Synthesis, biological investigation, calf thymus DNA binding and docking studies of the sulfonyl hydrazides and their derivatives

Article abstract of DOI:10.1016/j.molstruc.2015.11.046

The present study describes the syntheses and biological investigations of sulfonyl hydrazides and their novel derivatives. The detailed investigations involved the characterization of the newly synthesized compounds using FTIR, NMR, mass spectrometry and by single crystal X-Ray diffraction (XRD) analysis techniques. The binding tendencies of these compounds with CT-DNA (calf thymus DNA) have been explored by electronic absorption (UV) spectroscopy and viscosity measurement. The binding constant (K) and Gibb's free energy (ΔG) values were also calculated accordingly. In addition, we also investigated the biological activities such as antioxidant, antibacterial, enzyme inhibition and DNA interactions. The antioxidant activity was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, while antibacterial activity was investigated against four bacterial strains (viz. Escherichia coli, Crynibacteria bovius, Staphylococcus auras and Bacillus antherasis) by employing the common disc diffusion method. Enzyme inhibition activity of the synthesized compounds was examined against butyrylcholinestrase. The results of enzyme inhibition activity and the DNA binding interaction studies were also collected through molecular docking program using computational analysis. Our study reveals that the newly synthesized compounds possess moderate to good biological activities.

Full text of DOI:10.1016/j.molstruc.2015.11.046

Journal of Molecular Structure 1107 (2016) 99e108
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, biological investigation, calf thymus DNA binding and
docking studies of the sulfonyl hydrazides and their derivatives
,
Shahzad Murtaza a *, Saima Shamim a, Naghmana Kousar a, Muhammad Nawaz Tahir b,
,
**
Muhammad Sirajuddin c, Usman Ali Rana d
a Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
b Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
c Department of Physics, University of Sargodha, Sargodha, Pakistan
d Sustainable Energy Technologies Center, College of Engineering, PO-Box 800, King Saud University, Riyadh 11421, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes the syntheses and biological investigations of sulfonyl hydrazides and their
novel derivatives. The detailed investigations involved the characterization of the newly synthesized
compounds using FTIR, NMR, mass spectrometry and by single crystal X-Ray diffraction (XRD) analysis
techniques. The binding tendencies of these compounds with CT-DNA (calf thymus DNA) have been
explored by electronic absorption (UV) spectroscopy and viscosity measurement. The binding constant
Received 4 September 2015
Received in revised form
17 November 2015
Accepted 19 November 2015
Available online xxx
(K) and Gibb's free energy (DG) values were also calculated accordingly. In addition, we also investigated
the biological activities such as antioxidant, antibacterial, enzyme inhibition and DNA interactions. The
antioxidant activity was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, while
antibacterial activity was investigated against four bacterial strains (viz. Escherichia coli, Crynibacteria
bovius, Staphylococcus auras and Bacillus antherasis) by employing the common disc diffusion method.
Enzyme inhibition activity of the synthesized compounds was examined against butyrylcholinestrase.
The results of enzyme inhibition activity and the DNA binding interaction studies were also collected
through molecular docking program using computational analysis. Our study reveals that the newly
synthesized compounds possess moderate to good biological activities.
Keywords:
Sulfonyl hydrazide
Crystal structure
Enzyme inhibition
DNA interaction
Molecular docking
DPPH radical scavenging activity
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
done since long and the successful approach is the chemical com-
bination of biologically active moieties to develop new derivatives
The advancement of research reveals many hidden areas that
have the potential to address several known problems of modern
era. In medical science, many life threats have been efficiently
removed, a matter of great satisfaction for our future generation.
The strategy to control diseases with synthetic drugs is a remark-
able achievement of the combined efforts of the chemists, bi-
ologists and medical specialists. The major challenges in medical
science are the ability of the pathogens to develop resistance
against the medicine, the lack of antibiotic to kill bacteria and the
emergence of the new pathogens. It is imperative to develop new
materials to solve the health problems [1]. Intensive work has been
with subsequent screening of the biological activities.
In this research work, sulfonyl hydrazides have been explored to
synthesize new Schiff bases, as it consists of two chemically as well
as biologically important moieties viz. sulfonyl group and the hy-
drazine. These compounds also have similarity to some other
important chemical classes; a sulfonamide (O2SeN) and azome-
thine (C]N).
Sulfonyl hydrazides display diverse pharmacological activities
such as anti-inflammatory [2], antidiabetic [3], anticancer [4],
antitumour and analgesic activities [5]. Sulfonyl hydrazide and
sulfonyl semicarbazide derivatives exhibit wide range of bacterio-
static activity [6]. Isoniazid (isonicotinohydrazide) is anantitu-
berculosis drug [7], while Iproniazid (N'-isopropyliso-
nicotinohydrazide) possesses antidepressant activity [8]. Phe-
nelzine ((2-phenylethyl) hydrazine) is a good chemical to be used
as muscle relaxant. Similarly, Hydralazine is used to treat
* Corresponding author.
