Synthesis, structural properties, enzyme inhibition and molecular docking studies of (Z)-N'-(1-allyl-2-oxoindolin-3-ylidene) methanesulfono-hydrazide and (Z)-N'-(1-allyl-2-oxoindolin-3-ylidene)-3-nitrobenzenesulfono-hydrazide

Article abstract of DOI:10.1016/j.molstruc.2020.128880

Isatin and its derivatives exhibit broad range of biological and pharmacological applications. Keeping in view the importance of isatin and its derivatives, herein we report two isatin based new sulfono-hydrazides 4 & 5, synthesized in high yields and characterized by spectroscopic techniques. Their structures are confirmed unequivocally using X-ray diffraction crystallography, which revealed the presence of P2₁/c (4) and P2₁/n (5) space groups and unit cells stabilized through noncovalent interactions. Further details about geometric and electronic properties of compounds 4 and 5 are obtained by quantum mechanical approach based on density functional theory (DFT). These compounds are also evaluated for in vitro urease enzyme inhibition potential against Bacillus pasteurii. Both compounds inhibited the urease activity in μM concentration, however, compound 4 with IC₅₀ value of 15.26 ± 0.16 μM proved to be more potent than the standard thiourea having IC₅₀ value of 21.25 ± 0.15 μM. The higher inhibition activity of compound 4 might be associated with its stronger interaction as observed by in silico molecular docking studies using MOE, which showed that compound 4 interacts more closely to the binding site of enzyme (4UBP) via Ni²⁺ ions coordination as compared to its counterpart.

Full text of DOI:10.1016/j.molstruc.2020.128880

Journal Pre-proof
Synthesis, structural properties, enzyme inhibition and molecular
docking studies of (Z)-N’-(1-allyl-2-oxoindolin-3-ylidene)
methanesulfono-hydrazide and (Z)-N’-(1-allyl-2-oxoindolin-3-ylidene)-
3-nitrobenzenesulfono-hydrazide
Muhammad Arshad , Kainat Ahmed , Zafar Iqbal , Umer Rashid ,
Muhammad Nadeem Arshad , Abdullah M. Asiri , Tariq Mahmood
PII:
S0022-2860(20)31205-9
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128880
MOLSTR 128880
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
23 March 2020
8 July 2020
9 July 2020
Please cite this article as: Muhammad Arshad , Kainat Ahmed , Zafar Iqbal , Umer Rashid ,
Muhammad Nadeem Arshad , Abdullah M. Asiri , Tariq Mahmood , Synthesis, structural prop-
erties, enzyme inhibition and molecular docking studies of (Z)-N’-(1-allyl-2-oxoindolin-3-ylidene)
methanesulfono-hydrazide
and
(Z)-N’-(1-allyl-2-oxoindolin-3-ylidene)-3-nitrobenzenesulfono-
hydrazide, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128880
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Highlights
(i)
Synthesis of two allylisatin based sulfonyl hydrazides is performed.
(ii)
(iii)
(iv)
X-ray results are in strong correlation with the DFT studies.
Both are tested in vitro against Bacillus pasteurii urease.
Molecular docking studies further proved the mechanism of enzyme inhibition
1

Synthesis, structural properties, enzyme inhibition and molecular docking
studies of (Z)-N'-(1-allyl-2-oxoindolin-3-ylidene)methanesulfono-hydrazide and
(Z)-N'-(1-allyl-2-oxoindolin-3-ylidene)-3-nitrobenzenesulfono-hydrazide
Muhammad Arshada , Kainat Ahmeda, Zafar Iqbalb, Umer Rashidb, Muhammad
Nadeem Arshadc,d, Abdullah M. Asiric,d, Tariq Mahmoodb*
aInstitute of Chemistry, University of the Punjab, Quaid-i-Azam Campus, Lahore-54590, Pakistan.
bDepartment of Chemistry, COMSATS University, Abbottabad Campus, University Road, Tobe
Camp, Abbottabad-22060, Pakistan.
cChemistry Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah
21589, Saudi Arabia.
dCentre of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, P.
O. Box 80203, Jeddah 21589, Saudi Arabia.
*To whom correspondence should be addressed: E-mail: mahmood@cuiatd.edu.pk (T.M.)
2

Graphical Abstract
Molecular Docking
Crystallography
Urease inhibition
Quantum mechanical studies
Abstract
Isatin and its derivatives exhibit broad range of biological and pharmacological applications.
Keeping in view the importance of isatin and its derivatives, herein we report two isatin based new
sulfono-hydrazides 4 & 5, synthesized in high yields and characterized by spectroscopic
techniques. Their structures are confirmed unequivocally using X-ray diffraction crystallography,
3

which revealed the presence of P21/c (4) and P21/n (5) space groups and unit cells stabilized
through noncovalent interactions. Further details about geometric and electronic properties of
compounds 4 and 5 are obtained by quantum mechanical approach based on density functional
theory (DFT). These compounds are also evaluated for in vitro urease enzyme inhibition potential
against Bacillus pasteurii. Both compounds inhibited the urease activity in µM concentration,
however, compound 4 with IC50 value of 15.26 ± 0.16 µM proved to be more potent than the
standard thiourea having IC50 value of 21.25±0.15 µM. The higher inhibition activity of
compound 4 might be associated with its stronger interaction as observed by in silico molecular
docking studies using MOE, which showed that compound 4 interacts more closely to the binding
site of enzyme (4UBP) via Ni2+ ions coordination as compared to its counterpart.
Keywords: Enzyme inhibition; Isatin; Molecular docking; X-ray; DFT
1 INTRODUCTION
Isatin is a versatile heterocyclic scaffold with substantial significance for the synthesis of broad
range of biologically and pharmacologically attractive motifs. Since the commercial availability of
isatin in 1840 [1,2], it has been considered as synthetic for 140 years until the discovery of its
natural resources. Isatin has been employed as a valuable precursor for a plethora of heterocyclic
4

compounds, a number of which led to the construction of medicinally important building blocks
[1,3]. Over the time, isatin derivatives have been evaluated for several biological implications
including antimicrobial [4], antiangiogenic [5], antitubercular [6], anxiolytic [7], and
anticonvulsant agents [8]. Enzyme inhibition activity of isatin derivatives led to the discovery of
tyrosine kinase inhibitors (TKI) such as Sunitinib (approved by FDA in 2006) and Nintedanib.
These TKI are in clinical use for chemotherapeutic treatment of metastatic renal and
gastrointestinal stroma [9] and lung cancer [10].
Enzyme inhibition is an active area of medicinal chemistry where investigations have driven
towards the discovery of useful drugs against many diseases [11–13]. Urease enzyme catalyzes the
hydrolysis of urea into ammonium and carbamate ions, which eventually decompose into
ammonia and carbon dioxide [14,15]. The unusual rise of ammonia increases the blood pH
whereupon originating a number physiological complications such as urinary catheter
encrustation, gastric and peptic ulcers, hepatic coma urolithiasis and pyelonephritis [16,17].
Therefore, urease inhibitors are considered as effective remedies for controlling the damaging
effect of ureolytic bacterial infections, a very common problem in developed countries [18,19]. To
cater the enzymatic activity, a number of urease inhibitors including oxadiazoles [16], hydroxamic
acid derivatives [20], triazoles [21], coumarins [22], piperidines [23], urea and isatin derivatives
[24] have been developed, however, the quest for most potent urease inhibitors still exists.
In continuation to our efforts in the field of urease inhibition [24–26], herein, we report the
synthesis, structural properties, urease inhibition and molecular docking studies of the two new
allylisatin based sulfono-hydrazide derivatives.
2. Materials and Methods
5

2.1 Experimental
Chemicals purchased from sigma Aldrich are used further without any purification. All solvents
are distilled and dried before using in chemical reactions. Melting points of both compounds are
o
uncorrected and measured by using digital melting point apparatus (range is up to 300 C).
Absorption spectra of both derivatives are scanned by using t80 + UV/Vis spectrophotometer.
Vibrational spectra (FT-IR) are monitored by using Agilent FT-IR Cary 630 instrument, using
1
ATR sampling method. H and 13C-NMR spectra are recorded using deuterated chloroform
(CDCl3) as NMR solvent on Bruker’s 400 MHz NMR machine.
2.2 Synthesis
The synthesis of both compounds is accomplished starting form commercially available isatin. For
entire synthetic scheme see the Fig. 1.
Fig.1; Synthesis of N-allylisatin derived sulfono-hydrazides (4 & 5)
2.2.1 N-allylation of isatin
6

Isatin (1.47 g, 10 mmol) in DMF (20 ml) was taken in round bottom flask. Calcium hydride (0.63
g, 15 mmol) added to the reaction mixture and heated at 50 C for 30 minutes. Allylbromide (1.81

g, 15 mmol) was added and stirred the mixture for 3 hours at 4550 C. Upon completion as
indicated by TLC, ice cold water (100 ml) was added for precipitation of required product. The
precipitates were filtered, washed with water, dried in air and further purified by recrystallization
from ethanol to obtain compound 1 as reddish crystalline solid (1.46 g, 78%).
2.2.2 Synthesis of sulfonyl hydrazides (2 and 3)
Both intermediate compounds (2 & 3) were synthesized according to the slightly modified
procedure already described in literature [28]. Briefly, in a typical reaction, sulfonyl chloride (10
mmol) as solution in THF (10 ml) was taken in round bottom flask. Hydrazine monohydrate (0.88

ml, 15 mmol) was added dropwise to reaction mixture maintaining the temperature at 1015 C
followed by further stirring for 30 minutes at the same temperature. Ice cold water was added to
the reaction mixture for precipitation of respective compounds 2 & 3 as colorless solids.
Compound 2 was obtained as colorless solid; Rf (EtOAc:Hexane 1:1); 0.35 and yield 82%.
Compound 3 was obtained as colorless solid; Rf (EtOAc:Hexane 1:1); 0.22 and yield 71%.
(Z)-N'-(1-Allyl-2-oxoindolin-3-ylidene)methanesulfono-hydrazide (4)
Methanesulfonylhydrazide 2 (0.13 g, 1.2 mmol, 1.2 equiv.) and N-allylisatin 1 (0.187 g, 1.0 mmol)
were refluxed for 3 hours in ethanol (10 ml) containing five drops of acetic acid according to the
literature procedure [28] (for general procedure see supplementary material). Title compound 4
was obtained as yellow powder (0.223 g, 80%); Rf (EtOAc/Hexane, 1:4) 0.21; M.P. 162165 oC;
7

UV/Vis. (λmax, 0.1 mmol, acetonitrile) 338 nm; FT-IR (neat, cm1) 3169 (N-H, Str), 3006 (C-H,
Ar), 2926 (C-H, Alkane), 1690 (C=O, Str), 1616 (C=N, Str), 1468 (C-C, Ar), 1341, 1166 (S=O,
Str), 748 (C-H, (Bend) Ar). 1H NMR (400 MHz, CDCl3) δ 12.53 (s, 1H, NH), 7.87 (d, 1H, J = 7.8
Hz), 7.63 (t, 1H, J = 7.5 Hz), 7.30 (t, 1H, J = 7.8 Hz), 7.17 (d, 1H, J = 7.5 Hz), 5.83 (ddt, 1H, J =
16.0 Hz, 10.5 Hz, 5.2 Hz), 5.34 – 5.19 (m, 2H), 4.37 (d, 2H, J = 5.2 Hz), 2.83 (s, 3H, CH3).13C
NMR (100 MHz, CDCl3) δ 160.1 (C=O), 141.9 (C), 133.1 (C), 131.3 (CH), 130.7 (CH), 129.6
(CH), 121.5 (CH), 119.3 (=CH2), 118.2 (C), 109.8 (CH), 43.1 (N-CH2), 40.3 (CH3).
(Z)-N'-(1-Allyl-2-oxoindolin-3-ylidene)-3-nitrobenzenesulfono-hydrazide (5)
3-Nitrobezenesulfonylhydrazide (0.26 g, 1.2 mmol, 1.2 equiv.) and N-allylisatin 1 (0.187 g, 1.0
mmol) were refluxed for 3 hours in ethanol (10 ml) containing five drops of acetic acid
according to the reported procedure [28] (for general procedure see supplementary material).

Title compound 5 was obtained as orange powder (0.320 g, 83%); M.P. 178 180 C; Rf
(EtOAc/Hexane, 3:7), 0.40; UV/Vis. (λmax, 0.1 mmol, acetonitrile), 353 nm; FT-IR (neat, cm1)
3109 (N-H, Str), 3033 (C-H, Ar), 2922 (C-H, Alk), 1685 (NC=O, Str), 1616 (C=N, Str), 1533
(N-O, Str), 1470 (C-C, Ar), 1355, 1175 (S=O, Str), 762 (C-H, (Bend) Ar); 1H NMR (400 MHz,
CDCl3) δ 12.57(s, 1H, NH), 8.03(br.d, 2H, J = 7.3), 7.65–7.58 (m, 2H), 7.57– 7.50(m, 1H),
7.33(t, 1H, J = 7.8), 7.10(t, 1H, J = 7.6), 6.85(d, 1H, J = 7.8), 5.81(ddt, 1H, J = 15.9 Hz, 10.5 Hz,
5.3 Hz), 5.31–5.17(m, 2H), 4.34(d, 2H, J = 5.3 Hz); 13C NMR (100 MHz, CDCl3) δ 160.9
(C=O), 148.5 (C), 142.7 (C), 138.7(C), 135.5 (C), 133.6 (C), 133.1 (CH), 131.4 (CH), 130.6
(CH), 129.3 (CH), 128.0 (CH), 123.5 (CH), 121.4 (CH), 119.5 (=CH2), 118.5 (CH), 110.0 (CH),
42.1 (N-CH2).
8

2.3 Crystallography
Single crystal X-ray diffraction studies are executed on Agilent Super Nova (Dual source) Agilent
Technologies Diffractometer having microfocus Cu/Mo Kα radiation source. The final data
collection is made with the help of CrysAlisPro software [29]. SHELXL–97 method in-built with
X-Seed is used for final structure solution [30,31]. The all atoms other than hydrogen are refined
anisotropically by using full–matrix least squares methods [32]. The final structures of both
compounds 4 and 5 are accomplished through PLATON and ORTEP in built with WinGX
[33,34]. H atoms of benzene rings and terminal methylene are positioned geometrically and
treated as riding atoms with C–H = 0.93 Å and Uiso(H) = 1.2 Ueq(C) for carbons. The N-H
hydrogen atoms are located through fourier map and refined with N-H = 0.80(2) - 0.86(2)Å with
Uiso (H) = 1.2 Ueq for N atom. The methyl and methylene hydrogen atoms are also positioned
geometrically and treated as riding atoms with [C–H = 0.96 Å, Uiso(H) = 1.5 Ueq(C)] and [C–H =
0.97 Å, Uiso(H) = 1.2 Ueq(C)] for methyl and methylene carbon atoms, respectively. The crystal
data has been deposited at the Cambridge Crystallographic data center and the assigned CCDC
numbers are 1579181 and 1579182 for 4 and 5, respectively. Crystal data can be received free of
charge on application to CCDC 12 Union Road, Cambridge CB21 EZ, UK. (Fax: (+44) 1223 336-
033; e-mail: data_request@ccdc.cam. ac.uk.
2.4 DFT calculations
Computational simulations are performed by using GAUSSIAN 09 package [35], and geometries
are analyzed the help of GaussView 5.0 [36]. The geometry optimization of both derivatives (4
9

and 5) is performed by using DFT/B3LYP/6-31G (d,p) method. The X-ray crystal structures are
used as reference for the computational studies. Vibrational analysis is performed at the same level
of theory in order to confirm true optimization (all real vibrations are observed). FMOs and MEP
analyses are executed at the same level of theory.
2.5 Molecular Docking methods
Molecular Operating Environment (MOE 2016:0802) software package is used for the docking
simulations. Three-dimensional (3-D) structure of urease from Bacillus pasteurii is obtained from
Protein Data Bank (PDB accession code 4UBP). Acetohydroxamic acid (HAE) is the native
ligand. Molecular builder option of MOE is used to draw the structures of the molecules. The
drawn molecules are energy minimized using MMFF94 forcefield. The native ligand HAE is re-
docked in the binding site of the enzyme (4UBP) and root mean square deviation (RMSD) is
computed. The computed rmsd value is 0.89 Å. This confirms the reliability of our docking
program.
Finally, the prepared compounds are docked into the binding site of the enzyme using Triangular
Matching docking method. For each compound, 10 different conformations are allowed to be
generated. In order to obtain minimum energy, the ligands are allowed to be flexible. The
remaining parameters are maintained at their default settings. The best conformation of each
compound–enzyme complex is ranked by GBVI/WAS binding free energy calculation. The lowest
energy ligand-enzyme complex for each compound is selected and the resulting ligand-enzyme
complexes are used for two-dimensional (2-D) and three-dimensional (3-D) interaction studies. 2-
D docking results are viewed by using ligand interaction module implemented MOE. While, 3-D
interaction plots are generated by using Discovery Studio Visualizer [37].
10

2.6 Urease Inhibition Assay
Urease inhibition of the synthesized compounds is assayed using modified Berthelot method [38].
The experimental details are exactly same as we have reported in our previous paper [28]. The %
age inhibition is calculated with the help of mathematical expression given below:
IC50 values (conc. of inhibitor responsible for 50% inhibition of the enzymatic activity) of both
compounds are calculated by measuring activities at further dilutions after computing the data by
using EZ-Fit Enzyme software (Perrella Inc, USA) (units of absorbance of un-inhibited enzyme
are between 1.0 and 1.2).
3 Results and discussion
Synthesis of the both compounds 4 and 5 is accomplished according to the scheme, given in the
Fig. 1. First of all N-alkylation of the isatin is accomplished through nucleophilic substitution
using allyl bromide under basic conditions in DMF to afford N-allylisatin as intermediate 1 in high
yield. In parallel, two different sulfonyl chlorides are treated with hydrazine monohydrate in THF
to form respective sulfonylhydrazides 2 and 3 in good yields. Synthesis of final compounds 4 and
5 is carried out by condensing the N-allylisatin derivative 1 with sulfonyhydazides 2 and 3
separately, in boiling ethanol and catalytic acetic acid (5 drops).
11

3.1 Single crystal X-ray diffraction analysis
Single crystal X-ray diffraction analysis is not only helpful us to confirm and decide about the
final structures of compounds, but also provides the information about inter and intramolecular
interactions among the molecules in the unit cell which are vital to stability of the compounds
[39]. The single crystal X-ray parameters along with other structural information’s of both 4 and 5
are given in the Table 1. The ORTEP plots of both hydrazides are shown in the Fig. 2.
4
5
Fig. 2: The ORTEP plots of compounds 4 and 5, thermal ellipsoids were drawn at 50%
probability level
Both hydrazides (4 and 5) adopted the monoclinic crystal system and structures are solved with P
21/c and P21/n space groups, respectively. One independent molecule was observed in each case.
It is well established from previously reported crystallographic studies that < O-S-O value is
observed about 120º in sulfonamide containing molecules [40,41], and in the present both
molecules the almost similar values are observed i.e. O3-S1-O2 angles are 119.56 (1)º and
121.51(9)º in 4 and 5, respectively. This ultimately produced the distorted tetrahedral geometry
12

around S atom.
The root mean square (r.m.s.) deviation for the fitted atoms of indole ring (C1-C8/N1) in the both
4 and 5 are 0.0116(1) Å and 0.0112(1) Å, which suggest the planarity of the indole moiety. This
was further verified with the help of dihedral angles between the fused benzene (C1-C6) and
pyrrole (C1/C6/C7/C8/N1) rings which are 1.564(1)º and 1.471(1)º for molecule 4 and 5
respectively. Propylene (C10/C11/C12 for molecule 4 and C15/C16/C17 for molecule 5) side
chain which is attached to the nitrogen atom is oriented at dihedral angles of 77.25(2)º and
77.33(2)º with respect to the pyrrole (C1/C6/C7/C8/N1) rings in molecules 4 and 5, respectively.
In the molecule 5, the nitrophenyl ring (C9-C14) is oriented at dihedral angle of 61.37(5)º with
respect to indole functionality (C1-C8/N1). The nitro group N4/O4/O5 is twisted at dihedral angle
of 23.94(2)° with respect to its parent aromatic ring (C9-C14). Unit cell diagrams (Fig. 3) of both
compounds (4 and 5) show number of intra and inter non-covalent interactions. The oxygen atom
of carbonyl (O1) of pyrrole linked to the hydrogen of hydrazide to form N3-H3N…O1 type
intramolecular interaction and generate six membered ring motif S(6) in both compounds [42]. For
these interactions <D-H-A angles are 131.1(2)° and 136.9(2)°, respectively whereas D-A distances
are 2.77(2)Å and 2.73(2)Å while H-A distances are 2.19(2)Å and 2.06(2)Å for compound 4 and 5,
respectively. Due to presence of C-H…O type week interactions (atom numbering and other
values are provided in Table 2) the inversion dimers are formed and further produce ten membered
ring motif in molecule 5 while eighteen membered ring motif in molecule 4. These dimers further
connected through another C-H…O interactions to produce long zig-zag chains along c-axis in
both compounds (Table 2).
13

4
5
Fig. 3: Unit cell view of 4 and 5 showing non-covalent interactions
3.2 Molecular geometries
Experimental X-ray geometries provide basic guidelines for validating the theoretical method
through comparison of geometric parameters. Therefore, we have optimized the geometries of
compounds 4 and 5 at B3LYP/6-31G (d, p) method and compared with the geometric parameters
obtained from experiment [43,44]. The energy minima structures of both hydrazides 4 and 5 are
shown in the Fig. 4. Simulated and experimental bond lengths and bond angles of both derivatives
4 and 5 are given in Table 3 and Table 4, respectively.
14

4
5
Fig. 4: Energy minima geometries of compounds 4 and 5
X-ray diffraction values of vital bonds such as S1-N3, S1-O2, S1-O3, S1-C9, O1-C8, N2-N3, N2-
C7, C8-N1, N1-C1 and N1-C10 (atomic labels are according to the ORTEP plot shown in Fig. 2)
in (4) are 1.65Å, 1.41Å, 1.41Å, 1.74Å, 1.21Å, 1.36Å, 1.28Å, 1.36Å, 1.40Å and 1.44Å,
respectively. The quantum chemical values of same bonds are 1.71Å, 1.45Å, 1.46Å, 1.79Å,
1.23Å, 1.34Å, 1.29Å, 1.38Å, 1.41Å and 1.45Å, respectively and corroborated nicely with the
experimental results. The X-ray diffraction values of bonds C1-N1, C7-N2, C8-N1, C8-O1, C9-
S1, C11-N4, C15-N1, N4-O4, N4-O5, N2-N3, N3-S1, O2-S1 and O3-S1 bonds in 5 are 1.41Å,
1.28Å, 1.36Å, 1.22Å, 1.76Å, 1.46Å, 1.46Å, 1.22Å, 1.22Å, 1.35Å, 1.63Å, 1.42Å and 1.41Å,
respectively. Quantum chemical analysis of same bonds showed nice correlation with experiment
and are observed at 1.41Å, 1.29Å, 1.37Å, 1.23Å, 1.79Å, 1.47Å, 1.45Å, 1.22Å, 1.22Å, 1.33Å,
1.69Å, 1.45Å and 1.46Å, respectively.
15

The X-ray diffraction and quantum chemical results showed that vital bond angles of both
sulfonamides 4 and 5 have very excellent correlation with each other. The experimental values of
important bond angles of 4 such as N3-S1-C9, O2-S1-N3, O2-S1-C9, O3-S1-N3, O3-S1-O2, O3-
S1-C9, C7-N2-N3, N2-N3-S1, O1-C8-N1, O1-C8-C7, N1-C8-C7, C8-N1-C1, C8-N1-C10, C1-
N1-C10, C6-C1-N1, C2-C1-N1, N1-C10-C11, N2-C7-C8, and N2-C7-C6 (atomic labels are with
reference to Fig. 2) are 105.1o, 107.5o, 108.8o, 104.9o, 119.5o, 109.7o, 118.0o, 114.4o, 126.8o,
106.2o, 110.7o, 124.8o, 124.8o, 110.1o, 127.8o, 113.7o, 127.7o, and 125.9o respectively. The
theoretical values of respective bond angles are 100.9o, 110.5o, 107.9o, 102.9o, 121.8o, 110.4o,
118.2o, 117.5o, 126.3o, 127.2o, 106.3o, 110.4o, 123.0o, 126.3o, 107.9o, 128.6o, 115.2o, 126.3o, and
126.8o respectively. Again, the X-ray diffraction and quantum chemical results of vital bond
angles of hydrazide 5 reflect the excellent correlation with each other (for detailed values see
Table 4).
3.3 Frontier molecular orbitals and UV-vis analyses
FMOs analysis of both derivatives is performed for understanding the reactivity and kinetic
stability [45]. The FMOs surfaces of both compounds 4 and 5 are shown in the Fig. 5.
16

HOMO (4)
LUMO (4)
HOMO (5)
LUMO (5)
Fig. 5 FMOs surfaces of both compounds 4 and 5
It is cleared from the HOMO/LUMO surfaces of both compounds, that dispersion of isodensity is
different in both. In one the π-electronic cloud is mainly dispersed on isatin moiety, which reflects
17

that the electronic transitions occurs in isatin and enone functionality attached to it. In 5, the
dispersion of isodensity is totally different. The π-electronic cloud in HOMO is dispersed mainly
on isatin, whereas the in LUMO is shifted toward the nitro benzene ring, which is may be due to
the electron withdrawing nature of nitro group. In 5, the electronic transitions occur between isatin
and nitrobenzene moieties. The HOMO and LUMO energies of compound 4 are -6.12 eV and -
2.26 eV, respectively. The H-LG of compound 4 is 3.86 eV. The HOMO, LUMO energies and
respective H-LG of compound 5 are -6.22 eV, -2.81 eV and 3.41 eV, respectively. The absorption
analysis is performed by using TD-DFT level of theory at B3LYP/6-31G(d,p) method. Based on
DFT calculations the energy gap of 4 is 4.03 eV and corresponding λmax is 307 nm. For compound
5 the energy gap is 3.94 eV along the corresponding λmax is 314 nm. The energy gap values reflect
that 4 is more stable than the compound 5. The absorption analysis revealed that in 4 the major
excitation occurs from HOMO-1 to LUMO, whereas in 5 it occurs from HOMO-1 to LUMO+1
orbital.
3.4 Molecular electrostatic potential (MEP) analysis
MEP analysis is a global property and gives the idea about the electronic distribution in a
compound [46]. The MEP surfaces of both compounds are extracted from the energy minima
structures and shown in Fig. 6. The MEP analysis shows that both compounds are nucleophilic in
nature and have strong affinity for positively charged species. The electronegative potential on
compound 4 is localized on the sulfonamide and C=O moieties, whereas in 5 it is also located on
nitro group along with the previous moieties. The dispersion of electronic density of 4 is in the
range of -0.051 to 0.051 esu, whereas in 5 it is dispersed in the range of -0.045 to 0.045 esu.
18

Fig. 6 MEP surfaces of both compounds 4 and 5
3.5 Urease inhibition assay
In vitro enzyme inhibition bioassay of both 4 and 5 is performed against urease enzyme and
results are mentioned in Table 5. Both showed good to excellent urease inhibition efficiency,
determined at 0.5 mM concentration. Both compounds showed IC50 values in uM concentration
range. As mentioned in Table 5, compound 4 exhibited better efficacy against the enzyme with
least IC50 value of 15.26 ± 0.16 µM compared to the standard thiourea showing 21.25±0.15 µM.
On the other hand, compound 5 showed higher IC50 value (58.78±0.12 µM). % Inhibition of both
compounds is also measured at 0.5 mM concentration. The compound 4 has 92.75±0.22 %
inhibition, whereas 5 has 75.44±0.23 % inhibition as compared to the standard (98.45±0.87).
3.6 Molecular docking investigations
19

The binding orientation of all the synthesized isatin derivatives in the binding site of Bacillus
pasteurii was explored using docking simulations. Crystal structure of B. pasteurii urease with
acetohydroxamic acid (HAE) as a native ligand was obtained from Protein Data Bank (PDB code
4UBP) [47]. Docking simulations were carried out by using Molecular Operating Environment
(MOE 2016.08) [48]. The native ligand HAE was re-docked in the binding site of the enzyme
(4UBP) and root mean square deviation (RMSD) is computed. The computed rmsd value is 0.89
Å. This confirms the reliability of our docking program.
The visual inspection of the best-scored highly active isatin derivative 4 (IC50=15.26 ± 0.16 µM)
revealed that it coordinates with Ni798 and Ni799 via sulfonyl oxygen atoms. His275 is involved
in hydrogen bond interactions with one of the sulfonyl oxygen. While, His222 establishes
hydrogen bond interaction (HBI) with another sulfonyl oxygen and isatin oxygen. Another
important HBI was found between Asp363 and –NH of sulfonyl hydrazide (Fig. 7).
Fig. 7 (a) Three-dimensional modelled diagram of compound 4 into the binding site of 4UBP. Nickel ions
are shown in blue spheres. (b) Two-dimensional (2D) interactions plot of compound 4 into the binding site
of 4UBP.
In vitro activity data presented in Table 5 showed that moving from methanesulfonohydrazide 4 to
3-nitrobenzenesulfonohydrazide 5 (IC50=58.78 ± 0.12 μM) the activity decreases almost 4 times.
The binding orientation of compound 5 superimposed on native ligand HAE is shown in Fig. 8. It
20

can be seen that 5 binds away from the nickel bi-center and forms π-alkyl type of interaction with
Ala170 (Fig. 8). Isatin carbonyl group and –NH of sulfonyl hydrazide forms HBI with Cys322.
Nitro group forms HBI with Hus323 and Arg339.
Fig. 8 (a) Depiction of the lowest energy docking-poses of the compound 5 (pink) superimposed on the
native ligand HAE (yellow) into the binding site of B. pasteurii urease (PDB ID 4UBP). A ribbon model of
the enzyme is presented; (b) 2D interactions of 5; (c) 3-D interaction plot generated by Discovery Studio
Visualizer (hydrogen bond interactions are shown as green dotted line)
The docking results of the compounds 4 and 5 revealed that compound 4 binds deep into the
binding site of the 4UBP interacting with Ni2+ ions. However, our docking simulation results
showed that due to bulky nitro group, compound 5 is located 4.16 Å and 5.45 Å away from Ni798
and Ni799 respectively (Figure 9). It binds to the active site flap (gate-residues) residues. Apart
21

from the active site architecture of B. Pasteurii urase (BPU) containing two Ni ions and histidine
residues, residues at the flap of the active site also involves in its activation. Among them, Cys322
is an important flap residue and is involved in the positioning of other active site residues. Other
residues of flap region are: Arg339 and Ala170. It can be concluded here that compound 5 restrict
the mobility of the active site flap by interacting with Cys322.
Fig. 9: 3-D modelled ribbon diagram of compound 5 into the binding site of B. pasteurii urease (PDB ID
4UBP) showing the distance between two Ni ions and oxygen atom of nitro group (black solid lines). The
interaction with flap residues is also shown (dotted lines).
4 Conclusions
We have successfully accomplished the synthesis of two new sulfono-hydrazides 4 and 5 from N-
allylisatin and their structures are fully characterized with the help of different kind of
spectroscopic techniques. Both compounds have suitable crystals, which are subjected to the
single crystal X-ray diffraction analysis. The single crystal X-ray data of both 4 and 5 is compared
with the DFT calculations. The quantum chemical data is proved in full agreement with the X-ray
22

diffraction values. Both compounds showed urease inhibition activity in vitro with 4 showing
better results and thus making it more attractive motif for further studies in drug discovery and
development. Docking simulations revealed that highly active compound 4 coordinates deeply
with Ni798 and Ni799 of of Bacillus pasteurii.
Acknowledgements
The project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz
University, Jaddah, Saudi Arabia under grant no; (DF-614-130-1441). The authors, therefore,
gratefully acknowledge the DSR technical and financial support.
Authors declaration
We the undersigned declare that this manuscript is original, has not been published before and is not
currently being considered for publication elsewhere.
We wish to confirm that there are no known conflicts of interest associated with this publication and there
has been no significant financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all named authors and that there are no
other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order
of authors listed in the manuscript has been approved by all of us.
We confirm that we have given due consideration to the protection of intellectual property associated with
this work and that there are no impediments to publication, including the timing of publication, with
respect to intellectual property. In so doing we confirm that we have followed the regulations of our
institutions concerning intellectual property.
23

We understand that the Corresponding Author is the sole contact for the Editorial process (including
Editorial Manager and direct communications with the office). He is responsible for communicating with
the other authors about progress, submissions of revisions and final approval of proofs. We confirm that
we have provided a current, correct email address which is accessible by the Corresponding Author.
Credit author statement
All persons who meet authorship criteria are listed as authors, and all authors certify that they have
participated sufficiently in the work to take public responsibility for the content, including
participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore,
each author certifies that this material or similar material has not been and will not be submitted to
or published in any other publication before its appearance in the Optics and Laser Technology
Authorship contributions
Muhammad Arshad, Kainat Ahmed and Zafar Iqbal: Synthesis, Enzyme inhibition, Writing-
Original draft preparation. Visualization, Investigation
Umer Rashid: Molecular docking, Software
Muhammad Nadeem Arshad, Abdullah M. Asiri: X-ray analysis, Writing- Reviewing and
Editing
Tariq Mahmood: DFT, Writing- Reviewing and Editing, Supervision
24

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