Facile entry to germanate and stannate complexes [(η6-arene)RuCl(η2-dppm)]+[ECl3]- (E = Ge, Sn) as potent anti-cancer agents

Article abstract of DOI:10.1016/j.jorganchem.2020.121214

A series of arene Ru(II) salt complexes of the type [(η⁶-arene)RuCl(η²-dppm)]⁺[ECl₃]^- (arene = C₆H₆, p-cymene, 1,3,5-Me₃C₆H₃; E = Ge, Sn) bearing

Full text of DOI:10.1016/j.jorganchem.2020.121214

Journal Pre-proof
6
2
Facile entry to germanate and stannate complexes [(η -arene)RuCl(η -
+
-
dppm)] [ECl ] (E = Ge, Sn) as potent anti-cancer agents
3
Niccolo Aldeghi, Dario Romano, Christoph Marschner, Supratim Biswas, Suparna
Chakraborty, Sharon Prince, Siyabonga Ngubane, Burgert Blom
PII:
S0022-328X(20)30115-7
DOI:
https://doi.org/10.1016/j.jorganchem.2020.121214
JOM 121214
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 31 January 2020
Accepted Date: 4 March 2020
Please cite this article as: N. Aldeghi, D. Romano, C. Marschner, S. Biswas, S. Chakraborty, S.
6
2
Prince, S. Ngubane, B. Blom, Facile entry to germanate and stannate complexes [(η -arene)RuCl(η -
+
-
dppm)] [ECl ] (E = Ge, Sn) as potent anti-cancer agents, Journal of Organometallic Chemistry (2020),
3
doi: https://doi.org/10.1016/j.jorganchem.2020.121214.
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Facile Entry to Germanate and Stannate Complexes
[(η6-Arene)RuCl(η2-dppm)]+[ECl3]- (E = Ge, Sn) As
Potent Anti-Cancer Agents
Niccolo Aldeghi,a Dario Romano,b,c Christoph Marschner,d Supratim Biswas,e Suparna
f
Chakraborty,e Sharon Prince,e,* Siyabonga Ngubane, Burgert Blom,a,
∗
aMaastricht Science Programme, Faculty of Science and Engineering, Maastricht University, Kapoenstraat 2, PO Box
616, 6200 MD, Maastricht, The Netherlands
bDepartment of Biobased Materials, Faculty of Science and Engineering, Maastricht University, Brightlands Chemelot
Campus, Urmonderbaan 22, Geleen, 6167 RD, The Netherlands
cAachen-Maastricht Institute for Biobased Materials, Brightlands Chemelot Campus, Urmonderbaan 22, Geleen, 6167
RD, The Netherlands
dInstitut für Anorganische Chemie, Technische Universität Graz, Stremayrgasse 9, A-8010 Graz, Austria
eUniversity of Cape Town, Department of Human Biology, Observatory 7935, Cape Town, South Africa.
f University of Cape Town, Department of Chemistry, Rondebosch 7701, Cape Town, South Africa
Dedicated to F. R. Blom who passed away 29 October 2019 after a yearlong battle with cancer.
Abstract
A series of arene Ru(II) salt complexes of the type [(η6-arene)RuCl(η2-dppm)]+[ECl3]- (arene =
C6H6, p-cymene, 1,3,5-Me3C6H3; E = Ge, Sn) bearing trichlorogermanate and trichlorostannate
anions are reported. Starting from the known complexes: [(η6-C6H6)RuCl2(η1-dppm)] (1), [(η6-p-
cymene)RuCl2(η1-dppm)] (3) and the novel complex [(η6-1,3,5-Me3C6H3)RuCl2(η1-dppm)] (7)
(dppm = 1,1-bis(diphenylphosphino)methane), reactions with SnCl2 or GeCl2(dioxane) respectively
afforded, by halide abstraction at the ruthenium(II) centres in 1, 3 or 7 the salts: [(η6-C6H6)RuCl(η2-
-
-
-
dppm)]+ SnCl3 (2), [(η6-p-cymene)RuCl(η2-dppm)]+ SnCl3 (4), [(η6-C6H6)RuCl(η2-dppm)]+ GeCl3
-
-
(5), [(η6-p-cymene)RuCl(η2-dppm)]+ GeCl3 (6), [(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ SnCl3 (8)
-
and [(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ GeCl3 (9). The trichlorostannate complexes 2, 4 and 8
-
are extremely rare examples of ruthenium complexes bearing the SnCl3 counter anion, and the
complexes 5, 6 and 9 are the first examples of ruthenium trichlorogermanate complexes to be
reported. All compounds were isolated in high yields as air stable materials and were
spectroscopically characterized by multinuclear NMR: (1H, 31P{1H}, 13C{1H}), Infra-red (IR), UV-
Vis, and high resolution electrospray ionization mass spectrometry (HR-ESI-MS) , the latter both in
(+) and (-) mode. Additionally, single crystal X-ray diffraction analyses of salts 4 and 6 are
reported, revealing pseudotetrahedral Ru(II) centres with η6 bound p-cymene ligands and η2-bound
-
dppm ligands with statistical disorder on the ECl3 anions (E = Ge (6), Sn (4)). Density functional
theory calculations (B3LYP with the basis set 6-31+G(d,p) for H, C, P and Cl atoms; while for Ru,
1

Ge, and Sn atoms DGDZVP basis set) are reported for salts 4 and 6 revealing localization of the
LUMOs on the ruthenium-arene rings and some localization on the chloride atom. Finally, MTT in
vitro cytotoxicity assays for the MCF-7 and MDA-MB-231 breast cancer cell lines are reported for
all complexes and compared to cisplatin. All complexes show remarkable in vitro cytotoxic activity
and most are considerably more cytotoxic than cisplatin in both breast cancer cell lines: IC50 values
range from 2.25 µM (compound 2) to 5.97 µM (compound 9) (cisplatin = 5.74 µM) in MCF-7 cells;
2.20 µM (compound 2) to 6.39 µM (compound 5) (cisplatin = 13.98 µM) in MDA-MB-231.
Moreover, when compared to non-malignant breast epithelial cells (MCF12A), all complexes
exhibit promising selectivity indices (SI) with compound 5 having the highest SI in MCF-7 cells at
4.8; and compound 6 at 3.65 in MDA-MB-231, with most of the other compounds also being
considerably more selective than cisplatin on both cell-lines (SI = 2.26 on MCF-7 and 0.93 on
MDA-MB-231). A clonogenic assay was conducted for salts 5 and 6 and the results reveal that both
compounds inhibited long-term (14 days) survival in both breast cancer cell lines tested indicating
these drugs are very promising candidates for pre-clinical studies.
Keywords: Ruthenium anti-cancer agents, Ionic complexes, germanate and stannate
complexes.
TOC Graphic
*Corresponding authors email address: burgert.blom@maastrichtuniversity.nl,
sharon.prince@uct.ac.za.
2

Introduction
Cisplatin (Figure 1a) was discovered more than 50 years ago to exhibit antineoplastic properties in
the seminal work of Rosenberg et al.1 and has since been widely used in the treatment of various
types of cancer. Today, its derivatives carboplatin and oxaliplatin (Figure 1b and 1c) are used
routinely in chemotherapy and heptaplatin represents another promising analogue.2
Figure 1: Molecular Structures of Cisplatin (a), Carboplatin (b), Oxaliplatin (c).
Despite showing good anticarcinogenic properties, platinum based antineoplastics present
numerous challenges and deleterious side effects.3 Tumors can also develop resistance4 to the effect
of these drugs making their use in treatment ineffective. Platinum resistance in tumors has
precipitated extensive investigations into the anticancer properties of other metal complexes, most
notably ruthenium, in order to develop better and more efficient anticancer agents.5 Different
clinical and preclinical studies have proven the possibility of developing new antineoplastics based
on metal centres other than platinum. Ruthenium compounds such as KP1019 and NAMI-A (Figure
2a and 2b) have in this regard shown promise. 6,7
Figure 2: Molecular Structure of a) KP1019 and b) NAMI-A Anticancer Agents
3

