A Look at Novel Ruthenium (II)

Complexes
By: Rebekah Karadeema

A Look at Novel Ruthenium (II) Complexes

Rebekah J. Karadeema
Department of Chemistry, University of Central Florida, 4000 Central Florida Blvd. Orlando, FL, 32826
Cancer, Ruthenium, Antioxidant, Complex, DNA binding, X-ray, Cytotoxicity

ABSTRACT: Cancer treatment is a tricky, arduous science that seeks to balance therapeutic potential and toxic side
effects. Platinum-based cancer treatments have proven useful; however, tumor cells can adapted to elude their
potency. In addition, these methods exhibit relatively high toxicity to healthy cells resulting in unintended side effects.
Therefore, new ruthenium based compounds are under intensive investigation as they have shown promising results to
be more effective than platinum-based methods as well as less toxic, making them ideal for chemotherapy. One
advantage of ruthenium compounds is their ability to accumulate specifically in rapidly dividing cells, such as tumor
cells. In addition to cancer fighting ability, the antioxidant activity is studied due to antioxidants promise to promote
longer life, and ruthenium complexes have proven useful for this purpose. Two new ruthenium complexes
[RuH(HL)(PPh3)2(CO)] (1) and [RuH(HL)(AsPh3)2(CO)] (2) (HL=2,2-bipyridine-5,5-dicarboxylic acid) will be discussed.
Complexes 1 and 2 were synthesized, characterized, and tested for their biological activity based on their ability to bind
DNA, cytotoxicity, and antioxidant activity. Intense study of these ruthenium complexes through H 1 NMR proved the
effective synthesis of these complexes, and X-ray Diffraction methods determined the geometry of the complex to be a
slightly distorted octahedron. While the ruthenium complexes exhibited weak DNA binding capabilities, they have
demonstrated promising results for being cytotoxic agents as well as excellent antioxidants. This study improves
previous ruthenium-based agents by the addition of carboxylic acids on the ligand, giving these complexes superior
radical scavenging capabilities and therefore superb antioxidant activity.

Introduction
With the fight against cancer at an all-time high,
developing more effective chemotherapeutic agents is of
great importance. An optimum cancer fighting
chemotherapy drug would have toxicity toward tumor
cells with little-to-no effect on the surrounding healthy
tissue. Cisplatin is a well-studied platinum based
chemotherapeutic drug widely used to treat a variety of
cancers. The mechanisms of action involved binding to
DNA, causing crosslinking and eventually apoptosis.
While this square planar platinum complex has shown its
efficacy as a cancer fighting drug, it has drawbacks such
as having low selectivity toward cancer cells and a high
susceptibility towards the tumor developing drug
resistance, causing relapse of the cancer1. To combat
this, developments in inorganic, organic, and biological
chemistry have worked toward the goal of creating new
integrated anti-cancer drugs.

Figure 1. Structure of Cisplatin

In order to avoid the drawbacks of cisplatin analogs,

non-platinum based compounds have been developed
and tested by the scientific community2,3. Ruthenium is
among the most promising metals used, because its
complexes demonstrate extremely high cytotoxicity to
tumor cells along with the keen ability to target cancer
cells while leaving healthy cells unharmed3. Ruthenium,
being in the same family as iron has a special ability to
mimic iron, allowing for the binding to transferrin, a
protein enzyme that binds and transports iron throughout

the body4,5. Cancer cells exhibit high levels of

metabolism to sustain their unregulated division and
growth. Because iron is an essential nutrient to these
metabolic processes, iron accumulates at much higher
levels than is found in healthy cells. Due to the high iron
use, cancer cells exhibit higher frequency of transferrin
importer proteins to sustain a constant inflow of ferrous
ions. Therefore, transferrin can act as a delivery device
of these cancer fighting ruthenium complexes towards
the cancer cells directly while avoiding the toxic side
effects of killing healthy tissues5.
a)
b)

Figure 2. Structures of the promising ruthenium

chemotherapeutic complexes a) NAMI-A and b) KP1019.
Reduced cytotoxicity to healthy tissue and less
sensitivity to resistance mechanisms of the tumor cells
are the fundamental obstacles of developing new cancer
treatments. In addition, compounds used to fight disease
should be ideally stable in air and light as well as soluble
for ease of transmission of the drug. Two specific
ruthenium (II) complexes (seen in Figure 2) have proven
to be effective cancer fighting tools in the laboratory and
have since started clinical trials6,7.
When testing new prospective antitumor compounds,
many different modes of targeting cancer must be

explored and tested. Many traditional inorganic based

cancer treatments work by interfering with DNA.
Metallintercalators wedge their way into double stranded
DNA, binding through noncovalent stacking interactions.
By residing in between base pairs of DNA, the sequence
undergoes a structural modification, as it is margninally
unwound. Because structure is so impactful on DNA
function, this change often leads to inhibition of
transcription, replication, and DNA repair processes,
making these intercalators potent mutagenic agents.
Distortions in DNA structure alter the recognition of DNA
repairing proteins which leads to downstream apoptosis
and cancer cell death8. For this reason, DNA has been a
major target for chemotherapeutic drugs.
However, some drugs that show high cytotoxicity to
cancer cells do not bind or interfere with DNA and act by
other mechanisms such as interfering with the
mitochondria or other processes9.
Drugs that are developed for cancer activity can also
be tested to determine their use in other purposes. For
example, antioxidant activity is of heightened interest as
this property can help slow down the process of aging
and life-limiting chronic diseases. All cells produce high
amounts of natural oxidative damaging species through
the generation of free radicals. Oxidative damage from
these free radicals can affect lipids in the cellular
membrane, protein structure and function, as well as
cause DNA damage, resulting in a myriad of health
implications. Cells have natural processes to counteract
oxidative stress caused by free radicals including the
superoxide dismutase enzymes which muffle the harmful
effects of superoxide radicals10. Synthetic antioxidant
and natural compounds have become medicinally
popular as they promise to slow down the aging process.
Based on these requirements, synthesis and
characterization of new ruthenium (II) complexes was
performed and their DNA binding properties, cytotoxic
capabilities on healthy and cancer cell lines, and
antioxidant activity was studied11.
Synthesis and Characterization
The aim of the development of new dicarboxylate
ruthenium (II) complexes is to create new antioxidants
and agents for the treatment of cancer. Inorporationg
carboxylic acid groups in the coordination complexes
increase the solubility, because they allow for efficient
hydrogen bonding over complexes with only nonpolar
ligands. Increased solubility helps in cell transport as
well as increasing anticancer and antioxidant activity12.
To accomplish this, 2,2-bipyridine-5,5-dicarboxylic acid
was selected as the ligand system and reacted with
[RuHCl(CO)(PPh3)3]/ [RuHCl(CO)(AsPh3)3] to form
complex (see Scheme 1)11.

Scheme 1. Synthesis of new ruthenium complexes

where E=Phosphorous or Arsenic atom.
These complexes were characterized and validated
through IR spectroscopy, 1H NMR, and the solid state
structure of complex 1 was determined by single crystal
X-ray crystallography. The IR spectra asserted the
probable mode of coordination of the ligand (2,2bipyridine-5,5-dicarboxylic acid) to the ruthenium atom.
The IR spectra changes from the ligand alone to the
ligand in complex with the metal allow for determination
of the place of attachment of the ligand. This attachment
was determined to be through the bipyridine derivative
nitrogen atoms, as confirmed by the substantial
decrement of C=N vibrational frequency11.
1
H NMR spectra of the ruthenium complexes showed
chemical shift, multiplicity, and intensity of the signals
characteristic for diamagnetic Ru(II) complexes. One
interesting thing that was found through the NMR
spectra was that of the two carboxylic acids, one was
deprotonated to the carboxylate form, while the other
remained in the carboxylic acid form.

Figure 3. X-Ray crystal structure and atom numbering for

complex 1 as thermal ellipsoids at 50% probability level.
Data from reference 11.

Single crystal X-ray analysis was used to determine

geometrical parameters (inter atomic angles and
distances) and hydrogen bond distances in order to
determine the structure of complex 1. The geometrical
angles led to the conclusion that the coordination

charge is likely to be repelled from binding with DNA due

to electrostatic repulsions with the negatively charged
phosphodiester backbone.
Cytotoxic Activity
Even though these Ru(II) complexes did not show
good DNA-binding affinity, they were not excluded from
cytotoxicity screening because they could still be
effective anti-cancer agents due to acting by
mechanisms other than DNA interactions. As an
example, the ruthenium complex NAMI-A, mentioned
earlier, acts by mechanisms other than DNA interaction,
but has still shown to be effective against lung
metastasis in vivo and tumor cell invasion in vitro14. MTT
and SRB assays were used to determine the cytotoxicity
of these complexes as compared to the cytotoxicity of
the
clinically
approved
and
widely
used
chemotherapeutic drug cisplatin, which acted as a
positive control. The assays are designed to give
information about the in vitro cytotoxicity of the newly
synthesized ruthenium complexes and were tested
against six human cancer cell lines (HeLa, Hep-G2,
HEp-2, SKOV3, SKMel2, and HCT-15) and one healthy
cell line (NIH3T3). The results from these assays were
expressed as a quantitative value of the concentration at
which there was 50% inhibition of growth (IC50).

Cytotoxicity against Cancer Cell Lines

50

45.49

HCT-15

40

IC50(M)

31.6
30
22.1

HeLa

Hep-G2

33.5

32.56
25.1

25.53
16.2

20

12.5

10
0
Complex 1

Complex 2

Cisplatin

Figure 4. Cytotoxicity chart of Complex 1, 2, and cisplatin

against HCT-15, HeLa, and Hep-G2 (cancer) cells. Data
adapted from reference 11.

Cytotoxicity against Hep-2 and NIH3T3 Cell

Lines
Hep-2
2457

3000

IC50(M)

geometry around the Ru(II) ion is slightly distorted

octahedron.
Steric
considerations
predict
the
triphenylphosphines to reside in the axial positions, as
observed. These PPh3 groups interact with the bulky
ligand to give a bond angle of P(1)-Ru(1)-P(2) of
166.58. The five member ring formed from the bidentate
coordination of the bipyridine derivative ligand with Ru(II)
produces a N(1)-Ru(1)-N(2) angle of 75.70. Unit cell
dimensions revealed the crystal system is monoclinic
and a part of the P21/n space group with single unit cell
dimensions of 0.25 x 0.20 x 0.10.
A 1D intermolecular hydrogen bonding network can be
formed with neighboring molecules and protic solvents
through the carboxylic acid/carboxylate groups. This
shows how the addition of carboxylic acid groups to
ruthenium complexes aid in their solubility, a clear
advantage for medicinal use.
DNA Binding Properties
Many different testing methods were employed to
determine if and how these complexes interact with
DNA, the main mechanisms for chemotherapeutic
cytotoxicity. Electronic absorption spectroscopy can be
used to determine if a compound intercalates between
the base pairs of DNA by the observation of
hypochromism, or enhancement of absorbance, with the
possibility of a red or blue shift after DNA is added11. The
magnitude of the hypochromism is usually directly
related to the strength of interaction between DNA and
the complex13. Complexes 1 and 2 exhibited weak
association with the helix, leading to the conclusion that
these complexes are not good DNA intercalators11.
Another way to check for DNA binding properties is
through the ethidium bromide (EB) competition assay.
EB is a known DNA intercalator, so if the complex being
studied displaces EB from DNA-bound EB, then it can be
considered a good DNA intercalator or DNA groove
binder. The addition of 1 and 2 did not displace EB
bound to DNA, indicating poor DNA binding ability11.
Viscosity studies were performed to determine if the
complexes bound, thereby lengthening the DNA due to
intercalation (as is observed with EB) and increasing the
viscosity. It was found that increasing the concentrations
of the complexes did not alter the viscosity of the DNA,
again concluding that these complexes are not DNA
intercalators. DNA melting experiments were conducted
to determine if the complexes bind to DNA, increasing
the stabilization of double stranded duplex, thereby
raising the melting temperature (Tm). Complex 1 showed
a 1C increase in Tm compared to that of DNA with no
metal complex added, but 2 did not share this property.
The slight increase in Tm along with results from the
previous mentioned DNA studies is not enough evidence
to conclude that the mechanism of action by these
complexes is interfering with DNA properties.
Therefore, it must be concluded that complex 1 binds
to DNA through outside binding of either the major or
minor groove11. Complex 1 showed higher affinity to
DNA than complex 2 and this was attributed to AsPh3
hindering the strength of binding more than the PPh3
ligand. In addition, weak DNA binding might be predicted
when considering that the 1H NMR revealed that one of
the carboxylic groups is deprotonated, the negative

NIH3T3

2000

1414

1000
248

301

177

0
Complex 1

Complex 2

Cisplatin

Figure 5. Cytotoxicity chart of Complex 1, 2, and cisplatin

against Hep-2 (cancer) cells and NIH3T3 (healthy) cells.
Data adapted from reference 11.

The results from the cytotoxic tests revealed the Ru(II)

complexes to have low to moderate anticancer activities
against the tested cells. The complexes exhibited low

tumor inhibiting activity against HEp-2, SKOV3, SKMel2,

and JCT-15 cancer cell lines, however they exhibited
similar results to cisplatin against HeLa, HCT-15, and
HEp-G2 cell lines (See Figure 4). The ligand alone and
ruthenium precursors did not show any significant
cytotoxicity to any of the cell lines, so all cytotoxic effects
should be attributed to the properties of these molecules
when they are in complex.
The results are particularly promising because the
complexes showed to be very specific in targeting
cancer cells over healthy cells. IC50 values of the
complexes against the healthy NIH 3T3 cells were found
to be considerably higher than the IC50 values for
cisplatin against the healthy cells. This shows that in
order for the complexes to affect the healthy cells, they
must be at 8-14 times higher concentration than
cisplatin. However, the complexes proved to be less
effective in killing the cancer cell lines than cisplatin as is
seen in the higher IC50s for the complexes (See Figure
5). The cytotoxicity studies showed that complexes 1
and 2 have decent cancer fighting capabilities with the
key advantage of keen ability to select for cancer cell
lines over healthy cells.
Antioxidant Activity
Complexes 1 and 2 were tested for their antioxidant
activity as hydrogen donors or free radical scavengers 11.
The ability of the ruthenium complexes to act as effective
antioxidants was done through a series of in vitro
antioxidant assays against for the radicals DPPH, OH,
NO, H2O2, and O2-. In addition, the ability of complexes
to chelate ferrous atoms was determined through a
metal chelating activity assay. Complexes 1 and 2 were
tested alongside the ruthenium complex precursors
[RuHCl(CO)(PPh3)3],
[RuHCl(CO)(AsPh3)3],
2,2bipyridine-5,5-dicarboxylic acid (H2L ligand), and known
antioxidants including Vitamin C and butylated
hydroxytoluene (BHT).
500
400

IC50 (M)

300
200
100

Antioxidant Activity Chart

Metal Chelating
DPPH
OH
NO
O2-
H2O2

Figure 6. Antioxidant activity of the complexes 1, 2,

precursors, ligands, vitamin C and BHT against various
radicals. Data adapted from reference 11.

The data shows that complexes 1 and 2 exhibited

lower IC50 values and therefore better antioxidant activity
than the precursor complexes and the ligand alone. In
addition, complexes 1 and 2 demonstrated significantly
better antioxidant activity than known antioxidants
Vitamin C and BHT. This is likely due to the presence of
free carboxylic acid groups on complexes 1 and 2 that
make these complexes efficient hydrogen donors, aiding

in the stabilization of the unpaired electrons and thereby

scavenging the free radicals.
In addition, the complexes showed moderate to high
metal chelating abilities. The proposed mechanism is
chelation of Fe2+ ions by the electron rich COO- anion
and the uncoordinated COOH group. Although the
unattached ligands demonstrated similar chelation
ability, the increase in chelation of the complexes is likely
due to the coordination of the organic bidentate
carboxylic acid functionalized bipyridine ligand with the
Ru(II) ion.
Conclusion
Two new ruthenium(II) complexes have been studied
and exhibit significant cytotoxic and antioxidant activity.
The unique ligand allowed for the presence of carboxylic
acid/carboxylate groups in the ruthenium complex, aiding
in the solubility and allowing them to act as hydrogen
donors, increasing their effectiveness as antioxidants.
The complexes were characterized by analytical and
spectral methods, and X-ray diffraction was used to
characterize complex 1 in the solid state. The crystal
structure revealed a distorted octahedral geometry
around the ruthenium ion with the PPh3 groups in the
axial positions. The complexes were tested for their DNA
binding abilities; however, neither complex proved to be
effective at binding DNA, indicating that the complexes
are cytotoxic by an alternative mechanism. The
complexes did demonstrate strong selectivity towards
targeting cancer cell lines over healthy cell lines along
with moderate cytotoxicity, giving promise as
chemotherapeutic agents with reduced side effects. The
complexes show promise as chemotherapeutic agents
due to their reduced side effects, as evidenced by a
strong selectivity towards cancer cells over healthy cells.
The complexes were generally not as effective as the
standard antitumor drug, cisplatin, but did exhibit
comparable cytotoxicity against HeLa and Hep-G2
cancer cell lines. When tested for their antioxidant and
metal chelating abilities, the in vitro results showed
complexes 1 and 2 to have excellent free radical
scavenging capabilities, even outperforming standard
antioxidants such as Vitamin C. This study showed that
alternative ligand structures with functional groups
similar to carboxylic acids may be a good avenue for
further research in finding and developing cancer fighting
and antioxidant agents.

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