Synthesis of platinum(II) complexes of isatin thiosemicarbazones derivatives: In vitro anti-cancer and deoxyribose nucleic acid binding activities

Article abstract of DOI:10.1016/j.ica.2014.03.029

Six new platinum(II) complexes of a thiosemicarbazone Schiff base with isatin moiety [PtL1 to Pt(L6)₂] were synthesized by the reaction of platinum(II) with the following: (Z)-2-(2-oxoindolin-3-ylidene)-N- phenylhydrazinecarbothioamide [L1H], (

Full text of DOI:10.1016/j.ica.2014.03.029

Accepted Manuscript
Synthesis of platinum(II) Complexes of Isatin Thiosemicarbazones Derivatives:
In Vitro Anti-cancer and Deoxyribosenucleic Acid Binding Activities
Amna Qasem Ali, Siang Guan Teoh, Abdussalam Salhin, Naser Eltaher Eltayeb,
Mohamed B. Khadeer Ahamed, A.M.S. Abdul Majid
PII:
S0020-1693(14)00183-2
http://dx.doi.org/10.1016/j.ica.2014.03.029
ICA 15929
DOI:
Reference:
Inorganica Chimica Acta
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Received Date:
Revised Date:
Accepted Date:
14 November 2013
19 February 2014
16 March 2014
Please cite this article as: A.Q. Ali, S.G. Teoh, A. Salhin, N.E. Eltayeb, M.B. Khadeer Ahamed, A.M.S. Abdul
Majid, Synthesis of platinum(II) Complexes of Isatin Thiosemicarbazones Derivatives: In Vitro Anti-cancer and
Deoxyribosenucleic Acid Binding Activities, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.
2014.03.029
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Synthesis of platinum(II) Complexes of Isatin Thiosemicarbazones Derivatives : In Vitro
Anti-cancer and Deoxyribosenucleic Acid Binding Activities
Amna Qasem Ali a, Siang Guan Teoh a*, Abdussalam Salhin a, Naser Eltaher Eltayeb b,
Mohamed B. Khadeer Ahamed c and AMS Abdul Majid c
aSchool of Chemical Sciences, Universiti Sains Malaysia, Minden-11800, Pulau Pinang,
Malaysia,
bDepartment of Chemistry, Sciences & Arts College – Rabigh, King AbdullAziz University ,
Rabigh , Saudi Arabia,
cEMAN Research and Testing Laboratory, School of Pharmaceutical Sciences,
Universiti Sains Malaysia, 11800-Minden, Pulau Pinang, Malaysia
Corresponding Author:
* Corresponding Author: Prof. Dr. Siang Guan Teoh
Corresponding Address:
School of Chemical Sciences,
Universiti Sains Malaysia,
Minden, Pinang, Malaysia,
Email : sgteoh@usm.my
Tel : (604)-6577888 ext 3565
Fax: (604)-6574854
1

Abstract
Six new platinum(II) complexes of a thiosemicarbazone Schiff base with isatin
moiety [PtL1 to Pt(L6)2] were synthesized by the reaction of platinum(II) with the following:
(Z)-2-(2-oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide [L1H], (Z)-2-(5-methyl-2-
oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide
oxoindolin-3-ylidene)-N-phenylhydrazinecarbothioamide [L3H], (Z)-N-methyl-2-(5-nitro-2-
oxoindolin-3-ylidene)hydrazinecarbothioamide [L4H], (Z)-N-methyl-2-(5-methyl-2-
[L2H],
(Z)-2-(5-fluoro-2-
oxoindolin-3-ylidene)hydrazinecarbothioamide [L5H], and (Z)-N-ethyl-2-(5-methyl-2-
oxoindolin-3-ylidene)hydrazinecarbothioamide [L6H]. The structures of these complexes
1
were characterized by elemental analysis, IR, UV-vis, H NMR, and mass spectrometry
analyses. The structure of Pt(L6)2 was further characterized by single-crystal XRD. The
interaction of these complexes with calf thymus (CT) DNA exhibited a high intrinsic binding
constant (Kb = 3.5 × 104 to 3.29 × 106 M–1), which reflected the intercalative activity of these
complexes toward CT DNA. This result was also confirmed by viscosity data. Data obtained
from the in vitro anti-proliferative study clearly established the anticancer potency of PtL1,
PtL2, PtL3, PtL5, and Pt(L6)2 against the human colorectal carcinoma cell line HCT 116.
Keywords: Platinum(II) complexes, isatin moiety, intercalative activity, anti-proliferative
study
1.
Introduction
Cisplatin (cis-diamminedichloroplatinum(II) or cis-[PtCl2(NH3)2]) is one of the most
potent antitumor drugs available for the therapeutic management of solid tumors, such as germ
cell tumors, ovarian, lung, head and neck, and bladder cancers. Cisplatin promotes cancer cell
death by binding to DNA [1-3]. However, cisplatin has the shortcomings of nephrotoxicity, low
2

water solubility, and intrinsic resistance that limit its clinical application [4-7]. One of the
mechanisms inducing nephrotoxicity is the inactivation of some enzymes caused by the reaction
between platinum ions and sulfur-containing proteins. Hence, the synthesis of new types of
compounds whose structure and mode of action differ from those of cisplatin and bearing
nitrogen and sulfur mixed donor atoms is important to prevent adverse reactions. Anticancer
drugs that target DNA have two different interaction mechanisms, namely, covalent binding or
noncovalent interactions. Cisplatin is known to bind to DNA through covalent bonding through
chloride ligand exchange with a nitrogen base of DNA. Non-covalent interactions between DNA
and anticancer drugs are classified into three, i.e., electrostatic interactions with the negatively
charged phosphate backbone of DNA, binding interactions to the minor and major grooves of
DNA double helix, and intercalation of a planar aromatic ligand between two base pairs through
π-stacking [8]. One of most typical tridentate NNS chelate ligands is a-(N)-heterocyclic
thiosemicarbazones. Several mechanisms of antitumor action have been proposed for this class
of chelate ligands and their complexes. They could interact with DNA by formation of a metal-
DNA covalent bond, by interfering in DNA synthesis by ribonucleotide reductase inhibition, or
by interacting with DNA through non-covalent interactions including H-bonding and
intercalation [9]. Intercalation and groove binding are the most important DNA-binding modes
because they invariably lead to cellular degradation [10]. The current work describes the in vitro
anticancer activity of Pt(II) complexes of thiosemicarbazones of the combinatorial mixtures of
isatin derivatives. DNA binding of these complexes were conducted to evaluate the DNA
binding modes. The synthesis, physicochemical characterization and DNA binding activity of
mono platinum(II) complexes with bidentate ligands were reported. The in vitro cytotoxic
activities of compounds were tested against the human colorectal carcinoma cell line HCT 116.
3

2.
Experimental
2.1. Materials and methods
Isatin, 5-fluoroisatin, 5-methylisatin, 5-nitroisatin, 4-ethyl-3-thiosemicarbazide, 4-
methyl-3-thiosemicarbazide,
4-phenyl-3-thiosemicarbazide,
and
potassium
tetrachloroplatinate(II) were purchased from Aldrich Chemicals. 3-(4, 5-Dimethylthiazol-2-
yl)-2,5diphenyl tetrazolium bromide (MTT) was purchased from Sigma–Aldrich, Germany.
Commercial-grade solvents and reagents were used as supplied without further purification.
Calf thymus (CT) DNA, agarose (molecular biology grade), and ethidium bromide (EB) were
from Sigma (St. Louis, MO, USA). Elemental analysis was carried out using a PerkinElmer
2400 series-11 CHN/O analyzer (Waltham, MA, USA). Infrared, electronic, nuclear magnetic
resonance, and fluorescent spectra were recorded on PerkinElmer 2000, Perkin Elmer-
Lambda 25, and Bruker 500 MHz spectrometer at room temperature using DMSO-d6 using
as solvent and TMS as an internal standard. and Jasco FP-750 spectrophotometers,
respectively. Viscosity measurements were made using a Cannon Manning Semi-Micro
viscometer (State College, PA, USA). ESI-mass spectra were obtained using Liquid
Chromatography/Mass Spectrometry LC/MSD Trap VL (Agilent Technologies), it was
operated in either positive ion or negative ion detection mode.
2.2. Synthesis of ligands
Schiff bases (L1H to L6H) have been recently synthesized and characterized in our
laboratory [11-15]. In brief, Schiff base ligands were synthesized by refluxing the 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 (Scheme 1).
4

[Insert Scheme 1]
2.3.
Synthesis of complexes
The Pt(II) complexes were synthesized by refluxing the reaction mixture of hot ethanolic
solutions (30 ml each) of K2PtCl4 (0.01 mol) and appropriate ligand (0.01 mol) for 2 h. The
precipitates that formed during reflux were filtered and washed with cold ethanol (Scheme 2).
[Insert Scheme 2]
2.3.1.
C15H11ClN4OPtS (PtL1): Black; MP: >300 °C; yield: 72%; analytical
calculated values C (34.26%); H (2.11%); N (10.65%); Pt (37.10%); analytical results
(experimental): C (34.45%); H (2.42%); N (10.51%); Pt (37.22%); selected IR data (KBr
1
pellet, υmax/cm–1): 3309–3143 (NH), 1637 (C=O), 1614 (C=N), 767 (C-S); H NMR (500
MHz, DMSO-d6) ((δ (ppm)): 11.09 (s, 1 H, indole N-H), 10.62 (s, 1 H, CS-NH), 8.13 (d, 1 H,
indole C2-H, J = 5.3 Hz ), 7.50 (d, 2 H, thiosemicarbazide C11-H, C15-H, J = 7.4 Hz ), 7.45
to 7.41 (t, 2 H, thiosemicarbazide C12-H, C14-H, J = 7.8), 7.40 to 7.38 (dd, 1 H, indole C3-
H, J= 7.7, 1.1 Hz), 7.21 to 7.19 (t, 1 H, thiosemicarbazide C13-H, J= 7.3), 6.90 to 6.85 (q, 2
H, indole C4-H, C5, J = 7.6 Hz); UV-vis [DMSO λmax, nm]: 264, 409, 446 and ESI-MS (m/z):
523 (M-2H)2–.
2.3.2.
C16H13ClN4OPtS (PtL2): Black; MP: >300 °C; yield: 78%; analytical
calculated values: C (35.59%); H (2.43%); N (10.38%); Pt (36.13%); analytical results
(experimental): C (35.60%); H (2.17%); N (10.16%); Pt (36.20%); selected IR data (KBr
1
pellet, υmax/cm–1): 3326 to 3136 (NH), 1636 (C=O), 1613 (C=N), 750 (C-S); H NMR (500
MHz, DMSO-d6) (δ (ppm)): 11.95 (s, 1 H, indole N-H),10.56 (s, 1 H, CS-NH), 7.94 (s, 1 H,
5

indole C5-H), 7.51 (d, 2H, thiosemicarbazide C11-H, C15-H, J = 7.7 Hz), 7.45 to 7.42 (t, 2
H, thiosemicarbazide C12-H, C14-H, J = 7.8 Hz), 7.22 to 7.19 (t, 2 H, indole C3-H,
thiosemicarbazide C13-H, J = 7.1 Hz), 6.74 (d, 1 H, indole C2-H, J = 7.9), 2.13 (s, 3 H,
CH3);UV-vis [DMSO λmax, nm]: 265, 409, 450 and ESI-MS (m/z): 537.8 (M-2H)2–.
2.3.3.
C15H10ClFN4OPtS (PtL3): Brown; MP: >300 °C; yield: 72%; analytical
calculated values C (33.13%); H (1.85%); N (10.30%); Pt (35.87%); analytical results
(experimental): C (33.25%); H (1.63%); N (10.56%); Pt (14.20%); selected IR data (KBr
1
pellet, υmax/cm–1): 3308 (NH), 1637 (C=O), 1618 (C=N), 774 (C-S); H NMR (500 MHz,
DMSO-d6) (δ (ppm)): 11.08 (s, 1 H, indole N-H), 10.79 (s, 1 H, CS-NH), 7.91 (s, 1 H, indole
C5-H), 7.48 (d, 2 H, thiosemicarbazide C11-H, C15-H, J = 7.7 Hz), 7.44 to 7.14 (t, 2 H,
thiosemicarbazide C12-H, C14-H, J = 7.7 Hz), 7.28 to 7.21 (m, 2 H, indole C3-H,
thiosemicarbazide C13-H), 6.86 to 6.83 (dd, 1 H, indole C2-H, J = 8.5, 4.3 Hz); UV-vis
[DMSO λmax, nm]: 265, 406, 452 and ESI-MS (m/z): 540.7 (M-2H)2–.
2.3.4.
C10H8ClN5O3PtS (PtL4): Dark brown; MP: >300 °C; yield: 78%. analytical
calculated values C (23.61%); H (1.58%); N (13.76%); Pt (38.34%); analytical results
(experimental): C (23.58%); H (1.49%); N (13.44%); Pt (38.17%); selected IR data (KBr
1
pellet, υmax/cm–1): 3155 (NH), 1640 (C=O), 1625 (C=N), 741 (C-S); H NMR (500 MHz,
DMSO-d6) (δ (ppm)): 11.83 (s, 1 H, indole N-H), 9.56 to 9.54 (q, 1 H, CS-NH, J = 8.8, 4.3
Hz), 8.53 (d, 1 H, indole C5-H, J = 2.3 Hz), 8.28 to 8.26 (dd, 1 H, indole C3-H, J = 6.3, 2.4
Hz ), 7.13 (d, 1 H, indole C2-H, J = 8.7 Hz), 3.10 (d, 3H, thiosemicarbazide CH3, J = 4.5 Hz);
UV-vis [DMSO λmax, nm]: 259, 395, 450 and ESI-MS (m/z): 508.8 (M+H)+.
6

2.3.5.
C11H11ClN4OPtS (PtL5): Dark brown; MP: >300 °C; yield: 81%; analytical
calculated values C (27.65%); H (2.32%); N (11.73%); Pt (40.83%); analytical results
(experimental): C (27.95%); H (2.31%); N (11.84%); Pt (40.62%); selected IR data (KBr
1
pellet, υmax/cm–1): 3278 (NH), 1658 (C=O), 1615 (C=N), 765 (C-S); H NMR (500 MHz,
DMSO-d6) (δ (ppm)): 11.25 (s, 1 H, indole N-H), 9.25 to 9.23 (q, 1 H, CS-NH), 7.95 to 7.47
(dd, 1 H, indole C5-H), 7.25 to 7.16 (m, 1 H, indole C3-H), 6.89 to 7.77 (ddd, 1 H, indole C2-
H, J = 7.9 Hz), 3.10 (dd, 3 H, thiosemicarbazide CH3, J = 4.6 Hz), 2.27 (s, 3 H, indole CH3);
UV-vis [DMSO λmax, nm]: 265, 390, 460 and ESI-MS (m/z): 474.8 (M-2H)2–.
2.3.6. C24H26N8O2PtS2 (Pt(L6)2): The characterization data correspond to powder.
Brown; MP: >300 °C; yield: 75%; analytical calculated values C (40.16%); H (3.65%); N
(15.61%); Pt (27.78%); analytical results (experimental): C (40.21%); H (3.46%); N
(15.39%); Pt (27.57%); selected IR data (KBr pellet, υmax/cm–1): 3223 to 3180 (NH), 1693
1
(C=O), 1618 (C=N), 788 (C-S); H NMR (500 MHz, DMSO-d6) (δ (ppm)): 11.11 (s, 1H,
indole N-H ), 9.29 to 9.27 (t, 1H, CS-NH, J = 5.6 Hz), 7.50 (s, 1 H, indole C5-H), 7.18 to
7.16 (dd, 1 H, indole C2-H, J = 7.9 Hz ), 6.83 to 6.76 (dd, 1 H, indole C3-H, J = 7.9 Hz),
3.66 to 3.61 to (p, 2 H, thiosemicarbazide CH2, J = 6.6 Hz), 2.30 (s, 3H, indole CH3), 1.24 to
1.20 (t, 3H, thiosemicarbazide CH3, J = 7.1); and UV-vis [DMSO λmax, nm]: 266, 389, 455.
2.4.
X-ray crystallography
Single crystals of compound Pt(L6)2 suitable for diffraction were grown by slow
evaporation of 3:1 mixtures of acetone and DMF. Selected crystal data and data collection
parameters are presented in Table 1. Data of Pt(L6)2 were collected on a Bruker SMART
APEX II CCD diffractometer [16] equipped with graphite monochromatized Mo Kα radiation
(λ = 0.71073 ) at 100(1) K. Multi-scan absorption corrections were applied using the
7

SADABS program [16]. The structure was solved by the direct method using the SHELXS-
97 program [17]. Refinements on F2 were performed using SHELXL-97 by the full-matrix
least-square method with anisotropic thermal parameters for all non-hydrogen atoms. N-
bound H atoms were located in a difference Fourier map and were freely refined. The
remaining H atoms were geometrically positioned and refined using a riding model. The
program SHELXTL [17] was used to prepare Figs. 1 and 2.
[Insert Table1]
2.5.
DNA interaction studies
DNA binding experiments including absorption, emission spectral studies, and viscosity
measurements conformed to the standard methods [18- 20]. Concentrated CT DNA stock
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
absorption coefficient (6600 M-1 cm-1) at 260 nm [21]. The purity of the CT DNA was
verified by taking the ratio of the absorbance values at 260 and 280 nm in the respective
buffer, which was found to be >1.9, indicating that, the DNA was sufficiently free of protein.
The stock solutions were stored at 4 °C and used within 4 days. The DNA binding
experiments were performed at room temperature. All experiments were carried out by
keeping the concentration of platinum complexes constant (50 µM) while varying the DNA
concentration (0 to 200 µM). An equal amount of DNA was added to the platinum complexes
cuvette and the reference cuvette to eliminate the absorbance of CT DNA itself [22, 23], and
Tris buffer was subtracted through baseline correction. The UV–Vis and the emission spectra
were recorded after equilibration for 10 min after each addition. Viscosity experiments were
8

carried out using a Cannon Manning Semi-Micro viscometer (State College, PA, USA)
thermostated in a 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 Pt(II) complexes at various
concentrations (0–2.5 × 10–4M) were measured three times each with a digital stopwatch, and
the average flow time was calculated. Data were presented as (η/ηo)1/3 versus the ratio of the
concentration of the compound to CT DNA, where η is the viscosity of DNA in the presence
of complex, and ηo is that of DNA alone. Viscosity values were calculated from the observed
flow time of DNA containing solutions (t) corrected for that buffer alone (Tris–HCl/NaCl,
5:50 mM) (to), and η = (t − to).
2.6.
Anti-proliferative activity
2.6.1. Preparation of cell culture
HCT 116 cells were allowed to grow under optimal incubator conditions. Cells that had
reached a confluence of 70%–80% were chosen for cell plating. Old medium was aspirated
out of the plate, and then cells were washed using sterile phosphate buffered saline (PBS; pH
7.4) two to three times. PBS was completely discarded after washing. Trypsin was added and
evenly distributed onto the cell surface, and the cells were incubated at 37 °C in 5% CO2 for
1 min. Afterwards, the flasks containing the cells were gently tapped to aid cell segregation
and observed under an inverted microscope (if cell segregation was not satisfactory, the cells
were incubated for another minute). Trypsin activity was inhibited by adding 5 ml of fresh
complete medium (10% FBS). Cells were counted and diluted to get a final concentration of
2.5 × 105 cells/ml, and seeded into wells (100 µL cells/well). Finally, the cell-containing
plates were incubated at 37 °C with an internal atmosphere of 5% CO2.
2.6.2. MTT sssay
9

Cancer cells (100 µl cells/well, 1.5 × 105 cells/ml) were seeded on a 96-well microtitre
plate, which was incubated with CO2 overnight to allow cell attachment. The compounds
were diluted with media into the required concentrations from the stock. Various
concentrations (6.25–200 µM) of 100 µl of test substances were separately added to each
well containing the cells. Then, the plates were incubated at 37 °C with an internal
atmosphere of 5% CO2 incubator for 48 h. After this treatment period, the plates were treated
with 20 µl of MTT reagent and incubated again for 4 h. After this incubation period, 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 absorbance at 570 and 620 nm wavelengths was
measured using a standard Infinite 200 PRO multimode microplate reader (Tecan, USA).
Data were recorded and analyzed to estimate the effects of test compounds on cell viability
and growth inhibition. The percentage inhibition of cell proliferation was calculated from the
optical density obtained from the MTT assay. 5-Fluorouracil (5-FU) was used as the standard
reference drug [24]. Statistical difference between the treatments and control were evaluated
by one-way ANOVA followed by Tukey’s multiple comparison test. Differences were
considered significant at p < 0.05 and p < 0.01.
3.
Results and discussion
The platinum(II) complexes PtL1 to Pt(L6)2 were obtained in good yield from the
reaction of potassium tetrachloro platinate(II) with appropriate Schiff base (L1H to L6H) in
1:1 M ratio in the ethanol medium with reflux for 2 h (via Scheme 2). These complexes were
insoluble in common organic solvents, but soluble in DMF and DMSO.
3.1. Spectroscopic properties
3.1.1. IR studies
10

The characteristic IR bands recorded for the free ligands differ from those of the
related complexes and provided significant indications of the bonding sites of
thiosemicarbazone ligands. In comparison with the spectra of the Schiff base, the PtL1 to
PtL5 exhibited the band of ν(C=O) within 1637–1658 cm–1, showing a shift of the band to
lower wavenumbers. This finding indicates that the carbonyl oxygen is coordinated to the
metal ion. Meanwhile, the IR spectrum of the Pt(L6)2 complex shows the carbonyl oxygen
band as a strong band at 1693 cm–1, suggesting that the (C=O) group does not participate in
bonding. The band of ν(C=N) within 1613–1625 cm−1 in the metal complexes shows a shift
of the band to lower wavenumbers, which indicates that the nitrogen atom of the azomethine
group is coordinated to the metal ion. This finding is further supported by the band at around
741–788 cm−1 in the metal complexes because of ν(C−S) [25]. Thus, the IR spectral results of
PtL1– Pt(L6)2 provide strong evidence for the complexation of Schiff bases with metal ions
in tridentate mode, whereas the ligand L6 coordinates to the platinum ion in a bidenate
manner.
3.1.2. 1H NMR
1
The H NMR (d6-DMSO) spectra of Pt(II) complexes show approximately the same
peaks identical to those of the free ligands, except that the peak due to the NH group
resonance is absent. This finding is considered as additional evidence of the deprotonation of
NH.
3.1.3. UV-vis Studies
The UV-vis absorption spectra of the Pt(II) complexes were measured within the UV-
vis region (200–800 nm) using DMSO. Two absorption bands with varied intensity can be
observed. The bands observed within 389–409 nm) are attributable to metal-to-ligand charge
11

transfer transitions, whereas the bands observed at lower frequencies within 446–455 nm
correspond to d–d transitions [26, 27].
3.1.4. MS studies
The mass spectra of PtL1, PtL2, PtL3, and PtL5) recorded in the negative mode give
peaks at m/z 523, 537.8, 540.7, and 474.8, respectively. This result indicates the presence of
complexes [M-2H]2–, [M-2H]2–, [M-2H]2–, and [M-2H]2–, respectively. Meanwhile, the mass
spectrum of PtL4 recorded in the positive mode gives a peak at m/z 508.8, indicating the
presence of this complex as [M+H] +.
3.2. Crystal structure of Pt(L6)2
The molecular structure of Pt(L6)2 along with the atom numbering scheme is shown
in Fig. 1. Pt(L6)2 (cis-Pt(C12H13N4OS)2) presents a square-planar geometry that is slightly
distorted around the platinum atom, with the basal plane occupied by the S(1), S(1A), N(2)
and N(2A) atoms of two cis-thiosemicarbazone ligands. The distortion is evident from the
bond angles S(1)-Pt(1)-N(2) 82.46(7)°, S(1)-Pt(1)-S(1A) 95.71(3)°, S(1)-Pt(1)-N(2A)
174.46(6)°, S(1A)-Pt1-N2 174.46(6)°, N(2)-Pt(1)-N(2A) 99.84(10)°, and S(1A)-Pt(1)-N(2A)
82.46(7)°, as well as the bond lengths Pt(1)-S(1) 2.2537(8) Å, Pt(1)-N(2) 2.039(3) Å, Pt(1)-
S(1A) 2.2537(8) Å, and Pt1-N(2A) 2.039(3) Å. Fig. 2 shows the crystal packing of Pt(L6)2
viewed approximately along the a-axis. In the crystal, the molecules are connected together
through N-H…O intermolecular interactions into an infinite-one dimensional chain through
c-axis forming zigzag shape (Fig. 2).
[Insert Fig. 1 and Fig. 2]
3.3.
DNA interaction studies
3.3.1. Absorption spectrum studies
12

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
(bathchromism) as a consequence of the intercalation mode involving a strong stacking
interaction between an aromatic chromophore and the DNA base pairs [28]. It arises from the
contraction of CT DNA in the helix axis and its conformational changes [29, 30]. Meanwhile,
hyperchromism results from the secondary distortion of the DNA double-helix structure [31,
32]; the extent of hyperchromism indicates partial or non-intercalative binding modes [33].
The absorption spectra of 50 µM PtL3 and Pt(L6)2 in the absence and presence of CT DNA
(0 to 200 µM) are given in Figs. 3 and 4; the absorption spectra of PtL1, PtL2, PtL4, and
PtL5 are given in the Supplementary material (Figs. S1 –S4). The absorption spectra of PtL1
and Pt(L6)2 display clear hypochromism with a slight blue shift ~3–4 nm. After the
compounds intercalate to the DNA base pairs, the π* orbital of the intercalate compounds
couples with the π orbitals of the base pairs, thereby decreasing the π → π* transition
energies. These interactions result in the observed hypochromism [34]. The hypochromism of
these complexes follows the order of Pt(L6)2 > PtL1. Remarkably, PtL2, PtL3, PtL4, and
PtL5 exhibit both hypochromism and hyperchromism at ~402 – 444 and ~255 nm,
respectively, suggesting that the DNA double-helix structure is distorted after the complex
binds to DNA through the intercalation mode [35]. This behavior has been reported for chiral
Schiff base complexes [36, 37]. PtL1, PtL2, PtL3 and Pt(L6)2 show hypochromism with a
blue shift of ~3 nm, whereas PtL4 and PtL5 display a small blue shift of ~1 nm. The intrinsic
binding constant of all complexes with DNA was determined by observing the changes in
absorbance of the complexes with increased DNA concentration using the following equation
[38, 39]:
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb(εb - εf)
13

where εa, εf, and εb are the extinction coefficients observed for the absorption band at a
given DNA concentration for free and bound complex, respectively; [DNA] is the
concentration of DNA in base pairs; and the binding constant Kb was determined from the
_
slope-to-intercept ratio by plotting [DNA]/ (εa εf) vs. [DNA] (Figs. 3a and 4a). The
intrinsic binding constant (Kb) for PtL1, PtL2, PtL3, PtL4, PtL5, and Pt(L6)2 are found to be
1.59 × 106 , 6.3 × 105, 3.29 × 106, 3.5 × 104, 3.6 × 105, and 8.9 × 105 M–1, respectively. The
DNA binding abilities of PtL1, PtL2 and PtL3 are higher compared to other platinum
complexes. This is attributed to the effective 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 PtL3 is too high maybe due to the presence of fluorine
atom at C5 [40]. Comparison of the intrinsic binding constants of these complexes with
those of DNA-intercalative Pt(II) complexes such as [Pt(bpy)(pip)]2+ and [Pt(bpy)(hpip)]2+
[41] reveals that these complexes bind to DNA by intercalation, but proving the binding
mode requires more experiments.
[Insert Fig. 3 and Fig. 4]
3.3.2. Emission spectrum studies
To further study the interaction mode between PtL1–Pt(L6)2 and CT DNA,
fluorescence titration experiments were performed. These complexes are found to emit
luminescence in Tris–HCl/NaCl buffer at room temperature with maximum wavelengths of
about 379 nm (Supplementary material, Figs. S5–S8). The emission spectra of PtL2 and
Pt(L6)2 (50 µM) in the absence and presence of CT DNA (0 to 200 µM) are shown in Figs.
5 and 6. The emission intensity increases with increased CT DNA concentration. These
14

observations imply that these complexes can strongly interact with CT DNA and propose an
intercalative mode with the base pairs of the DNA helix [42].
[Insert Fig. 5 and Fig. 6]
3.3.3. Viscosity measurements
The most critical and least ambiguous tests of a binding in solution in the absence of
crystallographic data [43, 44] are hydrodynamic measurements (i.e., viscosity and
sedimentation), which are sensitive to length changes. A classical intercalation 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. Meanwhile, partial or nonclassical intercalation of the complex results in the bending
of the DNA helix that reduces the effective DNA length and viscosities. By contrast, an
electrostatic or groove binding mode has minimal effects on DNA viscosities [45, 46]. The
viscosity measurements of PtL1 to Pt(L6)2 and EB are shown in Figs. 7 and 8. The plot of
relative specific viscosity versus [complex]/[DNA] ratio shows a significant increase in the
ratio upon addition of the complex. These observations suggest the intercalative binding of
these complexes to the DNA double helix [47, 48], in agreement with the results of optical
absorption experiments.
[Insert Fig. 7 and Fig. 8]
3.4. Anti-proliferative activity
The in vitro anti-proliferative activity of PtL1–Pt(L6)2 was evaluated against HCT116
cells. Fig. 9 shows the effect of different concentrations of PtL1–Pt(L6)2 on HCT 116 cells
after 48 h of treatment. The anticancer efficiency of all tested compounds is listed in Table 2.
Results of the anti-proliferation test show that all tested compounds have a dose-dependent
15

effect. Fig. 10 shows the photomicrographic images of cancer cells treated with these
compounds for 48 h. First, cells treated with the standard drug 5-FU (IC50 = 7.3 µM) show
decreased viability. The cells reveal apoptotic characteristic features because cell membrane
blebbing, crescent-shaped nuclei, and nuclear condensation can be observed in the treated
cells. HCT 116 cells treated with PtL1 show a moderate cytotoxic effect (IC50 = 53.9 µM).
The treatment significantly reduces the doubling time of the cell population compared with
the negative control. Treatment with PtL2 shows a moderate inhibitory effect on cell
proliferation (IC50 = 61.3 µM), and the affected morphology of the treated cells can be clearly
visualized in the photomicrograph. The photomicrograph of HCT 116 cells treated with PtL3
shows the strong cytotoxic effect of the complex (IC50 = 2.8 µM). PtL3 is found to affect the
proliferation of almost all cells of the group and causes the cells to lose their spindle-shaped
structure and adherent property. Thus, except for a few live cells, only cellular debris can be
seen in the growth medium. The photomicrograph of HCT 116 cells treated with PtL4 show
poor anti-proliferative activity (IC50 = 149.9 µM). Treatment with PtL5 shows a considerable
anti-proliferative effect (IC50 = 22 µM) compared with the control. The photomicrograph
shows that treatment of HCT 116 cells with PtL5 reduces cell proliferation and renders the
cells less viable. Finally, treatment of HCT 116 cells with Pt(L6)2 demonstrates significant
anti-proliferative activity because the IC50 (7.8 µM) is found to be more or less similar to that
of the standard reference. The results presented in Table 2 revealed that as compared to the
compounds PtL1, PtL2, PtL3, PtL5 and PtL6, which has no substituent in the isatin scaffold
or substituent with methyl or fluoride, substitution of nitro group at its position-5 (PtL4; IC50
= 149 µM) reduced its activity [49].
16

[Insert Fig. 9, Table 2, and Fig. 10]
3.
Conclusion
Six novel square planar platinum(II) complexes, namely, PtL1 to Pt(L6)2, with a
tridentate ligands were synthesized and characterized using elemental analysis and various
spectroscopic techniques. The crystal structure of Pt(L6)2 shows that it is a four-coordinate
complex with a slightly distorted square-planar geometry. Results of binding studies showed
that these complexes have been found to interact with CT DNA through an intercalative
mode, which was supported by absorption studies, (the intrinsic binding constant (Kb) values
were found to be 3.5 × 104 to 3.29 × 106 M–1), fluorescence and viscosity
measurementtechniques, and their affinity to DNA follows the order Pt3 > PtL1 > PtL6 >
PtL2 > PtL5 > PtL4, which can be attributed to the presence of an extended aromatic phenyl
ring which allows an intercalating ligand to deeply penetrate into the DNA base pairs.
Finally, results of in vitro anti-proliferative activity against HCT 116 cells showed dose-
dependent cytotoxicity of the synthesized complexes, with significantly low IC50 values
ranging from 2.8 µM to 149.9 µM. The anti-proliferative effect of PtL3 was stronger than
that of the standard 5-FU, whereas Pt(L6)2, PtL5, PtL1, and PtL2 showed significant anti-
proliferation activity (less or equal to that of 5-FU). The strongest IC50 value for PtL3
compared to other complexes may be attributed to the presence of fluorine atom at C5 in
isatin moiety [50]. Therefore, the platinum(II) complexes of thiosemicarbazone Schiff bases
with isatin moiety can be promising anti-neoplastic agents. The toxic potentials of the most
active complexes and their anti-tumor efficacies are under investigation in animal models and
will be reported in due course.
17

Abbreviations
CT DNA Calf-thymus DNA
DMSO
EB
Dimethyl sulfoxide
Ethidium bromide
5-fluorouracil
5-FU
UV–Vis UV–visible
ROS
Reactive oxygen species
Acknowledgments
The authors thank the Malaysian Government and Universiti Sains Malaysia for the
FRGS research grant. AQA thanks the Ministry of Higher Education and the University of
Sabha (Libya) for a scholarship.
Appendixes Supplementary data
Crystallographic data for the structural analysis of the complex Pt(L6)2 has been deposited
with the Cambridge Crystallographic Data Center, CCDC No. CCDC909984. Copies of this
information may be obtained free of charge from The Director, CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK (fax: +44 1233336 033; e-mail: deposit@ccdc.cam.ac.uk).
18

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Scheme 1. Synthetic route and structures for compounds L1H-L6H.
Scheme 2. Synthetic route and structures for compounds PtL1-PtL6.
Fig.1. The structure of PtL6 complex, showing 50% probability displacement ellipsoids and
atomic numbering.
Fig.2. The crystal packing of PtL6, viewed approximately along the a axis.
Fig.3. Absorption spectra of PtL3 in the absence and in the presence of increasing amounts of
DNA in Tris HCl buffer (pH-7.2). [PtL3] = 50 µM, [DNA] = 0-163.58 x 10-6 M. The
arrow shows the effect of increasing of CT-DNA concentration on the absorption
intensity.
Fig. 3a. Comparative plot of [DNA]/(ɛa - ɛf) vs. [DNA] for the absorption titration of CT-DNA
with complex PtL3 in Tris HCl buffer (pH-7.2); Kp = 3.29 × 106 M-1(R = 0.9721, n = 6
points).
Fig.4. Absorption spectra of PtL6 in the absence and in the presence of increasing amounts of
DNA in Tris HCl buffer (pH-7.2). [PtL6] = 50 µM, [DNA] = 0-163.58 x 10-6 M. The
arrow shows the effect of increasing of CT-DNA concentration on the absorption
intensity.
Fig. 4a. Comparative plot of [DNA]/(ɛa - ɛf) vs. [DNA] for the absorption titration of CT-
DNA with complex PtL6 in Tris HCl buffer (pH-7.2); Kp = 3.6 × 105 M–1 (R =
0.9909, n = 6 points).
Fig.5. Emission spectra of PtL2 in the absence and in the presence of increasing amounts of
DNA in Tris HCl buffer (pH-7.2). [PtL2] = 50 µM, [DNA] = 0-163.58 x 10-6 M. The
arrow shows the emission intensity increases upon increasing the CT-DNA
concentration.
Fig.6. Emission spectra of PtL6 in the absence and in the presence of increasing amounts of
DNA in Tris HCl buffer (pH-7.2). [PtL6] = 50 µM, [DNA] = 0-163.58 x 10-6 M. The
arrow shows the emission intensity increases upon increasing the CT-DNA
concentration.
Fig.7. Effect of increasing amounts of PtL1 ( ), PtL2 (
viscosity of CT-DNA at 37.0 ( 0.1) °C.
) and PtL3 ( ) on the relative
23

Fig.8. Effect of increasing amounts of PtL4 ( ), PtL5 (
viscosity of CT-DNA at 37.0 ( 0.1) °C.
) and PtL6 ( ) on the relative
Fig.9. Effect of different concentrations of platinum(II) complexes of isatin
thiosemicarbazones derivatives (PtL1-PtL6) on human colorectal cancer cells
(HCT 116) after 48 hours treatment.
Fig.10. Picture of cancer cells treated with platinum(II) complexes of isatin
thiosemicarbazones derivatives (PtL1-PtL6) for 48 hr.
24

Figure(s)

Figure(s)

Figure(s)

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Figure(s)