** Corresponding author.
E-mail addresses: shahzad.murtaza@uog.edu.pk (S. Murtaza), urana@ksu.edu.sa
(U.A. Rana).
http://dx.doi.org/10.1016/j.molstruc.2015.11.046
0022-2860/© 2015 Elsevier B.V. All rights reserved.

100
S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
hypertension [9]. Sulfonyl hydrazide derivatives act as DNA modi-
fying agents and have antitumor activities against murine tumors,
including the B16 melanoma, M109 lung carcinoma, L1210 leuke-
mias, P388 and M5076 reticulum cell sarcoma [4]. Derivatives of
sulfonyl hyrazines also act as cancer chemotherapeutic agents such
as 1,2-bis(methylsulfonyl)-1-2(methylamino) carbonyl-hydrazine
(Cloretazine), which exhibits wide spectrum anticancer activity.
Cloretazine was found to inhibit enzymes containing thiols, such as
glutathione reductase [10]. Sulfonyl hydrazone derivatives of
safrole have potent analgesic action [5].
of drops and maintained at temperature < 5 ꢀC during addition.
This mixture was stirred for half an hour after the complete addi-
tion of hydrazine hydrate and was later transferred to a separating
funnel to obtain two separate layers. The upper layer containing
organic solvent (tetrahydrofuran) and the product (sulfonyl hy-
drazide) was separated and stirred vigorously with cold distilled
water, which precipitated out fluffy white crystalline needles of
sulfonyl hydrazide (1-3) that were later washed with distilled water
and dried at room temperature.
In view of wide range biological applications of hydrazine de-
rivatives, the present study focused on the synthesis of Schiff bases
of sulfonyl hydrazides in addition to evaluating their biological
activities such as antibacterial, antioxidant, enzyme inhibition and
DNA binding studies.
2.4. General procedure for the synthesis of Schiff bases of Sulfonyl
hydrazide
Schiff bases of sulfonyl hydrazides were prepared following the
Scheme 2. Stoichiometric amounts of ethyl acetoacetate and sul-
fonyl hydrazides (1-3) prepared earlier (Scheme 2) were dissolved
in ethanol (10 mL). The concoction of reactants was refluxed for 4 h
at 78 ꢀC, followed by the removal of solvent under reduced pres-
sure. This process yield white solid crystalline product that was
washed with distilled water and recrystallized from ethanol to get
colourless crystals of Schiff bases of sulfonyl hydrazide (4-6) [12].
The spectral data for the compounds (4-6) is given below:
2. Materials and methods
2.1. Reagents
Analytical grade Hydrazine hydrate, benzenesulfonyl chloride,
4-bromobenzenesulfonyl chloride, 4-toluensulfonyl chloride and
ethylacetoacetate were purchased from Aldrich (USA) and were
used as received. Sodium salt of calf thymus DNA (Arcos) was used
without any further process. Various solvents used in the present
study (E. Merck, Germany) were employed after drying following
the procedures described in literature [11].
2.4.1. Ethyl (3E)-3-[(phenylsulfonyl)hydrazono]butanoate (4)
Yield: 82%, Melting point: 108 ꢀC 1 ꢀC, IR (KBr): 3341 (NH, st),
3070 (CH, st), 2994 (CH, st), 1632 (eC]Oe, st), 1579 (eCH]Ne, st),
1433 (-SO2, symm),1360 (eSO2, asymm),1167 (eSO2, asymm),1033
(In plan CH, bend), 773 (out of plan CH, bend) cmꢁ1
;
1H NMR
(300 MHz, CDCl3, ppm):
d
¼ 1.19 (3H, t, J ¼ 7.0 Hz, CH3), 1.86 (3H, s,
2.2. Physical measurements
CH3), 3.25 (2H, s, CH2), 4.14 (2H, q, J ¼ 7.0 Hz, CH2), 7.3 (1H, m, CH),
7.54 (2H, m, 2CH), 7.93 (2H, m, 2CH), 13C NMR (300 MHz, CDCl3,
ppm): 13.7, 14.2, 49.2, 61.1, 127.3, 129.1, 132.0, 139.7, 153.9, 168.4, MS
(ESþ): m/z (%) ¼ (Mþ) 284 (15), 239 (37), 173 (42), 141 (90), 115 (15),
77 (100).
