrazides. In vitro urease enzyme inhibition assay revealed the compound INS-5 was
found to be the most potent (IC = 1.11±0.29µM) among the tested compounds. The compound INS-
5
0
2
was competitive inhibitor with Ki value 5.60 µM while the compounds INS-1 and INS-5 were
mixed type of inhibitors with Ki values 4.32 and 2.76 µM respectively. Ancillary to synthetic studies,
DFT and TDDFT calculations at B3LYP/6-311G(d,p) level of theory were performed for comparative
analysis of spectroscopic data, frontier molecular orbitals (FMOs), natural bond orbital (NBO)
analysis and molecule electrostatic potential (MEP) surface. Overall, experimental findings were
supported nicely by corresponding DFT computed results. The NBO analysis confirmed that the
presence of hyperconjugative interactions are pivotal cause for stability of investigated compounds.
Global reactivity descriptors were also calculated using the energies of FMOs energies. Molecular
docking studies were performed to identify the plausible binding mode of the competitive inhibitor.
Keywords: Hydrazide; Isonicotinoyl; Urease; DFT; Docking studies
1
. Introduction
Urease is a metalloenzyme (nickel based, urea amidohydrolase, EC 3.5.1.5) which belongs to super
family of aminohydrolase and phosphotriesterase that accelerate the transformation of urea into
carbon dioxide and ammonia. Canavaliaensiformis (jack bean) was first well characterized and
crystalize among the numerous ureases that is utilized in enzyme inhibition studies. Continuous
formation of ammonia enhances the gastric mucosa permeability that results in inflammation, ulcer,
adenocarcinoma and lymphoma [1-5]. Helicobacter pylori(H. pylori) relies on urease activity for
bacterial survival in low pH environment of the stomach, therefore targeting urease activity can
eradicate the bacterium in early stages of the infection[6, 7]. Since the structure, molecular weight,
amino acid sequence of urease greatly depends upon its origin. The bacterial ureases are
heteropolymeric molecules having three subunits, α, β and γ whereas the urease from jack beans are
homohexameric molecules having six α subunits. Despite the difference in the structure, the active
site of the enzyme, largely remains conserved. The active site is always located in α subunits having
binuclear nickel center[8, 9]. Due to involvement of urease in bacterial infections, designing of novel
urease inhibitors are of great research interest. The current eminence of urease inhibitors is quite
restricted. However mostly five different classes of inhibitors including barbituric acid analogues
[
10], thiourea derivatives [11], five and six-membered heterocyclics, natural products and metal
complexes are available in literature [12] but these are not efficient with regard their drug like
properties and the full potential of urease inhibition is yet to be discovered. Hydrazide moiety along
2

with heterocyclics and sulfoxide exhibits a wide range of biological activities including antibacterial,
antifungal, antinociceptive, anticonvulsant, enzyme inhibition and anticancer [10, 13-15]. In literature,
hydrazides as sulfonohydrazides [16], hydrazide-carbazoles [17] and hydrazide-hydrazones [18] are
reported for their cytotoxic activities. Sulfonohydrazides had shown the cytotoxic effect against brain
and ovarian tumor cells and inhibited the cell proliferation at < 10 µM concentration. Hydrazides
integrated carbazoles revealed a significant cytotoxic effect against the pancreatic cell lines(AsPC1
and SW1990) with IC50 values ranged between 3.42-22.42 µM whereas hydrazide-hydrazone
derivatives showed the cytotoxic effect against colon, gastric and hepatoma carcinoma cells and
displayed higher cytotoxic activity than 5-fluorouracil. So in struggle to identify lead molecules for
enzyme inhibition by our research group, we are reporting synthesis and urease inhibitory activities of
new hydrazides (Fig. 1) on the basis of their wide range biological activities. Due to the involvement
of ureases in different pathological conditions, the discovery of safe and potent urease inhibitors has
been an area of challenge in pharmaceutical research. Additionally density functional theory (DFT)
calculations have been performed to evaluate spectroscopic (FT-IR and UV-Visible) studies, frontier
molecular orbital (FMO) analysis, natural bond orbital (NBO) analysis, global reactivity parameters
and molecular electrostatic potential (MEP) surfaces properties of investigated compounds. Enzyme
kinetics and molecular modeling studies were also carried out to get insight into inhibition mechanism
and binding conformation of competitive inhibitor in urease enzyme.
CH3
CH3
CH3
O
O
S
O
O
O
O
S
O
O
O
O
S
S
S
O
O
O
O
O
O
N S
NH
NH
NH
NH
NH
N S
NH
CH3
N S
NH
O
O
O
O
O
N
INS-3
N
INS-4
N
INS-5
N
INS-1
N
INS-2
Fig. 1 Benzenesulfonohydrazide under investigation
2
. Experimental
In this research high purity chemicals were used and these were acquired from Falcon Scientific,
Lahore-Pakistan originate to Merck (Germany) whereas high purity water was produced in our own
®
lab using Milli-Q water system (UK). Spectral studies including IR (FTIR spectrophotometer,
1
13
Agilent Technologies, Carry 630), HNMR-500 MHz, CNMR-125 MHz (NMR spectrometer,
Bruker, USA) and elemental analysis (C, H, N and S by Flash elemental analyzer, HT+ Thermo
3

