Synthesis of isatin thiosemicarbazones derivatives: In vitro anti-cancer, DNA binding and cleavage activities

Article abstract of DOI:10.1016/j.saa.2014.01.086

New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The intera

Full text of DOI:10.1016/j.saa.2014.01.086

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis of isatin thiosemicarbazones derivatives: In vitro anti-cancer,
DNA binding and cleavage activities
a
b
Amna Qasem Ali a, Siang Guan Teoh a, , Abdussalam Salhin , Naser Eltaher Eltayeb ,
⇑
Mohamed B. Khadeer Ahamed c, A.M.S. Abdul Majid c
a School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia
b Department of Chemistry, Sciences & Arts College – Rabigh, King AbdullAziz University, Rabigh, Saudi Arabia
c EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
New derivatives of
thiosemicarbazone Schiff base with
isatin moiety were synthesized.
The interaction of these compounds
with calf thymus CT-DNA exhibited
high intrinsic binding constant.
The electrophoresis studies show the
higher cleavage activity of L1–L3.
Compounds (L3, L6 and L2) exhibited
good anticancer activity.
ꢀ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1–L6. The struc-
tures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was
further characterized by XRD single crystal. The interaction of these compounds with calf thymus
(CT-DNA) exhibited high intrinsic binding constant (kb = 5.03–33.00 ꢁ 105 Mꢂ1) for L1–L3 and L5 and
(6.14–9.47 ꢁ 104 Mꢂ1) for L4 and L6 which reflect intercalative activity of these compounds toward
CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the
higher cleavage activity of L1–L3 than L4–L6. The in vitro anti-proliferative activity of these compounds
against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2)
exhibited good anticancer potency.
Received 5 November 2013
Received in revised form 16 January 2014
Accepted 20 January 2014
Available online 29 January 2014
Keywords:
Thiosemicarbazone
Isatin moiety
CT-DNA binding
DNA cleavage
Ó 2014 Elsevier B.V. All rights reserved.
In vitro anti-proliferative activity
Introduction
metabolites and in the fruits of the cannon ball tree [3–5]. The syn-
thetic ingenuity of isatin has led to the wide use of this compound in
Isatin (1H-indole-2,3-dione) was first discovered by Erdmann
and Laurent in 1840 as a product of indigo oxidation [1,2]. Isatin
have also been recognized in natural products, such as in humans
as it is a metabolic derivative of adrenaline, marine molluscs, fungal
organic synthesis which reported to possess a variety of pharmaco-
logical properties such as anti-viral activities, antibacterial, antican-
cer activities, anticonvulsant activity, cytotoxicity antioxidant
activity [6–17]. Hence our studies have been focused towards the
synthesis of a series of isatin thiosemicarbazone derivatives with
empirical formula, C15H12N4OS (L1), C16H14N4OS (L2), C15H11FN4OS
(L3), C10H9N5O3S (L4), C11H12N4OS (L5) and C12H14N4OS (L6).
⇑
Corresponding author. Tel.: +60 4 6577888x3565; fax: +60 4 6574854.
E-mail address: sgteoh@usm.my (S.G. Teoh).
http://dx.doi.org/10.1016/j.saa.2014.01.086
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
441
There structures were proved by IR, 1H, 13C NMR and mass
analytical results (experimental):
C
(60.50%);
t
H
(4.19%);
N
spectroscopy. Single crystal X-ray structure of the Schiff base
[C12H14N4OS] (L6), was determined in order to confirm its structure.
Many techniques have been used to investigate the DNA binding
abilities of these compounds. These include: absorption, fluores-
cence spectrophotometry and viscosity measurements .The nucle-
ase activity with supercoiled pBR322 DNA have been also
examined. The in vitro cytotoxic activities of compounds were
tested against human colon cancer cell line (HCT 116).
(18.23%); selected IR data (KBr pellet,
max/cmꢂ1): 3300–3190
(NH), 1694 (C@O), 1594 (C@N); 1H NMR (500 MHz, DMSO-d6)
(d (ppm)): 12.79 (s, 1 H, thiosemicarbazide N-NH),11.26 (s, 1
H, indole NAH), 10.98 (s, 1 H, CSANH), 7.78 (d, 1 H, indole
C2AH, J = 7.5 Hz), 7.62 (d,
2 H, thiosemicarbazide C11AH,
C15AH, J = 7.7 Hz), 7.44–7.41 (t, 2 H, thiosemicarbazide C12AH,
C14AH, J = 7.8), 7.39–7.36 (dt, 1 H, indole C3AH, J = 7.7, 1.0 Hz),
7.29–7.26 (t, 1 H, thiosemicarbazide C13AH, J = 7.4), 7.13–7.10
(t,1 H, indole C4AH, J = 7.5 Hz), 6.95 (d, 1 H, indole C5AH, J = 7.8);
13C NMR (500 MHz, DMSO-d6 (d (ppm)): 176.33, 162.68, 142.47,
138.43, 132.26, 131.41, 128.36, 126.07, 125.65, 124.65, 122.34,
121.37, 119.89, 111.07 and m/z: 295 (Mꢂ).