Gallenkamp electrothermal melting point apparatus (U.K) was
used to determine the melting points of the synthesized com-
pounds using capillary tube and were uncorrected. Bruker-
300 MHz FT-NMR Spectrometer was used to record the NMR (1H
and 13C) and CDCl3 [1H ¼ 7.25 and 13C ¼ 77] was used as an internal
reference. Shimadzu 1800 UVeVisible Spectrophotometer was used
to collect the absorption spectra of all newly synthesized com-
pounds. Bruker KAPPA APEX CCD diffractometer was used to collect
the single crystal data, from which the structures of compounds
were resolved using the SHELXL program. All structural data was
collected at room temperature. A sealed ceramic diffraction tube
(SIEMENS) was used to generate the graphite monochromated Mo-
2.4.2. Ethyl (3E)-3-{[(4-methylphenyl)sulfonyl]hydrazono}
butanoate (5)
Yield: 85%, Melting point: 105 ꢀC 1 ꢀC, IR (KBr): 3342 (NH, st),
3010 (Aromatic CH, st), 2971 (CH, st), 1630 (eC]Oe, st), 1576
(eCH]Ne, st), 1431 (eSO2, symm), 1365 (eSO2, asymm), 1171
(eSO2, asymm), 1039 (In plan CH, bend), 763 (out of plan CH, bend)
cmꢁ1
;
1H NMR (300 MHz, CDCl3, ppm):
d
¼ 1.18 (3H, t, J ¼ 7.0 Hz,
CH3), 1.84 (3H, s, CH3), 2.39 (3H, s, CH3), 3.22 (2H, s, CH2), 4.08 (2H,
q, J ¼ 7.0 Hz, CH2), 7.26e7.80 (4H, m, 4CH), 13C NMR (300 MHz,
CDCl3, ppm): 13.4, 13.9, 24.3, 49.7, 61.3, 127.2, 129.4, 136.7, 141.6,
153.2, 167.9, MS (ESþ): m/z (%) ¼ (Mþ) 298 (15), 253 (30), 187 (35),
155 (60), 115 (15), 91 (100), 77 (62), 65 (35).
Ka radiation for intensity measurements. Generator was set to
generate 50 kV/35 mA of current. Direct methods were employed to
solve the crystal structures. Ultimate tuning on F2 was done by
using SHELXL-97 full-matrix least squares techniques. The packing
of structures were analyzed by the program PLUTO and PLATON.
2.4.3. Ethyl (3E)-3-{[(4-bromophenyl)sulfonyl]hydrazonobutanoate
(6)
2.3. General procedure for the synthesis of Sulfonyl hydrazide
Yield: 81%, Melting point: 116 ꢀC 1 ꢀC, IR (KBr): 3341 (NH, st),
3011 (Aromatic CH, st), 2968 (CH, st), 1631 (eC]Oe, st), 1570
(eCH]Ne, st), 1431 (eSO2, symm), 1364 (eSO2, asymm), 1169
(eSO2, asymm), 1029 (In plan CH, bend), 763 (out of plan CH, bend),
Sulfonyl hydrazides were prepared following the Scheme 1.
Weighed amount of aromatic sulfonyl chloride was first dissolved
in tetrahydrofuran (5 mL) in a round bottom flask and cooled to
below 5 ꢀC. Excess amount of Hydrazine hydrate (4:1) was
appended to the solution of aromatic sulfonyl chloride in the form
517 (CeBr) cmꢁ1; 1H NMR (300 MHz, CDCl3, ppm):
d
¼ 1.20 (3H, t,
J ¼ 7.0 Hz, CH3), 1.85 (3H, s, CH3), 3.24 (2H, s, CH2), 4.12 (2H, q,
J ¼ 7.0 Hz, CH2), 7.64e7.79 (4H, m, 4CH), 13C NMR (300 MHz, CDCl3,
ppm): 13.6, 14.1, 49.4, 61.0, 126.3, 129.5, 132.0, 138.7, 153.6, 168.1;
MS (ESþ): m/z (%) ¼ (Mþ) 363 (15), 318 (40), 290 (20), 252 (40), 220
(96), 156 (100), 77 (70).
O
O
O
S
S
O
Cl
NH NH2
H2N NH2
+
R
2.5. Antioxidant activity
R
R= H (1), R= CH3 (2), R= Br (3)
Free radical scavenging activities of the newly synthesized
Scheme 1.
compounds (1-6) were determined by using 2,2-diphenyl-1-

S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
101
O
S
H3C
O
O
O
CH3
O
O
S
NH NH2
NH N
O
+
CH3
O
O
H3C
R
R
R= H (4), R= CH3 (5), R= Br (6)
Scheme 2.
picrylhydrazyl (DPPH) scavenging method. In brief, approx. 100
mL
2.8. Molecular docking studies for enzyme inhibition activity
solution of the test compound was added to 2 mL of 0.2 mM
ethanolic solution of DPPH. After incubation (T ¼ 37 ꢀC for 20 min),
The relations of synthesized compounds with butyr-
ylcholinesterase were marked with the help of molecular docking
studies. The said protein was imported from Protein Data Bank
(PDB ID 1p0p). 3D molecular structures of all prepared compounds
were generated through ChemUltra3D 8.0. Synthesized derivatives
ligand and Protein receptor files were supplied through PatchDock
(Beta 1.3 version) molecular docking Algorithm using built-in set-
tings for all parameters. As counted by the program, the lowest
atomic connection energy (ACE) conformation was elected as the
binding mode. The protein structure was used after removing all
solvent molecules and excess ligands. The docked pose was
pictured by using UCSF Chimera 1.9 molecular representation sys-
tem [16].
absorbance of the mixture was recorded at
l
¼ 516 nm. For positive
control, 1 mM ascorbic acid was used. Free radical scavenging (%) of
the samples was calculated by using the following equation;
ꢀ
ꢁ
Ao ꢁ AT
% Scavenging ¼
ꢂ 100
(1)
Ao
In eq. (1), Ao
AT ¼ Absorbance of the sample solution.
¼
Absorbance of the DPPH solution,
2.6. Antibacterial activity
The newly synthesized sulfonyl hydrazides (1-3) and their Schiff
bases (4-6) were screened for their potential against the microbes
with the help of disc diffusion method [13]. Sterilized Nutrient agar
media was dispensed into pre-sterilized Petri plates. The media was
permitted to settle down and the plates were inoculated with 15 mL
suspension of bacterial growth culture one by one under sterilized
environment with the help of a micropipette. The plates were
2.9. Studies of DNA interaction by UVevisible spectroscopy
Single Stranded-DNA (50 mg) was dissolved in deionized water
by stirring it overnight (pH ¼ 7.0) and preserved at 4 ꢀC. The buffer
solution was prepared using deionized water (20 mM Phosphate
buffer (NaH2PO4eNa2HPO4), pH ¼ 7.2). To confirm that the DNA is
significantly free of proteins, a solution of ss-DNA in the buffer gave
1.8 absorbance value (A260/A280), when examined at 260 and 280
wavelengths [17]. The DNA concentration was determined
following BeereLambert law, according to which, the molar ab-
sorption coefficient of 6600 Mꢁ1 cmꢁ1 (260 nm) measured for ss-
DNA gave rise to the DNA concentration to be 1.4 ꢂ 10ꢁ4 M [18].
The compound was dissolved in 70% ethanol at a concentration of
1 mM. The UVeVis absorption measurements were carried out by
keeping the concentration of compounds fixed, while varying the
concentration of the DNA solution. Equal volumes of ss-DNA solu-
tion were added to both; the complex and the reference solutions
to get rid of the absorbance of DNA itself. Compound-DNA solutions
were permitted to incubate for about 10 min at room temperature,
prior to undertake the experiments. Absorption spectra were
recorded using glass cuvettes (1 cm) at room temperature
(25 1 ꢀC).
streaked by a bent glass rod. The stock solutions (10 mg/1.0 mL) of all
compounds (1-6) were prepared accordingly. Filter discs soaked
with drug solutions were set on inoculated plates by a sterilized
forceps against Escherichia coli, Crynibacteria bovius, Staphylococcus
auras and Bacillus antherasis. Triplicate plates of these strains were
then incubated overnight at 37 ꢀC. The results were determined by
the inhibition zone diameter values in mm.
2.7. Enzymatic activity
Butyrycholinesterase from Equine serum was used to estimate
the esterase activity by modified [14] Ellman method [15]. In brief, a
buffered DTNB solution was prepared by mixing 3.6 mL stock so-
lution containing 10 mM DTNB and 20 mM NaHCO3 in 0.1 M so-
dium phosphate buffer at pH 7, and with 96.4 mL of 0.1 M
phosphate buffer at pH 8. The reaction mixture was prepared by
mixing 1.35 mL of DTNB working solution at pH 8, 50
mL) of Butyrycholinesterase (EC3.1.1.8, Sigma) prepared in distilled
water and 50 L of 2 mM sample prepared in methanol. Then the
reaction was initiated by adding 50 L of 4 mM butyrylthiocholine
m
L (0.4 units/
2.10. Viscosity measurement
m
Ubbelodhe viscometer at a room temperature (25
used to measure the viscosity. The flow time was measured with a
1
ꢀC) was
m
chloride (Alorich, B3128-1G, UK). Final volume of the mixture was
made up to 1.5 mL. The absorbance was recorded in glass cuvette
(1 cm) at room temperature. The hydrolysis of butyrylthiocholine
chloride was examined by the formation of yellow coloured 5-thio-
2-nitrobenzoate anion upon reacting DTNB with thiocholines,
catalyzed by butyrylcholinesterase at 412 nm, using a spectro-
photometer (Humas Korea, Think Hs 3300) after 20 min. All ex-
periments were carried out in triplicate. Galantamine
hydrobromide from Lycorissp, an anticholinesterase compound
was used as a reference.
digital stopwatch with three concurrent readings, from which the
average flow time was calculated. The obtained data were pre-
1/3
sented as (
h
/ho)
vs. binding ratio (r) of [Compound]/[DNA],
where
h
is the relative viscosity of DNA in the presence of test
compounds, and ho is the relative viscosity of DNA itself. Viscosity
values were calculated from the observed flow time of DNA-
containing solutions, which was corrected for the flow time (to)
of 20 mM phosphate buffer solution (pH 7.2). The viscosity for DNA
in the presence and absence of the compound was calculated using
the following equations [19,20].

102
S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
+
.
h+ ¼ t ꢁ t+
(2)
O
O
O
O
S
N
CH3
S
N
CH3
H3C
NH
t ꢁ t+
NH
h ¼
(3)
O-
O
CH3
t+
C+
O
O
H3C
H3C
m/z = 298
m/z = 253
2.11. DNA molecular docking studies
All the synthesized compounds were evaluated for their DNA
binding affinity with the help of molecular docking studies; a
powerful computational analysis tool in the field of bio-informatics.