Scientific, UK) were performed to elucidate the structure of newly synthesized compounds. Melting
point was determined by Gallenkamp apparatus and PG-T80UV-Vis spectrophotometer (PG,
Instruments-UK) was used to calculate ꢀmax. Pre-coated silica plates (Merck, Germany) were spotted
for TLC analysis and spots were detected in UV light to confirm the purity of the synthesized
compounds.
2
.1.Protocol for benzenesulfonohydrazide synthesis
A most convenient route under dynamic pH control in an aqueous medium was used for the synthesis
of sulfonamides already described in our earlier work [19-21] and details are explained in
supplementary data. Details chemistry for synthesis of compounds 1-5 with physiochemical and
spectral data explained below.
Scheme 1. Synthesis of Benzenesulfonohydrazide
2
.1.1. N'-isonicotinoylbenzenesulfonohydrazide (INS-1)
◦
-1
White solid; Yield, 79.4 %; M. P., 112-114 C; R , 0.76. UV λ (nm): 242.IR (ATR, υ cm ): 3567,
f
max
3
247 (N-H), 3057 (CH-aromatic), 1681 (C=O), 1307 (-SO -NH
), 1154 (-SO -NH2-sym), 1091 (-
2-asym 2
2
1
S=O). H NMR (500 MHz, DMSO-d ): δ 8.89 (2H, d, J = 7.6 Hz, CH-pyridyl), 8.08 (1H, s, -NH-
6
H
1
3
CO), 7.56-7.84, (7H, m, ArH), 2.32 (1H, s, -NH). C NMR (125 MHz, DMSO-d ): 165.8, 149.0,
6
1
4
2
41.9, 140.3, 136.6, 131.0, 129.7, 127.7.Anal. Calc. for C H N O S (FW=277.3 g/mol): C, 51.98; H,
12 11 3 3
.0; N, 15.15; S, 11.56 %. Found: C, 51.81; H, 3.89; N, 15.37; S, 11.45%.
.1.2. N'-isonicotinoyl-4-methylbenzenesulfonohydrazide (INS-2)
◦
-1
White solid; Yield, 94.2 %; M. P., 180-182 C; R , 0.79. UV λ (nm): 242.IR (ATR, υ cm ): 3572,
f
max
3
244 (N-H), 3057 (CH-aromatic), 1676 (C=O), 1337 (-SO -NH
), 1157 (-SO -NH2-sym), 1090 (-
2-asym 2
2
1
S=O). H NMR (500 MHz, DMSO-d ): δ 8.89 (2H, d,J = 7.6 Hz, CH-pyridyl), 8.02 (1H, s, -NH-
6
H
13
CO), 7.74-7.84, (4H, m, ArH), 7.40 (2H, d, J = 7.5Hz,ArH), 2.32 (3H, s, CH ), 2.12 (1H, s, -NH). C
3
NMR (125 MHz, DMSO-d ): 165.8, 149.0, 141.9, 140.3, 136.6, 131.0, 129.7, 127.7, 21.3 (-CH ).
6
3
Anal. Calc. for C H N O S (FW=291.3 g/mol): C, 53.60; H, 4.50; N, 14.42; S, 11.01 %. Found: C,
1
3
13
3
3
5
3.86; H, 4.69; N, 14.57; S, 11.15 %.
4

2
.1.3. N'-isonicotinoyl-4-methyl-N-tosylbenzenesulfonohydrazide (INS-3)
◦
-
Yellowish white solid; Yield, 82.2 %; M. P., 174-176 C; R , 0.81. UV λ (nm): 235.IR (ATR, υ cm
f
max
1
)
: 3565, 3242 (N-H), 3054 (CH-aromatic), 1676 (C=O), 1336 (-SO -NH
), 1158 (-SO -NH2-sym),
2-asym 2
2
1
1
089 (-S=O). H NMR (500 MHz, DMSO-d ): δ 8.89 (2H, d,J = 7.6 Hz, CH-pyridyl), 8.02 (1H, s, -
6
H
1
3
NH-CO), 7.74-7.81, (6H, m, ArH), 7.40 (4H, d, J = 7.5Hz,ArH), 2.32 (6H, s, 2 x CH ). C NMR (125
3
MHz, DMSO-d ): 164.8, 149.0, 141.9, 140.3, 136.6, 131.0, 129.7, 127.7, 21.3 (2 x CH ). Anal. Calc.
6
3
for C H N O S (FW=445.5 g/mol): C, 53.92; H, 4.30; N, 9.43; S, 14.39 %. Found: C, 53.86; H,
20
19
3
5 2
4
.61; N, 9.54; S, 13.45 %.
2
.1.4. N'-isonicotinoyl-N-(phenylsulfonyl)benzenesulfonohydrazide (INS-4)
◦
-
Yellowish white solid; Yield, 75.5 %; M. P., 120-122 C; R , 0.67. UV λ (nm): 235.IR (ATR, υ cm
f
max
1
)
: 3572, 3244 (N-H), 3056 (CH-aromatic), 1676 (C=O), 1337 (-SO -NH
), 1157 (-SO -NH2-sym),
2-asym 2
2
1
1
090 (-S=O). H NMR (500 MHz, DMSO-d ): δ 8.87 (2H, d, J = 7.6 Hz, CH-pyridyl), 8.02 (1H, s, -
6
H
13
NH-CO), 7.81-7.86, (6H, m, ArH), 7.71 (2H, d, J = 7.4Hz,ArH), 7.62 (2H, d, J = 7.5Hz,ArH). C
NMR (125 MHz, DMSO-d ): 164.0, 149.0, 141.0, 140.0, 139.6, 131.0, 129.7, 127.0. Anal. Calc. for
6
C H N O S (FW=417.5 g/mol): C, 51.79; H, 3.62; N, 10.07; S, 15.36 %. Found: C, 51.90; H, 3.51;
18
15
3
5 2
N, 10.14; S, 15.48%.
2
.1.5. N'-isonicotinoyl-4-methyl-N-(phenylsulfonyl)benzenesulfonohydrazide (INS-5)
◦
-
Yellowish white solid; Yield, 93.8 %; M. P., 162-164 C; R , 0.86. UV λ (nm): 242.IR (ATR, υ cm
f
max
1
)
: 3565, 3248 (N-H), 3055 (CH-aromatic), 1676 (C=O), 1336 (-SO -NH
), 1156 (-SO -NH2-sym),
2-asym 2
2
1
1
090 (-S=O). H NMR (500 MHz, DMSO-d ): δ 8.88 (2H, d,J = 7.6 Hz, CH-pyridyl), 8.04 (1H, s, -
6
H
1
3
NH-CO), 7.74-7.81, (9H, m, ArH), 7.40 (2H, d, J = 7.5Hz,ArH), 2.32 (3H, s, CH ). C NMR (125
3
MHz, DMSO-d ): 164.2, 149.0, 141.0, 140.3, 136.2, 131.0, 129.6, 127.7, 21.3 (CH ). Anal. Calc. for
6
3
C H N O S (FW=431.5 g/mol): C, 52.89; H, 3.97; N, 9.74; S, 14.86 %. Found: C, 52.86; H, 3.71;
19
17
3
5 2
N, 7.58; S, 14.65 %.
.2.% Urease inhibition assay, IC and kinetics study protocol
2
5
0
Urease inhibition assay was performed as reported in our earlier studies [22] and details are provided
in supplementary data.
2
.3.Computational studies
The overall quantum chemical calculations are performed with the help of DFT employing Gaussian
9 program package[23]. Geometrical optimization was done without symmetry restrictions by
0
applying B3LYP level of DFT and 6-311G (d,p) basis set combination. The frequency analysis based
on DFT/B3LYP/6-311G (d,p) level of theory was used for confirmation of stability associated with
the optimized geometries. The absence of negative eigen values among all calculated frequencies
reflected that optimized geometries of INS-1-INS-5 correspond to true minimum at potential energy
surfaces. The frontier molecular orbital and molecule electrostatic potential analysis was conducted at
5

