Synthesis and evaluation of series of diazine-bridged dinuclear platinum(II) complexes through in vitro toxicity and molecular modeling: Correlation between structure and activity of pt(II) complexes

Article abstract of DOI:10.1021/jm5017686

Polynuclear Pt(II) complexes are a novel class of promising anticancer agents with potential clinical significance. A series of pyrazine (pz) bridged dinuclear Pt(II) complexes with general formulas {[Pt(L)Cl]₂(μ-pz)}²⁺ (L, ethylenediamine, en; (±)-1,2-propylenediamine, 1,2-pn; isobutylenediamine, ibn; trans-(±)-1,2-diaminocyclohexane, dach; 1,3-propylenediamine, 1,3-pd; 2,2-dimethyl-1,3-propylenediamine, 2,2-diMe-1,3-pd) and one pyridazine (pydz) bridged {[Pt(en)Cl]₂(μ-pydz)}²⁺ complex were prepared. The anticancer potential of these complexes were determined through in vitro cytotoxicity assay in human fibroblasts (MRC5) and two carcinoma cell lines (A375 and HCT116), interaction with double stranded DNA through in vitro assay, and molecular docking study. All complexes inhibited cell proliferation with inhibitory concentrations in the 0.5-120 μM range. While {[Pt(1,3-pd)Cl]₂(μ-pz)}²⁺ showed improved activity and {[Pt(en)Cl]₂(μ-pydz)}²⁺ showed comparable activity to that of clinically relevant cisplatin, {[Pt(en)Cl]₂(μ-pydz)}²⁺ was less toxic in an assay with zebrafish (Danio rerio) embryos, causing no adverse developmental effects. The in vitro cytotoxicity of all diazine-bridged dinuclear Pt(II) complexes is discussed in correlation to their structural characteristics.

Full text of DOI:10.1021/jm5017686

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Series of Diazine-Bridged Dinuclear
Platinum(II) Complexes through in Vitro Toxicity and Molecular
Modeling: Correlation between Structure and Activity of Pt(II)
Complexes
Lidija Senerovic,†,∥ Marija D. Zivkovic,‡,∥ Aleksandar Veselinovic,§ Aleksandar Pavic,† Milos I. Djuran,‡
Snezana Rajkovic,*,‡ and Jasmina Nikodinovic-Runic*,†
†Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia
‡Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12, P.O. Box 60, 34000 Kragujevac, Serbia
§Faculty of Medicine, Department of Chemistry, University of Nis,
̌ ̌
18000 Nis, Serbia
S
* Supporting Information
ABSTRACT: Polynuclear Pt(II) complexes are a novel class
of promising anticancer agents with potential clinical
significance. A series of pyrazine (pz) bridged dinuclear Pt(II)
complexes with general formulas {[Pt(L)Cl]2(μ-pz)}2+ (L,
ethylenediamine, en; ( )-1,2-propylenediamine, 1,2-pn; iso-
butylenediamine, ibn; trans-( )-1,2-diaminocyclohexane,
dach; 1,3-propylenediamine, 1,3-pd; 2,2-dimethyl-1,3-propyle-
nediamine, 2,2-diMe-1,3-pd) and one pyridazine (pydz)
bridged {[Pt(en)Cl]2(μ-pydz)}2+ complex were prepared.
The anticancer potential of these complexes were determined through in vitro cytotoxicity assay in human fibroblasts
(MRC5) and two carcinoma cell lines (A375 and HCT116), interaction with double stranded DNA through in vitro assay, and
molecular docking study. All complexes inhibited cell proliferation with inhibitory concentrations in the 0.5−120 μM range.
While {[Pt(1,3-pd)Cl]2(μ-pz)}2+ showed improved activity and {[Pt(en)Cl]2(μ-pydz)}2+ showed comparable activity to that of
clinically relevant cisplatin, {[Pt(en)Cl]2(μ-pydz)}2+ was less toxic in an assay with zebrafish (Danio rerio) embryos, causing no
adverse developmental effects. The in vitro cytotoxicity of all diazine-bridged dinuclear Pt(II) complexes is discussed in
correlation to their structural characteristics.
centers linked by a bridging diamine ligand. These linkers are
commonly flexible linear aliphatic molecules with variable chain
length,12 which enables them to form long-range cross-links
with DNA.13,14 Some of the polynuclear complexes possess
rigid linkers such as hydrazine and azoles.15−17 These
complexes are developed to minimize distortion of the DNA
double helix in a cross-link.18 Recently, another class of
dinuclear platinum(II) complexes with six-membered hetero-
cyclic diazines, namely, pyridazine (pydz), pyrimidine (pm),
and pyrazine (pz) as bridging ligands, has been synthesized and
they present a combination of the two above-mentioned types
of linker ligands.15,19 Two dinuclear platinum(II) complexes,
INTRODUCTION
■
After Rosenberg’s discovery of the antitumor activity of
cisplatin (cis-[PtCl2(NH3)2], also known as cis-DDP and
CDDP) it became one of the most often used metal-containing
drugs in cancer chemotherapy.1,2 Unfortunately, the side effects
of cisplatin are severe and include dose-limiting toxicity such as
nephrotoxicity, neurotoxicity, ototoxicity, and emetogenesis.3
The so-called second-generation of platinum based drugs, such
as carboplatin and oxaliplatin, were developed with the aims of
obtaining better antitumor activity, increased solubility, and
lower toxic side effects.4,5 Interactions of platinum-based drugs
with DNA, through covalent binding or intercalation of the
aromatic ligands to cellular DNA, are responsible for their
antitumor activity. This modified tertiary structure of DNA
results in the death of the cancer cell through apoptosis.6−8
Despite all the toxic side effects, platinum(II)-based cytotoxic
agents are a part of more than 50% of clinically applied
anticancer regimens and first-line chemotherapy for 12 different
neoplasms.9−11
20
namely, {[Pt(en)Cl]2(μ-pz)}Cl2 and {[Pt(en)Cl]2(μ-pydz)}-
Cl2,21 have been synthesized in our laboratory and charac-
1
terized by application of H NMR spectroscopy and single-
crystal X-ray diffraction. The obtained crystallographic results
showed that in {[Pt(en)Cl]2(μ-pz)}Cl2 complex, pyrazine acts
as linear bridge between metal ions separated by ∼7 Å,20 while
in the pyridazine analogue, the two Pt(II) ions are only ∼3 Å
Polynuclear platinum complexes represent a novel class of
promising antitumor agents with potential clinical signifi-
cance.4,11 These complexes contain two, three, or four platinum
Received: November 14, 2014
© XXXX American Chemical Society
A
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apart.21 As a continuation of our ongoing interest toward the
coordination chemistry of platinum(II) with bridging nitrogen-
containing heterocyclic ligands20,21 in light of the facts that
polynuclear Pt(II) complexes represent promising antitumor
agents, in the present paper we report the synthesis,
spectroscopic characterization, and biological evaluation of a
whole series of {[Pt(L)Cl]2(μ-pz)}2+ type complexes (3−9), all
differing in the chelated diamine ligand L (L is ethylenedi-
amine, en (3); ( )-1,2-propylenediamine, 1,2-pn (4); iso-
butylenediamine, ibn (5); trans-( )-1,2-diaminocyclohexane,
dach (6); 1,3-propylenediamine, 1,3-pd (7); 2,2-dimethyl-1,3-
propylenediamine, 2,2-diMe-1,3-pd (8); and pz is bridging
pyrazine ligand), as well as {[Pt(en)Cl]2(μ-pydz)}2+ complex
(9) (pydz is bridging pyridazine ligand) (Figure 1).
During this study for the first time in vitro cytotoxic activity
of complexes 3−9 against human cell lines (fibroblast and
cancer) was evaluated and their interaction with double
stranded circular and chromosomal DNA was examined
through in vitro activity assay and through molecular modeling
with direct comparison with those for cis-[PtCl2(NH3)2] (1)
and {cis-[PtCl(NH3)2]2(μ-pz)}Cl2 (2). In addition zebrafish
embryo toxicity of potentially useful anticancer leads was
evaluated.
RESULTS AND DISCUSSION
■
Synthesis and Structural Characteristics of Diazine-
Bridged Dinuclear Pt(II) Complexes. Six pyrazine-bridged
dinuclear {[Pt(L)Cl]2(μ-pz)}2+ complexes (3−8) (L is ethyl-
enediamine, en; ( )-1,2-propylenediamine, 1,2-pn; isobutyle-
nediamine, ibn; trans-( )-1,2-diaminocyclohexane, dach; 1,3-
propylenediamine, 1,3-pd; and 2,2-dimethyl-1,3-propylenedi-
amine, 2,2-diMe-1,3-pd) and one pyridazine-bridged {[Pt(en)-
Cl]2(μ-pydz)}2+ complex (9) have been synthesized (Figure 1).
The structures of all these complexes were confirmed by
elemental microanalyses and NMR (1H and 13C) spectroscopy,
and these data were correlated with those previously reported
for the same complexes.20−22 Moreover, the crystal structures
of 3 and 9 were also confirmed by single-crystal X-ray
diffraction analysis.20,21 The schematic presentation of the
reaction for the syntheses of these complexes is shown in
Scheme 1. The mononuclear Pt(II) complexes of the type
[Pt(L)Cl2] were prepared from equimolar amounts of K2PtCl4
and corresponding diamine ligand (L) according to a procedure
published in the literature (Scheme 1A).23−25 The mono-
nuclear [Pt(L)Cl2] complex was reacted with an equivalent
amount of AgNO3 to replace one chloride by a DMF molecule.
In order to obtain diazine-bridged dinuclear Pt(II) complex, the
corresponding monoactivated ([Pt(L)Cl)DMF)]+ species was
reacted with an equivalent amount of pyrazine or pyridazine
ligand at room temperature (Scheme 1B). All complexes were
crystallized as chloride salts from water solution in an excess of
LiCl. Complexes 3−6 have the same pyrazine-bridged ligand
but differ in the structure of the five-membered diamine ring
(en, 1,2-pn, ibn, and dach) (Figure 1). Recently reactions
between aqua derivatives of 3−6, {[Pt(L)(H2O)]2(μ-pz)}4+,
and different methionine-containing peptides have been
20,23
1
Figure 1. Structural representation of cisplatin (1) and diazine-bridged
Pt(II) dinuclear complexes (2−9) analyzed in this study.
investigated by H NMR spectroscopy.
This study showed
that all investigated Pt(II) aqua complexes bind to the
methionine side chain of the peptide and promote the cleavage
Scheme 1. Reactions for Preparation of Dinuclear Pt(II) Complexes (3−9)
B
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of the amide bond involving the carboxylic group of
methionine. Moreover, it was demonstrated that the substituent
in the ethylenediamine skeleton of these complexes, one methyl
group in 1,2-pn (4), two methyl groups in ibn (5), or
cyclohexane ring in dach (6), has influence on this hydrolytic
reaction. Thus, the amount of hydrolyzed peptide was
decreased by increasing the steric bulk of the corresponding
dinuclear Pt(II) complexes (3 > 4 > 5 > 6). In comparison with
the above-mentioned 3−6 all having five-membered ethyl-
enediamine ring, complexes 7 and 8 contain six-membered 1,3-
propanediamine ring (Figure 1). Finally, in complex 9 pyrazine-
bridged ligand is replaced with less symmetrical pyridazine, for
which we can assume that it also contributes to different
reactivity of 9 in comparison with its analogue 3. Additionally,
the studies of the reactions between some dinuclear Pt(II)
complexes and sulfur- or nitrogen-containing biomolecules
showed that these complexes are quite stable in the broad pH
range (2.0 < pH < 8.0) for more than 24 h.20−22 According to
the above-discussed results for different reactivity of dinuclear
Pt(II) complexes with peptides in connection with their
structural characteristics and those related to the facts that
these types of complexes showed reasonable stability in the
reactions with sulfur- and nitrogen-containing biomolecules in
the broad pH range, in the second part of this work in vitro
toxicity of 3−9 has been evaluated and discussed.
In Vitro Cytotoxicity and DNA Cleavage (Unwinding)
Ability. The antiproliferative (cytotoxic) potential of diazine-
bridged dinuclear Pt(II) complexes in comparison to cisplatin
was tested on human lung fibroblasts (MRC5) and two
carcinoma cell lines, melanoma (A375) and colon cancer
(HCT116), by MTT assay after a 48 h treatment (Figure 2).
Although all complexes showed the cytotoxic effect in all cell
lines in concentrations of up to 50 μM, HCT116 appeared to
be more sensitive in comparison to fibroblasts and melanoma,
indicating some cell-line specificity (Figure 2). The cytotoxic
profiles of each complex were constant for different cell lines
but slightly different among complexes. Dose-dependent
cytotoxicity exhibited by 1 was comparable to that of 2, 3,
and 7−9, while complexes 4−6 exhibited cytotoxicity in less
dose-dependent manner. Indeed, the least cytotoxic was 5 with
IC50 values being from 45- to 22-fold higher against fibroblasts
and colon cancer in comparison to cisplatin, respectively (Table
1).
Diazine-bridged dinuclear Pt(II) complexes (3−9) generally
appear to be less cytotoxic in comparison to cisplatin (1) and
azine-bridged complex 2, apart from the 7 and 9. In fact, the
only complex that showed 2-fold lower IC50 values in
comparison to those of cisplatin for both melanoma and
colon cancer was complex 7 (Table 1). Interestingly,
structurally similar complex 8 showed desirable 3- to 5-fold
lower activity against fibroblasts in comparison to carcinoma
cell lines but slightly lower activity in comparison to complex 7.
This can be correlated with the fact that complex 7 is less
sterically demanding with respect to complex 8 having two
methyl groups attached on the middle carbon atom of 1,3-
propanediamine ring. Also, higher activity of 3 in comparison
with its analogues 4−6 contributed to the presence of different
substituents in the ethylenediamine skeleton of the latter three
complexes, i.e., one methyl group in 4, two methyl groups in 5,
or cyclohexane ring in 6. However, from the present
investigation it can be concluded that complexes 3−6 with
five-membered ethylenediamine ring showed lower IC50 values
in comparison to those with six-membered 1,3-propanediamine
Figure 2. In vitro cytotoxicity (antiproliferative effect) profile of
cisplatin and Pt(II) complexes 2−9 against (A) human lung fibroblast
(MRC5), (B) melanoma (A375), and (C) colon cancer (HCT116)
cell lines ( , 0.5 μM; gray box, 5 μM; , 50 μM concentration tested
compound).
□
■
ring (7 and 8) (Table 1). All these complexes have the same
pyrazine bridged ligand, but they differ in the ring size of the
chelating diamine ligand. In all these complexes coordination
around two Pt(II) ions is square planar but they differ in
conformation of diamine rings. The diamine rings in complexes
3−6 adopt the usual twist conformation with an approximate 2-
fold axis passing through the C1−C2 bond.20 On the other
hand, the six-membered chelate ring in complexes 7 and 8 is in
a chair conformation. This conformation was established by X-
ray crystallography for Pt(II) and Pd(II) complexes with
different 1,3-propanediamine ligands.26 From the above facts it
can be assumed that different conformations between five-
membered and six-membered rings in the above-mentioned
complexes play crucial roles in their different activity. It is worth
mentioning that complexes 7 and 8 contained 2 equiv of LiCl
C
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Table 1. In Vitro Antiproliferative Activity Given as IC50
SD in μM of the Cisplatin (1) and Complexes 2−9
cell line
compd
MRC5
A375
0.1
HCT116
Figure 3. Agarose gel electrophoresis patterns of supercoiled pUC18
plasmid DNA (200 ng) incubated with cisplatin (1) and Pt(II)
complexes 2−9 in the buffer at pH 7.4 (50 mM Tris-HCl, 50 mM
NaCl) at 37 °C for 12 h.
cisplatin
4.5 0.1
14.3 0.5
20 0.9
45 0.8
6
1
0.1
2
3
4
5
6
7
8
9
20 0.4
25 0.4
3.2 0.1
3
0.1
50
1
30 0.8
100
40 0.8
0.2
15 0.4
0.1
2
120
3
45
4
1
45 0.9
0.1
a
a
4
3
5
5
0.1
0.2
0.1
0.5 0.1
0.3
3.3 0.2
complexes 2−6 and 8 could exhibit irreversible DNA
degradation more readily than 1, 7, and 9, which may lead to
cell death by necrosis, therefore indicating potentially higher
general toxicity of these compounds. However, all of these
compounds exhibited lower antiproliferative activities in
cytotoxicity assays in comparison to cisplatin (Table 1).
Complex 7, although with the highest activity against human
cell lines, was mostly able to unwind supercoiled to circular
form at concentration tested. Therefore, from the high
interaction with circular double stranded DNA in vitro,
cytotoxicity against human cell lines could not be predicted.
In Silico DNA Binding Ability of Diazine-Bridged
Dinuclear Platinum(II) Complexes. Molecular docking is a
powerful computational technique that can be used for the
investigation of DNA−drug interactions, the identification of
binding place (minor or major groove), and the prediction of
complex binding affinities.29,30 Molecular docking study was
carried out to gain information about in silico DNA binding
affinity for studied compounds 1−9. The predicted top-ranking
pose with the complex with lowest energy was applied for
suggesting the best possible geometry of compounds inside the
DNA double helix and the highest binding affinity of DNA.
MolDock, Docking, Rerank, and Hbond scoring functions were
used for the assessment of complexes DNA binding affinity.
Top-ranked poses according to used scoring functions are
presented in Table 2.
3
6
a
Statistically significantly lower (p < 0.05) as compared to cisplatin.
as contaminant that itself in the applied concentrations did not
have antiproliferative effect on the used cell lines (data not
shown). Difference in the activity of pyrazine-bridged complex
3 with respect to the analogue pyridazine-bridged Pt(II)
complex 9 can be explained from crystallographic results of
these two complexes.21 The X-ray results of 3 showed that the
Pt···Pt distance in this complex is 6.7890(3) Å, comparable
with the mean value of 6.815(8) Å obtained from 17
observations in the crystal structures containing a discrete
pyrazine-bridged Pt(II) dimer, deposited in the CSD.27
However, this distance for complex 9 having two ortho-
arranged nitrogen donor atoms of pyridazine is much shorter
(3.2535(4) Å) than in the closely related {[Pt(en)Cl]2(μ-
pz)}2+ cation (3).20 From the difference in the intramolecular
distances of two Pt(II) ions of 9 and 3, it can be assumed that
the shorter distance in the first complex contributes to its better
ability to form DNA adducts and higher cytotoxic activity.
Cytotoxicity of diazine-bridged Pt(II) complexes (3−9) was
not previously examined, while in vitro cytotoxicity of the
azine-bridged complex 2 was previously examined in several
human tumor cell lines including melanoma (M19), colon
cancer (WIDR), breast cancer (MCF7 and EVSA-T), renal
cancer (A498), and non-small-cell lung cancer (H226).15,19
Generally, 2 and its nitrate salt showed IC50 values higher than
those of cisplatin, between 1.6- and 30-fold, with only
comparable IC50 exhibited against ovarian cancer, indicating
cell line and cancer-type specificity.15 This is in agreement with
data obtained in this study. On the other side, 2 showed
improved activity with IC50 values 1.8- and 5.8-fold lower in
comparison to cisplatin when tested against murine leukemia
cell lines sensitive (L1210(0)) and resistant (L1210(cisPt)) to
cisplatin, respectively, with the induction of apoptosis in this
experimental system also reported.19
Since DNA is the primary target of Pt(II)-based antitumor
complexes,28 the ability of Pt(II) complexes to bind, unwind,
and cleave DNA was investigated following the conversion of
the supercoiled form of pUC18 plasmid DNA to the open
circular and/or linear forms using agarose gel electrophoresis.
Electrophoresis patterns of supercoiled pUC18 plasmid DNA
in the presence of 50 μM 1−9 showed that all compounds had
the ability to interact with DNA in vitro at physiological pH 7.4
(Figure 3). The supercoiled form of plasmid DNA was
completely converted to circular and linear forms in the
presence of 2−6 and 8, while cisplatin (1) and Pt(II) dinuclear
complexes 7 and 9 had only partially converted supercoiled
DNA to other forms exhibiting quite similar pattern of plasmid
DNA unwinding (Figure 3). Obtained results imply that
Table 2. Score Values (kcal/mol) for Cisplatin and Diazine-
Bridged Pt(II) Dinuclear Complexes
MolDock
(kcal/mol)
Docking
(kcal/mol)
Rerank
(kcal/mol)
HBond
(kcal/mol)
compd
a
b
cisplatin
cisplatin
−55.738
−48.392
−97.320
−54.669
−52.322
−94.349
−33.556
−31.583
−56.667
−48.246
−81.746
−62.883
−76.378
−66.401
−81.047
−83.910
−90.716
−76.170
−83.667
−66.648
−85.893
−76.749
−58.2179
0
−2.176
0
a
2
b
2
−96.699
−96.416
−2.583
0
a
3
−137.564
−126.189
−141.673
−138.476
−136.797
−134.686
−154.521
−149.530
−137.554
−121.797
−145.241
−133.637
−105.609
−135.115
−125.065
−139.506
−135.245
−135.985
−133.195
−152.912
−146.665
−134.041
−121.334
−141.051
−131.236
−105.210
b
3
−0.051
−0.222
−1.071
−0.546
−2.329
−0.917
−0.991
0
a
4
b
4
a
5
b
5
a
6
b
6
a
7
b
7
−2.994
0
a
8
b
8
−4.976
−2.57
ab
,
9
a
Best complex pose according to MolDock, Docking, and Rerank
b
scoring functions. Best complex pose according to Hbond scoring
function.
D
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According to MolDock, Docking, and Rerank score values,
the highest binding affinity has compound 6. However,
hydrogen bonds formed between complex and DNA are
important for metal complex DNA binding and for further
possible cytotoxic activity.31,32 According to Hbond values the
highest binding affinities have complexes 2, 7, 8, and 9. The
more negative is the relative binding energy, greater is the
binding propensity of the complex with DNA, which correlated
well with the experimental in vitro DNA binding studies
(Figure 3) and IC50 values for the cytotoxic activity of complex
(Table 1). According to molecular modeling results complex 1
(cisplatin) formed eight hydrogen bonds with DNA (seven
hydrogen bonds had binding energy values of −2.5 kcal/mol
with bond lengths 2.62, 2.77, 2.82, 2.96, 3.03, 3.09, and 3.10 Å,
respectively; one hydrogen bond had binding energy value of
−0.84 kcal/mol with bond length value of 3.43 Å). Complex 2
formed nine hydrogen bonds (seven hydrogen bonds had
binding energy values of −2.5 kcal/mol with bond lengths 2.62,
2.73, 2.75, 3.046, 3.058, 3.098, and 3.10 Å, respectively; one
hydrogen bond had binding energy value of −2.44 kcal/mol
with bond length value of 3.11 Å, and one hydrogen bond had
binding energy value of −0.77 kcal/mol with bond length value
of 3.44 Å). Complex 7 formed five hydrogen bonds with
binding energy values of −2.5, −2.26, −2.21, −0.856, and
−0.79 kcal/mol with bond length values of 3.098, 3.148, 2.60,
3.43, and 2.58 Å, respectively. Complex 8 formed four
hydrogen bonds with binding energy values of −2.5, −2.5,
−2.48, and −2.35 kcal/mol with bond length values of 2.66,
3.10, 2.92, and 3.13 Å, respectively. Complex 9 formed six
hydrogen bonds with binding energy values of −2.5, −2.5,
−1.79, −1.45, −1.18, and −1.13 kcal/mol with bond length
values of 2.75, 2.99, 3.24, 2.9, 3.36, and 2.43 Å, respectively.
Best poses for complexes 7−9 according to Hbond values are
presented in Figure 4. The minimum energy docked pose
in the AT stretches of the minor groove was indicative of an
extensive H-bonding network. Structural analysis of the docking
positions gave insight into the binding pattern of the
complexes. Comparable semiempirical level of theory was
successfully applied to metal complexes geometry optimization
in similar docking studies.34,35
Mechanistic Insights of Cytotoxicity of Complexes 7
and 9 in Comparison to Cisplatin. It was established that
Pt(II) complexes bind to DNA via coordination bonds
distorting normal three-dimensional structure of double
helix.36 This subsequently induces cell death through
apoptosis.37 The ability to cause cellular chromosomal DNA
damage was examined for Pt(II) complexes 7 and 9 and
compared to that of cisplatin (Figure 5A). Because of specific
Figure 5. Cytotoxic effect of cisplatin and diazine-bridged Pt(II)
complexes 7 and 9 on melanoma (A375) cells. (A) Cellular DNA
degradation is induced by complexes 7 and 9 in comparison to
cisplatin (1) and untreated cells (C). DNA molecular weight marker is
in lane M (1 kb ladder, Nippon Genetics). (B) Cell viability was
determined by MTT (white diamond) and CV (black diamond) assays
upon treatment with a range of cisplatin and complex 7 and 9
concentrations for 48 h. Data are an average of three independent
determinations.
structural characteristics, we assumed that 9 may exhibit a
cytotoxic pathway in human cells different from that of
cisplatin. Indeed, electrophoresis pattern of DNA extracted
from melanoma cells (A375) treated with cisplatin and complex
7 was similar and revealed DNA damage, while DNA from the
cells treated with complex 9 showed more resemblance to
untreated cells with DNA degradation occurring to low extent
(Figure 5A).
Figure 4. Computational docking model illustrating interactions
between (A) cisplatin 1, (B) complex 2, (C) complex 7, (D) complex
8, and (E) complex 9 and DNA.
Cisplatin generally causes DNA damage subsequently
followed by activation of mitochondrial cell death pathway
which can also be observed as significant difference between
IC50 values calculated from MTT and CV assays (Figure 5B).
MTT viability screen indicates the number of metabolically
active cells, while CV assay indicates the total number of live
adherent cells; therefore, from the result, an agreement
conclusion can be drawn if the cell mitochondria were the
initial target of the tested compound. Complex 7 induced DNA
fragmentation and affected mitochondrial respiration similar to
cisplatin itself, while complex 9 most likely employs different
revealed that complexes 7 and 9 are very well fitted into the
DNA minor groove. Docking poses suggest that complexes and
DNA base pairs are arranged in such a way that they have
effective π−π stacking interactions. These interactions can lead
to higher van der Waals interaction with the DNA functional
groups which define the stability of groove, making the AT
regions more preferable regions of dodecamer.33 Complexes
exhibited additional stabilization through the strong intermo-
lecular hydrogen bonding interaction between the C-2 carbonyl
oxygen of T and the N-3 nitrogen of A. The best binding pose
E
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mechanisms of action. From the shorter distance of two Pt(II)
ions of 9 its better ability to form DNA adducts and high
cytotoxic activity was proposed. It was previously shown that
multinuclear Pt(II) complexes, such as a complex of two
monofunctional [trans-PtCl(NH3)2] platinum units bridged by
a platinum tetraamine unit [trans-Pt(NH3)2(NH2(CH2)-
6NH2)2]2+ (BBR3436),38 maintain bifunctional binding mode
on DNA and forms long-range cross-linking adducts, but when
the cisplatin moieties were bridged by aromatic linkers,
preferentially interstrand DNA cross-linking adducts were
formed such as in the case of {[cis-PtCl(NH3)2]2(4,40-
methylenedianiline)}2+.39 Cytotoxicity screening of numerous
multinuclear Pt(II) complexes revealed the importance of the
ligands, as well as that of tailoring groups.1,40
Toxicity of Cisplatin (1) and Diazine-Bridged Dinu-
clear Platinum(II) Complexes (7 and 9) on Zebrafish
Embryos. The effects of cisplatin (1) and complexes 7 and 9
on development of Danio rerio embryos were examined at
concentrations close to IC50 values determined in the
cytotoxicity assessment (Table 1). None of tested substances,
applied at concentrations from 0.5 to 50 μM, showed lethal or
adverse developmental effects on treated embryos until 96 hpf
(hpf = hours post fertilization) (Figure 6A). Low mortality rate
7, and 9 were not teratogenic (neither skeletal nor
cardiovascular abnormalities were observed), 9 was significantly
less toxic than 1 and 7, displaying no adverse effect on the
embryos development (hatching) (Figure 6B). Hatching
success is supposed to be a sensitive end point of the zebrafish
embryo in toxicity assay, since no hatched embryos had a lethal
outcome.41 While no reference data are available in the
literature on cisplatin and dinuclear Pt(II) complexes, it has
been demonstrated that platinum and platinum nanoparticles
had adverse impact on the hatching success on developing
zebrafish embryos.41,42
The zebrafish allows high-quality in vivo validation of drug
targets before clinical trials, serving as a very useful bioassay
platform for toxicology studies.43,44 Cisplatin was approved by
Food and Drug Administration in 1978 in spite of some dose
limiting side effects; hence, the data on cisplatin toxicology in
wild type zebrafish that emerged as model later are not readily
available in the literature. Likewise, there are no reports on
embryotoxic effects on zebrafish of cisplatin derivatives such as
oxaliplatin, carboplatin, tetraplatin or of any Pt(II) dinuclear
complexes. In a few studies, concentration-dependent ototoxic,
neurotoxic, and nephrotoxic effects of ciplatin and oxaliplatin
were reported including work done on transgenic zebrafish
(Brn3C: EGFP).45 In these experiments, toxic effects were
evaluated on 5-day-old embryos that were exposed to cisplatin,
oxaliplatin, and related compounds for 24 h.46,47 It was shown
that 50 and 100 μM cisplatin significantly reduced hair-cell
survival up to 81% and 55%, respectively, while at
concentration of 400 μM cisplatin induced a lethal outcome
for embryos.46
CONCLUSION
■
Over the past few years, modern medicinal chemistry has been
providing us with new and alternative chemotherapeutic
compounds with high cytotoxicity toward tumor cells, with
reduced side effects in cancer patients. However, anticancer
platinum complexes with their unique physicochemical proper-
ties still prevail in the field of metals in cancer chemotherapy
with their use escalated and being sustained over decades.11,48
In summary, a series of diazine-bridged dinuclear platinum-
(II) complexes was developed and evaluated in bioassays for
potential further development toward novel anticancer agents.
From these, although {[Pt(1,3-pd)Cl]2(μ-pz)}2+ showed higher
cytotoxic potential in comparison to cisplatin, {[Pt(en)Cl]2(μ-
pydz)}2+ stood out exhibiting similar in vitro cytotoxicity profile
against different cell lines and desirable properties of being less
embryotoxic in comparison to cisplatin. Therefore, this
complex could serve as baseline for further derivatizations
and development of new anticancer drug candidates.
Figure 6. Effects of cisplatin (1) and two diazine-bridged dinuclear
platinum(II) complexes (7 and 9) on development of zebrafish
embryos: (A) at 0.5, 5, and 50 μM concentration from 24 to 96 h after
fertilization ( , normal embryos; , lethal embryos; ∗, at 72 hpf no
hatching of embryos exposed to 50 μM; ∗∗, at 96 hpf prevented
hatching of embryos was scored as lethal effect) and (B) images of
zebrafish embryos at 96 hpf exposed to 1, 7, and 9 (50 μM).
EXPERIMENTAL SECTION
■
Synthesis and Characterization of Pt(II) Complexes. Materi-
als. Distilled water was demineralized and purified to a resistance
greater than 10 MΩ cm−1. Ethylenediamine (en), ( )-1,2-
propylenediamine (1,2-pn), isobutylenediamine (ibn), trans-( )-1,2-
diaminocyclohexane (dach), 1,3-propylenediamine (1,3-pd), 2,2-
dimethyl-1,3-propylenediamine (2,2-diMe-1,3-pd), pyrazine or 1,4-
diazine (pz), pyridazine or 1,2-diazine (pydz), and K2[PtCl4] were
obtained from Sigma-Aldrich. The cis-[PtCl2(NH3)2] (1) and {cis-
[PtCl(NH3)2]2(μ-pz)}Cl2 (2) were synthesized according to a
procedure published in the literature.15,49
■
□
(total 1 out of 24 embryos) observed at 50 μm of 9
corresponded to those observed in negative control (results
not shown). At 96 hpf, all embryos treated with 50 μM of
cisplatin and 7 have been prevented from hatching, while all
embryos exposed to 50 μM of 9 hatched up to 72 hpf (Figure
6A). These data showed that while at tested concentrations 1,
Measurements. The 1H and 13C NMR spectra were recorded on a
Varian Gemini 2000 spectrometer (1H at 200 MHz, 13C at 50 MHz)
using 5 mm NMR tubes. The NMR samples were prepared in D2O as
F
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Journal of Medicinal Chemistry
Article
solvent, and the total volume was 0.6 mL. Sodium trimethylsilylpro-
pane-3-sulfonate (TSP) was used as an internal reference. Elemental
microanalyses for carbon, hydrogen, and nitrogen parameters were
performed using standard techniques by the Microanalytical
Laboratory, Faculty of Chemistry, University of Belgrade.50
(s, 4H, pz). 13C NMR (50 MHz, D2O): δ = 17.83, 54.52, 59.86, 153.44
ppm.
{[Pt(ibn)Cl]2(μ-pz)}Cl2 (5). Yield 35% (41.39 mg, 0.05 mmol).
Elemental analysis calculated (%) for C12H28N6Cl4Pt2 (FW = 788.36):
C 18.28, H 3.58, N 10.66. Found: C 17.84, H 3.63, N 10.47. 1H NMR
(200 MHz, D2O): δ = 1.42−1.48 (m, 6H, ibn), 2.66 (s, 2H, ibn), 9.13
ppm (s, 4H, pz). 13C NMR (50 MHz, D2O): δ = 25.96, 60.09, 63.61,
153.36 ppm.
The mononuclear platinum(II) complexes of the type [Pt(L)Cl2]
(L is en, 1,2-pn, ibn, dach, 1,3-pd, and 2,2-diMe-1,3-pd) were prepared
according to a procedure published in the literature.23−25 To
K2[PtCl4] (207.56 mg, 0.5 mmol) dissolved in 10 mL of water was
added potassium iodide (332.02 mg, 2.0 mmol), and the mixture was
heated at 50 °C for 5 min. Subsequently, an equimolar amount
(1equiv, 1 mmol) of diamine ligand (L) was added to the obtained
reaction mixture with heating (50 °C) and stirring continued for 30
min. All [Pt(L)I2]-type complexes were crystallized from water at
room temperature. The [Pt(L)I2] complexes were converted into the
corresponding aqua derivatives by treatment with 1.98 equiv of
AgNO3 according to a previously published method.51 The mixture
was stirred overnight at room temperature in the dark. In each case,
the formed solid AgCl was removed by filtration in the dark, and in the
fresh solutions of the aqua complexes an excess of potassium chloride
was added. The pale-yellow precipitate of [Pt(L)Cl2] complexes were
removed by filtration, washed with methanol and then ether, and air-
dried. The yield was between 80% and 90%. The experimental results
of the elemental analysis for C, H, and N parameters for all Pt(II)
complexes are in accordance with theoretical values calculated for
[Pt(L)Cl2] type complexes. These complexes were used for further
synthesis of the corresponding dinuclear platinum(II) complexes.
Synthesis of {[Pt(L)Cl]2(μ-X)}Cl2 Complexes (3−9). The
dinuclear platinum(II) complexes of the type {[Pt(L)Cl]2(X)}Cl2
(X is pyrazine, pz, or pyridazine, pydz) were synthesized by
modification of the procedure published in the literature.14,15,18,19
The mononuclear [Pt(L)Cl2] complex was converted into the
corresponding monodimethylformamide (DMF) complex [Pt(L)Cl-
(DMF)]NO3 by treatment with 0.98 equiv of AgNO3. To a solution of
AgNO3 (49.26 mg, 0.29 mmol) in 5 mL of DMF was added a
suspension of [Pt(L)Cl2] (0.30 mmol) in 10 mL of DMF. The mixture
was stirred overnight at room temperature in the dark. The
precipitated AgCl was removed by filtration, and the resulting pale
yellow DMF solution of [Pt(L)Cl(DMF)]NO3 was used as the
starting material for the preparation of the required pyrazine or
pyridazine-bridged platinum(II) complexes.
DMF solution of the X ligand (10.01 mg, 0.15 mmol) was added
dropwise to the solution of [Pt(L)Cl(DMF)]NO3. The mixture was
stirred at room temperature in the dark for 24 h. The solvent was then
rotary evaporated and the residue washed with ether. The crude
product was dissolved in a minimal amount of 0.5 M LiCl aqueous
solution. The obtained solution was left overnight in the dark. The
pale-yellow precipitate of dinuclear Pt(II) complex was removed by
filtration, washed with methanol and then ether, and air-dried.
Complexes containing six-membered diamine ring L crystallized
with two molecules of LiCl and two water molecules ({[Pt(L)Cl]2(μ-
X)}Cl2·2LiCl·2H2O) (7 and 8), which differs from complexes with
five-membered chelate ring (L) crystallizing in {[Pt(L)Cl]2(μ-X)}Cl2
(4, 6, and 9) molecular form. Depending of the type diamine (L) and
bridging (X) ligand, the yield of {[Pt(L)Cl]2(μ-X)}Cl2 complex was
between 35% and 40%. The purity of the complexes was checked by
elemental microanalysis and NMR (1H and 13C) spectroscopy. All
these data confirmed >95% purity of the tested compounds and were
in accordance with those previously reported for the same
complexes.20−22
{[Pt(dach)Cl]2(μ-pz)}Cl2 (6). Yield 32% (40.34 mg, 0.048 mmol).
Elemental analysis calculated (%) for C16H32N6Cl4Pt2 (FW = 840.43):
1
C 22.87, H 3.84, N 10.00. Found: C 22.56, H 3.94, N 9.56. H NMR
(200 MHz, D2O): δ = 1.27−1.62 (m, 4H, dach), 1.76−2.08 (m, 4H,
dach,), 2.45−2.61 (m, 2H, dach,), 9.00 ppm (s, 4H, pz). 13C NMR (50
MHz, D2O): δ = 26.55, 34.56, 63.61, 65.15, 153.31 ppm.
{[Pt(1,3-pd)Cl]2(μ-pz)}Cl2·2LiCl·2H2O (7). Yield 32% (44.94 mg,
0.051 mmol). Elemental analysis calculated (%) for
C10H28N6Cl6O2Li2Pt2 (FW = 881.12): C 13.63, H 3.20, N 9.54.
1
Found: C 14.80, H 3.03, N 10.07. H NMR (200 MHz, D2O): δ =
1.84−1.95 (m, 2H, 1,3-pd), 2.71−2.88 (m, 4H, 1,3-pd), 9.03 ppm (s,
4H pz). 13C NMR (50 MHz, D2O): δ = 29.83, 44.56, 45.66, 153.57
ppm.
{[Pt(2,2-diMe-1,3-pd)Cl]2(μ-pz)}Cl2·2LiCl·2H2O (8). Yield 30%
(42.18 mg, 0.045 mmol). Elemental analysis calculated (%) for
C14H36N6Cl6O2Li2Pt2 (FW = 937.23): C 17.94, H 3.87, N 8.97.
Found: C 18.04, H 3.72, N 9.17. 1H NMR (200 MHz, D2O): δ = 1.00
(s, 6H, 2,2-diMe-1,3-pd), 2.38 (2s, 2H, 2,2-diMe-1,3-pd,), 2.49 (2s,
4H, 2,2-diMe-1,3-pd,), 9.05 ppm (s, 4H pz). 13C NMR (50 MHz,
D2O): δ = 25.57, 36.14, 53.97, 54.99, 153.58 ppm.
{[Pt(en)Cl]2(μ-pydz)}Cl2 (9). Yield 40.0% (43.93 mg, 0.06 mmol).
Elemental analysis calculated (%) for C8H20N6Cl4Pt2 (FW = 732.25):
C 13.12, H 2.75, N 11.48. Found: C 13.16, H 2.98, N 11.19. 1H NMR
(200 MHz, D2O): δ = 2.78−2.82 (m, 8H, en), 8.14 (m, 2H, pz), 9.58
ppm (m, 2H, pz). 13C NMR (50 MHz, D2O): δ = 51.10, 137.33,
164.36 ppm.
Cell Viability Assays. Cell viability was tested by (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
crystal violet (CV) colorimetric assays as previously described.52,53
Assays were carried out after 48 h of cell incubation in the medium
containing test compounds at concentrations ranging from 0.25 to 150
μM (including 0.5, 5, 50 μM). The results are presented as percentage
of the control (untreated cells) that was arbitrarily set to 100%. The
percentage viability values were plotted against the log of
concentration and a sigmoidal dose−response curve was calculated
by nonlinear regression analysis using GraphPad Prism software,
version 5.0, for Windows (GraphPad Software, CA, USA). From these
curves, IC50 values were obtained.
Gel Electrophoreses Study. The ability of 1−9 to cleave DNA
was examined by following the conversion of the supercoiled form of
pUC18 plasmid DNA (FI) to the open circular (FII) and/or linear
forms (FIII), using agarose gel electrophoresis.54 For the gel
electrophoresis experiments, pUC18 plasmid DNA (200 ng) was
treated with the cisplatin and Pt(II) complexes (50 μM) in buffer, and
the contents were incubated for 12 h at 37 °C, then subjected to
electrophoresis on a 1% (w/v) agarose gel containing 0.1 μg/mL
ethidium bromide in TAE buffer (40 mM Tris acetate/1 mM EDTA,
pH 7.4) buffer at 60 V for 2 h.
For the analysis of cellular DNA degradation, A375 cells (2 × 105
cells) were exposed to the concentrations equal to IC50 of cisplatin and
Pt(II) complex 7 and 9 for 48 h. The DNA was subsequently extracted
from the cells and analyzed by agarose electrophoresis (2% (w/v)
agarose gel in a TAE buffer (40 mM Tris acetate/1 mM EDTA, pH
7.4) at 60 V for 2 h) as described previously.19
Molecular Docking. In this work, the geometry optimization of
platinum(II) complexes has been carried out using semiempirical
quantum chemistry method (PM6),55,56 because of its excellent
compromise between computational time and description of electronic
correlation.57 The calculations were performed with the Gaussian 09
molecular package.58 The structure of B-DNA dodecamer
(CGCGAATTCGCG)2 (PDB code 1BNA)59 was used as a model
to study the interaction between the metal complex and DNA.60 The
{[Pt(en)Cl]2(μ-pz)}Cl2 (3). Yield 40.0% (43.93 mg, 0.06 mmol).
Elemental analysis calculated (%) for C8H20N6Cl4Pt2 (FW = 732.25):
C 13.12, H 2.75, N 11.48. Found: C 13.16, H 2.98, N 11.19. 1H NMR
(200 MHz, D2O): δ = 2.68−2.79 (m, 8H, en), 9.03 ppm (s, 4H, pz).
13C NMR (50 MHz, D2O): δ = 52.34, 153.46 ppm.
{[Pt(1,2-pn)Cl]2(μ-pz)}Cl2 (4). Yield 33.3% (38.02 mg, 0.05
mmol). Elemental analysis calculated (%) for C10H24N6Cl4Pt2 (FW
= 760.31): C 15.80, H 3.18, N 11.05. Found: C 15.45, H 3.19, N
1
10.82. H NMR (200 MHz, D2O): δ = 1.34 (d, 3H, J = 6.6 Hz, 1,2-
pn), 2.45−2.98 (m, 2H, 1,2-pn), 3.11−3.32 (m, H, 1,2-pn), 9.01 ppm
G
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flexible compounds (platinum(II) complexes) were docked into rigid
DNA structure using the Molegro Virtual Docker (MVD, version
2013.6.0.1).61 MVD has been successfully applied in docking studies of
metal complexes in DNA.12 Hydrogen bonds and hydrophobic
interactions between platinum(II) complexes and DNA were
calculated. The binding site was computed with a grid resolution of
0.3 Å. The MolDock SE as a search algorithm was used with the
number of runs set to 100. The parameters of docking procedure were
the following: population size 50, maximum number of iterations
1500, energy threshold 100.00, and maximum number of steps 300.
The number of generated poses was 5. The estimation of platinum(II)
complexes and DNA interactions was described by the MVD-related
scoring functions: MolDock, Docking, Rerank, and Hbond. A
maximum population of 100 and maximum number of iterations of
10 000 were used for each run, and the 5 best poses were retained.
Visualization of the docked pose has been done by using the
CHIMERA (http://www.cgl.ucsf.edu/chimera/) molecular graphics
program.
Toxicity against Zebrafish Embryos. For zebrafish embryo-
toxicity assessments general rules of the OECD Guidelines for the
Testing of Chemicals were followed.62 Adult wild type zebrafish
(Danio rerio) obtained from a commercial supplier (Pet Centar,
Belgrade, Serbia) and maintained under laboratory conditions for a
several months were used in this study. Collected eggs at the 4- to 16-
cell stage were exposed to three different concentrations of 1, 7, and 9
(0.5, 5, 50 μM) prepared by diluting the stock solution of each
compound in the fish medium. Appropriate no-treatment controls
were also included. Embryos were placed into 96-well plates
containing 200 μL of solution, one embryo per well, and incubated
at 27 °C. Twenty-four embryos have been used per group.
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All embryos were staged and evaluated using standard procedures as
previously described.62−64 Apical endopoints (Table S1) were
recorded at 24, 48, 72, and 96 h post fertilization (hpf) using an
inverted microscope (CKX41; Olympus, Tokyo, Japan).
ASSOCIATED CONTENT
* Supporting Information
NMR spectra of complexes 2−9 and table of lethal and
teratogenic effects in zebrafish. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
■
*S.R.: phone, +381-34300251; fax, +381-34335040; e-mail,
snezana@kg.ac.rs. ; jasmina.nikodinovic@imgge.bg.ac.rs.
*J.N.-R.: phone, +381-113976034; fax, +381-113975808; e-
mail, jasmina.nikodinovic@gmail.com.
Author Contributions
∥L.S. and M.D.Z. contributed equally.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(18) Komeda, S.; Ohishi, H.; Yamane, H.; Harikawa, M.; Sakaguchi,
K.-I.; Chikuma, M. An NMR study and crystal structure of [{cis-
Pt(NH3)2(9EtG-κN7)}2(μ-pz)][NO3]3 (9EtG = 9-ethylguanine) as a
model compound for the 1,2-intrastrand GG crosslink. Dalton Trans.
1999, 2959−2962.
Notes
The authors declare no competing financial interest.
(19) Kalayda, G. V.; Komeda, S.; Ikeda, K.; Sato, T.; Chikuma, M.;
Reedijk, J. Synthesis, structure, and biological activity of new azine-
bridged dinuclear platinum(II) complexesa new class of anticancer
compounds. Eur. J. Inorg. Chem. 2003, 4347−4355.
(20) Asanin, D. P.; Zivkovic, M. D.; Rajkovic, S.; Warzajtis, B.;
Rychlewska, U.; Djuran, M. I. Crystallographic evidence of anion···π
interactions in the pyrazine bridged {[Pt(en)Cl]2(μ-pz)}Cl2 complex
and a comparative study of the catalytic ability of mononuclear and
binuclear platinum(II) complexes in the hydrolysis of N-acetylated L-
methionylglycine. Polyhedron 2013, 51, 255−262.
(21) Rajkovic, S.; Rychlewska, U.; Warzajtis, B.; Asanin, D. P.;
Zivkovic, M. D.; Djuran, M. I. Disparate behavior of pyrazine and
pyridazine platinum(II) dimers in the hydrolysis of histidine- and
ACKNOWLEDGMENTS
■
̌
Dr. Zeljko Vitnik (Department of Chemistry, IChTM
Institute of Chemistry, Technology and Metallurgy, University
of Belgrade, Belgrade, Serbia) is greatly acknowledged for
providing the G09 and computing facilities. This work has been
financially supported by the Ministry of Education and Science,
Republic of Serbia, under Grants 172036 and 173048.
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