The DNA sequence d(CGCGAATTCGCG)2 of B-form DNA was fetched
from the protein databank using PDB ID of 1bna. 3D models for all
newly synthesized compounds were generated with the help of
ChemUltra 3D 8.0 program. PatchDock molecular docking program
was used to provide the files for the receptor and the ligand mol-
ecules. The program was used with its default settings such as
RMSD 4.0. The docking program came up with more than 100
conformations, out of which lowest atomic energy conformation
was opt for the binding mode [16].
CH3
HC+
O
O
S
NH+3
O
CH3
NH
O
m/z = 115
H3C
m/z = 187
C+
m/z = 77
O
O
S+
CH+
3. Results and discussion
H2C+
H3C
The sulfonyl hydrazide and their derivatives were prepared by
following Schemes 1 and 2 respectively. The compounds (1-3) were
crystallized and were used for the preparation of compounds (4-6)
in the Schiff derivative forms. The synthesized derivatives were
crystallized, dried and subjected to further analysis. The sharp
melting points, FTIR, NMR spectra, MS results and single crystal
analysis confirmed the formation and purity of the newly synthe-
sized compounds.
m/z = 65
m/z = 155
m/z = 91
Scheme 3.
z ¼ 115, correspond to the cleavage from imine (eN]Cꢁ) bond. A
peak at m/z ¼ 187 related to toluenesulfonohydrazinium ion, a
continuation of fragmentation from m/z ¼ 253, which further
fragmented at m/z ¼ 155 (removal of N2H4), at m/z ¼ 91 (removal of
SO2), corresponding to tropylium ion. This fragment upon loosing
CH2 group gave the mass spec peak at m/z ¼ 77 and upon loosing
C2H2 group gave rise to the peak at m/z ¼ 65. Similar patterns were
observed for other compounds (4-6) with a difference of the lack of
CH3 (15 atomic mass units) in compound 4 and with the presence of
Bromine (79Br and 81Br having atomic mass units of 79 and 81
respectively) in 6.
3.1. NMR
The 1HNMR spectra of compounds 4-6 were recorded in anhy-
drous CDCl3 solvent. The detailed interpretation of the spectra is
given in the experimental section. The presence of triplet
(
d
¼ 1.18 ppm) and a quartet ( ¼ 4.12 ppm) for ethyl protons both
d
with coupling constant around J ¼ 7 Hz confirmed the formation of
Schiff derivative forms of the compounds. In 1HNMR spectra of 4ꢁ6,
a sharp singlet in the range of
d
¼ 1.84e1.85 ppm is related to the
methyl protons attached to the carbon involved in eC]N bonding
3.3. XRD analysis
and confirm the products in Shiff bases forms. The 1HNMR peak ca.
d
¼ 2.39 ppm is ascribed to the methyl protons in compound 5,
The molecular structures of compounds 4-5 are shown in Fig. 1.
The details for the molecular structures of 6 were reported earlier
[12]. The compounds 4 and 5 exist as dimers, where the dimer
formation occurred by the hydrogen bonding between carbonyl
group and the sulfonamide moiety. In compound 5, a dimeredimer
interaction was also observed, where the oxygen of ester group
interacts with the hydrogen of aromatic ring (Table 1).
while no such peak was found in the spectra of compounds 4 and 6
due to the absence of methyl group that is present in compound 5.
The integral value for the peaks ca.
d
d
¼ 7.26e7.95 ppm and
¼ 7.64e7.79 ppm (shifted to low field due to the presence of Br)
for 5 and 6 indicated four protons each, while five protons were
observed from the 1HNMR spectrum of compound 4. In the 13CNMR
spectra of compounds 4-6, the peaks ca.
d
¼ 153.9, 153.2 and
153.6 ppm respectively can be ascribed to the azomethine carbon.
The other peaks are portraying in the positions as deliberated by
incremental methods [21].
3.4. Antioxidant activities
The scavenging by DPPH radical is generally carried out to es-
timate antioxidant activities of the compounds in short times. The
compounds having the potential to reduce the DPPH counter this
radical to form 1,1-diphenyl-2-picrylhydrazine [22]. The odd elec-
tron of DPPH is responsible for its deep violet colour, which leads to
3.2. Mass
The mass spectra of 4ꢁ6 exhibited molecular ion peaks viz. m/
z ¼ 284, 298 and 263, respectively. The details of fragmentation is
discussed for compound 5 (Scheme 3), whose molecular ion peak
was observed ca. m/z ¼ 298. The molecular ion initially fragmented
through two pathways i.e. the loss of eOC2H5 group, giving frag-
mented ion peak at m/z ¼ 253 and the other fragment with m/
an intense absorption at
l
¼ 517 nm [23]. Due to pairing off elec-
trons by reacting with antioxidant agent, the absorption disap-
pears. The results obtained during the reaction of the test
compounds with the DPPH are shown in Table 2.

S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
103
Fig. 1. Oak Ridge Thermal Ellipsoid Plot (ORTEP) drawings of compounds 4 and 5 with atomic numbering scheme and molecular packing. The dotted lines show the intermolecular H-
bonding. The figure ‘a’ and ‘b’ represent the compound 4, while the figures c’ and ‘d’ represent the compound 5.
Table 1
Single Crystal XRD Analysis data for the compounds 4 and 5.
