Synthesis, molecular structure, quantum mechanical studies and urease inhibition assay of two new isatin derived sulfonylhydrazides

Article abstract of DOI:10.1016/j.molstruc.2016.11.065

Two new isatin derivatives (E)-N′-(1-allyl-2-oxoindolin-3-ylidene)-4-methylbenzenesulfono-hydrazide (5) and (E)-N′-(1-allyl-2-oxoindolin-3-ylidene)-4-chlorobenzenesulfono-hydrazide (6) were synthesized in good yields by adopting two component synthetic methodology. The structure elucidation was accomplished with the help of UV–vis., FT-IR and NMR (¹H and ¹³C) spectroscopic techniques. Suitable crystals were grown by slow evaporation method and structures were confirmed unequivocally with the help of single crystal X-ray diffraction analysis. Both isatin derivatives 5 and 6 exist in triclinic crystal packing having space group P-1. Crystal structures of both compounds showed that the geometries are stabilized by several intermolecular hydrogen bonds. Quantum mechanical calculations performed at density functional theory (DFT) level confirmed the experimental spectroscopic (UV–vis., FT-IR and ¹H NMR) as well as X-ray diffraction results. Kinetic stability, reactivity, electrophilicity and nucleophilic behavior of both the derivatives was elaborated using frontier molecular orbitals (FMOs) and molecular electrostatic potential (MEP) analyses. Enzyme inhibition potential of both compounds was tested in vitro against Bacillus pasteurii urease and both compounds retarded the enzymatic activity with IC₅₀ values of 39.46 ± 0.12 μM and 148.35 ± 0.16 μM respectively.

Full text of DOI:10.1016/j.molstruc.2016.11.065

Accepted Manuscript
Synthesis, molecular structure, quantum mechanical studies and urease inhibition
assay of two new isatin derived sulfonylhydrazides
Muhammad Arshad, Mehwish Jadoon, Zafar Iqbal, Mehwish Fatima, Muhammad Ali,
Khurshid Ayub, Ashfaq Mahmood Qureshi, Muhammad Ashraf, Muhammad Nadeem
Arshad, Abdullah M. Asiri, Amir Waseem, Tariq Mahmood
PII:
S0022-2860(16)31251-0
10.1016/j.molstruc.2016.11.065
MOLSTR 23166
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 28 September 2016
Revised Date: 16 November 2016
Accepted Date: 22 November 2016
Please cite this article as: M. Arshad, M. Jadoon, Z. Iqbal, M. Fatima, M. Ali, K. Ayub, A.M. Qureshi, M.
Ashraf, M.N. Arshad, A.M. Asiri, A. Waseem, T. Mahmood, Synthesis, molecular structure, quantum
mechanical studies and urease inhibition assay of two new isatin derived sulfonylhydrazides, Journal of
Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.11.065.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Spectroscopy
Crystallography
Urease inhibition
Quantum mechanical studies

ACCEPTED MANUSCRIPT
Synthesis, molecular structure, quantum mechanical studies and urease
inhibition assay of two new isatin derived sulfonylhydrazides
Muhammad Arshada , Mehwish Jadoonb, Zafar Iqbalb, Mehwish Fatimaa, Muhammad
Alib, Khurshid Ayubb, Ashfaq Mahmood Qureshic, Muhammad Ashrafd, Muhammad
Nadeem Arshade,f, Abdullah M. Asirie,f, Amir Waseemg, Tariq Mahmoodb*
aInstitute of Chemistry, University of the Punjab, Quaid-i-Azam Campus, Lahore-54590, Pakistan.
bDepartment of Chemistry, COMSATS Institute of Information Technology, University Road,
Tobe Camp, Abbottabad-22060, Pakistan.
cInstitute of Chemical Sciences, Bahauddin Zakariya University, Multan-60800, Pakistan.
dDepartment of Chemistry, The Islamia University of Bahawalpur, Baghdad-ul-Jadeed Campus,
Bahawalpur-63100, Pakistan.
eChemistry Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah
21589, Saudi Arabia.
fCentre of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, P.
O. Box 80203, Jeddah 21589, Saudi Arabia.
gDepartment of Chemistry, Quaid-e-Azam University, Islamabad-45320, Pakistan.
*To whom correspondence should be addressed: E-mail: mahmood@ciit.net.pk (T. M)
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Abstract
Two new isatin derivatives (E)-N'-(1-allyl-2-oxoindolin-3-ylidene)-4-methylbenzenesulfono-
hydrazide (5) and (E)-N'-(1-allyl-2-oxoindolin-3-ylidene)-4-chlorobenzenesulfono-hydrazide (6)
were synthesized in good yields by adopting two component synthetic methodology. The structure
elucidation was accomplished with the help of UV-vis., FT-IR and NMR (1H and 13C)
spectroscopic techniquies. Suitable crystals were grown by slow evaporation method and
structures were confirmed unequivocally with the help of single crystal X-ray diffraction analysis.
Both isatin derivatives 5 and 6 exist in triclinic crystal packing having space group P-1. Crystal
structures of both compounds showed that the geometries are stabilized by several intermolecular
hydrogen bonds. Quantum mechanical calculations performed at density functional theory (DFT)
1
level confirmed the experimental spectroscopic (UV-vis., FT-IR and H-NMR) as well as X-ray
diffraction results. Kinetic stability, reactivity, electrophilicity and nucleophilic behavior of both
the derivatives was elaborated using frontier molecular orbitals (FMOs) and molecular
electrostatic potential (MEP) analyses. Enzyme inhibition potential of both compounds was tested
in vitro against Bacillus pasteurii urease and both compounds retarded the enzymatic activity with
IC50 values of 39.46 ± 0.12 µM and 148.35 ± 0.16 µM respectively.
Keywords: (a) Isatin (b) X-ray (c) DFT (d) Enzyme inhibition
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1. INTRODUCTION
Isatin 1 (Fig. 1), also known as (1H-indole-2,3-dione) [1], is a valuable nitrogen bearing
heterocyclic motif. It was, first synthesized in 1840 from oxidation of indigo mediated by nitric
and chromic acids [2]. Naturally, isatin is found in plants of genus Isatis [3], Calanthe discolor
LINDL [4] and Couroupita guianensis AUBL [5]. It is also present in the secretion of parotid gland
of Bufo frogs [6], in human adrenaline [7] and in coal tar [8]. Isatin is a versatile synthetic
precursor, which has been employed for the construction of a wide variety of heterocyclic
compounds [9], their metal complexes [10] and synthetic drugs [11]. Isatin based drugs have
already been pursued in the pharmaceutical industry, e. g. Suntinib [12] and Nintedanib [13] are
approved for the treatment of metastatic renal cell carcinoma and as anti-cancer agent,
respectively.
Isatin and its metabolites are important structural backbone of many natural products as well as
numerous synthetic compounds. Isatin derivatives have been explored for a wide range of
biological properties including anti-viral, anti-tumor, anti-angiogenic [14], anxiolytic [15], anti-
fungal, anti-bacterial, anti-tubercular and anti-convulsant activity [16].
In recent years quantum mechanical methods by using GAUSSIAN software have been applied on
wider level to study and compare the spectroscopic properties and geometric parameters of
synthetic and natural products [17,18]. These are also applied to investigate the structural
properties of compounds, such as frontier molecular orbitals (FMOs) [19], molecular electrostatic
potential (MEP) [20], Mulliken charge analysis, natural bond orbital (NBO) analysis and non-
linear optics properties [21]. Among all quantum mechanical methods, density functional theory
(DFT) is very famous and has wide range applicability due to its computational cost and accuracy
[22–24]. In continuation to our previous work of synthesis and DFT investigations of diverse types
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of hetrocyclic compounds [25–27] and keeping in view of the biological effectiveness of the isatin
derivatives we intended to explore the antienzymatic activities of its hydrazone derivatives. To
accomplish the aim we synthesized two new phenylsulfonyl hydrazones of intermediate allylic
isatin derivative, performed the quantum mechanical studies and determined their anti-urease
activity in vitro.
2. Materials and Methods
2.1 Experimental
All chemicals and reagents of synthetic grade were purchased from Alfa Aesar and Acros
Organics and used without further purifications. The reaction progress was monitored by thin
layer chromatography (TLC) using aluminum plates pre-coated with silica gel. Solvents used were
of either synthetic grade or commercial grade, which were purified and properly dried before use.
Melting points were monitored by digital melting point apparatus having temperature range up to
o
300 C and are reported as uncorrected. UV-vis. spectroscopic measurements were carried out
using t80 + UV-vis. spectrophotometer. FT-IR spectra were measured using Agilent FTIR Cary
630 instrument, and by applying ATR sampling technique. NMR spectra were recorded using
1
13
Bruker’s (300 MHz for H and 75 MHz for C) NMR machine. Deuterated solvent was used for
NMR analysis and chemical shift values are reported in ppm. Coupling constants (J values) are
1
reported in Hertz and multiplicity of the signals in H-NMR is reported as doublet (d), triplet (t),
quartet (q), doublet of doublet (dd) and multiplet (m).
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2.2 Synthesis
Isatin 1 which is a commercially available and very inexpensive heterocyclic precursor, was
subjected to allylation of nitrogen atom [28] to generate the N-allyl isatin 2, which in turn was
reacted with sulfonylhydrazides (3 and 4, obtained by reacting sulfonyl chloride with hydrazine
hydrate) to afford the target compounds 5 and 6 in good yields (Scheme 1).
2.2.1 General Procedure for the synthesis of 1-allylindoline-2,3-dione (2)
Calcium hydride (1.26 g, 30 mmol) was added portion wise to a solution of isatin 1 (1.47 g, 10
mmol) in DMF (10 mL) and the resulting mixture heated at 45 οC. After 30 minutes, allyl bromide
(0.78 mL, 15 mmol) was added to the above mixture followed by heating at the same temperature
for 3 hrs. After completion of the reaction as monitored by TLC, the mixture was cooled to room
temperature. 1 M HCl (100 mL) was added slowly with continuous stirring to neutralize the
excess of CaH2. Product was extracted with ethyl acetate (3 × 30 mL) and combined organic layer
was washed with brine, dried over MgSO4, filtered and evaporated to afford crude product.
Purification of crude product was accomplished by flash column chromatography, using
EtOAc/Hexane (2:8) mixture as eluant. Title compound 2 was obtained as pure orange red solid
(0.77 g, 41% yield); Rf = 0.4 (EtOAc/Hexane, 3:7).
2.2.2 General procedure for the synthesis of arylsulfonyl hydrazides (3 and 4)
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Hydrazine mono hydrate (1.40 mL, 15 mmol) was slowly added to a 10−15 οC cooled and stirred
solution of appropriate arylsulfonyl chloride (10 mmol) in THF (10 mL). After complete addition,
the resulting mixture was stirred for 15 minutes in ice bath maintaining the same low temperature.
Clear one spot for the product on TLC indicated the completion of reaction. Cold water (100 mL)
was added to the reaction mixture, which precipitated out the product. Precipitates were filtered,
washed with cold water and dried in air. Sulfonylhydrazides 3 or 4 were obtained as pure white
solid.
4-Methylbenzenesulfonyl hydrazide (3): White solid, (1.50 g, 83% yield), Rf = 0.51(Ethyl
o
acetate/n-hexane 3:7); M. P. = 101−105 C, FT-IR (ATR, cm−1): 3385, 3252, 1600, 1421, 1301,
1154, 1091, 815.
4-Chlorobenzenesulfonyl hydrazide (4): White powder (1.00 g, 50% yield), Rf = 0.63(Ethyl
o
acetate/n-hexane 3:7), M. P. = 86−90 C, FT-IR (ATR, cm−1): 3394, 3351, 3294, 1588, 1477,
1325, 1154, 1086, 1015, 828, 754.
2.2.3 General procedure for the synthesis of final products (5 and 6)
Arylsulfonyl hydrazide (3 or 4) (1.20 mmol) was added to a solution of 1-allylindoline-2,3-dione 2
(0.18 g, 1.0 mmol) in ethanol (10 mL). Glacial acetic acid (1 mL) was added to the reaction
mixture, which was refluxed for 2 hrs. After completion of the reaction, cold water (100 mL) was
added to the reaction mixture. The precipitates formed were collected by filtration, washed with
cold water and dried in air. Target compounds 5 and 6 were obtained as pure yellow solids.
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2.2.3.1 (E)-N'-(1-Allyl-2-oxoindolin-3-ylidene)-4-methylbenzenesulfono-hydrazide (5)
ο
Title compound 5 was obtained as pure yellow powder (0.29 g, 82%); M. P., 177−180 C; Rf =
0.21 (EtOAc/Hexane, 3:7); UV-vis. (λmax.), 260 nm, 320 nm; FT-IR (ATR, cm−1), 3088, 1677,
1613, 1469, 1399, 1346, 1399, 1346, 1181, 1028, 1010, 741, 720; 1H-NMR (300 MHz, DMSO-d6)
δ 12.45 (s, 1H, NH), 7.88 (d, J = 8.3 Hz, 2H, ArH × 2), 7.48 (d, J = 7.5 Hz, 1H, ArH), 7.44 (d, J =
8.3 Hz, 2H, ArH × 2), 7.38 (dd, J = 7.8, 1.1 Hz, 1H, ArH), 7.09 (t, J = 7.5 Hz, 1H, ArH), 7.02 (d, J
= 7.8 Hz, 1H, ArH), 5.83 (ddt, J = 17.1, 10.2, 5.1 Hz, 1H, N-CH2CH=CH2), 5.20 (dd, J = 17.1, 1.3
Hz, 1H, N-CH2CH=CHH), 5.15 (dd, J = 10.2, 1.3 Hz, 1H, N-CH2CH=CHH), 4.33 (d, J = 5.1 Hz,
2H, N-CH2CH=CH2), 2.37 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-d6) δ 159.6 (N-C=O),
144.5(C), 142.8 (C), 136.4 (C), 135.0 (C), 131.6(C), 131.4(CH), 129.9 (CH × 2), 127.8 (CH × 2),
123.1 (CH), 120.4 (CH), 118.8 (CH2), 117.5 (CH), 110.4 (CH), 41.4 (N-CH2), 21.1 (CH3).
2.2.3.2 (E)-N'-(1-Allyl-2-oxoindolin-3-ylidene)-4-chlorobenzenesulfono-hydrazide (6)
Title compound 6 was obtained as dark yellow powder (0.29 g, 78%); Rf (EtOAc/Hexane, 3:7)
0.25; M.P. 175−178 oC; UV-vis. (λmax.) 255 nm, 320 nm; FT-IR (ATR cm−1), 1691, 1618, 1469,
1384, 1290, 1180, 1081, 1002, 754, 724; (1H NMR (300 MHz, DMSO-d6) δ 12.49 (s, 1H, NH),
8.18 – 7.88 (m, 2H, ArH × 2), 7.83 – 7.61 (m, 2H, ArH × 2), 7.49 (d, J = 7.4 Hz, 1H, ArH), 7.40
(td, J = 7.8, 1.1 Hz, 1H, ArH), 7.10 (t, J = 7.4 Hz, 1H, ArH), 7.03 (d, J = 7.8 Hz, 1H, ArH), 5.83
(ddt, J = 17.1, 10.2, 5.0 Hz, 1H, N-CH2CH=CH2 ), 5.20 (dd, J = 17.1, 1.3 Hz, 1H, N-
CH2CH=CHH), 5.15 (dd, J = 10.2, 1.3 Hz, 1H, N-CH2CH=CHH), 4.33 (d, J = 5.0 Hz, 2H, N-
CH2CH=CH2); 13C NMR (75 MHz, DMSO-d6) δ 159.3 (N-C=O), 143.0 (C), 138.9 (C), 137.1 (C),
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136.8 (C), 131.8 (C), 131.4 (CH), 129.8 (CH × 2), 129.6 (CH × 2), 123.1 (CH), 120.6 (CH), 118.8
(CH2), 117.5 (CH), 110.4 (CH), 41.4 (N-CH2).
2.3 Crystallography
Suitable crystals were mounted on Agilent SuperNova (Dual source) Agilent Technologies
Diffractometer, equipped with microfocus Cu/Mo Kα radiation for data collection. The data
collection was accomplished using CrysAlisPro software [29] at 296 K under the Cu Kα radiation.
The structure solution was performed using SHELXS–97 method and refined by full–matrix least–
squares methods on F2 using SHELXL–97 method [30] in-built with X-Seed [31]. All non–
hydrogen atoms were refined anisotropically by full–matrix least squares methods [30]. The
figures were generated through PLATON [32] and ORTEP [33] in built with WinGX [34].
All aromatic hydrogen atoms were positioned geometrically and treated as riding atoms with C–H
= 0.93 Å and Uiso(H) = 1.2 Ueq(C) carbons. Accordingly, the methyl (5) and methelene
hydrogens were also positioned geometrically with Cmethyl-H = 0.96 Å and Cmethelene-H = 0.97 Å
and Uiso(H) = 1.5 Ueq(C) and Uiso(H) = 1.2 Ueq(C) for methyl and methylene carbon atoms,
respectively. The hydrogen atom fixed on nitrogen [N-H = 0.80 (2) and 0.88(3) for 5 and 6,
respectively] was located with difference fourier map and refined using riding model with Uiso
(H) = 1.2 Ueq(N). The Crystal data was deposited at the Cambridge Crystallographic Data Centre
and following deposition numbers have been assigned 1501255 and 1501254 for molecules 5 and
6, respectively.
2.4 Theoretical calculations
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Computational studies has been performed at DFT level by using GAUSSIAN 09 package [35].
The geometric/graphics analysis was executed with the help of Gauss View 5.0 [36]. First of all
optimization of both isatin derivatives 5 and 6 was executed by adopting B3LYP/6-31G (d, p)
level of theory without any symmetry constraint. Vibrational analysis was performed by using the
same basis set, in order to confirm the energy minima structure and to extract the theoretical
vibrational spectrum. Electronic studies were performed at same level by using time delay TD-
DFT method. For generation of solvent affect CPCM model was adopted. Theoretical nuclear
magnetic resonance (1H ) chemical shifts were calculated by using higher basis set i.e. B3LYP/6-
311+G (2d, p) which normally works better for NMR chemical shift calculations [21], and by
adopting GIAO formalism. Molecular electrostatic potential (MEP) and frontier molecular orbital
(FMOs) analyses were executed by using B3LYP/6-31G (d, p) basis set.
2.5 Urease Inhibition Assay
Modified Berthelot assay was implemented for evaluating the anti-urease potential of both
compounds (5 and 6) [37]. The current assay deals the urea hydrolysis to produce ammonia, which
on reaction with phenol-hypochlorite produces light blue complex showing λmax. at 625 nm. A
mixture of 10 µL of 50 mM phosphate buffer (pH 7.0), 10 µL of sample solution and 25 µL of
Bacillus pasteurii urease (Sigma) solution (0.015 units) placed in a 96- well plate, was incubated
for 10 minutes at 37 oC. 40 µL of urea stock solution (20 mM) was added to each well, incubated
ο
for further 10 min at 37 C and pre-read at 625 nm using the 96-well plate reader Synergy HT
(Biotek Inc.). Phenol hypochlorite (115 µL) reagent was added in each well (newly prepared by
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adding 70 µL of alkali reagent to 45 µL phenol reagent). Incubation was carried out for another 10
ο
min at 37 C for color development. Absorbance was measured again at 625 nm. The % age of
enzyme inhibition was calculated by applying the formula given below:
IC50 values (sample conc. at 50% inhibition of the enzyme) of samples were calculated by
measuring activities at further dilutions followed by computing the data using EZ-Fit Enzyme
software (Perrella Inc, USA). The units of absorbance of un-inhibited enzyme were between 1.0
and 1.2.
3. Results and Discussion
The synthesis, structural investigations and enzyme inhibition assay of two new N-alkylated isatin
based sulfonylhydrazide derivatives 5 and 6, have been executed successfully. The proposed
synthetic route consists of nucleophilic substitutions and condensation reactions. The synthesis
was performed by adopting two component synthetic methodology (the details about synthetic
scheme have been already discussed above in experimental section). After finishing the successful
synthesis, both compounds were characterized with the help of spectro-analytical techniques such
1
as H, 13C-NMR, FT-IR, UV-vis., and final structures were confirmed unequivocally by single
crystal X-ray diffraction analysis. Quantum mechanical investigations were executed to compare
the experimental spectroscopic results and to understand the reactivity and dispersion of electronic
density.
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3.1 X-ray Diffraction analysis
The both isatin derivatives (5 and 6) were crystallized from methanol (slow evaporation method)
and suitable crystals were obtained for crystallographic analysis. Molecular structures of 5 and 6
consists on [C18H17N3O3S] and [C17H14N3O3SCl] entities having formula weights 355.40
grams/mol and 375.82 grams/mol, respectively. The ORTEP diagrams of 5 and 6, where thermal
ellipsoids are drawn at 50% probability level are shown in Fig. 2 and crystallographic data
collection parameters are given in Table 1. The basic skeleton of compounds consist of indole
based sulfonyl hydrazine moiety. The aromatic and pyrole rings are twisted at 1.98 (1)º and 1.31
(9)º in 5 and 6, respectively. Indole ring in both compounds is oriented at dihedral angles of 89.57
(6)º and 73.66 (7)º with respect to p-tolyl and 4-chlorophenyl rings, depicts that both (5 and 6)
confer L shape configuration. The sulphur atoms adopted distorted tetrahedral geometry with
respect to O2-S1-O3 = 120.08 (9)º and 122.1 (9)º in 5 and 6. Both molecules are stabilized by
adopting cis geometry around the C=N imine functionality. The intra-molecular classical N-H...O
hydrogen bonding is observed to form six membered rings, those are connected through another
non-classical C-H...O hydrogen bonding and formed dimers Fig. S1, (supplementary information).
In compound 5, the dimers further linked through another C-H...O hydrogen bonding to form
infinite long chain along a axes.
3.2 Molecular structure
Density functional theory (DFT) simulations were executed to validate geometric parameters
(bond lengths and bond angles) obtained through single crystal X-ray diffraction studies. The
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geometries of both compounds 5 and 6 were simulated by using B3LYP/6-31G(d,p) basis set to
estimate the geometric parameters, and given in the Fig. 3. Theoretical and experimental values of
bond lengths and bond angles are narrated in Table 2 (bond lengths) and Table 3 (bond angles).
The experimental and theoretical bond lengths of both isatin derivatives (5 and 6) corroborated
nicely to each other. Experimental values of bond lengths such as C1-N1, C9-S1, C16-N1, C7-N2,
C8-N1, C8-O1, N2-N3, N3-S1, O2-S1 and O3-S1 in compound (5) are observed at 1.41Å, 1.75Å,
1.45Å, 1.28Å, 1.37Å, 1.21Å, 1.36Å, 1.64Å,1.42Å and 1.42Å respectively, whereas the theoretical
values of respective bond lengths are found at 1.40Å, 1.78Å, 1.46Å, 1.29Å, 1.38Å, 1.23Å, 1.33Å,
1.71Å, 1.45Å and 1.46Å. Similarly the experimental bond lengths of (6), such as C1-N1, C7-N2,
C8-N1, C8-O1, C9-S1, C15-N1, N2-N3, N3-S1, O2-S1, O3-S1 and C12-Cl1 are depicted at
1.41Å, 1.28Å, 1.36Å, 1.22Å, 1.75Å, 1.45Å, 1.35Å, 1.64Å, 1.42Å, 1.41Å and 1.73Å respectively.
Theoretical values of respective bond lengths are observed at 1.41Å, 1.29Å, 1.38Å, 1.23Å, 1.70Å,
1.45Å, 1.33Å, 1.70Å, 1.45Å, 1.46Å and 1.75Å, respectively.
The experimental and theoretical values of different bond angles in both derivatives showed very
tight correlation to each other. The X-ray values of bond angles such as C2-C1-N1, C6-C1-N1,
N1-C16-C17, C1-N1-C16, N2-C7-C6, N2-C7-C8, N1-C8-C7, O1-C8-N1, C10-C9-S1, C14-C9-
S1, C8-N1-C1, C8-N1-C16, C7-N2-N3, N2-N3-S1, N3-S1-C9, O2-S1-C9, O2-S1-N3, O2-S1-O3,
O3-S1-C9 and O3-S1-N3 (atomic labels are with reference to Fig. 2) are found at 129.3o, 109.4o,
111.2o, 124.9o, 124.9o, 128.1o, 105.4o, 126.8o, 119.0o, 120.1o, 111.1o and 123.4o, 117.9o, 114.8o,
105.6o, 108.0o, 108.0o, 120.0o, 110.2o and 103.8o respectively. Their theoretical counterparts are
observed at 128.8o, 109.7o, 113.0o, 126.4o, 126.8o, 126.2o, 106.3o, 126.3o, 119.2o, 119.4o, 110.5o,
122.9o, 118.1o, 119.2o, 104.6o, 108.1o, 107.9o, 123.1o, 109.1o and 102.2o respectively. Similarly,
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the experimental and theoretical values of all bond angles in 6 showed an excellent correlation to
each other (for individual values see Table 3).
3.3 Vibrational analysis
Vibrational spectroscopy is very significant tool for identification of the structures of compounds
[38]. Experimental FT-IR spectra of isatin derivatives 5 and 6 were measured under neat
conditions (ATR method), and simulated vibrational spectra were calculated by using B3LYP/6-
31G(d,p) basis set. Experimental and simulated spectra of both compounds are provided in Fig. 4
and Fig. 5. To minimize the theoretical error, theoretical values were scaled by multiplying with
scaling factor 0.9627 [39], and detailed comparison of prominent vibrations
(experimental/theoretical) is narrated in Table 4. Both compounds have aromatic, amide,
sulphonamide, C=N, CH2, and CH3 functional groups. The data provided in Table 4, clearly
indicates that an outstanding agreement exists between the experimental and theoretical values.
The experimental values of CH and NH stretching vibrations generally appear in the range 2800-
3100 cm-1, and some times are hard to identify because of low intensity [40]. Theoretical CH3 and
CH2 stretching (symmetric/asymmetric) vibrations of 5 and 6 depicted at 3015 cm-1 and 2915 cm-
1, whereas the experimental values of these appeared at 3088 cm-1. Among all, the carbonyl group
gives strong signal of stretching vibration in the range of 1600-1750 cm-1 [41] and can be
identified easily in FT-IR spectrum. In both derivative 5 and 6, the simulated value of stretching
vibration of cyclic amide found at 1695 cm-1 and 1698 cm-1 and corroborate with experimental
ones those appeared at 1677 cm-1 and 1691 cm-1, respectively.
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The simulated vibrational spectra of both derivatives showed several signals of stretching and
bending aromatic ring vibrations (both C=C and CH), correlated with experimental values nicely.
The asymmetric stretching C=Carom. vibrations are appeared at 1605 cm-1 (5), 1451 cm-1 (5), 1606
cm-1 (6) and 1453 cm-1 (6) theoretically, showed good correlation with the experimental values
those are found at 1613 cm-1 (5), 1469 cm-1 (5), 1618 cm-1 (6) and 1469 cm-1 (6), respectively.
Apart from stretching vibrations of C=Carom., some important bending vibrations of aromatic CH
groups were identified in both compounds (for detailed values see Table 4). The S=O is another
functional which can be easily identified and compared by using vibrational spectroscopy. In
computed spectra of both 5 and 6, stretching vibrations (symmetric/asymmetric) of S=O are
depicted at 1105 cm-1 (5), 1048 cm-1 (5), and 1107 cm-1 (6), respectively. The simulated values
showed strong agreement with experimental values that appeared at 1028 cm-1 and 1081 cm-1 for
compounds 5 and 6, respectively.
3.4 Nuclear magnetic resonance (NMR) studies (1H)
Since last one decade scientists are extensively applying the computational methods to measure
the chemical shifts of different protons in any compound and to compare with the experimental
1
values [42,43]. Experimental H-NMRs of both compounds 5 and 6 were measured in deuterated
dimethyl sulphoxide (see electronic supplementary information Fig. S2-S3). Theoretical 1H-NMRs
were calculated using GIAO method, applying B3LYP/6-311+G (2d, p) basis set. The detailed
experimental and theoretical 1H-NMR chemical shifts of both compounds are narrated in Table 5.
1
Both compounds mainly have NH, aromatic, CH and CH2 protons, in the experimental H-NMR
spectrum of both 5 and 6, the NH protons found at 12.45 ppm and 12.49 ppm respectively. The
14

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theoretical chemical shifts of respective protons appeared at 13.06 ppm and 13.10 ppm.
Experimental values of NH protons may not correlate with that of theoretical ones since the
chemical shift of NH protons is highly dependent on the nature of solvent and electronic
environment of the neighboring atoms [21]. Experimental chemical shift values of aromatic
protons found in the range 8.30–7.02 ppm for compound 5 and ranging from 8.18 ppm to 7.03
ppm for 6, whereas theoretical values appeared in the range 8.35-7.06 ppm for 5 and that for
compound 6 at 8.32-6.83 ppm, respectively. The experimental chemical shifts of CH protons in
both compounds appeared at 5.83 ppm however theoretical values were slightly shifted at 6.22
ppm and 6.10 ppm for 5 and 6 respectively. Experimental chemical shifts of CH2 group attached
to the aromatic ring depicted at 4.33 ppm for both compounds 5 and 6, respectively. The
theoretical values of same protons were found at 4.38 ppm for derivative 5 and at 4.44 ppm for
compound 6. In compound 5 the methyl group attached to the benzene correlated nicely in terms
of theoretical value (2.50 ppm) and experimental value (2.37 ppm). Again the experimental and
simulated chemical shifts of CH2 protons attached to the double bond revealed outstanding
correlation to each other (detailed values are given in the Table 5).
3.5 Frontier molecular orbitals (FMOs) analysis
The biological potential, optical activity and reactivity of compounds can be expressed in terms of
frontier molecular orbitals (HOMO/LUMO) [44]. If the band gap between frontier orbitals is low,
the molecule is chemically more active and kinetically less stable and vice versa [45]. The HOMO
and LUMO surfaces of both compounds (5 and 6) were analyzed by using 6-31G(d,p) basis set
and graphics are shown in Fig. 6. The spreading of isodensity on frontier orbital two orbitals are
15

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very vital to understand the reactivity and optical activity. It can be under stood clearly from the
surfaces shown in Fig. 6, that isodensity in both HOMO/LUMO orbitals is delocalized mainly
over the isatin skeleton along with N=NH functionality, which reflects that the electronic
transitions from HOMO to LUMO are confined to the benzene and indol moieties. Canonical
frontier orbitals (HOMO/LUMO) extracted from the G09 reflect that these molecular orbitals
mainly consist of p orbitals, and could facilitate π to π* electronic transitions. The HOMO and
LUMO band gap of both derivatives 5 and 6 is found 3.83 eV and 3.81 eV, respectively. The low
band gap in both compounds means that both are believed to be soft in nature and chemically
more active.
3.6 Comparison between experimental and theoretical UV-vis. studies
The experimental UV-vis. absorption spectra of both products 5 and 6 were measured in the range
of 200-600 nm and chloroform as a solvent. Theoretical absorption spectra were calculated with
the aid of TD-DFT/B3LYP/6-31G(d,p) method. Experimental and theoretical absorption spectra
of 5 and 6 are showing in Fig. 7. The absorption maxima (λmax.), absorption value, oscillating
strength (f) and assignments have been mentioned in Table 6.
In the experimental absorption spectrum of 5, two absorption maxima appeared at 260 nm and 320
nm, whereas their theoretical counter parts found at 261nm and 324 nm and corroborate to each
other excellently. Similarly the experimental and theoretical absorption spectra of 6, showed
excellent correlation to each other. Experimentally λmax. values of 6 appeared at 255 nm and 320
nm, whereas theoretically appeared at 246 nm and 328 nm. These electronic transitions in both
compounds are in complete association with frontier orbitals i.e. HOMO/LUMO analysis. As it is
16

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clear from the Fig. 7, that HOMO and LUMO electronic density is delocalized mainly over the
isatin moiety which reflects that transition of electrons are confined at the isatin moiety. The shape
of orbitals suggest that these are consist of p orbitals, therefore the transitions are π to π* in nature
through extended conjugations.
3.7 Molecular electrostatic potential (MEP)
Molecular electrostatic potential (MEP) analysis by using different computational methods is an
important tool to understand the spreading of electronic density over the compounds. MEP
analysis helps to predict the electrophilic as well as nucleophilic centers in any molecule and sites
for hydrogen bonding as well. In MEP analysis, dispersion of electronic density is expressed with
the help of appearance of different colors, electronegative potential is represented by red color,
whereas electropositive potential is represented by blue color [46]. MEP analysis of both isatin
derivatives 5 and 6 was simulated by using B3LYP/6-31G(d,p) basis set and surfaces are provided
in the Fig. 8.
MEP graphics are indicating that, both derivatives (5 and 6) are nucleophilic and negative region
is concentrated on sulfonamide and amide moiety of triazol ring, which are preferred
electropositive species. MEP values are ranged from -0.05485 a. u. to 0.05485 a. u. for 5 and -
0.05027 a. u. to 0.05027 a. u. for compound 6.
3.8 Enzyme inhibition assay
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In vitro urease enzyme inhibition bioassay was performed for both compounds 5 and 6 which
showed good to excellent inhibition results. Compound 5 showed 88.58 ± 0.21 % inhibition.
Whereas compounds 6 showed 82.27 ± 0.23 % inhibition of the enzyme at 0.5 mM concentration
for all compounds, compared to the standard thiourea, which showed 98.45 ± 0.87 % inhibition.
IC50 (Inhibitor’s concentration at 50% inhibition) values were also measured as 39.46 ± 0.12 µM
and 148.35 ± 0.16 µM respectively for compound 5 and 6, compared to the standard thiourea
showing 21.25±0.15 µM.
4. Conclusions
We have successfully synthesized two new sulfonylhydrazides (5 and 6) from N-allylisatin and
their structures were fully characterized by detailed spectroscopic techniques like UV-vis., FT-IR
and NMR spectroscopy. Structures of both the target compounds were further confirmed by single
crystal X-ray diffraction method. The spectroscopic data for these compounds was compared with
the data obtained theoretically by DFT calculations. Experimental spectroscopic data was found to
be in good agreement with the corresponding simulated values for each compound. Theoretically
calculated low band gap values of both compounds suggest the high reactivity of these
compounds. Therefore, the compounds may be used as reactive intermediates for biologically
valuable end products. Both compounds show promising urease inhibition activity in vitro thus
making them attractive motifs for further studies in drug discovery and development.
Acknowledgements
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The authors are highly thankful to COMSATS Institute of Information Technology Abbottabad,
University of the Punjab Lahore for internal research grant and Higher Education Commission of
Pakistan (Grant no. 20-3013/NRPU/R&D/HEC/14/525) for financial support.
Supplementary material
Cartesian co-ordinates of optimized geometries and check cif files of isatin derivatives 5 and 6 are
1
pasted in supporting information. Unit cell diagrams, experimental H and 13C-NMR of 5 and 6
are also pasted in supporting information as Fig. S1-S5. Supplementary data associated with this
article can be found, in the online version, at http://dx.doi.org/
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Captions
Table 1: X-ray and structural parameters of compounds (5 and 6).
Table 2: Comparison of some important X-ray and simulated bond lengths (Å) of compounds 5
and 6 (Atomic labels are with reference ORTEP plots Fig. 2).
Table 3: Comparison of some important X-ray and simulated bond angles (o) of compounds (5
and 6 (Atomic labels are with reference ORTEP plots Fig. 2).
Table 4: Prominent experimental and simulated vibrational (cm-1) frequencies of 5 and 6.
1
Table 5: Comparison of experimental and simulated H-NMR of 5 and 6 (ppm) (proton labelling
is according to the Fig. 3).
Table 6: Experimental and simulated UV-vis. λmax. (nm) values of 5 and 6.
Scheme 1: Synthetic scheme of compounds (5 and 6).
Figure 1: General structure of Isatin.
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Figure 2: Labelled ORTEP diagrams of 5 and 6 (thermal ellipsoids were drawn at 50% probability
level).
Figure 3: Optimized geometries of both compounds 5 and 6.
Figure 4: Experimental FT-IR spectra of compounds, 5 (above) and 6 (below).
Figure 5: Simulated spectra of compounds 5 and 6.
Figure 6: HOMO/LUMO orbitals of both compounds 5 and 6.
Figure 7: Overlaid UV-vis. spectra of both compounds 5 and 6 measured in CHCl3.
Figure 8: MEP surfaces of both compounds 5 and 6.
26

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Table 2: Comparison of some important X-ray and simulated bond lengths (Å) of compounds 5
and 6 (Atomic labels are with reference ORTEP plots Fig. 2).
(5)
C1-N1 1.41 (3)
C9-S1 1.75 (19)
C16-N1 1.45 (3)
C7-N2 1.28 (2)
C8-N1 1.37 (3)
C8-O1 1.21 (2)
N2-N3 1.36 (2)
X-ray
Calc.
1.40
(6)
X-ray
Calc.
C1-N1
C7-N2
C8-N1
C8-O1
C9-S1
1.41 (2)
1.28 (3)
1.36 (3)
1.22 (2)
1.75 (19)
1.41
1.29
1.38
1.23
1.70
1.45
1.33
1.70
1.45
1.46
1.75
1.78
1.46
1.29
1.38
1.23
1.33
1.71
1.45
1.46
C15-N1 1.45 (3)
N2-N3
N3-S1
O2-S1
O3-S1
1.35 (2)
N3-S1
O2-S1
O3-S1
1.64 (17)
1.42 (15)
1.42 (15)
1.64 (19)
1.42 (14)
1.41 (15)
C12-Cl1 1.73 (2)

ACCEPTED MANUSCRIPT
Table 3: Comparison of some important X-ray and simulated bond angles (o) of compounds (5
and 6 (Atomic labels are with reference ORTEP plots Fig. 2).
(5)
X-ray
Calc.
(6)
X-ray
Calc.
C2-C1-N1
C6-C1-N1
N1-C16-C17 111.2 (2)
129.3 (2)
109.4 (18)
128.8
109.7
113.0
126.4
126.8
126.2
106.3
126.3
119.2
119.4
110.5
122.9
118.1
119.2
104.6
108.1
107.9
123.1
109.1
102.2
C2-C1-N1
C6-C1-N1
N2-C7-C6
N2-C7-C8
N1-C8-C7
O1-C8-C7
O1-C8-N1
C10-C9-S1
C14-C9-S1
127.8 (19)
109.7 (17)
126.2 (18)
127.5 (19)
106.1 (17)
126.8 (19)
127.0 (19)
119.3 (16)
120.1 (15)
128.6
109.7
126.8
126.2
106.3
127.1
126.4
119.1
119.3
115.2
126.2
110.4
123.1
118.0
118.7
104.6
107.8
107.9
108.7
102.6
C1-N1-C16
N2-C7-C6
N2-C7-C8
N1-C8-C7
O1-C8-N1
C10-C9-S1
C14-C9-S1
C8-N1-C1
C8-N1-C16
C7-N2-N3
N2-N3-S1
N3-S1-C9
O2-S1-C9
O2-S1-N3
O2-S1-O3
O3-S1-C9
O3-S1-N3
124.9 (18)
124.9 (19)
128.1 (19)
105.4 (18)
126.8 (2)
119.0 (15)
120.1 (16)
111.1 (17)
123.4 (2)
117.9 (18)
114.8 (14)
105.6 (9)
108.0 (10)
108.0 (9)
120.0 (9)
110.2 (9)
103.8 (10)
N1-C15-C16 114.1 (18)
C1-N1-C15
C8-N1-C1
C8-N1-C15
C7-N2-N3
N2-N3-S1
N3-S1-C9
O2-S1-C9
O2-S1-N3
O3-S1-C9
O3-S1-N3
124.4 (17)
111.0 (16)
124.4 (17)
117.2 (17)
117.8 (15)
105.8 (9)
107.8 (9)
107.0 (9)
109.2 (9)
103.5 (9)