Another seminal example of an active ruthenium complex is RAPTA-C: [(η6-p-
cymene)RuCl2(PTA)]8 which is synthesized using 1,3,5-triaza-7- phosphaadamantane (PTA) as a
ligand. PTA is responsible for the hydrophilicity of this agent, which is one of the reasons why
RAPTA (Ruthenium Arene PTA) agents show good antiproliferative properties towards cancer
-
cells. Cationic Ru(II) complexes [(η6-p-cymene)RuCl(η2-phosphine)]+ with PF6 counter anions
have also been reported to exhibit high cytotoxicity towards several human cell lines, including
cervical cancer (SIHA), breast cancer (MCF7), prostate cancer (PC3), ovarian cancer (A2780) and
haptocellular carcinoma (HEPG2) with some selectivity.9
One research area has been the development of bi and multimetallic complexes, incorporating
multiple metals into one compound10 as potential anti-cancer agents. Among the aspects of this
work, one was the incorporation of Sn into the coordination sphere of a ruthenium centre, showing
that the presence of a SnCl3 ligand can enhance cytotoxicity.11 From our research, we found that
complexes bearing σ-trichlorogermyl (GeCl3) ligands bound to RAPTA like complexes (complexes
of the general formula [(η6-arene)RuCl(GeCl3)(PR3)] (PR3 = phosphane or phosphite ligand, or
PTA)), significantly retards cytotoxicity. We postulated that this was a result of enhanced aquation
kinetics, potentially rendering the complexes inactive to bind to the necessary bimolecular targets.12
On the other hand, we found that incorporation of a trichlorostannyl (SnCl3) moiety into the
coordination sphere of a RAPTA like complex can enhance cytotoxicity if the complexes are ionic
−
as exemplified by the complex [Ru(η6-C6H6)(PPh3)(SnCl3)(DMAP)]+BF4 which is more cytotoxic
on A2780 and A2780cisR than its analogue [(RuCl(η6-C6H6)(PPh3)(DMAP)]+BF4 which does not
−
contain tin.13 In our studies, broadly speaking, neutral Ru(II) complexes which contain the SnCl3
moiety tend to be less cytotoxic than their non-stannylated counterparts, possibly due to decreased
solubility.14 Based on these studies, we envisaged that the incorporation of SnCl3 or GeCl3 into a
ruthenium complex may enhance cytotoxicity, as long as the solubility is not compromised. Hence
ionic complexes, where the counter anion contains tin or germanium might be beneficial to
enhancing cytotoxicity while not hindering solubility. Continuing in our quest for highly active
metal-based anti-cancer agents, building on lessons from the past, we report herein a series of
readily accessible and air stable trichlorogermanate and trichlorostannate salt-complexes of the type
[(η6-arene)RuCl(η2-dppm)]+[ECl3]- (arene = C6H6, p-cymene or 1,3,5-Me3C6H3; E = Ge, Sn)
representing a new class of anti-cancer agents. These are all shown to be very potent and with
promising selectivity profiles on the MCF-7 and MDA-MB-231 breast cancer cell lines through in
vitro studies.
4

Results and Discussion
We recently employed some known and novel complexes of the type [(η6-arene)RuCl2(η1-dppm)]
as easily accessible starting materials to access heterobimetallic Ru,Fe and homobimetallic Ru,Ru
complexes, by facile coordination of the pendant PPh2 moiety to another metal centre by dimer
cleavage (Scheme 1).15 In this work the known [(η6-arene)RuCl2(η1-dppm)] (arene = C6H6 (1); p-
cymene (3)) and novel [(η6-1,3,5-Me3C6H3)RuCl2(η1-dppm)] (7) were employed as starting
materials.
Scheme 1: Dimer cleavage reaction to obtain ruthenium piano stool complexes bearing η1-bound dppm.
We initially reasoned that reactions of the precursor complexes 1, 3 and 7 with SnCl2 or
GeCl2(dioxane), respectively, would either afford stannyl or germyl complexes of the type [(η6-
arene)RuCl(SnCl3)(η1-dppm)] or [(η6-arene)RuCl(GeCl3)(η1-dppm)], as observed in our earlier
work,13,10 or alternatively, that the pendant :PPh2 moiety would coordinate via the lone pair at the
phosphorus atom to the vacant p-orbital at the Sn(II) or Ge(II) centre affording dinuclear complexes
of the type [(η6-arene)RuCl2(µ-Ph2CH2Ph2P:→)ECl2] (E = Ge, Sn). However in all cases the
reactions of the precursor complexes 1, 3 and 7 with SnCl2 or GeCl2(dioxane) afforded selectively
and in good to high yields the ionic compounds of the type [(η6-arene)RuCl(η2-dppm)]+[ECl3]-
(Scheme 2), by halide abstraction of the Cl atom at the ruthenium centre, promoted by the Lewis
-
acidity of the respective SnCl2 and GeCl2. Arene Ru(II) complexes featuring SnCl3 as counter
-
anions are very rare16,17 and ruthenium complexes featuring a GeCl3 anion to our knowledge are
not known yet, making the trichlorogermanate complexes reported here the first examples.18
Accompanying these transformations is the change in hapticity of the dppm ligand from η1 to η2 at
the Ru(II) centre. We prepared a full series of these complexes (Scheme 2) varying the nature of the
arene ring from benzene, to p-cymene and 1,3,5-Me3C6H3 since the nature of the ring has been
shown to be important in the cellular uptake of cytotoxic agents.19
5

Scheme 2: Reaction of piano stool complexes to obtain ionic stannate or germanate complexes
Table 1 shows the yields and appearances of all isolated salts. The salts were isolated in good to
excellent yields (68 – 93 %), typically as yellow to orange solids and were found in all cases to be
O
thermally robust with melting points typically above 100 C (with the exception of 9 which
decomposes at 90 OC). The complexes are air-stable and for prolonged periods and no
decomposition was observed, despite the propensity of Ge-Cl bonds to undergo hydrolysis.20 This is
akin to our earlier studies of Ru trichlorogermyl complexes, which were also robust in air and
impervious to hydrolysis or oxidation.12
Complex
[(η6-C6H6)RuCl2(η1-dppm)] 1
[(η6-C6H6)RuCl(η2-dppm)]+ SnCl3
Yield (%)
Appearance
Bronze-orange
70
93
60
85
61
73
95
87
68
-
Gold-yellow
Red-orange
Yellow-orange
Yellow
2
[(η6-p-cymene)RuCl2(η1-dppm)] 3
[(η6-p-cymene)RuCl(η2-dppm)]+ SnCl3
4
6
-
[(η6-C6H6)RuCl(η2-dppm)]+ GeCl3
5
-
[(η6-p-cymene)RuCl(η2-dppm)]+ GeCl3
[(η6-1,3,5-Me3C6H3)RuCl2(η1-dppm)] 7
[(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ SnCl3
-
Yellow
Orange-bronze
Shiny orange
Yellow
-
8
9
[(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ GeCl3
-
Table 1: Summary of yields and colours of complexes 1-9.
The trichlorostannate and germanate salt complexes 2, 4, 5, 6, 8, and 9 all feature a singlet
resonance signal in their corresponding 31P{1H} NMR spectra due to the η2 coordination of the
dppm in the cation. This renders both P atoms equivalent due to a mirror plane through the
molecule, in contrast to their precursors 1, 3 and 7 which feature two distinct doublet resonance
signals corresponding to Ru bound (low field shifted) and uncoordinated P atoms (high-field
shifted).
6