Experimental
General and instrumental
(Z)-2-(5-methyl-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbo-
thioamide (L2)
The materials used in this study such as isatin, 5-fluoroisatin,
5-methylisatin, 5-nitroisatin, 4-ethyl-3-thiosemicarbazide, 4-
methyl-3-thiosemicarbazide and 4-phenyl-3-thiosemicarbazide
were purchased from Aldrich Chemicals. Commercial grade sol-
vents and reagents were used as supplied without further purifica-
tion. Supercoiled (SC) pBR322 DNA and loading dye were
purchased from Fermentas. Calf thymus (CT-DNA), agarose
(molecular biology grade), and ethidium bromide (EB) were
from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,
5diphenyl tetrazolium bromide (MTT) was purchased from
Sigma–Aldrich, Germany. The elemental analysis was carried out
using Perkin–Elmer 2400 series-11 CHN/O analyzer (Waltham,
MA, USA). The infrared, electronic, and fluorescent spectral were
recorded on Perkin–Elmer 2000, Perkin Elmer-lambda 25, and
Jasco FP-750 spectrophotometers, respectively. Viscosity measure-
ments were made using a cannon manning semi-micro viscometer
(State College, PA, USA). 1H and 13C NMR spectra were recorded on
Bruker 500 MHz spectrometer at room temperature using DMSO-
d6. Mass spectra were obtained using liquid chromatography/mass
spectrometry LC/MSD trap VL (Agilent Technologies).
Orange crystals; MP: 238.6–239.1 °C; yield: 80%; analytical
calculated values for
(18.05%); analytical results (experimental): C (61.89%); H (4.65%);
N (18.01%); selected IR data (KBr pellet,
max/cmꢂ1): 3308–3169
(NH), 1691 (C@O), 1593 (C@N); 1H NMR (500 MHz, DMSO-d6)
(d (ppm)): 12.77 (s, H, thiosemicarbazide NANH), 11.15
C16H14N4OS: C (61.92%); H (4.55%); N
t
1
(s, 1 H, indole NAH),10.81 (s, 1 H, CSANH), 7.61 (d, 3 H, indole
C5AH, thiosemicarbazide C11AH, C15AH, J = 7.7 Hz), 7.44–7.41
(t, 2 H, thiosemicarbazide C12AH, C14AH, J = 7.8 Hz), 7.29–7.26
(t, 1 H, thiosemicarbazide C13AH, J = 7.4 Hz), 7.19 (d, 1 H, indole
C3AH, J = 8 Hz), 6.84 (d, 1 H, indole C2AH, J = 7.9), 2.3 (s, 3 H,
CH3); 13C NMR (500 MHz, DMSO-d6 (d (ppm)): 176.30, 162.75,
140.25, 138.43, 132.37, 131.82, 131.36, 128.35, 126.06, 125.64,
121.74, 119.92, 110.82, 20.59 and m/z: 309 (Mꢂ).
(Z)-2-(5-fluoro-2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbo-
thioamide (L3)
Orange crystals; MP: 244.2–244.8 °C; yield: 77%; analytical cal-
culated values for C15H11FN4OS: C (57.31%); H (3.52%); N (17.82%);
analytical results (experimental):
(17.76%); selected IR data (KBr pellet,
C
(57.42%);
t
H (3.31%); N
Synthesis of ligands
max/cmꢂ1): 3313–3183
(NH), 1691 (C@O), 1594 (C@N); 1H NMR (500 MHz, DMSO-d6) (d
(ppm)): 12.67 (s, 1 H, thiosemicarbazide NANH), 11.26 (s, 1 H,
indole NAH), 10.85 (s, 1 H, CSANH), 7.65–7.63 (dd, 1 H, indole
C3AH, J = 8.1, 2.6 Hz), 7.61 (d, 2 H, thiosemicarbazide C11AH,
C15AH, J = 7.9 Hz), 7.45–7.42 (t, 2 H, thiosemicarbazide C12AH,
C14AH, J = 7.8 Hz), 7.30–7.27 (t, 1 H, thiosemicarbazide C13AH,
General procedure: The thiosemicarbazone derivatives (L1–L6)
were prepared by refluxing equimolar quantities of unsubstituted,
5 and 5-substituted isatin with thiosemicarbazide derivatives in
ethanol for 2 h (Scheme 1). The resulting precipitates were filtered
off, washed with cold ethanol. The structures of the synthesized
compounds were proved by mass-spectrometry, FTIR and NMR-
spectroscopy. The elemental analyses data were within 0.4% of
the theoretical values. The structures were further confirmed
through X-ray diffraction studies.
J = 7.4 Hz), 7.23–7.19 (dt,
1 H, indole C5AH, J = 7.7, 2.5 Hz),
6.95–6.93 (dd,
1
H, indole C2AH, J = 8.6, 4.1 Hz); 13C NMR
(500 MHz, DMSO-d6 (d (ppm)): 176.79, 163.26, 159.69, 132.12,
128.92, 126.70, 126.12, 121.88, 121.81, 118.21, 118.01, 112.88,
112.61, 108.88, 108.68 and m/z: 313 (Mꢂ).
These Schiff bases were insoluble in common organic solvents,
but soluble in DMF and DMSO.
(Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide
(L1)
(Z)-N-methyl-2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinecarbo-
thioamide (L4)
Yellow crystals; MP: 237.2–238.6 °C; yield: 90%; analytical cal-
culated values for C15H12N4OS: C (60.79%); H (4.08%); N (18.91%);
Orange crystals; MP: 306.6–307.1 °C; yield: 80%; analytical cal-
culated values for C10H9N5O3S: C (43.01%); H (3.25%); N (25.08%);
Scheme 1. Synthetic route and structures for compounds L1–L6.

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A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
analytical results (experimental):
(24.92%); selected IR data (KBr pellet,
C
(43.12%);
t
H
(3.17%);
N
thiosemicarbazide CH3, J = 7); 13C NMR (500 MHz, DMSO-d6 (d
(ppm)): 176.70, 162.66, 139.99, 131.70, 131.52, 131.29, 121.14,
119.98, 110.78, 40.11, 20.56, 14.00 and m/z: 263 (M+).
max/cmꢂ1): 3342 (NH),
1702 (C@O), 1551 (C@N); 1H NMR (500 MHz, DMSO-d6) (d (ppm)):
12.32 (s, 1 H, thiosemicarbazide NANH), 11.80 (s, 1 H, indole
NAH), 9.53–9.50 (q, 1 H, CSANH, J = 8.5, 4.6 Hz), 8.50 (d, 1 H, indole
C5AH, J = 2.3 Hz), 8.25–8.23 (dd, 1 H, indole C3AH, J = 8.6, 2.5 Hz),
7.10 (d, 1 H, indole C2AH, J = 8.6 Hz), 3.40 (d, 3H, thiosemicarba-
zide CH3, J = 4.6 Hz); 13C NMR (500 MHz, DMSO-d6 (d (ppm)):
177.54, 162.86, 147.19, 142.70, 129.47, 126.72, 120.92, 115.97,
111.15, 31.33 and m/z: 278 (Mꢂ).
X-ray crystallography
The data of the ligand L6 was collected on a Bruker SMART APEX
II CCD diffractometer [18] equipped with a graphite monochroma-
tized Mo Ka radiation (k = 0.71073) at 100(1) K. Multi-scan absorp-
tion corrections were applied using the SADABS program [18]. The
structure was solved by the direct method using the SHELXS-97
program [19]. Refinements on F2 were performed using SHELXL-
97 by the full-matrix least-squares method with anisotropic
thermal parameters for all non-hydrogen atoms. N-bound H atoms
were located in a difference Fourier map and were refined freely.
The remaining H atoms were positioned geometrically and refined
using a riding model. The program SHELXTL [19] was used in the
preparation of Figs. 1 and 2. Table 1 lists crystallographic details.
(Z)-N-methyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbo-
thioamide (L5)
Yellow crystals; MP: 278.7–279.2 °C; yield: 94%; analytical
calculated values for C11H12N4OS:
(22.56%); analytical results (experimental): C 53.45; (H 4.21%); N
(22.51%); selected IR data (KBr pellet,
max/cmꢂ1): 3284–3248
C (53.21%); H (4.87%); N
t
(NH), 1693 (C@O), 1561 (C@N); 1H NMR (500 MHz, DMSO-d6) (d
(ppm)): 12.56 (s, 1 H, thiosemicarbazide NANH), 11.09 (s, 1 H, in-
dole NAH), 9.23–9.21 (q, 1 H, CSANH, J = 9.0, 4.6 Hz), 7.50 (s, 1 H,
indole C5AH), 7.16–7.14 (dd, 1 H, indole C3AH, J = 7.9, 0.9 Hz), 6.82
(d, 1 H, indole C2AH, J = 7.9 Hz), 3.09 (d, 3 H, thiosemicarbazide
CH3, J = 4.6 Hz), 2.30 (s, 3 H, indole CH3); 13C NMR (500 MHz,
DMSO-d6 (d (ppm)): 177.68, 162.65, 136.97, 131.64, 131.50,
131.31, 121.02, 119.98, 110.78, 31.27, 20.57 and m/z: 247 (Mꢂ).
DNA binding studies
DNA binding experiments which include absorption, emission
spectral studies and viscosity measurements, were performed in
accordance with the standard methods [20,21]. An equal amount
of CT-DNA was added to the Schiff base cuvette and the reference
cuvette to eliminate the absorbance of the CT-DNA itself [22–24],
and Tris buffer was subtracted through baseline correction. Viscos-
ity experiments were conducted using a cannon-manning semi-
micro viscometer (State College, PA, USA), which was thermostated
in a water bath maintained at 37.0 0.1 °C. The flow rates of the
(Z)-N-ethyl-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazinecarbo-
thioamide (L6)
Yellow crystals; MP: 260.0–261.8 °C; yield: 80%; analytical cal-
culated values for C12H14N4OS: C (54.94%); H (5.38); N (21.36%);
analytical results (experimental): C 54.83; H (5.41%); N (21.46%);
Tris–HCl buffer (pH 7.2), CT-DNA (200 lM), and DNA in the pres-
selected IR data (KBr pellet,
t
max/cmꢂ1): 3343–3190 (NH), 1691
ence of Schiff base at various concentrations (0–2.5 ꢁ 10ꢂ4 M)
(C@O), 1545 (C@N); 1H NMR (500 MHz, DMSO-d6) (d (ppm)):
12.53 (s, 1H, thiosemicarbazide NANH), 11.09 (s, 1H,
indole NAH), 9.28–9.25 (t, 1H, CSANH, J = 5.8 Hz), 7.50 (s, 1 H,
indole C5AH), 7.17 (d, 1 H, indole C2AH, J = 7.9 Hz), 6.83 (d, 1 H,
indole C3AH, J = 7.9 Hz), 3.68–3.62 to (p, 2 H, thiosemicarbazide
CH2, J = 6.8 Hz), 2.31 (s, 3H, indole CH3), 1.21–1.19 (t, 3H,
were measured three times each with a digital stopwatch; from
these, the average flow time was calculated. The resulting data
1/3
are presented as (
g
/go)
vs [Schiff base]/[DNA], where g is the
viscosity of DNA in the presence of the Schiff base, and go refers
to the viscosity of DNA alone. The viscosity values of DNA-
containing solutions were calculated from the observed flow time
Fig. 1. The structure of L6 ligand, showing 50% probability displacement ellipsoids and atomic numbering.

A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
443
the plate. The cells were then washed (twice to thrice) using sterile
phosphate buffered saline (PBS) (pH 7.4). The PBS was discarded
after washing. Trypsin was then added and distributed evenly onto
the cell surfaces. Next, the cells were incubated at 37 °C in 5% CO2
for 1 min. Following this step, the flasks that contained the cells
were gently tapped to aid cell segregation. These flasks were
observed under inverted microscope; if cell segregation was not
satisfactory, the cells were incubated for another minute. Trypsin
activity was inhibited by adding 5 ml of 10% fresh complete media.
The cells were counted and diluted to obtain a final concentration
of 2.5 ꢁ 105 cells/ml, and then inoculated into wells (100
ll cells/
well). Finally, the plates containing the cells were incubated at
37 °C with an internal atmosphere of 5% CO2.
MTT assay
Initially, the cancer cells (100
were inoculated in a 96-well microtitre plate, which was incubated
l
l cells/well, 1.5 ꢁ 105 cells/ml)
overnight under CO2 to allow the attachment of the cells. Next,
100 ll of the test substance was added into each well, and the
compounds were diluted with a media into the required concen-
trations from the stock. The plates were then incubated at 37 °C
with an internal atmosphere of 5% CO2 incubator for 72 h. After
Fig. 2. The crystal packing of L6, viewed approximately along the a axis.
this treatment period, the plates were treated with 20
reagent and then incubated for another 4 h. After this incubation
period, 50 l of MTT lysis solution (DMSO) was added into the
ll of MTT
Table 1
l
Crystallographic data for ligand L6.
wells, and the plates were further incubated for 5 min in the CO2
incubator. Finally, the absorbance rates at 570 and 620 nm wave-
lengths were measured using a standard ELISA microplate reader
(Ascent Multiskan). The data were recorded and analyzed to esti-
mate the effects of the test compounds on cell viability and growth.
