Indole-7-carbaldehyde thiosemicarbazone as a flexidentate ligand toward ZnII, CdII, PdII and PtII ions: Cytotoxic and apoptosis-inducing properties of the PtII complex

Article abstract of DOI:10.1039/c3dt53032a

A new thiosemicarbazone (LH₂) derived from indole-7-carbaldehyde was synthesized and reacted with Zn^II, Cd^II, Pd ^II and Pt^II salts. The reactions with zinc and cadmium salts in 2:1 (ligand-metal) molar ratio afforded complexes of the type MX ₂(LH₂)₂, (X = Cl, Br or OAc), in which the thiosemicarbazone acts as a neutral S-monodentate ligand. In the presence of potassium hydroxide, the reaction of LH₂ with ZnBr₂ resulted in deprotonation of the thiosemicarbazone at the hydrazine and indole nitrogens to form Zn(L)(CH₃OH). The reaction of LH₂ with K₂PdCl₄ in the presence of triethylamine, afforded Pd(L)(LH₂) which contains two thiosemicarbazone ligands: one being dianionic N,N,S-tridentate while the other one is neutral S-monodentate. When PdCl₂(PPh₃)₂ was used as the Pd^II ion source, Pd(L)(PPh₃) was obtained. In a similar manner, the analogous platinum complex, Pt(L)(PPh₃), was synthesized. The thiosemicarbazone in the latter two complexes behaves in a dianionic N,N,S-tridentate fashion. The platinum complex was found to have significant cytotoxicity toward four cancer cells lines, namely MDA-MB-231, MCF-7, HT-29, and HCT-116 but not toward the normal liver WRL-68 cell line. The apoptosis-inducing properties of the Pt complex was explored through fluorescence microscopy visualization, DNA fragmentation analysis and propidium iodide flow cytometry.

Full text of DOI:10.1039/c3dt53032a

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Indole-7-carbaldehyde thiosemicarbazone as a
flexidentate ligand toward ZnII, CdII, PdII and PtII
ions: cytotoxic and apoptosis-inducing properties
of the PtII complex†
Cite this: Dalton Trans., 2014, 43,
3850
Abeer A. Ibrahim,a Hamid Khaledi,*a Pouya Hassandarvish,b Hapipah Mohd Alia and
Hamed Karimianb
A new thiosemicarbazone (LH2) derived from indole-7-carbaldehyde was synthesized and reacted with
ZnII, CdII, PdII and PtII salts. The reactions with zinc and cadmium salts in 2 : 1 (ligand–metal) molar ratio
afforded complexes of the type MX2(LH2)2, (X = Cl, Br or OAc), in which the thiosemicarbazone acts as a
neutral S-monodentate ligand. In the presence of potassium hydroxide, the reaction of LH2 with ZnBr2
resulted in deprotonation of the thiosemicarbazone at the hydrazine and indole nitrogens to form Zn(L)-
(CH3OH). The reaction of LH2 with K2PdCl4 in the presence of triethylamine, afforded Pd(L)(LH2) which
contains two thiosemicarbazone ligands: one being dianionic N,N,S-tridentate while the other one is
neutral S-monodentate. When PdCl2(PPh3)2 was used as the PdII ion source, Pd(L)(PPh3) was obtained. In
a similar manner, the analogous platinum complex, Pt(L)(PPh3), was synthesized. The thiosemicarbazone
in the latter two complexes behaves in a dianionic N,N,S-tridentate fashion. The platinum complex was
found to have significant cytotoxicity toward four cancer cells lines, namely MDA-MB-231, MCF-7,
HT-29, and HCT-116 but not toward the normal liver WRL-68 cell line. The apoptosis-inducing properties
of the Pt complex was explored through fluorescence microscopy visualization, DNA fragmentation
analysis and propidium iodide flow cytometry.
Received 28th October 2013,
Accepted 22nd November 2013
DOI: 10.1039/c3dt53032a
www.rsc.org/dalton
coordination (e.g. salicylaldehyde thiosemicarbazone), a D,N,S-
tricoordination normally takes place (3);5–9 however examples
Introduction
Thiosemicarbazones constitute a class of ligands that have are found in the literature where the potential O donor atom
attracted a great deal of attention owing to their versatile from a salicylaldehyde thiosemicarbazone does not bind to the
coordination behavior toward metal ions.1 Fig. 1 presents a metal and the ligand shows an N,S-bidentate mode (4 and
variety of coordination modes observed in thiosemicarbazone 5).9–11 In addition, the S-donor atoms of thiosemicarbazones
metal complexes. The coordination usually occurs through the can bridge metal ions to form dinuclear,12,13 or multinuclear
sulfur atom and either the azomethine N atom to create a five- metal complexes (6).14–16 Other coordination modes observed
membered chelate ring (1),2 or the hydrazine N atom to make in thiosemicarbazone metal complexes include S-monodentate
a more strained four membered ring (2).3,4 If the ligand (7),17–19 and N(thioamide),S-bidentate (8).20 The applications
contains a third donor site (D) appropriately located for of these metal complexes range from biological to material
sciences fields. Examples are cytotoxic, antibacterial and
antimalarial activities of thiosemicarbazone PdII and PtII
complexes,5–8,21 and non-linear optical properties associated
aDepartment of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia.
with thiosemicarbazone ZnII and CdII complexes.17–19
E-mail: hamid.khaledi@gmail.com
bDepartment of Molecular Medicine, University of Malaya, 50603 Kuala Lumpur,
The present paper introduces a new thiosemicarbazone
ligand (LH2, Fig. 1) derived from indole-7-carbaldehyde. The
Malaysia
†Electronic supplementary information (ESI) available: X-ray crystallographic structure of the molecule mimics that of salicylaldehyde thio-
files in CIF format, tables for the selected geometrical parameters for the crystal
semicarbazone: both thiosemicarbazones have the third poten-
tial donor atom (D) linked to the carbonylic carbon via two
intervening atoms; however, the third donor in LH2 is the
structures, tables for crystal data and refinement parameters, 1H, 13C NMR,
HSQC and HMBC spectra of selected compounds. CCDC 939908–939913 for
ZnBr2(LH2)2·2MeOH, CdCl2(LH2)2·MeOH, Cd(OAc)2(LH2)2·2MeOH, Pd(L)(LH2),
softer N atom vs. the harder O atom in the salicylaldehyde
derivative (4 and 5). The complexation behavior of LH2 toward
Pd(L)(PPh3)·DMF and Pt(L)(PPh3)·0.75 MeOH. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c3dt53032a
3850 | Dalton Trans., 2014, 43, 3850–3860
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Fig. 1 Various coordination modes observed in metal complexes of thiosemicarbazone ligands (1–8); the chemical diagram of LH2.
ZnII, CdII, PdII and PtII was studied. Furthermore, the syn- compounds do not show any shift from those of the free
thesized PtII complex was evaluated for its cytotoxicity and ligand suggesting that decomposition into the free ligand and
apoptosis-inducing properties.
metal salts in DMSO-d6 solution occurs. In contrast, the reac-
tion of LH2 with ZnBr2 in a 1 : 1 ratio and in the presence of
KOH afforded a complex with the formula of Zn(L)(CH3OH).
1
The H NMR spectrum of this complex confirms the deproto-
Results and discussion
nation of the indole NH and thioamide NNH of the ligand
while its 13C NMR spectrum shows an upfield shift of the CS
signal (∼4 ppm) and a downfield shift of the azomethine CHN
signal (∼4 ppm) from those in the spectrum of LH2. Attempts
to grow an X-ray quality crystal from this compound failed;
however, based on elemental analysis and NMR spectroscopy
the ligand in this compound is believed to act as a dianionic
tridentate chelate, coordinating the metal atom through its
sulfur, azomethine N and indole N atoms.
The condensation reaction of indole-7-carboxaldehyde with
thiosemicarbazide resulted in the formation of the corres-
ponding thiosemicarbazone, LH2. Similar to most thio-
semicarbazones, the predominant form of LH2 in both the
solid state and solution is the thione tautomer (rather than
the thiol tautomer). It is indicated by the absence of the
S–H band in the IR spectrum (at ca. 2700 cm−1) and any
signal attributable to the thiol proton in the 1H NMR spec-
trum. The NNH signal of the thione is observed at δ =
11.25 ppm.
The synthesized thiosemicarbazone, LH2, was reacted with
Zn, Cd, Pd and Pt salts under various conditions to afford the
corresponding metal complexes in moderate to high yield
(Scheme 1). Upon reaction of LH2 with MX2 (M = Zn or Cd; X =
Cl, Br or OAc) in a ligand–metal ratio of 2 : 1, metal complexes
with the formula of MX2(LH2)2 were obtained. As revealed by
X-ray crystallographic analysis, in the solid state structures of
these complexes, LH2 acts as a neutral S-monodentate ligand;
however, the solution 1H and 13C NMR signals of the
The reaction of LH2 with K2PdCl4 in a 2 : 1 (ligand–metal)
ratio and in the presence of triethylamine, led to the formation
of Pd(L)(H2L). In agreement with the X-ray crystal structure,
1
the solution H and 13C NMR spectra of this complex clearly
shows the existence of two types of the thiosemicarbazone
ligand in the structure, one being doubly deprotonated while
the other one is neutral. The upfield shift (5–6 ppm) of the two
CS signals in the 13C NMR spectrum of the complex compared
to that of the free LH2 suggests that the S atoms of both
ligands remains coordinated to the PdII center in the DMSO-d6
solution.
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Scheme 1 Reactions of LH2 with the selected metal salts.
The reaction of LH2 with PdCl2(PPh3)2 in a 1 : 1 ratio and in same spectra as before, indicating the stability of the complex
the presence of triethylamine, yielded Pd(L)(PPh3). The plati- in DMSO solution.
num analog, Pt(L)(PPh3), was similarly obtained. The IR
Crystal structure of ZnBr2(LH2)2·2MeOH
spectra of the two complexes closely resemble each other and
agree with reported peaks for coordinated PPh3 (e.g., 745, 691
The molecular structure of ZnBr2(LH2)2 is shown in Fig. 2 and
the selected bond lengths and angles are listed in Table S1.†
Two thiosemicarbazone molecules, acting as neutral mono-
dentate ligands, coordinate the metal center through their
sulfur atoms. The two ligands are almost planar with r.m.s.
deviation from the planes of S1 and S2 containing ligands
being 0.0503 and 0.0284 Å respectively; the dihedral angle
between the planes is 60.92(7)°. Two Br atoms complete the
distorted tetrahedral geometry around the Zn atom with
coordination angles in the range of 100.26(3)–113.80(2)°. The
Zn–S bond lengths are comparable to those observed in
similar structures,17,19,22 and so are the Zn–Br distances.19
There is intramolecular hydrogen bonding between hydrogens
1
and 537 for the Pd complex).6 The solution H and 13C NMR
spectra of the Pd complex do not show any characterizable
species, suggesting the instability of the compound in DMSO
solution. In contrast, the 1D- and 2D-NMR spectra of the Pt
complex in DMSO-d6 is in accordance with the solid state
1
structure. In the H NMR spectrum of the platinum complex
the azomethine CHN signal (δ = 8.80 ppm) appears as a
doublet due to the coupling with the phosphorus atom.5,10
The phosphorus atom is also coupled with some of the carbon
atoms so that their signals appear as doublets in the 13C NMR
spectrum. It is worth mentioning that the NMR solution of the
Pt complex, kept at room temperature for a month, gave the
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Fig. 4 The molecular structure and atom labeling scheme of Cd-
(OAc)2(LH2)2 (25% probability ellipsoids). Methanol solvate molecules are
not shown.
Fig. 2 The molecular structure of ZnBr2(LH2)2 with thermal ellipsoids
drawn at the 30% probability level. Methanol solvate molecules are not
shown.
coordination geometry can be determined using the index τ =
(β − α)/60 where β is the largest angle and α is the second one
around the metal center.24 For an ideal square pyramidal geo-
metry τ = 0, while it is 1 for a perfect trigonal bipyramidal geo-
metry. In the present structure τ computes to 0.23 which
indicates a distorted square-pyramidal geometry. The r.m.s.
deviation from planarity for the S1 and S2 containing ligands
are 0.068 and 0.095 Å, respectively, and the two planes make a
dihedral angle of 4.96(12)°. There is intramolecular hydrogen
bonding between thioamide nitrogens N4 and N8 and the
monodentate acetate ligand (Table S3†). In the crystal, the
complexes are connected through intermolecular N–H⋯O and
N–H⋯N interactions to form infinite chains along the crystal-
lographic a direction. The methanol solvate molecules are H-
bonded to the formed chains.
bonded to N3 and N7 and the Br ligands (Table S3†). More-
over, intermolecular N–H⋯Br interactions occur in the crystal
to connect the adjacent molecules into chains along the a axis.
The crystal structure contains methanol solvent molecules
which are H-bonded to these chains.
Crystal structure of CdCl2(LH2)2·MeOH
The molecular structure is depicted in Fig. 3 and Table S1†
lists the selected bond lengths and angles. The structure is
similar to that of the Zn complex. The thiosemicarbazone
molecules are nearly planar (r.m.s. deviation = 0.0236 and
0.0715 Å for S1 and S2 containing ligands, respectively) and
make a dihedral angle of 80.97(6)°. The Cd–S distances are
compatible with those in similar structures.18,23 Unlike the
zinc complex structure, the intramolecular H-bonding occurs
between the thioamide hydrogens, N4 and N8, and the halide
ligands (Table S3†). The crystal packing shows interdigitating
layers in the bc plane formed by intermolecular N–H⋯Cl inter-
actions. Disordered solvent methanol molecules are N–H⋯O
bonded to these layers.
Crystal structure of Pd(L)(HL2)
The crystal structure of Pd(L)(HL2) is depicted in Fig. 5. The Pd
center is coordinated by two different thiosemicarbazone
ligands, one of them being dianionic N,N′,S-tridentate, while
the other one acts as a neutral S-monodentate ligand. The two
ligands are almost planar with r.m.s. deviation from planarity
for their non-H atoms of 0.087 and 0.034 Å for the tridentate
and monodentate ligands respectively. The two planes make a
dihedral angle of 77.67(3)°. The selected bond lengths and
angles for the structure are collected in Table S2.† The longer
S1–C10 bond length than S2–C20 distance points out the
higher thiolic character of the tridentate ligand compared to
the monodentate ligand. The two ligands differ also in their
configuration about N3–C10 (Z) and N8–C20 (E) bonds, in
Crystal structure of Cd(OAc)2(LH2)2·2MeOH
The asymmetric unit of the crystal structure consists of two
crystallographically independent Cd complexes and four
methanol molecules. The two complexes have only slightly
different geometrical parameters; the weighted r.m.s. fit for
the superposition of the non-H atoms in both molecules is
0.103 Å. The structure of one of the complex molecules is
depicted in Fig. 4 and selected bond lengths and angles are
listed in Table S1.† The CdII atom is coordinated by two thio-
semicarbazone molecules which behave as neutral S-donor
monodentate ligands. One monodentate and one bidentate
acetate groups complete a five-coordinate metal complex. The
Fig. 3 The molecular structure of CdCl2(LH2)2 with thermal ellipsoids
drawn at the 30% probability level. Methanol solvate molecules are not
shown.
Fig. 5 The molecular structure of Pd(L)(HL2) with thermal ellipsoids
drawn at 30% probability level.
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keeping with that observed in the structures of similar Pd com- Crystal structure of Pt(L)(PPh3)·0.75MeOH
plexes.25,26 The metal atom is in a distorted square-planar geo-
The molecular structure of the platinum(II) complex (Fig. 7)
resembles that of Pd(L)(PPh3). The planar thiosemicarbazone
(r.m.s. deviation = 0.0812 Å) is doubly deprotonated and co-
ordinated to the platinum atom through its indolic N, azo-
methine N and thioamide S atoms to form a five- and a six-
membered chelate ring. A triphenylphosphine ligand at the
fourth coordination site completes a distorted square-planar
PtII complex; the metal center is displaced from the coordi-
nation plane, N1/N2/S1/P1, by 0.0578 Å. Table S2† compiles
the selected bond lengths and angles for the structure. The Pt–
P is slightly shorter and Pt–N2 slightly longer than corres-
ponding bond lengths in similar structures.27,28 In the crystal,
the molecules are connected through C–H⋯π interactions to
form hexagonal channels along the c-axis (void size = 264 Å3)
within which there is no evidence for included solvent (Fig. 8).
The outer-shell of the channel is solvated by the partially
occupying methanol molecules (Table S3†).
metry and is displaced by 0.0575 (6) Å out of the coordination
plane (N1/N2/S1/S2). In the crystal, pairs of the metal complex
molecules are doubly connected through N8–H8A⋯N3 hydro-
gen bonding into centrosymmetric dimers (Table S3†). The
resulting dimers are connected to each other via C–H⋯π and
N–H⋯π interactions to form a three-dimensional network with
voids of the size of 331 Å3 wherein disorder solvent molecules
reside.
Crystal structure of Pd(L)(PPh3)·DMF
The molecular structure of Pd(L)(PPh3) and the labeling
scheme is depicted in Fig. 6. The doubly deprotonated thio-
semicarbazone ligand coordinates the Pd atom in an N,N′,S-tri-
dentate manner to create five- and six-membered chelate
rings. A triphenylphosphine group occupies the fourth coordi-
nation site, making a square-planar palladium complex with
the metal atom being 0.0236 Å out of the coordination plane,
N1/N2/S1/P1. The ligand is almost planar with an r.m.s. devi-
ation of 0.0536 Å. Table S2† lists the selected bond lengths
and angles for the structure. While the Pd–N1 and Pd–S dis-
tances are comparable to those in Pd(L)(LH2), the Pd–N2 dis-
tance in the present structure is 0.0431 Å longer than that in
the former structure. This can be attributed to the trans-influ-
ence of the PPh3 ligand as was observed in similar palladium
complex structures.7,8 In the crystal, pairs of the metal
complex molecule are connected into centrosymmetric dimers
through N4–H4B⋯N3 hydrogen bonds and these are linked
via C–H⋯π interactions to form a three-dimensional polymeric
structure (Table S3†). In the crystal, disordered DMF solvate
molecules exist as hydrogen bond acceptors in N4–H4A⋯O
interactions.
Cytotoxicity assay
Preliminary cytotoxicity screening for LH2 and its Pd and Pt
complexes against four cancer cell lines, revealed significant
activity for Pt(L)PPh3. Table 1 lists the IC50 values of the plati-
num complex after treating the cell lines for 24, 48 and 72 h.
As shown by the results, for 24 h treatments the IC50 values lie
between 2.08–22.97 µM. These values dropped when the treat-
ments were prolonged, thus the IC50 values for 48 h treatments
range from 1.49 to 3.34 µM and for 72 h treatments are
between 0.77–2.77 µM. The effect of the platinum complex on
normal liver cell lines (WRL-68) was also examined. As shown
in Fig. 9, minimal toxicity was observed at the concentrations
below 40 µM, attesting to cancer-selective cytotoxicity of Pt(L)-
(PPh3) at low doses. For the sake of comparison, cells were also
Fig. 6 The molecular structure of Pd(L)(PPh3) with thermal ellipsoids
drawn at 50% probability level. The DMF solvate molecules are not
shown.
Fig. 7 The molecular structure and atom labeling scheme of Pt(L)-
(PPh3) (50% probability ellipsoids). Methanol solvate molecules are not
shown.
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Fig. 9 Cytotoxicity effect of Pt(L)(PPh3) on normal liver WRL-68 cells.
propidium iodide (PI) is a fluorochrome mixture for nuclear
staining which enables distinction between viable, apoptotic
and necrotic cells. In this work, the four cancer cell lines were
subjected to AO/PI staining after treatment with respective IC50
concentrations of Pt(L)PPh3 for 48 h. Fluorescence microscopy
reveals that in all the cancer lines the platinum complex
induced cell death mainly via apoptosis (Fig. 10).
Fig. 8 Crystal packing view of Pt(L)(PPh3)·0.75MeOH, showing channels
along the c-axis. Hydrogen bonds are depicted as red dashed lines.
treated with cisplatin, a clinical anticancer drug. As presented
in Table 1, compared to cisplatin, Pt(L)(PPh3) exerted higher
or comparable cytotoxicity against the cancer cell lines and
higher selectivity for the cancer cells over normal cells.
Fluorochrome-labelled Annexin V staining is another way
for microscopic visualization of the apoptotic cells. Normal
cells have the phospholipid phosphatidylserine (PS) on their
inner-leaflet of the membranes, but upon undergoing apopto-
sis, PS becomes exposed on the external side of the cellular
membrane. Annexin V binds to cell membranes containing
exposed PS, thus labelled annexin V can be used to detect apo-
ptotic cells at early stages. In this study FITC-annexin V was
applied to target the apoptotic cells after treatment of the
cancer cells with IC50 concentrations of Pt(L)PPh3 for 48 h.
The fluorescence microscopic examination of the treated cells
when compared to those grown under the control conditions
attest to the binding of FITC-annexin V to the externalized PS
in the treated cells, thus induction of apoptosis by the plati-
num complex in the four cancer cell lines (Fig. 11).
Apoptosis assay
Apoptosis induction is an anti-proliferative mechanism by
which the cancer cells undergo programmed death. Cells
undergoing apoptosis are characterized by morphological and
biochemical changes including cell shrinkage, chromatin
condensation and DNA fragmentation. Based on the results of
the in vitro cytotoxicity assay for Pt(L)PPh3, it was considered
worthwhile to explore the apoptotic properties of the com-
pound. Apoptosis in the four cancer cell lines was examined
through fluorescence microscopy visualization, DNA fragmen-
tation analysis and propidium iodide flow cytometry.
DNA Fragmentation analysis
Fluorescence microscopy visualization
The degradation of nuclear DNA into nucleosomal units is a
Apoptotic cells exhibit increased plasma membrane per- biochemical feature of apoptosis. To evaluate the apoptotic
meability to certain fluorescent dyes. Acridine orange (AO)/ DNA fragmentation in the cancer cells, the four cell lines were
Table 1 IC50 (µM) values SEM of Pt(L)(PPh3) and cisplatin on different cancer cell lines
24 hours
48 hours
72 hours
MDA-MB-231
MCF-7
Pt(L)(PPh3)
Cisplatin
Pt(L)(PPh3)
Cisplatin
Pt(L)(PPh3)
Cisplatin
Pt(L)(PPh3)
Cisplatin
22.97 4.13
18.09 1.32
2.23 0.13
14.76 0.58
2.08 0.12
72.68 0.92
3.71 0.10
38.73 8.23
52.58 0.71
>80
3.34 0.18
16.37 0.65
1.49 0.04
11.89 0.71
1.74 0.29
36.71 3.48
2.41 0.24
23.95 5.18
48.01 0.49
13.58 2.34
2.27 0.28
15.64 0.77
0.77 0.045
9.13 0.62
0.94 0.02
5.76 1.49
1.52 0.04
5.76 1.49
48.53 0.67
10.32 1.62
HT-29
HCT-116
WRL-68
Pt(L)(PPh3)
Cisplatin
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Fig. 10 Fluorescent microscopy of AO/PI stained cancer cells. The first row shows the cells without any treatment and the second row shows the
cells after treatment with IC50 concentrations of Pt(L)PPh3 for 48 h. Cells with intact membranes are stained green. Apoptotic cells appear in
orange–yellow and the necrotic cells are stained red.
Fig. 11 Fluorescence microscopy of the cancer cells stained by FITC–annexin V. The first row shows the cells without any treatment and the
second row shows the cells treated with Pt(L)PPh3 at IC50 concentrations for 48 h. FITC–annexin V positive cells show bright green fluorescence.
incubated with the corresponding IC50 values of Pt(L)PPh3 for Cell cycle analysis
48 h. The cells were then lysed, the nuclear DNA was extracted,
and subjected to gel electrophoresis. Inspection of the electro- The DNA content of cells duplicates during the S phase, there-
phoretic profiles revealed a ladder formation (fragments range fore cells in the G0 and G1 phases (before the S phase) have
from 400 to 1000 bp), attesting to the occurrence of apoptosis unreplicated DNA, while those in the G2 and M phases (after
(Fig. 12).
the S phase) have replicated DNA. Analysis of cell cycles by
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arrested in the G0/G1 phase whereas the HT-29 and MCF-7 cell
cycle arrests occurred in the S phase. Moreover, the histogram
shows gradual increases in the populations of sub-G0/G1 cells
during the incubation periods. Overall flow cytometry suggests
the induction of apoptosis by the platinum complex in the
cancer cells through G0/G1 or S phase cell cycle arrest.
Conclusion
LH2, a new thiosemicarbazone whose structure resembles that
of salicylaldehyde thiosemicarbazone, is a polydentate ligand
having flexidentate character toward metal ions. In the
examples in this study it exhibits two coordination modes, i.e.
S-monodentate (in its neutral form) and N,N,S-tridentate (in a
dianionic form). The latter creates a five- and a six-membered
chelate ring with the metal ion. It would appear that the
coordination mode is ruled by the basicity of the reaction
medium, ligand/metal ratio and the identity of the metal salt.
The indole N atom is either deprotonated and coordinated to
the metal center or is intramolecularly H-bonded to the azo-
methine N atom. In all the examples, regardless of the charge
and denticity of the ligand and the coordination geometry of
the complex, the thiosemicarbazone ligands keep their
planarity.
Fig. 12 Gel electrophoresis of apoptotic DNA fragmentation. Lane A:
1000 bp molecular weight marker; lane B: untreated MDA-MB-231 cells;
lane C: treated MDA-MB-231 cells; lane D: untreated MCF-7 cells; lane
E: treated MCF-7 cells; lane F: untreated HT-29 cells; lane G: treated
HT-29 cells; lane H: untreated HCT-116 cells; lane I: treated HCT-116
cells.
flow cytometry enables us to distinguish and quantify the cells
in different phases of the cell cycle. On the flow cytometry
DNA histograms cells with degraded and thus hypodiploid
DNA are represented in so-called “sub-G0/G1” peaks therefore,
sub-G0/G1 is a specific marker of cell death by apoptosis.29
Flow cytometry analysis of the four cancer cell lines after treat-
ments with respective IC50 concentrations of the Pt(L)PPh3 for
24 and 48 h was carried out. The DNA histograms (Fig. 13)
suggest that the HCT-116 and MDA-MB-231 cell cycles were
The platinum complex introduced in this article has shown
a remarkable and cancer-selective cytotoxicity against a variety
Fig. 13 Flow cytometric analysis of cell cycle distribution in the four cancer cell lines treated with IC50 concentrations of Pt(L)PPh3 for 24 and 48 h.
Data were shown as mean SEM. * ρ < 0.05 vs. controls.
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of cell lines as inferred from the MTT-based IC50 values and mixture was refluxed for 4 h and then set aside at room temp-
different apoptotic assay methods thus, can be considered as erature overnight whereupon X-ray quality crystals of Cd-
a potential anticancer drug.
(LH2)2(OAc)2·2MeOH were obtained. After the crystal structure
determination, the crystals were ground and dried at 50 °C
and used for further analysis. Yield: 0.169 g, 51%. Anal. Calcd
For C24H26CdN8O4S2: C, 43.21; H, 3.93; N, 16.80. Found: C,
42.66; H, 4.66; N, 16.54%. IR (ATR): 3415, 3265, 3159, 1610,
Experimental
Materials and measurements
1542, 1372, 1339, 1294, 829, 793, 730, 693 cm−1
.
Indole-7-carbaldehyde was purchased from Sigma-Aldrich
Company. Ethanol was distilled prior to use. The IR spectra
were obtained using a Perkin-Elmer Spectrum 400 ATR-FTIR
spectrometer. The NMR spectra were recorded with a JEOL
Lambda 400 MHz FT-NMR spectrometer.
Zn(L)(CH3OH). To a solution of LH2 (0.218 g, 1 mmol)
and KOH (0.14 g, 2.5 mmol) in methanol (10 mL), a solution
of ZnBr2·2H2O (0.261 g, 1 mmol) in the same solvent (10 mL)
was added and the mixture was stirred at room temperature
for 24 h. The resulting precipitate was filtered, washed
with 50% aqueous ethanol and dried over silica gel. Yield:
0.23 g, 74%. Anal. Calcd For C20H20Br2N8S2Zn: C, 42.12; H,
Synthesis
LH2. A mixture of indole-7-carbaldehyde (1.45 g, 10 mmol) 3.86; N, 17.86. Found: C, 42.13; H, 3.42; N, 18.05%. IR
and thiosemicarbazide (0.91 g, 10 mmol) in ethanol (25 mL) (ATR): 3424, 3252, 3142, 1604, 1538, 1372, 1330, 1280, 896,
containing a few drops of acetic acid was refluxed for 3 h. The 824, 795, 734 cm−1 1H NMR (DMSO-d6): δ 3.13 (d, 3H,
.
solution was then partially evaporated followed by addition of CH3OH); 4.12 (q, 1H, CH3OH); 6.29 (s, 2H, NH2); 6.37 (d, 1H,
distilled water to precipitate the yellowish product. It was fil- Ar-H); 6.83 (t, 1H, Ar-H); 7.03 (d, 1H, Ar-H); 7.49 (d, 1H, Ar-
tered, washed with 30% aqueous ethanol and dried over silica H); 7.51 (d, 1H, Ar-H); 8.40 (s, 1H, CHN) ppm. 13C NMR
gel. Yield: 1.9 g, 87%. Anal. Calcd for C10H10N4S: C, 55.02; H, (DMSO-d6): δ 49.13 (CH3OH); 100.74, 116.52, 119.15, 122.16,
4.62; N, 25.67; S, 14.69. Found: C, 55.13; H, 4.45; N, 25.33%. IR 123.72, 131.53, 137.74, 140.61 (Ar); 150.22 (CHN); 173.23 (CS)
(ATR): 3445, 3407, 3383, 3234, 3145, 1610, 1526, 1363, 1335, ppm.
1
1290, 944, 897, 825, 795, 728, 712 cm−1. H NMR (DMSO-d6): δ
Pd(L)(H2L). A solution of K2PdCl4 (0.163 g, 0.5 mmol) in
6.53 (dd, 1H, Ar-H); 7.06 (t, 1H, Ar-H); 7.17 (d, 1H, Ar-H); 7.37 methanol (5 mL) was added to a solution of LH2 (0.218 g,
(t, 1H, Ar-H); 7.65 (d, 1H, Ar-H); 8.25 (s, 1H, NH2); 8.28 (s, 1H, 1 mmol) in the same solvent (10 mL) followed by addition of a
NH2); 8.33 (s, 1H, CHN); 10.98 (s, 1H, indole NH); 11.25 (s, 1H, few drops of triethylamine. The mixture was refluxed for 5 h,
NNH) ppm. 13C NMR (DMSO-d6): δ 102.42, 117.59, 119.63, and then set aside at room temperature for one day whereupon
123.60, 126.19, 127.03, 129.23, 131.17 (Ar); 146.47 (CHN); brownish crystals of the product were formed. X-ray crystallo-
177.64 (CS) ppm.
graphic analysis of the crystals showed inclusion of disordered
ZnBr2(LH2)2. A solution of ZnBr2·2H2O (0.13 g, 0.5 mmol) in solvent molecules in the crystal, therefore for the other analy-
methanol (5 mL) was added to a solution of LH2 (0.218 g, sis purposes the crystals were ground and dried at 50 °C. Yield:
1 mmol) in the same solvent (10 mL). The mixture was 0.194 g, 72%. Anal. Calcd For C20H18N8PdS2: C, 44.41; H, 3.35;
refluxed for 4 h and then set aside at room temperature for 5 N, 20.71. Found: C, 44.41; H, 3.69; N, 20.32%. IR (ATR): 3420
days whereupon crystals of ZnBr2(LH2)2·2MeOH were formed. (sh), 3309, 1600, 1550, 1506, 1304, 1231, 793, 732 cm−1 1H
.
After the structure determination by X-ray crystallography, NMR (DMSO-d6): δ 6.48 (d, 1H, Ar-H); 6.57 (dd, 1H, Ar-H); 6.62
the crystals were ground and dried at 50 °C for the other (br, 2H, NH2); 6.95 (t, 1H, Ar-H); 7.10 (t, 1H, Ar-H); 7.30 (d, 1H,
analysis purposes. Yield: 0.225 g, 68%. Anal. Calcd For Ar-H); 7.38 (d, 1H, Ar-H); 7.43 (m, 1H, Ar-H); 7.73 (m, 3H, Ar-
C20H20Br2N8S2Zn: C, 36.30; H, 3.05; N, 16.93. Found: C, 36.69; H); 8.38 (s, 1H, CHN); 8.53 (s, 1H, CHN); 9.04 (s, 1H, NH2); 9.31
H, 3.13; N, 16.77%. IR (ATR): 3441, 3329, 3227, 1603, 1546, (s, 1H, NH2); 11.14 (s, 1H, indole NH); 12.18 (s, 1H, NNH)
1375, 1337, 1284, 950, 814, 792, 725, 717 cm−1
CdCl2(LH2)2. A solution of CdCl2·2H2O (0.11 g, 0.5 mmol) 117.59, 119.74, 124.82, 124.89, 125.89, 127.21, 127.40, 129.34,
in methanol (5 mL) was added to methanolic 129.93, 131.30, 133.45, 135.29 (Ar); 147.52, 151.63 (CHN);
solution (10 mL) of LH2 (0.218 g, 1 mmol). The mixture 171.68, 172.92 (CS) ppm.
.
ppm. 13C NMR (DMSO-d6): δ 101.69, 102.69, 116.59, 116.83,
a
was refluxed for 4 h, then set aside at room temperature for
Pd(L)(PPh3). A solution of trans-PdCl2(PPh3)2 (0.702 g,
one day whereupon X-ray quality crystals of CdCl2(LH2)2. 1 mmol) in methanol (10 mL) was added to a solution of LH2
MeOH were formed. After the structure determination, the (0.218 g, 1 mmol) in the same solvent (10 mL) followed by
crystals were ground and dried at 50 °C. Yield: 0.216 g, 79%. addition of a few drops of triethylamine. The mixture was
Anal. Calcd For C20H20CdCl2N8S2: C, 38.75; H, 3.25; N, 18.08. refluxed for 5 h and then set aside at room temperature for a
Found: C, 38.57; H, 3.36; N, 17.98%. IR (ATR): 3399, 3285, few days whereupon the orange crystals of the product
3187, 1605, 1549, 1371, 1337, 1287, 953, 893, 815, 797, 731, appeared. The crystals were ground and dried at 50 °C. Yield:
715 cm−1
.
0.480 g, 82%. Anal. Calcd For C28H23N4PPdS: C, 57.49; H, 3.96;
Cd(OAc)2(LH2)2. A solution of Cd(OAc)2·2H2O (0.133 g, N, 9.58. Found: C, 57.23; H, 4.04; N, 9.27%. IR (ATR): 3442 (w),
0.5 mmol) in methanol (10 mL) was added to a solution 3320 (w), 1603, 1556, 1517, 1435, 1306, 1233, 792, 745, 691,
of H2L (0.218 g, 1 mmol) in the same solvent (10 mL). The 537 cm−1
.
3858 | Dalton Trans., 2014, 43, 3850–3860
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X-ray quality crystals of Pd(L)(PPh3)·DMF were obtained on a plate for each concentration) for 24, 48 and 72 h. Treated
from a DMF solution of the complex on standing for four days and untreated cells were inspected qualitatively using an
at room temperature.
inverted light microscope (100×). Then, 10 μL of MTT (5 mg
Pt(L)(PPh3). A solution of cis-PtCl2(PPh3)2 (0.791 g, 1 mmol) mL−1) was added to each well and the plates were incubated at
in methanol (10 mL) was added to a solution of LH2 (0.218 g, 37 °C for 4 h. The media was then gently aspirated, and 100 μL
1 mmol) in the same solvent (10 mL) followed by addition of a DMSO was added to dissolve the formazan crystals. The
few drops of triethylamine. The mixture was refluxed for 6 h amount of formazan product was measured spectrophoto-
and then set aside at room temperature overnight whereupon metrically at 570 nm using a microplate reader (Power Wave
X-ray quality crystals of the product were obtained. After the X 340). The concentration of the test compound required for
crystal structure determination, the crystals were washed with 50% inhibition of cell growth (IC50) was determined by inter-
50% aqueous ethanol, ground and dried at 50 °C. Yield: polation of regression analysis.
0.512 g, 76%. Anal. Calcd For C28H23N4PPtS: C, 49.92; H, 3.44;
N, 8.32. Found: C, 49.69; H, 3.04; N, 8.28%. IR (ATR): 3441(w),
Apoptosis detection with fluorescence microscopy
3318 (w), 1603, 1556, 1520, 1434, 1304, 1233, 791, 743, 689,
AO/PI staining assay. The cancer cells were seeded in a
538 cm−1. H NMR (DMSO-d6): δ 6.13 (d, 1H, Ar-H); 6.65 (br s, 25 mL culture-flask (1 × 106 cells per mL) and treated with the
2H, NH2); 6.72 (d, 1H, Ar-H); 7.10 (t, 1H, Ar-H); 7.47–7.55 (m. Pt complex at the corresponding IC50 concentrations for 48 h
9H, Ar-H); 7.61 (d, 1H, Ar-H); 7.68–7.73 (dd, 6H, Ar-H); (control cells were treated with 0.5% DMSO vehicle). The cells
7.75–7.77 (d, 1H, Ar-H); 8.80 (d, 1H, CHN) ppm. 13C NMR were then washed with phosphate buffered saline (PBS)
(DMSO-d6): δ 102.75, 117.21, 117.57, 125.86, 127.48, 128.72 and suspended in 500 μL of PBS followed by addition of a
(Ar); 129.13 (d, J [31P–13C] = 10.49 Hz, Ar); 130.14, 130.75 (Ar); 1 : 1 mixture of AO (10 μg mL−1) and PI (10 μg mL−1). A drop of
132.21 (d, J [31P–13C] = 5.72 Hz, Ar); 135.15 (d, J [31P–13C] = 11.45 the suspension was placed on a glass slide and covered with a
Hz, Ar), 139.90 (d, J [31P–13C] = 6.8 Hz, Ar); 147.70 (CHN); cover slip. Images of the cells were taken by a UV-fluorescence
1
171.28 (d, J [31P–13C] = 11 Hz, CS) ppm.
microscope within 30 min.
Annexin V staining. The cancer cells (1 × 106 cells) were
seeded on a chamber slide and were treated with the respective
X-ray crystallography
Diffraction data were measured with a Bruker SMART Apex II IC50 concentrations of the Pt complex for 48 h (control cells
CCD area-detector diffractometer (graphite-monochromated were treated with 0.5% DMSO vehicle). The medium was then
Mo-Kα radiation, λ = 0.71073 Å). The orientation matrix, unit- removed and the slide was washed with PBS. Treated and
cell refinement, and data reduction were all handled by the control cells were exposed to FITC–annexin V for 15 min
Apex2 software (SAINT integration, SADABS absorption correc- according to the manufacturer’s protocol (BD Pharmingen,
tion).30 The structures were solved using direct or Patterson USA). After a 488 nm excitation, green fluorescence (emission
methods in the program SHELXS-97 and were refined by the at 515 nm) was visualized by an inverted fluorescence
full matrix least-squares method on F2 with SHELXL-97.31 All microscope.
the non-hydrogen atoms were refined anisotropically. The
structure of Pd(L)(LH2) contains disordered solvent molecules
DNA fragmentation analysis
which could not be satisfactorily modeled. The data were The cancer cells were seeded in a 25 mL culture-flask (1 × 106
therefore treated with the SQUEEZE routine of PLATON.32 cells per mL) and treated with the respective IC50 concen-
Drawings of the molecules were produced with XSEED.33 trations of the test compound for 48 h (control cells were
Crystal data and refinement details are summarized in Tables treated with 0.5% DMSO vehicle). The cells were then washed
S4 and S5.†
with PBS. The treated and control cells were washed with PBS
and harvested. Cellular DNA was extracted using a Genomic
DNA purification kit (Promega, Madison, WI). The DNA frag-
Cytotoxicity assay
Cell culture. The cell lines were provided by Faculty of Medi- ments were then separated by a 1.5% agarose gel electrophor-
cine, University of Malaya. All the cells were cultured in either esis at 50 V for 1.5 h. The gels were then stained with ethidium
Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 bromide and visualized on a UV-illuminator.
supplemented with 10% fetal bovine serum (Sigma Aldrich)
Cell cycle analysis
and L-glutamine at 37 °C in a 5% CO2 humidified atmosphere.
MTT cytotoxicity test. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- Cell cycle analysis was carried out by propidium iodide flow
diphenyltetrazolium bromide) assay was carried out as cytometry as described elsewhere.35 Briefly, the cancer cells
described by Mosmann,34 with some modifications. The cells (1 × 106) were seeded in three culture flasks and were treated
were seeded into 96-well plates (5000 cells per well) and with the respective IC50 concentrations of the Pt complex for
allowed to adhere overnight. Pt(L)PPh3 was pre-dissolved in 24 h and 48 h. The cells were then washed with PBS, sus-
dimethyl sulfoxide (DMSO) and diluted to the desired concen- pended in 90% ethanol and kept overnight (4 °C). The pellets
trations (eight concentrations, 0.39–50 μg mL−1), such that the were further washed with PBS and suspended in PBS at 37 °C.
final concentrations of DMSO did not exceed 0.5%. Each cell 25 µl of RNase A (10 µg mL−1) and 50 µl of propidium iodide
line was treated with the test compound solutions (three wells (10 µg mL−1) were added to the mixture and the cells were
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Dalton Transactions
incubated at 37 °C for 30 min. The distribution of the cell 12 A. Nunez-Montenegro, R. Carballo and E. M. Vazquez-
cycle was measured by a FACsCANTO II flow cytometer, and
the data were analyzed with Prism 5.
Lopez, Polyhedron, 2009, 28, 3915.
13 M. D. Revenko, P. N. Bourosh, E. F. Stratulat, I. D. Corja,
M. Gdaniec, Y. A. Simonov and F. Tuna, Russ. J. Inorg.
Chem., 2009, 54, 530.
14 D. Kovala-Demertzi, N. Kourkoumelis, M. A. Demertzis,
J. R. Miller, C. S. Frampton, J. K. Swearingen and
D. X. West, Eur. J. Inorg. Chem., 2000, 727.
15 P. N. Yadav, M. A. Demertzis, D. Kovala-Demertzi,
A. Castineiras and D. X. West, Inorg. Chim. Acta, 2002, 332,
204.
16 F. Hueso-Urena, N. A. Illan-Cabeza, M. N. Moreno-Carre-
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Acknowledgements
We would like to thank Maysam Hafezparast Maradat,
Shahram Golbabapour, Maryam Zahedi Fard and Javad Paydar
from Faculty of Medicine, University of Malaya for their assist-
ance with the biological data collection and analysis. Financial
Support from the University of Malaya (HIR Grant Number
UM.C/625/1/HIR/151, UMRG Grant Number RG066/12BIO and
PPP Grant Number PV048/2012A) and the Center for Natural
Products and Drug Research, CENAR, (FL001-13BIO) is highly
appreciated.
18 Y. P. Tian, C. Y. Duan, C. Y. Zhao, X. Z. You, T. C. W. Mak
and Z. Y. Zhang, Inorg. Chem., 1997, 36, 1247.
19 C. Y. Duan, Z. H. Liu, Y. C. Shi and X. Z. You, J. Coord.
Chem., 1999, 47, 433.
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This journal is © The Royal Society of Chemistry 2014