Synthesis, structure and in vitro anticancer, DNA binding and cleavage activity of palladium (II) complexes based on isatin thiosemicarbazone derivatives

Article abstract of DOI:10.1002/aoc.3813

Six novel palladium(II) complexes of a thiosemicarbazone Schiff base with isatin moiety (PdL1 to PdL6) were synthesized by the reaction of palladium(II) with the following: (Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide (L1H), (Z)-2-(5-met

Full text of DOI:10.1002/aoc.3813

Received: 22 October 2016
DOI: 10.1002/aoc.3813
Revised: 4 February 2017
Accepted: 26 February 2017
F U L L PA P E R
Synthesis, structure and in vitro anticancer, DNA binding and
cleavage activity of palladium (II) complexes based on isatin
thiosemicarbazone derivatives
1
2
3
4
Amna Qasem Ali | Siang Guan Teoh | Naser Eltaher Eltayeb
| Mohamed B. Khadeer Ahamed |
4
1
A.M.S. Abdul Majid | Arabya A.A. Almutaleb
1
Chemistry Department, Faculty of Science,
Six novel palladium(II) complexes of a thiosemicarbazone Schiff base with isatin
moiety (PdL1 to PdL6) were synthesized by the reaction of palladium(II) with the
following: (Z)‐2‐(2‐oxoindolin‐3‐ylidene)‐N‐phenylhydrazinecarbothioamide (L1H),
Sebha University, Sebha, Libya
2
School of Chemical Sciences, Universiti
Sains Malaysia, Minden‐11800, Pulau
Pinang, Malaysia
(
Z)‐2‐(5‐methyl‐2‐oxoindolin‐3‐ylidene)‐N‐phenylhydrazinecarbothioamide (L2H),
Z)‐2‐(5‐fluoro‐2‐oxoindolin‐3‐ylidene)‐N‐phenylhydrazinecarbothioamide (L3H),
3
Department of Chemistry, College of
(
Sciences and Arts, King Abdullaziz
University, Rabigh, Saudi Arabia
(Z)‐N‐methyl‐2‐(5‐nitro‐2‐oxoindolin‐3‐ylidene)hydrazinecarbothioamide (L4H),
Z)‐N‐methyl‐2‐(5‐methyl‐2‐oxoindolin‐3‐ylidene)hydrazinecarbothioamide (L5H)
4
EMAN Research and Testing Laboratory,
(
School of Pharmaceutical Sciences,
Universiti Sains Malaysia, Minden‐11800,
Pulau Pinang, Malaysia
and (Z)‐N‐ethyl‐2‐(5‐methyl‐2‐oxoindolin‐3‐ylidene)hydrazinecarbothioamide (L6H).
The structures of these complexes were characterized using elemental analysis and
1
infrared, UV–visible, H NMR and mass spectroscopies. The structure of PdL5 was
Correspondence
further characterized using single‐crystal X‐ray diffraction. The interaction of these
Naser Eltaher Etayeb, Department of
Chemistry, College of Sciences and Arts, PO
Box 344, King Abdulaziz University, Rabigh
complexes with calf thymus DNA was characterized with a high intrinsic binding
4
6
−1
constant (K = 5.78 × 10 to 1.79 × 10 M ), which reflected the intercalative activity
b
2
1911, Saudi Arabia.
of these complexes towards calf thymus DNA. This result was also confirmed from
viscosity data. Electrophoresis studies revealed that complexes PdL1 to PdL6 could
cleave DNA via an oxidative pathway in the presence of an external agent. Data
obtained from an in vitro anti‐proliferative study clearly established the anticancer
potency of these compounds against the human colorectal carcinoma cell line HCT
Email: netaha@kau.edu.sa
Funding information
Malaysian Government and Universiti Sains
Malaysia for the RU research grant, Grant/
Award Number: 1001/PKIMIA/815067;
Ministry of Higher Education and the Uni-
versity of Sabha (Libya), Grant/Award
Number: scholarship for AQA
116.
KEYWORDS
activityisatin, anti‐proliferative, complexes, moietyoxidative, pathwaypalladium(II), studyintercalative
1
| INTRODUCTION
coordination sphere, such as organometallic complexes and
complexes with metallic elements other than platinum.
Square planar diamminedichloroplatinum(II), cis‐[PtCl2
NH ) ] or cisplatin, is one of the most potent anti‐tumour
drugs available for the therapeutic management of solid
Among the transition metal complexes, palladium(II)
complexes are very interesting candidates for alternative
metal‐based drugs because the coordination geometry and
complex‐forming processes of palladium(II) are highly
(
3
2
tumours, such as germ cell tumours and ovarian, lung, head,
[1,2]
[3]
neck and bladder cancers.
Given the numerous shortcom-
similar to those of platinum(II). Palladium complexes show
4
5
ings of platinum complexes, anti‐tumour drug research has
been developing new compounds outside the usual
approximately 10 to 10 times higher reactivity, whereas
their structural and equilibrium behaviours are very similar
Appl Organometal Chem. 2017;e3813.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 12
https://doi.org/10.1002/aoc.3813

2
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ALI ET AL.
to those of platinum complexes. Similarity between the
coordination chemistry of platinum(II) and palladium(II)
compounds also supports the theory that palladium
ionization (ESI) mass spectra were obtained using an LC/
MSD Trap VL (Agilent Technologies), which was operated
in either positive ion or negative ion detection mode.
Synthesis of Ligands.
In brief, the Schiff base ligands were synthesized by
refluxing a reaction mixture of hot ethanolic solutions
(30 ml each) of 4‐substituted 3‐thiosemicarbazide
(0.01 mol) and 5‐substituted isatin (0.01 mol) for 2 h. The
precipitates that formed during reflux were filtered and
washed with cold ethanol.
[
4]
complexes can act successfully as anti‐tumour drugs. Thus,
some palladium complexes with aromatic N‐containing
Schiff bases have shown very promising anti‐tumour
[
3,5–7]
characteristics.
With similar structural features, a study
in 2015 investigated the interaction between isatin β‐
thiosemicarbazone and calf thymus DNA (CT DNA) using
UV–visible absorption and fluorescence spectroscopy, as
well as viscosity measurements. From the absorption
spectrum of isatin β‐thiosemicarbazone, as the concentration
of DNA increases, a large degree of hypochromism develops
in the spectrum which reflects the strong stacking interaction
Synthesis of Complexes.
The Pd(II) complexes were synthesized by refluxing a
reaction mixture of hot ethanolic solutions (30 ml each) of
PdCl (0.01 mol) and appropriate ligand (0.01 mol) for 2 h.
2
[
8]
between the aromatic chromophore and the base pairs.
The precipitates that formed during reflux were filtered and
washed with cold ethanol.
Recently Patange et al. reported the synthesis and
antimicrobial activity of palladium(II) complexes of
thiosemicarbazone and semicarbazone derived from 5‐
bromoisatin. Those authors concluded that sulfur‐containing
ligands as well as their complexes are more active than their
2
.1.1 | C H ClPdN OS (PdL1)
15 11 4
Brown powder; m.p. > 300 °C; yield 88%. Anal. Calcd (%):
C, 41.21; H, 2.54; N, 12.81; Pd, 24.34. Found (%): C, 41.53;
H, 2.50; N, 12.85; Pd, 24.41. Selected IR data (KBr pellet,
[9]
oxygen‐containing counterparts.
The current paper describes the synthesis, physicochemi-
cal characterization and DNA binding activity of
monopalladium(II) complexes with tridentate ligands. The
in vitro cytotoxic activities of the complexes were tested
against the human colorectal carcinoma cell line HCT 116.
−
1
ν , cm ): 3306 to 2995 (NH), 1644 (C═O), 1550 (C═N),
ma
1
769 (C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.99
6
(s, 1 H, indole N‐H), 11.28 (s, 1 H, CS‐NH), 7.59 (s, 3 H,
indole C2‐H, thiosemicarbazide C11‐H, C15‐H), 7.48 to
7
.43 (t, 3 H, indole C3‐H, thiosemicarbazide C12‐H, C14‐
H, J = 6.5 Hz), 7.24 to 7.15 (dt, 2 H, indole C4‐H,
thiosemicarbazide C13‐H, J = 7.0 Hz), 7.03 (d, 1 H, indole
C5‐H, J = 7.5 Hz). ESI‐MS (m/z): 435.9 (M ).
2
2
| EXPERIMENTAL
−
.1 | Materials and methods
Schiff base ligands (L1H to L6H) were prepared as previ-
2
.1.2 | C H ClPdN OS (PdL2)
[
10–15]
16 13 4
ously reported.
Palladium(II) chloride was purchased
from Aldrich Chemicals. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide (MTT) was purchased from
Sigma‐Aldrich, Germany. Commercial‐grade solvents and
reagents were used as supplied without further purification.
Supercoiled pBR322 DNA and loading dye were purchased
from Fermentas. CT DNA, agarose (molecular biology
grade) and ethidium bromide (EB) were obtained from Sigma
Brown powder; m.p. > 300 °C; yield 80%. Anal. Calcd (%):
C, 42.59; H, 2.90; N, 12.42; Pd, 23.58. Found (%): C, 42.71;
H, 2.88; N, 12.73; Pd, 23.45. Selected IR data (KBr pellet,
−
1
ν , cm ): 3332 to 3134 (NH), 1643 (C═O), 1586 (C═N),
ma
1
753 (C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.83
6
(s, 1 H, indole N‐H), 11.22 (s, 1 H, CS‐NH), 7.63 to 7.56
(dd, 2 H, C11‐H, C15‐H, J = 7.6 Hz), 7.46 to 7.44 (dd, 3
H, indole C5‐H, thiosemicarbazide C12‐H, C14‐H,
J = 8.3 Hz), 7.25 to 7.22 (t, 2 H, indole C3‐H,
thiosemicarbazide C13‐H, J = 7.5 Hz), 6.90 (d, 1 H, indole
(
St Louis, MO, USA). Elemental analysis was carried out
using a PerkinElmer 2400 series‐11 CHN/O analyser
Waltham, MA, USA). Quantitative determination of metal
(
ions was carried out with a PerkinElmer model 3100 atomic
absorption spectrometer. Infrared (IR), electronic (UV–visi-
ble), NMR and emission spectra were recorded with a
PerkinElmer 2000 spectrometer; PerkinElmer Lambda 25
spectrometer; Bruker 500 MHz spectrometer at room temper-
C2‐H, J = 7.9), 2.27 (s, 3 H, CH ). ESI‐MS (m/z): 449.0
(M − H) .
3
−
2
.1.3 | C H ClFPdN OS (PdL3)
15 10 4
ature using deuterated dimethylsulfoxide (DMSO‐d ) as
Brown powder; m.p. > 300 °C; yield 79%. Anal. Calcd (%):
C, 39.58; H, 2.21; N, 12.31; Pd, 23.38. Found (%): C, 39.70;
H, 2.32; N, 12.39; Pd, 23.41. Selected IR data (KBr pellet,
6
solvent and tetramethylsilane as internal standard; and a
JASCO FP‐750 spectrophotometer, respectively. Viscosity
measurements were made using a Cannon Manning Semi‐
Micro viscometer (State College, PA, USA). Electrospray
−
1
ν , cm ): 3415 to 3136 (NH), 1650 (C═O), 1552 (C═N),
ma
1
768 (C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.91
6

ALI ET AL.
3 of 12
(
3
s, 1 H, indole N‐H), 11.30 (s, 1 H, CS‐NH), 7.58 to 7.52 (dd,
H, indole C5‐H, thiosemicarbazide C11‐H, C15‐H,
2.2 | X‐ray crystallography
Single crystals of PdL5, suitable for diffraction, were grown
by slow evaporation of 3:1 mixtures of acetone and DMSO.
Selected crystal data and data collection parameters are pre-
sented in Table 1.
J = 8.0), 7.47 to 7.44 (t, 2 H, thiosemicarbazide C12‐H,
C14‐H, J = 7.8 Hz), 7.31 to 7.27 (dt, 2 H, indole C3‐H,
thiosemicarbazide C13‐H, J = 8.9, 2.0 Hz), 7.02 to 7.00
(dd, 1 H, indole C2‐H, J = 8.6, 4.2 Hz). ESI‐MS (m/z):
2+
Data for the PdL5 complex were collected using a Bruker
4
55.0 (M + 2H) .
[
16]
SMART APEX II CCD diffractometer
graphite monochromatized Mo
λ = 0.71073 Å) at 100(1) K. Multi‐scan absorption correc-
equipped with
Kα radiation
(
[
16]
2
.1.4 | C H ClPdN O S (PdL4)
tions were applied using the SADABS program.
The
10
8
5
3
structure was solved by the direct method using the
Brown powder; m.p. > 300 °C; yield 84%. Anal. Calcd (%):
C, 28.59; H, 1.92; N, 16.67; Pd, 25.33. Found (%): C, 28.33;
H, 1.65; N, 16.43; Pd, 25.61. Selected IR data (KBr pellet,
[
17]
SHELXS‐97 program.
Refinements on F2 were per-
formed using SHELXL‐97 by the full‐matrix least‐squares
method with anisotropic thermal parameters for all non‐
hydrogen atoms. Nitrogen‐bound hydrogen atoms were
located in a difference Fourier map and refined freely. The
remaining hydrogen atoms were positioned geometrically
−1
ν , cm ): 3290 (NH), 1641 (C═O), 1527 (C═N), 748
ma
1
(
(
C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.20
s, 1 H, indole N‐H), 9.59 to 9.56 (q, 1 H, CS‐NH, J = 9.7,
6
4
2
2
.8 Hz), 8.30 to 8.28 (dd, 1 H, indole C5‐H, J = 8.3,
.2 Hz), 8.26 to 8.24 (dd, 1 H, indole C3‐H, J = 8.5,
.4 Hz), 7.21 to 7.10 (t, 1 H, indole C2‐H, J = 8.5 Hz), 3.10
[
17]
and refined using a riding model. The SHELXTL program
was used for the pictorial representation of the structure.
(
4
d, 3 H, thiosemicarbazide CH , J = 4.9 Hz). ESI‐MS (m/z):
18.0 (M ).
3
−
TABLE 1 Crystallographic data for complex PdL5
Formula
11 4 6
C H11ClN OPdS (C2.92H OS0.08)
Formula weight
T (K)
448.86
2
.1.5 | C H ClPdN OS (PdL5)
11 11 4
100(1)
Brown crystals; m.p. > 300 °C; yield 88%. Anal. Calcd (%):
C, 33.95; H, 2.85; N, 14.40; Pd, 27.35. Found (%): C, 33.77;
H, 2.59; N, 14.36; Pd, 27.52. Selected IR data (KBr pellet,
Crystal system
Space group
a (Å)
Triclinic
P‐1
−1
ν , cm ): 3336 to 3245 (NH), 1635 (C═O), 1538 (C═N),
8.4311(1)
10.4885(1)
10.5347(1)
109.588(1)
103.743(1)
100.822(1)
ma
1
764 (C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.81
6
b (Å)
(s, 1 H, indole N‐H), 9.35 to 9.32 (q, 1 H, CS‐NH, J = 9.2,
c (Å)
4.6 Hz), 7.41 (d, 1 H, indole C5‐H), 7.22 to 7.16 (dd, 1 H,
α (°)
indole C3‐H, J = 8 Hz), 6.89 to 6.88 (d, 1 H, indole C2‐H,
J = 8.0 Hz), 3.07 (d, 3 H, thiosemicarbazide CH3,
J = 5.0 Hz), 2.30 (d, 3 H, indole CH , J = 5.0 Hz). ESI‐
β (°)
γ (°)
3
+
3
MS (m/z): 388.0 (M + H) .
V (nm )
815.334(18)
Z
2
D
c
(mg m−
3
)
1.828
450
2
.1.6 | C H ClPdN OS (PdL6)
12 13 4
F(000)
Brown powder; m.p. > 300 °C; yield 80%. Anal. Calcd (%):
C, 35.75; H, 3.25; N, 13.90; Pd, 26.39. Found (%): C, 35.65;
H, 3.55; N, 13.73; Pd, 26.60. Selected IR data (KBr pellet,
Crystal dimensions (mm)
θ range (°)
0.09 × 0.12 × 0.31
2.2–32.5
hkl ranges
−12 < h < 12
−15 < k < 15
−15 < l < 15
−1
ν , cm ): 3385 to 3342 (NH), 1636 (C═O), 1513 (C═N),
ma
1
765 (C─S). H NMR (500 MHz, DMSO‐d , δ, ppm): 11.78
6
(
s, 1H, indole N‐H), 9.40 to 9.38 (t, 1H, CS‐NH,
Data/parameters
5811/223
1.05
J = 5.3 Hz), 7.39 (d, 1 H, indole C5‐H, J = 5.6 Hz), 7.22
to 7.17 (dd, 1 H, indole C2‐H, J = 8.5 Hz), 6.89 (d, 1 H,
indole C3‐H, J = 7.9 Hz), 3.53 to 3.49 (p, 2 H,
thiosemicarbazide CH , J = 6.5 Hz), 2.29 (s, 3H, indole
CH ), 1.20 to 1.17 (t, 3H, thiosemicarbazide CH ,
2
Goodness‐of‐fit on F
Final R indices [I > 2σ(I)]
R
wR
1
= 0.0261
2
= 0.0609
2
Δρmax = 0.79 e Å− /Δρmin = −0.68
3
Highest peak/deepest hole
3
3
e Å−
3
−
J = 7.6 Hz). ESI‐MS (m/z): 401.0 (M − H) .

4
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ALI ET AL.
2
.3 | DNA interaction studies
2.5 | Anti‐proliferative activity
2.5.1 | Preparation of cell culture
DNA binding experiments, including absorption and emis-
sion spectral studies and viscosity measurements, conformed
to the standard methods.
solution was prepared in 5 mM Tris–HCl/50 mM NaCl in
water at pH = 7.2. The DNA concentration per nucleotide
was determined by absorption spectroscopy using the molar
HCT 116 cells were allowed to grow under optimal incubator
conditions. Cells that had reached a confluence of 70 to 80%
were chosen for cell plating. Old medium was aspirated out
of the plate, and cells were washed using sterile phosphate‐
buffered saline (pH = 7.4) two to three times. Phosphate‐
buffered saline was completely discarded after washing.
Trypsin was added and evenly distributed onto the cell sur-
[18–21]
Concentrated CT DNA stock
−
1
−1
[22]
absorption coefficient (6600 M cm ) at 260 nm.
The
purity of the CT DNA was verified using the ratio of the
absorbance values at 260 and 280 nm in the respective buffer,
which was found to be greater than 1.9. This result indicates
that DNA was sufficiently free of protein. The stock solutions
were stored at 4 °C and used within four days. The DNA
binding experiments were performed at room temperature.
All experiments were carried out by keeping the concentra-
tion of palladium complexes constant (50 μM), whereas the
DNA concentration was varied (0 to 200 μM). An equal
amount of DNA was added to the palladium complex cuvette
and reference cuvette to eliminate the absorbance of CT DNA
face, and the cells were incubated at 37 °C in 5% CO for
2
1
min. The flasks containing the cells were gently tapped to
aid cell segregation, and observed under an inverted micro-
scope (if cell segregation was unsatisfactory, the cells were
incubated for another minute). Trypsin activity was inhibited
by adding 5 ml of fresh complete medium (10% foetal bovine
serum). Cells were counted and diluted to obtain a final con-
5
−1
centration of 2.5 × 10 cells ml , and seeded into wells
100 μl of cells per well). Finally, the cell‐containing plates
(
were incubated at 37 °C with an internal atmosphere of 5%
CO2.
[
23]
itself.
Tris buffer was subtracted through baseline correc-
tion. UV–visible and emission spectra were recorded after
equilibration for 10 min after each addition. Viscosity exper-
iments were carried out using a thermostatically controlled
water bath maintained at 37.0 ꢀ 0.1 °C. The flow rates of
Tris–HCl buffer (pH = 7.2), DNA (200 μM) and DNA in
the presence of Pd(II) complexes at various concentrations
2.5.2 | MTT assay
5
−1
Cancer cells (100 μl of cells per well, 1.5 × 10 cells ml )
were seeded on a 96‐well microtiter plate, which was incu-
0 to 2.5 × 10−4 M) were measured three times each with a
digital stopwatch, and the average flow time was calculated.
bated with CO overnight to allow cell attachment. The com-
2
(
pounds were diluted with media into the required
concentrations from the stock. Various concentrations (6.25
to 200 μM) of 100 μl of test substances were separately added
to each well containing the cells. The plates were then incu-
1
/3
Data are presented as (η/η0) versus the ratio of the concen-
tration of complex to CT DNA, where η is the viscosity of
DNA in the presence of complex and η is that of DNA alone.
Viscosity values were calculated from the observed flow time
of DNA‐containing solutions (t) corrected for that of buffer
alone (5 mM Tris–HCl/50 mM NaCl) (t ) using η = (t – t ).
0
bated at 37 °C with an internal atmosphere of 5% CO for
2
4
8 h. The plates were then treated with 20 μl of MTT reagent
and incubated again for 4 h. Approximately 50 μl of MTT
lysis solution (DMSO) was added to the wells, and the plates
were further incubated for 5 min at room temperature.
Finally, the absorbances at 570 and 620 nm were measured
using a standard Infinite 200 PRO multimode microplate
reader (Tecan, USA). Data were recorded and analysed to
estimate the effects of test compounds on cell viability and
growth inhibition. The percentage inhibition of cell prolifera-
tion was calculated from the optical density obtained from
MTT assay. 5‐Fluorouracil (5‐FU) was used as a standard ref-
0
0
2
.4 | DNA cleavage studies
Cleavage experiments of supercoiled pBR322 DNA (0.5 μg
μl) were performed at pH = 7.2 in 5 mM Tris–HCl/50 mM
NaCl buffer. Oxidative DNA cleavage was monitored by
treating pBR322 DNA with varying concentrations of Pd(II)
complexes (0.1 to 1 mM) and H O , followed by dilution
with 5 mM Tris–HCl/50 mM NaCl buffer to a total volume
of 20 μl. The experiment was carried out by adding scavenger
[24]
2
2
erence drug. Statistical differences between the treatments
and control were evaluated by one‐way ANOVA, followed by
Tukey's multiple comparison test. Differences were consid-
ered significant at p < 0.05 and p < 0.01.
(DMSO) for the hydroxyl radical species to a complex–DNA
mixture to investigate the mechanism of DNA cleavage pro-
moted by these complexes. The samples were incubated for
2
h at 37 °C. A loading dye was added, and electrophoresis
3 | RESULTS AND DISCUSSION
was carried out at 50 V for 1 h in Tris–HCl buffer using 1%
agarose gel. The resulting bands were stained with EB before
being photographed under UV light.
Complexes PdL1 to PdL6 were obtained in good yield from
the reaction of Pd(II) with an appropriate Schiff base (L1H

ALI ET AL.
5 of 12
to L6H; Scheme 1) in a 1:1 molar ratio in ethanol medium
with reflux for 2 h (Scheme 2). These complexes were
slightly soluble in common organic solvents, but soluble in
dimethylformamide and DMSO.
Spectroscopic Analysis.
3
.1 | IR spectra
[
10]
The characteristic IR bands recorded for the free ligands
differed from those of the related complexes, and provided
significant indications of the bonding sites of
thiosemicarbazone ligands. Compared with the spectra of
the Schiff bases, PdL1 to PdL6 exhibited the ν(C═O) band
SCHEME 2 Synthetic route and structures for the compounds
−
1
from 1635 to 1650 cm , showing a shift to lower
wavenumbers. This finding indicates that the carbonyl oxy-
gen was coordinated to the metal ion. The ν(C═N) band from
PdL1–PdL6. PdL1: X = H, Y = Ph; PdL2: X = CH , Y = Ph; PdL3:
3
X = F, Y = Ph; PdL4: X = NO , Y = CH ; PdL5: X = CH , Y = CH ,
2
3
3
3
3 3 2
PdL6: X = CH , Y = CH CH
−
1
1
513 to 1586 cm in the spectra of the metal complexes
showed a shift to lower wavenumbers, which indicates that
the nitrogen atom of the azomethine group was coordinated
to the metal ion. This finding was further supported by the
MS (m/z) of 455.0 and 388.0, respectively, indicating the
presence of these complexes as [M + 2H] and [M + H] .
+
+
−
1
band at approximately 748 to 769 cm for the metal com-
3.4 | Crystal structure of PdL5
[25]
plexes because of ν(C─S).
Thus, the IR spectral results
for PdL1 to PdL6 provide strong evidence for the complexa-
tion of Schiff bases with metal ions in a tridentate mode as
reported for similar structures.
The molecular structure of compound PdL5 and the atom
numbering scheme are shown in Figure 1. The PdL5 complex
presented a square planar geometry, which was slightly
distorted around the palladium atom. The basal plane was
occupied by the S(1), O(1) and N(2) atoms of the ligand
and Cl(1) ion. Distortion from square planar geometry was
evident from the bond angles S(1)–Pd(1)–N(2) = 83.92(5)°,
[7]
1
3
.2 | H NMR spectra
1
The H NMR (DMSO‐d ) spectra of the Pd(II) complexes
6
showed approximately the same peaks identical to those of
[10]
the free ligands,
except that the signal of
thiosemicarbazide N─NH proton in the spectra of the ligands
disappeared in the spectra of the complexes. This finding pro-
vides additional evidence of ligand deprotonation during
metal chelation.
3
.3 | Mass spectra
The mass spectra of PdL1, PdL2, PdL4 and PdL6 recorded in
the negative mode showed the following peaks at ESI‐MS
(
m/z): 435.9, 449.0, 418.0 and 401.0, respectively. These
−
−
−
indicate the presence of [M ], [M − H] , [M ] and
−
[M − H] , respectively. The mass spectra of PdL3 and
PdL5 recorded in the positive mode showed peaks at ESI‐
SCHEME 1 Synthetic route and structures for the Schiff base
ligands L1H–L6H. L1: X = H, Y = Ph; L2: X = CH , Y = Ph; L3:
X = F, Y = Ph; L4: X = NO , Y = CH ; L5: X = CH , Y = CH ; L6:
X = CH , Y = CH CH
3
2
3
3
3
FIGURE 1 Structure of complex PdL5, showing 50% probability
displacement ellipsoids and atomic numbering
3
3
2

6
of 12
ALI ET AL.
Cl(1)–Pd(1)–S(1) = 98.24(2)°, Cl(1)–Pd(1)–O(1) = 94.14(4)°,
O(1)–Pd(1)–N(2) = 83.72(6)°, S(1)–Pd(1)–O(1) = 167.60(4)°
and Cl(1)–Pd(1)–N(2) = 177.40(5)°; and bond lengths
Pd(1)–S(1) = 2.2314(5) Å, Pd(1)–N(2) = 1.9726(15) Å,
Pd(1)–O(1) = 2.1339(14) Å and Pd(1)–Cl(1) = 2.3030(5)
Å. The unit cell contained a non‐integer number of atoms
(
C2.92H OS0.08) because of partially occupied (solvent) sites
6
and substitutional disorder. In crystal packing (Figure 2).
the molecules were interconnected through intermolecular
hydrogen bonds. A weak bond between S(2) and Pd(1) with
Pd–S = 3.794 Å was also observed, which changed the
coordination geometry around Pd(1) from square planar to
pseudo square pyramidal.
3
3
.5 | DNA interaction studies
FIGURE 3 Absorption spectra of PdL3 in the absence and in the
presence of increasing amounts of DNA in Tris–HCl buffer (pH = 7.2).
.5.1 | Absorption spectral studies
−
5
−5
[
[PdL3] = 50 μM; [DNA]: A, 2.861 × 10 ; B, 5.666 × 10
;
One of the most useful techniques for studying the DNA
binding of molecules is electronic absorption spectroscopy.
DNA usually results in hypochromism and red shifting
−5
−4
−4
−4
C, 8.417 × 10 ; D, 1.1115 × 10 ; E, 1.3761 × 10 ; F, 1.6358 × 10
M
(bathchromism) as a consequence of the intercalation mode
involving a strong stacking interaction between an aromatic
[26]
chromophore and the DNA base pairs.
This phenomenon
arises from the contraction of CT DNA in the helix axis
[27,28]
and conformational changes.
Hyperchromism results
from the secondary distortion of the DNA double‐helix struc-
[
29,30]
ture.
The extent of hyperchromism indicates partial or
[31]
non‐intercalative binding modes.
The absorption spectra
of 50 μM PdL3 and PdL5 in the absence and presence of
CT DNA (0 to 200 μM) are shown in Figures 3 and 4. The
absorption spectra of PdL1, PdL2, PdL4 and PdL6 are given
in the supporting information (Figures S1–S4). The absorp-
tion spectra of PdL2, PdL5 and PdL6 displayed clear
hypochromism with a slight red shift of approximately 1 to
3
nm. After the compounds intercalated to the DNA base
pairs, the π* orbitals of the intercalated compounds coupled
FIGURE 4 Absorption spectra of PdL5 in the absence and in the
presence of increasing amounts of DNA in Tris–HCl buffer (pH = 7.2).
−
5
−5
[
PdL5] = 50 μM; [DNA]: A, 2.861 × 10 ; B, 5.666 × 10
;
−5
−4
−4
−4
C, 8.417 × 10 ; D, 1.1115 × 10 ; E, 1.3761 × 10 ; F, 1.6358 × 10
M
with the π orbitals of the base pairs, thereby decreasing the
π ! π* transition energies. These interactions result in the
[
32]
observed hypochromism.
The hypochromism of these
complexes followed the order of PdL2 = PdL5 > PdL6. By
contrast, PdL1, PdL3 and PdL4 exhibited both
hypochromism and hyperchromism at approximately 382 to
4
42 nm and 243 to 358 nm, respectively; this phenomenon
FIGURE 2 Crystal packing of PdL5, viewed approximately along
the c‐axis
suggests the distortion of the DNA double‐helix structure
after the complex binds to DNA through the intercalation

ALI ET AL.
7 of 12
[
33]
mode.
This behaviour has been reported for chiral Schiff
[
34,35]
[36]
base complexes
and PdCl (LL).
Complexes PdL1
2
and PdL6 showed hypochromism with a small red shift of
approximately 1 nm, whereas PdL2, PdL3 and PdL5
displayed red shifts of approximately 3 nm, and PdL4 showed
a high red shift of approximately 5 nm.
The intrinsic binding constant of all complexes with
DNA was determined by observing the changes in absor-
bance of the complexes with increased DNA concentration
[30,37]:
using the following equation
FIGURE 5 Emission spectra of PdL2 in the absence and in the
presence of increasing amounts of DNA in Tris–HCl buffer (pH = 7.2).
[PdL2] = 50 μM; [DNA] = 0–163.58 × 10− M. The arrow shows the
emission intensity increasing upon increasing the CT DNA concentration
½
DNAꢁ ½DNAꢁ
1
6
¼
þ
εa−εf
εb−εf
Kbðεb−εfÞ
<
NI>where ε , ε and ε are the extinction coefficients
a f b
observed for the absorption band at a given DNA concentra-
tion for free and bound complexes, respectively; [DNA] is the
DNA concentration in base pairs; and K is the intrinsic bind-
b
ing constant determined from the slope‐to‐intercept ratio of a
plot of [DNA]/(ε − ε ) versus [DNA] (Figures S3 and S4).
a
f
The K values for PdL1, PdL2, PdL3, PdL4, PdL5 and
b
6
5
6
4
PdL6 were 1.79 × 10 , 2.69 × 10 , 1.55 × 10 , 5.78 × 10 ,
.14 × 10 and 4.93 × 10 M , respectively. The K values
5
5
−1
3
b
of the complexes PdL1, PdL2, PdL3, PdL5 and PdL6 were
[
38]
close to those reported for typical classical intercalators,
whereas that of complex PdL4 was much smaller. This
behaviour reflects that the DNA binding affinities of PdL1,
PdL2, PdL3, PdL5 and PdL6 were stronger than that of
PdL4. The DNA binding abilities of PdL1 and PdL3 were
higher than those of the other complexes. This result was
attributed to the effective stacking interaction in the former
because of the presence of an extended aromatic phenyl ring,
which allowed an intercalating ligand deep in the DNA base
pairs. A comparison of the intrinsic binding constants of
these complexes with those of DNA‐intercalative Pd(II)
FIGURE 6 Emission spectra of PdL4 in the absence and in the
presence of increasing amounts of DNA in Tris–HCl buffer (pH = 7.2).
6
[
PdL4] = 50 μM; [DNA] = 0–163.58 × 10− M. The arrow shows the
emission intensity increasing upon increasing the CT DNA concentration
[39]
complexes, such as [Pd(dmphen)(CO )]H O
and
3
2
[40]
[Pd(bpy)(pip‐dtc)]NO3,
revealed that these complexes
interact with CT DNA, and indicate an intercalative mode
bind to DNA by intercalation, but the binding mode requires
more experiments.
[41]
with the base pairs of the DNA helix.
3
.5.2 | Emission spectral studies
3.5.3 | Viscosity measurements
Fluorescence titration experiments were performed to further
study the interaction mode between PdL1–PdL6 and CT
DNA. These complexes could emit luminescence in Tris–
HCl/NaCl buffer at room temperature with maximum wave-
length of approximately 379 nm (supporting information,
Figs S5–S8). The emission spectra of PdL2 and PdL4
The most critical and least ambiguous tests of binding in
[
42,43]
solution in the absence of crystallographic data
are
hydrodynamic measurements (i.e. viscosity and sedimenta-
tion), which are sensitive to length changes. A classical inter-
calation molecule, such as EB, lengthens the DNA helix,
leading to increased DNA viscosity caused by an increase
in the separation of base pairs at the interaction site and an
increase in the overall double‐helix length. Partial or non‐
classical intercalation of a complex results in the bending of
the DNA helix that decreases the effective DNA length and
(50 μM) in the absence and presence of CT DNA (0 to
200 μM) are shown in Figures 5 and 6. The emission inten-
sity increased with increasing CT DNA concentration. These
observations imply that these complexes could strongly

8
of 12
ALI ET AL.
[
46,47]
double helix,
which agree with the results of the optical
absorption experiments.
3.5.4 | DNA cleavage studies
The degree to which complexes PdL1–PdL6 could function
as DNA cleavage agents was examined using supercoiled
−
1
pBR322 DNA (0.5 μg μl ) as the target. The cleavage
efficiency of these complexes was investigated through
agarose gel electrophoresis using different concentrations of
complexes (supporting information, Figs S9–S12) in 1%
DMSO/5 mM Tris–HCl/50 mM NaCl buffer at pH = 7.2,
with and without H O , and incubation of 2 h. The activity
FIGURE 7 Effect of increasing amounts of EB, PdL1, PdL2 and
PdL3 on the relative viscosity of CT DNA at 37.0 ꢀ 0.1 °C
2
2
of the complexes was estimated by the conversion of DNA
from Form I to Forms II and III. The fastest migration is
detected in the supercoiled form (Form I). If only one
strand is cleaved, the supercoils relax to convert into a
slower‐moving nicked form (Form II). If both strands are
cleaved, a linear form (Form III) is produced, which migrates
[
48]
between Forms I and II.
3.5.5 | Oxidative cleavage
This experiment was conducted in the presence of H O as an
2
2
oxidizing agent. The control experiment did not show any
apparent cleavage of DNA (lane 2). In the presence of the
complexes at different concentrations (lanes 4 to 12), the
plasmid DNA was converted from Form I to Forms II and
III at 0.1 mM (lane 4 for PdL1 (Figure 9).and PdL3),
FIGURE 8 Effect of increasing amounts of EB, PdL4, PdL5 and
PdL6 on the relative viscosity of CT DNA at 37.0 ꢀ 0.1 °C
0
.3 mM (lane 6 for PdL2) and 0.6 mM (lane 8 for PdL4).
viscosity. By contrast, an electrostatic or groove binding
The plasmid DNA was converted from Form I to Form II at
0.1 mM (lane 4 for PdL5 and PdL6). DNA was completely
degraded above these concentrations. The reactions were
allowed to proceed in the presence of DMSO as hydroxyl
radical scavenger (lane 3) to investigate the DNA cleavage
[44,45]
mode has minimal effects on DNA viscosity.
The
viscosity measurements of PdL1–PdL6 and EB are shown
in Figures 7 and 8. A plot of relative specific viscosity versus
the [complex]/[DNA] ratio showed a significant increase
upon addition of the complexes. These observations suggest
the intercalative binding of these complexes to the DNA
[
49]
mechanism promoted by these complexes.
The addition
of the hydroxyl radical scavenger completely inhibits the
FIGURE 9 Cleavage of supercoiled
pBR322 (0.5 μg μl− ) at different
1
concentrations of PdL1 in Tris–HCl buffer
(pH = 7.2) for 2 h at 37 °C. Lane 1: DNA
ladder; lane 2: DNA + H ; lane 3:
2 2
O
DNA + PdL1 (1 mM) + DMSO; lanes 4–12:
DNA with increasing the concentrations of
2 2
PdL1 + H O (0.1–1 mM) + buffer

ALI ET AL.
9 of 12
DNA cleavage activity (PdL4, Figure 10), which is induced
by these complexes. This observation suggests the involve-
[50]
ment of the hydroxyl radical in cleavage.
3
.5.6 | Anti‐proliferative activity
The in vitro anti‐proliferative activity of PdL1–PdL6 was
evaluated against HCT 116 cells. The effects of various con-
centrations of the complexes on HCT 116 cells after 48 h of
treatment are shown in Figure 11. The anticancer efficiencies
of all tested compounds are summarized in Table 2. Results
of the anti‐proliferation test show that all tested compounds
had a dose‐dependent effect. Figure 12 shows photomicro-
graphic images of cancer cells treated with these compounds
for 48 h. First, cells treated with the standard drug 5‐FU
FIGURE 11 Effect of different concentrations of PdL1–PdL6 on
human colorectal cancer cells (HCT 116) after 72 h treatment
(IC50 = 7.3 μM) showed decreased viability. The HCT 116
cells treated with PdL1 exhibited the most potent anti‐prolif-
erative activity (IC50 = 11 μM) among all the synthesized
compounds tested. Cell growth was clearly affected by the
treatment. The results could be compared with the standard
TABLE 2 Anticancer efficiencies of tested compounds
Compound
IC50 value (μM)
[
5]
reference ‐FU. Except for a few affected cells, the activity
was so pronounced that only cellular debris remained in the
growth medium. HCT 116 cells treated with PdL2 showed
considerable cytotoxicity with IC50 = 51.4 μM. The reduc-
tion in the population doubling time could be clearly visual-
ized from the reduced number of viable cells. Treatment
with PdL3 showed a significant inhibitory effect on the pro-
liferation of HCT 116 cells (IC50 = 16 μM). The effect could
PdL1
PdL2
PdL3
PdL4
PdL5
PdL6
11.0
51.4
16.0
112.0
46.0
28.0
[5]
be compared with the standard reference ‐FU. The treat-
ment caused an abnormality in cellular morphology of
treated cells, and rendered cells with round shapes. The pho-
tomicrographic images of HCT 116 cells show the effect of
PdL4. The compound displayed moderate cytotoxicity as
the IC50 value (112 μM) was higher than that of the other
samples tested. HCT 116 cells treated with PdL5 showed a
significant cytotoxic effect (IC50 = 46 μM). The treatment
significantly (p < 0.05) affected cellular morphology and
growth compared with the control. Finally, the treatment with
PdL6 showed a significant (p < 0.05) cytotoxic effect on the
proliferation of HCT 116 cells. The cells showed an abnor-
mal cellular membrane with affected pseudopodial projec-
tions. Thus, cell growth was severely affected and presented
a significantly low IC50 value (28 μM).
FIGURE 10 Cleavage of supercoiled
−1
pBR322 (0.5 μg μl ) at different
concentrations of PdL4 in Tris–HCl buffer
(pH = 7.2) for 2 h at 37 °C. Lane 1: DNA
ladder; lane 2: DNA + H ; lane 3:
2 2
O
DNA + PdL4 (1 mM) + DMSO; lanes 4–12:
DNA with increasing the concentrations of
2 2
PdL4 + H O (0.1–1 mM) + buffer

1
0 of 12
ALI ET AL.
FIGURE 12 Images of cancer cells treated with complexes PdL1–PdL6 for 48 h
4
| CONCLUSIONS
mode, which was investigated using absorption (K
b
ranged
4
6
−1
from 5.78 × 10 to 1.79 × 10 M ) and fluorescence spectra
and viscosity measurements. Their affinity to DNA followed
the order of PdL1 > PdL3 > PdL6 > PdL5 > PdL2 > PdL4,
which could be attributed in some instances to the presence
of an extended aromatic phenyl ring that allows an intercalat-
Six novel square planar palladium(II) complexes (i.e. PdL1–
PdL6) with tridentate ligands were synthesized and character-
ized using elemental analysis and various spectroscopic tech-
niques. The crystal structure of PdL5 showed that it was a
four‐coordinate complex with a slightly distorted square pla-
nar geometry. The results of binding studies show that these
complexes interacted with CT DNA through an intercalative
[
50]
ing ligand deep in the DNA base pairs.
The results of gel
electrophoresis experiments indicate that these complexes
could induce cleavage of plasmid DNA. Cleavage of DNA

ALI ET AL.
11 of 12
by these complexes was dependent on concentration. All
complexes showed nuclease activity in the presence of an
oxidant, which may be due to free radical reaction (OH )
with DNA. Finally, the results of in vitro anti‐proliferative
activity against HCT 116 cells show dose‐dependent cytotox-
icity of the synthesized complexes, with significantly low
IC50 ranging from 11.0 to 112.0 μM. Thus, the palladium(II)
complexes of thiosemicarbazone Schiff bases with isatin
moiety are promising anti‐neoplastic agents.
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Sains Malaysia for the RU research grant (1001/PKIMIA/
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