1H NMR spectroscopy revealed two sets of resonance signals corresponding to the diastereotopic
set of protons on the bridgehead carbon atom of the η2-coordinated dppm (Ph2PCHAHBPPh2) in all
complexes 2, 4, 5, 6, 8, and 9. For each of the two protons the signals appeared as a doublet of
2
2
triplets corresponding to J(Ha,Hb) and J(H,P) coupling, respectively, and was well resolved. This
suggests conformational locking of the dppm to the metal and fluxional processes can be rules out
on the time-scale of the measurement. Furthermore, the η6 bound arene rings of the complexes
show, in the case of cations bearing C6H6, a sharp resonance at δ = 6.16 ppm, and in the case of the
p-cymene systems a series of doublets in the same region, slightly high field shifted from the free
arene, consistent with coordination to ruthenium. The cations bearing a 1,3,5-Me3C6H3 ligand
exhibit a singlet at δ = 5.79 ppm, corresponding to the three equivalent hydrogen atoms on the arene
ring. 13C{1H} NMR typically revealed a signature triplet resonance signal for the bridgehead carbon
atom of the dppm ligand in accordance with 1J(C,P) coupling, typically at δ = 41.1 – 44.8 ppm for
the compounds. The characteristic resonance signals of the metal bound arene rings were also found
in the typical regions, consistent with coordination (see experimental section).
All complexes were also subjected of High Resolution Mass Spectroscopy (ESI), in both positive
and negative mode, which was highly instructive in unambiguously confirming the constitution of
the salts. The precursor complexes 3 and 7 both show the mass fragments of [M+H]+, while the
stannate and germanate complexes 4, 5, 6, 8, and 9 all show the mass fragments of both [M]+ in the
positive mode and [ECl3]- (E= Sn or Ge) in the negative mode with excellent agreement to the
theoretical isotope patterns and exact masses in all cases (Table 2).
4
5
6
8
9
ESI-MS (theor.) (+)655.1024
(-)224.8088
(+)599.0398
(-)178.8277
(+)655.1024
(-)178.8277
(+)641.0868
(-)224.8088
(+)641.0868
(-)178.8277
ESI-MS (expt.)
(+)655.0990
(-)224.8133
(+)599.0533
(-)178.8294
(+)655.0990
(-)178.8294
(+)641.1112
(-)224.0878
(+)641.1112
(-)178.8245
Table 2: Comparison of ESI-MS of selected complexes. In each case the line of highest intensity in the isotope pattern
is presented.
To gain insight into the redox behavior of the stannate and germanate salt complexes, cyclic
voltammetry of selected complexes 2, 4, 5, and 6 was carried out in acetonitrile. Whereas reversible
metal-centered Ru(II)/Ru(III) oxidations of the stannate salt adducts, 2 and 4 are observed at 0.84 V
and 1.24 V half-wave potentials respectively, the germanate adducts 5 and 6 are quasi-reversible at
7

1.13 V and 1.20 V respectively. In both cases a cathodic shift is the oxidation potential is observed
for the arene complexes 4 and 5. This is indicative of the electron donating capabilities of the arene
ring to the metal center rendering the metal center slightly harder to oxidize. It is also interesting to
note the presence of oxidation peaks at lower potentials attributed to the coordination of the
acetonitrile solvent to the complex. To validate this, similar experiments were carried out in a non-
coordinating solvent, CH2Cl2. No metal-centered oxidation peaks were observed unless a few drops
of CH3CN are added into the solution.
Crystals, suitable for single crystal X-ray diffraction analysis of salts 4 and 6 were obtained by slow
O
evaporation of a dilute solution of the salt in chloroform at 7 C further confirming their structure
which is in agreement with all the spectral data. Both compounds crystallize isotypic in the
-
monoclinic space group Cc and feature the identical cation with statistically disordered anions ECl3
(E = Ge and Sn) in the asymmetric unit (Figure 3). Compound 6 is the first structurally
characterized trichlorogermanate ruthenium complex to date. Both complexes feature fully
separated ion pairs judging from the large distance between Ru and Sn in 4 (6.224 Å) and the Ru-
Ge distance in 6 (6.228 Å).
Figure 3: ORTEP representations of salts 4 (left) and 6 (right) at the 30 % probability level. H atoms omitted for
-
-
clarity. The side occupancies in the disordered SnCl3 in 4 and GeCl3 in 6 have also been removed for clarity. The Ru-
Sn distance in 4 is 6.224 Å and the Ru-Ge distance in 6 is 6.228 Å.
The cation of salt 4 is depicted in Figure 4. The closely related compound [(η6-p-cymene)RuCl (η2-
-
dppm)]+ BF4 has been reported by Dyson and co-workers previously.21
8

Figure 4: ORTEP Representation of the cation in salt 4; H Atoms omitted for clarity, thermal ellipsoids set at the 30 %
probability level. Selected bond lengths [Å] and angles [°] for sample 4: Ru(1)-Cl(1) 2.3893(8), Ru(1)-P(1) 2.3059(9),
Ru(1)-P(2) 2.3257(10), P(1)-C(10) 1.844(3), P(2)-C(10) 1.847(3), P(1)-Ru(1)-P(2) 71.07(3), P(1)-C(10)-P(2) 93.68(15),
Sn(1)-Cl(2) 2.484(9), Sn(1)-Cl(3) 2.497(9), Sn(1)-Cl(6) 2.427(2), Cl(6)-Sn(1)-Cl(2) 95.85(17), Cl(6)-Sn(1)-Cl(3)
94.1(2), Cl(2)-Sn(1)-Cl(3) 95.0(3).
The solid-state structure of the two salts (4 vs 6) are very similar when looking at the metrical
parameters of the identical cations in both structures, and slight differences in the values may be
attributed to solid state packing effects of the anions GeCl3 vs SnCl3. For example, the bite angle of
the coordinated dppm P(2)-Ru(1)-P(1) in the cation of 4 is 71.03(3) and the one of 6 is 71.43(6).
Most of the other metrical parameters in the cations were found to be very similar, as expected,
within narrow limits across both structures. The solid-state structure of the cation in both structures
reveals η6 coordination of the arene moiety and a distorted tetrahedral geometry at the ruthenium
-
centre. The Sn-Cl bond lengths of SnCl3 had an average value of 2.469 Å, while the average Ge-Cl
-
bond in GeCl3 is 2.291 Å, in 4 and 6 respectively in accordance with the increased single bond
covalent radius of Sn vs Ge.22 The anions, despite being disordered reveal a trigonal pyramidal
structure in both cases, as supported by our density functional theory (DFT) calculations
investigations (see below).
Short-term and Long-term Cytotoxicity against Breast Cancer Cells
To investigate the anticancer activity of the germanate and stannate compounds (compounds 2, 4, 5,
6, 7, 8 and 9) in breast cancer cells (MCF-7 and MDA-MB-231), the 3-(4,5 dimethylthiazol-2-yl)-
2,5- diphenyltrazolium bromide (MTT) assay was performed and cisplatin was included as a
control. Furthermore, to determine the selectivity of the compounds for the breast cancer cells,
normal breast epithelial (MCF-12A) cells were included in these assays. 23,24 Figure 5 and Table 2
9

(summary of the IC50 concentrations and selectivity indices (SI)) show that all the compounds tested
severely inhibited breast cancer cell viability at concentrations lower than (compounds 2, 4-8), or
comparable to (compound 9), cisplatin. While all the compounds showed selectivity for the breast
cancer cells, compound 5 was the most selective for MCF-7 cells (SI = 4.804) and compound 6 was
the most selective for MDA-MB-231 cells (SI = 3.651). Cisplatin showed no selectivity for the
-
-
MDA-MB-231 cells (SI = 0.93). It is instructive to note that the GeCl3 and SnCl3 counter anions
play a distinct role in the overall cytotoxicity profiles of the compounds and somewhat large
variations in the in vitro activity is noted across the data series comparing the stannate and
germanate analogues to each other. For example, compound 2 and its germanium analogue, 5 show
noticeable differences in cytotoxic behavior on MCF-7 and MDA-MB-231 and differences in
selectivity (see Table 3). In addition, the neutral compound 7 exhibits a different profile across all
-
-
cell-lines, compared to its GeCl3 and SnCl3 salts, again indicating that the presence of the stannate
and germanate anions can tune the activity and selectivity and are not innocent in terms of
cytotoxicity.
Cell lines
Compounds
MCF 7
MDA-MB-231
IC50
MCF 12 A
IC50
IC50
2.25
3.86
2.7
SI
SI
2.97
2.02
4.80
3.45
3.34
3.41
1.89
2.26
2.2
2.73
6.39
3.56
5.51
4.44
4.25
13.98
3.04
2.85
2.03
3.65
1.94
2.54
2.63
0.93
6.7
2
7.78
4
12.98
13
5
3.76
3.2
6
10.71
11.31
11.2
7
3.31
5.95
5.74
8
9
12.98
Cisplatin
Table 3: IC50 values and selectivity indices (SI) of the tested compounds and cisplatin.
To determine the ability of compounds 5 and 6 to inhibit long-term survival of the breast cancer
cells, clonogenic assays were performed over a 14 day period. These compounds were chosen based
on their SI values which indicated that they were the most promising of the compounds tested.
Figure 6 indicates that compounds 5 and 6 were able to inhibit the colony forming ability of both
breast cancer cell lines in a dose-dependent manner. Together the results from the biological studies
strongly suggest that compounds 5 and 6 are worth taking forward to pre-clinical studies.
10

Figure 5: Compounds (2, 4-9) show potent and selective inhibition of breast cancer cells. (a-g) MTT cell viability
assays of MCF-7 and MDA-MB-231 breast cancer lines as well as non-tumorigenic breast epithelial cells (MCF-12A)
treated with 2 - 10 µM of compounds or vehicle for 48 h. The graph shows mean cell viability as a percentage of
vehicle control ± SEM for each concentration of the compounds tested from at least three independent experiments each
performed in quadruplicate. The concentrations of drug required to kill 50% of the cells (IC50) in each cell line were
calculated by fitting a curve in Graph Pad Prism. (i) Selectivity indices (SIs) were determined for each breast cancer cell
line by dividing the IC50 of the non-tumorigenic cell line by the IC50 of each breast cancer cell line.
11

Figure 6: In order to determine the long-term cytotoxic effects of compound 5 and 6 on MCF-7 and MDA-MB-231
breast cancer cells, clonogenic assays were carried out. Each breast cancer cell line was treated with the vehicle or ¼
IC50, ½ IC50, IC50 and 2 IC50 concentrations of compounds 5 or 6 for 24 h after which the cells were re-plated at low
densities (1000 cells) in drug-free medium and allowed to grow and form colonies for 14 days. The graph represents the
mean colony area ± SEM of each treatment as a percentage of the vehicle control for experiments done in triplicate.
In order to gain an insight on the electronic and geometrical features of the salt complexes 4 and 6,
DFT calculations were performed (level of theory used for all calculations is B3LYP with the basis
set 6-31+G(d,p) for H, C, P and Cl atoms; while for Ru and Ge, Sn atoms the DGDZVP basis set
was used). Structural features of the optimized geometries were compared with the experimental
data available from the single crystal X-ray diffraction analyses (see above). In each case three
geometry optimizations were performed: the cations were optimized separately from the anions
(ECl3-), and finally the optimized cations and anions were merged together and optimized and
compared to the X-ray structures of the salts 4 and 6 in the solid state (Table 4).
12

Complex
Bond
Exp.
(Å)
Calc.
(Å)
Diff.
(%)
Ru-Sn
Ru-Ge
6.114
6.056
6.718
6.578
9.4
8.3
4
6
Angle
Exp.
(Å)
Calc.
Diff.
(%)
< 1
< 1
(Å)
71.32
71.31
P-Ru-P
P-Ru-P
71.03
71.43
4
6
Table 4: Structural comparison between predicted and measured geometries of complexes 4 and 6.
The deviation between the experimental and calculated (most important) bond distances and angles
for 4 and 6 is within a maximum of 8 % for the bond length and < 1 % for the angles indicating a
good quality of the optimized structures achieved with the methodology used (DFT, B3LYP and the
two basis sets used).
The electronic nature of complexes 4 and 6 were, in addition calculated to gain possible insights
into the mechanism of action of the complexes. In particular, Figure 7 summarizes the LUMO and
HOMO, with relative energies, of the salt compound 6 (cation, salt complex and anion). The
analogous orbitals for complex 4 can be found in the supporting information (Figure S35).
13

Figure 7: LUMO (top) and HOMO (bottom) with corresponding energy values (in hartree) for the cation of salt
compound 6 (left), compound 6 (middle) and anion of compound 6 (right). (See SI for the analogues 4).
From Figure 7 it is possible to visualize the location of the HOMO and LUMO of salt 6 and the
corresponding cation and anion separately. The salt most likely undergoes dissociation in vitro and
hence the HOMO and LUMOs of the separated ions (left, Figure 7, and right Figure 7) are most
instructive. It is very unlikely that the anion and cation stay closely associated with each other in
vitro (centre, Figure 7). Both the HOMO and LUMOs are delocalized on the η6-bound arene, Ru
centre and Cl atom in the cation of complex 6, in the separated cation. Both the HOMO and LUMO
are extensively delocalized over the entire GeCl3- unit in the separated anion. In the compound
(anion and cation together), the HOMO is localized on the GeCl3- moiety and the LUMO on the
Ru- η6-bound arene-Cl (centre, Figure 6). The latter is not instructive due to dissociation of the salt
in vitro. The locality of the frontier orbitals in the separated cation of 6 are consistent with previous
studies on the cytotoxic action of Ru-based complexes which suggest that the arene ligand provides
a hydrophobic face to facilitate uptake across the cell membranes. 25 After crossing the cell
membrane and prior to binding the DNA, the Ru-complex is proposed to undergo hydrolysis of the
Ru-Cl bond in the cell media facilitating binding to DNA.26 This would be consistent with our DFT
results which show that both HOMO and LUMO are localized to an extent over the Ru-Cl atom,
suggesting a similar mechanism of action might be possible. Such a ligand exchange mechanism is
also observed for the aqueous intracellular activation of cisplatin.27 Hence, the calculated LUMOs
14

of complexes 4 and 6 delocalized on the arene-Ru-Cl are in agreement with the interaction between
the arene and the cell membrane and a nucleophilic substitution of water with the chlorine in the
Ru-Cl bond.
Experimental
General Section
Standard Schlenk and glove-box techniques were employed, and solvents were dried by passage
over a column of alumina under nitrogen. Chemicals and starting materials were obtained from
commercial sources and used as received. Bis(diphenylphosphino)methane (97%), benzene-
ruthenium(II)chloride
dimer,
dichloro(p-cymene)ruthenium(II)dimer,
dichloro(1,3,5-
trimethylbenzene)ruthenium(II)dimer and silica gel (technical grade, 60 Å pore size, 70-230 mesh,
63-200 µm) were purchased from Sigma-Aldrich. AR grade diethyl ether (stab. BHT) was
purchased from Biosolve Chimie SARL as well as dichloromethane (stab. Amylene). Neutral TLC
Silica 60 F254 sheets were used for thin layer chromatography and the aforementioned silica gel was
used for column chromatography. A SMP10 apparatus (Stuart equipment) was used to measure
melting points and those reported are uncorrected. The melting points were carried out in triplicate.
The rotary evaporator consisted of 3 parts: the pump was a SC920 model (KNF lab), the rotational
part was the RV10 digital (VWR by IKA®), and the water bath was the model HB10 digital (VWR
by IKA®). A RCT basic heating plate (IKA®) and a laboport vacuum pump (KNF lab) were used.
A 300 MHz/54 mm Ultrashield™ Magnet System (Bruker) was used for NMR experiments at
ambient temperature. Chemical shifts for 1H and 13C NMR were measured relative to
tetramethylsilane (TMS), which was in the solvent. For 31P NMR, the chemical shifts were
measured relative to phosphoric acid (85%), and the software for data workup was Topspin
3.5pl7. A MIRacle 10 (single reflection ATR accessory, Shimadzu) was used to acquire FTIR
spectra (Happ-Genzel apodisation, 128 scans, resolution 2) with the software IR solution. A UV-
188 spectrophotometer (Shimadzu), and quartz suprasil precision cells (type 100-QS, 10 mm light
path; Hellma Analytics) were used to obtain UV-Vis spectra. 30 µL of a 1mM solution containing
the sample were dissolved in 2.5mL of C2H3N for UV-vis spectra and the maximum absorption
peak is quoted. Electrochemical measurements were carried out on a BASi® Epsilon Eclipse
potentiostat/galvanostat. All experiments were carried out at room temperature under an inert
atmosphere (Ar). A three-electrode system was used on a C-3 cell stand equipped with glassy
carbon working electrode, Pt wire auxiliary electrode and Ag/Ag+ (0.1 M AgNO3) reference
15

electrodes. All Experiments were carried out in CH3CN containing either 0.10 M 0.20 M
[NBu4][PF6] as a supporting electrolyte. A Ferrocene/Ferrocenium internal standard was used as an
internal standard with a Fe(II)/Fe(III) oxidation potential of 0.042 V vs Ag/Ag+ in CH3CN. The ESI
(+) parameters for High Resolution Mass Spectroscopy were capillary: 3.5kV, sampling cone: 50,
extraction cone: 2, source temp 100, dissolvation temp: 200, Flow rate: 5ul/min; while for ESI (-):
capillary: 1.5kV, sampling cone: 40, extraction cone: 5, source temp 100, dissolvation temp: 200,
Flow rate: 10ul/min. Only the peaks corresponding to the cation in ESI(+) and anion in ESI(-) of the
salt-compounds are reported and for neutral compounds the [M+H]+ and [M+Na]+ signals are
reported. These were carefully checked and compared against theoretical isotope patterns and the
line of highest intensity in the isotope pattern is reported. Samples were sent to UCT Medical
School, South Africa for in vitro anticarcinogenic testing, to TU Graz, Austria for crystallography
and to Maastricht Randwyck for High Resolution Mass Spectroscopy.
X-ray structure determination
For X-ray structure analysis of complexes 4 and 6 the crystals were mounted onto the tip of glass
fibers, and data collection was performed with a BRUKER-AXS SMART APEX CCD
diffractometer using graphite-monochromated Mo Kα radiation (0.71073 Å). The data were
reduced to F2 and corrected for absorption effects with SAINT28 and SADABS 29,30 respectively.
0
Structures were solved by direct methods and refined by full-matrix least-squares method
31
(SHELXL97 and SHELX2013).
All non-hydrogen atoms were refined with anisotropic
displacement parameters. Hydrogen atoms were placed in calculated positions to correspond to
standard bond lengths and angles.
Cell Culture and treatments
The human breast adenocarcinoma cells MDA-MB-231 (triple negative) and MCF-7 (oestrogen
receptor positive) were maintained in DMEM (Highveld Biologicals, Lyndhurst, United Kingdom
(UK) and RPMI 1640 (Highveld Biologicals, Lyndhurst, United Kingdom (UK) respectively. The
non-tumorigenic breast epithelial MCF 12A cells were maintained in a 1:1 mixture of DMEM and
Ham’s F 12 (Highveld Biologicals, Lyndhurst, United Kingdom (UK), supplemented with 20
ng/mL of human epidermal growth factor, 100 ng/mL of cholera toxin, 0.01 mg/mL of bovine
insulin and 500 ng/mL of hydrocortisone. When treated with the test compounds, the MCF-12A
16

cells were cultured in non-supplemented medium. Cells were maintained at 37 ºC in a 5% CO2–
95% air-humidified incubator.23,24
Cell Treatments
The compounds 2, 4, 5, 6, 7, 8 and 9 were dissolved in dimethyl sulfoxide (DMSO) (Merck
48856212719) to achieve a final stock concentration of 5 mM and heated at 100° C for 2 min and
stored at room temperature for a maximum of 5 days before use. The 5 mM stock solutions were
further diluted using cell culture medium to attain final experimental concentrations (2, 4, 6, 8 and
10 µM) and a vehicle control (DMSO) of the same concentration was prepared simultaneously.
Cisplatin was used as the positive control at its reported IC50 values for MCF-7, MDA-MB-231 and
MCF-12A cells. 23, 24
Cytotoxicity Assay
The MCF-7 and MDA-MB-231 breast cancer cells were plated in 96 well plates and treated 48 h
and 24 h respectively with a range of concentrations (2, 4, 6, 8 and 10 µM) of the compounds or
vehicle (DMSO) at a concentration of 10 µM for a period of 48 h. Cytotoxicity of these compounds
were evaluated using the 3-(4,5 dimethylthiazol-2-yl)-2,5- diphenyltrazolium bromide (MTT) assay
(M21281G, Sigma-Aldrich) according to the manufacturer’s instructions. The mean cell viability
was calculated as a percentage of the mean of the vehicle control. At least three independent
experiments in quadruplicate were performed from which the half-maximal inhibitory concentration
(IC50) was determined using graph prism version 6.0. The selectivity index (SI) was calculated
using the following formula (IC50 of normal breast epithelial cells) / (IC50 of tumorigenic cell
line).23, 24
Clonogenic Assay
Cells were seeded in 6-well plates and treated the following with ¼ IC50, ½ IC50, IC50 and 2 IC50 of
compounds 5 and 6 and vehicle. After 24 h, 1000 cells were seeded in 35 mm dishes in drug free
medium. The formation of colonies was monitored and after 14 days the surviving cells were fixed
and stained with crystal violet (Sigma-Aldrich, USA). Colonies were imaged and the percentage
17

colony area was determined using Image J v 1.50i software. Colony area was determined for each
concentration of compound and expressed as a percentage of vehicle-treated control.23, 24
Density Functional Theory Calculations
DFT calculations were performed to model the complexes 4 and 6. Gaussian09 software package
was used.32 For all the calculations, the level of theory used is B3LYP with the basis set 6-
31+G(d,p) for H, C, P and Cl atoms; while for Ru, Ge, and Sn atoms DGDZVP basis set was used.
Geometry optimizations were calculated without any constrains. All the optimized geometries show
no imaginary frequencies. Energies, bonds and angles were calculated on the optimized structures.
Atom coordinates for the optimized structures of complexes 4 and 6 are available in the supporting
info.
Synthesis and characterization of complexes
-
Synthesis of [(η6-C6H6)RuCl(η2-bis(diphenylphoshino)methane)]+ SnCl3 2
0.100g (1.0 eq, 0.16 mmol) of the starting material 1 were weighed into a 100mL round bottom
flask together with 0.036g (1.2 eq, 0.19 mmol) of ground SnCl2. The mixture was stirred at room
temperature in 20mL of dichloromethane until complete dissolution. The resulting bronze-red
solution was then placed under reflux at 60°C for 3 hours. The solution’s color changed to a murky
yellow suspension as the reaction proceeded. Once the reaction was finished, the excess tin was
filtered out by vacuum filtration, the filtrate was collected and the solvent was completely
o
evaporated. Golden crystals formed in the flask and were washed (3 x 10mL) with cold ) (0 C)
diethyl ether. Properties: sparingly soluble in CDCl3 and DMSO, air stable. Melting point: 190°C +
dec. Yield: 0.122g (93 %). 1HNMR (300 MHz, CDCl3, 299K, ppm): δ= 7.73-7.28 (m, 20H, dppm),
2
2
6.17 (s, 6H, C6H6), 4.83 (dt, J(H,H)= 15.0Hz, 2J(H,P)= 10.5Hz, 1H, PCHA), 4.46 (dt, J(H,H)=
2
27.6Hz, J(H,P)= 13.4Hz, 1H, PCHB). 31P{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 2.5 (s).
1
13C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.4 (pst, J(C,P)= 5.2Hz), 132.2 (s, 4C, C1-
dppm or C4-dppm), 131.8 (pst, J= 5.2Hz), 131.6 (s, 4C, C1-dppm or C4-dppm), 129.9 (pst, J=
2
1
5.2Hz), 129.1 (pst, J= 6.0Hz), 93.0 (t, J(C,P)= 2.5Hz, 6C, C6H6), 41.1 (t, J(C,P)= 26.8Hz, 1C,
P2CH2(Ph)4). FTIR v(cm-1): 3080 (vw), 1571 (w), 1541 (vw), 1481 (m), 1433 (m), 1309 (w), 1263
(w), 1186 (w), 1157 (w), 1095 (m), 1024 (w), 999 (m), 812 (m), 775 (w), 729 (s), 688 (s), 607 (w)
534 (m). UV-vis (C2H3N, 299K, nm): 385.0. Too insoluble for ESI-MS.
18

-
Synthesis of [(η6-p-cymene)RuCl(η2-bis(diphenylphosphino)methane)]+ SnCl3 4
0.100g (1 eq, 0.14 mmol) of 3 was weighed out and placed in a 100mL round bottom flask together
with 0.032g (1.2 eq, 0.17mmol) of ground SnCl2. The two complexes were dissolved in 20mL of
dichloromethane and stirred until dissolution at room temperature. The pale orange solution was put
under reflux at 60°C for 3 hours, the color of the solution gradually changed to dark yellow-orange
during this process. The remaining tin (white deposit in the reaction solution) was filtered out by
Buchner vacuum filtration, and the filtrate was collected in a different flask. Subsequently the
solvent was removed in vacuo, which left a yellow-orange residue in the RBF. This material was
washed (3 x 10mL) with cold Et2O, yielding pale yellow-orange crystals. Properties: Sparingly
soluble in CDCl3 and DMSO. Air stable. Melting point: 210°C + dec. Yield: 0.105g (85 %).
3
1HNMR: (300 MHz, CDCl3, 299K, ppm): δ= 7.70-7.33 (m, 20H, dppm), 5.92 (d, J(H,H)= 6.5Hz,
3
2
2
2H, C6H4), 5.71 (d, J(H,H)= 6.2HZ, 2H, C6H4), 4.80 (dt, J(H,H)= 14.8Hz, J(H,P)= 10.3Hz, 1H,
2
2
3
PCHA), 4.56 (dt, J(H,H)= 14.8Hz, J(H,P)= 12,5Hz, 1H, PCHB), 2.54 (sept, J(H,H)= 6.9Hz, 1H,
3
CH(CH3)2), 1.94 (s, 3H, CH3), 1.10 (d, J(H,H)= 6.9Hz, 6H, CH(CH3)2). 31P{1H}NMR: (300MHz,
CDCl3, 299K, ppm): δ= 2.6 (s). 13C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.4 (pst, J=
5.2Hz), 132.2 (pst, J= 5.2Hz), 132.0 (s, 4C, C1-dppm or C4-dppm), 131.4 (s, 4C, C1-dppm or C4-
2
dppm), 129.7 (pst, J= 5.4Hz), 128.8 (pst, J= 5.6Hz), 119.8 (t, J(C,P)= 2.9Hz, 1C, C1 or C4, η6-p-
2
cymene), 102.1 (t, C4 or C1, η6-p-cymene), 91.8 (t, J(C,P)= 2.2Hz, 2C, C2,6 or C3,5, η6-p-cymene)
2
1
91.0 (t, J(C,P)= 3.7Hz, 2C, C3,5 or C2,6, η6-p-cymene), 41.4 (t, J(C,P)= 1.3Hz, 1C, P2CH2(Ph)4),
30.1 (s, 1C, CH(CH3)2), 20.9 (s, 2C, CH(CH3)2), 16.6 (s, 1C, CH3). FTIR: v(cm-1)= 3053 (w), 2966
(w), 2929 (w), 2870 (w), 1886 (vw), 1573 (w), 1541 (w), 1502 (w), 1481 (m), 1471 (m), 1435 (s),
1384 (m), 1346 (w), 1328 (w), 1307 (m), 1276 (vw), 1261 (w), 1215 (vw), 1186 (w), 1157 (w),
1097 (m), 1056 (w), 1026 (w), 999(m), 864 (m), 800 (w), 742 (s), 727 (s), 713 (s), 702 (s), 690 (s)
634 (vw). UV-vis (C2H3N, 299K, nm): 224.0. ESI-MS (+) 655.1024 (theor.), 655.0990 (expt.)
[C35H36ClP2Ru]+ .ESI-MS (-) 224.8088 (theor.), 224.8133 (expt.) [SnCl3]-.
-
Synthesis of [(η6-C6H6)RuCl(η2-bis(diphenylphosphino)methane)]+ GeCl3 5
0.00470g (1 eq, 0.74mmol) of 1 were weighed out and held in a sample vial. In the meantime,
dichloromethane was being dried inside an alumina column, with N2 flow to avoid contact with air
moisture. 0.0180g (1.05 eq, 0.78mmol) of GeCl2(dioxane) were carefully weighed out inside a pre-
weighed sample vial, inside the glove box. The two complexes were then quickly transferred to a
19

100mL two-necked round bottom flask, filled with 20mL of the dry solvent under nitrogen. The
reaction solution was refluxed for 1 hour at 60°C. It could be clearly seen how the reaction’s color
changed from orange to bright yellow as the reaction proceeded. Once the refluxing step was
completed, the solvent was evaporated at reduced pressure, yielding yellow crystals. These were
then washed (3x 10mL) with cold diethyl ether and the washings were discarded. The resulting
product were bright yellow crystals. Properties: Soluble in CDCl3 and DMSO. Air stable. Melting
1
point: 190°C+ dec. Yield: 0.0350g (61 %). HNMR (300MHz, CDCl3, 299K, ppm): δ= 7.72-7.29
(m, 20H, dppm), 6.16 (s, 6H, C6H6), 4.81 (dt, 2J(H,H)= 14.8Hz, 2J(H,P)= 10.4Hz, 1H, PCHA), 4.45
2
2
31
(dt, J(H,H)= 14.8Hz, J(H,P)= 12.7Hz, 1H, PCHB). P{1H}NMR (300MHz, CDCl3, 299K, ppm):
δ= 2.3 (s). 13C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.4 (pst, J= 5.2Hz), 132.2 (s, 4C, C1-
dppm or C4-dppm ), 131.7 (pst, J= 5.4Hz), 131.6 (s, 4C, C1-dppm or C4-dppm), 129.9 (pst, J=
2
1
5.6Hz), 129.1 (pst, J = 5.6Hz), 92.9 (t, J(C,P)= 11.0Hz, 6C, C6H6), 41.0 (t, J(C,P)= 27.0Hz, 1C,
P2CH2(Ph)4). FTIR v(cm-1): 3064 (w), 2960 (w), 2926 (w), 2856 (w), 1720 (m), 1571 (vw), 1481
(w), 1435 (m), 1408 (w), 1381 (w), 1307 (w), 1263 (m), 1186 (w), 1099 (ms), 1018 (m), 999 (m),
887 (vw), 871 (w), 808 (m), 729 (s), 715 (ms), 688 (s), 638 (w), 611 (w). UV-vis (C2H3N, 299K,
nm): 228.0. ESI-MS (+) 599.0398 (theor.), 599.0533 (expt.) [C31H28ClP2Ru]+.ESI-MS (-) 178.8277
(theor.), 178.8294 (expt.) [GeCl3]-.
Synthesis of [(η6-p-cymene)RuCl(η2-bis(diphenylphosphino)methane)]+ GeCl3 6
-
0.186g (1 eq, 0.27mmol) of starting material 3 was weighed and transferred to a two-necked round
bottom flask, under nitrogen. In the meantime dichloromethane was dried through an alumina
column and purged under nitrogen gas to eliminate all traces of oxygen and moisture. 0.0750g (1.2
eq, 0.32mmol) of germanium chloride dioxane adduct were carefully placed in a pre-weighed vial
in the glovebox. The germanium chloride was quickly added to the orange ruthenium solution, and
the reaction was left to reflux under nitrogen. After one and a half hours the solution color changed
from bright orange to bright yellow and the reaction solution was removed from the reflux. The
yellow solvent was evaporated in vacuo, leaving yellow crystals inside the reaction vessel. They
were washed (3x 10mL) first with toluene and then (3x 10mL) with cold diethyl ether to further
purify the end product. Yield: 0.165g (73%). Properties: Sparingly soluble in CDCl3 and soluble in
1
DMSO. Air stable. Melting point: 170°C + dec. HNMR (300MHz, CDCl3, 299K, ppm): δ= 7.68-
7.31 (m, 20H, dppm), 6.23 (m, 4H, C6H4), 4.82 (dt, 2J(H,H)= 14.7Hz, 2J(H,P)= 10.2Hz, 1H, PCHA),
2
2
3
4.56 (dt, J(H,H)= 14.7Hz, J(H,P)= 12.7Hz, 1H, PCHB), 2.53 (sept, J(H,H)= 7.0Hz, 1H,
20

3
31
CH(CH3)2), 1.56 (s, 3H, CH3), 1.09 (d, J(H,H)= 7.0Hz, 6H, CH(CH3)2). P{1H}NMR (300MHz,
CDCl3, 299K, ppm): δ= 2.5 (s). 13C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.4 (pst, J=
4.9Hz), 132.1 (pst, J= 5.3Hz), 131.4 (s), 129.7 (pst, J= 5.3Hz), 128.8 (pst, J= 5.6Hz), 102.1 (d,
2
2J(C,P)= 2.6Hz, 1C, C1 or C4, η6-p-cymene), 101.0 (d, J(C,P)= 2.6Hz, 1C, C4 or C1, η6-p-cymene),
91.8 (t, 2J(C,P)= 1.9Hz, 2C, C2,6 or C3,5, η6-p-cymene), 90.9 (t, 2J(C,P)= 3.9Hz, 2C, C3,5 or C2,6, η6-
1
p-cymene), 41.5 (t, J(C,P)= 1.86Hz, 1C, P2CH2(Ph)4), 31.2 (s, 1C, CH(CH3)2), 22.6 (s, 2C,
CH(CH3)2), 22.0 (s, 1C, CH3). FTIR v(cm-1): 3064 (w), 2966 (w), 2929 (w), 2872 (w), 1541 (w),
1483 (w), 1471 (mw), 1435 (m), 1381 (mw), 1327 (w), 1307 (w), 1186 (w), 1157 (w), 1099 (m),
1056 (mw), 1026 (mw), 999 (mw), 866 (w), 773 (w), 742 (ms), 727 (s), 713 (m), 702 (ms), 692 (s),
669 (m). UV-vis (C2H3N, 299K, nm): 222.0. ESI-MS (+) 655.1024 (theor.), 655.0990 (expt.)
[C35H36ClP2Ru]+. ESI-MS (-) 178.8277 (theor.), 178.8294 (expt.) [GeCl3]-.
Synthesis of [(η6-1,3,5-Me3C6H3)RuCl2(η1-bis(diphenylphosphino)methane)] 7
0.150g (1 eq, 0.26mmol) of the starting material [(η6-1,3,5-Me3C6H3)RuCl2]2 was weighed out,
together with 0.196g (2 eq, 0.51mmol) of bis(diphenylphosphino)methane; the two were quickly
dissolved into the dry dichloromethane, under a positive N2 pressure. The solution turned murky
orange as the compounds were dissolved. The solution was left to reflux for 3 hours at 60°C. The
color of the solution changed throughout the reaction, it went from murky orange to cabernet red,
and it eventually became red-orange. Once the reflux was done, the solvents were removed in
vacuo. The deposit that was left in the reaction vessel was washed (3x 10mL) with cold diethyl
ether and scratched in order to obtain crystals. Shiny orange-bronze crystals were obtained from this
reaction with a yield of 0.328g (95%). Properties: Soluble in CDCl3. Air stable. Melting point:
175°C + dec. 1HNMR (300MHz, CDCl3, 299K, ppm): δ= 7.33-6.91 (m, 20H, P2CH2(Ph)4), 4.64 (s,
2
3H, C6H3), 3.43 (d, J(H,H)= 8.1Hz, 2H, P2CH2(Ph)4), 1.75 (s, 9H, (CH3)3). 31P{1H}NMR
2
2
(300MHz, CDCl3, 299K, ppm): δ= 27.4 (d, J(P,P)= 29.8Hz, Ru-P), -27.2 (d, J(P,P)= 29.9Hz,
13
1
3
PPh2). C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 137.3 (dd, J(C1,PA)= 14.0Hz, J(C1,PB)=
5.5Hz, 1C, PAC1-dppm), 132.4 (dd, 1J(C1,PB)= 8.6Hz, 3J(C1,PA)= 2.6Hz, 1C, PBC1-dppm), 131.7 (s,
2
1C, PAC4-dppm), 131.4 (s, 1C, PBC4-dppm), 130.7 (d, J(P,C)= 2.8Hz, 4C, PAC2,6-dppm), 130.1 (d,
2
2J(P,C)= 2.8Hz, 4C, PBC2,6-dppm), 127.1-126.8 (m, PA+BC3,5-dppm), 100.0 (d, J(C,P)= 2.9Hz, 3C,
2
1
C6H3), 86.0 (d, J(C,P)= 3.4Hz, (CCH3)3), 22.0 (dt, J(C,P)= 28.3Hz, 1C, P2CH(Ph)4), 17.3 (s, 3C,
(CCH3)3). FTIR v(cm-1): 3055 (w), 2964 (w), 2036 (w), 1965 (m), 1541 (w), 1481 (mw), 1433 (m),
1386 (w), 1261 (mw), 1159 (w), 1097 (m), 1029 (mw), 999 (w), 798 (w), 731 (ms), 704 (ms), 690
21

(s). UV-vis (C2H3N, 299K, nm): 248.0. ESI-MS (+) 677.0629 (theor.), 677.1344 (expt.)
[C34H34Cl2P2Ru+H]+, 699.0454 (theor.), 699.1158 (expt.) [C34H34Cl2P2Ru+Na]+.
-
Synthesis of [(η6-1,3,5-Me3C6H3)RuCl(η2-bis(diphenylphosphino)methane)]+ SnCl3 8
0.120g (1 eq, 0.18mmol) of starting material 7 were weighed out and placed in a round bottom
flask, together with 0.040g (1.2 eq, 0.21mmol) of ground tin chloride. The two reagents were
dissolved in 20mL of dichloromethane. The solution appeared red-orange in color. The solution
mixture was left to reflux at 60°C for 3 hours. The solution color quickly changed to a light and
bright yellow-orange. After 3 hours the solvent was evaporated out and an orange oily deposit
formed inside the vessel. This deposit was washed (3x 10mL) with cold diethyl ether and the
resulting product were orange shiny crystals, which were collected. Properties: Very soluble in
1
CDCl3. Air stable. Melting point: 107°C + dec. Yield: 0.136g (87%). HNMR (300MHz, CDCl3,
2
299K, ppm): δ= 7.65-7.29 (m, 20H, dppm), 5.79 (s, 3H, C3H6), 4.69 (dt, J(H,H)= 28.2Hz, 1H,
2
P2CH2(Ph)4), 4.55 (dt, J(H,P)= 13.6Hz) 2.13 (s, 9H, (CH3)3). 31P{1H}NMR (300MHz, CDCl3,
13
299K, ppm): δ= 3.58 (s). C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.7 (pst, J= 5.1Hz),
132.4 (pst, J= 5.1Hz), 132.1 (s, 2C, PAC1-dppm or PAC4-dppm), 131.4 (s, 2C, PBC1-dppm or PBC4-
2
dppm), 129.5 (pst, J=5.2Hz), 128.8 (pst, J= 5.6Hz), 110.6 (t, J(C,P)= 2.2Hz, 3C, C6H3), 90.1 (t,
1
3J(C,P)= 2.6Hz, 3C, (CCH3)3), 44.8 (t, J(C,P)= 26.7Hz, 1C, P2CH2(Ph)4), 19.6 (s, 3C, (CCH3)3).
FTIR v(cm-1): 3051 (w), 2983 (w), 2922 (w), 1585 (w), 1571 (w), 1533 (mw), 1481 (mw), 1450
(mw), 1435 (m), 1377 (mw), 1350 (w), 1300 (w), 1263 (w), 1186 (w), 1159 (w), 1095 (m), 1029
(m), 999 (mw), 916 (w), 893 (w), 844 (w), 775 (w), 738 (ms), 727 (s), 713 (ms), 690 (s), 617 (w),
534 (m). UV-vis (C2H3N, 299K, nm): 224.0. ESI-MS (+) 641.0868 (theor.), 641.1112 (expt.)
[C34H34ClP2Ru]+. ESI-MS (-) 224.8088 (theor.), 224.0878 (expt.) [SnCl3]-.
-
Synthesis of [(η6-1,3,5-Me3C6H3)RuCl(η2-bis(diphenylphosphino)methane)]+ GeCl3 9
The reaction was performed using dichloromethane dried inside an alumina column and purged
with N2. 0.0410g (1.2 eq, 0.18mmol) of GeCl2(dioxane) adduct were carefully weighed inside a
glovebox and sealed. 0.100g (1 eq, 0.15mmol) of the starting material 7 was weighed and quickly
dissolved in the dry DCM. The GeCl2 was also added quickly to the red-orange stirring solution
under nitrogen. As the reaction went to completion, the solution color changed to a bright yellow-
orange. The solvent was pumped out in vacuo and an orange deposit formed inside the reaction
vessel. This deposit was first washed (3x 10mL) of toluene, and subsequently (3x 10mL) with cold
diethyl ether. This process yielded yellow crystals. Properties: Not very soluble in CDCl3. Air
22

stable. Melting point: 90°C + dec. Yield: 0.100g (68%). 1HNMR (300MHz, CDCl3, 299K, ppm): δ=
2
7.37-7.21 (m, 20H, dppm), 5.80 (s, 3H, C6H3), 4.65 (dt, J(H,H)= 10.1Hz, 1H, PCHA), 4.60 (dt,
2J(H,P)= 11.1Hz, 1H, PCHB), 2.14 (s, 9H, (CH3)3).31P{1H}NMR (300MHz, CDCl3, 299K, ppm): δ=
3.6 (s). 13C{1H}NMR (300MHz, CDCl3, 299K, ppm): δ= 132.8 (pst, J= 4.9Hz), 132.4 (pst, J=
4.9Hz), 132.2 (s, 4C, C1-dppm or C4-dppm), 131.5 (s, 4C, C4-dppm or C1-dppm), 129.6 (pst, J=
2
2
5.4Hz), 128.8 (pst, J= 5.4Hz), 110.7 (t, J(C,P)= 1.9Hz, 3C, C6H3), 90.0 (t, J(C,P)= 2.7Hz, 3C,
(CCH3)3), 19.6 (s, 3C, (CCH3)3). FTIR v(cm-1): 3053 (w), 2970 (w), 2920 (w), 1602 (w), 1585 (w),
1571 (w), 1531 (mw), 1483 (mw), 1450 (w), 435 (m), 1379 (mw), 1348 (w), 1334 (w), 1300 (w),
1261 (w), 1186 (w), 1159 (w), 1093 (m), 1028 (mw), 999 (mw), 916 (w), 873 (w), 775 (w), 727 (s),
713 (ms), 690 (s), 615 (w), 570 (w). UV-vis (C2H3N, 299K, nm): 225.0. ESI-MS (+) 641.0868
(theor.), 641.1112 (expt.) [C34H34ClP2Ru]+.ESI-MS (-) 178.8277 (theor.), 178.8245 (expt.) [GeCl3]-.
Conclusion
A series of novel arene ruthenium(II) trichlorostannate and trichlorogermanate salt compounds have been
prepared and fully characterized by spectroscopic means and in two cases by single crystal X-ray diffraction
analysis. Their in vitro cytotoxicity has been studied on the two breast cancer cell-lines: MCF-7 and MDA-
MB-231 as well as non-tumorigenic breast epithelial cells (MCF-12A) and compared to cisplatin as positive
control. All complexes show remarkable cytotoxic activity and promising selectivity on both cell lines
compared to cisplatin. A clonogenic assay on two of the complexes show long-term (14 day) stability and
dose dependent behavior providing some impetus for further pre-clinical trials of this novel class of
compounds. These salt complexes represent a promising new entry into ruthenium based agents that are
active against breast cancer cell-lines.33 We are currently exploring the extension of these results to other
group 8 metals and will report on these endeavors in due course.
Acknowledgements
The authors wish to thank the Maastricht Science Programme, and the Faculty of Science and Engineering
(FSE) at Maastricht University (UM) for financial support of this research. This work was supported by
grants from the South Africa Medical Research Council (SA MRC), the South Africa National Research
Foundation (NRF), Cancer Association of South Africa (CANSA) and the University of Cape Town (UCT).
We also thank Maarten Honing and Darya Hadavi (M4I, FHML (Maastricht MultiModal Molecular
Imaging; Faculty Health, Medicine & Life Sciences, Maastricht University) for measuring and interpreting
the HRMS of all the compounds.
Supporting Information
23

A file containing additional experimental details, experimental spectra of the compounds reported, X-ray
data and tables of cartesian coordinates of the DFT studied compounds is available. CCDC 1975891-
1975892 contain the supplementary crystallographic data for 4 and 6. This data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: +44 1223 336033.
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26

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: