Platinum(ii) O,S complexes as potential metallodrugs against Cisplatin resistance

Article abstract of DOI:10.1039/c6dt01388k

We report on platinum(ii) complexes with different cinnamic acid derivatives as ligands with cytotoxic activity against Cisplatin resistant ovarian cancer cell line subcultures of SKOV3 and A2780. A typical mechanism of action for platinum(ii) complexes a

Full text of DOI:10.1039/c6dt01388k

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Dalton Trans., 2016, DOI: 10.1039/C6DT01388K.
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Platinum(II) O,S complexes as potential metallodrugs against
Cisplatin resistance
Jana Hildebrandt,a Norman Häfner,b Helmar Görls,a Daniel Kritsch,b Giarita Ferraro,c Matthias Dürst,b
Ingo B. Runnebaum,b * Antonello Merlino,c,d * and Wolfgang Weiganda *
* Prof. Dr. W. Weigand EꢀMail: wolfgang.weigand@uniꢀjena.de
* Prof. Dr. A. Merlino EꢀMail: antonello.merlino@unina.it
a Institut für Anorganische und Analytische Chemie FriedrichꢀSchillerꢀUniversität Jena Humboldtstraße
8, 07743 Jena, Germany
bDepartment of Gynecology, Jena University Hospital ꢀ Friedrich Schiller University Jena
cDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte
Sant’ Angelo, Via Cintia, Iꢀ80126, Napoli, Italy
dCNR Institute of Biostructures and Bioimages, Via Mezzocannone 16, Iꢀ80100, Napoli, Italy
AUTHOR EMAIL ADDRESSES (wolfgang.weigand@uniꢀjena.de antonello.merlino@unina.it)
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
* To whom correspondence should be addressed. Eꢀmail: wolfgang.weigand@uniꢀjena.de (W.W.), Fax: +49
3641 948102, Tel: +49 3641 948160, antonello.merlino@unina.it (A.M), Fax: +39 081674090, Tel: +39
081674276
1

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Abstract We report on platinum(II) complexes with different cinnamic acid derivatives as ligands with
cytotoxic activity against Cisplatin resistant ovarian cancer cell line subcultures of SKOV3 and A2780.
Typical mechanism of actions for platinum(II) complexes as Cisplatin itself are binding to the DNA and
inducing doubleꢀstrand breaks. We examined the biological behavior of these potential drugs with 9ꢀ
methylguanine using NMR spectroscopic methods and their DNA damage potential including γH2AXꢀfoci
analyses. Xꢀray diffraction methods have been used to elucidate the molecular structures of the platinum(II)
complexes. Interactions with the model protein lysozyme have been evaluated by different techniques
including UvꢀVis absorption spectroscopy, fluorescence and Xꢀray crystallography.
Keywords Cisplatin / 9ꢀmethylguanine /Cisplatin resistance / γH2AXꢀfoci analyses / protein platination/ Xꢀ
ray crystallography
Introduction
Cisplatin was first synthesized by Michele Peyrone in 1845 and its square planar configuration was
demonstrated by Alfred Werner ca. 50 years later. 1ꢀ4 Its mechanism of action results in binding to the DNA,
especially guanine, introducing strand crosslinks and causing DNA breaks during replication in proliferating
cells (Figure 1). This results in an arrest of the cell cycle and to apoptosis of the affected cells as one of the
main effects.5ꢀ10 After intravenous application of the drug the delivery into the cells can be achieved by
active transport via copper transporter or by passive diffusion.11ꢀ13
Figure 1. Behavior of Cisplatin after i.v. application: extracellular inactivation via binding to proteins (like
albumin), active transport via copper transporter (CTR1) or passive diffusion into the cell and activation
inside depending on lower Clꢀ concentration [Clꢀ] to cationic aqua species which bind to genomic DNA.4ꢀ12
2

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The lower intracellular chloride concentration compared to the blood plasma leads to ligand exchange
reactions, in which the chlorido ligands of Cisplatin are substituted by aqua ligands.14 This reaction generates
the active platinum(II) species being able to bind to the DNA (Figure 1).15, 16 Nevertheless, there are certain
drawbacks in the anticancer therapy using Cisplatin: 17ꢀ27
•
•
•
•
low selectivity of this drug for tumor cells, resulting in severe side effects
binding to extracellular albumin that leads to inactivation of the drug
activity of DNA repair mechanisms may destroy the DNA Cisplatin adduct
further principles of drug resistance of tumor cells.
The drug resistance, especially if caused by pꢀglycoprotein, is a major problem in treating diseases by
pharmacological therapy.28, 29 Moreover, it was shown that epigenetic changes in the cancer cells, if treated
with sublethal concentrations of Cisplatin may contribute to the resistance phenotype.30 All resistance
mechanisms result in the lowering of Cisplatin effects and, in most cases, reduce the efficacy of a second
treatment using Cisplatin. To solve these problems many substances of biological or synthetic origin were
investigated concerning their use in cancer therapy. Targeted therapeutics offer an increased selectivity, but
they are only suitable for specific cancer harboring the target. For other types of cancer, especially those that
occur rarely, the use of Cisplatin is still the best way to treat them. It is noteworthy that even more selective
drugs can lose their selectivity by changes of the genetic material of the tumor or of the tumor
environment.21, 26
This work deals with the design of platinum(II) based drugs to circumvent Cisplatin resistance. One problem
that has to be solved is the inactivation of Cisplatin by binding to sulfurꢀcontaining molecules like albumin in
the blood plasma. It is known that platinum(II) exhibits a high affinity to sulfur atoms in organic
molecules.20, 31, 32 By binding to these sulfur sites, the drug molecules are excreted before they can bind to the
DNA or even before they can pass the cell wall. For this reason there is a necessity to investigate the
interactions of new platinum based drugs to proteins as well as the interactions with the DNA.33
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Substance
Code
Rest
ꢀR
Alkylgroup
ꢀAlk
ꢀ
o
ꢀOCH3
ꢀmethyl
ꢀmethyl
ꢀmethyl
ꢀethyl
ꢀethyl
ꢀethyl
ꢀmethyl
ꢀmethyl
ꢀethyl
ꢀethyl
ꢀmethyl
ꢀethyl
L1/Pt1
L2/Pt2
L3/Pt3
L4/Pt4
L5/Pt5
L6/Pt6
L7/Pt7
L8/Pt8
L9/Pt9
L10/Pt10
L11/Pt11
L12/Pt12
ꢀmꢀOCH3
ꢀp
ꢀOCH3
ꢀOCH3
ꢀo
ꢀm
ꢀOCH3
ꢀp
ꢀOCH3
ꢀ
m
ꢀOH
ꢀOH
ꢀOH
ꢀOH
ꢀH
ꢀH
ꢀp
ꢀ
m
ꢀp
Figure 2. Scheme of synthesized ligands and platinum(II) complexes and its substance code with new
platinum(II) complexes Pt1ꢀPt6.
Different types of βꢀhydroxyꢀdithiocinnamic acid derivatives were used as ligands. The properties of this
class of compounds can be modified by changing the substitution pattern as well as the chain length of the
alkyl substituent. As substituents for the aromatic moiety hydroxy and methoxy groups were used to vary the
polarity of the ligand as well as of the platinum(II) complex. To investigate the effect of Cisplatin resistance,
the cytotoxic activity of all compounds (βꢀhydroxy dithiocinnamic acid derivatives and the corresponding
platinum(II) complexes) was tested with normal and Cisplatin resistant cell lines.
Figure 2 shows the used βꢀhydroxy dithiocinnamic acid derivatives and their corresponding platinum(II)
complexes. Ligands L1ꢀL12 can be found in the literature, their cytotoxic effects were not determined until
now.33ꢀ39 Platinum(II) complexes Pt7ꢀPt12 were reported earlier, this works presents first time biological
significance of these compounds depending on their antitumor activity and reinvestigated what was reported
earlier.33, 40
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Results and Discussion
Synthesis
Scheme 1. Reagents and conditions: (a) (i) 2 equiv imidazole, 1 equiv TBDMS, DMF, rt, 24 h; (ii) H2O/
NaHCO3; (b) (i) 2 equiv ꢀBuOK, Et2O, ꢀ70 °C, 0.5 h; (ii) 1.4 equiv CS2, ꢀ70 °C, 1 h; (iii) rt, 1.5 h; (iv) 1
t
equiv AlkꢀI, rt, 24 h; (v) H2SO4/H2O, rt, 0.5 h; (c) (i) 2 equiv TBAF, THF, rt, 72 h; (ii) H2SO4/H2O, rt, 0.5 h.
37ꢀ39
Cinnamic acid derivatives L1ꢀL12 were synthesized according to modified literature methods.34,
A
general pathway is shown in Scheme 1.
For the synthesis of the platinum(II) complexes (Scheme 2) two pathways differing in the amount of tꢀBuOK
can be applied. In absence of an aromatic ꢀOH substituent one equivalent is used to deprotonate the cinnamic
acid derivative. This intermediate is reacted with in situ generated K[PtCl3(dmso)] complex (prepared from
K2PtCl4 and dmso).33, 40ꢀ43 In presence of an aromatic ꢀOH substituent the use of two equivalents of base
results in higher yield of the desired platinum(II) complexes. This pathway needs an additional protonation
step in the end.
Scheme 2. Reagents and conditions: (a) (i) 1 equiv tꢀBuOK, THF, rt, 0.5 h; (ii) 1.1 equiv K[PtCl3(dmso)], rt,
H2O/THF, 96 h; (b) (i) 2 equiv
(iii) H2SO4/H2O, rt, 0.5 h.
t
ꢀBuOK, THF, rt, 0.5 h; (ii) 1.1 equiv K[PtCl3(dmso)], rt, H2O/THF, 96 h;
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Molecular Structures
Platinum(II) complexes Pt1ꢀPt3, Pt5ꢀPt7, Pt9 and Pt10 as well as ligands L1 and L7 were characterized by
means of single crystal Xꢀray structure determination, whereas the molecular structures of Pt12, L3, L8 and
L11 are already known.[32, 36, 43] For compounds Pt2, Pt3, Pt6, Pt9 and L7 there are at least two
independent molecules in their unit cells, but just one is shown. Bond lengths and angles are very similar for
the molecules in a unit cell so that only one molecule is discussed below. All molecular structures are
displayed in supporting information (Figure S1), Figure 3 shows as an example Pt1 and Pt7.
In Figure 4 selected bond lengths and angles of the ligands L1 and L7 are presented. Both compounds offer a
cis enol configuration due to intramolecular hydrogen bonding between the OH group and the adjacent
thiocarbonyl moiety. Ligand L1 displays a OH¬…S distance of 2.08 Å, which is quite similar to that of
ligand L7 with 2.05 Å. Results confirm data that were reported earlier.[36, 43] The C(1)ꢀS(1) distance is a
bit longer than that of a typical ꢀC=S double bond (1.61 Å), C(2)ꢀC(3) shows typical double bond character,
whereas C(1)ꢀC(2) distance is typical for a single bond. The bond lengths C(3)ꢀO(1) are determined as
1.334(3) for L1 and 1.3308(6) for L7. For the meta hydroxy substituted ligand L7 the formation of
intermolecular hydrogen bondings can be assumed (2.77 Å for molecule A, 2.74 Å for molecule B) (Figure
5). Furthermore in the molecular structure of L1 an interaction of the methoxy substituent and the methine
proton can be detected, which is expressed by a short distance (2.33 Å) of the oxygen atom of the methoxyꢀ
group and the methine proton.
Figure 3. Molecule Structures (50% propability) of Pt1 (left) and Pt7 (right).
This proves an intramolecular interaction which results in a low field shift of the resonance signal of the
methine proton in the 1H NMR spectra in contrast to other βꢀhydroxy dithiocinnamic acid alkyl esters. Due
to the steric demand of the metaꢀmethoxy substituent in L1 a torsion angle between the aryl ring and the
planar enol moiety (C3/C2/C1/O1/S1/S2/C10) of 29° is detected, whereas in L7 the same angle was
determined as 4° (Figure 5).
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L1
L7
1.681(2)
1.334(3)
1.424(3)
1.368(3)
1.364(3)
1.439(3)
1.476(3)
1.739(3)
0.94(3)
1.662(5)
1.330(6)
1.435(6)
1.361(6)
1.376(6)
C(1)ꢀS(1)
C(3)ꢀO(1)
C(1)ꢀC(2)
C(2)ꢀC(3)
C(9/8)ꢀO(2)
O(1)ꢀC(11)
C(3)ꢀC(4)
C(1)ꢀS(2)
C(2)ꢀH(2)
1.477(6)
1.746(5)
0.950(0)
125.2(4)
123.4(4)
126.29(19)
123.2(2)
S(1)ꢀC(1)ꢀC(2)
O(1)ꢀC(3)ꢀC(2)
Figure 4. Molecule Structures (50% propability) of L1 (left) and L7 (right) with specific bond angles [Å]
and bond lengths [ppm].
Figure 5. Left: Molecular packing of L7, intermolecular hydrogenꢀbonding; Right: Steric demand of
methoxyꢀgroup resulting in nonꢀplanarity behaviour of L1 (top) in comparison to the planar molecule L7
(below).
The bond lengths and angles in the platinum(II) complexes Pt1ꢀPt3, Pt5ꢀPt7, Pt 9 and Pt10 are in good
agreement with values reported earlier.33 The platinum(II) center shows a distorted square planar
environment with LꢀPtꢀL angles of around 90° (see also Figure 3 and Table 1). The dmso coordination is cis
to the sulfur atom of the bidendate O,Sꢀligand because of the πꢀdonorꢀfunction of the sulfur atom. The bond
lengths of platinum (here for example Pt7) and their four neighbor atoms are decreasing in the order of
Cl(1)ꢀPt(1) (2.347(2)) > S(1)ꢀPt(1) (2.234(2)) ≈ S(3)ꢀPt(1) (2.189(2)) > O(1)ꢀPt(1) (2.014(6)). Moreover, the
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angles of these coordination spheres are in the range of 90°. The bond lengths of the oxygen substituted
moiety at the aromatic ring O(2)ꢀC(9/8/7) are in the same range.
Table 1. Specific bond angles [Å] and bond lengths [ppm] for all characterized platinum(II) compounds.
Pt1
Pt2
Pt3
Pt5
2.015(7)
2.251(6)
2.347(6)
2.195(6)
1.274(3)
1.710(3)
1.363(3)
175.10(5)
91.20(2)
84.13(5)
89.51(2)
1.985(10)
2.251(3)
2.338(3)
2.198(3)
1.282(18)
1.690(14)
1.298(17)
174.9(3)
90.05(12)
85.3(3)
1.998(3)
2.007(4)
O(1)ꢀPt(1)
S(1)ꢀPt(1)
Cl(1)ꢀPt(1)
S(3)ꢀPt(1)
O(1)ꢀC(3)
2.2556(13) 2.2520(14)
2.3595(13) 2.3463(13)
2.1846(12) 2.1934(14)
1.283(6)
1.694(5)
1.355(6)
1.287(7)
1.701(6)
1.368(7)
S(1)ꢀC(1)
O(2)ꢀC(9/8/7)
O(1)ꢀPt(1)ꢀS(3)
S(3)ꢀPt(1)ꢀCl(1)
Cl(1)ꢀPt(1)ꢀO(1)
S(1)ꢀPt(1)ꢀS(3)
174.44(11) 175.75(12)
89.98(5)
84.48(11)
90.11(5)
89.84(5)
84.91(12)
89.84(5)
89.10(13)
Pt6
Pt7
Pt9
Pt10
O(1)ꢀPt(1)
S(1)ꢀPt(1)
Cl(1)ꢀPt(1)
S(3)ꢀPt(1)
O(1)ꢀC(3)
2.003(5)
2.2521(17)
2.3430(17)
2.1865(17)
1.280(8)
1.700(7)
1.361(8)
2.014(6)
2.234(2)
2.347(2)
2.189(2)
1.278(11)
1.704(9)
1.362(12)
2.017(3)
2.2439(10)
2.3520(10) 2.3359(13)
2.1900(10) 2.1985(13)
1.284(5)
1.700(4)
1.351(6)
176.34(9)
90.96(4)
85.77(8)
87.96(4)
2.011(4)
2.449(13)
1.286(7)
1.696(6)
1.362(7)
175.11(11)
90.48(5)
84.99(11)
88.62(5)
S(1)ꢀC(1)
O(2)ꢀC(9/8/7)
O(1)ꢀPt(1)ꢀS(3)
S(3)ꢀPt(1)ꢀCl(1)
Cl(1)ꢀPt(1)ꢀO(1)
S(1)ꢀPt(1)ꢀS(3)
174.14(15) 174.97(19)
90.08(6)
84.28(14)
89.63(6)
90.21(7)
85.96(17)
88.77(8)
Table 2 shows torsion angles (aryl planes and C(1)ꢀC(2)ꢀC(3) planes) which are resulting of the steric claim
of different substitution pattern at the aromatic ring. The values show clearly that the smallest angle can be
observed in case of the para substituted molecules.
Coordination of all O,Sꢀchelating ligands to platinum(II) results in elongation of the C(1)ꢀS(1) and
shortening of the C(3)ꢀO(1) bonds. This tendency can be also observed in 13C{1H}ꢀNMR spectra resulting in
a high field shift of the signal of the ꢀC=Sꢀresonance and low field shift of the ꢀCꢀOꢀgroup. For Pt1 an
interaction between the methoxy group and the methine proton is observed as for L1. The short O(2)ꢀH(2)
distance of 2.20 Å is indicative for the intramolecular relationship in these ortho substituted molecules.
The molecular structure of L7 reveals that four molecules are connected through four hydrogen bonds
forming an almost perfect square Pt10 is able to form an intermolecular hydrogen bonding system (Figure
6). In the crystal there is a short contact between the hydroxyꢀgroup of one molecule and the oxygen of the
dmso ligand of another molecule with a OꢀO distance of 2.73 Å.
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Table 2. Torsion angles: angles between the aryl planes and C(1)ꢀC(2)ꢀC(3) planes in dependence of the
substitution pattern.
Pt1
Pt2
Pt3
Pt5
Pt6
Pt7
Pt9
Pt10
30°
24°
17°
35°
7°
21°
31°
16°
Torsion angle
Figure 6. Intermolecular hydrogenꢀbonding observed in the crystals of Pt10.
Spectroscopic Characterization
All compounds were characterized by NMR spectroscopy, mass spectrometry and elemental analysis (see
1
experimental part). The chemical shifts in H NMR and 13C{1H} NMR spectra for ligands L1ꢀL12 are in
good agreement with the values reported earlier, Table 3.37 Interestingly the interaction of the ortho methoxy
substituent and the methine proton that was found in the molecular structure of L1 can be also detected in the
1H NMR spectrum. The resonance signal of this proton is shifted to low field compared to those resonances
of all other ligands, in which such interactions are not possible due to steric reasons. Another characteristic
resonance signal in the 1H NMR spectra of this type of compounds is caused by the enolic OH group and can
be observed in some cases at very low frequencies at around 15 ppm. This also indicates the intramolecular
hydrogen bondings to the sulfur atom of the thiocarbonyl moiety.
Table 3. Specific signals in NMR spectra of L1ꢀL12 and Pt1ꢀPt12 in ppm.
1H
NMR
L1
Pt1
L2
Pt2
L3
Pt3
L4
Pt4
L5
Pt5 Pt6
L6
L7
Pt7
L8
Pt8
L9
Pt9
L10
L11
L12
Pt10 Pt11 Pt12
15.08
15.21 15.21 15.18 15.21 15.09
15.14 15.20 15.02 15.16
ꢀCꢀOH
7.20 6.97
7.35 7.35
6.98 7.23 6.91
7.04 7.35 7.06
6.91
7.07
L6
6.96 6.88 6.91
7.15 7.16 7.15
6.91
7.16
L10
6.89 6.90
7.28 7.27
ꢀCꢀH
13C{1H}
NMR
L1
L2
L3
L4
L5
L7
L8
L9
L11
L12
Pt1
Pt2
Pt3
Pt4
Pt5
Pt6
Pt7
Pt8
Pt9
Pt10 Pt11 Pt12
167.6 169.1 169.6 167.8 169.4 169.9 169.3 169.2 169.6 170.1 169.2 169.4
ꢀCꢀOH
ꢀCꢀCꢀH
ꢀC=S
112.9 112.9 107.1 112.8 112.9 106.8 113.8 106.8 113.8 106.9 107.8 107.7
116.3 112.9 111.0 116.5 112.9 111.0 110.8 109.9 111.0 110.3 111.6 111.8
217.2 217.3 215.7 216.3 217.3 214.9 217.8 215.8 217.1 215.2 217.3 216.3
199.0 180.9 177.9 199.0 177.6 177.9 177.6 178.7 196.8 177.6 177.6 181.5
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1
Specific shifts of 13C{1H} NMR and H NMR resonance signals observable for the platinum(II) complexes
are shown in Table 3. After complexation of the ligands via oxygen and sulfur atoms to platinum(II) the
1
enolic OH signals in the H NMR spectra disappear. Interestingly the signals of the methine protons are
shifted to low frequencies as a result of their complexation to platinum(II). In the 13C{1H} NMR spectra the
13C signal of the ꢀC=S group can be observed at high frequencies for the platinum(II) compounds compared
to those of the ligands. The protons of the dmso ligand are observed as a singlet accompanied with 195Pt
satellites in 1H NMR spectra. The averages for these signals, which are presented with respect to the carbon
side chains are shown in supporting information (Table S2).
Mass spectra are in common with earlier reported results.33, 37, 40
Stability Determination
1
Stability of compounds Pt7 and L8 in dmso as solvent were determined using H NMR spectroscopy. For
biological testing compounds were dissolved in dmso (see experimental part). Figure 7 shows exemplary
aromatic signals for compound Pt7 from start point (blue) and after 48 hours (red). It was observed that all
compounds are stable under these conditions (for L8: Figure S2 in supporting information).
Figure 7. Stability determination of Pt7 using 1H NMR spectroscopy, Conditions: 600 MHz, 37 °C, dmsoꢀ
d6. Blue: first measurement (starting point), red: after 48 h. All compounds are stable under these conditions.
However, since it is well known that the behavior of metal based drugs in organic solvent like dmso could be
different when compared to that observed in the solutions used for the biological studies, the stability of the
platinum complexes here studied were assessed also in aqueous solutions (from 10 to 100 % dmso) using
UV–Vis absorption spectroscopy.44 In particular, the spectra of the compounds Pt1ꢀPt6 were collected at t =
0 h and after 24 h. Analyses of the spectra show that the compounds Pt1ꢀPt6 with the exception of Pt2, are
highly stable in pure dmso (Figure S3A), and rather stable in 70ꢀ90% dmso. The reason why Pt2 is not so
stable in pure dmso are unknown. On the contrary, they are less stable at low dmso concentrations (Figure
S3C). For example Pt1 is stable in 70ꢀ90% and pure dmso, in agreement with NMR data, whereas it presents
a significant decrease in the intensity of the UvꢀVis absorption bands after 24 h in aqueous solutions with
dmso ranging from 10 to 60%. This decrease is coincident with precipitation of the sample.
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The stability of Pt complexes Pt1ꢀPt6 was also assessed in a saline solution (0.9% NaCl, 1%<[dmso]<2%),
and in the reference physiological buffer solution (10 mM PBS pH 7.4) (Figure S4). Under these
experimental conditions, the complexes seem only marginally stable, since they rapidly precipitate, as occurs
in aqueous solutions containing low dmso concentrations. These findings indicate that under these
experimental conditions the integrity of the compounds could be compromised.
DNAꢀbinding Behavior
H
H
N
H3N
H
Pt
Cl
O
N
NH
N
N
NH2
sugar
Figure 8. DNAꢀbinding behaviour of Pt7 with model base 9ꢀmethylguanine shows formation of new
compound after 24 h, 37 °C, 600 MHz, dmsoꢀd6; blue: start of measurement, red: after 24 h.
The cytotoxic behavior of Cisplatin and analogues is a result of binding to DNA bases i.e. guanine,
influencing the DNA structure and causing DNA damage during genome replication. Preferred binding
position is N7 of guanine because of a stabilization effect via hydrogenꢀbonding of Cisplatin ꢀNH3 group and
ꢀC=Oꢀgroup of the DNA base (Figure 8).5, 31 As shown in Figure 1, mechanism of activation contains a
1
ligandꢀexchange of the chlorido ligands. Using H NMR spectroscopy the binding properties of a 3.23ꢀfold
stoichiometric excess of the model base 9ꢀmethylguanine to platinum(II) compounds should be investigated.
As shown in Figure 7, platinum(II) complexes are stable under these conditions, so all changes in the NMR
spectra results from interaction with the model base. This NMR spectroscopic experiment carried out for
compound Pt7 monitored over 24 h at 37 °C in dmsoꢀd6 results in significant changes in this spectra. After
24 h the 1H NMR spectra show in the aromatic region that a new compound is generated slowly, which can
be seen by occurrence of a new set of proton signals for the methine proton as well as for all aromatic
protons. The spectra show after 24 h a second proton group high field shifted (red spectrum, 0.05 ppm)
compared to the signal set at t = 0 h (blue spectrum). In contrast, a down field shift (red spectrum) can be
observed for the methine proton of Pt7 after 24 h. These results give hint for a successful reaction of Pt7
with 9ꢀmethylguanine however it seems that the reaction is slow and not completed after 24 h.
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Biological Behavior
A further aim of our investigations was to characterize all of these compounds for their cytotoxic activity
against a panel of cell lines enabling an interrogation of the structure – activity relationship. Used cell lines
differed in their sensitivity against Cisplatin. Therefore candidate platinum(II) complexes potentially
effective against Cisplatin resistant cells could be identified.
Figure 9. Influence of dmso depending on %/well for each cell line.
Analyzed ligands as well as platinum(II) complexes exhibit a low solubility in water independent from
ligand structure. DMSO is used as a solvent for preparation of dilution series in cell culture experiments. The
toxic influence of a range of dmso concentrations was measured via MTT assay under identical conditions as
for substance tests (Figure 9). Results show that concentrations of dmso down to 1% have high cytotoxic
effects. Therefore a dmso concentration as low as possible to reach required Pt(II) complex concentrations
was used in the experiments and was identical for each substance and concentration (0.5% dmso in cell
culture media). To exclude side effects from this dmso concentration all IC50 determination experiments
used 0.5% dmso as reference samples. Additional experimental conditions can bias cell cultivation based
IC50 determinations i.e. cell density, cultivation time and drug exposure time influence measured effects.
High cell numbers (>5000 cells per well in 96ꢀwell plate) combined with long cultivation times (>72 h) may
result in inhibitory effects on cell proliferation for samples with a low drug concentration and for control
cells. Importantly any bias for cells even treated with drug concentrations different from IC50 will affect
IC50 determination because regression analyses will interrogate all data. Thus we measured drug effects for
48 h exposure time after 24 h seeding of 5000 cells per well resulting in unsaturated cell density and
absorbance measurements for untreated cells (data not shown). Both, constantly low dmso concentrations
and controlled cultivation procedures represent important improvements in comparison to earlier data.33, 40
Detailed experimental procedures are described in experimental part.
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Table 4. IC50 values and resistance factors (RF) for all substances.
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The tested cell line panel consisted of A549 (lung cancer) and two pairs of isogenic ovarian cancer cell lines
with different Cisplatin sensitivity.30
Resistant ovarian cancer cells derived from A2780 and SKOV3 exhibited a 4.7 and 3.6 times higher IC50
value for Cisplatin, respectively. These cell cultures were established by repeated rounds of incubation with
increasing Cisplatin concentrations starting with nonꢀlethal concentrations.30 Table 4 shows that the new
substances show cytotoxic activity against all cell lines, not only for ovarian carcinoma cell lines SKOV and
A2780. Determination for all pairs of ligand and platinum(II) complex prove that dithiocinnamic acid
derivatives show low cytotoxic behavior itself and corresponding platinum(II) complex elevated
cytotoxicity. It can be concluded, that the increased cytotoxic behavior of platinum(II) complexes is a result
of the metal itself. An exception seems to be the cell line A2780cis exhibiting IC50 values similar between
some ligands and their complexes (Table 4). Moreover a significant but weak correlation was identified
between the IC50 of the ligand and the complex (Pearson correlation coefficient 0.301; p<0.01; Figure 10).
Figure 10. Correlation of ligand and platinum(II) complex IC50 values in all cell lines. The weak but
significant correlation (Pearson r=0.301, p<0,001) is depicted by the regression line.
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Figure 11. Structure activity relationship for analyzed platinum(II) complexes. Substances were ordered
with increasing mean IC50 values.
Figure 12. Detection of γH2AX foci in parental and Cisplatin resistant A2780 (A) and SKOV3 (B) after
mock treatment or incubation with Cisplatin, Pt9 or Pt10 at IC50 concentrations for the resistant cells.
Identical results were obtained in an independent experiment.
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Thus a limited part of the platinum(II) complexes activity is maybe directly contributable to the ligand
properties. One aim of the present study was to analyze structureꢀactivityꢀrelationships (SAR). Two
structures within the analyzed ligands were systematically changed to vary complex properties (substituent at
the benzyl ring (ꢀR) and alkylꢀchain at the sulfur residue (ꢀAlk)). Plotting the IC50 values for all substances
of each cell line in the order of increasing mean IC50 enabled the identification of SAR (Figure 11).
Interestingly most active complexes exhibited an increased polarity of ꢀR (ꢀOH > ꢀOCH3 > ꢀH) but increased
lipophilicity at the alkyl chain (Et > Me). The exceptional cases of orthoꢀOCH3 may be explained by
intramolecular interactions between the methoxy group and the methine proton influencing the complex
structures (see part molecular structures). Increased activity of complexes with longer alkyl chains confirms
earlier studies.33, 40 Nevertheless differences in the mean IC50 were small and substances exhibited cell line
specific activities (i.e. Pt9, Figure 11). Interestingly, we did observe an inverse association between the
platinum(II) complex stability and the cytotoxic activity. Pt1 exhibiting a lower stability in aqueous solutions
showed a higher cytotoxicity than Pt4 (Figure 11, Table 4). Nevertheless the general impact should be
determined in the in-vivo situation taking effects of the bioavailability into account.
The second aim was to test new platinum(II) complexes on Cisplatinꢀresistant cell lines to evaluate the
possibility to overcome the resistance phenotype. The two most active platinum(II) complexes (Pt9, Pt10)
showed an increased or at least equal cytotoxic activity against the Cisplatinꢀresistant ovarian cancer cell
lines (Table 4). Moreover the activity was similar or even higher against the resistant cell lines in comparison
to the parental cells. Calculated resistance factors between parental and resistant ovarian cancer cell lines
(Table 4) illustrate clear differences between Cisplatin and some of the new platinum(II) complexes (i.e. RF
Cisplatin 4.7 and 3.6; Pt9 1.0 and 0.4 for A2780 and SKOV3, respectively). These data let assume that the
mechanism of action is different between the new compounds and Cisplatin. Moreover, a different kinetic
cannot be excluded and is already seen within the guanine binding studies (see part DNA bonding behavior).
Albeit we detected a high cytotoxic activity of the new complexes in-vitro an inactivation mechanism by
binding to the tripeptide glutathion could not be excluded ꢀ for more information see supporting information
(Figure S10). To gain insights into the mode of action we exemplarily analyzed Pt9 and Pt10 for the
induction of DNA damage in the ovarian cancer cell line pairs in two independent experiments. Cells were
incubated with IC50 concentrations of the resistant cell culture of A2780 or SKOV3 for 24 hours on cover
slips. Afterwards cells were washed, fixed and antibodyꢀstained for γH2AX histone. Nuclear foci of γH2AX
are indicative for dsDNA break regions.45 Cisplatin induced DNA damage and γH2AX foci formation clearly
correlates with cytotoxic effects (Figure 12). Resistant cells showed decreased numbers of γH2AX foci.
SKOV3 cells that are more resistant to Cisplatin than A2780 as illustrated by different IC50 values of 13.5
µM and 6.1 µM for the resistant cells, respectively, can tolerate increased γH2AX foci. The amount of
γH2AX foci after treatment with Pt9 or Pt10 was much lower in comparison to the Cisplatin treatment
despite all substances were used with IC50 concentrations (Figure 12). Therefore a different mode of action
for the new platinum(II) complexes is likely and may include nonꢀgenomic targets. Otherwise it cannot be
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excluded that the platinum(II) complexes show different kinetic behavior or target DNA differently from
Cisplatin leading to other DNA damage responses not including γH2AX.
In conclusion platinum(II) complexes were proven to exhibit cytotoxic activities partially correlated to the
ligand properties. The small differences in mean IC50 values enable a selection of the best candidates with
both cytotoxic activity and superior bioavailability in future experiments (i.e. in vivo). Furthermore a high
activity was detected for some substances against Cisplatinꢀresistant cell lines. Additional experiments will
clarify the underlying mode of action for these platinum(II) complexes. If general differences to Cisplatin
can be detected the efficacy of a combination treatment should be evaluated.
Interactions with proteins
Previous data show that the here investigated compounds show cytotoxic activity, especially Pt9 and Pt10
circumvent Cisplatin resistance (Table 4) but show decreased DNA damages (Figure 12). For this reason the
discovery of other targets for their mechanism of action is necessary. The interaction of metallodrugs with
proteins is very important, since it affects their pharmacokinetics, toxicity and tissue distribution. The
binding of metalꢀbased drugs with transferrin for example is directly involved in the transport of drugs into
the cell. We have already demonstrated that compounds Pt7 and Pt9 are able to bind the model protein hen
egg white lysozyme (HEWL) and the Xꢀray structure determinations of the adducts forming upon drugꢀ
protein interaction have been determined.40 The structures demonstrate that the compounds can act as
monofunction drugs, retaining dmso ligand upon macromolecule binding. To further characterize the binding
properties of the compounds here studied with proteins, we have investigated the reactivity of compounds
Pt1ꢀPt6 with hen egg white lysozyme (HEWL), a protein that is very frequently used as a prototype for
protein metalation studies.46, 47
First, fluorescence spectra of HEWL in the presence of different concentrations of the platinum complexes
were collected. Intrinsic fluorescence of HEWL arise from the intrinsic structural features of the protein and
is mainly due to the presence of six tryptophan residues. Figure S5 and Figure S6 show the modifications to
the fluorescence emission of HEWL, upon excitation at 280 and 295 nm, respectively, when the Pt1ꢀPt6
concentrations were increased. The binding of the drugs to the protein induces a significant quenching of the
emission. Differences in the fluorescence quenching are probably related to different solubility of the
samples rather than to differences in the binding properties of the compounds.
Finally, to identify the type of interaction that occur between HEWL and the Pt compounds we have tried to
obtain structural information on the adducts formed by the protein with Pt1ꢀPt4 by Xꢀray crystallography.
Attempts to crystallize the adducts formed upon the binding of the compounds to the protein under the same
conditions used to crystallize the HEWLꢀPt7 and HEWLꢀPt9 adducts failed.40 This is probably due to the
lower solubility of Pt1, Pt2, Pt3, and Pt4 in ethylene glycol when compared to the compounds Pt7 and Pt9,
which present aꢀOH in meta position. Thus, the reactivity of the compounds with HEWL was explored under
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different experimental conditions and the structures of some adducts formed in the presence of concentrated
NaCl solutions were solved (see Supporting information Figure S7ꢀS9). Under these experimental conditions
Pt compounds degrade and the products of this degradation react with the protein forming an adduct with a
Pt atom bound to ND1 atom of His15 side chain (see for example Figure S8), as in the case of many Pt
compounds, including Cisplatin, Pt7 and Pt9 (see Supporting Information for further details).40, 48ꢀ51
Conclusion
The mechanism of action for standard drug Cisplatin is realized by DNA binding and induction of replicative
stress resulting in DNA damage. This mechanism can be disturbed especially by the activity of DNA repair
mechanism but also other mechanisms ending in the resistance of cancer cells. To circumvent Cisplatin
resistance by the use of the effective Pt(II) pharmacophore was one aim of this work. Different ligands with
varying properties and corresponding Pt(II) complexes were tested on several cell lines. Results show that
the platinum(II) center is necessary for an effective anticancer activity. Examination of the mechanism of
action shows that the typical DNAꢀbinding behavior of Cisplatin and analogues and the induction of the
DNA damage is decreased for the new Pt(II) complexes. Nevertheless some compounds have a high activity
against Cisplatin resistant cell lines.
In summary, this work presents new platinum(II) complexes with O,Sꢀbidendate ligands which are well
characterized by different analytical techniques, especially with Xꢀray structure determinations. These
complexes show cytotoxic activity partially overcoming Cisplatin resistance of cell lines which may be
explained by a different mechanism of action ꢀ specifically the bonding to proteins.
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Experimental Part
Materials and Techniques
All reactions were performed using standard Schlenk and vacuumꢀline techniques under nitrogen
atmosphere. The NMR spectra were recorded with a Bruker Avance 200 MHz, 400 MHz or 600 MHz
spectrometer. Chemical shifts are given in ppm with reference to SiMe4. Mass spectra were recorded with a
Finnigan MAT SSQ 710 instrument. Elemental analysis was performed with a Leco CHNSꢀ932 apparatus.
Silica gel 60 (0.015ꢀ0.040 mm) was used for column chromatography and TLC was performed using Merck
TLC aluminium sheets (Silica gel 60 F254). Chemicals were purchased from Fisher Scientific, Aldrich or
Acros and were used without further purification. All solvents were dried and distilled prior to use according
to standard methods.
Synthesis
General procedure 1: platinum(II)ꢀcomplexes with βꢀhydroxy dithiocinnamic acid alkyl esters,
chlorido and dmso as ligands (Pt1ꢀPt12).
Pathway A: The βꢀhydroxy dithiocinnamic acid alkyl ester (1 equiv) was solved in tetrahydrofurane (THF,
20 ml) and tꢀBuOK (1 equiv) was added to the the solution and stirred for one hour at room temperature.
Potassium tetrachloroplatinate (K2PtCl4, 1.1 equiv) was solved in degassed water (10 ml) and
dimethylsulfoxide (dmso, 2 equiv) was added, the mixture was stirred for 30 minutes at room temperature.
The solution of the deprotonated ligand was added drop wise to the suspension of the platinum complex and
stirred at room temperature for 4 days. After adding water (25 ml) to the solution, the mixture was extracted
with dichlormethane (DCM, 3x 30 ml), the combined organic phases were washed with water (3x20 ml),
dried over sodium sulfate and after filtration and evaporation of the solvent the crude product was purified
with column chromatography. Pathway B: Experimental procedure is similar to pathway A, for
deprotonation 2 equiv of tꢀBuOK was used. Before extracting with DCM, sulfuric acid (20 ml, 2M) was
added and mixture was stirred one hour at room temperature followed by extraction and purification.
Chloroꢀ(1ꢀ(2ꢀmethoxyphenyl)ꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt1).
Synthesis was performed according to general procedure 1, pathway A. L1 (367 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatographie mobile phase: DCM 4:hexane 1 ꢀ
DCM ꢀ acetone. Yield: 520 mg (62.1%) as red crystals. 1H NMR (600 MHz, acetoneꢀd6): δ = 2.65 (s, 3H, ꢀSꢀ
3
3
CH3); 3.66 (s w/Pt satellites JPt-H=22.5 Hz, 6H, CH3 (DMSO)); 3.94 (s, 3H, ꢀOCH3); 7.04 (ddd, JH-H=7.6
4
3
3
Hz, JH-H=1.0 Hz, 1H, ꢀArꢀmꢀH); 7.13 (d, JH-H=8.4 Hz, 1H, ꢀArꢀoꢀH); 7.35 (s, 1H, =CH); 7.53 (ddd, JH-
4
3
4
H=7.6 Hz, JH-H=1.0 Hz, 1H, ꢀArꢀpꢀH); 7.80 (dd, JH-H=7.8 Hz, JH-H=1.8 Hz, 1H, ArꢀmꢀH). 13C{1H} NMR
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(101 MHz, acetoneꢀd6): δ = 17.7 (ꢀSꢀCH3); 46.7 (DMSO); 56.4 (ꢀOCH3); 113.3 (ꢀArꢀoꢀC); 116.3 (=CH);
121.7 (ꢀArꢀmꢀC); 128.0 (ꢀArꢀC1); 132.1 (ꢀArꢀmꢀC); 133.8 (ꢀArꢀpꢀC); 158.3 (ꢀArꢀOCH3); 175.0 (ꢀCꢀOꢀ);
199.0 (ꢀC=S). MS (DEI): m/z = 548, 546, 386, 341, 284, 152, 135, 105, 78, 63. Elemental analysis:
calculated for C13H17ClO3PtS3ꢂ2/3 acetone C: 30.71%; H: 3.60%; S: 16.40%, found: C: 30.82%; H: 3.32%;
S: 16.71%.
Chloroꢀ(1ꢀ(3ꢀmethoxyphenyl)ꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt2).
Synthesis was performed according to general procedure 1, pathway A. L2 (367 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatographie mobile phase: DCM 4:hexane 1 ꢀ
DCM ꢀ acetone. Yield: 350 mg (41.8%) as orange crystals. 1H NMR (400 MHz, CDCl3): δ = 2.70 (s, 3H, ꢀSꢀ
CH3); 3.68 (s w/Pt satellites 3JPt-H=22.5 Hz, 6H, CH3 (DMSO)); 3.88 (s, 3H, ꢀOCH3); 7.10 (dd, 3JH-H=8.2 Hz,
4JH-H=0.8 Hz, 1H, ꢀArꢀpꢀH); 7.28 (s, 1H, =CH); 7.32 (t, 1H, ꢀArꢀmꢀH); 7.54 (m, 2H, ꢀArꢀoꢀH). 13C{1H} NMR
(101 MHz, CDCl3): δ = 17.6 (ꢀSꢀCH3); 46.9 (DMSO); 55.5 (ꢀOCH3); 112.9 (=CH); 118.3 (ꢀArꢀoꢀC); 129.7 (ꢀ
ArꢀmꢀC/ꢀArꢀC1); 135.7 (ꢀArꢀpꢀH); 159.9 (ꢀArꢀOCH3); 174.2 (ꢀCꢀOꢀ); 180.9 (ꢀC=S). MS (ESI): m/z = 565,
536, 512, 445, 101. Elemental analysis: calculated for C13H17ClO3PtS3ꢂ1/5 acetone C: 29.19%; H: 3.27%; S:
17.19%, found: C: 29.46%; H: 2.90%; S: 17.10%.
Chloroꢀ(1ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt3).
Synthesis was performed according to general procedure 1, pathway A. L3 (367 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatographie mobile phase: DCM 4:hexane 1 ꢀ
DCM ꢀ acetone. Yield: 350 mg (41.8%) as orange crystals.. 1H NMR (600 MHz, CDCl3): δ = 2.60 (s, 3H, ꢀSꢀ
CH3); 3.59 (s w/Pt satellites 3JPt-H=22.5 Hz, 6H, CH3 (DMSO)); 3.78 (s, 3H, ꢀOCH3); 6.84 (d, 3JH-H=9.0 Hz,
2H, ꢀArꢀoꢀH); 7.04 (s, 1H, =CH); 7.91 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀmꢀH). 13C{1H} NMR (101 MHz, CDCl3): δ
= 22.3 (ꢀSꢀCH3); 46.8 (DMSO); 55.5 (ꢀOCH3); 111.0 (=CH); 114.1 (ꢀArꢀoꢀC); 129.4 (ꢀArꢀmꢀC); 130.3 (ꢀArꢀ
mꢀC); 163.0 (ꢀArꢀOCH3); 174.2 (ꢀCꢀOꢀ); 177.9 (ꢀC=S). MS (ESI): m/z = 565, 536, 512, 445, 101. Elemental
analysis: calculated for C13H17ClO3PtS3ꢂ1/2 acetone C: 30.18%; H: 3.49%; S: 16.67%, found: C: 30.57%; H:
3.18%; S: 16.18%.
Chloroꢀ(1ꢀ(2ꢀmethoxyphenyl)ꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt4).
Synthesis was performed according to general procedure 1, pathway A. L4 (389 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatographie mobile phase: DCM 4:pentane 1 ꢀ
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DCM ꢀ DCM 2:acetone1ꢀ ꢀacetone. Yield: 516 mg (60.0%) as red oil. 1H NMR (600 MHz, acetoneꢀd6): δ =
3
1.43 (t, 3H, ꢀSꢀCH2ꢀCH3); 3.23 (q, 2H, ꢀSꢀCH2ꢀ); 3.65 (s w/Pt satellites JPt-H=22.5 Hz, 6H, CH3 (DMSO));
3.94 (s, 3H, ꢀOCH3); 6.99 (ddd, 3JH-H=7.6 Hz, 4JH-H=1.0 Hz, 1H, ꢀArꢀmꢀH); 7.14 (d, 3JH-H=8.4 Hz, 1H, ꢀArꢀoꢀ
3
4
3
4
H); 7.35 (s, 1H, =CH); 7.50 (ddd, JH-H=7.6 Hz, JH-H=1.0 Hz, 1H, ꢀArꢀpꢀH); 7.62 (dd, JH-H=7.8 Hz, JH-
H=1.8 Hz, 1H, ArꢀmꢀH).13C{1H} NMR (101 MHz, acetoneꢀd6): δ = 13.7 (ꢀSꢀCH2ꢀCH3); 26.1 (ꢀSꢀCH2ꢀ); 46.7
(DMSO); 56.4 (ꢀOCH3); 112.9 (ꢀArꢀoꢀC); 116.5 (=CH); 121.7 (ꢀArꢀmꢀC); 129.3 (ꢀArꢀC1); 130.6 (ꢀArꢀmꢀC);
133.8 (ꢀArꢀpꢀC); 158.3 (ꢀArꢀOCH3); 175.3 (ꢀCꢀOꢀ); 199.0 (ꢀC=S).MS (ESI): m/z = 525, 448, 393, 337, 331,
245, 205, 173, 151.
Elemental analysis: calculated for C14H19ClO3PtS3ꢂ2/3 pentane C: 33.62%; H: 5.86%; S: 15.54%, found: C:
33.23%; H: 5.39%; S: 15.25%.
Chloroꢀ(1ꢀ(3ꢀmethoxyphenyl)ꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt5).
Synthesis was performed according to general procedure 1, pathway A. L5 (389 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 4:hexane 1 ꢀ
DCM ꢀ DCM 2:acetone1ꢀ ꢀacetone. Yield: 160 mg (18.6%) as yellow crystals. 1H NMR (400 MHz, CDCl3):
δ = 1.43 (t, 3H, ꢀSꢀCH2ꢀCH3); 3.26 (q, 2H, ꢀSꢀCH2ꢀ); 3.64 (s w/Pt satellites 3JPt-H=22.5 Hz, 6H, CH3 (dmso));
3.84 (s, 3H, ꢀOCH3); 7.06 (m, 2H, ꢀArꢀpꢀH/=CH); 7.29 (t, 1H, ꢀArꢀmꢀH); 7.50 (m, 2H, ꢀArꢀoꢀH).13C{1H}
NMR (400 MHz, CDCl3): δ = 14.0 (ꢀSꢀCH2ꢀCH3); 22.3 (ꢀSꢀCH2ꢀ); 46.9 (dmso); 55.4 (ꢀOCH3); 112.9 (=CH);
118.3 (ꢀArꢀoꢀC); 129.7 (ꢀArꢀmꢀC/ꢀArꢀC1); 135.7 (ꢀArꢀpꢀC); 159.9 (ꢀArꢀOCH3); 174.5 (ꢀCꢀOꢀ); 177.6 (ꢀC=S).
MS (ESI): m/z = 579, 413, 393, 301.Elemental analysis: calculated for C14H19ClO3PtS3ꢂ1/2 acetone C:
31.5%; H: 3.75%; S: 16.27%, found: C: 32.02%; H: 3.75%; S: 16.76%.
Chloroꢀ(1ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt6).
Synthesis was performed according to general procedure 1, pathway A. L6 (389 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 4:pentane 1 ꢀ
DCM ꢀ DCM 2:acetone1ꢀ ꢀacetone. Yield: 150 mg (17.5%) as yellow crystals. 1H NMR (400 MHz, CDCl3):
δ = 1.42 (t, 3H, ꢀSꢀCH2ꢀCH3); 3.24 (q, 2H, ꢀSꢀCH2ꢀ); 3.63 (s w/Pt satellites 3JPt-H=22.5 Hz, 6H, CH3 (dmso));
3.83 (s, 3H, ꢀOCH3); 6.89 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀoꢀH); 7.07 (s, 1H, =CH); 7.95 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀ
mꢀH).
13C{1H} NMR (101 MHz, CDCl3): δ = 13.3 (ꢀSꢀCH2ꢀCH3); 22.3 (ꢀSꢀCH2ꢀ); 46.8 (dmso); 55.5 (ꢀOCH3);
111.0 (=CH); 114.1 (ꢀArꢀoꢀC); 129.4 (ꢀArꢀC1); 130.2 (ꢀArꢀmꢀC); 163.0 (ꢀArꢀOCH3); 174.2 (ꢀCꢀOꢀ); 177.9 (ꢀ
C=S). MS (ESI): m/z = 579, 413, 393, 301.Elemental analysis: calculated for C14H19ClO3PtS3ꢂ1/3 pentane C:
32.11%; H: 3.96%; S: 16.41%, found: C: 32.43%; H: 3.73%; S: 16.52%.
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Chloroꢀ(1ꢀ(3ꢀhydroxyphenyl)ꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt7).
Synthesis was performed according to general procedure 1, pathway A and B. L7 (350 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.70 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM ꢀ acetone
20:DCM 1 ꢀ acetone 10:DCM 1 — acetone 2:DCM 1. Yield: 380 mg (46.4%) as orange crystals.
3
1H NMR (600 MHz, acetoneꢀd6): δ = 2.63 (s, 3H, ꢀSꢀCH3); 3.61 (s w/Pt satellites JPt-H=22.5 Hz, 6H, CH3
(dmso)); 7.02 (dd, 3JH-H=8.2 Hz, 4JH-H=0.8 Hz, 1H, ꢀArꢀpꢀH); 7.15 (s, 1H, =CH); 7.24 (t, 1H, ꢀArꢀmꢀH); 7.47
(m, 2H, ꢀArꢀoꢀH); 8.73 (s, 1H, ꢀArꢀOH).13C{1H} NMR (101 MHz, acetoneꢀd6): δ = 16.7 (ꢀSꢀCH3); 45.9
(dmso); 110.8 (=CH); 114.6 (ꢀArꢀoꢀC); 118.6 (ꢀArꢀoꢀC); 119.3 (ꢀArꢀC1); 130.1 (ꢀArꢀmꢀC); 138.6 (ꢀArꢀpꢀC);
157.9 (ꢀArꢀOH); 174.2 (ꢀCꢀOꢀ); 177.6 (ꢀC=S). MS (ESI): m/z = 516, 471, 413, 359, 301, 215. MS (DEI): m/z
= 279, 167, 149, 121, 113, 83, 71, 57, 43. Elemental analysis: calculated for C12H17ClO3PtS3ꢂ1/3 acetone C:
28.22%; H: 3.09%; S:17.38%, found: C: 28.53%; H:2.82%; S: 17.95%.
Chloroꢀ(1ꢀ(4ꢀhydroxyphenyl)ꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt8).
Synthesis was performed according to general procedure 1, pathway A and B. L8 (248 mg, 1.10 mmol) was
dissolved in THF, tꢀBuOK (123 mg, 1.10 mmol) was added. K2PtCl4 (500 mg, 1.21 mmol) was dissolved in
water, dmso (160 ꢁL, 2.20 mmol) was added. Column chromatography mobile phase:DCM 2:pentane 1 ꢀ
DCM 1 ꢀ acetone 20:DCM 1 ꢀ acetone 10:DCM 1 ꢀ acetone 2:DCM 1. Yield: 210 mg (35.8%) as yellow
1
3
crystals. H NMR (600 MHz, acetoneꢀd6): δ = 2.61 (s, 3H, ꢀSꢀCH3); 3.60 (s w/Pt satellites JPt-H=22.5 Hz,
6H, CH3 (dmso)); 6.85 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀoꢀH); 7.16 (s, 1H, =CH); 7.95 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀmꢀ
H).
13C{1H} NMR (101 MHz, acetoneꢀd6): δ = 16.7 (ꢀSꢀCH3); 45.9 (dmso); 109.9 (=CH); 115.7 (ꢀArꢀoꢀC); 128.3
(ꢀArꢀC1); 130.5 (ꢀArꢀmꢀC); 161.7 (ꢀArꢀCꢀOH); 174.2 (ꢀCꢀOꢀ); 178.7 (ꢀC=S). MS (ESI): m/z = 530, 471, 413,
359, 301, 194, 121. MS (DEI): m/z = 528, 382, 327, 279, 194, 179, 167, 136, 121, 78. Elemental analysis:
calculated for C12H17ClO3PtS3ꢂ1/3 pentane C: 29.42%; H: 3.43%; S:17.24%, found: C: 29.93%; H:3.01%; S:
16.80%.
Chloroꢀ(1ꢀ(3ꢀhydroxyphenyl)ꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt9).
Synthesis was performed according to general procedure 1, pathway A and B. L9 (369 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.70 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 2:pentane 1 ꢀ
DCM 1 ꢀ acetone 20:DCM 1 ꢀ acetone 10:DCM 1 ꢀ Acetone 2:DCM 1. Yield: 130 mg (15.5%) as yellow
crystals. 1H NMR (600 MHz, acetoneꢀd6): δ = 1.37 (t, 3H, ꢀSꢀCH2ꢀCH3); 3.22 (q, 2H, ꢀSꢀCH2ꢀ); 3.61 (s w/Pt
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satellites 3JPt-H=22.5 Hz, 6H, CH3 (dmso)); 7.03 (d, 3JH-H=8.1 Hz, 1H, ꢀArꢀpꢀH); 7.15 (s, 1H, =CH); 7.24 (t,
1H, ꢀArꢀmꢀH); 7.46 (m, 2H, ꢀArꢀoꢀH); 8.74 (s, 1H, ArꢀCꢀOH).13C{1H} NMR (101 MHz, acetoneꢀd6): δ =
12.8 (ꢀSꢀCH2ꢀCH3); 25.9 (ꢀSꢀCH2ꢀ); 45.9 (dmso); 111.0 (=CH); 114.4 (ꢀArꢀoꢀC); 119.6 (ꢀArꢀC1); 120.2 (ꢀArꢀ
oꢀC); 129.7 (ꢀArꢀmꢀC); 138.8 (ꢀArꢀpꢀC); 156.9 (ꢀArꢀCꢀOH); 174.5 (ꢀCꢀOꢀ); 196.8 (ꢀC=S). MS (ESI): m/z =
510, 434, 359, 301, 289, 197, 78. MS (DEI): m/z = 298, 268, 239, 213, 208, 181, 179, 167, 138, 136, 98, 93,
78, 63, 61, 29. Elemental analysis: calculated for C12H17ClO3PtS3ꢂ1/10 pentane C: 29.2%; H: 3.30%; S:
17.33%, found: C: 29.44%; H: 3.02%; S: 17.38%.
Chloroꢀ(1ꢀ(4ꢀhydroxyphenyl)ꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀ
platinum(II) (Pt10).
Synthesis was performed according to general procedure 1, pathway A and B. L10 (369 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.70 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 1 ꢀ acetone
20:DCM 1 ꢀ acetone 10:DCM 1 ꢀ Acetone 2:DCM 1. Yield: 130 mg (15.5%) as yellow crystals.
1H NMR (600 MHz, acetoneꢀd6): δ = 1.35 (t, 3H, ꢀSꢀCH2ꢀCH3); 3.20 (q, 2H, ꢀSꢀCH2ꢀ); 3.60 (s w/Pt satellites
3JPt-H=22.5 Hz, 6H, CH3 (dmso)); 6.85 (m, 2H, ꢀArꢀoꢀH); 7.16 (s, 1H, =CH); 7.82 (d, 3JH-H=9.0 Hz, 2H, ꢀArꢀ
mꢀH); 9.24 (s, 1H, ArꢀCꢀOH). 13C{1H} NMR (101 MHz, acetoneꢀd6): δ = 12.9 (ꢀSꢀCH2ꢀCH3); 28.1 (ꢀSꢀCH2ꢀ
); 45.9 (dmso); 110.3 (=CH); 115.0 (ꢀArꢀoꢀC); 128.4 (ꢀArꢀC1); 129.7 (ꢀArꢀmꢀC); 161.7 (ꢀArꢀCꢀOH); 174.5 (ꢀ
CꢀOꢀ); 177.6 (ꢀC=S). MS (ESI): m/z =, 471, 413, 359, 301, 194, 139. MS (DEI): m/z = 530, 359, 267, 167,
149, 136, 121, 93, 78, 63, 45, 29. Elemental analysis: calculated for C12H17ClO3PtS3ꢂ2,5 acetone C: 35.52%;
H: 4.65%; S: 13.88%, found: C: 35.71%; H: 4.08%; S: 14.68%.
Chloroꢀ(1ꢀphenylꢀ3ꢀ(methylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀS)ꢀplatinum(II)
(Pt11).
Synthesis was performed according to general procedure 1, pathway A. L11 (322 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 4:hexane 1 ꢀ
1
DCM ꢀ DCM 1:acetone 1 — acetone. Yield: 330 mg (41.5%) as yellow crystals. H NMR (600 MHz,
3
acetoneꢀd6): δ = 2.70 (s, 3H, ꢀSꢀCH3); 3.68 (s w/Pt satellites JPt-H=22.5 Hz, 6H, CH3 (dmso)); 7.28 (s, 1H,
=CH); 7.49 (m, 2H, ꢀArꢀoꢀH); 7.62 (m, 1H, ꢀArꢀpꢀH); 8.08 (m, 2H, ꢀArꢀmꢀH). 13C{1H} NMR (101 MHz,
acetoneꢀd6): δ = 17.8 (ꢀSꢀCH3); 46.9 (dmso); 111.6 (=CH); 128.2 (ꢀArꢀmꢀC/ꢀArꢀpꢀC); 128.8 (ꢀArꢀoꢀC); 133.0
(ꢀArꢀC1); 175.1 (ꢀCꢀOꢀ); 177.6 (ꢀC=S). MS (DEI): m/z = 518, 517, 504, 341, 209, 207, 105, 78, 63, 45.
Elemental analysis: calculated for C12H15ClO2PtS3ꢂ1/3 acetone/1/3 CHCl3 C: 27.75%; H: 3.02%; S: 16.67%,
found: C: 27.88%; H:2.72%; S: 17.02%.
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Chloroꢀ(1ꢀphenylꢀ3ꢀ(ethylthio)ꢀ3ꢀthioxoꢀpropꢀ1ꢀenꢀ1ꢀolateꢀO,S)ꢀ(dimethylsulfoxideꢀ
S)ꢀplatinum(II)
(Pt12).
Synthesis was performed according to general procedure 1, pathway A. L12 (343 mg, 1.53 mmol) was
dissolved in THF, tꢀBuOK (172 mg, 1.53 mmol) was added. K2PtCl4 (700 mg, 1.69 mmol) was dissolved in
water, dmso (220 ꢁL, 3.07 mmol) was added. Column chromatography mobile phase: DCM 4:hexane 1 ꢀ
DCM ꢀ DCM 1:acetone 1 ꢀ acetone. Yield: 470 mg (57.5%) as red crystals. 1H NMR (600 MHz, acetoneꢀd6):
3
δ = 1.43 (t, 3H, ꢀCH2ꢀCH3); 3.29 (q, 2H, ꢀSꢀCH2ꢀ); 3.67 (s w/Pt satellites JPt-H=22.5 Hz, 6H, CH3 (dmso));
7.27 (s, 1H, =CH); 7.49 (m, 2H, ꢀArꢀoꢀH); 7.62 (m, 1H, ꢀArꢀpꢀH); 8.07 (m, 2H, ꢀArꢀmꢀH). 13C{1H} NMR
(101 MHz, acetoneꢀd6): δ = 13.7 (ꢀSꢀCH2ꢀCH3); 29.2 (ꢀSꢀCH2ꢀ); 46.8 (dmso); 111.8 (=CH); 128.1 (ꢀArꢀoꢀC);
128.7 (ꢀArꢀmꢀC/ꢀArꢀpꢀC); 138.2 (ꢀArꢀC1); 175.3 (ꢀCꢀOꢀ); 181.5 (ꢀC=S). MS (DEI): m/z = 518, 517, 504,
341, 209, 207, 105, 78, 63, 45. Elemental analysis: calculated for C12H15ClO3PtS3ꢂ2/3 acetone/1/3 CHCl3 C:
30.17%; H: 3.52%; S: 15.76%, found: C: 30.38%; H: 2.93%; S: 15.68%.
Crystal structure determination
The intensity data for the compounds were collected on a Nonius KappaCCD diffractometer using graphiteꢀ
monochromated MoꢀKα radiation. Data were corrected for Lorentz and polarization effects; absorption was
taken into account on a semiꢀempirical basis using multipleꢀscans.52ꢀ54
The structures were solved by direct methods (SHELXS) and refined by fullꢀmatrix least squares techniques
against Fo2 (SHELXLꢀ97).55 All hydrogen atoms of compound L1, Pt1 and molecule B of L7, the hydrogen
atoms bounded to the hydroxyꢀgroups of molecule A of L7 and Pt10 were located by difference Fourier
synthesis and refined isotropically. All other hydrogen atoms were included at calculated positions with
fixed thermal parameters. The crystal of Pt7 was a nonꢀmerohedral twin. The twin law was determined by
PLATON to (1.0 0.0 0.129) (0.0 ꢀ1.0 0.0) (0.0 0.0 1.0).56 The contribution of the main component was
refined to 0.776(1). Crystallographic data as well as structure solution and refinement details are summarized
in Table S1. XP (SIEMENS Analytical Xꢀray Instruments, Inc.) was used for structure representations.
Supporting Information available: Crystallographic data (excluding structure factors) has been deposited
with the Cambridge Crystallographic Data Centre as supplementary publication CCDCꢀ1446182 for L1,
CCDCꢀ1446183 for L7, CCDCꢀ1446184 for Pt1, CCDCꢀ1446185 for Pt2, CCDCꢀ1446186 for Pt3, CCDCꢀ
1446187 for Pt5, CCDCꢀ1446188 for Pt6, CCDCꢀ1446189 for Pt7, CCDCꢀ1446190 for Pt9, and CCDCꢀ
1446191 for Pt10. Copies of the data can be obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK [Eꢀ mail: deposit@ccdc.cam.ac.uk].
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Stability determinations
NMRꢀspectra were measured via NMRꢀspectroscopy on Bruker Avance 600 MHz. Substances were solved
in dmsoꢀd6 and measured directly at 37 °C. NMR tubes were incubated at 37 °C and after 24 hours measured
again under same conditions.
UV–vis spectra of the compounds at different dmso concentrations and under the conditions used to obtain
crystals of HEWLꢀPt compounds adducts were registered using a Jasco spectrophotomer at room
temperature in cuvette with optical length of 0.1 cm. Spectra of the compounds in 10 mM PBS pH 7.4 and
0.9% NaCl were registered using Caryꢀwin 50 spectrophotomer in cuvette with optical length of 1 cm.
DNAꢀbinding behavior
DNAꢀbinding behavior were measured via NMRꢀspectroscopy on Bruker Avance 600 MHz by 37 °C. Model
base 9ꢀmethylguanine was applied from Sigma Aldrich, both compounds (model base as well as
platinum(II)ꢀcomplex) were solved separately in dmsoꢀd6,quickly mixed and quickly transferred in a NMR
tube. Measurements starts directly after the transfer of the single molecules for 24 hours and 30 minutes (NS
= 64 (3 minutes and 38 seconds), pause: 390 seconds, 145 experiments). We used 6 mg from complex as
well as for the model base, which results in a high molecular excess of the 9ꢀmethylguanine, to imitate
excesses in the biological system and because of intensity of the signals.
Biological Assays
Ovarian cancer cell lines were cultured under standard conditions (5 % CO2, 37 °C, 90 % humidity) in
RPMI medium supplemented with 10 % FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (Life
Technologies, Germany). Cisplatin (Sigma, Germany) was freshly dissolved at 1 mg/ml in 0.9 % NaCl
solution and diluted appropriately. New Platinum(II) complexes and ligands were dissolved in dmso.
Platinumꢀresistant A2780 and SKOV3 cells were established by repeated rounds of 3 day incubations with
increasing amounts of Cisplatin starting with 0.1 µM. The concentration was doubled after 3 incubations
interrupted by recovery phases with normal medium. Cells that survived the third round of 12.8 µM Cisplatin
were defined as resistant cultures. Determinations of IC50 values were carried out using the CellTiter96 nonꢀ
radioactive proliferation assay (MTT assay, Promega). After seeding 5000 cells per well in a 96ꢀwell plate
cells were allowed to attach for 24h and were incubated for 48h with different concentrations of the
substances ranging from 0 to 1000 µM for Platinum and 0 to 1000 µM for ligand tests (0, 1, 10, 50, 100, 500,
1000 µm), for Cisplatin from 0 to 100 µM (0.1, 1, 5, 10, 50, 100 µM). Each measurement was done in
triplicate and repeated 3ꢀtimes. The proportion of live cells was quantified by the MTT assay and after
background subtraction relative values compared to the mean of medium controls were calculated. Nonꢀ
linear regression analyses applying the Hillꢀslope were run in GraphPad 5.0 software.
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For the determination of DNA damage induced by the treatment with different substances histone γH2AX
foci were visualized by immunocytochemical staining. Cells were seeded on coverslips to reach 60ꢀ70%
confluence after 24h. After incubation (24h) with different substances at IC50 concentrations for the resistant
cells, cells were washed 3x with PBS and fixed for 10min in 4% paraformaldehyde. Cells were again
washed 3ꢀtimes and then permeabilised by incubation with 0.25% TritonꢀX in PBS for 5min. Primary
antibody against γH2AX (clone JBW301, Millipore, diluted 1:2000) was incubated for 1h at RT and
coverslips were washed 3ꢀtimes afterwards. Alexa488ꢀlabelled secondary antiꢀmouse antibody (life
technologies) was used in a 1:1000 dilution in PBS and applied for 1h at RT. Cells were washed 3ꢀtimes,
counterstained with DAPI, washed again and embedded in mounting medium (Vectorshield, Vector
Systems). Slides were stored at 4°C in darkness until microscopic evaluation was done using a Zeiss LSM
710 laser scanning microscope.
Fluorescence
Intrinsic fluorescence spectra of HEWL (0.1 mg x mLꢀ1) in the presence of increasing concentrations of the
Pt compounds, at fixed dmso concentration (1.4%), were measured at 25 °C with a Horiba Fluoromax 4,
using 5 nm/5 nm slit widths. The excitation wavelength was 280 nm, and the emission wavelength was read
at 295–450 nm. The temperature of the sample was maintained by a Peltierꢀthermostat.
Crystallization of proteinꢀPt compound adducts
Crystals of hen egg white lysozyme (HEWL)ꢀPt compound adducts appeared in solutions consisting of 1.1
M NaCl, 0.1 M sodium acetate pH 4.4. Single crystals suitable for Xꢀray experiments were grown by the
hanging drop vaporꢀdiffusion method using a 1:1 ratio of reservoir solution and protein adducts solution with
a protein concentration of ∼
15 mg ml−1.
Data collection, structure determination and refinement
Crystals were flaskꢀcooled in nitrogen in the absence of cryoprotectant, as done in other works (see for
example 57) and then screened for diffraction quality. Xꢀray data were collected at
∼100 K on at the CNR
Institute of Biostructure and Bioimages. Data were indexed, integrated and scaled with HKL2000.52 Details
of data processing are reported in Table S3.
The structures were solved using the coordinated from PDB code 4J1A, without water and ligands and the
molecularꢀreplacement method as implemented in Phaser.47, 58 All the models were refined independently
using Refmac of the CCP4 suite (see Table 1).59, 60 Model building and electron density map fitting were
performed using WinCoot.61 Structural figures were prepared using PyMOL (http://www.pymol.org/ )
Structures were deposited in the Protein Data Bank.
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Acknowledgements
The authors would like to thank Ralf Trautwein for fruitful discussions. A.M. would like to thank G.
Sorrentino and M. Amendola at CNR Institute of Biostructures and Bioimages for technical assistance.
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