Formula
Compound 4
12H16N2O4S
Compound 5
C13H18N2O4S (5)
C
Formula weight
284.13
298.35
a, b, c (Å)
8.1947 (8), 17.3607 (15), 20.0751 (17)
10.9202 (7), 12.0083 (8), 13.0828 (8)
a
,
b
,
g
(deg)
90, 90, 90
2856.0(4)
6
76.478 (3), 73.028 (3), 72.445 (3)
1544.33 (17)
4
V (Å3)
Z
Temperature (K)
Radiation type
296(2)
296(2)
Mo Ka
0.41
Mo K
a
m
(mmꢁ1
)
0.19
No. of measured, independent and observed
13446, 3398, 1918
22950, 6736, 5182
[I > 2
Rint
(sin
R[F2 > 2
s
(I)] reflections
0.048
0.659
0.059, 0.176, 1.04
0.023
0.645
0.051, 0.218, 0.88
q
/
l
)
(Åꢁ1
)
max
s
(F2)], wR(F2), S
No. of reflections
No. of parameters
H-atom treatment
3398
174
6736
380
H atoms treated by a mixture of independent and
constrained refinement
0.45, ꢁ0.43
H atoms treated by a mixture of independent and
constrained refinement
0.67, ꢁ0.33
Drmax
,
Drmin (e Åꢁ3
)
3.5. Antibacterial activity
concentration of 1 mg/mL was performed against four different
bacterial strains Staphylococus aurus (SA), B. antherasis (BA), E. coli
(EC) and C. bovius (CB). Disc diffusion method was executed for
In vitro antibacterial activity of the compounds 1-6 at

104
S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
Table 2
Percentage scavenging of compounds 1-6 at 1
m
g/
mL concentration.
Compounds
Absorbance
Percentage scavenging (%)
1
2
3
4
5
6
0.797
0.379
0.565
0.345
0.302
0.104
0.101
14.39
59.29
39.31
62.94
67.56
88.82
89.15
þve Control
Table 3
Inhibition zone diameter values against four different bacterial strains.
Microorganisms (mm)a
Fig. 2. Graphical representation of percentage inhibition of butyrylcholinesterase by 1-
6.
Compounds
1
2
3
4
5
6
SA
BA
EC
CB
Inactive
24 0.2
14 0.4
Inactive
14 0.5
23 0.4
34 0.4
25 0.5
29 0.3
28 0.2
20 0.4
16 0.5
26 0.3
39 0.3
15 0.4
26 0.3
13 0.4
17 0.4
10 0.4
14 0.3
12 0.5
Inactive
which indicate moderate to strong binding interaction between the
synthesized compounds and the enzyme (Fig. 3). The promising
results (Table 5) obtained for the Schiff base derivatives suggest
that the enzymatic activity is good due to the presence of eC]N-
linkage.
8
0.6
8
0.3
21 0.5
42.5 0.3
18 0.3
31.5 0.2
Cefixime
a
IZD ¼ Inhibition Zone Diameter.
antibacterial studies and each test was conducted in triplicate.
Mean inhibition zone diameter (IZD) was measured from which the
standard deviation was calculated. Cefixime, a commercial antibi-
otic, was used as the reference. The results reveal that the com-
pound 1 was active against BA, compound 2 showed good activities
against SA, BA and EC, compound 3 exhibited good activity against
BA and the compound 6 displayed good activities against all four
strains. However, the compounds 4 and 5 showed no appreciable
activity against the tested bacterial strain (Table 3).
3.8. UVevisible absorption study of complex-DNA interaction
Electronic absorption spectra showed how the compounds
(1¡4) interact with the DNA structures (Fig. 4, Fig. 5, Fig. 6 and
Fig. 7). The compound 1 displayed one strong absorption peak at
216.80 nm with a shoulder peak at 271.9 nm (Fig. 4), while for the
compound 3, a strong absorption peak at 229.30 nm with a
shoulder peak at 277.10 nm was observed (Fig. 6). The compound 1
showed blue shifts ~1 nm at
l
¼ 216.80 nm and 2.9 nm at
l
¼ 271.9 nm. In the case of compound 3, Fig. 6 displayed blue shifts
~1 nm at
l
¼ 229.30 nm and 11.2 nm at
l
¼ 277.10 nm. Fig. 5 shows
3.6. Enzyme assay
that the compound 2 displayed a strong peak at 265.30 nm, with a
shoulder peak at 273 nm. Yet different from compounds 1, 2 and 3,
Fig. 7 displayed three peaks for the compound 4 ca. 272.30 nm,
265.30 nm and 258.50 nm respectively. A common behaviour for all
compounds can be observed from the gradual decrease in the in-
tensities of the absorption peaks with increasing amounts of DNA,
while the wavelengths showed minor to significant blue shift
effects.
This shifting of absorption peaks and the variation in intensities
can help estimate the binding mode between the ligand molecules
and the DNA. The intercalative mode of binding is associated with a
hypochromic effect tied with obvious bathochromism for the
characteristic peaks of the small molecules. This is believed to
happen due to the strong stacking between the chromophore and
the base pairs of DNA [24]. A crescent shape molecule usually
shows minor-groove-binding by supporting' van der Waals in-
teractions. Such molecules may also form hydrogen bonds to bases,
typically to N2 of guanine, N3 of adenine and O2 of thymine. DNA
interaction studies of compounds (1-4) revealed that this interac-
tion could be partial intercalative binding as well as groove binding
The butyrylcholinesterase (BChE, EC 3.1.1.8) is intrinsically pre-
sent in the mammalian tissue sand and is responsible for Alzheimer
disease (AD) by breakdown of butyrylcholine (BCh) neurotrans-
mitters. At 2 mM concentration, all six compounds were examined
for anti-butyrylcholinesterase activity. The obtained results out-
lined in Table 4 and displayed in Fig. 2 revealed that the compound
1 exhibited highest activity ~38.74%, followed by the compound 6,
which showed the activity ~33.64% and compound 5 that showed
31.68% activity. The compounds 2 and 3 displayed 23.26% and
18.76% inhibition activity respectively, while the compound 4
showed lowest activity of all ~15.07% (Table 4).
3.7. Molecular docking studies for enzyme inhibition activity
The obtained results from PatchDock molecular docking pro-
gram showed average to good shape complementarity scores,
Table 4
via H-bonding. After intercalating the base pairs of DNA, the
orbital of the intercalated ligand could couple with orbital of base
pairs, thus decreasing the * transition energy, and further
resulting in the bathochromism. On the other hand, the coupling of
orbital with partially filled electrons decreases the transition
probabilities, hence results hypochromic shift [17]. In case of
compound 4, first the compound probably makes aggregation with
DNA and then undergoes intercalation into base pair of DNA. Based
upon the variation in absorbance, the intrinsic binding constant of
p*
Percentage inhibition of Butyrylcholinesterase SEM by test compounds (1-6).
p
Compounds
Percentage inhibition SEM
pep
1
2
3
4
5
6
23.26 1.94
18.76 1.28
38.74 0.93
31.68 1.63
15.07 0.82
33.64 0.89
70.68 0.41
a
p
GalantamineHydrobromide (GH)

S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
105
Fig. 3. Molecular docked pose for 5 (enzyme inhibition studies).
Table 5
Where K is the association/binding constant, A0 and A are the
absorbance of the compound and its complex with DNA, respec-
tively, and εG and εHꢁG are the absorption coefficients of the com-
pound and the compound-DNA complex, respectively. The
association constants were obtained from the intercept-to-slope
Shape complementarity scores for enzyme inhibition Activity.
Compound
ACE
Shape complementarity score
1
2
3
4
5
6
ꢁ200.16
ꢁ206.08
ꢁ189.79
ꢁ275.73
ꢁ286.10
ꢁ277.70
2310
2660
2524
3854
4146
3602
ratios of A0/(A-A0) vs. 1/[DNA] plots. The Gibb's free energy (
was determined from the equation:
DG)
DG ¼ ꢁRT lnK
ACE ¼ Atomic Connection Energy.
Fig. 4. Absorption spectrum of 1 mM compound 1 in the absence (a) and presence of 4.6 (b), 9.2 (c), 13.8 (d), 18.4 (e), 23.0 (f), 27.6 (g), 32.2 (h), 36.8 (i) and 41.4 (j)
m
M DNA. The
arrow indicates the increasing conc. of DNA. The inset graph shows the plot of Ao/A-Ao vs. 1/[DNA] (
m
M)ꢁ1 for the calculation of binding constant (K) and Gibb's free energy (
D
G).
the compound with DNA were determined according to Benesi-
Where R is general gas constant (8.314 J Kꢁ1 molꢁ1) and T is the
Hildebrand equation:
temperature (298 K).
The binding constants for the compounds (1-4) were found to
be 5.4 ꢂ 104 Mꢁ1, 6.6 ꢂ 103 Mꢁ1, 1.13 ꢂ 104 Mꢁ1 and 2.46 ꢂ 105 Mꢁ1
A0
εG
εG
1
¼
þ
ꢂ
respectively,
while
the
Gibb's
free
energies
A ꢁ A0 εHꢁG ꢁ εG εHꢁG ꢁ εG K½DNAꢃ
were ꢁ21.3 KJ molꢁ1
,
ꢁ21.8 KJ molꢁ1
,
ꢁ21.3 KJ molꢁ1

106
S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
Fig. 5. Absorption spectrum of 1 mM compound 2 in the absence (a) and presence of 4.6 (b), 9.2 (c), 13.8 (d), 18.4 (e), 23.0 (f), 27.6 (g), 32.2 (h), 36.8 (i) and 41.4 (j)
m
M DNA. The
G).
arrow indicates the increasing conc. of DNA. The inset graph shows the plot of Ao/AꢁAo vs. 1/[DNA] (
m
M)ꢁ1 for the calculation of binding constant (K) and Gibb's free energy (
D
Fig. 6. Absorption spectrum of 1 mM compound 3 in the absence (a) and presence of 4.6 (b), 9.2 (c), 13.8 (d), 18.4 (e), 23.0 (f), 27.6 (g), 32.2 (h), 36.8 (i) and 41.4 (j)
m
M DNA. The
G).
arrow indicates the increasing conc. of DNA. The inset graph shows the plot of Ao/AꢁAo vs. 1/[DNA] (
m
M)ꢁ1 for the calculation of binding constant (K) and Gibb's free energy (
D
and ꢁ30.75 KJ molꢁ1 respectively.
intercalation model can best explain the increase in DNA viscosity
due to the separation of base pair to lodge the binding complex. In
non-classical intercalation (partial intercalation), a bend (or kink)
in the DNA helix may reduce its effective length. The effect of
intercalation can be seen by a noticeable increase in the viscosity of
DNA upon adding test compound, which leads to the separation of
3.9. Viscosity measurements
Viscosity measurements were carried out for the further eluci-
dation of binding mode of compounds with DNA. The classical

S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
107
Fig. 7. Absorption spectrum of 1 mM compound 4 in the absence (a) and presence of 4.6 (b), 9.2 (c), 13.8 (d), 18.4 (e), 23.0 (f), 27.6 (g), 32.2 (h), 36.8 (i) and 41.4 (j)
arrow indicates the increasing conc. of DNA. The inset graph shows the plot of Ao/AꢁAo vs. 1/[DNA] (
mM DNA. The
m DG).
M)ꢁ1 for the calculation of binding constant (K) and Gibb's free energy (
measured with addition of the compounds 1¡4 to the DNA solu-
tion, symptomatic of mainly intercalating binding nature of the
compounds.
1.8
1.5
1.2
0.9
0.6
0.3
3.10. DNA molecular docking studies
Table 6 contains the results obtained from the molecular dock-
ing studies. The values of geometrical shape complementarity score
(SCS) were used to infer the best results for all tested compounds. A
large value for SCS indicates the greater stability of the drug/DNA
complex.
As described in the UVeVisible absorption studies above, as a
result of electrostatic interactions, the crescent shaped molecules
likely underwent groove binding with the DNA backbone [17].
Almost all synthesized compounds exhibited this type of binding
with the DNA receptor molecule, though some of the compounds
specially compounds 1, 2, 4 and 6 showed lesser degree of binding
(due to intercalation) too.
The binding of compound 4 with DNA is of special interest, as it
resembles the way cisplatin binds by crosslinking both chains of
DNA double helix through the formation of two hydrogen bonds
(Fig. 9). The compound sits in the minor groove of DNA and forms
two weak hydrogen bonds with the guanidine units of opposite
chains. One hydrogen bond is formed between the sulfonyl oxygen
of the compound and guanidine (16) of one strand of DNA, where
the O atom acts as hydrogen bond donor and N2 of the phosphate
unit acts as hydrogen bond acceptor (O(5)dH … N(2): 3.24 Å). The
other hydrogen bond is formed between the same oxygen with
guanidine (10) of other strand of double helix (O(5)eH … N(2):
3.50 Å).
The results of DNA binding studies as obtained from the UVeVis
absorption and viscosity measurements are yet not truly in-line
with the results of the docking studies. There could be a number
of possible reasons, some of which include manual error and the
use of un-optimized structures for molecular docking. However, the
mode of binding of synthesized compounds is same in both; the
0
5
10
15
20
25
30
r = [Compound]/[DNA]
Fig. 8. Plot of relative viscosity of ss-DNA against binding ratio (r) of the compounds
(1-4): 1 (:), 2 ( ꢂ ), 3 (-), 4 ( ) at 25 0.1 ꢀC. [DNA] ¼ 7.2
M, r ¼ 0, 6.9, 13.9, 20.8,
m
A
27.8.
Table 6
Geometrical shape complementarity scores for compounds 1-6.
Compound
aACE
bSCS
1
2
3
4
5
6
ꢁ236.08
ꢁ242.03
ꢁ220.09
ꢁ350.50
ꢁ334.71
ꢁ322.21
2488
2092
2638
2984
3710
3704
a
ACE ¼ Atomic Contact Energy (cal/mole).
SCS ¼ Shape Complementarity Score.
b
the DNA base pairs [17]. The plot of (
provides a measure of the viscosity changes (Fig. 8) that occurred
due to intercalation. An increase in the relative viscosity was
h
/ho
)
1/3 vs. [Compound]/[DNA]

108
S. Murtaza et al. / Journal of Molecular Structure 1107 (2016) 99e108
Fig. 9. A pictorial view of DNA interaction with compound 4.
experimental and the computational analysis i.e., the groove
binding and the smaller intercalatory binding. The results also
strongly suggest that the Schiff bases bind more strongly to the
DNA as compared to their corresponding hydrazide forms.
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intermolecular interaction. Such characteristics are valuable for
biological applications. The results of the biological activities
including antioxidant, antibacterial and anti-enzymatic studies
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Crystallographic data for the compounds 4-5 has been deposited
with the Cambridge Crystallographic Data Centre, CCDC 1005157
for 4 and 1005158 for 5. The information may be obtained from The
Director, CCDC, 12, Union Road, Cambridge CB2 1EZ [Fax: þ44 1223
336 033] or deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk.