Facile synthesis of heterobimetallic [FeII(μ-diphosphine)RuII] and homobimetallic [FeII(μ-diphosphine)FeII] complexes and their in vitro cytotoxic activity on cisplatin-resistant cancer cells

Article abstract of DOI:10.1016/j.ica.2020.119731

A series of heterobimetallic [Fe,Ru] and homobimetallic [Fe,Fe] complexes, featuring μ -diphosphine bridges between the two metal centres, are reported along with their in vitro cytotoxicity activity on the A2780cisR cell-line. The known starting material [CpFe(CO)₂(CH₃)] (Cp = η⁵-C₅H₅) (1) was reacted with dppe (1,2-bis(diphenylphosphino)ethane) forming the known mononuclear κ¹-dppe complex: [CpFe(CO)(COCH₃)(κ¹-dppe)] (2) and the known dinuclear complex: and [[CpFe(CO)(COCH₃)]₂(μ-dppe)] (3). Dimer cleavage reactions between complex 2 and two ruthenium arene complexes [(η⁶-p-cymene)RuCl₂]₂ and [(η⁶-benzene)RuCl₂]₂ formed two new heterobimetallic complexes: [CpFe(CO)(COCH₃)(μ-dppe)Ru(η⁶-cymene)Cl₂] (4) and [CpFe(CO)(COCH₃)(μ-dppe)Ru(η⁶-C₆H₆)Cl₂] (5). A homobimetallic system, [[CpFe(CO)(I)]₂(μ-dppb)] (6) was obtained by the facile reaction between of [CpFe(CO)₂I] and 1,4-bis(diphenylphosphino)butane (dppb). Complex 6 was methylated using MeLi to generate the more lipophilic complex [[CpFe(CO)(CH₃)]₂(μ-dppb)] (7). All of the complexes were fully characterized by spectroscopy (¹H, ¹³C {¹H}, ³¹P {¹H} NMR; FTIR, UV–Vis), and high resolution mass spectrometry (HRMS). Density functional theory calculations (DFT) (Level of theory B3LYP, basis set for H, C, P, O, Cl is 6-31+G(d,p) and for Ru, Fe, I is DGDZVP) on complexes 5 and 6 are also reported. An excellent agreement between the DFT calculated infra-red (IR) spectra of the optimised geometries of 5 and 6 was found with the experimentally determined spectra. In vitro cytotoxicity studies of all complexes was carried out on A2780cisR (cisplatin resistant ovarian cancer cell-line) and compared to cisplatin as a positive control and RAPTA-C as a negative control. Although limited to only one cell-line, the results show that the heterobimetallic complexes 4 and 5 are the most cytotoxic (IC₅₀ = 4.9 ± 0.4 μM and 6.5 ± 0.1 μM respectively), whilst the dinculear [Fe,Fe] complexes exhibit no, or very low cytotoxicity.

Full text of DOI:10.1016/j.ica.2020.119731

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Research paper
Facile Synthesis of Heterobimetallic [FeII(µ-diphosphine)RuII] and Homobi‐
metallic [FeII(µ -diphosphine)FeII] Complexes and their in vitro Cytotoxic Ac‐
tivity on Cisplatin-resistant Cancer Cells
Danya BenYosef, Dario Romano, Mouna Hadiji, Paul J. Dyson, Burgert Blom
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DOI:
Reference:
S0020-1693(20)30851-3
https://doi.org/10.1016/j.ica.2020.119731
ICA 119731
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1 May 2020
Please cite this article as: D. BenYosef, D. Romano, M. Hadiji, P.J. Dyson, B. Blom, Facile Synthesis of
Heterobimetallic [FeII(µ-diphosphine)RuII] and Homobimetallic [FeII(µ -diphosphine)FeII] Complexes and their in
vitro Cytotoxic Activity on Cisplatin-resistant Cancer Cells, Inorganica Chimica Acta (2020), doi: https://doi.org/
10.1016/j.ica.2020.119731
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Facile Synthesis of Heterobimetallic [FeII(-diphosphine)RuII] and
Homobimetallic [FeII(-diphosphine)FeII] Complexes and their in
vitro Cytotoxic Activity on Cisplatin-resistant Cancer Cells
Danya BenYosef,a Dario Romano,b,c Mouna Hadiji,d Paul J. Dyson,d Burgert Bloma,∗
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
d Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH1015
Lausanne, Switzerland
Abstract
A series of heterobimetallic [Fe,Ru] and homobimetallic [Fe,Fe] complexes, featuring -diphosphine
bridges between the two metal centres, are reported along with their in vitro cytotoxicity activity on the
A2780cisR cell-line. The known starting material [CpFe(CO)2(CH3)] (Cp = 5-C5H5) (1) was reacted with
dppeꢀꢀ(1,2-bis(diphenylphosphino)ethane) forming the known mononuclear ??1-dppe complex:
[CpFe(CO)(COCH3)(??1-dppe)] (2) and the known dinuclear complex: and [[CpFe(CO)(COCH3)]2(??-
dppe)] (3). Dimer cleavage reactions between complex 2 and two ruthenium arene complexes ([(6-p-
cymene)RuCl2]2 and [[(6-benzene)RuCl2]2] formed two new heterobimetallic complexes:
[CpFe(CO)(COCH3)(µ-dppe)Ru(η6-cymene)Cl2] (4) and [CpFe(CO)(COCH3)(µ-dppe)Ru(6-C6H6)Cl2]
(5). A homobimetallic system, [[CpFe(CO)(I)]2(??-dppb)] (6) was obtained by the facile reaction between
of [CpFe(CO)2I] andꢀ1,4-bis(diphenylphosphino)butane (dppb). Complex 6 was methylated using MeLi to
generate the more lipophilic complex [[CpFe(CO)(CH3)]2(??-dppb)] (7). All of the complexes were fully
13
31
characterized by spectroscopy (1H, C {1H}, P {1H} NMR; FTIR, UV-Vis), and high resolution mass
spectrometry (HRMS). Density functional theory calculations (DFT) (Level of theory B3LYP, basis set for
H, C, P, O, Cl is 6-31+G(d,p) and for Ru, Fe, I is DGDZVP) on complexes 5 and 6 are also reported. An
excellent agreement between the DFT calculated infra-red (IR) spectra of the optimised geometries of 5
and 6 was found with the experimentally determined spectra. In vitro cytotoxicity studies of all complexes
was carried out on A2780cisR (cisplatin resistant ovarian cancer cell-line) and compared to cisplatin as a
positive control and RAPTA-C as a negative control. Although limited to only one cell-line, the results
1

show that the heterobimetallic complexes 4 and 5 are the most cytotoxic (IC50 = 4.9±0.4 M and 6.5±0.1
M respectively), whilst the dinculear [Fe,Fe] complexes exhibit no, or very low cytotoxicity.
Keywords: Heterobimetallic complexes, Homobimetallic complexes, Anti-cancer activity, -
Phosphine Bridged Complexes
*Corresponding author email: burgert.blom@maastrichtuniversity.nl
TOC Graphic
Highlights:

Synthesis and characterisation of novel heterobimetallic iron-ruthenium and homobimetallic iron-
iron complexes and their in vitro cytotoxicity on A2780cisR.



First cytotoxic investigations of diphosphine bridged () diiron complexes.
DFT studies of the homo and heteronuclear complexes.
Comparison of DFT calculated and experimentally determined IR spectra.
2

Introduction
In 2020 the WHO estimates the number of new cancer cases to rise to 15 million and currently the
disease is responsible for 12 % of mortality world-wide.1 Chemotherapeutic interventions
including metal based drugs, are widely employed for treatment,2 to induce cell apopotosis in
cancer cells.3,4 Cisplatin, found to exhibit anticancer properties in the 1960s by Rosenberg,
heralded a new era in cancer chemotherapy.5 The well-studied mechanism of action of this drug
involves impairing cell function by binding to two guanine bases in DNA distorting DNA and
inhibiting replication.6-8 DNA.platinum adducts are recognized by certain proteins which either
initiate DNA repair mechanisms, or initiate apoptotic cell death. While cisplatin is a highly
effective drug, it causes nephrotoxicity and neurotoxicity and other side effects including hair loss,
nausea and vomiting.9 Other limitations of cisplatin include intrinsic drug resistance or resistance
after exposure to some types of tumours, as well as low solubility in aqueous media.10 Thus,
platinum analogues have been developed and studied in order to circumvent these limitations.11-15
In addition, complexes based on other metals such as gold,16,17 titanium,16 ruthenium18-28 and iron
have also been reported to exhibit anti-cancer properties.29-32 One area that has attracted the
attention is the synthesis heterobimetallic systems where potential cooperative behaviour can lead
to the potentiation of cytotoxic activity. Systems containing gold(I) and platinum(II) centres, for
example, display improved antiproliferative activity, as well as overcome drug resistance issues
seen in cisplatin.34 A study by Fernandez-Gallardo et al. shows that ruthenium-gold
heterobimetallic systems reveal higher selectivity and cytotoxicity towards renal and colon cancer
cell lines than their mononuclear analogs.35 Serra et al. elucidated the enhanced redox properties
that iron-ruthenium heterobimetallic systems have for catalytic applications, which served as
inspiration for our own work.33
We have focussed on the development of heterobimetallic complexes and explored their use in
anti-cancer applications. Our initial focus was arene-ruthenium(II) half-sandwich complexes with
one or two trichlorostannyl ligands -bound to the Ru(II) centre. The compounds showed poor
cytotoxicity due to low solubility in aqueous media, which could be overcome in ionic compounds
3

or by increasing solubility in aqueous media.36,37 Ruthenium(II) arene complexes bearing a -
bound germyl group were also prepared and showed low to negligible cytotoxic activity, which
was postulated to be due to rapid aquation kinetics.38 We have also focussed on homo and
heterobimetallic systems bearing two group 8 metals and their respective cytotoxcity (Figure 1).
In this regard [Fe,Ru] and [Ru,Ru] complexes bridged with μ-dppm (dppmꢀ=ꢀ1,1-
bis(diphenylphosphino)methane)) were synthesized with the expectation that the presence of iron
in the bimetallic compound may increase drug uptake.39a The study revealed that a heteronuclear
[Fe,Ru] system was indeed substantially more cytotoxic than cisplatin, on A2780 and A2780cisR
cell-lines, while a comparable dinuclear [Ru,Ru] system showed a decrease in cytotoxicity.
In the present study, we report on our latest findings in this area, in which we explored the effects
of increasing the lipophilicity at the iron centre by replacing the iodo ligand with an acyl group
and explored the effect of increasing the spacer length between the Fe and Ru centres to observe
changes in cytotoxicity. Studies by Hartinger, for example have shown that spacer length plays a
role in cytotoxic action. 39e Moreover, the preparation and cytotoxic testing of homobimetallic -
diphosphine [Fe,Fe] complexes is also reported (Figure 1).
4

Figure 1. Heterobimetallic and homobimetallic complexes reported which exhibit promising cytotoxic
profiles and cancer cell selectivity and the compounds reported in the present study.
Results and Discussion
Our strategy to prepare heterobimetallic [Fe,Ru] complexes is similar to the one we employed
previously,39a Requiring a suitable mononuclear iron complex, bearing a 1 metal bound
bisphosphine ligand with a pendant uncoordinated phosphorus atom of the type LnFe←:P⁔P:. The
pendant uncoordinated phosphine could then, in a second synthetic step, cleave Ru dimer
complexes of the type [RuCl2(η6-arene)]2 (arene = benzene and p-cymene),40 to afford the desired
dinuclear complexes. We sought complexes bearing
a
1 dppe (dppeꢀ=ꢀ1,1-
bis(diphenylphosphino)ethane)) ligand, as previously we employed dppm (dppeꢀ=ꢀ1,1-
bis(diphenylphosphino)methane)),39a and wanted to increase the spacer length between the Fe and
Ru centres. Moreover, we were also interested in obtaining an iron centre which was more
lipophilic than in our earlier studies, since we envisioned higher cellular uptake and potential
efficacy. A suitable agent for these purposes was the known complex [CpFe(CO)(COCH3)(??1-
dppe)] (Cp= 5-C5H5) (2).43,44 Complex 2 is accessible via a facile migratory insertion route using
dppe starting from the known complex [CpFe(CO)2(CH3)] 1.41,42 Complexes
[CpFe(CO)(COCH3)(??1-dppe)] 2 and [[CpFe(CO)(COCH3)]2(µ-dppe)] 3,45 the latter which exists
as a mixture diastereoisomers, are obtained from this reaction (Scheme 1).43,44 While compounds
2 and 3 are known, their cytotoxicity has not been determined, and we decided to include them in
our
in
vitro
cytotoxicity
screening
studies
(see
below).
5

Scheme 1. Reaction scheme for the synthesis of products [CpFe(CO)(COCH3)(??1-dppe)] 2 and
[[CpFe(CO)(COCH3)]2(??-dppe)] 3. Complexes 2 and 3 possess asymmetric centres (Fe). For complex 2
this exists as a mixture of enantiomers (rac-2) (only one is depicted), while for complex 3 three
stereoisomers are possible, i.e. the meso form and a set of enantiomers (only one is depicted in Scheme 1).46
Employing complex 2 as starting material (which is chiral and exists as a mixture of enantiomers:
rac-2) the reaction thereof with the Ru dimer complexes [RuCl2(η6-p-cymene)]2 and [RuCl2(η6-
C6H6)]2 in a 2:1 ratio afforded the heterobimetallic complexes [CpFe(CO)(COCH3)(µ-
dppe)Ru(η6p-cymene)Cl2] 4 and [CpFe(CO)(COCH3)(µ-dppe)Ru(η6-C6H6)Cl2] 5, respectively
(Scheme 2) in a straightforward manner. The -dppe bridged [Fe,Ru] complexes with no Fe-Ru
bond are somewhat rare in the literature.39b-d Complex 4 was isolated as a clay red/orange powder
in an excellent yield of 85%, while complex 5 required column chromatographic purification using
an eluent mixture of 1:1 n-hexane/ethyl acetate. The latter afforded a light brown powder with a
suboptimal yield of 34 %. Both complexes exhibit high decomposition temperatures: 4 at 191 °C,
and complex 5 at 130 °C.
Scheme 2. Reaction scheme for the synthesis of complex [CpFe(CO)(COCH3)(µ-dppe)Ru(η6-p-
cymene)Cl2] 4 and [CpFe(CO)(COCH3)(µ-dppe)Ru(η6-C6H6)Cl2] 5. The iron centre in both complexes is
asymmetric and complexes 4 and 5 exist as a racemic mixture (rac-4 and rac-5). Only one stereoisomer is
depicted for clarity in both cases.
6

31
13
The full spectroscopic characterization consisting of multinuclear NMR (1H, P{1H}, C{1H})
FTIR, and ESI-MS, was carried out for both complexes. In complex 4, a series of unresolved
multiplet resonances in the range δ = 7.84 to 7.05 ppm correspond to the 20 aromatic protons in
dppe. Four distinct doublets are observed from δ = 5.24 to 4.95 ppm, corresponding to the 4
inequivalent protons in the p-cymene ring, due to the asymmetry at the iron centre. The singlet
resonance for η5-C5H5 is observed at δ = 4.39 ppm, and the multiplet signals that follow from δ =
2.70 to 2.45 ppm correspond to the inequivalent protons in the dppe bridge. The consecutive
singlets represent the methyl groups in the compound: the COCH3 resonates at δ = 2.25 ppm and
the CH3 from the p-cymene is seen at δ = 1.84 ppm. Finally, the isopropyl methyl groups from
the p-cymene substituent appear as 2 distinct doublet resonances at δ = 0.88 and 0.80 ppm
respectively confirming the diastereotopic nature of the iPr groups. The aromatic signals for
complex 5 appear at similar ppm values to 4. A singlet is observed at δ = 5.32 for complex 5, and
corresponds to 6 equivalent protons from the benzene (η6-C6H6) substituent. Complex 5 shows the
η5-C5H5 resonance at δ = 4.39 ppm, as well as similar ppm values for the protons in the dppe bridge
as with 4. There is only one singlet resonance for the only methyl group in the compound (COCH3),
the latter which is seen at δ = 2.35 ppm. The 31P{1H} NMR spectra for complexes 4 and 5 are very
similar and each contain two doublet resonances. A low-field shifted doublet signal for both
complexes is seen at δ = 74.3 ppm, representing the Fe-PPh2 bond, while the second high-field
shifted signal, also seen in both complexes, appears at δ = 25.1 ppm which corresponds to the Ru-
PPh2 bond. The 13C{1H} NMR data for complex 4 shows a series of unresolvable multiplets from
δ = 135.5 to 126.7 ppm for the carbon atoms in the aromatic substituents of dppe. Singlets which
appear at δ = 107.4 and 93.4 ppm correspond to two C atoms on p-cymene ring, while the
remaining carbon atoms within the p-cymene ring are represented by two sets doublets: one
doublet appears at δ = 89.9 ppm due to 2J(C,P) coupling. The singlets at δ = 83.6 ppm pertains to
the equivalent carbons in the η5-C5H5 group, and the doublet at δ = 50.9 ppm the methyl carbon
from COCH3 coupling to phosphorus atom.
Complex 5 shows similar 13C{1H} NMR features as 4. The spectrum displays a doublet resonance
at δ = 219.4 ppm with a 2J(C,P) coupling constant of 30 Hz, for the Fe-CO. A singlet at δ = 87.6
ppm corresponds to the benzene substituent and the signal at δ = 83.7 ppm corresponds to η5-C5H5.
The FTIR spectra for complexes 4 and 5 reveal the strong stretching vibrations for both the Fe-
7

(CO) and the Fe-(C=O)CH3: For complex 4, these are visible at = 1901 and 1581 cm-1
respectively, while for complex 5, the stretching vibrations appear at 1897 and 1597 cm-1. Both
complexes were subjected to high resolution ESI-MS (+) and exhibit signals corresponding the
radical cation [M]‧+ and [M+Na]+, both with matching isotope patterns confirming their
constitution. In order to test the stability of the complexes in (DMSO, see below), 31P{1H} NMR
spectra were recorded of complex 5 dissolved in DMSO-d6 (containing water) after different time
intervals.47 The sample was tested each hour during the first 7 hours, after 24 hours, and finally
after 72 hours. When observing the spectra, it is evident that there are no changes in this time
period.
We next targeted the homobimetallic [Fe,Fe] complex [[CpFe(CO)(I)]2(??-dppb)] (complex 6),
bearing the longer tether (dppb as opposed to dppe). We decided to use dppb to explore the effect
of a longer spacer between the two metal centres, and to exclude formation of a 2-phosphine
product. Surprisingly, homobimetallic complexes of the type [Fe(-phosphane)Fe], although
highly prevalent in the literature and used inter alia as iron hydrogenase models, have altogether
escaped investigation as potential anti-cancer agents, and herein we report the first such study (see
below).48 The complex [CpFe(CO)2I] and dppb (dppbꢀ=ꢀ1,1-bis(diphenylphosphino)butane)) was
dissolved in toluene and refluxed at 85 °C for 7 hours. The crude reaction mixture was purified by
column chromatography using a 3:2 eluent mixture of dichloromethane/n-hexane which exhibited
three separable bands. The third band corresponded to [[CpFe(CO)(I)]2(??-dppb)], which was
isolated as a light green powder in a 37 % yield (Scheme 3) as a mixture of stereoisomers.
8

Scheme 3. Reaction scheme for the synthesis of complex 6 [[CpFe(CO)(I)]2(??-dppb)] ⋅ 0.5 hexane
Complex 6 exists as a mixture of three stereoisomers: a pair of enantiomers and a meso form akin
to complex 3. The complex was also subjected to full spectroscopic characterisation. The 1H NMR
spectrum reveals multiplet resonances from δ = 7.71 to 7.35 ppm correspond to the 20 protons of
the phenyl rings of dppb, and a singlet at δ = 4.32 ppm with an integration of 10 protons
corresponds to two equivalent η5-C5H5 groups in the dinuclear compound. Two multiplet
resonances at δ = 2.37 and 0.95 ppm respectively represent the protons of the dppb bridge. The
31
remaining signals correspond to half a molecule of hexane included in the solid. In the P{1H}
NMR spectrum, two singlets appear at δ = 61.7 ppm and δ = 61.6 ppm, akin to complex 3;
indicating the diasteriosomers in the meso form and pair of enantiomers in D,L form. The FTIR
confirms the presence of one CO stretching vibration with a medium strong intensity at 1932 cm-
1. The ESI-MS spectra of 6 show a complex fragmentation pattern including the [M]‧+ signal at
977.9068 m/z and a [M+Na]+ signal at 1000.9033 m/z, which can be compare well to the
‧
+
31
theoretical values ([M] 977.9129; for [M+Na]+ 1000.9028) confirming constitution. P{1H}
NMR spectra were also recorded of complex 6 in wet DMSO-d6 after different time intervals,
showing stability. 47 The compound, as with the [Fe,Ru] complexes shows stability over this time
period (see SI for the spectra).
The related complex 7, [[CpFe(CO)(CH3)]2(??-dppb)] ⋅ 0.5 hexane, could readily be isolated upon
reaction of complex 6 with MeLi (Scheme 4). After column chromatographic purification using
9

CH2Cl2, the compound was successfully isolated as a vermilion powder with a yield of 46.5%. The
complex is thermally robust and onset of colour change starts at 134 °C with full decomposition
at 146 °C.
Scheme 4. Reaction scheme for the synthesis of complex 7 [[CpFe(CO)(CH3)]2(??-dppb)] ⋅ 0.5 hexane
1
The H NMR reveals the Fe-CH3 resonance at δ = -0.29 ppm as a doublet of doublets due to
31
coupling to P. In contrast to the precursor 6 the P{1H}NMR spectrum shows only one singlet
signal at δ = 75.5 ppm for the Fe-PPh2 although two signals are expected due to the
diasteriosomerism possibly due to isochronicity. In the FTIR spectrum, the CO stretching vibration
appears at  = 1892 cm-1, shifted by 40 wavenumbers from its precursor, 6, due to enhanced
electron density at the iron centre afforded by the methyl group.
Complexes 2-7 were subjected to in vitro cytotoxicity studies on the cisplatin resistant ovarian
cancer cell line A2780cisR and compared to cisplatin as positive control and RAPTA-C as
negative control (Table 1). In the series, the heterobimetallic [Fe,Ru] complexes 4 and 5 exhibit
promising cytotoxic activity and are the most active in the series, with the cymene analogue 4
being slightly more cytotoxic than the benzene analogue 5. This is in keeping with our earlier
findings of related complexes bearing a dppm bridge between the Fe and Ru centres, i.e. [(6-
C6H6)RuCl2(-dppm)Fe(CO)I(5-C5H5)] (IC50 = 1.5 M on A2780cisR) and [(6-p-
cymene)RuCl2(-dppm)Fe(CO)I(5-C5H5)] (IC50 = 1.2 M or A2780cisR).39a
Interestingly, the bimetallic [Fe,Fe] complexes 3 and 6 are inactive with IC50 values > 100 M,
while the mononuclear iron complex 2 exhibits moderate activity. Complex 7, the methylated
analogue of 6, exhibits some, albeit it low, cytotoxicity. These latter results suggest that increasing
the lipophilicity of the ligand at the iron centre (I in 6 vs CH3 in 7) does enhance cytotoxicity, but
overall the dinuclear [Fe,Fe] complexes are rather inactive.
10

Complex
IC50 (M)
8.5 ± 0.1
>100
2
3
4
4.9 ± 0.4
6.5 ± 0.1
> 100
5
6
7
61 ± 3
Cisplatin
RAPTA-C
35 ± 3
> 200
Table 1. IC50 (M) values of complexes 2-7 on the A2780cisR cell-line after 72 h exposure to the
compound. The values represent the mean between four replicates and ± the std. deviation.
Structural, electronic and spectroscopic characteristics for complexes 5 and 6 were calculated
using density function theory methods (DFT). For all the calculations, the level of theory employed
was B3LYP with the basis set 6-31+G(d,p) for H, C, O, P and Cl atoms; while for I, Ru and Fe
atoms DGDZVP basis set was used. Since we were unable to obtain suitable single crystals for X-
ray diffraction analysis of the complexes, despite several attempts and crystallisation techniques,
we obtained structural information of the complexes via DFT methods. Moreover, the calculated
IR spectra of the optimised structures (in the gas phase), were compared to those obtained by
experiment (solid state IR). These showed an excellent agreement, in both 5 and 6, suggesting
reasonable structural models for both complexes using DFT methods. Full identification and
assignment of the vibrational modes in the IR spectra was also possible for 5 (see SI Fig. 5.5) and
6 (see SI Fig. 6.6) in light of this. Figure 2 shows the optimised structures of complex 5 and 6 with
some key metrical parameters obtained from DFT.
11

Figure 2. DFT optimised structure of complex 5 (left). Selected bond lengths [Å]: Fe-P2 2.260, Ru-P1
2.387, Fe-C1 1.960, Fe-C2 1.749, Ru-Cl1 2.440, Ru-Cl2 2.466. Fe-P2-P1-Ru dihedral angle: 145o. DFT
optimised structure of complex 6 (right). Selected bond lengths [Å] Fe1-I2/Fe2-I2 2.696, Fe1-P1/Fe2-P2
2.330, Fe1-Ccarbonyl/Fe2-Ccarbonyl= 2.696. Fe1-P1-P2-Fe2 dihedral angle: 163o.
The most striking structural feature of both complexes is the zig-zag arrangement of the heavy
atoms, with the two metal centres being somewhat anti with respect to each other. In complex 5 a
Fe-P-P-Ru dihedral angle of 145o is observed, while complex 6 exhibited an Fe1-P1-P2-Fe2
dihedral angle of 163o (Figure 3).
Figure 3. Perspective of 5 (left) and 6 (right) viewed down the P-P vector. All atoms except the Fe,Ru and
P are omitted for clarity.
12

The electronic nature of complexes 5 and 6 was also studied. Figure 4 shows the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) with the
corresponding molecular orbitals’ energies of the two complexes.
Figure 4. LUMO (top) and HOMO (bottom) of the optimized structures of complex 5 (left) and 6 (right).
The energies of the LUMOs (top) and HOMOs (bottom) are expressed in hartree. Blue = iron, red = oxygen,
grey = carbon, orange = phosphorus, green = chlorine,
Clear differences can readily be observed for the localizations of the HOMO and LUMO when
comparing complexes 5 and 6. For complex 5, due to its asymmetrical nature, the HOMO and the
LUMO are asymmetrically localized on the complex. In particular, the HOMO of complex 5 is
delocalized on the iron metal and the ligands (Cp, CO, one phosphorous of the dppe and acetyl),
while the LUMO is delocalized on the ruthenium terminus over the ligands (arene and chlorides)
excluding the phosphorus atoms of the bridging dppe. This is in contrast to our earlier studies of
related heterobimetallic [Fe,Ru] complexes, where both HOMO and LUMO were located on the
iron terminus of the complex in [(6-Arene)RuCl2(-dppm)Fe(CO)I(5-C5H5)] (Ar = C6H6 and p-
13

cymene, dppm = 1,1-bis(diphenylphosphinomethane)).39a The symmetric complex 6 shows the
calculated HOMO and LUMO symmetrically localized on both metal centres with their
corresponding ligands.
Conclusions
A series of heterobimetallic [Fe,Ru] and homobimetallic [Fe,Fe] complexes, bridged with a dppe
ligand, were prepared and fully characterised spectroscopically and their structures, electronic
nature and IR spectra investigated by DFT methods, the latter showing excellent agreement with
experiment. The DFT study revealed that for the heterobimetallic complexes, the HOMO and
LUMO are not both located on the iron terminus of the complex, in contrast to earlier findings.39a
An in vitro study of all dinuclear complexes was carried out on the A2780cisR cisplatin resistant
ovarian cancer cell-line, and compared to the known mononuclear iron complex
[CpFe(CO)(COCH3)(??1-dppe)] 2. While limited to only one-cell line, it can be seen that the
heterobimetallic [Fe,Ru] complexes exhibit promising cytotoxicity profiles, while the
corresponding [Fe,Fe] complexes exhibit very low activity; and the mononuclear complex 2 only
moderate activity. Hence such homodinuclear [Fe,Fe] complexes, appear not be suitable
candidates for further pursuit, whereas the [Fe,Ru] complexes have promising cytotoxicity.39a The
increased spacer length, however, seems to slightly reduce cytotoxic action in the latter with the
previously reported [Fe,Ru] complexes bearing a dppm bridge being somewhat more cytotoxic.
Experimental Section
All reactions, unless stated otherwise, were performed under a nitrogen atmosphere using Schlenk
procedures and/or glove-box manipulations. All solvents were degassed by bubbling nitrogen gas
for several minutes through them and dried by passage over activated alumina. The starting
materials were obtained from commercial suppliers and used as received.
Dicarbonylcyclopentadienyliodoiron(II) ([CpFe(CO)2I]) (97%), anhydrous tetrahydrofuran
(99.9%), methyllithium (1.6 M in diethyl ether), dppe, dppb (99%) and silica gel (60 Å pore size,
70-230 mesh, 63-200 μm) were purchased from Sigma-Aldrich/Merck. Dichloromethane (stab.
Amylene), as well as n-hexane and diethyl ether (stab. BHT), were obtained from Biosolve
14

Chemicals. Ethyl acetate (99.5%) was purchased from VWR, as well as chloroform-d +0.03%
TMS and dimethyl sulfoxide-d6 (99.80%). Dry toluene (99.85%) was obtained from Acros
Organics. Dichloro(p-cymene)ruthenium(II) dimer ([(p-cymene)RuCl2]2) (98%) was purchased
from STREM, while benzeneruthenium(II)chloride dimer ([(benzene)RuCl2]2) (95%) was
purchased from TCI. The known complexes 2 and 3 were prepared by modified literature
procedures and can be found in the SI. 43-45The apparatus used to measure the melting points was
a Stuart SMP10 and are uncorrected. NMR experiments at room temperature were recorded using
a Bruker Ultrashield 300 MHz/54 mm magnet system. Chemical shifts were observed relative to
1
13
the residual solvent peak. The H and C {1H} NMR chemical shifts were observed relative to
tetramethylsilane (TMS), while for 31P {1H} NMR, they were measured relative to phosphoric acid
(85%). The software for spectral analysis was Bruker Topspin 4.0.6. FTIR spectra were retrieved
using a MIRacle 10 Shimadzu, with a single reflection ATR accessory. Per sample, 64 scans were
collected over a range of 400-4000 cm-1, with a spectral resolution of 1 cm-1; these were analysed
using the IR solution software. UV-Vis spectra were obtained using a UV-188 spectrophotometer
(Shimadzu) and quartz suprasil precision cells (100- QS, 10 mm light path, from Hellma
Analytics). Finally, high resolution mass spectrometry (HR-ESI-MS) was recorded using a Bruker
SolariX XR FT-ICR-MS in the Maastricht MultiModal Molecular Imaging Institute (M4I). The
samples were dissolved in CHCl2-CH3CN- CH3COOH (49.95 – 49.95 – 0.1 %) and then diluted
100 times. The MS parameters were set in ionization + mode, with a capillary of 4.4 kV, end plate
offset of -800V, a nebulizer at 1bar, dry gas at 4 L/min, a dry temperature of 200 ℃, and a mass
range of 300-1300 m/z. The resolution of all measurements was above 100000. Only the relevant
signals are quoted in the experimental section. All isotope patterns were checked using online
software; the isotope line with the highest intensity was reported for each sample.
Cytotoxicity Tests
Human ovarian carcinoma (A2780cisR) cell lines were obtained from the European Collection of
Cell CulturesPenicillin streptomycin, RPMI 1640 GlutaMAX (where RPMI = Roswell Park
Memorial Institute), and DMEM GlutaMAX media (where DMEM = Dulbecco’s modified Eagle
medium) were obtained from Life Technologies, and fetal bovine serum (FBS) was obtained from
Sigma. The cells were cultured in RPMI 1640 GlutaMAX (A2780cisR) and DMEM GlutaMAX
(HEK-293) media containing 10% heat-inactivated FBS and 1% penicillin streptomycin at 37 °C
15

and CO2 (5%). The A2780cisR cell line was routinely treated with cisplatin (2 μM) in the media
to maintain cisplatin resistance. The cytotoxicity was determined using the 3-(4,5-dimethyl 2-
49
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
Cells were seeded in flat-
bottomed 96-well plates as a suspension in a prepared medium (100 μL aliquots and approximately
4300 cells/well) and pre-incubated for 24 h. Stock solutions of compounds were prepared in
DMSO and were diluted in medium. The solutions were sequentially diluted to give a final DMSO
concentration of 0.5% and a final compound concentration range (0−200 μM). Cisplatin and
RAPTA-C were tested as a positive (0−100 μM) and negative (200 μM) controls respectively. The
compounds were added to the preincubated 96-well plates in 100 μL aliquots, and the plates were
incubated for a further 72 h. MTT (20 μL, 5 mg/mL in Dulbecco’s phosphate buffered saline) was
added to the cells, and the plates were incubated for a further 4 h. The culture medium was
aspirated and the purple formazan crystals, formed by the mitochondrial dehydrogenase activity
of vital cells, were dissolved in DMSO (100 μL/well). The absorbance of the resulting solutions,
directly proportional to the number of surviving cells, was quantified at 590 nm using a
SpectroMax M5e multimode microplate reader (using SoftMax Pro software, version 6.2.2). The
percentage of surviving cells was calculated from the absorbance of wells corresponding to the
untreated control cells. The reported IC50 values are based on the means from two independent
experiments, each comprising four tests per concentration level.
Density Functional Theory Calculations
DFT calculations were performed to model the complexes 5 and 6. Guassian09 software package
was used. For all the calculations, the level of theory used for all calculations is B3LYP with the
basis set 6-31+G(d,p) for H, C, O, P and Cl atoms; while for I, Ru and Fe atoms DGDZVP basis
set was used. Geometry optimizations were calculated without any constrains. All the optimized
geometries show not imaginary frequency. Energies and infrared spectra were calculated on the
optimized structures.50
Synthesis of [CpFe(CO)(COCH3)(??-dppe)Ru(η6-p-cymene)Cl2] (4)
0.048 g of [CpFe(CO)(COCH3)(??1-dppe)] (2) (0.081 mmol, 1 eq.) and 0.024 g of [(η6-p-
cymene)RuCl2]2 (0.040 mmol, 0.5 eq) were added to a 100 mL round bottom Schlenk flask. The
reactants were dissolved in approximately 20 mL of dichloromethane; the solution was left to stir
16

at room temperature for 2 hours. The solvent was evaporated under reduced pressure, the residue
washed with hexane, and the hexane washings discarded. The resulting solid was dried in vacuo
affording a clay red/orange powder (0.0616g, 0.068 mmol, 85%) with a decomposition
temperature of 191 °C. 1H NMR (300 MHz, CDCl3, 298 K): δ 7.84-7.05 (20H, m, C-H dppe), 5.24
(1H, d, 3J(H,H)= 6.20 Hz, p-cymene C-HD), 5.20 (1H, d, 3J(H,H)= 6.20 Hz, p-cymene C-HC), 5.13
(1H, d, 3J(H,H)= 6.20 Hz, p-cymene C-HB), 4.95 (1H, d, 3J(H,H)= 6.20 Hz, p-cymene C-HA), 4.39
(5H, s, η5-C5H5), 2.70 (1H, m, CHMe2), 2.49 (1H, m, PCHC bridge), 2.47 (1H, m, PCHB bridge),
2.45 (1H, m, PCHA bridge), 2.25 (3H, s, COCH3), 1.84 (3H, s, p-cymene CH3), 0.88 (3H, d, 3J(H,
H)= 7.02 Hz, p-cymene CH(CH3)B), 0.80 (3H, d, 3J(H,H)= 7.02 Hz, p-cymene CH(CH3)A) ppm;
31P{1H} NMR (CDCl3, 298 K): δ 74.3 (d, 3J(P,P)= 34 Hz, Fe-PPh2), 25.1 (d, 3J(P,P)= 34 Hz, Ru-
PPh2) ppm; 13C {1H} NMR (CDCl3, 298 K): δ 135.5-126.7 (m, dppe), 107.4 (s, p-cymene C1 or 4
H), 93.4 (s, p-cymene C1 or 4-H), 89.9 (d, 2J(C,P)= 4.7 Hz, p-cymene CA-H), 88.6 (d, 2J(C,P)= 3.8
-
2
2
Hz, p-cymene CB-H), 84.8 (d, J(C,P)= 6.0 Hz, p-cymene CC-H), 84.1 (d, J(C,P)= 5.7 Hz, p-
3
cymene CC-H), 83.6 (s, η5-C5H5), 50.9 (d, J(C,P)= 5.7 Hz, COCH3), 28.9 (s, p-cymene
A
B
CH(CH3)2), 23.9 (m, dppe), 21.6 (s, p-cymene iPr CH3 ), 20.6 (s, p-cymene iPr CH3 ), 19.7 (d, xJ
(C,P)= 28.8 Hz, dppe ),16.3 (s, p-cymene CH3) ppm; FTIR (cm-1): = 3059 (vw), 1948 (w), 1901
(m, CO stretch), 1886 (m), 1581 (m, CO stretch), 1481 (w), 1435 (m), 1321 (w), 1261 (vw), 1178
(w), 1093 (w), 1060 (w), 999 (w), 906 (w), 889 (w), 819 (w), 748 (m), 717 (m), 694 (ms) cm-1;
ESI-MS: m/z Calcd. For [M]‧+ 896.0745, found 896.0702. Calcd. For [M+Na]+ 919.0635, found
919.0607.
Synthesis of [CpFe(CO)(COCH3)(??-dppe)Ru(η6-C6H6)Cl2] (5)
0.100 g of [CpFe(CO)(COCH3)(??1-dppe)] (2) (0.169 mmol, 1 eq.) and 0.042 g of [(η6-
benzene)RuCl2]2 (0.084 mmol, 0.5 eq.) were added to a 100 mL Schlenk flask. The reactants were
dissolved in approximately 20 mL of dichloromethane; the solution was left to stir at room
temperature for 2 hours. The solvent was evaporated under reduced pressure, and then column
chromatography was performed in which a 1:1 n-hexane/ethyl acetate eluent was utilized. The
purification afforded a light brown/red powder (0.0477 g, 0.056 mmol, 34%) with a decomposition
1
temperature of 130 °C. H NMR (300 MHz, CDCl3, 298 K): δ 7.73- 7.14 (20H, m, C-H dppe),
5.32 (6H, s, η6-C6H6), 4.39 (5H, s, η5-C5H5), 2.70 (1H, m, PCHD bridge), 2.46 (1H, m, PCHC
bridge), 2.40 (1H, m, PCHB bridge), 2.35 (3H, s, COCH3), 2.15 (1H, m, PCHA bridge) ppm;
17

31P{1H} NMR (CDCl3, 298 K): δ 74.3 (d, 3J(P,P)= 38.5 Hz, Fe-PPh2), 25.1 (d, 3J(P,P)= 38.5 Hz,
Ru-PPh2) ppm; 13C{1H} NMR (CDCl3, 298 K): δ 219.4 (d, 2J(C,P)= 30 Hz, Fe-CO), 135.6-127.0
(m, dppe), 87.6 (d, 2J(C,P)= 3.5 Hz, η6-C6H6) 83.7 (s, η5-C5H5), 50.7 (d, 3J(C,P)= 5 Hz, COCH3),
1
1
24.9 (d, J(C,P)= 27 Hz, CBH2PPh2 bridge), 21.1 (d, J(C,P)= 27 Hz, CAH2PPh2 bridge) ppm;
FTIR: = 3072 (vw), 2962 (vw), 2897 (vw), 2474 (vw), 2312 (vw), 1897 (ms, CO stretch), 1734
(vw), 1597 (m, CO stretch) 1500 (vw), 1481 (w), 1433 (mw), 1411 (w), 1373 (vw), 1319 (vw),
1259 (w), 1176 (w), 1095 (s), 1055 (s), 1028 (ms), 1014 (ms), 898 (w), 887 (w), 842 (w), 821 (ms),
800 (ms), 746 (ms), 725 (ms), 717 (s), 692 (s), 621 (w), 611 (w), 584 (w), 574 (w), 561 (w), 414
(w) cm-1 (Full assignment of all stretching vibrations can be found in SI, secured by DFT); ESI-
MS: m/z Calcd. For [M]‧+ 840.0117, found 840.0085. Calcd. For [M+Na]+ 863.0015, found
862.9983.
Synthesis of [[CpFe(CO)(I)]2(??-dppb)] ⋅ 0.5 hexane (6)
0.100 g of [CpFe(CO)2I] (0.329 mmol) and 0.121 g of 1,4-Bis(diphenylphosphino)butane (dppb)
(0.284 mmol) were added to a 100 mL round bottom Schlenk flask. The reactants were dissolved
in 30 mL of dry toluene. The reaction was left to stir under a reflux at 85 °C for 7 hours. The
solution was then cooled to room temperature and filtered under gravity. The remaining mixture
was dried in vacuo and was purified using column chromatography with an eluent consisting of
3:2 dichloromethane/n-hexane. The first band was a light brown colour, which was discarded. The
second band was light green, which was contaminated with starting dppb. The desired product,
[[CpFe(CO)(I)]2(??-dppb)], was concentrated in the third dark green band. The latter was dried in
vacuo, which afforded a light green powder with a yield of 0.101 g (0.103 mmol, 37%) and a
decomposition temperature of 81-85 °C. 1H NMR (300 MHz, CDCl3, 298 K): δ 7.71- 7.35 (20H,
m, C-H dppb), 4.32 (10H, s, η5-C5H5), 2.37 (4H, m, PCHA bridge), 1.25 (5H, br s, CH2 hexane),
0.95 (4H, m, PCHB bridge), 0.87 (1H, m, CH3 hexane) ppm (assigned by HSQC/HMBC
experiments); 31P{1H} NMR (CDCl3, 298 K): δ 61.7 (s, Fe-PPh2, meso stereoisomer and/or D,L
13
form), 61.6 (s, Fe-PPh2, meso stereoisomer and/or D,L form) ppm; C{1H} NMR (CDCl3, 298
K): δ 220.7 (s, Fe-CO), 133.1-131.7(m, C-H dppb), 130.4 (s, C quaternary dppb), 128.6- 128.2
(m, C-H dppb), 82.4 (s, η5-C5H5), 33.3 (d, 1J(C,P)= 17.5 Hz, CAH2PPh2 bridge), 32.9 (d, 1J(C,P)=
17.5 Hz, CBH2PPh2 bridge), 31.6 (s, CH2(3) hexane), 25.2 (s, CAH2 bridge), 25.1 (s, CBH2 bridge),
22.7 (s, CH2(2) hexane), 14.1 (s, CH3 hexane) ppm; FTIR (cm-1): = 2953 (w), 2924 (w), 2854
18

(w), 1932 (ms, CO stretch), 1587 (vw), 1571 (vw), 1479 (w), 1433 (w), 1359 (vw), 1307 (vw),
1259 (vw), 1180 (w), 1157 (w), 1093 (w), 1070 (w), 1026 (w), 999 (w), 839 (w), 821 (w), 740
(m), 719 (w), 694 (s), 617 (vw), 596 (w), 569 (w), 553 (w), 536 (w), 447 (vw), 418 (vw), 412 (vw)
cm-11 (Full assignment of all stretching vibrations can be found in SI, secured by DFT); UV-Vis
(nm)/ dichloromethane λmax: λ 615 (minor), 436 (major); ESI-MS: m/z Calcd. For [M]‧+ 977.9135,
found 977.9068. Calcd. For [M+Na]+ 1000.9033, found 1000.8967.
Synthesis of [[CpFe(CO)(CH3)]2(??-dppb)] ⋅ 0.5 hexane (7)
0.243 g of [[CpFe(CO)(I)]2(??-dppb)] (0.248 mmol) were added to a 100 mL round bottom flask,
containing 50 mL of degassed toluene. The dark green solution was cooled to approximately -80
°C, and MeLi (0.341 mL, 0.546 mmol, 2.2 eq) was carefully added in a dropwise fashion, turning
the solution brown. The mixture was removed from the cooling bath and allowed to warm to
ambient temperature, and then was left to stir for 2 hours, which darkened the colour of the brown
solution. The solvent was evaporated under reduced pressure. The air-stable dark brown solid was
purified via column chromatography using CH2Cl2 as the eluent, and then washed with hexane.
The latter yielded 0.087 g of a vermilion-coloured powder (0.115 mmol, 46.5%) with a
decomposition temperature of 134-146 °C. 1H NMR (300 MHz, CDCl3, 298 K): δ 7.62- 7.29 (20H,
m, C-H dppb), 4.12 (10H, s, η5-C5H5), 1.94 (4H, m, bridge), 1.25 (4H, s, CH2 hexane), 1.16 (2H,
m, bridge), 0.92 (2H, m, bridge), 0.88 (1H, m, CH3 hexane), -0.29 (6H, dd, 3J(H,P)= 8.0 Hz & 6.40
31
Hz, CH3) ppm; P {1H} NMR (CDCl3, 298 K): δ 75.5 (s, Fe-PPh2) ppm; FTIR (cm-1): = 3059
(vw), 2924 (w), 2870 (w), 1892 (s, CO stretch), 1718 (vw), 1684 (vw), 1653 (vw), 1647 (vw),
1586 (vw), 1571 (vw), 1479 (w), 1458 (w), 1432 (m), 1360 (vw), 1329 (vw), 1306 (w), 1291 (vw),
1260 (w), 1181 (w), 1170 (w), 1157 (w), 1093 (m), 1070 (m), 1058 (m), 1027 (m), 1011 (m), 999
(m), 891 (vw), 840 (m), 818 (m), 801 (m), 743 (m), 738 (m), 723 (m), 693 (vs), 618 (vw), 603 (w),
593 (w), 576 (w), 545 (vw), 535 (vw), 462 (vw) cm-1. ESI-MS: m/z Calcd. For [M]‧+ 754.1515,
found 754.1524 (17 %). Calcd. For [M-CpFe(CO)(CH3)]+ 590.1591, found 590.1558 (100 %).
Acknowledgement
We thank the Maastricht Science Programme (MSP) and Maastricht University (Faculty of Science and
Engineering) for financial support of this research. We also thank Maarten Honing, Peilang Han and Darya
Hadavi (M4I, FHML (Maastricht MultiModal Molecular Imaging; Faculty Health, Medicine & Life
19

Sciences, Maastricht University) for measuring the HRMS of all the compounds. Dr. Christian Bahn (MSP)
is also thanked for useful discussions on the stereochemistry of the complexes.
Associated Content
The supporting information is available free of charge. Supporting Information is available
containing the synthesis and characterisation of known complexes, Multinuclear NMR, IR
(experimental and calculated) and ESI-MS spectra of the compounds reported here and Cartesian
coordinates for the complexes studied by DFT methods.
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