Ruthenium(II)–Arene Thiocarboxylates: Identification of a Stable Dimer Selectively Cytotoxic to Invasive Breast Cancer Cells

Article abstract of DOI:10.1002/cbic.201900676

Ru^II-arene complexes provide a versatile scaffold for novel anticancer drugs. Seven new Ru^II-arene-thiocarboxylato dimers were synthesized and characterized. Three of the complexes (2 a, b and 5) showed promising antiproliferative activities in MDA-MB-231 (human invasive breast cancer) cells, and were further tested in a panel of fifteen cancerous and noncancerous cell lines. Complex 5 showed moderate but remarkably selective activity in MDA-MB-231 cells (IC₅₀=39±4 μm Ru). Real-time proliferation studies showed that 5 induced apoptosis in MDA-MB-231 cells but had no effect in A549 (human lung cancer, epithelial) cells. By contrast, 2 a and b showed moderate antiproliferative activity, but no apoptosis, in either cell line. Selective cytotoxicity of 5 in aggressive, mesenchymal-like MDA-MB-231 cells over many common epithelial cancer cell lines (including noninvasive breast cancer MCF-7) makes it an attractive lead compound for the development of specifically antimetastatic Ru complexes with low systemic toxicity.

Full text of DOI:10.1002/cbic.201900676

Accepted Article
Title: Ruthenium(II)-Arene Thiocarboxylates: Identification of a Stable
Dimer Selectively Cytotoxic to Invasive Breast Cancer Cells
Authors: Liam Stephens, Aviva Levina, Iman Trinh, Victoria Blair,
Melissa Werrett, Peter Lay, and Philip Andrews
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Ruthenium(II)-Arene Thiocarboxylates: Identification of a Stable
Dimer Selectively Cytotoxic to Invasive Breast Cancer Cells
Liam J. Stephens,[a] Aviva Levina,[b] Iman Trinh,[a] Victoria L. Blair,[a] Melissa V. Werrett,[a]
Peter A. Lay,*[b] and Philip C. Andrews,*[a]
Abstract: Ru(II)-arene complexes provide a versatile scaffold for
novel anti-cancer drugs. A new series of Ru(II)-arene-thiocarboxylato
dimers 1-7 were synthesized and characterized. Three of the
complexes (2a,b and 5) showed promising anti-proliferative activities
in MDA-MB-231 (human invasive breast cancer) cells, and were
further tested in a panel of fifteen cancerous and non-cancerous cell
lines. Complex 5 showed moderate but remarkably selective activity
in MDA-MB-231 cells (IC50 = 39 ± 4 µM Ru). Real-time proliferation
studies showed that 5 induced apoptosis in MDA-MB-231 cells but
had no effect in A549 (human lung cancer, epithelial) cells. By
contrast, 2a and b showed moderate anti-proliferative activity, but no
apoptosis, in either cell line. Selective cytotoxicity of 5 in aggressive,
mesenchymal-like MDA-MB-231 cells over many common epithelial
cancer cell lines (including non-invasive breast cancer MCF-7) makes
it an attractive lead compound for the development of specifically anti-
metastatic Ru complexes with low systemic toxicity.
Ru-chlorido ligand substitution in biological media compared to
those of Pt(II) complexes leads to predominant binding of Ru(II)
or Ru(III) anti-metastatic drugs to biomolecules in the extracellular
space, or at the cell surface, rather than to intracellular targets.[8,
The resultant disruption in communication between the cell
surface and extracellular matrix is thought to be the main cause
10-13]
of anti-metastatic activities of NAMI-A, RAPTA-T and related Ru
10-13]
complexes.[8,
However, the high reactivity of NAMI-A in
biological media has also led to Ru binding to non-target tissues,
which has caused excessive side effects that led to withdrawal of
the drug from Phase II clinical trials.[11] The only Ru complex
remaining in active clinical trials, IT-139 (Figure 1, also known as
NKP-1339)[14] is thought to have predominantly cytotoxic
activity.[13, 15, 16] However, the mode of action differs from that of
conventional cytotoxicity of Pt anti-cancer drugs that are used in
the clinic as there is little difference in activity between the Pt
resistant phenotype and the non-resistant phenotype of related
cell lines.[17] This experience emphasizes the need for new metal
complexes as drugs with anti-metastatic activities.[11, 13]
Introduction
Ruthenium(II) arene complexes of “piano-stool” geometry
(including RAPTA-T, Figure 1)[18] are among the most extensively
studied metal-based anti-cancer drugs, due to their
straightforward synthesis from commercially available precursors
and a great variety of available ligands.[19-21] Although such
Despite significant advances in cancer treatment, metastasis
remains the main obstacle for successful therapy and the main
cause of cancer-related mortality.[1, 2] Metastasis is caused by a
subpopulation of cancer cells that are not only able to invade
surrounding tissues, but are often resistant to conventional
chemotherapy.[1, 2] Specific targeting of aggressive cancer cells
remains challenging, as shown by a recent screening of 301
known anti-cancer drugs against a panel of sixteen breast cancer
cell lines.[3]
complexes predominantly contain N, O, or P-donor ligands,[19]
a
notable series of thiolato-bridged Ru(II) arene dimers has been
developed by Furrer, Süss-Fink and co-workers.[20, 21] In particular,
trithiolato dimers, such as those shown in Figure 1, are stable
under biological conditions, and are highly cytotoxic (IC50 values
in nanomolar range) in common cancer cell lines.[22-25] The
absence of easily hydrolysable ligands in these dimers (Figure 1)
means that they are likely to act by a different mechanism to those
of classical Pt(II) drugs,[6] or the Ru drugs that have entered
clinical trials, which also undergo rapid ligand exchange
reactions.[8, 26] A proposed mechanism is based on their efficient
cellular uptake (probably by passive diffusion through cell
membranes due to the hydrophobic nature of the complexes),[8, 9]
followed by catalytic oxidation of cellular thiols by oxygen, leading
to production of reactive oxygen species that cause cell death.[21,
As part of a global effort towards the design and development of
specifically anti-metastatic drugs,[2] certain Ru complexes, such
as NAMI-A and RAPTA-T (Figure 1, Ru(III) coordination and
Ru(II) organometallic complexes, respectively), have attracted
5]
attention.[4,
Like well-established Pt(II) anti-cancer drugs
(cisplatin, carboplatin and oxaliplatin),[6] these complexes contain
relatively labile chlorido ligands that can be exchanged for donor
groups of proteins and nucleic acids.[7-9] The greater affinity for
23, 27-29]
Generally, these complexes were not selective for a
particular cell type,[22-24] although in one instance lower toxicity in
lung cancer cells compared with breast cancer, or leukaemia cells
was observed.[25] The reasons for such selectivity were not further
explored.
[a]
[b]
Dr. L. J. Stephens, Dr. I. Trinh, Dr V. L. Blair, Dr. M. V. Werrett, Prof.
P. C. Andrews
School of Chemistry, Monash University
Clayton, Melbourne, Vic, 3800 (Australia)
E-mail: phil.andrews@monash.edu
Dr. A. Levina, Prof. P. A. Lay
School of Chemistry, University of Sydney
Eastern Avenue, Sydney, NSW 2006 (Australia)
E-mail: peter.lay@sydney.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cbic.201900676
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FULL PAPER
line (MDA-MB-231),[31] while having little or no effect in most non-
invasive cancer or non-cancer cell lines. These results show
considerable promise in the challenging area of treating
aggressive types of breast cancer.[2, 3]
Results and Discussion
Synthesis of
a
and
b
-mercaptocarboxylic acids. a-
Mercaptocarboxylic acids were derived from the D- and L-
isomeric forms of valine and phenylalanine respectively, via a
three-step reaction process (Scheme 1-A).[32] The first step in this
sequence involved the substitution of the amino acid amine
functionality with bromine using HBr and NaNO2. Once in hand,
the a-bromo carboxylic acids were exposed to a nucleophilic
attack from potassium thioacetate, before subsequent
deprotection of this moiety resulted in the formation of the desired
a-mercaptocarboxylic acids in good overall yields.
The b-mercaptocarboxylic acids were derived from a,b-
unsaturated carboxylic acids via a two- (Scheme 1-B) or three-
step reaction process (Scheme 1-C).[33] The first step in this
sequence involved the Michael addition of either potassium
thioacetate or HBr to the a,b-unsaturated acid. The intermediate
bromo compound formed from cinnamic acid then underwent
nucleophilic attack using potassium thioacetate resulting in the
formation of the thioacetate functionality in excellent yield. The 3-
(acetylthio)carboxylic acids were then deprotected under basic
conditions, to give the desired b-mercaptocarboxylic acids in good
overall yields.
Figure 1. Typical structures of Ru complexes tested as anti-cancer drugs.[4, 5, 15,
18, 21, 24, 25, 30]
In this work, we present the synthesis, purification, and
comprehensive characterisation of a novel series of hydrolytically
stable
Ru(II)-arene-thiocarboxylato
complexes,
[Ru(p-
cymene)O2C-CH(-S)-R]2 (1-3) and [Ru(p-cymene)O2C-CH2-CH(-
S)-R]2 (4-7) (Figure 1 and 2), derived from bifunctional a- and b-
mercaptocarboxylic acids; thiolactic acid (TLA-H2), (R)-2-
mercapto-3-phenylpropanoic acid ((R)-MPA-H2), (S)-2-mercapto-
3-phenylpropanoic acid ((S)-MPA-H2), (R)-2-mercapto-3-
methylbutanoic
acid
((R)-MMA-H2),
(S)-2-mercapto-3-
methylbutanoic acid ((S)-MMA-H2), 3-mercaptobutanoic acid
(MBA-H2), 3-mercapto-3-phenylpropanoic acid (MPP-H2), 3-
mercapto-3-(4-methoxyphenyl)propanoic acid (MMPA-H2) and
thiosalicylic acid (TSA-H2) (Figure 2). The anti-proliferative
activities of all the complexes (1-7) were studied in various types
of cultured mammalian cells.
Scheme 1. General synthesis of α-(A) and β-mercaptocarboxylic acids (B and
C).
Synthesis of ruthenium complexes. A previous study by
Henderson et al. demonstrated that the dimeric complex, [Ru(p-
cymene)(TSA)]2, could be formed from the treatment of [RuCl2(p-
cymene)]2 with thiosalicylic acid (TSA).[34] This method, which
involved treatment of [RuCl2(p-cymene)]2 with the acid in
methanol and excess Et3N, under reflux for one hour, was initially
applied in the synthesis of our target [Ru(p-cymene)L]2 complexes,
using the broader range of mercaptocarboxylic acids (Scheme 2).
Figure 2. The five a-mercaptocarboxylic acids (left) and four b-
mercaptocarboxylic acids (right) used for complexation with [RuCl2(p-cymene)]2
and their corresponding complex formula and code.
Although these complexes mostly showed low to moderate
activities in the studied cell lines, remarkably one of them (5) was
selectively cytotoxic to a highly invasive human breast cancer cell
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Structure elucidation. Single crystal X-ray diffraction revealed
that complexes 1a, 2b, 3b, 4a, 5, 6 and 7a (obtained from the first
eluted band) are dimeric in the solid-state and adopt a pseudo
octahedral ‘piano-stool’ geometry consisting of central Ru2S2
units formed by two bridging thiolates.[34] Unfortunately, the
structures of complexes 1b, 4b and 7b, complexes obtained from
the second eluted band, have not been amenable to producing
crystals suitable for X-ray crystallography, as yet. The use of both
2D NMR and Raman spectroscopy did not elicit any additional
information with regards to their particular structure and chemical
composition.[35] First reported by Deacon et al., IR spectroscopy
and in particular the carbon-oxygen stretching frequencies can be
used to diagnose the nature of carboxylate coordination to a metal
centre.[36] Resulting from the inequivalence of the two oxygen
atoms, it has been demonstrated that unidentate coordination of
the carboxylate ligand results in an increase in uasym (CO2), a
decrease in usym (CO2) and hence the separation (D) between the
two C-O stretching frequencies is larger in comparison to the free
carboxylate ion.[36] Deacon et al. report that D values of separation
for unidentate coordination are typically larger than 200 cm-1,
whilst bidentate coordination typically give much lower values.
The spectra obtained for complexes 4a and 4b suggest a different
carboxylate coordination to the Ru centre, with the Dvalue of
separation for complex 4a observed as 215 cm-1 (unidentate)
whilst the equivalent value for complex 4b was observed as 151
cm-1 (bidentate). As a result of this finding, we hypothesise that
complexes 1b, 4b and 7b exist as dimers in which each p-cymene
Ru centre is bound to an O,O’ chelate and a thiolate (Figure 4).
To further probe this hypothesis, multiple scattering analysis of
XAS data may be able to differentiate between different possible
structures. This will be the subject of future works.[37]
Scheme 2. General synthesis of ruthenium (II) (p-cymene) thiocarboxylato
complexes 1-7.
However, attempts to isolate complexes of high purity and in high
yield proved problematic; 1H NMR spectra indicated a number of
presently unidentified species in the crude solution. The crude
mixtures were therefore purified using silica gel chromatography.
For each of the crude products, two separate light-orange bands
were eluted off the column (Rf ~ 0.90 and 0.26) and a dark brown
band was retained on the column at Rf = 0 (Figure S1). Mass
spectrometry on each of the two light-orange bands collected,
showed them to have the same molecular mass, composition, and
fragmentation pattern, and to exist as dimers in the solution state,
[Ru(p-cymene)L]2. However, 1H and 13C NMR spectra on the two
bands showed significant chemical shift differences (see Figure 3
as a representative example); both of which were consistent with
previously reported Ru(II) p-cymene complexes, in that the
aromatic p-cymene signals appeared as four independent
doublets, whilst the iso-propyl methyl signals appeared as two
inequivalent doublets arising from the unsymmetrical Ru centre
(see Supporting Information for NMR spectra for all complexes).
Figure 4. Proposed structure of complex 4b.
X-Ray crystallography. Of the twelve Ru(II)-arene
thiocarboxylato complexes synthesized, 1a, 2b, 3b, 4a, 5, and 6
were amenable to single crystal diffraction studies on crystals
grown from methanol/ethanol layered water or DMSO solutions.
The crystal structure of complex 7a has been described
previously.[34] Pertinent bond lengths and distances are outlined
in Table 1 and a full summary of the crystallographic data is
provided in the Experimental section. All six Ru-thiocarboxylato
complexes are isostructural with the ligand being dianionic having
doubly deprotonated at the thiol and hydroxy functionalities to give
chelated dimeric complexes; [Ru(p-cymene)(TLA)]2 1a, [Ru (p-
cymene)(MPA)]2 2b, [Ru(p-cymene)(MMA)]2 3b, [Ru(p-
Figure 3. 1H NMR spectra for complex 4a (bottom) and complex 4b (top), both
synthesized from the b-mercaptocarboxylic acid; MBA-H2.
Despite the use of prep-HPLC as an additional purification step,
1H NMR spectroscopy on the two bands obtained on the
complexes derived from MPA-H, MMA-H, MPP-H and MMPA-H,
showed only one to be an analytically pure complex with the
‘second band’ composed of traces of a number of presently
unidentified species. Hence, the ‘second band’ of these
complexes were discarded for use in biological studies.
cymene)(MBA)]2 4a, [Ru(p-cymene)(MPP)]2
5 and [Ru(p-
cymene)(MMPA)]2 6. The solid-state structures of complexes 1a
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and 5 are shown in Figures 5 and 6, whilst complexes 2b, 3b, 4a
and 6 are shown in the Supporting Information (Figures S2-S5).
Table 1. Selected comparative bond lengths (Å) and angles (°) of complexes 1-
7.
1a
2b
3b
4a
5
6
7a
Ru-
S
2.375
3(9)
2.375
3(11)
2.391
3(12)
2.094(
3)
2.379
4(14)
2.377
4(14)
2.099
4(4)
2.381
8(16)
2.384
6(16)
2.111(
4)
2.394
0(8)
2.376
8(4)
2.384
8(4)
Ru-
S’
2.391
4(10)
2.109(
2)
2.388
6(8)
2.385
3(4)
2.417
7(4)
Ru-
O
2.111
4(2)
2.104
4(10)
1.691
2.093
0(12)
Ru-
Ccen
troid
S-
1.969
1.687
1.679
2.186
2.210
(3)
81.35(
4)
80.67(
4)
81.34(
5)
81.41(
6)
80.95
(3)
79.79(
13)
80.71
4(15)
Ru-
S’
S-
80.88(
7)
80.67(
8)
81.43(
10)
86.87(
14)
87.15
(6)
87.27(
3)
87.80(
4)
Figure 5. Molecular structure of [Ru(p-cymene) (TLA)]2Ÿ2H2O 1a. Hydrogen
atoms (except C-H backbone ones) and uncoordinated solvent molecules
omitted for clarity. Thermal ellipsoids show at 40 % probability. Symmetry
operator: -x, 1-y, 1-z.
Ru-
O’
S’-
Ru-
O
80.42(
7)
80.31(
8)
81.25(
9)
77.77(
14)
76.53
(6)
76.14(
3)
77.80(
4)
Stability and anti-proliferative activity of complexes 1 - 7.
Activity of complexes 1-7 against MDA-MB-231 and A549 cell
lines. Preliminary studies compared the effects of 1-7 (10-100 µM
Ru, 72-96 h) on the proliferation rates of two contrasting human
cancer cell lines: (i) MDA-MB-231 (breast cancer, mesenchymal-
like, highly invasive)[3, 31] and (ii) A549 (lung cancer, epithelial,
non-invasive in the absence of growth factor stimulation),[38] using
real-time observations with IncuCyte Zoom imaging system.[39, 40]
Typical results (Figures S6 and S7, Supporting Information)
indicated that only 2a,b and 5 (i.e., the complexes that contained
phenyl groups in the ligands, Schemes 1 and 2) had promising
anti-proliferative activities at ~50 µM Ru. All the complexes with
aliphatic substituents in the ligands (1a,b, 3a,b and 4a,b) had little
or no activity at ~100 µM Ru, while the use of 6 and 7a,b was
limited by their low solubility in organic solvents (DMF or DMSO)
that were used to prepare stock solutions for cell assays.[41] All the
mercaptocarboxylic acids used as precursors to 1-7 (Figure 2)
had no significant anti-proliferative effect on either MDA-MB-231
or A549 cells at 100 µM for 72 h (data not shown).
Complexes 1-7 retained dimeric structures under biomimetic
conditions (50 µM Ru, 10 mM aqueous NH4HCO3, pH 7.5, stored
for up to a week at 295 K),[40, 42] as shown by ESI-MS (Figure S8
in Supporting Information). These results contrast those for typical
metal complexes that rapidly decompose under cell culture
conditions.[9, 40, 42, 43] Stock solutions of 1-7 used in the ESI-MS
studies (2-20 mM Ru in DMF) were stored for about a month in
the dark at 295 K prior to the dilution with 10 mM NH4HCO3. This
observation confirms that the DMF stock solutions of Ru(II) arene
thiocarboxylato dimers were also stable, which is an important
consideration for cell culture assays.[9, 44]
Figure 6. Molecular structure of [Ru(p-cymene)(MPP)]2 5. Hydrogen atoms and
uncoordinated solvent molecules omitted for clarity. Thermal ellipsoids show at
40 % probability. Symmetry operator: -x, -y, -z.
Complexes 1-3 and 4-6 can be viewed as [Ru(p-cymene)(L)] (L =
TLA, MBA, MPA, MBA, MPP or MMPA respectively) monomers
connected by bridging Ru-S bonds (average bond length = 2.3859
Å), with the Ru metal atom O,S-chelated to form a RuSOC2 five-
membered ring in the α-thiocarboxylato complexes 1-3 or a six-
membered RuOSC3 ring in β-thiocarboxylato 4-6. Dimerisation
through Ru-S bonds leads to a planar central Ru2S2 core, while
the coordination sphere of the Ru(II) atom is completed by an η6-
p-cymene coordinated molecule giving an overall distorted
tetrahedral geometry. The bond parameters and geometries in 1-
3
and 4-6 are comparable with the previously reported
thiocarboxylato Ru complex [Ru(p-cymene)(TSA)]2, 7a (Table
1).[34]
Activity of 2a,b and 5 in various cell types. Based on the
preliminary results (Figure S6), the anti-proliferative activities of
2a,b and 5 were compared in a broader range of cell lines (Table
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10.1002/cbic.201900676
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2). An endpoint colorimetric MTT assay (MTT = 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)[45] was
used instead of IncuCyte observations, because it allowed to test
the non-adherent and semi-adherent cell lines, such as THP-1,
HL-60 and HepG2 (Table 2). This or similar assays have been
widely used previously to assess the anti-proliferative activity of
metal complexes, including Ru-arene-thiolato dimers.[22-25] All the
cell lines were treated for 72 h with 50 ± 5 µM Ru (verified by
atomic absorption spectroscopy, see Experimental Section). A
summary of results is presented in Figure 7. Compound 1b that
showed a weak but statistically significant activity in A549 cells in
preliminary experiments (Figure S6) was also tested in all the cell
lines listed in Table 2, but the resultant cell viabilities were ≥80%
of control and are not included into Figure 7.
Most strikingly, 5 showed the greatest proliferation inhibition in
mesenchymal-like[31] MDA-MB-231 cells (No. 3 in Figure 7) while
having little effect in all the studied epithelial and fibroblastic cells,
with the exception of A2780 (ovarian cancer, No. 6 in Figure 7).
Concentration-dependent studies of 5 in MDA-MB-231 cells
(Figure S7) have shown that the complex did not change its
activity (IC50 = 39 ± 4 µM Ru) after 72 h pre-incubation with fully
supplemented cell culture medium (Experimental Section) at 310
K and 5% CO2. This result, in conjunction with ESI-MS data
(Figure S8), confirms that 5 was stable for the duration of cell
culture assays. Differences in action of 2a,b in various cell types
were less prominent (Figure 7), and no consistent differences
were observed between the activities of these two complexes that
were derived from enantiomeric ligands.
Figure 7. Comparison of anti-proliferative activities (MTT assays) of 2a,b and 5
in a panel of cancer and non-cancer cell lines (Table 2). Stock solutions of Ru
complexes in DMF (20 mM Ru, DMF) were diluted 400-fold with growth medium
(Advanced DMEM with 2% vol. FCS) immediately before the 72 h treatments.
Final Ru concentrations in the medium were 50 ± 5 µM (GFAAS). Error bars
represent mean values and standard deviations of two independent
experiments, each including six replicate wells (n = 12). The # signs represent
highly significant differences (P < 0.001) of the effect of 5 from the average effect
of 2a and 2b.
Anti-proliferative vs. Cytotoxic activity of 2a,b and 5. Typical
cell proliferation assays, including MTT assays, are based on
measuring bulk cell metabolism,[45] and do not differentiate
between anti-proliferative and cytotoxic activities of anti-cancer
drugs.[3] Preliminary morphological observations using IncuCyte
Zoom imaging system (Figure S9) indicated that 5 (~50 µM Ru)
caused death of MDA-MB-231 cells after 24 h incubation, while
2a and 2b slowed their growth, but the remaining cells were alive
after 72 h incubations. This difference was explored further using
fluorescent dyes that were designed for IncuCyte assays: (i)
green fluorescent (excitation/emission = 500/530 nm) Caspase
3/7 reagent that is specific for early stage apoptotic cells;[50] and
(ii) red fluorescent (excitation/emission = 610/630 nm) Cytotox
Red reagent that stains late stage apoptotic and necrotic cells with
damaged membranes.[51] A well-known cytotoxic Ru(III) complex,
KP1019 (Figure 1)[13, 30, 52] was used as a positive control. Figure
S10 shows typical morphologies of viable (unstained), apoptotic
(green)[53] and late apoptotic or necrotic (red)[54] MDA-MB-231 (a)
and A549 (b) cells after 24-h treatments with 50 µM Ru (5 or
KP1019, respectively). Figure 8 shows the results of quantitative
processing of cell confluence (a,b) and fluorescence (c-f) data.
Fluorescence results in Figure 8 are presented as a proportion of
green or red fluorescent cell area relative to total cell area
(determined from phase contrast images), but similar time profiles
were obtained using integrated green or red fluorescence
intensities (Figure S11, Supporting Information).[39]
Generally, higher activities of 2a,b and 5 were observed in blood-
46, 47]
derived or mesenchymal-like[31,
cells compared with
epithelial or fibroblastic cells (Nos. 1-5 vs. 6-15 in Figure 7 and
Table 2). Such selectivity is remarkable since mesenchymal cells
and fibroblasts share many morphological and biochemical
characteristics.[48] No major differences in activity were observed
between cisplatin-resistant and sensitive A2780 cells (Nos. 5 and
6 in Figure 7 and Table 2).[47, 49] Like previous results for Ru(II)-
arene-trithiolato dimers,[22] this suggests that 2a,b and 5 act by a
different mechanism compared to Pt(II) drugs.[6, 9, 21] However, the
mechanisms of action of these two types of Ru(II) dimers are
unlikely to be the same, since typical trithiolato complexes were
more toxic in MCF-7 (epithelial, non-invasive breast cancer
cells)[31] than in MDA-MB-231 cells,[24, 25] while the opposite trend
was observed for 2a,b and 5 (Nos. 3 and 9 in Figure 7 and Table
2).
Among the epithelial cell lines studied (Nos. 6-13 in Figure 7 and
Table 2), there were no consistent differences in the effects of
2a,b and 5 in immortalized non-cancer (HEK-293 and BEAS-2B,
Nos. 7 and 10) compared with cancer cells. Similarly, previous
studies[23] showed no differences in the effects of Ru(II)-arene-
trithiolato dimers in A2780 and HEK-293 cells. However, 2a,b and
5 showed low toxicity in normal rodent fibroblasts (V79-4 and 3T3-
L1 cell lines, Nos. 14 and 15 in Figure 7 and Table 2), which points
to possible selectivity for cancer versus rapidly growing non-
cancer cells.[26]
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In agreement with results from MTT assays (Nos. 3 and 11, Figure
7), treatment with 5 (50 µM Ru) completely stopped the growth of
MDA-MB-231 cells after 24 h, but had no significant effect on the
growth rate of A549 cells compared with the control (black and
red lines in Figure 8a,b). By contrast, 2a and 2b caused partial
growth inhibition in both cell lines (green and blue lines in Figure
8a,b). In agreement with published data,[52] KP1019 (50 µM,
freshly added to cell culture medium) completely stopped the
proliferation of both cell lines (purple lines in Figure 8a,b).
Concomitantly with the halt in MDA-MB-231 cell growth caused
by 5, there was a significant increase in the proportion of apoptotic
cells (red lines in Figure 8a,c), while the treatments with 2a or 2b
did not cause a significant degree of apoptosis (green and blue
lines in Figure 8c). Treatment of MDA-MB-231 cells with KP1019
caused rapid growth in the proportion of apoptotic cells in the first
4 h of incubation, followed by slower decrease due to cell death
and detachment (purple line in Figure 8c). In A549 cells, only
treatment with KP1019 led to significant apoptosis (Figure 8d). As
expected, time-dependent cell death profiles in both cell lines
were similar but slower to proceed compared to those due to
apoptosis (Figure 8e,f). Slow decays in red fluorescence
registered by IncuCyte (Figure 8e,f) were due to the detachment
of dead cells. In summary, the data within Figure 8 confirmed the
striking difference in the effects of structurally similar compounds,
2a,b and 5 (Figure 1 and Scheme 1), in two well-established
human cancer cell lines (Table 2). Namely, 5 (50 µM Ru)
completely stopped the proliferation and triggered apoptosis in
MDA-MB-231 cells but had no significant effect in A549 cells
under the same conditions, while 2a and 2b caused partial
inhibition of cell growth, but not apoptosis, in either cell line. The
Ru concentration used is clinically relevant, as 0.10-0.40 mM Ru
were detected in the blood plasma of patients receiving KP1019
during clinical trials.[55] Notably, most of the known anti-cancer
drugs are unable to cause apoptosis in aggressive breast cancer
cell lines, including MDA-MB-231, at clinically relevant
concentrations.[3]
Figure 8. Typical time profiles of cell proliferation (a and b, phase contrast
images), apoptosis (c and d, green fluorescence with Caspase 3/7 reagent)[50]
and cell death (e and f, red fluorescence with Cytotox reagent)[51] in MDA-MB-
231 or A549 cells in the presence or absence of 50 ± 5 µM Ru (2a,b, 5 or
KP1019 as a positive control, IncuCyte Zoom imaging).[52] Other cell culture
conditions correspond to those in Figure 7. Error bars represent mean values
and standard deviations of six replicate wells.
Uptake of Ru complexes by MDA-MB-231 and A549 cells.
Figure 9 compares the short-term (a, 50 µM Ru for 4 h) and long-
term (b, 20 µM Ru for 72 h) uptake of 1b (non-toxic in both cell
lines), 2a,b (anti-proliferative in both cell lines) and 5 (cytotoxic in
MDA-MB-231, non-toxic in A549 cells). Treatment conditions
were chosen based on the data contained within Figures 7, S5, 6
and 7, to avoid extensive cell death and detachment during the
treatments. Both conditions led to significantly higher uptake of
dimers that have phenyl residues in the ligands (2a,b and 5)
compared with 1b that had methyl residue (Figure 1 and Figure
9). This feature is readily explained by the increased lipophilicity
of aromatic ligands in 2a,b and 5 and is likely to cause lower anti-
proliferative activity of 1b compared with the other complexes.[8, 9,
52]
The uptakes of 2a,b or 5 by A549 cells were 5-30-fold lower
than those by MDA-MB-231 cells under the same conditions
(Figure 9), which is consistent with the lower toxicity of these
compounds in the former cell line (Figures 7 and 8). Long-term
uptake of lower Ru concentrations (Figure 9b) was 4-8-fold less
efficient than short-term uptake of higher Ru concentrations
(Figure 9a). This feature points to the existence of an active Ru
efflux mechanism, probably through lysosomal uptake and
exocytosis, which was previously described for Pt(II) complexes
and other anti-cancer drugs.[56] More intriguingly, the uptake of 2a
and 2b by MDA-MB-231 cells was higher than that of 5, although
the difference was statistically significant only for the longer
treatment time (Figure 9), which shows that the higher toxicity of
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10.1002/cbic.201900676
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5 in this cell line compared with 2a,b (Figures 7 and 8), has a
specific mechanistic aspect that is unrelated to the level of Ru
uptake in these complexes.
membrane permeability,[62] which is likely to lead to different rates
of cellular Ru uptake and efflux in various cell types. However,
this factor does not account for the higher activity of 5 compared
with 2a,b in MDA-MB-231 cells (No. 3 in Figure 7, and Figure
8a,c), since its accumulation in this cell line was lower (Figure 9).
Another consideration is the difference in cellular thiol
concentrations in various cell types,[26] since 2a,b and 5 are likely
to act through catalytic cellular thiol depletion,[28] similarly to other
hydrolytically stable Ru(II) arene complexes.[21, 27] MDA-MB-231
cells are more susceptible to cellular thiol oxidation compared with
MCF-7 cells.[63] Still, if 5 was a more efficient redox catalyst than
2a,b, it would show consistently higher activity across the panel
of cell lines, which was not the case (Figure 7). The mechanisms
behind the unusual selectivity of 5 in MDA-MB-231 cells (Figures
7 and 8) will require further investigation with the use of more
advanced in vitro models, including hypoxic environment and
three-dimensional cell culture systems.[64]
While the search for new metal-based anti-cancer drugs has long
been focused on highly cytotoxic compounds,[21] the current
emphasis is on the selectivity and low systemic toxicity.[29]
Deliberate choice of moderately active but less toxic compounds
was behind the clinical success of some established anti-cancer
drugs, such as histone deacetylase inhibitors.[65] In addition, the
only Ru complex that was so far successful in clinical trials, IT-
139 (or NKP-1339, Figure 1),[14, 15] showed weak to moderate
anti-proliferative activities in cell culture assays (typical IC50
values, 20-100 µM Ru after 96 h treatment).[16] In this respect, 5
can provide an attractive lead in the search of compounds that
selectively target more aggressive cancer cells.[3] Unusually high
stability of 5 in biological media for a metal complex (Figures 8
and S7),[9] particularly compared with the previously studied
potentially anti-metastatic Ru complexes such as NAMI-A,[4, 10] is
likely to reduce the probability of side effects that hampered the
clinical use of this and other metal anti-cancer drugs.[6, 11]
Figure 9. Ru uptake by MDA-MB-231 or A549 cells after short-term (a) or long-
term (b) treatments with selected Ru arene thiocarboxylato complexes. Error
bars represent means and standard deviations of three replicate samples. The
P values indicate statistically significant differences in Ru uptake compared with
compound 5 in the same cell line.
Implications for the use of 5 in cancer treatment. The
mesenchymal-like MDA-MB-231 (invasive breast cancer) cell
line[31] is a widely used in vitro model for the detection and
treatment of metastatic cancers, particularly breast cancer.[3, 57]
High anti-proliferative activities in MDA-MB-231 cells (IC50 < 1 µM
Ru, 72 h treatment) have been reported for Ru(II)-arene-trithiolato
Conclusions
25]
dimers[24,
and some monomeric Ru(II) and Os(II) arene
complexes,[58] which far surpassed the highest activity reported
here for 5 (Figures 7 and 8). However, 5 is among a few reported
metal complexes that were selective to MDA-MB-231 cells over
common epithelial cancer cell lines,[59] such as MCF-7 (non-
invasive breast cancer),[60] as well as over immortalized non-
cancer epithelial cells,[58] or normal fibroblasts[61] (Table 2 and
Figure 7). To our knowledge, there were no previous reports of
metal complexes that showed selectivity for MDA-MB-231 cells
over all three mentioned cell types. This selectivity is particularly
remarkable, since it was not observed for two other Ru(II)-arene-
thiocarboxylato dimers that are closely structurally related to 5
(2a,b in Figure 1 and Figure 7). Unlike for 2a and b, 5 was able to
trigger apoptosis in MDA-MB-231 (but not in A549) cells (Figure
8b,d). All three complexes are likely to retain their dimeric
structures during cell culture assays (Figures 8 and S7) and to
enter cells intact through passive diffusion.[9]
We have synthesized and characterized a new series of Ru(II)
sulfidocarboxylate complexes of general formula [Ru(p-
cymene)L]2 from a series of α- and β-mercaptocarboxylic acids
(L-H2). Of the twelve Ru(II)-arene thiocarboxylato complexes
synthesized, 1a, 2b, 3b, 4a, 5, and 6 were amenable to single
crystal diffraction studies. The solid state structures of the six
complexes revealed isostructural dimeric compositions in which
the parent ligand has been doubly deprotonated at the thiol and
hydroxyl functionalities allowing O,S-chelation to the Ru(II) metal
center.
The cytotoxicity of complexes 1-7 in cultured human cancer cell
lines was determined by real-time observations with an IncuCyte
Zoom imaging system and in standard MTT assays. In general,
the particular activities of these complexes appear to be highly
structure dependant. Three of the complexes (2a,b and 5), all of
which contain a phenyl substituent in the ligand, showed
promising anti-proliferative activities. Complex 5, [Ru(p-
cymene)(MPP)]2, showed moderate but remarkably selective
activity in highly invasive MDA-MB-231 cells (IC50 = 39 ± 4 µM
Part of the reason for the higher toxicity of 2a,b and 5 in blood-
derived and mesenchymal-like cells over epithelial cells and
fibroblasts (Figure 7 and Table 2) is related to variations in
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10.1002/cbic.201900676
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Ru). 5 induced apoptosis and cell death in MDA-MB-231 cells
after 24 h incubation with 50 µM Ru, but had no effect in A549
(human lung cancer, epithelial) cells under the same conditions.
By contrast, 2a and b showed moderate anti-proliferative activity,
but no apoptosis, in either cell line. Given the high selectivity of 5
towards highly invasive, mesenchymal-like MDA-MB-231 cells
over many common epithelial and fibroblast cells, shows this is
an important lead complex for further pre-clinical studies.
The choice of ligand has a significant effect on anti-cancer
activities and from the promising results in this study, these Ru(II)
sulfidocarboxylate complexes warrant further investigation and
may emerge one day as new cancer treatment candidates.
32.5, 23.9, 22.3, 20.4, 18.9.ꢀIR (film, cm-1): n = 1609 (C=O), 2924
(C-H), 2959 (C-H). MS (ESI) calculated for C26H36O4Ru2S2: 680.0
(M + H)+, 703.0 (M + Na)+. Yield: 67%. HPLC conditions: isocratic
(40% methanol). Melting point: 141 ºC (dec.).
[Ru(p-cymene)(TLA)]2 (1b): 1H NMR (400 MHz, Methanol-d4) δ
5.47 (td, J = 6.7, 1.5 Hz, 2H), 5.34 – 5.26 (m, 2H), 3.06 (q, J = 7.7
Hz, 1H), 2.84 (sept, J = 6.9 Hz, 1H), 2.14 (s, 3H), 1.35 – 1.25 (m,
9H).ꢀ13C NMR (101 MHz, Methanol-d4) δ 187.9, 108.4, 102.2, 87.7,
86.5, 86.2, 83.9, 41.7, 31.9, 23.1, 22.9, 20.4, 18.3.ꢀIR (film, cm-1):
n = 1620 (C=O), 2922 (C-H), 2961 (C-H). MS (ESI) calculated for
C26H36O4Ru2S2: 703.0 (M + Na)+. Yield: 11%. HPLC conditions:
isocratic (52.5% methanol). Melting point: 133 ºC (dec.).
[Ru(p-cymene)(MPA)]2 (2a): 1H NMR (400 MHz, Methanol-d4) δ
7.38 – 7.34 (m, 4H), 7.28 (ddt, J = 6.6, 5.2, 2.6 Hz, 1H), 5.14 –
5.09 (m, 2H), 5.03 (dd, J = 5.6, 1.5 Hz, 1H), 4.81 (s, 13H), 3.36
(dd, J = 10.7, 3.5 Hz, 2H), 3.09 (dd, J = 13.7, 3.5 Hz, 1H), 2.68
(dd, J = 13.8, 10.7 Hz, 1H), 2.50 (sept, J = 6.9 Hz, 1H), 2.00 (s,
3H), 1.13 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, Methanol-d4) δ
186.9, 140.9, 131.0, 129.3, 127.8, 108.1, 102.0, 87.1, 87.1, 86.2,
83.4, 40.4, 31.6, 23.0, 22.9, 18.4. IR (film, cm-1): n = 1605 (C=O),
2929 (C-H), 2960 (C-H). MS (ESI) calculated for C38H44O4Ru2S2:
831.0 (M + H)+, 852.9 (M + Na)+. Yield: 46%. HPLC conditions:
gradient (60% - 75% methanol). Melting point: 114 ºC (dec.).
Experimental Section
Synthesis and Characterization of 1-7. Unless preparative
details are included, solvents and reagents were purchased from
commercial suppliers; Sigma Aldrich and ThermoFischer
Scientific. Proton (1H NMR) and carbon magnetic resonance (13C
NMR) spectra were recorded on a Bruker AV400 spectrometer at
400 MHz and 100 MHz respectively. Chemical shifts are quoted
in parts per million (d/ppm) and are referenced to the residual
solvent peak. Infrared spectroscopy measurements were carried
out with a Cary 630 FTIR in the range of 4000-500 cm-1. HPLC
purification was conducted using an Agilent Technologies 1260
Infinity Preparative pump with a 1260 DAD VL UV detector (280
nm) on a Phenomenex Onyx Monolithic 100 x 10 mm C18 column.
The mobile phase was a mixture of MilliQ water and analytical
grade methanol using either isocratic conditions or a linear
gradient over 15 minutes and a flow rate of 4 mL/min. Mass
spectrometry was performed on a Micromass Platform QMS
spectrometer with an electrospray source and cone voltage of 35
eV. Melting points were obtained using a Stuart Scientific SMP 3
melting point machine.
[Ru(p-cymene)(MPA)]2 (2b): 1H NMR (400 MHz, Methanol-d4) δ
7.39 – 7.34 (m, 4H), 7.30 – 7.25 (m, 1H), 5.12 (td, J = 5.8, 5.3, 1.2
Hz, 2H), 5.03 (d, J = 5.2 Hz, 1H), 4.82 (d, J = 5.6 Hz, 6H), 3.36
(dd, J = 10.7, 3.5 Hz, 1H), 3.09 (dd, J = 13.7, 3.5 Hz, 1H), 2.68
(dd, J = 13.7, 10.7 Hz, 1H), 2.50 (sept, J = 7.0 Hz, 1H), 2.00 (s,
3H), 1.13 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, Methanol-d4) δ
186.9, 140.9, 131.0, 129.3, 127.8, 108.1, 102.0, 87.1, 87.1, 86.2,
83.4, 49.7, 49.5, 49.3, 49.1, 48.4, 40.4, 31.6, 23.0, 22.9, 18.4. IR
(film, cm-1): n =1606 (C=O), 2927 (C-H), 2962 (C-H). MS (ESI)
calculated for C38H44O4Ru2S2: 831.0 (M + H)+, 852.9 (M + Na)+.
Yield: 46%. HPLC conditions: gradient (60% - 75% methanol).
Melting point: 114 ºC (dec.).
(Full experimental details for the synthesis of a- and b-
mercaptocarboxylic acids can be found in the Supporting
Information).
[Ru(p-cymene)(MMA)]2 (3a): 1H NMR (400 MHz, Methanol-d4) δ
5.47 (d, J = 7.2 Hz, 1H), 5.33 (d, J = 7.4 Hz, 1H), 5.26 (d, J = 7.1
Hz, 1H), 5.14 (d, J = 7.3 Hz, 1H), 3.17 (d, J = 4.1 Hz, 1H), 2.82
(sept, J = 6.9 Hz, 1H), 2.28 (s, 3H), 2.20 (pd, J = 6.8, 4.2 Hz, 1H),
1.31 (dd, J = 6.9, 1.6 Hz, 6H), 1.07 (d, J = 6.9 Hz, 3H), 0.92 (d, J
= 6.7 Hz, 3H). 13C NMR (101 MHz, Methanol-d4) δ 187.7, 108.4,
101.4, 88.6, 87.9, 82.1, 51.4, 32.7, 32.0, 23.4, 23.1, 21.3, 19.2,
18.6. IR (film, cm-1): n = 1601 (C=O), 2927 (C-H), 2957 (C-H). MS
(ESI) calculated for C30H44O4Ru2S2: 735.9 (M + H)+, 758.0 (M +
Na)+. Yield: 48%. HPLC conditions: gradient (60% - 70%
methanol). Melting point: 146 ºC (dec.).
Preparation of [Ru(p-cymene)L]2 complexes: General
procedure. To a solution of [RuCl2(p-cymene)]2 (1 equiv.) in
methanol was added the a/b-mercaptocarboxylic acid (2.5 equiv.)
and excess triethylamine (6 equiv.) The solution was stirred at
reflux for 1 h before being concentrated under vacuo. The
resulting crude material was first purified by silica gel column
chromatography to remove bulk impurities (SiO2, 100% DCM –
20% methanol/DCM), before being further purified using reverse
phase prep-HPLC (C18 column Phenomenex Monolithic).
Analysis by MS identified fractions that contained the desired
compound.
[Ru(p-cymene)(MMA)]2 (3b): 1H NMR (400 MHz, Methanol-d4) δ
5.46 (d, J = 7.1 Hz, 1H), 5.33 (d, J = 7.4 Hz, 1H), 5.26 (d, J = 7.2
Hz, 1H), 5.14 (d, J = 7.3 Hz, 1H), 3.17 (d, J = 4.1 Hz, 1H), 2.82
(sept, J = 7.0 Hz, 1H), 2.28 (s, 3H), 2.20 (pd, J = 6.8, 4.1 Hz, 1H),
1.31 (dd, J = 6.9, 1.5 Hz, 6H), 1.07 (d, J = 6.9 Hz, 3H), 0.92 (d, J
= 6.8 Hz, 3H).ꢀ13C NMR (101 MHz, Methanol-d4) δ 187.7, 108.4,
[Ru(p-cymene)(TLA)]2 (1a): 1H NMR (400 MHz, Methanol-d4) δ
5.76 (d, J = 6.9 Hz, 1H), 5.42 (d, J = 5.7 Hz, 1H), 5.36 (d, J = 6.2
Hz, 1H), 5.08 (d, J = 7.0 Hz, 1H), 3.65 (q, J = 7.2 Hz, 1H), 2.77
(sept, J = 6.9 Hz, 1H), 2.11 (s, 3H), 1.52 (d, J = 7.3 Hz, 3H), 1.33
(d, J = 6.9 Hz, 4H), 1.28 (d, J = 7.0 Hz, 3H).ꢀ13C NMR (101 MHz,
Methanol-d4) δ 187.8, 107.6, 97.8, 88.6, 88.1, 86.5, 79.0, 50.9,
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101.4, 88.6, 87.9, 82.1, 51.4, 32.7, 32.0, 23.4, 23.1, 21.3, 19.2,
18.6. IR (film, cm-1): n = 1601 (C=O), 2927 (C-H), 2955 (C-H). MS
(ESI) calculated for C30H44O4Ru2S2: 735.9 (M + H)+, 758.0 (M +
Na)+. Yield: 52%. HPLC conditions: gradient (60% - 70%
methanol). Melting point: 147 ºC (dec.).
8.14 (dd, J = 7.7, 1.5 Hz, 1H), 7.67 (dd, J = 7.7, 1.0 Hz, 1H), 7.39
(td, J = 7.5, 1.2 Hz, 1H), 7.24 (dd, J = 7.6, 1.7 Hz, 1H), 5.10 (d, J
= 5.9 Hz, 1H), 4.82 – 4.73 (m, 2H), 4.62 (d, J = 5.6 Hz, 1H), 2.51
(sept, J = 6.9 Hz, 1H), 1.84 (s, 3H), 1.15 (d, J = 7.0 Hz, 3H), 0.86
(d, J = 6.8 Hz, 3H).ꢀ13C NMR (101 MHz, Chloroform-d) δ 171.6,
139.4, 133.1, 132.3, 128.9, 128.5, 107.5, 99.6, 84.0, 83.4, 83.3,
83.3, 29.9, 23.3, 21.3, 17.7. IR (film, cm-1): n = 1587 (C=O), 2926
(C-H), 2971 (C-H). MS (ESI) calculated for C34H36O4Ru2S2: 777.0
(M + H)+, 800.0 (M + Na)+. Yield: 65%. HPLC conditions not
required.
[Ru(p-cymene)(MBA)]2 (4a): 1H NMR (400 MHz, Methanol-d4) δ
5.59 (d, J = 5.7 Hz, 1H), 5.41 (d, J = 6.2 Hz, 1H), 5.29 (d, J = 6.2
Hz, 1H), 4.96 (d, J = 5.7 Hz, 1H), 3.17 (ddd, J = 11.7, 6.8, 3.6 Hz,
1H), 2.74 – 2.66 (m, 2H), 2.46 (dd, J = 13.2, 11.6 Hz, 1H), 2.15 (s,
3H), 1.38 (d, J = 6.8 Hz, 3H), 1.29 (dd, J = 15.1, 6.9 Hz, 6H).ꢀ13C
NMR (101 MHz, Methanol-d4) δ 182.8, 107.9, 98.6, 88.2, 88.2,
85.9, 79.6, 48.9, 40.5, 32.0, 25.1, 23.4, 22.3, 18.6.ꢀIR (film, cm-1):
n = 1579 (C=O), 2918 (C-H), 2960 (C-H). MS (ESI) calculated for
C28H40O4Ru2S2: 707.9 (M + H)+, 730.9 (M + Na)+. Yield: 66%.
HPLC conditions: gradient (40% - 60% methanol). Melting point:
112 ºC (dec.).
[Ru(p-cymene)(TSA)]2 (7b): 1H NMR (400 MHz, Methanol-d4) δ
8.19 – 8.15 (m, 1H), 7.60 – 7.56 (m, 1H), 7.43 – 7.37 (m, 2H),
5.52 (d, J = 5.9 Hz, 1H), 5.40 (dd, J = 12.1, 5.9 Hz, 2H), 5.28 (d,
J = 6.0 Hz, 1H), 3.35 (s, 1H), 2.12 (sept, J = 6.9 Hz, 1H), 1.60 (s,
3H), 0.85 (d, J = 6.9 Hz, 3H), 0.67 (d, J = 6.8 Hz, 3H).ꢀ13C NMR
(101 MHz, Methanol-d4) δ 173.6, 142.7, 137.6, 136.0, 131.0,
129.0, 128.9, 108.9, 101.2, 86.7, 86.3, 85.2, 85.1, 31.6, 23.4, 21.4,
17.8.ꢀ IR (film, cm-1): 2961 (C-H). MS (ESI) calculated for
C34H36O4Ru2S2: 800.0 (M + Na)+. Yield: 12%. HPLC conditions:
gradient (50% - 80% methanol). Melting point: 258 ºC (dec.).
[Ru(p-cymene)(MBA)]2 (4b): 1H NMR (400 MHz, Methanol-d4) δ
5.36 (dd, J = 8.3, 5.9 Hz, 2H), 5.22 (d, J = 6.0 Hz, 1H), 5.17 (d, J
= 5.8 Hz, 1H), 2.92 (sept, J = 6.9 Hz, 1H), 2.76 (ddp, J = 10.3, 6.8,
3.5 Hz, 1H), 2.47 – 2.32 (m, 2H), 2.23 (s, 3H), 1.32 (dd, J = 6.9,
6.2 Hz, 6H), 1.17 (d, J = 6.8 Hz, 3H).ꢀ 13C NMR (101 MHz,
Methanol-d4) δ 183.2, 111.3, 102.7, 86.8, 86.7, 85.6, 84.3, 47.8,
31.4, 29.0, 23.9, 23.0, 22.6, 18.0.ꢀIR (film, cm-1): n = 1604 (C=O),
2916 (C-H), 2959 (C-H). MS (ESI) calculated for C28H40O4Ru2S2:
707.9 (M + H)+, 730.9 (M + Na)+. Yield: 27%. HPLC conditions:
gradient (50% - 65% methanol). Melting point: 123 ºC (dec.).
KP1019 was synthesized and characterized as described
previously.[52]
X-Ray Crystallography. Crystallographic data of compounds 1-
3 was collected at the MX1 beamline at the Australian
Synchrotron, Melbourne, Victoria, Australia (λ = 0.71070 Å). All
data were collected at 100 K, maintained using an open flow of
nitrogen. The software used for data collection and reduction of
the data were BluIce and XDS. Crystallographic data for
complexes 5 and 6 were obtained on a Bruker X8 APEXII CCD
diffractometer equipped with an OXFORD Cryosystems 700
Cryostream and cooled to 123(1) K. Data were collected with
[Ru(p-cymene)(MPP)]2 (5): 1H NMR (400 MHz, Methanol-d4) δ
7.55 – 7.47 (m, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.28 – 7.23 (m, 1H),
5.33 – 5.27 (m, 2H), 5.17 (d, J = 5.2 Hz, 1H), 5.11 (d, J = 4.9 Hz,
1H), 3.90 (dd, J = 13.1, 1.4 Hz, 1H), 3.05 (dd, J = 15.0, 13.1 Hz,
1H), 2.67 – 2.57 (m, 2H), 2.02 (s, 3H), 1.14 (d, J = 6.9 Hz, 3H),
1.01 (d, J = 6.9 Hz, 3H).ꢀ13C NMR (101 MHz, Methanol-d4) δ 183.5, monochromatic (graphite) Mo Kα radiation (λ = 0.71073 Å) and
144.8, 129.6, 129.3, 128.1, 111.8, 104.3, 86.4, 86.0, 85.5, 84.5,
46.8, 35.9, 31.5, 23.1, 22.3, 17.8. IR (film, cm-1): n = 1641 (C=O),
2998 (C-H). MS (ESI) calculated for C38H46O4Ru2S2: 832.0 (M +
H)+, 854.9 (M + Na)+. Yield: 43%. HPLC conditions: gradient (65%
- 75% methanol). Melting point: 134 ºC (dec.).
processed using the Bruker Apex2 v2012.2.0 software; Lorentz,
polarization and absorption corrections (multi-scan – SADABS)
were applied. Crystallographic data for compound 4a were
obtained on Rigaku Synergy S diffractometer Mo Kα radiation (λ
= 0.71073 Å) HYpix_6000HE. Compounds 1-6 were solved and
refined with SHELX-97. All non-hydrogen atoms were refined with
anisotropic thermal parameters unless otherwise indicated and
hydrogen atoms were placed in calculated positions using a riding
model with C-H = 0.95-0.98 Å and Uiso(H)=xUiso(C), x = 1.2 or
1.5. Data for 1a, 2b, 3b, 4a, 5 and 6 has been deposited with the
Cambridge Crystallographic Database with CCDC number
1046084, 1046085, 1046086, 1901567, 1901574 and 1046087
respectively.
[Ru(p-cymene)(MMPA)]2 (6): 1H NMR (400 MHz, Methanol-d4) δ
7.44 – 7.41 (m, 2H), 6.91 – 6.88 (m, 2H), 5.30 – 5.27 (m, 2H),
5.14 (d, J = 5.7 Hz, 1H), 5.10 (d, J = 5.7 Hz, 1H), 3.84 (dd, J =
13.2, 1.5 Hz, 1H), 3.81 (s, 3H), 3.02 (dd, J = 15.0, 13.1 Hz, 1H),
2.65 – 2.60 (m, 2H), 2.03 (s, 3H), 1.15 (d, J = 6.9 Hz, 3H), 1.04
(d, J = 6.9 Hz, 3H).ꢀ13C NMR (101 MHz, Methanol-d4) δ 183.7,
160.3, 136.7, 130.3, 115.0, 111.7 104.2, 86.4, 85.8, 85.4, 84.5,
55.8, 47.0, 35.4, 31.4, 23.1, 22.4, 17.8. IR (film, cm-1): n = 1599
(C=O), 2909 (C-H), 2955 (C-H).MS (ESI) calculated for
C40H48O6Ru2S2: 892.1 (M + H)+, 915.0 (M + Na)+. Yield: 52%.
HPLC conditions: gradient (65% - 75% methanol). Melting point:
138 ºC (dec.).
Crystal Data for 1a: C26H36O6Ru2S2; Mr = 710.81; triclinic; space
group: P-1; a = 9.167 (18), b = 10.208 (2), c = 15.506 (3); α =
83.32 (3); β = 87.39 (3); γ = 73.83 (3); V = 1384.0(5) Å3; Z = 2,
reflections collected/unique: 6012/6012 (Rint = 0.0327); R1 values
(I > 2σ(I)) = 0.0405; wR(F2) values (I > 2σ(I)) = 0.1091; R1 values
(all data) = 0.0417; wR(F2) values (all data) = 0.1102; GOF =
1.052.
[Ru(p-cymene)(TSA)]2 (7a): All analytical data, such as NMR
spectra and MP, were consistent with the literature reported
values for compound 7a.[34] 1H NMR (400 MHz, Chloroform-d) δ
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10.1002/cbic.201900676
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Crystal Data for 2b: C30H44O7Ru2S2; Mr = 782.91; monoclinic;
space group: P21/c; a = 13.682 (3), b = 15.375(3), c = 17.054 (3);
β = 110.61(3); V = 3357.8 (12) Å3; Z = 4, reflections
collected/unique: 55310/7958 (Rint = 0.0738); R1 values (I > 2σ(I))
= 0.0469; wR(F2) values (I > 2σ(I)) = 0.1171; R1 values (all data)
= 0.0572; wR(F2) values (all data) = 0.1227; GOF = 1.022.
Cell culture and MTT proliferation assays. Pre-sterilized media
and sterile plasticware used in cell culture were purchased from
Life Technologies Australia. Cell lines (listed in Table 2) were
purchased from American Type Culture Collection (ATCC,
www.atcc.org), or European Collection of Authenticated Cell
Cultures (ECACC, www.phe-culturecollection.org.uk). The cells
were cultured using standard techniques[67] in Advanced DMEM
(Thermo Fisher 12491-015), supplemented with L-glutamine (2.0
mM), antibiotic-antimycotic mixture (100 U mL-1 penicillin, 100 mg
mL-1 streptomycin and 0.25 mg mL-1 amphotericin B) and fetal calf
Crystal Data for 3b: C42H54O6Ru2S2; Mr = 921.11; triclinic; space
group: P-1; a = 8.8340 (18), b = 10.281 (2), c = 12.880 (3); α =
72.96 (3); β = 78.96 (3); γ = 65.53 (3); V = 1014.6(4) Å3; Z = 1,
reflections collected/unique: 19968/5246 (Rint = 0.0322); R1
values (I > 2σ(I)) = 0.0521; wR(F2) values (I > 2σ(I)) = 0.1457; R1
values (all data) = 0.0552; wR(F2) values (all data) = 0.1474; GOF
= 1.145.
serum (FCS; heat-inactivated;
2 % vol). For proliferation
experiments, cells were seeded in 96-well plates in 0.10 mL
medium per well, and incubated overnight (310 K, 5% CO2) before
replacing the growth medium with fully supplemented medium
containing Ru complexes. Non-adherent cells (Nos. 1 and 2,
Table 2) were seeded at 1.0×104 viable cells per well in round-
bottom plates, while adherent cells (Nos. 3-15, Table 2) were
seeded at 1.0×103 viable cells per well in flat-bottom plates. Cell
counting and viability tests were performed using Countess
automatic counter (Thermo Fisher) with chamber slides and
Trypan blue dye.[67]
Crystal Data for 4a: C28H46O7Ru2S2; Mr = 760.91; triclinic; space
group: P-1; a = 10.9622 (2), b = 11.3748 (3), c = 13.5843 (4); α =
69.997 (2); β = 81.661 (2); γ = 80.084 (2); V = 1561.10 (7) Å3; Z
= 2, reflections collected/unique: 47943/6475 (Rint = 0.0896); R1
values (I > 2σ(I)) = 0.0666; wR(F2) values (I > 2σ(I)) = 0.1609; R1
values (all data) = 0.0777; wR(F2) values (all data) = 0.1677; GOF
= 1.097.
Stock solutions of the complexes were 1-12 mM Ru in DMSO
(preliminary experiments, Figure S6), or 20 mM Ru in DMF (main
experiments, Figures 7 and S7). These solutions were stable for
at least a month when stored in the dark at 295 K (verified by ESI-
MS, Figure S8). Unless stated otherwise, stock solutions were
diluted 100-400-fold with fully supplemented cell culture medium
immediately before cell treatments. All treatments contained the
same amount of organic solvent (0.25% vol. DMF or 1.0% vol.
DMSO), which was also added to the medium in control wells. At
these concentrations, the organic solvents did not have any
significant effect on cell viability over 72-96 h treatments.[41] Each
treatment included six replicate wells and two background wells
that contained the same components except the cells. After 72 h
incubations (310 K, 5% CO2), MTT reagent (Sigma M5655) was
added (50 µL per well of freshly prepared 2.0 mg mL-1 solution in
complete medium), and incubation was continued for 4-6 h. After
that, the medium was removed, the blue formazan crystals were
dissolved in 0.10 mL per well of DMSO, and the absorbance at
600 nm was measured using Victor V3 plate reader.[45]
Calculation of IC50 values and statistical analysis of the results
were performed using Origin software.[68] For all the cell assays,
consistent results were obtained in at least two independent
experiments, using different passages of cells and different stock
solutions of the treatment compounds.
Crystal Data for 5: C38H44O4Ru2S2; Mr = 831.02; triclinic; space
group: P-1; a = 8.8674 (9), b = 10.1893 (9), c = 10.3615 (9); α =
72.345 (3); β = 89.989 (4); γ = 65.774 (3); V = 805.17 (12) Å3; Z
= 1, reflections collected/unique: 20079/4976 (Rint = 0.0832); R1
values (I > 2σ(I)) = 0.0447; wR(F2) values (I > 2σ(I)) = 0.0775; R1
values (all data) = 0.0641; wR(F2) values (all data) = 0.0857; GOF
= 1.075.
Crystal Data for 6: C44H60O8Ru2S2; Mr = 983.21; triclinic; space
group: P-1; a = 9.2472 (5), b = 11.2364 (6), c = 11.6790 (3); α =
115.282 (3); β = 104.523 (3); γ = 97.263 (3); V = 1023.72 (9) Å3;
Z = 2, reflections collected/unique: 15961/5867 (Rint = 0.0205); R1
values (I > 2σ(I)) = 0.0208; wR(F2) values (I > 2σ(I)) = 0.0479; R1
values (all data) = 0.0233; wR(F2) values (all data) = 0.0492; GOF
= 1.027.
Stability studies of 1-7 in aqueous media. Electrospray
ionization mass spectrometry (ESI-MS) data for 1-7 under
biomimetic conditions[9, 40, 42] were collected on a Bruker amaZon
SL spectrometer, using the following parameters: nebulizer
pressure, 27.3 psi; spray voltage, 4.5 kV; capillary temperature,
453 K; N2 flow rate, 4 L min-1; m/z range, 100-2000 (alternating
positive and negative ion modes). Analyzed solutions (5.0 µL)
were injected into a flow of MeOH (flow rate, 0.30 mL min-1).
Acquired spectra were the averages of 100-200 scans (scan time,
10 ms). Solutions for mass spectrometry were prepared by
diluting stock solutions of 1-7 (1.0 µL of 2-20 mM in DMF) with
aqueous NH4HCO3 (350 µL of 10 mM, pH 7.5). The resultant
solutions were stored for several hours or days at 295 K, then
mixed with MeOH (50 µL) immediately prior to collecting the ESI-
MS data. Simulations of the mass spectra were performed using
IsoPro software.[66]
IncuCyte proliferation and cytotoxicity assays. MDA-MB-231
and A549 cells (Table 2) were seeded at 2.0×103 cells per well in
flat-bottom 96-well plates (Corning 3599) and left to attach
overnight (310 K, 5% CO2), after which the growth medium was
replaced with fully supplemented treatment medium containing
Ru complexes (50 µM Ru), Caspase 3/7 reagent (3.0 µM)[50] and
Cytotox Red (0.30 µM).[51] The plates were placed into an
IncuCyte Zoom imaging system[39] that was maintained at 310 K,
5% CO2, and phase contrast and fluorescent images were
collected every 2 h for 80-100 h, using x10 objective. Green and
red fluorescence acquisition times were 0.40 s and 0.80 s,
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10.1002/cbic.201900676
ChemBioChem
FULL PAPER
respectively. Image analyses were performed with IncuCyte
software,[39] using the following parameters: cell over background
threshold, 1.0; minimal object size, 300 µm2; green threshold, 2.0;
red threshold, 0.5; 5% of green fluorescence removed from the
red channel. Further analysis and plotting of IncuCyte data was
performed using Origin software.[68]
(0.10-1.0 mg mL-1) were used for calibration. The remaining
undiluted lysate was brought to 1.0 mL with 0.10 M HCl, left
overnight at 295 K, centrifuged for 2 min at 16,000 g to remove
denatured protein, and used for GFAAS measurements. The
results were presented in nmol Ru per mg protein (Figure 9).
Measurements of Ru in cell culture media and cell lysates.
Analyses were performed by graphite furnace atomic absorption
spectrometry (GFAAS), using an Agilent Technologies series 200
spectrometer, equipped with Zeeman background correction.
Standard Ru solution (Aldrich 207446) was diluted with 0.10 M
HCl (prepared from 35% aqueous solution, trace pure grade,
Merck Cat. No. 1.15186) for calibration (0-500 ppb Ru). Aliquots
(50 µL) of Ru-containing media from the cell assays were diluted
to 1.0 mL with 0.10 M HCl and left overnight at 295 K before the
GFAAS measurements.
Cellular Ru uptake was measured following our published
method.[40, 52, 69] Cell treatments were performed in triplicate in six-
well plates, either by growing MDA-MB-231 or A549 cells to ~80%
confluence and then incubating them with 50 µM Ru for 4 h
(Figure 9a), or seeding 1.0×105 viable cells per well and growing
them to near-confluence for 72 h in the presence of 20 µM Ru
(Figure 9b). After the treatments, the medium was removed, the
cell layers were washed three times with PBS, collected by
trypsinization, pelleted (3 min at 600 g), washed again with PBS,
and digested with 0.10 mL of 0.10 M NaOH by pipetting up and
down and storing overnight at 277 K. Trypsinization and washing
of cell pellets was used to remove Ru that was adsorbed on
plastic ware and on the cell surface.[70] Since the resultant lysate
was viscous, an aliquot (10 µL) was taken with a positive
displacement pipette, diluted 10-fold with 0.10 M NaOH, and used
for protein determination. For this, aliquots of the dilute lysate (3
× 10 µL) were mixed with Bradford reagent (90 µL, Sigma B6916),
and the absorbance at 600 nm was measured using a Victor V3
plate reader. Freshly prepared BSA solutions in 0.10 M NaOH
Acknowledgements
Financial support for this work was provided by Australian
Research Council (ARC) grants to PAL for an ARC Senior
Research Associate position for AL (DP130103566,
DP160104172). The authors acknowledge the facilities and the
scientific and technical assistance of the Mass Spectrometry
Facility, School of Chemistry, University of Sydney (Nicholas
Proschogo); Australian Microscopy & Microanalysis Research
Facility at the Australian Centre for Microscopy & Microanalysis at
the University of Sydney (Minh Huynh and Ellie Kable) for the use
of cell culture laboratory; and Molecular Biology Facility of the
Bosch Institute, University of Sydney (Donna Lai and Sheng Hua)
for the use of IncuCyte. The authors also acknowledge the
scientific and technical assistance of Mr. Phillip Holt with HPLC at
Monash University.
Keywords: Anti-cancer agents • Anti-metastatic • Apoptosis •
Breast cancer • Ruthenium • Selectively cytotoxic
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10.1002/cbic.201900676
ChemBioChem
FULL PAPER
Table 2. Cell lines used for testing the anti-proliferative activities of Ru(II)-arene-thiocarboxylato dimers.
No.a
Cell line
THP-1
ATCC b No.
Species
human
human
human
human
human
human
human
human
human
human
human
human
human
hamster
mouse
Tissue
Disease
Phenotype
1
TIB-202
peripheral blood
peripheral blood
mammary gland f
connective tissue
ovaries
acute monocytic leukemia
acute promyelocytic leukemia
adenocarcinoma
non-adherent
non-adherent
mesenchymal g
2
HL-60
CCL-240
HTB-26
3
MDA-MB-231
HT-1080
A2780cis c
A2780 d
HEK-293
PC-3
4
CCL-121
93112517 e
93112519 e
CRL-1573
CRL-1435
HTB-22
fibrosarcoma
mesenchymal g
mixed h
5
adenocarcinoma
6
ovaries
adenocarcinoma
epithelial
epithelial
epithelial
epithelial
epithelial
epithelial
epithelial
epithelial
fibroblasts
fibroblasts
7
embryonic kidney
prostate
normal (virus-immortalized)
adenocarcinoma
8
9
MCF-7
mammary gland f
lung, bronchus
lung
adenocarcinoma
10
11
12
13
14
15
BEAS-2B
A549
CRL-9609
CCL-185
CRL-1469
HB-8065
CCL-93
normal (virus-immortalized)
carcinoma
PANC-1
HepG2
pancreas/duct
liver
epithelioid carcinoma
hepatocellular carcinoma
normal
V79-4
lung
3T3-L1
CL-173
embryo
normal
a Numbering corresponds to that in Figure 7. b American Type Culture Collection (ATCC). cCisplatin-resistant clone, IC50 for cisplatin 5.4 ± 0.5 µM (72 h treatment, MTT assay).59 d
Cisplatin-sensitive clone, IC50 for cisplatin 0.93 ± 0.05 µM (72 h treatment, MTT assay).59 e European Collection of Authenticated Cell Cultures (ECACC). f Derived from lung metastases.
g Although classified as epithelial by ATCC, these cell lines exhibit mesenchymal-like properties, including elongated morphology, low expression of E-cadherin and high expression of
vimentin, formation of protrusions, and invasion through extracellular matrix in the absence of added growth factors.35, 55 h Cisplatin resistance in A2780 cells is acquired by partial
conversion from epithelial to mesenchymal phenotype.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201900676
ChemBioChem
FULL PAPER
FULL PAPER
A sulfidocarboxylato Ru(II) (p-cymene)
complex; [Ru(p-cymene)(MPP)]2,
derived from 3-mercapto-3-
phenylpropanoic acid (MPP),
demonstrates strong ligand
dependency in displaying selective
anti-metastatic activity towards a
highly aggressive breast cancer cell
line.
L. J. Stephens, A. Levina, I. Trinh, V. L
Blair, M. V. Werrett, P. A. Lay* and P. C.
Andrews*
Page No. – Page No.
A Stable Ruthenium(II)-Arene
Thiocarboxylato Dimer is Selectively
Cytotoxic to Invasive Breast Cancer
Cells
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