B3LYP level of DFT and 6-311G (d,p) basis set conjunction. The NBO analysis was carried out at
B3LYP/6-311G (d,p) level of theory using NBO 3.1 program package. The UV-Vis analysis was
performed employing time dependent density functional theory (TDDFT) calculations at B3LYP/6-
3
11+G (d,p) level of theory for estimation of photophysical characteristics of INS-1-INS-5.
Furthermore, the frequency analysis based on DFT/B3LYP/6-311G(d,p) level of theory was used for
confirmation of stability associated with the optimized geometries. The absence of negative eigen
values among all calculated frequencies reflected that optimized geometries of INS-1-INS-5
correspond to true minimum at potential energy surfaces. The input files were organized with the help
of Gauss View 5 [24]. The Gauss View 5.0, Avogadro [25] and Chemcraft [26] programs were
employed for interpreting output files.
2
2
.4.Molecular Docking studies
.4.1. Protein structure and newly synthesized compounds preparation
In molecular docking protocol, the X-ray structure of Jack bean urease (PDB ID 4H9M) was retrieved
from Protein Data Bank (PDB) (https://www.rcsb.org/), prepared by using protein preparation wizard
embedded in Schrodinger software (www.schrodinger.com). Protein preparation includes structure
refinement, removal of unnecessary ligands, co-factors, water molecules, addition of missing residues
(
using Prime), and polar hydrogen atom. Ionization and states were also generated at PH 7. Finally
structure was minimized by applying force field OPLS 2005 to remove steric hindrance and allowed
deviation of 0.3 RMS from its native conformation. Synthesized compounds were drawn in Chem
draw ultra and prepared using LigPrep tool in Schrodinger Release 2018-2. Stereoisomers and
possible ionization states were generated by keeping at most 32 possible conformers of each
compound.
2
.4.2. Grid generation and Docking Protocol
Before docking the synthesized compounds, Grid box was generated around the Nickel ions present in
the active site. Size of cubic box was set at 20Å in each dimension and X, Y, Z coordinates setting
parameters which include cutoff radius scaling of 0.25 and Van der Waal factor 1.0 were defined at
1
7.5, 36.56, 20.48 respectively. After grid generation, the candidate compound was docked into the
receptor grid using GLIDE standard precision mode (SP) to predict best binding mode of the
competitive inhibitor. Initially 200 conformations were generated in the active site, out of which top
five were minimized and finally best was selected based on the glide score.
3
. Results and Discussion
.1.Chemistry
3
The target benzenesulfonohydrazides (INS1-INS-5) were prepared by interaction of INS with p-
toluenesulfonyl chloride and benzenesulfonyl chloride respectively and were resulted in good yield
(
75.5-94.2 %, Table 1). Synthetic route is depicted in scheme 1. All the synthesized compounds were
6

1
13
characterized by performing IR, UV, HNMR, and CNMR studies. In IR spectra, a broad absorption
-1
band at 3572-3242 cm indicating the presence of -NH moiety of sulfonamides in while a strong
-1
-1
absorption band in 1154-1158 cm (symmetrical) while 1307-1337 cm (asymmetrical)for all
synthesized compounds confirm the presence of -NH-S=O group. Moreover, characteristics sulfoxide
-1
1
absorption band appeared in region 1089-1091cm for all synthetic compounds. In H NMR spectra,
the peaks for –NH proton of –CONH- group appeared at δ 8.02-8.08 ppm whereas proton of –CH-
pyridyl appeared at δ 8.89 ppm. The singlet at δ 2.32 ppm assigned to proton of –CH linked to aryl.
3
In addition, all the other aliphatic and aromatic protons appeared in appropriate values of chemical
13
shifts. In C NMR spectra, characteristics peaks at δ 164.2-165.8 ppm assigned to amide carbon (-
13
CONH-) while signals for methyl carbon in all the compounds were appeared at δ 21.3 ppm. So, C
NMR spectral analyses were consistent with assigned structure of all compounds.
3
.2.Urease inhibition
All the synthesized benzenesulfonohydrazides were evaluated for urease inhibitory activity using well
known indophenol method. By using serial dilution method, 250-0.49µM concentrations were used of
each synthetic compound, thiourea (standard inhibitor for urease) for inhibitory assay and also IC of
5
0
each compound was calculated under investigation (Table 1). Compound INS-5 was found to be more
potent (IC = 1.11±0.29µM) among the tested compounds, whereas INS-3 and INS-4 with same
5
0
terminal aryl ring with and without –CH group respectively exhibited equal urease inhibition.
3
Table 1. IC and kinetics parameters of benzenesulfonohydrazides
50
a
b
c
IC (µM); mean±SEM
Vmax (app)
Km (app)
(mM)
6.19
Ki
Mode of
inhibition
Mixed
5
0
Compound
INS-1
(% inhibition)
(µM/min)
8.68
(µM)
4.32
5.60
1.79±0.18 (90.9)
1.33±0.14 (86.8)
INS-2
7.21
3.07
Competitive
INS-3
INS-4
INS-5
4.89±0.09 (81.1)
4.86±0.11 (81.3)
1.11±0.29 (81.8)
15.51±0.11 (92.1)
-
-
-
-
-
-
-
-
7.00
11.05
2.76
Mixed
d
Thiourea
18.61
2.18
18.18 Competitive
a
Vmax (app)= Maximum velocity of enzymatic activity at 20 µM inhibitor concentration
b
Km(app)= Michaelis–Menten constant at 20 µM inhibitor concentration
c
d
K (µM)= Calculated from Dixon plot, Standard inhibitor of urease
i
7

Inhibition mechanism was also investigated by performing the kinetic studies on three most potent
compounds INS-1, INS-2 and INS-5 with different concentration of compounds (0-20 µM) and
substrate (0.5-4.0 mM). Mode of inhibition and inhibition constant (K ) were determined by
i
enzymatic kinetics. Lineweaver Burk plots were used to assess the mode of inhibition by determining
the effect of inhibitors (compounds) on Vmax and K . V of jack bean urease enzyme was not
m
max
effected in the presence of inhibitor (INS-2) while K of enzyme increase which indicate the
m
competitive inhibition. The enzymatic kinetics of most active compound is presented below in Fig. 2-
4
.
Fig. 2 Enzymatic kinetics of compound INS-1, (a) Lineweaver–Burk plot (b) Secondary replot
Lineweaver–Burk) (c) Dixon plot
(
Fig. 3 Enzymatic kinetics of compound INS-2, (a) Lineweaver–Burk plot (b) Secondary replot
Lineweaver–Burk) (c) Dixon plot
(
Fig. 4 Enzymatic kinetics of compound INS-5, (a) Lineweaver–Burk plot (b) Secondary replot
Lineweaver–Burk) (c) Dixon plot
(
8

Both Vmax (decreased) and K (increased) of jack bean urease enzyme were affected in the presence of
m
inhibitors (INS-1 and INS-5) which indicate the mixed type inhibition and these compounds could
interact at the allosteric site or active site of the enzyme. Lineweaver Burk secondary plots were
drawn between the slope of each line and different concentrations of inhibitors to calculate K values.
i
Dixon plots were drawn between the reciprocal of rate of reaction and different concentration of
inhibitors to confirm the K value of each inhibitor. It was concluded from kinetic studies that
i
compound INS-2 was competitive inhibitor with Ki values (Table 1) 5.60 µM while the compounds
INS-1 and INS-5 were mixed type of inhibitors with Ki values 4.32 and 2.76 µM respectively (Table
1
).
Hydrazide moiety along with other cyclics, heterocyclics and sulfoxide explored for antiurease
activities in literature. Isatin derived sulfonyl hydrazides [27], phosphorehydrazides [28] and
benzohydrazides [29] revealed the IC values ranged between 39-148 µM, 1.611-7.251 µM and 0.87-
50
1
1
9 µM respectively. But in the benzohydrazides series the compounds with IC values 1.64, 1.82 and
50
.97 µM showed the competitive mode of inhibition. Whereas in this work the compound INS-2
showed competitive mode of inhibition with IC50 value 1.33 µM which is lower than reported
compounds with same mode of inhibition.
3
.3.Computational studies
All synthesized compounds (INS1-INS-5) are optimized by using density functional theory (DFT) at
B3LYP level of theory and 6-311G(d,p) basis set conjunction. The optimized coordinates and dihedral
angles of optimized geometries are presented in Supplementary data (Table S1-S6). The fully
optimized geometries of all compounds are present in Fig.5a.
Fig. 5 Optimized geometries (a) calculated at B3LYP/6-311G(d,p) level of DFTand frontier
molecular orbital (FMO’s) diagrams (b) of synthesized compounds (INS-1-INS-5)
9

3
.3.1. Frontier molecular orbital (FMO) analysis
Frontier molecular orbitals of investigated molecules are calculated at B3LYP/6-311G (d,p) level of
theory and computed results are given in Fig. 5b and results are placed in Table S7 (supplementary
data). In addition the discussion related to chemical reactivity and frontier molecular orbitals is also
present in supplementary data. The energy gaps values of investigated compounds INS-1-INS-5 are
found close to each other with only a minute difference. The smallest energy gap value among
investigated compounds (INS-1-INS-5) is found to be in INS-1 with ∆E value of 5.16 eV. This might
due to the planar configuration and absence of any additional functional group in INS-1 as compared
to other architectures of compounds. In contrast, computational findings reflect that the highest ∆E
value of 5.33 eV is observed in INS-2. Overall, the increasing order of HOMO-LUMO energy gap is
noticed as: INS-1< INS-3 = INS-4 =INS-5 <INS-2. The pictorial representation is shown in Fig. 6 in
which red color specifies the negative phase while green color indicates the positive phase of
molecular orbitals respectively.
3
.3.2. Global reactivity descriptors
Result of ionization potential and electron affinity mentioned in Table S8 (Supplementary
Information) reveal that our compounds of interest INS-1-INS-5 show higher value of ionization
potential as compared to electron affinity indicating the greater electron donating aptitude of INS-1-
INS-5 as compared to their accepting nature. Overall, A value is found positive in all compounds
which is a good indication for the possible utilization of these compounds in charge transfer reactions.
-
Electrophilicity results also evident that electron donor capability (ω ) values of all compounds are
+
very large as compared to the electron accepting capability (ω ) values. The global softness value is
found to be greater and with third digit difference in INS-3, INS-4, INS-5, while smaller and one digit
difference in INS-1, INS-2. Following decreasing order of electronegativity is observed among INS-
1
-INS-5 compounds INS-1>INS-4 >INS-2 >INS-5>INS-3. The studied compounds INS-1-INS-5 are
found to be chemically hard in nature owing to larger values of global hardness as compared to global
softness. These results unveil the great chemical stability and less reactivity. The value of chemical
potential µ shows a direct relation with chemical stability and inverse relation with reactivity of the
compounds under observation (INS-1-INS-5). In compounds under our investigation INS-1-INS-5,
the following decreasing order of chemical potential is found: [INS-1 (µ = -4.762 eV)] > [INS-4 (µ = -
4
.724 eV)] > [INS-2 (µ = -4.683 eV)] > [INS-5 (µ = -4.636 eV)] > [INS-3 (µ = -4.558 eV)]. This order
points out that compound INS-1 has lowest value of chemical potential (µ) which prove that it is the
least stable and most reactive compound. This result is also in fine agreement with the HOMO-
LUMO energy gap proving the fact that molecules with smaller ∆E value are considered as soft
molecules with small kinetic stability, large reactivity. Overall findings of global reactivity parameters
indicate that all investigated compounds contain more donating capability, stability and less accepting
1
0

aptitude. All studied compounds are suitable and can participate in charge transfer reactions.
Furthermore, the actual reason for performing density functional theory (DFT) calculations is to
support experimental and molecular docking studies and also countercheck the experimental findings
along with evaluation of stability, reactivity, intra or intermolecular charge transfer that are useful
parameters to provide clue for potential biological activities of studied compounds [32, 33]. For
instance, Tables S7 and S8 results provide evident about softness, reactivity, accepting and donating
aptitude of the investigated molecules which have good relationship with potential biological
activities.
3
.3.3. Natural bond orbital (NBO) analysis
The natural bond orbital (NBO) analysis is widely performed to investigate the different
hyperconjugative and non-covalent interactions in the molecules [34, 35]. The NBO analysis is
proved to be a helpful tool for examine the hydrogen bonding originates from hyper-conjugation. The
NBO analysis is also used to determine the transfer of densities from filled orbitals (Lewis type NBOs
orbitals) to unfilled or vacant orbitals (non-Lewis NBOs orbitals) of investigated molecules. In
present study, we have made an attempt to investigate all above mentioned interactions in 5
compounds by using DFT calculations at B3LYP/6-311G (d,p) level of theory and results are
summarized in Table 2. Following equation 1 is proved to be helpful in exploring the interactions and
second order Fock Matrix.
2
(
F )
i. j
2
E = q
(1)
i
ε j
− ε i
2
Here E represents stabilization energy, q represents donor orbital occupancy, F describes off
i
i,j
diagonal NBO Fock matrix elements and ε j , i
ε
represents diagonal elements. The results obtained
from NBO analysis is tabulated in Table 2. And atoms numbering of INS-1-INS-5 according to NBO
charge is also present in Figure S1 (Supplementary data).
1
1

Table 2. Second-order perturbation theory analysis of Fock matrix on NBO basis for all compounds
a
b
c
Compound
Donor (i)
C -C
Type
Acceptor (j) Type
E(2) [Kcal/mol]
0.53
E(j) E(i) [a.u.]
F(I;j) [a.u.]
σ
C -N
σ*
π*
π*
σ*
σ*
π*
σ*
π*
π*
σ*
π*
π*
σ*
π*
π*
σ*
π*
π*
σ*
π*
π*
σ*
σ*
π*
σ*
π*
π*
σ*
π*
σ*
1.08
0.32
0.28
1.14
0.61
0.38
0.75
0.29
0.27
1.15
0.39
0.36
1.29
0.26
0.27
0.65
0.34
0.32
1.29
0.27
0.28
1.14
0.58
0.32
1.15
0.29
0.27
0.65
0.41
0.32
0.022
0.081
0.077
0.051
0.161
0.093
0.019
0.072
0.076
0.051
0.014
0.098
0.024
0.076
0.076
0.044
0.013
0.110
0.024
0.074
0.076
0.051
0.016
0.110
0.020
0.073
0.077
0.042
0.014
0.087
6
7
7
9
N -C
π
C -C
2
25.14
26.49
2.90
3
4
1
INS-1
C -C
π
N -C
5
6
3
4
C -C
σ
C -H
5
1
6
23
O12
N9
L.P[2]
N -N
9
0.51
10
L.P[1]
C -O
7
28.26
0.53
8
N -H
σ
N -S
10 11
1
0
26
19
17
C -C
π
C -C
16
22.11
26.72
2.87
1
8
17
INS-2
C -C
π
C -C
14
1
6
15
C -C
σ
C -H
1
5
6
21
N10
N9
L.P[1]
C -C
14
0.55
15
L.P[1]
C -O
7
33.23
0.51
8
S -O
σ
C -C
1
1
13
3
4
C -C
π
C -C
14
26.67
26.97
2.99
1
7
18
27
18
19
C -C
π
C -C
24
2
6
25
INS-3
INS-4
C -C
σ
C -H
20
1
7
24
N10
L.P[1]
C -C
24
0.57
25
N9
L.P[1]
C -O
7
47.19
0.50
8
S -O
σ
C -C
3 4
1
1
13
C -C
π
C -C
23
25.06
26.27
2.85
2
6
27
28
C -C
π
N -C
5
6
3
4
C -C
σ
C -H
5
1
6
32
N9
N9
L.P[1]
S -O
0.53
11
13
23
L.P[1]
C -O
7
46.84
0.52
8
S -O
σ
S -O
21
2
1
22
C -C
π
C -C
5 6
22.47
27.71
2.76
1
2
INS-5
C -C
π
C -C
24 15
1
6
17
C -C
σ
C -H
20
1
6
17
21
N10
O13
L.P[1]
L.P[3]
C -C
0.54
5
6
N -S
10
27.38
11
a
Mean energy of hyper conjugative,
b
c
Energy difference between donor and acceptor i and j NBO orbitals
Fock matrix element between i and j NBO orbitals
1
2

*
*
NBO analysis suggests four different types of transitions in molecules which are σ→σ , π→π ,
*
*
L.P→σ and L.P→π . The intra-molecular hydrogen bonding also plays a vital role in stabilizing the
*
*
molecule. The π→π transition in molecules confirms the conjugations. The dominant π→π
*
*
transitions in INS-1-INS-5 are found to be as π(C -C )→π (N -C ), π(C -C )→π (C -C ), π(C -
5
6
3
4
16
17
14
15
26
*
*
*
C ) →π (C -C ), π(C -C )→π (N -C ) and π(C -C )→π (C -C ) with stabilization energy values
27
24
25
5
6
3
4
16
17
24
15
of 26.49, 26.72, 26.97, 26.27 and 26.71 Kcal/mol respectively. In case of resonance large values of
stabilization energy is seen, which suggests the delocalization of electrons of oxygen and nitrogen
*
*
atoms to the entire system. These transitions are L.P (N ) → π (C -O ) in INS-1, L.P (N ) → π (C -
9
7
8
9
7
*
*
*
O ) in INS-2, L.P (N )→ π (C -O ) in INS-3, L.P(N )→ π (C -O ) in INS-4 and L.P (O )→ σ (N -
8
9
7
8
9
7
8
13
10
S ) in INS-5 with stabilization energy of 28.26, 33.23, 47.19, 46.84 and 27.38 Kcal/mol respectively.
11
Moreover, in all compounds hydrogen bonding due to hyper conjugation is also present and relevant
data is enclosed in Table 2. Similarly, Table 2 results confirmed that successful intramolecular charge
transfer (ICT) due to delocalization of electrons on whole systems and hyper-conjugative interactions
(
HCIs) among bonds provide stability to the investigated compounds INS-1-INS 5.Furthermore, Table
results suggested that ICT and HCIs plays a crucial role for potential biological activities. In this
2
way, Tables 2 results are supportive analysis for biological potential of investigated molecules. From
preceding discussion, it can be concluded that extended conjugation is present in all compounds INS-
1
-INS-5. Successful intramolecular charge transfer due to delocalization of electrons on whole
systems and hyper-conjugative interactions (HCIs) among bonds provide stability to the investigated
compounds INS-1-INS-5.
3
.3.4. FT-IR analysis
Nowadays, modern vibrational spectroscopy is proved to be helpful tool for identifying the modes of
vibration in investigated compounds (experimental as well as theoretical)[36]. Experimentally FT-IR
-
1
spectrum is recorded in the range of 4000-600 cm while theoretically spectrum with harmonic
frequencies, nature of vibrational modes and intensities of the vibrational bands for compounds INS-
1
-INS-5was examined by utilizing DFT/B3LYP/6-311G(d,p) level of theory. The most important
experimental and theoretical FT-IR descriptions with concerning assignment are presented in Table
S9 (Supplementary data).
3
.3.4.1. C–H vibrations
DFT computed and experimentally recorded frequency bands unveils that the C-H vibrational bands
are mostly observed due to wagging, scissoring, twisting and rocking mode of vibrations. In INS-1,
-1
twisting and rocking mode of vibration is observed at wave numbers 898, 1007, 1089 and 1215 cm
-1
in benzene unit and 669 cm in pyridine unit which show a good agreement with experimentally
-1
-1
reported band at 907, 1001, 1091, 1236 cm in benzene and 670 cm in pyridine unit respectively. In
-1
INS-2 compound, rocking mode of vibration is observed at wave number 1141, 1228 cm in benzene
-1
unit and 1092, 3225 cm in pyridine unit which correlate with experimentally reported C-H band
1
3

-1
-1
found at 1157, 1223 cm in benzene and 1090, 3244 cm in pyridine unit correspondingly. In INS-3,
-1
-1
C-H vibrational band in benzene is recorded at 1336 cm and in pyridine ring at 1092, 1226 cm
-1
which show a good harmony with experimentally reported C-H band at 1336 cm in benzene unit and
-1
1
089, 1225 cm in pyridine ring respectively. In INS-4 and INS-5 compound, theoretically C-H
-1
-1
vibrational band is recorded at 1528 cm and 1143, 3228 cm in benzene and pyridine rings
respectively. These theoretically calculated values show a good harmony with experimentally
-1
-1
reported band at 1540 cm and 1156, 3248 cm in compound INS-4 and INS-5 respectively. In
-1
pyridine unit C-H vibrational band is recorded at wave numbers 1095, 1528 cm in INS-4 and at
-
1
wave numbers 1094, 1337 cm in INS-5. These values show correlation with experimentally recorded
-1
-1
C-H vibrational bands at wave number 1090, 1528 cm (INS-4) and 1090, 1337 cm (INS-5)
respectively.
3
.3.4.2. C-C stretching vibration
In current report, C-C stretching vibrations are found mostly in benzene rings and pyridine units. The
C-C vibrational bands are seen in the benzene ring with stretching frequencies at wave number 1042
-
1
-1
-1
-1
-
cm (INS-1), 1141, 1339, 1615 cm (INS-2), 847, 1336 cm (INS-3), 628 cm (INS-4) and 1137 cm
1
(
INS-5) which are associated nicely with experimental analyzed bands appeared at wave number
-1
-1
-1
-1
1
058 cm (INS-1), 1157, 1337, 1603 cm (INS-2), 845, 1336 cm (INS-3), 634 cm (INS-4) and
-1
1
136 cm (INS-5) respectively. Similarly, in pyridine units of all compounds, vibrational bands with
-1
-1
-1
stretching frequencies observed in 1324 cm (INS-1), 1013 cm (INS-2), 1010 cm (INS-3), 1010
-
1
-1
cm (INS-4) and 1596 cm (INS-5) show excellent consistency with experimental vibrational band
-1
appeared at 1307, 1001, 1001, 1001 and 1595 cm respectively.
3
.3.4.3. N-H band vibration
-1
N-H stretching vibrational mode in INS-1 are noticed with wave number at 669 cm , in INS-2 at 902,
-1
-1
-1
-1
3
500 cm , in INS-3 at 3571 cm , in INS-4 at 3572 cm and in INS-5 at 631 cm which shows a nice
-1
concurrence with experimental observed frequencies at 670, 906, 3537, 3565, 3572 and 632 cm
respectively. From preceding discussion, it is well evident that the experimental and computed FT-IR
results are in very good agreement with each other.
3
.4.UV-Visible study
Ultraviolet-Visible (UV–Vis)spectroscopy is a very useful tool for estimating the nature of electronic
transition, probability of charge shifting within the compound and molecular assignment according to
transition[37]. UV-Visible study is performed experimentally as well as theoretically by using specific
tools. Theoretically UV-Visible study is performed by using time-dependent DFT (TDDFT) at
B3LYP/6-311G(d,p) level of theory. TDDFT computed spectral results with experimental absorption
spectral results, oscillator strength (f) and concern molecular orbital contributions in compounds
INS1-INS5 is tabulated in Table 3. Results of maximum absorption for all compounds obtained from
experimental and computational analysis are found very close to each other. The computed UV-
1
4

Visible spectra of INS1 highlighted
λ
max at 242.84 nm with oscillating strength value of 0.05. In
max is observed at 248.40 nm and 248.09 nm with oscillating strength
λ
values of 2.5800 and 0.0008 respectively. The simulated max and f in
compound INS-2 and INS-3
λ
DFT
value 0.01, 0.01 and E
compound INS-4 are found closer to INS-1, INS-2 and INS-3 with values of 243.92 nm, 0.01
respectively. The computed UV-Visible spectra of INS-5 showed max at 244.54 nm with oscillating
strength value of 0.06. Overall, calculated UV-Visible spectrum of INS, INS2, INS-3, INS-4 and INS-
showed absorption maximum at 242.84, 248.40, 248.09, 243.92 and 244.54 nm which is in
reasonable agreement with the experimental UV-Visible spectrum valued observed at 242, 242, 235,
35, 242 nm respectively. HOMO to LUMO electronic transition which is the characteristic of soft
λ
5
2
molecules, hence, may have potential of utilization in biological applications.
Table 3. Absorption values (λmax), oscillator strength (f), are calculated at TDDFT/B3LYP/6-31G(d,p)
level of theory in the methanol solvent
Compounds
Cal.
λ
max (nm)
Exp.
λ
max (nm)
f (oscillator strength) MO contributions
INS-1
242.84
248.40
242
0.05
0.01
H→L+1(81%)
H-3→L (88%)
INS-2
242
235
235
242
H-6→L (24%), H-5→L (25%),
H-3→L (11%)
INS-3
INS-4
INS-5
248.09
243.92
244.54
0.01
0.01
0.06
H-3→L (20%), H-2→L (42%)
H-3→L (25%), H-2→L (31%),
H→L (12%)
3
.5.Molecular electrostatic potential (MEP) analysis
Three dimensional plot of electron density on whole compound is investigated through molecular
electrostatic potential analysis using following equation 2.
Z
A
/
/
/
V (r) = ∑( − r) − ∫( p(r )/ r − r)dr
R
A
(
2)
In above equation V(r) represents molecular electrostatic potential and Z represents charge density
A
over nucleus. A placed at RA, p(r’) defines the electronic density function and r’ is the integration
variable[38, 39]. The color in MEP analysis actually defines the sites for electrophilic and
nucleophilic attack. Electrostatic potential magnitude is increasing in following order; red < orange <
yellow <green < blue[40]. The red color represents the best site for electrophilic attack and blue color
indicates the site which is best for nucleophilic attack. So, for the MEP analysis were formed at
1
5

B3LYP/6-311G(d,p) functional for INS-1-INS-5and results are portrayed in Fig. 6. It can be seen that
negative potential (red color) is shown by oxygen atoms, while blue area shows positive potential
exhibited mostly by nitrogen and partially by hydrogen and carbon atoms. But green area shows mean
potential i.e. the area between two extremes. From red and blue colors it is cleared that different
reaction sites are presents in all molecules.
Fig. 6 Molecular electrostatic potential of compounds at DFT/B3LYP/6-311G(d,p) basis set
3
.6.Binding mode prediction using molecular docking studies
The competitive inhibitor INS-2 was docked in the active site of urease enzyme as shown in Fig. 7.
The pyridine ring directed towards the zinc metal ion is making an electrostatic interaction shown in
Fig. 7b. The MEP analysis also show that N present in the pyridine has strong tendency for
nucleophilic attack which might be responsible for making observed pose of INS-2 compound as
shown in Fig. 7a. The tail part containing the toluene moiety is exposed towards the solvent side,
contributing to push the compound in active site. The compound has gorged well in the binding site
also making t-type π-π interactions with HIS-519 and HIS-492 residues which might be responsible
for making it competitive inhibitor.
Fig. 7 Binding mode of competitive inhibitor (INS-2) in urease enzyme. a) Best docked pose of INS-2
compound (green sticks) in urease active site (cyan sticks), Nickle ions are mentioned with brown
sphere. b) 2D view of INS-2 docking pose in active site.
1
6

Conclusion
In this study, five hydrazides were successfully synthesized and their structures were confirmed by
physiochemical and spectral techniques. All the compounds were assayed for in vitro urease
inhibition studies. Among the tested compounds, compound INS-5 exhibited lowest IC50 value
(
1.11±0.29 µM) and other compounds in the series showed good antiurease activity. Inhibition
mechanisms (competitive, mixed or non-competitive) of biologically assayed compounds were also
investigated by Lineweaver Burk and Dixon plots to determine the Vmax, K and K values. Among
m
i
the already reported benzohydrazides [29], compounds with IC50 values 1.64, 1.82 and 1.97 µM
showed the competitive mode of inhibition whereas in this work the compound INS-2 showed
competitive mode of inhibition with IC values 1.33 µM which is lower than reported compounds
5
0
with same mode of inhibition. The experimental FT-IR and UV-Visible spectroscopic analysis
showed reasonable agreement to corresponding DFT based results. The NBO analysis confirmed that
the presence of hyperconjugative interactions and intramolecular charge transfer are pivotal cause for
the existence of stability of investigated compounds. Global reactivity parameters indicate that INS-1-
INS-5 contain more donating capability, stability and less accepting aptitude. MEP descriptor
indicates that oxygen atoms in all title compounds are prone to electrophilic attack, while nitrogen and
carbon atoms are prone to nucleophilic attack which is also evident in docking studies of competitive
inhibitor. We hope that this synergistic experimental-computational study may provide new ways for
the utilization of INS-1-INS-5 in biological chemistry.
Acknowledgment
The authors are thankful to HEC-Pakistan for providing financial support to purchase software and
hardware for computational studies vide Projects No. 6804/Federal/NRPU/R&D/HEC/2016 &
8
094/Balochistan/NRPU/R&D/HEC/2017.
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2
0

Highlights
1
. New Benzenesulfonohydrazide were synthesized and urease enzyme inhibition was evaluated
through in vitro assays.
2
. All compounds displayed potent urease inhibitory activity with IC50 values of 1.11–4.89
compound INS-2 was competitive inhibitor with Ki values 5.60 µM.
ꢀµM, the
3
4
. Molecular docking studies were established to elucidate the binding energy and interaction.
. DFT and TDDFT calculations at B3LYP/6-311G(d,p) level of theory were performed, overall,
experimental findings were supported nicely by corresponding DFT computed results.

Compliance with Ethical Standards
Informed consent: Not applicable
Conflict of interest: We wish to confirm that there are no known conflicts of interest associated with this
publication.
Funding: There has been no financial support for this work that could have influenced its outcome.