The percentage of inhibition of cell proliferation was calculated
from the optical density, which was obtained from the MTT assay.
In the current work, 5-fluorouracil was used as the standard
reference drug [25]. Statistical differences between the treatments
and the control were evaluated using ANOVA, followed by Tukey’s
multiple comparison tests. The differences were considered
significant at p < 0.05 and p < 0.01.
Formula
Fw
T (K)
Cryst syst
Space group
a (Å)
C12H14N4OS
262.34
100 (1)
Orthorhombic
P212121
4.6215(1)
12.5592(3)
21.9352(5)
1273.17(5)
4
b (Å)
c (Å)
V (nm3)
Z
Dc (Mg mꢂ3
)
1.369
552
F (000)
Cryst dimens (mm)
h range (°)
0.07 ꢁ 0.11 ꢁ 0.51
1.9–30.0
hkl ranges
ꢂ6 < h < 6
ꢂ14 < k < 17
ꢂ30 < l < 28
3710/177
1.05
Results and discussion
Data/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
Spectral characterization
R1 = 0.0413
wR2 = 0.0900
IR studies
Highest peak/deepest hole
D
q
max = 0.28 e Åꢂ3
/
D
q
min = ꢂ0.25 e Åꢂ3
IR spectra of thiosemicarbazone derivatives (L1–L6) showed
absorption bands in the 3437–3169 and 1702–1691 cmꢂ1 regions
resulting from the NH and C@O functions, respectively [26,27].
The various absorption bands in the region 1669–1594, 1594–
1535 cmꢂ1 may be assigned due to (C@N), (C@C) respectively
[28]. A characteristic strong bands at 1212–1202 cmꢂ1 are assigned
(t) corrected from that of the buffer alone (Tris–HCl/NaCl,
5:50 mM) (to),
= (tꢂto).
g
DNA cleavage studies
to m(C@S) [29].
The cleavage experiments of supercoiled pBR322 DNA (0.5 lg/
l
l) by ligands (L1–L6 mM) in Tris–HCl/NaCl (5:50 mM) buffer at
NMR studies
The 1H NMR spectra of the compound L6 (atom numbering used
pH 7.2 was carried out using agarose gel electrophoresis. The sam-
ples were incubated for 2 h. at 37 °C. A loading dye was added and
electrophoresis was carried out at 50 V for 1 h in Tris–HCl buffer
using 1% agarose gel .The resulting bands were stained with
ethidium bromide before being photographed under UV light.
for description of these spectroscopic data is given in Scheme 1)
exhibited two separate singlets at d 12.53, 11.09 attributed N-NH
of thiosemicarbazide and indole NAH moiety, respectively, and
one separate triplet at d 9.26 attributed to CSANH proton of the
thiosemicarbazide moiety. The indole C2AH and C3AH appeared
as a doublet at d 7.17–6.83, while the indole C5AH appeared at d
7.50 as a singlet. The methyl group of indole appeared as a singlet
at d 2.31 whereas the methyl group of thiosemicarbazide moiety
appeared as a triplet at d 1.20. Finally The CH2 group of thiosemi-
carbazide appeared as a quartet at the range d 3.68–3.62. 13C NMR
spectra of this compound revealed the thiocarbamide carbon peak
In vitro anti-proliferative activity
Preparation of cell culture
First, HCT 116 cells were allowed to grow under optimal
incubator conditions. Cells that reached a confluence of 70–80%
were chosen for cell plating. Old medium was aspirated out of

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A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
at 176.70 whereas, the carbonyl and imine carbons observed at
162.66, 139.99, respectively.
MS studies
The mass spectra of the Schiff bases (L1–L5), which were
recorded in negative mode, exhibit peaks at m/z: 295, 309, 313,
278 and 247, respectively, thus indicating the presence of
[M–H]ꢂ Schiff bases. By contrast, the mass spectrum of L6 which
was recorded in positive mode, exhibited peak at m/z: 263, thereby
indicating the presence of [M + H]+ Schiff base.
Crystal structure description
The thiosemicarbazone derivatives (L1–L5) have been character-
ized structurally by X-ray crystallography and have been previously
reported [27–31]. The single crystal X-ray structure of L6 [C12H14-
N4OS] is shown in Fig. 1. Crystallographic data and refinement
statistics are given in Table 1. In this compound (Fig. 1), the chain
N2/N3//C9/S1/N4/C10/C11 connected to the nine-membered
5-methylindolin-2-one ring system in C7. In this chain, C7AN2A
N3AC9 and C9AN4AC10AC11 have torsion angle ꢂ179.64(15)
and ꢂ144.62(18)°, respectively. The bonds length N2@C7 and
N3AC9 are 1.298 Å and 1.382 Å indicated double and single bond,
respectively. The bond length of C9AS1 = 1.669 Å which is compa-
rable with the similar compounds [30–34]. In the crystal, molecules
are linked via an in N1AH1N1ꢃ ꢃ ꢃO1 hydrogen bond into an infinite
one-dimensional chain along the a axis (Fig. 2).
Fig. 3. Absorption spectra of L1 in the absence and in the presence of increasing
amounts of DNA in Tris HCl buffer (pH-7.2). [L1] = 50
lM, [DNA] = 0–163.58 ꢁ
10ꢂ6 M. The arrow shows the effect of increasing of CT-DNA concentration on the
absorption intensity.
DNA binding studies
Absorption spectral studies
One of the most useful techniques for DNA binding studies of
molecules is electronic absorption spectroscopy. DNA usually re-
sults in hypochromism and red shift (bathchromism) as a result
of the intercalation mode involving a strong stacking interaction
between an aromatic chromophore and the base pairs of DNA
[35]. Hypochromism arises from the contraction of CT-DNA (calf
thymus) in the helix axis, as well as its conformational changes
[36]. On the other hand, hyperchromism results from the second-
ary damage of DNA double helix structure [37,38], in which the ex-
tent of hyperchromism is indicative of partial or non-intercalative
Fig. 4. Absorption spectra of L4 in the absence and in the presence of increasing
amounts of DNA in Tris HCl buffer (pH-7.2). [L4] = 50
lM, [DNA] = 0–163.58 ꢁ
10ꢂ6 M. The arrow shows the effect of increasing of CT-DNA concentration on the
absorption intensity.
binding modes [39]. The absorption spectra of 50 lM of ligands L1
and L4 in the absence and presence of CT-DNA were given in Figs. 3
and 4, absorption spectra of L2, L3, L5 and L6 in the Supplementary
Material (Figs. S1–S4). The absorption spectra of L1–L6 display
clear hypochromism with slight red shift ꢄ2–4 nm are observed.
After the intercalation of the compounds into the base pairs of
DNA, the
couple with the
the
pꢅ transition energies [40]. These interactions result in
p
h orbital of the intercalated compounds are able to
p
orbitals of the base pairs, thereby decreasing
p
?
the observed hypochromism [41]. The hypochromism of the com-
pounds with aromatic thiosemicarbazone side is in the order of
L2 > L3 > L1, whereas the hypochromism of the compounds with
aliphatic thiosemicarbazone side in the order of L4 > L5 > L6 this
is may be due to a strong stacking interaction between the aro-
matic chromophore of the ligand and the base pairs of the DNA
[42]. Remarkably, ligand L3 exhibited both hypochromism and
hyperchromism at ꢄ365 nm and 460 nm, respectively. This behav-
ior reported for chiral Schiff base complexes [43,44]. The intrinsic
binding constant of all the ligands with DNA was obtained by
observation the changes in absorbance of ligands with increasing
concentration of DNA using the following equation [39,45].
Fig. 5. Emission spectra of L2 in the absence and in the presence of increasing
amounts of DNA in Tris HCl buffer (pH-7.2). [L2] = 50
lM, [DNA] = 0–163.58 ꢁ
10ꢂ6 M. The arrow shows the emission intensity increases upon increasing the
CT-DNA concentration.
ligand, respectively, [DNA] is the concentration of DNA in base
pairs. The binding constant kb was determined from the ratio of
slope to intercept by plot of [DNA]/(ea–ef) vs [DNA]. The intrinsic
binding constant (kb) for L1–L6 were found to be 10.31 ꢁ 105,
9.28 ꢁ 105, 33 ꢁ 105, 9.47 ꢁ 104, 5.03 ꢁ 105 and 6.14 ꢁ 104 Mꢂ1
respectively. The DNA binding abilities of L1–L3 are higher
compared to other Schiff bases. This is attributed to the effective
½DNAꢆ=ðea
where ea
ꢂ
ef Þ ¼ ½DNAꢆ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
,
ef, and eb are extinction coefficients observed for the
absorption band at a given DNA concentration, free and bound

A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
445
Fig. 9. Cleavage of supercoiled pBR322 (0.5
l
g/lL) by L6 at different concentrations
in Tris–HCl buffer pH (7.2) for 2 h at 37 °C. Lane 1: DNA ladder; lane 2:
DNA + DMSO; lane 3: DNA + L6 + 90% DMSO; lane 4: DNA + H2O2 + buffer; lanes:
(5–12), DNA with increasing the concentrations of L6 (1–6 mM) + H2O2 + 1% DMSO.
Fig. 6. Emission spectra of L5 in the absence and in the presence of increasing
amounts of DNA in Tris HCl buffer (pH-7.2). [L5] = 50
M. The arrow shows the emission intensity increases upon increasing the CT-DNA
concentration.
l
M, [DNA] = 0ꢂ163.58 ꢁ 10ꢂ6
-
Fig. 10. Cleavage of supercoiled pBR322 (0.5 lg/lL) by L3 at different concentra-
tions in Tris–HCl buffer pH (7.2) for 2 h at 37 °C. Lane 1: DNA ladder; lane 2:
DNA + DMSO; lane 3: DNA + L3 + 90% DMSO; lane 4: DNA + H2O2 + buffer. Lanes:
(5–12), DNA with increasing the concentrations of L3 (1–6 mM) + H2O2 + 1% DMSO.
Fig. 7. Effect of increasing amounts of EB ( ), L1 ( ), L2 (
relative viscosity of CT-DNA at 37.0 ( 0.1) °C.
) and L3 ( ) on the
Fig. 11. Effect of different concentrations of isatin thiosemicarbazones derivatives
(L1–L6) on human colorectal cancer cells (HCT 116) after 72 h treatment.
experiments were performed. These compounds emit lumines-
cence in Tris–HCl/NaCl buffer at room temperature with maximum
wavelengths of about 399 nm for (L1–L3), 379 nm for (L4–L6), see
Supplementary Material, Figs. S5–S8. The emission spectra of L2
and L5 in the absence and presence of CT-DNA are shown in Figs. 5
and 6. The emission intensity of (L1–L6) increased with increasing
CT-DNA concentrations. These observations implied these Schiff
bases can strongly interact with CT-DNA and propose intercalative
mode with the base pairs of the DNA helix [46].
Fig. 8. Effect of increasing amounts of EB ( ), L4 ( ), L5 ( ) and L6 (
relative viscosity of CT-DNA at 37.0 ( 0.1) °C.
) on the
stacking interaction in the former due to the presence of an
extended aromatic phenyl ring which allows an intercalating ligand
deeply into the DNA base pairs. The Kp for L3 is too high maybe due
to the presence of fluorine atom at C5 [16]. We can assume initially
that these ligands bind to DNA by intercalation, but the binding
mode need to be proved through more experiments.
Viscosity measurements
Hydrodynamic measurements (i.e. viscosity and sedimentation)
that are sensitive to length changes are regarded as the most-
critical and the least-ambiguous tests of a binding in solution in
the absence of crystallographic data [47,48]. A classical intercalator
molecule like EB cause a significant increase in the viscosity of the
DNA solution due to an increase in the separation of base pairs at
the interaction site and an increase in overall double helix length.
Whereas, molecule that bind exclusively in the DNA grooves,
groove-face or electrostatic interactions typically cause a bend in
Emission spectral studies
To further investigate the interaction mode between the Schiff
bases under studying (L1–L6) and CT-DNA fluorescence titration

446
A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
DNA helix reducing its effective length and as a result increase in
its viscosity [49–51]. The viscosity measurements of Schiff bases
(L1–L6) and EB are shown in Figs. 7 and 8. Plot of relative specific
viscosity vs [ligand]/[DNA] ratio shows strongly increased upon
addition of ligand. These observations suggest intercalative bind-
ing of these ligands to the double helix [52].
DNA cleavage studies
The cleavage of supercoiled pBR322 DNA (0.5 lg/lL) was stud-
ied by agarose gel electrophoresis using thiosemicarbazone ligands
(see Supplementary Material, Figs. S8–S11) in 1% DMSO/5 mM
Tris–HCl/50 mM NaCl buffer at pH 7.2 with and without H2O2
Fig. 12. Picture of cancer cells treated with isatin thiosemicarbazones derivatives (L1–L6) for 48 h.

A.Q. Ali et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 440–448
447
and incubated for 2 h. The activity of the ligands was evaluated by
the conversion of DNA from Form I to Form II and Form III. The fast-
est migration will be detected for the supercoiled form (Form I). If
only one strand is cleaved, the supercoils will relax to give a
slower-moving nicked form (Form II). If both strands are cleaved,
a linear form (Form III) will be produced that migrates between
two forms [53].
rendered the cell less viable. The compound L2 demonstrated
considerable anti-proliferative activity with IC50 = 66.6 M and
the treatment with this compound significantly (p > 0.001)
l
reduced the doubling time of cell population. Compound L1 and
L5 exhibited moderate anti-proliferative activity (IC50 = 149.9
202.1 M resp.). Finally the compound L4 showed least cytotoxic
effect (IC50 = 338.8 M). The treatment did not affect much signif-
lM,
l
l
icantly than compared to the negative control.
Oxidative cleavage
The results indicate that with increasing concentration of thio-
semicarbazone ligands, supercoiled form (Form I) of DNA de-
creases gradually while the amount of nicked form (Form II)
increases. Ligands (L1–L3 and L6) in the presence of H2O2 at
(2 mM) concentration shows more cleavage activity in which
supercoiled DNA (Form-I) cleaved and converted to open circular
form (Form-II) compared to ligands (L4 and L5) which cleaved
and converted to (Form II) at (3 mM) concentration. At (3 mM)
concentration the ligands (L1–L3) resulting the conversion of some
supercoiled form (Form-I) into circular form (II) as well as to the
linear form (Form-III) whereas ligands (L4 and L5) show that
conversion at 6 and 5 mM respectively while ligand L6 does not
shows linear form (Fig. 9). The supercoiled DNA was completely
degraded at (6, 3.5 and 4 mM) for ligands L1–L3, respectively.
Therefore, DNA cleavage promoted by these entire Schiff base
should occur via an oxidative pathway [54]. The decreased
intensity of the bands in the gel diagram indicates the presence
of radical cleavage [55].
Conclusion
New thiosemicarbazone derivatives incorporating the isatin
moiety (L1–L6) have been synthesized and their structures have
been confirmed by elemental and spectrometry analyses. Single
crystal X-ray structure of L6 has also been determined. The new
Schiff bases were evaluated for their DNA binding, cleavage and
in vitro anti-proliferative activities against human colon cancer cell
line (HCT 116). The results of the binding studies by absorption,
emission spectral studies and viscosity measurements reveal the
intercalative mode of binding. Results of gel electrophoresis exper-
iments indicate that these Schiff base can induce cleavage of plas-
mid DNA. Cleavage of DNA by these Schiff base has been found to
be concentration dependent. L1, L2, L4–L6 demonstrated oxidative
behavior toward DNA, while L3 Schiff base revealed oxidative as
well as hydrolytic behaviors. Finally, the results of in vitro anti-
proliferative activity against human colon cancer cell line (HCT
116) suggested that, L3 exhibited a good cytotoxicity against colon
cancer cell line tested in which it shows cytotoxic effect
Hydrolytic cleavage
(IC50 = 31.4 lM) followed by L6 (IC50 = 59.5) whereas L2 shows
The roles of active radicals in the DNA cleavage by Schiff base
ligands (L1–L6), were also investigated. The reactions were allowed
to proceed in the presence of DMSO as hydroxyl radical scavengers
(lane 3). The addition of hydroxyl radical scavenger completely
inhibits DNA cleavage activity, which is induced by L1, L2, L4–L6
(Fig. 9). This observation suggests the involvement of the hydroxyl
radical in the cleavage [55], thereby confirming the oxidative path-
ways of these ligands toward DNA. In other hand, ligand L3
(Fig. 10) shows totally degraded in the presence of trapping agent
DMSO (a known ÅOH radical scavenger), which can inhibit the
cleavage activity [56]. This confirms the hydrolytic cleavage of
DNA by this ligand as well as the oxidative pathways (as shown
in last section). This behavior may be due to the introducing of
fluorine atom in the isatin moiety in L3 [57]. The results revealed
that the ligands (L1–L3) have more cleavage activities than L4–L6
ligands; probably this may be due to the presence of phenyl group
in thiosemicarbazide side [58].
considerable activity (IC50 = 66.6).
Acknowledgments
The authors thank the Malaysian Government and Universiti
Sains Malaysia for the RU research Grant (1001/PKIMIA/815067).
Appendix A. Supplementary material
The crystallographic data for structural analysis has been
deposited in the Cambridge Crystallographic Data Center, CCDC
909983 for L6. This data can be obtained free of charge from CCDC
via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.01.086.
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