Half-sandwich Ru(II) and Os(II) Bathophenanthroline complexes containing a releasable dichloroacetato ligand

Article abstract of DOI:10.3390/molecules23020420

We report on the preparation and thorough characterization of cytotoxic half-sandwich complexes [Ru(η⁶-pcym)(bphen)(dca)]PF₆ (Ru-dca) and [Os(η⁶-pcym)(bphen)(dca)]PF₆ (Os-dca) containing dichloroacetate(1–) (dca

Full text of DOI:10.3390/molecules23020420

molecules
Article
Half-Sandwich Ru(II) and Os(II) Bathophenanthroline
Complexes Containing a Releasable
Dichloroacetato Ligand
Pavel Štarha 1 ID , Zdeneˇk Trávnícˇek 1,
*
ID , Ján Vancˇo 1 and Zdeneˇk Dvorˇák 2
ID
1
Department of Inorganic Chemistry & Regional Centre of Advanced Technologies and Materials,
Faculty of Science, Palacký University in Olomouc, 17. listopadu 12, 771 46 Olomouc, Czech Republic;
pavel.starha@upol.cz (P.Š.); jan.vanco@upol.cz (J.V.)
Department of Cell Biology and Genetics & Regional Centre of Advanced Technologies and Materials,
Faculty of Science, Palacký University in Olomouc, Šlechtitelu˚ 27, 783 71 Olomouc, Czech Republic;
zdenek.dvorak@upol.cz
2
*
Correspondence: zdenek.travnicek@upol.cz; Tel.: +42-058-563-4352
Received: 15 January 2018; Accepted: 12 February 2018; Published: 14 February 2018
Abstract: We report on the preparation and thorough characterization of cytotoxic half-sandwich
6-pcym)(bphen)(dca)]PF6 (Ru 6-pcym)(bphen)(dca)]PF6 (Os
complexes [Ru(η -dca) and [Os(η -dca)
containing dichloroacetate(1–) (dca) as the releasable O-donor ligand bearing its own cytotoxicity;
pcym = 1-methyl-4-(propan-2-yl)benzene (p-cymene), bphen = 4,7-diphenyl-1,10-phenanthroline
(bathophenanthroline). Complexes Ru-dca and Os-dca hydrolyzed in the water-containing media,
which led to the dca ligand release (supported by 1H NMR and electrospray ionization mass spectra).
Mass spectrometry studies revealed that complexes Ru-dca and Os-dca do not interact covalently
with the model proteins cytochrome c and lysozyme. Both complexes exhibited slightly higher
in vitro cytotoxicity (IC50 = 3.5
ovarian carcinoma cells than cisplatin (IC50 = 5.9
hepatocytes was found to be IC50 = 19.1 M for Ru-dca and IC50 = 19.7
µ
M for Ru-dca, and 2.6
M), while their toxicity on the healthy human
M for Os-dca. Despite
µM for Os-dca) against the A2780 human
µ
µ
µ
comparable cytotoxicity of complexes Ru-dca and Os-dca, both the complexes modified the cell
cycle, mitochondrial membrane potential, and mitochondrial cytochrome c release by a different way,
as revealed by flow cytometry experiments. The obtained results point out the different mechanisms
of action between the complexes.
Keywords: ruthenium; osmium; half-sandwich; dichloroacetate(1–); cytotoxicity; flow cytometry
1. Introduction
Platinum(II) complexes cisplatin, carboplatin, and oxaliplatin represent the worldwide used metal-based
anticancer chemotherapeutics [1]. It is well known that their application in clinics is connected with multiple
drawbacks including nephrotoxicity, myelosuppression, and resistance of many tumors. Various strategies
can be employed to improve the potency of the conventional platinum-based drugs and to circumvent
the mentioned drawbacks [
using their oxidation to Pt(IV) and coordination of bioactive ligands to the axial sites [
complex of this type, cis,cis,trans-[Pt(NH3)2Cl2(dca)2] (mitaplatin) contains the orphan drug dichloroacetate
(dca) coordinated to cisplatin [ ]. The biological impact of mitaplatin is dual and affects both nuclear DNA
2
,
3
]. One of them is based on the chemical modification of platinum(II) drugs,
4–7]. The first Pt(IV)
4
(cisplatin) and mitochondria (dca) [4].
Osmium and especially ruthenium complexes have been intensively studied for their anticancer
activity [8,9]. They act through different mechanisms of action than platinum-based drugs,
resulting in different cytotoxicity profiles and ability to overcome both the intrinsic and acquired
Molecules 2018, 23, 420; doi:10.3390/molecules23020420
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Molecules 2018, 23, 420
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resistance of various tumors against platinum-based drugs. Importantly, several ruthenium complexes
have entered the clinical trials as prospective non-platinum anticancer drugs [ 10]. Among the
numerous types of anticancer ruthenium and osmium complexes, the half-sandwich complexes of the
general formula [M(
6-ar)(XY)Z]0/+ represent a structural type showing the encouraging biological
8,
η
properties [11,12]; ar = six-membered arene ligand (e.g., p-cymene, pcym), XY = bidentate-coordinated
ligand, Z = monodentate (typically halogenido) ligand. The mechanism of action is most likely based
on a modulation of the redox status of the treated cancer cells [13,14]. Based on the extensive literature
research, it is quite surprising that to date only one work has been dealing with the studies of
cytotoxicity of half-sandwich Ru(II) or Os(II) complex containing the monodentate releasable bioactive
ligand (in particular deprotonated ethacrynic acid, a known inhibitor of glutathione S-transferase
isoenzymes [15], instead of the chlorido one [16]. By adopting the same premise, that it might be of
great interest to investigate an impact of another type of simple bioactive carboxylate on cytotoxicity
of half-sandwich non-platinum complexes, and having in mind the considerable anticancer effect of
above-mentioned mitaplatin [4], we chose dichloroacetate as a suitable monodentate bioactive ligand
for this research. Sodium dichloroacetate is an approved drug for the treatment of lactic acidosis,
with promising cytotoxic properties [17]. The cytotoxic action of dichloroacetate is connected with
an inhibition of pyruvate dehydrogenase kinase (PDK) resulting in a Warburg effect reversion, damage
to the mitochondria and induction of apoptosis [18,19].
Concerning the metal species used in this work, we utilized the formerly reported anticancer
Ru(II) chlorido complex [Ru(
A2780 cells (IC50 = 0.5 M) was ca. 2-fold higher as compared with cisplatin (IC50 = 1.1
= 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline) [20]. Herein, its new Os(II) analogue,
[Os(
6-pcym)(bphen)Cl]PF6 (Os-Cl), was prepared and both Ru(II) and Os(II) chlorido complexes were
modified by the replacement of the chlorido ligand by dichloroacetato ligand, providing complexes of
the composition [Ru(
6-pcym)(bphen)(dca)]PF6 (Ru-dca) and [Os( 6-pcym)(bphen)(dca)]PF6 (Os-dca
η
6-pcym)(bphen)Cl]PF6 (Ru-Cl) whose in vitro cytotoxicity against the
µ
µM); bphen
η
η
η
)
(Figure 1). Complexes Ru-dca and Os-dca represent the first examples of the half-sandwich Ru(II) and
Os(II) complexes containing dca.
Figure 1. General structural formula of the [M(η
6-pcym)(bphen)(dca)]PF6 complexes, where
dca = dichloroacetate M = Ru for Ru-dca and Os for Os-dca), given with the atom numbering scheme.
2. Results and Discussion
2.1. Synthesis and Characterization
The chlorido complexes Ru-Cl (known from the literature [20]) and Os-Cl (newly prepared) were
synthesized in the microwave reactor and subsequently used for the syntheses of the dichloroacetato

Molecules 2018, 23, 420
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complexes Ru-dca and Os-dca (Figure 1), representing the first examples of half-sandwich Ru(II) and
Os(II) complexes containing the dca ligand. The complexes were characterized by elemental analysis (C,
H, N), electrospray ionization (ESI+) mass spectrometry, and 1H and 13C NMR, and IR spectroscopies.
The ESI+ mass spectra of complexes Ru-dca and Os-dca contained the peaks of 100% relative
intensity, belonging to the complex [M(pcym)(bphen)(dca)]+ cations (Figure 2), which directly
proved the formation of complexes Ru-dca and Os-dca from the Ru-Cl and Os-Cl starting
compounds. Further, the peaks, assignable according their m/z values and isotopic distribution
to the {[M(pcym)(bphen)]–H}+ species, were detected in the mass spectra of both complexes Ru-dca
and Os-dca (Figure 2).
Figure 2. Electrospray ionization (ESI+) mass spectra of complexes Ru-dca (top) and Os-dca (bottom)
given together with the details of the experimental and simulated peaks of the [M(pcym)(bphen)(dca)]+
species (M = Ru or Os).
1
Although measured in different solvents, H NMR results of complex Ru-Cl (dissolved in
DMSO-d6 or CDCl3 in this work; Figure 3 and Figure S1) were consistent with those previously
reported for this complex (dissolved in acetone-d6) [20]. Complex Os-Cl had a similar pattern on
the 1H NMR spectrum as the Ru-Cl complex, with the characteristic signals of the pcym aromatic
hydrogens (C22–H and C23–H) shifted to the lower fields by ca. 0.2 ppm than for the Ru-Cl analogue.
The replacement of the chlorido ligand of the precursors Ru-Cl and Os-Cl by the dca one became
evident by a new 1H NMR signal (DMSO-d6 solutions) of the C32–H hydrogen atom of dca, detected at
5.81 ppm (for Ru-dca; Figure 3) and 5.86 ppm (for Os-dca). The C32–H signal of Na(dca) (measured in
DMSO-d6) was found at 6.65 ppm, proving a coordination shift ∆δ~0.8 ppm. Concerning the 13C NMR
spectroscopy, the obtained spectra of the dca-complexes contained two new signals as compared with
their chlorido analogues. These signals were detected at 168.3 ppm and 67.4 ppm (for Ru-dca), and at
168.1 ppm and 66.8 ppm (for Os-dca), and can be unambiguously assigned to the C31 and C32 atoms
of the dca ligand. These signals were shifted by 2.4 ppm (for C31) and 1.5 ppm (for C32) for Ru-dca
,
and by 2.2 ppm (for C31) and 0.9 ppm (for C32) for Os-dca, as compared with Na(dca) (measured in
the same solvent; δ (ppm) = 65.9 (C31), 165.9 (C32)).
The purity of complexes Ru-dca and Os-dca was >97% and >99%, respectively, based on the
results of elemental analysis and 1H NMR spectroscopy (Figure 3). Regarding the stability in the used
solvent (DMSO-d6), no new signals or chemical shift changes were observed in the 1H NMR spectra of
the studied complexes Ru-dca and Os-dca even after 72 h of standing at ambient temperature.

Molecules 2018, 23, 420
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Figure 3. 1H NMR spectra (DMSO-d6 solutions) of Na(dca) (top), complexes Ru-Cl (middle) and
Ru-dca (bottom) given together with the assignment of the detected signals (see Figure 1 for the atom
numbering scheme). Note: the impurity visible in the 1H NMR spectrum of complex Ru-dca can be
assigned to the [Ru(η6-pcym)(bphen)(OH)]+ species (violet asterisks).
1
2.2. H NMR Spectroscopy and ESI+ Mass Spectrometry Studies of Hydrolytic Stability
Since the studied complexes Ru-dca and Os-dca were designed as prodrugs of two
mechanistically independent cytotoxic species (i.e., metal-based species and released dca), the solution
behavior in water-containing medium was studied by relevant techniques (NMR, ESI-MS) to
show, whether the dca ligand releases under the used conditions. Both the complexes Ru-dca
and Os-dca (1 mM concentration) hydrolyzed in the used water-containing mixture of solvents
(20% MeOD-d4/80% D2O), where methanol ensured the sufficient solubility to reach relevant
concentration (1 mM in this work) for 1H NMR experiments. Hydrolysis of the studied complexes is
connected with the release of the dca ligand (Figure 4). A 1H NMR signal of C32–H of the coordinated
dca ligand showed at 5.47 ppm for Ru-dca and at 5.48 ppm for Os-dca, while the released dca anion
showed at 5.91 ppm in the 1H NMR spectra of both complexes (Figure S2). A position of this signal
correlated well for the released dca and free dca (measured for Na(dca) in the same mixture of solvents)
detected at 5.93 ppm. Complex Ru-dca hydrolyzed completely, while the hydrolysis rate was only
ca. 65% for its Os(II) analogue after standing at room temperature for next 24 h (Figure 4).
The ESI+ mass spectra of both complexes Ru-dca and Os-dca (10 µM concentration) contained the peaks
assignable to the hydrolytic products of the parent complexes of the composition [M(pcym)(bphen)(OH)]+.
As for Ru-dca, a ratio of intensities between the [Ru(pcym)(bphen)(dca)]+ (695.1 m/z) and hydrolyzed
[Ru(pcym)(bphen)(OH)]+ (584.8 m/z) species equaled ca. 3:2 after standing at room temperature for 1 h,
while a peak of the parent [Ru(pcym)(bphen)(dca)]+ species almost disappeared (<5% relative intensity)
with ongoing hydrolysis after 24 h (Figure S3). In the case of complex Os-dca, the change of the ratio
of intensities between the [Os(pcym)(bphen)(dca)]+ (785.3 m/z) and [Os(pcym)(bphen)(OH)]+ (675.3 m/z)
species detected after 1 h (ca. 6:1) and 24 h (ca. 5:1) was negligible (Figure S3). Thus, the ESI+ mass
spectrometry results, consistently with 1H NMR spectra (see above), provided proofs revealing a higher
hydrolytic stability of the Os-dca complex as compared to its Ru(II) analogue.

Molecules 2018, 23, 420
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Figure 4. The results of the time-dependent 1H NMR studies showing the progress of the dca ligand
release for complexes Ru-dca (brown solid squares) and Os-dca (green solid squares). Note: The lines
are only guides for eyes.
2.3. Mass Spectrometry Studies of Interactions with Sulfur-Containing Biomolecules
To investigate better the behavior of the studied complexes in the presence of selected
biomolecules, we performed the ESI+ MS experiments for the mixtures of complexes Ru-dca and
Os-dca (10
µM final concentration) with the sulfur-containing biomolecules L–cysteine (CySH) and
reduced glutathione (GSH) at their average levels in human plasma [21
–
23]. No peaks assignable
to the covalent adducts with CySH or GSH were detected in the ESI+ mass spectra of the samples
containing complex Ru-dca and the mixture of CySH and GSH after 24 h of incubation. However,
the peaks assignable to the adducts of {[Ru(pcym)(bphen)]–H}+ with either two CyS (or one cystine
(CySSCy); 807.2 m/z) or with CyS and GS (or their disulfide CySSG; 993.2 m/z) appeared in
the ESI+ mass spectrum of the reaction system after 24 h of incubation (Figure S4). Moreover,
a peak whose mass-to-charge ratio and isotopic pattern suggests the formation of adduct of
{[Ru(pcym)(bphen)]+(HL1)–H}+ with deaminated cysteine (i.e., 3-sulfanylpropanoic acid; HL1), showed
at 673.0 m/z in the ESI+ mass spectrum recorded after 24 h (Figure S4). Although the deamination
reaction is not very common in connection with the electrospray ionization process, similar processes,
such as the deamination of various anilines by the Ru-based catalyst [24] or cysteine deamination to
β-mercaptopyruvate [25] were already described in the literature.
As for the Os-dca complex, the peaks of similar adducts described above for the the Ru-dca
complex were also detected by ESI+ mass spectrometry in the mixture containing Os-dca complexes
(i.e., {[Os(pcym)(bphen)]–H}+ with either two CyS or one CySSCy at 897.2 m/z, or with CyS and GS
or disulfide CySSG at 1083.3 m/z), however their assignability is uncertain due to very low intensity.
On the other hand, the ESI+ mass spectrometry experiments confirmed higher stability of the Os-dca
complex in the presence of GSH. Specifically, the intensity ratio of [Os(pcym)(bphen)(dca)]+ and
[Os(pcym)(bphen)(OH)]+ was ca. 8:1 after 1 h and ca. 6:1 after 24 h. Contrary to complex Ru-dca
,
no peak indicating the cysteine deamination was detected in the ESI+ mass spectra recorded on the
mixture containing complex Os-dca.
2.4. Mass Spectrometry Studies of Interactions with Model Proteins
The affinity of the complexes Ru-dca and Os-dca towards the model proteins cytochrome c
(Cytc) and lysozyme (HEWL) was studied by ESI+ mass spectrometry. Both proteins were formerly
reported as interacting with platinum-based drugs [26–28] as well as with similar half-sandwich
Ru(II) [28–33] and Os(II) [34] complexes, thus they represent the suitable models to shed a light
on whether the interactions with proteins should be taken into account as a possible aspect of
mechanism of action/transportation/activation of the Ru-dca and Os-dca complexes. However,
any peaks assignable to the adducts of the [M(pcym)(bphen)(dca)]+ complex cations or their fragments

Molecules 2018, 23, 420
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were not detected in the deconvoluted mass spectra of the mixtures of the studied dca-complexes with
neither Cytc nor HEWL, even after 120 h after preparation of the reaction systems. The only adduct,
visible after 24 h of incubation of Cytc with the studied complexes Ru-dca or Os-dca corresponded
–
to the mass difference of 145 Da, assignable to the adduct of Cytc with the PF6 ion (Figure S5).
–
Such adducts appear commonly in the protein-metal complex systems containing the PF6 anions
studied by electrospray-ionization mass spectrometry as minor adducts, accompanying the major
metalated ones [35,36]. However, in our case, we did not observe formation of such products.
2.5. In Vitro Cytotoxicity
The in vitro cytotoxicity of complexes Ru-dca and Os-dca was screened at the A2780 human
ovarian carcinoma cell line. The dca-complexes were slightly more cytotoxic (IC50 = 3.5
2.6 0.4 M for Ru-dca, and Os-dca, respectively) against the used A2780 cells than the clinically
used platinum-based drug cisplatin (IC50 = 5.9 1.2 M); note: IC50 of Hdca equaled 5.0 mM with the
±
0.3 µM and
±
µ
±
µ
same experimental conditions used. The relative activity (RA = IC50(cisplatin)/IC50(complex)) equaled
ca. 1.7 and 2.3 for complexes Ru-dca, and Os-dca, respectively, which implied approximately 2-fold
higher potency than determined for conventional cisplatin. Even if no direct comparison should be
made between the IC50 values obtained by our experiments and the published results of cytotoxicity,
it is important to note that the well-known Ru(II) complex KP1019, which have entered into the
clinical trials, showed lower potency (IC50 = 77.8 µM) as compared to cisplatin (IC50 = 9.5 µM) at the
A2780 cells (24 h exposure with no recovery, MTT assay), thus showing the RA value of ca. 0.1 [37].
Similarly, mitaplatin, a Pt(IV) complex containing the dca ligand (see Introduction), is also less effective
(IC50 = 1.1 µM) than the reference drug cisplatin (IC50 = 0.6 µM) at the same A2780 cell line (72 h
exposure with no recovery, MTT assay), resulting in the RA value of ca. 0.5 [4].
The cellular accumulation of the Ru-dca and Os-dca complexes equaled 366.1
±
14.0,
and 273.1 ± 6.4 fmol/106 cells, respectively, indicating that the Os-dca complex was less accumulated
within the cancer cells than its Ru(II) analogue. Taking into account the similar results of cytotoxicity
of both complexes and on the other hand the differences in their cellular accumulation, these findings
may indicate dissimilarity in molecular mechanisms of cytotoxicity of both complexes. In addition,
both the dca-complexes exceeded the cell uptake level of their chlorido precursors (see Table 1).
Table 1. Cellular accumulation of complexes Ru-Cl, Ru-dca, Os-Cl and Os-dca (calculated as the metal
content) at the A2780 cells after the treatment for 24 h by the equipotent (IC50) concentrations. The data
are given as arithmetic means from three independent experiments.
ng/106 Cells
fmol/106 Cells
Ru-Cl
Ru-dca
Os-Cl
26.5 ± 0.7
37.0 ± 1.4
33.5 ± 1.4
52.5 ± 1.2
262.2 ± 7.0
366.1 ± 14.0
174.3 ± 7.4
273.1 ± 6.4
Os-dca
In vitro toxicity of the Ru-dca and Os-dca complexes was studied on the primary culture of human
hepatocytes. The obtained results indicated that the toxic effect of complexes Ru-dca (IC50 = 19.1 µM)
and Os-dca (IC50 = 19.7 µM) is lower than their cytotoxicity against the A2780 ovarian carcinoma cells.
2.6. Cell Cycle Analysis
The Ru-dca and Os-dca complexes modified the cell cycle of the A2780 human ovarian carcinoma
cells differently, when applied at equipotent (IC50) concentration (Figure 5, Table 2). In particular,
the Ru-dca complex strongly increased the sub-G1 cell population (16.8% of cells) as comparted to
the Os-dca complex (0.7% of cells). The increase in the sub-G1 population is often connected with
the appearance of late-apoptotic and necrotic cells. Simultaneously, the Ru-dca complex caused the
decline in the G0/G1 cell cycle phase population (37.0% of cells) as compared with the negative control

Molecules 2018, 23, 420
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(69.2% of cells) as well as with complex the Os-dca complex (62.9% of cells). In the S-phase, the effect
of the Ru-dca complex is much closer to that of cisplatin (24.3% vs. 30.9% of cells) as compared with the
Os-dca complex (13.6% of cells). In the G2/M cell phase, both the complexes showed the similar effect
(20.9% vs. 22.2% of cells). Overall, the results of cell cycle modifications revealed that the cellular effects
of the Ru-dca complex are much more similar to the effects of cisplatin, while the Os-dca complex
showed only a small effect on the cell cycle in the A2780 cells as compared to the negative control.
Just for the comparative purposes we may show that similar human cancer cell cycle perturbations
were recently reported for half-sandwich complexes [Ru(
containing 1-(4-methoxybenzyl)-4-phenyl-1H-1,2,3-triazole (HL2) [38].
η η
6-pcym)(L2)Cl] and [Os( 6-pcym)(L2)Cl]
Table 2. The A2780 cell populations (%) in the cell cycle phases after the treatment for 24 h by the IC50
concentrations of complexes Ru-Cl Ru-dca Os-Cl and Os-dca, given together with negative control
,
,
(untreated cells) and cisplatin (for comparative purposes). The data are given as arithmetic means from
three independent experiments.
Sub-G1
G0/G1
S
G2/M
Ru-Cl
Ru-dca
Os-Cl
Os-dca
Cisplatin
Control
0.7 ± 0.2
16.8 ± 1.9
0.8 ± 0.3
0.7 ± 0.2
8.0 ± 0.9
0.8 ± 0.3
62.4 ± 1.8
37.0 ± 2.7
59.0 ± 2.3
62.9 ± 1.3
26.1 ± 2.1
69.2 ± 2.2
13.1 ± 2.8
24.3 ± 3.3
14.5 ± 0.9
13.6 ± 4.7
30.9 ± 4.9
15.7 ± 1.4
23.2 ± 2.9
20.9 ± 1.1
25.1 ± 1.5
22.2 ± 4.5
34.5 ± 3.5
13.9 ± 1.1
Figure 5. The A2780 cell cycle phase populations (%) after the 24 h treatment by the IC50 concentrations
of complexes Ru-dca and Os-dca, given together with untreated cells (negative control) and the
cells treated with platinum-based drug cisplatin. The data are given as arithmetic mean from three
independent experiments.
2.7. Induction of Mitochondrial Membrane Potential Changes and Cytochrome c Release
In general, the cytotoxic effect of anticancer agents is often connected with the mitochondrial
membrane potential alternation [39
the dca-containing Pt(IV) complex mitaplatin [
reported complexes [13]. Further, it is generally accepted that the depolarization of mitochondrial
membrane is usually connected with the Cytc release [39 41]. The released Cytc subsequently activates
,40], as formerly described for various cytotoxic agents including
4] or half-sandwich complexes similar to the herein
,
various proapoptotic signals in the cytosol (like formation of apoptosome), making Cytc an ultimate
factor in the programmed cell death regulation. The Cytc release also most likely plays a crucial
role in the mechanism of cancer cell resistance against the therapeutic action of anticancer drugs
such as cisplatin [39,40]. Additionally, sodium dichloroacetate is well-known clinically studied
mitochondria-targeting anticancer active small molecule [19]. This implied a possibility of the

Molecules 2018, 23, 420
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synergistic effect of the metal-based species and the dca ligand, both released from the studied
complexes Ru-dca and Os-dca.
Concerning the Ru-dca and Os-dca complexes, these compounds induced only low mitochondrial
membrane depolarization while 89.3% of cells, and 93.2% of cells, respectively, stayed intact. This result
is also comparable with the conventional platinum-based drug cisplatin (88.8% of intact cells) (Table 3).
Table 3. The results (given as % cell populations) of flow cytometry studies of the mitochondrial
membrane potential changes, studied at the A2780 cells using the 24 h treatment by the IC50
concentrations of complexes Ru-Cl
, Ru-dca, Os-Cl and Os-dca (and cisplatin for comparative
purposes). The untreated A2780 cells were employed as the negative control, while the CCCP-treated
cells represent the positive control. The data are given as arithmetic means
independent experiments.
±
SD from three
% of Cells Showing High PE-Channel Fluorescence
Ru-Cl
Ru-dca
Os-Cl
Os-dca
Cisplatin
94.0 ± 1.4
89.3 ± 1.4
95.2 ± 0.7
93.2 ± 2.3
88.8 ± 2.6
38.6 ± 1.7
99.1 ± 0.3
Positive control
Negative control
Although the above-mentioned results revealed that both the complexes caused only slight
depolarization of mitochondrial membranes, the further flow-cytometry studies of cytochrome c (Cytc)
cytosolic release [39,41], which is a causal pre-condition of apoptosome activation, were performed.
The Ru-dca complex showed considerable induction of Cytc release from the mitochondria (94.6% of all
cells were positive), while only the moderate Cytc release was induced by the Os-dca complex
(39.9% of all cells were positive), as compared with the negative control (only 12.3% of all cells were
Cytc positive) (see Figure 6). The effect of the Ru-dca complex almost reached the level of the positive
control (Figure 6 and Table 4) and significantly exceeded both reference drugs staurosporine (47.1% of
positive cells) and cisplatin (53.0% of positive cells). Furthermore, the induction of the Cytc release
from the mitochondria in the A2780 cells was significantly higher for both dca-complexes as compared
with their chlorido-analogues (see Table 4).
The studies of the ability of half-sandwich Ru(II) or Os(II) complexes to induce the Cytc release
are quite rare in the literature. In particular, it has been reported that complexes RAPTA-C (Western
blot analysis) [42] and [Os(η
6-pcym)(L2)I]PF6 (flow cytometry studies) [43] induced the Cytc release in
the treated human cancer cells; L2 = N,N-dimethyl-4-[(E)-pyridin-2-yl-diazenyl]-aniline.
Table 4. The results (given as % cell populations) of the cytochrome c release studied by flow cytometry
at the A2780 cells treated for 24 h by the IC50 concentrations of complexes Ru-Cl, Ru-dca, Os-Cl and
Os-dca (cisplatin and staurosporine were used for comparative purposes). The untreated A2780 cells were
employed as the negative control, while the cells applied with the Anti-IgG1-FITC Isotype represented
the positive control. The data are given as arithmetic means
±
SD from three independent experiments.
% of Cells Showing Shift in FITC-Channel Fluorescence
Ru-Cl
Ru-dca
Os-Cl
53.0 ± 5.7
94.6 ± 0.6
16.5 ± 4.1
39.9 ± 4.6
47.1 ± 6.1
53.0 ± 6.0
99.3 ± 0.1
12.3 ± 2.3
Os-dca
Staurosporine
Cisplatin
Positive control
Negative control

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Figure 6.
treated for 24 h by the IC50 concentrations of complexes Ru-Cl (dashed blue), Ru-dca (blue), Os-Cl
(dashed red) and Os-dca (red); white = negative control ( Ctrl), grey = positive control (+Ctrl),
(Left) The results of the flow cytometry studies of cytochrome c release at the A2780 cells
−
p < 0.005 (***). (Right) The representative histogram of the flow cytometry studies of cytochrome c
release, showing the fluorescence intensities in FITC-H channel in the A2780 cells treated by complexes
Ru-dca (pink) and Os-dca (blue), given together with the negative (green) and positive (red) controls.
Orange line segments show the edge between low and high fluorescence populations.
A lack of correlation between the flow cytometry studies of the mitochondria membrane potential
depletion and Cytc release induced by the studied complexes (Tables 3 and 4) could be connected with
various aspects of the Cytc release from mitochondria [44,45]. In other words, the Cytc release does not
have to be necessarily a consequence of a ∆ψm loss, because it was reported that (a) the Cytc release
occurs independently from the mitochondria membrane status [44], and (b) Cytc releases mitochondria
through the multiple pathways [45].
3. Materials and Methods
3.1. Materials
The starting salts RuCl3
(University of Wollongong, Wollongong, Australia) and Sigma-Aldrich (Prague, Czech Republic)
respectively. The chemicals 1-methyl-4-(propan-2-yl)cyclohexa-1,3-diene ( -terpinene), 4,7-diphenyl-
·
xH2O and OsCl3
·
xH2O were supplied by Precious Metals Online
,
α
1,10-phenanthroline (bathophenanthroline, bphen), dichloroacetic acid (Hdca), sodium hydroxide,
silver(I) trifluoromethanesulfonate (AgOTf), ammonium hexafluorophosphate, L–cysteine (CySH),
reduced glutathione (GSH), cytochrome c from bovine heart (Cytc) and hen egg-white lysozyme
(HEWL) were purchased from VWR International (Strˇíbrná Skalice, Czech Republic) and Sigma-Aldrich
(Prague, Czech Republic), while the solvents (methanol, diethyl ether, n-hexane, dichloromethane)
were supplied by Litolab (Chudobín, Czech Republic). The deuterated solvents for the NMR
experiments (DMSO-d6, CDCl3, MeOD-d4, D2O, DClO4 and KOD) were purchased from Sigma-Aldrich
(Prague, Czech Republic).
[Ru(
µ
-Cl)(
η
6-pcym)Cl]2 and [Os(
µ
-Cl)(
η
6-pcym)Cl]2 were prepared in a microwave reactor
47].
Monowave 300 (Anton PaarGmbH, Graz, Austria), according to the reported protocols [46
,
Silver(I) dichloroacetate, Ag(dca), was prepared as white solid from Hdca, which was neutralized by
stoichiometric amount of 1 M NaOH (in methanol) providing Na(dca), which subsequently interacted
in situ (15 min of stirring at ambient temperature in the dark) with 1 molar equiv. of AgOTf.
3.2. Synthesis
The formerly reported protocol for the preparation of complex Ru-Cl [20] was modified,
as described in Supplementary Materials, and this modification was used also for new Os(II) analogue
Os-Cl. Complexes Ru-Cl and Os-Cl (0.05 mmol) were dissolved in MeOH (15 mL) and an excess
of Ag(dca) (0.25 mmol) was added to both solutions. The mixtures were stirred overnight without

Molecules 2018, 23, 420
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heating under the aluminum foil. Thereafter, the formed AgCl and unreacted Ag(dca) was removed,
and NH4PF6 (0.5 mmol) was added to the filtrates, followed by the solvent evaporation combined with
the addition of excess diethyl ether, until the solid formed. The crude products were recrystallized
from the mixture of dichloromethane and n-hexane to give complexes [Ru(
Ru-dca) and [Os(
6-pcym)(bphen)(dca)]PF6 (Os-dca) (Figure 1). The products were collected by
filtration, washed (1 0.5 mL of MeOH and 3 1 mL of diethyl ether) and dried under vacuum.
η
6-pcym)(bphen)(dca)]PF6
(
η
×
×
The yields were 45% (for Ru-dca) and 35% (for Os-dca), related to the starting Ru(II) and Os(II) dimers.
Anal. Calcd. for C36H31N2O2Cl2RuPF6 (Ru-dca): C, 51.44; H, 3.72; N, 3.33%; found: C, 51.06; H, 3.63;
N, 3.14%. 1H NMR (DMSO-d6, ppm):
δ
10.19 (d, J = 5.5 Hz, C2–H, 2H), 8.17 (d, J = 5.5 Hz, C3–H, 2H)
8.08 (s, C5–H, 2H), 7.67 (m, C9–H, C10–H, C11–H, 10H), 6.63 (d, J = 6.4 Hz, C23–H, 2H),
6.33 (d, J = 6.4 Hz, C22–H, 2H), 5.81 (s, C32–H, 1H), 2.66 (sep, J = 7.3 Hz, C25–H, 1H), 2.14 (s, C27–H, 3H)
1.01 (d, J = 7.3 Hz, C26–H, 6H). 1H NMR (CDCl3, ppm): δ 9.90 (d, J = 5.5 Hz, C2–H, 2H)
8.03 (s, C5–H, 2H)
,
,
,
,
7.98 (d, J = 5.5 Hz, C3–H, 2H), 7.59 (m, C9–H, C10–H, C11–H, 10H), 6.31 (d, J = 6.4 Hz, C23–H, 2H),
6.07 (d, J = 6.4 Hz, C22–H, 2H), 5.36 (s, C32–H, 1H), 2.70 (sep, J = 6.6 Hz, C25–H, 1H), 2.19 (s, C27–H,
3H), 1.16 (d, J = 6.4 Hz, C26–H, 6H). 13C NMR (DMSO-d6, ppm):
δ 168.3 (C31), 156.4 (C2), 150.4 (C4),
146.2 (C7), 134.9 (C12), 130.0–127.5 (C6, C9, C10, C11), 125.4 (C5), 102.6 (C24), 102.4 (C21), 86.3 (C23),
83.3 (C22), 67.4 (C32), 30.6 (C25), 22.0 (C26), 18.1 (C27). ESI+ MS (methanol, m/z): 567.2 (calc. 567.1; 30%;
{[Ru(pcym)(bphen)]–H}+), 695.1 (calc. 695.1; 100%; [Ru(pcym)(bphen)(dca)]+). IR (ATR, cm–1): 401, 489, 556,
636, 669, 702, 736, 765, 834, 927, 1000, 1020, 1029, 1055, 1077, 1191, 1231, 1325, 1403, 1420, 1426, 1445, 1470,
1494, 1518, 1537, 1562, 1598, 1659, 2846, 2923, 2965, 3057.
Anal. Calcd. for C36H31N2O2Cl2OsPF6 (Os-dca): C, 46.51; H, 3.36; N, 3.01%; found: C, 46.60; H,
3.20; N, 3.16%. 1H NMR (DMSO-d6, ppm):
δ 10.12 (d, J = 5.5 Hz, C2–H, 2H), 8.15 (s, C5–H,
2H), 8.13 (d, J = 5.5 Hz, C3–H, 2H), 7.70 (m, C9–H, C10–H, C11–H, 10H), 6.88 (d, J = 5.5 Hz,
C23–H, 2H), 6.53 (d, J = 5.5 Hz, C22–H, 2H), 5.86 (s, C32–H, 1H), 2.56 (sep, J = 6.4 Hz, C25–H, 1H),
2.23 (s, C27–H, 3H), 0.97 (d, J = 6.4 Hz, C26–H, 6H). 1H NMR (CDCl3, ppm): δ 9.84 (d, J = 5.5 Hz
,
C2–H, 2H), 8.08 (s, C5–H, 2H), 7.95 (d, J = 5.5 Hz, C3–H, 2H), 7.59 (m, C9–H, C10–H, C11–H,
10H), 6.56 (d, J = 5.8 Hz, C23–H, 2H), 6.28 (d, J = 5.8 Hz, C22–H, 2H), 5.33 (s, C32–H, 1H),
2.57 (sep, J = 6.8 Hz, C25–H, 1H), 2.26 (s, C27–H, 3H), 1.08 (d, J = 6.8 Hz, C26–H, 6H).
13C NMR (DMSO-d6, ppm): δ 168.1 (C31), 156.4 (C2), 150.8 (C4), 147.5 (C7), 134.7 (C12), 130.0–127.8
(C6, C9, C10, C11), 126.5 (C5), 101.6 (C21), 92.8 (C24), 85.1 (C23), 77.4 (C22), 66.8 (C32), 30.8 (C25),
22.4 (C26), 18.2 (C27). ESI+ MS (methanol, m/z): 657.3 (calc. 657.2; 45%; {[Os(pcym)(bphen)]–H}+),
785.1 (calc. 785.1; 100%; [Os(pcym)(bphen)(dca)]+). IR (ATR, cm–1): 401, 491, 557, 638, 669, 702, 735,
766, 835, 928, 971, 1000, 1021, 1028, 1054, 1134, 1232, 1268, 1322, 1407, 1420, 1427, 1445, 1470, 1495, 1519,
1558, 1601, 1628, 1660, 2871, 2927, 2967, 3065.
3.3. Methods
The 1H, 13C and 1H–1H gs-COSY spectra were recorded using a JEOL JNM-ECA 600II spectrometer
(JEOL USA, Inc., Peabody, MA, USA) at 600.00 MHz (for 1H) and 150.86 MHz (for 13C) in the DMSO-d6
or CDCl3 solutions. The obtained 1H and 13C NMR spectra were calibrated against the residual signals
(2.50 ppm for 1H NMR, 39.52 ppm for 13C NMR) of the used solvent [48]. The splitting of the 1H NMR
signals is defined as s = singlet, d = doublet, sep = septet and m = multiplet. Mass spectrometry experiments
were performed using a LCQ Fleet Ion Trap spectrometer (Thermo Scientific; Qual Browser software,
version 2.0.7; Waltham, MA, USA) in the positive electrospray ionization mode (ESI+) in the methanolic
solutions, and methanol/water (1:1, v/v) for interaction experiments, respectively. The protein mass spectra,
measured in ESI+ mode, were deconvoluted by Promass for Xcalibur ver. 3.0, rev. 10 software (Novatia LLC,
Newtown, PA, USA) to obtain the neutral mass spectra. Infrared spectra (400–4000 cm–1, ATR technique)
were acquired by a Nexus 670 FT-IR (Thermo Nicolet; Waltham, MA, USA). Elemental analyses (C, H, N)
were carried out by a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific; Waltham, MA, USA).

Molecules 2018, 23, 420
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1
3.4. H NMR and ESI+ MS Studies of Hydrolytic Stability
The appropriate amount (for 600
µ
L of 1 mM solutions) of complexes Ru-dca and Os-dca were
dissolved in MeOD-d4 (120 L) and diluted by 480
µ
µ
L of D2O. 1H NMR spectra were recorded on
the fresh solutions (t = 0 h), and after 0.5, 1, 2, 3, 4, 6, 8 and 24 h of standing at ambient temperature.
1
The obtained H NMR spectra were calibrated against the residual signal of D2O (4.85 ppm) [48].
1
For comparative purposes, the H NMR spectrum was also recorded for sodium dichloroacetate
(Na(dca)) in 20% MeOD-d4/80% D2O.
Similar experiments were performed by ESI+ mass spectrometry for complexes Ru-dca and
Os-dca (10 µM concentration) dissolved in the MeOH/H2O mixture (1:1, v/v). The spectra were
recorded immediately after the sample preparation, and then after 1 h and after 24 h of standing at
ambient temperature.
3.5. Studies of Interactions with Biomolecules
The ESI+ MS studies of interactions of complexes Ru-dca and Os-dca with relevant biomolecules
GSH and CySH were performed as follows: complexes (either Ru-dca or Os-dca) were dissolved in
MeOH (500
µL, 10
µM final concentration) and 500
µL of the mixture of GSH (6 µM final concentration)
and CySH (290
µM final concentration) in H2O was added. The obtained solutions were mixed
properly and the ESI+ mass spectra were recorded immediately after preparation and after 1 and 24 h
of standing at laboratory temperature.
Similarly, the interactions of complexes Ru-dca and Os-dca with the model protein lysozyme
and cytochrome c were studied by means of ESI+ mass spectrometry. The samples contained the
mixtures of either complex Ru-dca or Os-dca (at the 10
µM final concentration) with HEWL or Cytc
(at the 3 M final concentration) in the mixture of MeOH/H2O (1:1, v/v). The ESI+ mass spectra
µ
were recorded immediately after preparation and then after 1 and 24 h of standing of the mixtures at
laboratory temperature. The specific experimental conditions were as follows: the sample solutions
were introduced by HPLC (Dionex UltiMate 3000; Thermo Scientific; Waltham, MA, USA) in 50 µL
spikes into the continual flow of methanol (0.2 mL/min flow rate), 5.3 kV spray voltage, 110 V
and 275 ◦C capillary voltage and temperature. The obtained spectra were acquired in the range of
150–2000 m/z and the raw ESI+ spectra of proteins were deconvoluted by Promass for Xcalibur ver.
3.0, rev 10 software (Novatia LLC, Newtown, PA, USA) using the 0.1 Da mass step size.
3.6. Cell Culture
The A2780 human ovarian carcinoma cell line (European Collection of Cell Cultures; Salisbury, UK)
and primary culture of human hepatocytes (Hep, Biopredic Intl., Saint-Grégoire, France) were cultured
according to the suppliers’ instructions, using RPMI-1640 medium supplemented with 10% of fetal calf
serum, 1% of 2 mM glutamine and 1% penicillin/streptomycin (for the A2780 cells) and chemically
defined medium consisting of a mixture of William’s E and Ham’s F-12 (1:1, v/v) (for the Hep
◦
cells), respectively. All cell lines were grown as adherent monolayers at 37 C and 5% CO2 in
a humidified atmosphere.
3.6.1. In Vitro Cytotoxicity
The 100 mM stock solutions of complexes Ru-dca and Os-dca were prepared by dissolving
of appropriate amounts of these agents in 500
µL of DMF. Cisplatin and Hdca were involved
in the testing for comparative purposes, and their stock solutions were of 80 mM and 10 M
concentrations, respectively.
The cultured A2780 cells were seeded to the 96-well culture plates, while the 96-well culture plates
with the Hep cells were received from the supplier. The A2780 and Hep cells were pre-incubated in
◦
◦
drug-free media at 37 C for 24 h and then treated for the next 24 h at 37 C with different concentrations
of Ru-dca Os-dca, cisplatin, and Hdca prepared from their stock solutions by dilution with RPMI-1640
,

Molecules 2018, 23, 420
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medium (note: Hdca was studied only at the A2780 cells). After 24 h drug exposure, the supernatants
were removed and the cells were washed with drug-free PBS followed by 72 h recovery in drug-free
medium at 37 ◦C. The MTT assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) was used to determine the cell viability. A concentration of the formazan formed from
MTT was determined spectrophotometrically at 540 nm using an Infinite 200 PRO microplate reader
(Tecan Group Ltd., Männedorf, Switzerland).
The data were expressed as the percentage of viability, where 100% and 0% represents the
treatments with negative (0.1% DMF in medium) and positive (1% Triton X-100) controls, respectively.
The data from the cancer cells were acquired from three independent experiments (conducted in
triplicate) using cells from different passages. The resulting IC50 values (µM) were calculated from the
viability curves and the results are presented as arithmetic mean ± standard deviation (SD).
3.6.2. Cellular Accumulation
The A2780 cells were seeded in 6-well culture plates (1
(37 ◦C and 5% CO2 in a humidified incubator). Then the solutions, containing complexes Ru-Cl
Ru-dca and Os-dca at the concentrations corresponding to IC50 from cytotoxicity testing, were added for the
24 h treatment, followed by washing with PBS (2 2 mL). The cells were harvested by trypsin, resuspended
×
106 cells per well) and incubated overnight
Os-Cl
,
,
×
in PBS and centrifuged. The cell pellets were, immediately after the supernatant suction, digested in 500 µL
of nitric acid (70 ◦C, overnight) to give the fully homogenized solutions. Then 4.5 mL of water was added
and the final Ru and Os contents were determined by inductively coupled plasma mass spectrometry
(ICP-MS; 7700
×
ICP-MS device, Agilent Technologies; Santa Clara, CA, USA) by external calibration
using the 100 mg/L TraceCERT® Transition metal mix 3 for ICP (Sigma Aldrich, Prague, Czech Republic).
The obtained values were corrected for adsorption effects. The experiments were conducted in triplicate
and the results are presented as arithmetic mean ± SD.
3.7. Flow Cytometry Studies
For all the performed flow cytometry studies (cell cycle analysis, mitochondrial membrane
potential assay and cytochrome c release), the A2780 cells were pre-incubated in the 6-well plates
(1.0 × 106 cells per well) for 24 h at 37 ◦C and 5% CO2 atmosphere. Complexes Ru-Cl
, Os-Cl,
Ru-dca and Os-dca (cisplatin was involved for comparative purposes) were added at the equipotent
concentrations (equal to their IC50 concentrations from cytotoxicity testing). After 24 h exposure
time, the cells were harvested using trypsin, washed twice with PBS and isolated by centrifugation.
After that, the cells were stained as described below, and analyzed by a CytoFLEX Flow Cytometer
(Beckman Coulter, Brea, CA, USA). In all cases, the untreated cells were used as a negative control,
the experiments were conducted in triplicate (ca. 3
×
105 cells per sample) and the obtained data were
analyzed using CytExpert Software (Beckman Coulter, Brea, CA, USA).
3.7.1. Cell Cycle Analysis
The washed cells were resuspended, stained by a propidium iodide (PI) solution supplemented
◦
with RNase A (25 C, in the dark, 30 min) and analyzed by flow cytometry (excitation at 535 nm,
emission detected at 617 nm).
3.7.2. Mitochondrial Membrane Potential Assay
The A2780 cells were processed using the MITO-ID® Membrane potential detection kit (Enzo
Life Sciences, Farmingdale, NY, USA), with the emission maxima detected at 525 nm (green dye)
and 590 nm (orange dye). The positive control included the A2780 cells exposed to carbonyl cyanide
3-chlorophenylhydrazone (CCCP; 2 µM final concentration).

Molecules 2018, 23, 420
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3.7.3. Cytochrome c Release
The cell staining procedure was performed according to the instructions supplied in the
FlowCellect Cytochrome c Kit (Millipore, Burlington, MA, USA; Catalog No. FCCH100110)
containing the Anti-IgG1-FITC Isotype Control (positive control) and Anti-Cytochrome c-FITC
™
Antibody dye. For this experiment, the apoptosis inducer staurosporine (1
involved for comparative purposes as well.
µg/mL concentration) was
3.8. Statistical Analysis
An ANOVA test was used for statistical analysis with the values of p < 0.05 (*), 0.01 (**) and
0.005 (***) considered to be statistically significant. QC Expert 3.2 Statistical software (TriloByte Ltd.;
Pardubice—Staré Hradišteˇ, Czech Republic) was used to perform the analyses.
4. Conclusions
Half-sandwich complexes [Ru(
Os-dca) showed good in vitro cytotoxicity (IC50 = 3.5
human ovarian carcinoma cells, slightly exceeding the clinically-used platinum-based drug cisplatin
(IC50 = 5.9 µM) Hydrolysis of the Ru-dca and Os-dca complexes is connected with the release of
η
η
6-pcym)(bphen)(dca)]PF6 (Ru-dca) and [Os( 6-pcym)(bphen)(dca)]PF6
(
µ
M, and 2.6 M, respectively) against the A2780
µ
.
dichloroacetate (dca). The Ru-dca and Os-dca complexes do not interact with the model proteins cytochrome
c (Cytc) and lysozyme. The results of the detailed flow cytometry studies (cell cycle perturbation,
depolarization of mitochondrial membrane, Cytc release) were indicative for considerable differences
in biological effects of the Ru-dca and Os-dca complexes at the used A2780 cells. The results point out the
fact that the structural optimization of half-sandwich Ru(II) and Os(II) complexes represents a promising
approach for the development of highly potent anticancer agents.
Supplementary Materials: Supplementary materials are available online: synthesis of Ru-Cl and Os-Cl
,
1H NMR spectra of Ru-dca and Os-dca dissolved in CDCl3 Figure S1), 1H NMR (Figure S2) and ESI+ MS (Figure S3)
studies of solution behavior in water-containing media, ESI+ MS (Figure S4) studies of the interaction of Ru-dca and
Os-dca with GSH and CySH and mass spectra of cytochrome c and its mixture with Ru-dca (Figure S5).
Acknowledgments: The authors gratefully thank the Ministry of Education, Youth and Sports of the
Czech Republic (projects LO1305 and CZ.1.05/2.1.00/19.0377), the Czech Science Foundation (a Grant No.
17-08512Y) and Palacký University in Olomouc (a Grant No. PrF_2017_018) for the financial support. The authors
also thank Prˇemysl Lexa for his help with syntheses of complexes Ru-Cl, Os-Cl, Ru-dca and Os-dca,
Katerˇina Kubešová for performing the in vitro cytotoxicity experiments and preparing the cell pellets, Peter Antal
for recording the NMR spectra, Bohuslav Drahoš for performing ESI+ MS experiments and Pavla Richterová for
performing elemental analysis.
Author Contributions: P.S., Z.T., J.V. and Z.D. conceived and designed the experiments; P.S. and J.V. performed the
experiments; P.S., Z.T., J.V. and Z.D. analyzed the data; Z.T. and Z.D. contributed reagents/materials/analysis tools;
P.S., J.V. and Z.T. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds Ru-Cl, Os-Cl, Ru-dca and Os-dca are available from the authors.
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