Current Medicinal Chemistry, 2006, 13, 1085-1107
1085
Ruthenium Complexes as Anticancer Agents
Irena Kostova*
Department of Chemistry, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria
Abstract: Cancer is one of the major cases of death in the world. Current treatment of cancer is limited to surgery,
radiotherapy, and the use of cytotoxic agents, despite their well known side effects and problems associated with the
development of resistance. For most forms of disseminated cancer, however, no curative therapy is available, and the
discovery and development of novel active chemotherapeutic agents is largely needed. Since the development of cisplatin,
an inorganic platinum complex, numerous platinum and non-platinum metal complexes were synthesized and tested for
anticancer activity. Very few match the clinical efficacy of cisplatin. Ruthenium complexes were prepared to ameliorate
cisplatin activity, particularly on resistant tumours, or to reduce host toxicity at active doses. Since many years a lot of
scientific groups have actively worked in the field of inorganic antitumor drugs and have developed a number of Ru(II)
and Ru(III) complexes, which were shown to possess good antitumor and, above all, antimetastatic properties against
animal models. Ruthenium complexes are presently an object of great attention in the field of medicinal chemistry, as
antitumor agents with selective antimetastatic properties and low systemic toxicity. Ruthenium compounds appear to
penetrate reasonably well the tumor cells and bind effectively to DNA. In this review, the achievements in the field of
medicinal chemistry, DNA binding modes, and the development status of Ru(II) and Ru(III) complexes as anticancer
agents are discussed. The aim of this review is therefore that of critically examining the past and the actual work on
ruthenium compounds with emphasis on their proposed role in cancer therapy.
Keywords: Anticancer agents, ruthenium, coordination complexes, cytotoxic activity, tumor cell lines.
INTRODUCTION
Cancer is a genetic disease resulting from faulty DNA.
Mutations can occur in genes, causing normal cells to
become cancerous. More specifically, a defective gene
leading to increased cellular proliferation in one cell can be
passed down to a daughter cell. The accumulation of
mutations in subsequent generations of daughter cells can
cause cells to proliferate even more rapidly and eventually
undergo structural changes to become malignant. Cancer
cells are believed to result from at least two genetic
mutations to a normal cell. These mutations cause the cells
to divide uncontrollably. This uncontrolled cell growth may
be lethal if it is not treated. It is important to stress that
metastases of solid tumors represent the main reason of
failure in cancer therapy. In fact, while surgery and/or
radiotherapy may successfully cure the primary lesions,
many human tumors develop distant metastases that bring
almost invariably to death. Because of the scattered location
of metastases, drug therapy appears to be the best choice
and, from the therapeutic standpoint, the development of
new compounds endowed with a specific antimetastatic
activity is a topic of paramount importance. New drugs are
constantly being screened for their potential anticancer
properties. Among the category of new drugs that are
receiving much attention are metal based drugs.
At present, the development of new drugs against cancer
belong among priorities of the development of science and
fundamental research. A successful development of these
drugs is conditioned by the existence of extensive theoretical
*Address correspondence to this author at the Department of Chemistry,
Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000,
Bulgaria; Tel: 92 36 569; Fax: 987 987 4; E-mail: irenakostova@yahoo.com
0929-8673/06 $50.00+.00
background, which contains in particular data on the
mechanism of action of these compounds. In recent years
metal-based antitumor drugs have been playing a relevant
role in antiblastic chemotherapy. Especially cisplatin is
regarded as one of the most effective anticancer drugs used
in the clinic. Thus, the research, development and production
of metal-based drugs is at present a very active international
field. In spite of the great efficacy of cisplatin, carboplatin or
oxaliplatin against ovarian, bladder and testicular cancers,
these drugs display limited activity against some of the most
common tumors, such as colon and breast cancers. In
addition, a variety of adverse effects and acquired resistance
are observed in patients receiving cisplatin chemotherapy.
The great success of c i splatin on one hand and these
limitations on the other have initiated efforts to develop new
agents that will display improved therapeutic properties. The
goals for the synthesis of new platinum complexes thus
remain activity in cisplatin-resistant cells, an altered
spectrum of antitumor activity and reduced adverse effects.
Drug modifications may further allow for reduced
cytotoxicity and altered mode of delivery. Another approach
in the search for new, metal-based anticancer agents is to
examine complexes that would contain another transition
metal. In the design of these new drugs, ruthenium
complexes have raised great interest.
The most important aspect is to examine and compare the
mechanisms of action of metal complexes structurally
distinct from cisplatin. Notwithstanding the widespread
applications of platinum anticancer drugs, there is still a
large need for the development of novel metal-based
compounds with unprecedented features. The search for
“non-classical” metal antitumor drugs has since long
stimulated investigations into the field of non-platinum metal
drugs. Non-platinum active compounds are likely to have
mechanism of action, biodistribution and toxicity which are
© 2006 Bentham Science Publishers Ltd.
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Current Medicinal Chemistry, 2006, Vol. 13, No. 9
Irena Kostova
different from those of platinum drugs and might therefore
be active against human malignancies that are resistant, or
have acquired resistance, to them. They might also show
reduced host toxicity. Ruthenium seems to be the most
promising among the several metals investigated.
platinum. These metals may differ in oxidation state, ligand
affinity, and substitution kinetics. Because of their variation
in chemical characteristics, the mode of action and spectrum
of activity of these compounds can differ significantly from
cisplatin.
The antitumor activity of platinum and ruthenium drugs
is generally accepted to involve binding to DNA. These
drugs form adducts with DNA which block DNA and RNA
synthesis and induce programmed cell death. Thus,
intracellular interactions with these adducts are likely to be
of central importance in explaining their toxicity towards
rapidly dividing tumor cells. Development of structurally
novel drugs derived from the platinum-group metals is
focused on those complexes which may act by different
mechanisms than c i splatin. The structure-activity
relationships originally delineated for platinum complexes
stressed the necessity for the cis-[PtX2(amine)2] structure,
where X is a leaving group such as chloride and amine
represents ammonia or a primary or secondary amine group.
The trans isomer of cisplatin (transplatin) and monodentate
charged complexes (for instance chlorodiethylenetriamineplatinum(II) chloride) are considered inactive, but this need
not to be true for their analogues. These relationships have
always been accepted to be empirical in nature but initially
allowed the synthesis and study of a manageable number of
complexes, albait of similar structure. However, the DNA
adducts formed by all simple cis-diammine(Pt) analogues are
similar to those of cisplatin and it is unclear whether these
compounds will have activity complementary or superior to
cisplatin in the clinic.
The transition elements, to which the Group 8, 9 and 10
metals belong, show typical chemical behavior that is not
shared by the main-group elements. All are metals with
partly filled d or f subshells that conduct heat and electricity
well, with very few exceptions they exhibit variable valence,
and their ions and compounds are colored in most oxidation
states. Because of their partly filled subshells, they form
complexes easily, and bonding is usually of a more ionic
than covalent nature. Groups 8, 9 and 10 are composed of
iron, cobalt and nickel in the first transition series, in which
the 3d subshell is partly filled. Ruthenium, rhodium and
palladium make up the second transition series, in which the
4d subshell is partly filled, and osmium, iridium and
platinum form the third transition series, with a partly filled
5d subshell. Especially the extended 4d and 5d orbitals give
rise to good complex-forming abilities, often strengthened by
-bond formation (back-donation). Therefore, Groups 8, 9
and 10 are subdivided into the iron group (first transition
series) and the platinum group (second and third transition
series). As the metals of Groups 8, 9 and 10 have many
different valences and form complexes easily, a multitude of
compounds can be synthesized with a wide variety of
ligands. Thus far, a lot of new compounds of these metals
have been proven active in vitro against different types of
tumor cell lines and can thus be marked as potential new
anticancer agents. Among them, ruthenium complexes seem
very promising.
The long term goal of the studies in this field is to
develop a systematic approach to the synthesis of metalbased drugs with unique DNA binding activities capable of
overcoming the problem of cellular resistance to cisplatin
and of limited activity against the most common tumors,
such as gastrointestinal and breast cancers. An important
aspect of this approach is to find the DNA adduct of
platinum and ruthenium most likely relevant to their
antitumor effect and the factors controlling its formation.
The interactions of the active Ru complexes with their likely
biological targets, i.e. DNA and proteins (in particular
transferrin and albumin) were largely investigated [1]. In
general, Ru(III) complexes bind DNA but much more
weakly than platinum complexes. Thus the structural and
conformational modifications produced on the DNA double
helix are significantly smaller. Under physiological
conditions, antitumor Ru(III) complexes bind tightly plasma
proteins (albumin and transferrin), with a marked preference
for surface imidazole groups; thus, very likely, protein
binding of ruthenium(III) complexes has a large impact on
the biodistribution, the pharmacokinetics and the mechanism
of action of these experimental drugs.
RUTHENIUM CHEMISTRY
The success of cisplatin [cis-diamminedichloroplatinum
(II)] as an anticancer agent has stimulated the search for
cytotoxic compounds with more acceptable toxicity profiles,
but retention, and if possible expansion, of activity. This has
generated interest in molecules containing other heavy
metals of Groups 8, 9, and 10 (formerly known as Group
VIII) of the periodic table, which have similar properties to
The chemistry of ruthenium complexes, with special
attention to their electron-transfer properties, has been
receiving continuous attention for the latest decades.
Ruthenium offers a wide range of oxidation states which are
accessible chemically and electrochemically (from oxidation
state -2 in [Ru(CO)4]2- to +8 in RuO4). Therefore, the
complexes of ruthenium are redox-active and their
application as redox reagents in different chemical reactions
is of much current interest. The kinetic stability of ruthenium
in several different oxidation states, the often reversible
nature of its redox couples, and the relative ease with which
mixed-ligand complexes can be prepared by controllable
stepwise methods, all make ruthenium complexes
particularly attractive targets of study.
Ruthenium complexes exhibit a great deal of applications
in many fields of chemistry. Clear correlations can be
observed between their properties and the nature of the
ligands bound to the central ion. Thus, ruthenium sulfoxide
complexes have been extensively investigated in the last two
decades because of their properties and usefulness,
particularly in catalysis and chemotherapy. Ruthenium
complexes with polypyridyl ligands have received much
attention owing to their interesting spectroscopic,
photophysical, photochemical and electrochemical
properties, which lead to potential uses in diverse areas such
as photosensitizers for photochemical conversion of solar
energy, molecular electronic devices and as photoactive
DNA cleavage agents for therapeutic purposes. Ruthenium
complexes are also known to perform a variety of inorganic
Ruthenium Complexes as Anticancer Agents
and organic transformations. Their synthetic versatility, high
catalytic performance under relatively mind reaction
conditions, and high selectivity make these complexes
particularly well suited for this purpose.
A wide range of ruthenium agents has been synthesized
and tested for their antitumor properties in the past 30 years.
Most of these agents, independent of the ligands attached to
the ruthenium ion, have shown relatively low cytotoxicity
and are less toxic than cisplatin, correspondingly requiring a
higher therapeutic dose. Extensive binding to many cellular
and extracellular components may account for this fact.
Despite their low cytotoxic potential, many ruthenium
complexes increase the lifetime expectancy in tumor-bearing
hosts. Ruthenium(III) complexes likely remain in their
(relatively inactive and unreactive) Ru(III) oxidation state
until they reach the tumor site. In this environment, with its
lower oxygen content and pH than healthy tissue, reduction
to the more reactive Ru(II) oxidation state takes place. In this
manner (termed “activation by reduction”), ruthenium(III)
compounds may provide selective toxicity. However, to be
active in vivo, the complexes must have a biologically
accessible reduction potential, which can vary considerably
with the ligands present. A second mechanism that could
explain the observed antitumor activity is the high affinity of
ruthenium(III) for the transferrin iron-binding sites. This
binding capacity provides a possibility to target
ruthenium(III) complexes to tumors with high transferrin
receptor densities. A large number of ruthenium complexes
(both in the II and III oxidation states) with in vitro
antitumor activity have been synthesized.
CHARACTERIZATION OF TUMOR-INHIBITING
RUTHENIUM COMPLEXES-COMPARISON WITH
CISPLATIN
25 years after the first approval of cisplatin in the clinic
against a number of cancer diseases, cisplatin and related
compounds continue to be among the most efficient
anticancer drugs used so far. However, direct structural
analogs of c i splatin have not shown greatly improved
clinical efficacy in comparison with the parent drug. The
explanation for this finding is that all cis-[PtX2(amine)2]
compounds have shown similar DNA-binding modes,
thereby resulting in similar biological consequences. One
approach is to look beyond structure-activity on the basis of
cisplatin analogs antitumor agents, by identifying novel
materials that can be utilized as building blocks. These may
have DNA binding modes quite different from that of
cisplatin. The introduction of such aromatic N-containing
ligands as pyridine, imidazole and 1,10-phenanthroline, and
their derivatives (whose donor properties are somewhat
similar to the purine and pyrimidine bases) to antitumor
agents is drawing attention. Many platinum and nonplatinum metal complexes with these aromatic N-containing
ligands, have shown very promising antitumor properties in
vitro and in vivo in cisplatin-resistant model systems or
against cisplatin-insensitive cell lines.
Efforts are focused to develop novel platinum- and nonplatinum-based antitumor drugs to improve clinical
effectiveness, to reduce general toxicity and to broaden the
spectrum of activity. In the field of non-platinum compounds
exhibiting anticancer properties, ruthenium complexes are
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1087
very promising, showing activity on tumors which developed
resistance to cisplatin or in which cisplatin is inactive.
Furthermore, general toxicity was found to be very low.
Metal complexes of ruthenium were subjected to a number
of studies concerning their chemical behaviour and their
potential role in medical applications [2-7]. Particular
emphasis was given to the examination of the antineoplastic
properties of ruthenium complexes with a number of ligands
of biological interest. The possibility of obtaining
compounds of potential value in the chemotherapeutic
approach to neoplastic disease is supported by observations
that ruthenium compounds could interact with tumor cells
better than with normal tissues. This interaction can be
considered the result of the chemical characteristics of
ruthenium ions which can confer much more selectivity than
do the actually available and clinically used organic
compounds or cis-dichlorodiammineplatinum (II). Thus,
ruthenium-based compounds represent the way for
introducing a new class of antitumor drugs endowed with a
great potential for the management of human tumors.
A number of ruthenium complexes showed in vitro and
in vivo antitumor activity in different models including a
Cisplatin-resistant cell lines, many of them reduce metastasis
formation [8, 9]. Ru(III)-complexes exhibited the best results
in antitumor tests, as well as in an autochthonous colorectal
carcinoma of rats, a model that resembles the colon cancer of
humans in its histological appearance and its behavior
against chemotherapeutics [10]. In comparison, the well
established antitumor drug Cisplatin, cis-[PtCl2(NH3)2], was
completely inactive in this model. If one is aware of the high
percentage of cancer mortality caused by tumors of the colon
and the fact that no satisfactory chemotherapy exists, it is
clear that development of these ruthenium-based drugs is of
outstanding importance.
Although many groups are working on platinum based
antitumor drugs since the discovery of the tumor-inhibiting
qualities of Cisplatin by Barnett Rosenberg in 1969, a
conclusive mode of action has not been found as yet. Even
less is known about non platinum antitumor drugs like Rucomplexes and their mode of action. It should be of special
interest to enhance knowledge in that field as it might lead to
a better understanding of the differences between these
metalbased drugs concerning toxicity or selectivity for
different tumors. Therefore galenic formulation, hydrolysis
reactions, interaction with serum proteins and reactions
taking place in the cell (redox reactions, binding towards
DNA, RNA and other occurring target molecules) have been
investigated [11]. The obtained results could lead to new
improved drugs with enhanced activity and selectivity and
reduced side effects.
Hydrolysis Reactions
The knowledge of the rate of hydrolysis and occurring
hydrolysis products is also of interest with regard to storage
and clinical application of this and other complexes as well
as to further reactions of the drugs in blood and cell.
Cisplatin is usually administered intravenously rather
than orally because of solubility problems. Once in the
bloodstream, cisplatin diffuses across the cell membranes
into the cytoplasm. The intracellular Cl- concentration is less
than that beyond the cell walls, so a complex equilibrium
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Current Medicinal Chemistry, 2006, Vol. 13, No. 9
Irena Kostova
cis-Pt(NH3)2Cl2
+Cl-
-Cl-
+H+
cis-Pt(NH3)2Cl(H2O)+
cis-Pt(NH3)2Cl(OH)
-H
+
+Cl+H+
cis-Pt(NH3)2(OH)2
-Cl-
+H+
cis-Pt(NH3)2(H2O)(OH)+
-H+
cis-Pt(NH3)2(H2O)22+
-H+
Oligomers
Scheme 1. Equilibrium process for cisplatin in cancer cells.
process is set up (see Scheme 1 ). Cationic platinum
complexes, such as [Pt(NH3)2(OH2)Cl]+, are formed when a
water molecule attacks the platinum metal centre, thus
eliminating a chloride ion which acts as a non-coordinating
anion. The cell essentially traps the cisplatin by transforming
it into a cationic component of a neutral molecule. After
losing two Cl- ions, hydrolysed cisplatin reacts with DNA,
forming coordinative bonds to nitrogen atoms of the
nucleobases. The active species in the cell is thus (NH3)2Pt2+,
not cisplatin. The binding of (NH3)2Pt2+ to DNA leads to
changes in the DNA structure. NMR studies indicate that the
Pt2+ binds to N7 atoms of a pair of guanine bases on adjacent
strands of DNA. (NH3)2Pt2+ creates a unique junction
between the strands. This 'local distortion' leads to an
impairment in the processing of DNA in tumour cells. The
distortion is 'characterised' by a High Mobility Group
(HMG), i.e. is an 80 amino acid sequence found in many
proteins that bend DNA dramatically [12].
In the patent [13] fifteen medicaments were described
which contain ruthenium(III) complexes with a monocyclic
or multi-cyclic basic heterocycle. The compounds
investigated were tested on P 388 Leukemia model;
Leukemia 1210 model; Melanoma B16 model and AMMNinduced autochthonous carcinoma of the colon. The
complexes had an advantageous antitumoral activity coupled
with favorable toxicity. They were therefore suitable as
chemotherapeutics for the treatment of cancers. As
chemotherapeutic agents with few side effects, they were
suitable for the treatment of tumors, for example ovarian
tumors, mammary tumors, stomach tumors, prostate tumors,
lung tumors, bladder tumors and, in particular, colorectal
tumors, and other malignant neoplasms. The compounds
were accordingly useful for alleviating pain and suffering
associated with cancer therapy, for inhibition and regression
of tumors and for alleviating symptoms and increasing life
expectancy. The complexes were suitable for cancer therapy,
but were difficult to dissolve in water and were therefore not
lyophilisable.
In the ruthenium metal class, HIm trans- [RuCl4(im)2],
Fig. 1 and HInd trans-[RuCl4(ind)2], Fig. 2 are two of the
few promising anticancer complexes to date. In the first
complex a ruthenium atom is coordinated to two imidazole
ligands and is also bonded to 4 chloride atoms. The second is
HInd trans-[RuCl4(ind)2], which has indazole ligands in
place of the imidazoles. The synthesis and anticancer activity
of these metal complexes was first described by Keppler et
al. (1989) [9]. Keppler et al. investigated the hydrolysis of
these Ru(III)-complexes under different conditions, like
varying temperature, pH or NaCl concentration by means of
UV/VIS, NMR-spectrometry, HPLC, pH- and conductivity
measurements [14-16]. Both the complexes possess
significant anticancer activity against the Walker 256
carcinosarcoma, MAC 15A colon tumour, B16 melanoma
and solid sarcoma 180. These compounds were more
superior in their action against an autochthonous chemicallyinduced colorectal adenocarcinoma in rats compared to even
5-fluorouracil, which is an established cytostatic drug against
human gastrointestinal carcinomas. Fruhauf and Zeller
(1991) [17] observed that HInd trans-[RuCl4(ind)2] brings
about antitumour activity by interacting with DNA and
inhibiting DNA synthesis.
NH
H
N
N
Cl
Cl
Ru
Cl
HN
Cl
N
HN
Fig. (1). Chemical structure of HIm trans- [RuCl4(im)2].
The complex HIm trans- [RuCl4(im)2] is an active agent
with a new mechanism effect and concerning to other current
standard therapies shows in vivo a plain predominance
against colorectal carcinoma cells and indicate activity
against cisplatin resistant tumours with very low side effects
[13]. This complex is the most promising cytostatic drug
among several analogous metal complexes which were
developed in the last years. Among these compounds it has
Ruthenium Complexes as Anticancer Agents
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
trans-[RuCl4(ind)2] suggest that hydrolysis proceeds slower
but leads temporary also to aquacomplexes. The crystal
structure of a monoaqua complex of the corresponding 1methylindazole complex could be resolved [16]. As the
formation of aqua complexes results in leaving chloride ions,
chloride ion (physiological saline) concentration is another
parameter to play a role in hydrolysis equilibria. Further
hydrolysis products are unknown but could be -oxocomplexes. Formation of such di- or polynuclear Rucomplexes is pH dependent, with hydrolysis proceeding
faster at higher pH, leading to precipitation. Scheme 2 shows
some possible hydrolysis reactions and decomposition
compounds of the complex anion trans-[RuCl4(ind)2]–.
the highest therapeutic index due to its strong antitumour
activity and very slight toxicity. The tumour inhibiting effect
has been proved in vitro on human colorectal carcinoma cell
lines and on a series of freshly explanted human tumours
[13]. Most remarkably, the effect of HIm trans- [RuCl4(im)2]
significantly exceeded that of 5-fluorouracil which is still the
standard agent in chemotherapy of colorectal cancer at
present. First results of a clinical trial phase I with this
compound and encouraging phase II trials in several solid
tumour indications, including liver, colon, head and neck and
endometrial cancers showed that this complex has been well
tolerated in the doses studied. The rights of HIm trans[RuCl4(im)2] are protected by the patent [13]. The patent
covers the anionic ruthenium(III) complexes with different
counter ions and their use for the treatment of cancers.
Interaction of Ru(III) Complexes with Serum Proteins
Interaction of drugs with serum proteins plays an
important role in distribution of the drug in the body and
affects features like toxicity and biological activity. Animal
experiments in the autochthonous colon cancer model in rats
have shown that HInd trans-[RuCl4(ind)2] was far less toxic
than HIm trans-[RuCl4(im)2] but also exhibited a slightly
higher antitumor activity. This difference in toxicity might
be related to the different protein binding ability of the two
compounds, assuming that the free complex is responsible
for systemic toxicity. In comparison, over 90% of the
platinum found in blood 3-4 h after administration of
Cispaltin (25% in the case of Carboplatin) is bound
irreversibly to plasma proteins, and the protein bound
species have no significant antitumor activity and are not as
toxic as Cisplatin. The loss of activity of Cisplatin when
bound to plasma proteins is probably due to the irreversible
binding to cysteines of plasma proteins such as albumin.
H
N
NH
HN
N
Cl
Cl
Ru
Cl
Cl
N
HN
Fig. (2). Chemical structure of HInd trans-[RuCl4(ind)2].
The complex salt HIm t r a n s- [RuCl4(im)2] was
investigated in water and solvents like DMSO and ethanol
by UV [14] and NMR [15]-spectroscopy. Aquation of the
imidazole complex leads to mono- and diaqua complexes
and eventually to a trisimidazole complex (reaction with the
imidazolium counterion). Initial aquation to a monoaqua
complex seems to play a crucial role for further reactions,
since reaction with biological substrates is much faster in
“aged” solutions of HIm trans-[RuCl4(im)2] than in “fresh”
solutions. First investigations into the hydrolysis of HInd
Cl
Cl
Ru
L
Cl
Cl
Ru
L
OH2
OH2
Cl
H2 O
Cl-
Cl
Cl
Cl
Ru
OH2
L
OHClH2O H+
L
Cl
First investigations showed that Hind trans-[RuCl4(ind)2]
binds within a few minutes to the serum proteins albumin
(Mr: 66.5 kDa) and transferrin (Mr: 80 kDa, responsible for
iron transport) [18]. Interpretation of the results suggests that
apo-transferrin (the “iron-free” form of the protein)
specifically binds two equivalents of HInd t r a n s [RuCl4(ind)2] . Albumin seems to bind five equivalents. Xray crystallographic structure analysis of crystals of
structurally transferrin-related human apo-lactoferrin that
L
L
OR
OH2
Cl
L
Ru
L
Cl
OH2
L
Cl
Cl
Ru
Cl
OH
L
-H2O
L
polynuclearcomplexes
1089
Cl
Cl
Ru
L
L
Cl Cl
O
Ru
2
Cl
Cl
L
Scheme 2. Possible hydrolysis pathways and decomposition species of the complex anion trans-[RuCl4L2]-.
1090
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
were treated (soaked) with solutions of HInd trans[RuCl4(ind)2] and HIm trans-[RuCl4(im)2], was used to
obtain binding sites for the Ru-complexes by difference
Fourier analysis [19]. These investigations show that binding
can not only specifically occur at the C- and N-lobe ironbinding cleft of the protein, but also at histidine residues on
the surface of the protein.
It is assumed that the Ru-complexes can be transported to
the tumor cell via the transferrin cycle. Because tumor cells
have an increased requirement of iron , they have a higher
number of transferrin receptors than normal cells. This
enables accumulation of the Ru-complexes selectively in
tumor cells (indirect drug targeting). Thus, further
investigations into protein binding of different Ru-complexes
should focus on different serum proteins, like albumin and
transferrin, as well as on separation of protein fractions of
whole serum samples, incubated with Ru-compounds.
Besides mode and rate of binding, release of ruthenium from
the protein, especially from transferrin, is of interest, because
the free, maybe structurally transformed, Ru-complex should
be responsible for antitumor activity and further reactions in
the tumor cell.
Interaction of Ru(III) Complexes with Cytochrome c
While the initial DNA binding site of many ruthenium
comlexes is the same as that of cisplatin, i.e. the N7 site of
Gua, their antitumor mechanism is most likely distinctly
different [20, 21]). HInd trans-[RuCl4(ind)2] complex, Fig. 2
for a long time have been investigated by the group of
Bernard Keppler, showing encouraging pharmacological
properties and low toxicity (Keppler et al. 1990 [22]). This
complex has now entered phase I clinical trials.
The disclosure of the complex HInd trans-[RuCl4(ind)2]
has been presented and the rights of it are protected by the
patents [23, 24]. The object of these inventions was to avoid
the disadvantages of [13] and to provide a composition
which is easily water-soluble and which exhibits a high
effectiveness in the treatment of cancer illnesses. The
complex was tested on the human lines of tumor cells
SW480 (colon carcinoma), CH1 (ovarian carcinoma) and
SW480 (colon carcinoma) in the MTT assay with continuous
96 hour exposure to the active substance. Further
experiments gave insight into the manner in which an excess
of indazol affected the cytotoxicity. It has been surprisingly
shown that the tumor inhibiting activity can be further
increased by the addition of an excess of indazol.
In preclinical investigations, the activity of HInd trans[RuCl4(ind)2] complex was observed against non-small cell
lung, breast and renal cancers (Depenbrock et al. 1997 [25]).
This has inspired considerable interest in the study on the
biochemical behavior of the HInd trans-[RuCl4(ind)2]
complex including its interactions with proteins. The binding
to proteins might result in drastic modifications or even loss
of the biological activity of the starting biomolecules [18,
19]. The major fraction of ruthenium(III) species (80–90%)
is bound to albumin and a much smaller amount to
transferrin. It has been proposed that cell surface transferrin
receptors bind ruthenium-loaded transferrin with high
affinity; the transferrin-receptor complexes are subsequently
endocytosed and transferred to acidic non-lysosomal
compartments where ruthenium is released (Klausner et al.
Irena Kostova
1983 [26]). Binding of ruthenium(III) species has a strong
impact on albumin structure and influences considerably its
binding of other molecules including drugs (Trynda-Lemiesz
et al. 2000 [27]).
The preferred binding sites for Ru(III) complexes are
histidine residues of the proteins (Smith et al. 1996 [19];
Yocom et al. 1982 [28]). It is known that at pH 7 a stable
complex between Ru(III) and histidine-33 in ferricytochrome
c is formed [28]. In previous investigations (Tian et al. 2000
[29]) ruthenium-cytochrome c derivatives were used to
define the interaction domain for cytochrome c on the
cytochrome bc1 complex. Cytochrome c is a mitochondrial
peripheral membrane protein functioning in the respiratory
chain in the inner mitochondrial membrane, shuttling
electrons from cytochrome c reductase to cytochrome c
oxidase. However, in 1996 it was found that cytochrome c,
when released from mitochondria to the cytosol, activates a
programmed cell death cascade (apoptosis) (Cai et al. 1998
[30]). The binding of the ruthenium complex to cytochrome
c may change considerably the structure of the protein and
affect its biological function.
The effect of HInd trans-[RuCl4(ind)2] on the
conformation of cytochrome c , its heme state, the
interactions with apocytochrome c and cytochrome c dimer
formation have been examined [20]. For this purpose, gelfiltration chromatography, absorption second derivative
spectroscopy, circular dichroism (CD) and inductively
coupled plasma atomic emission spectroscopy (ICP (AES))
methods have been used. The data reveal that binding of
ruthenium complexes (a potential new metallopharmaceutical) to cytochrome c can induce a conformational
change of the protein with a loss of organized tertiary
structure, change of the heme group state, and increase in the
-helical content of apocytochrome c. It seems plausible that
the coordination of the HInd trans-[RuCl4(ind)2] complex
and the consequent conformational changes can influence the
biological function of cytochrome c.
Binding of Ru-Complexes to Oligo- and Polynucleotides
As in the case of platinum complexes, interaction of Ru
complexes with DNA is assumed to be responsible for
antitumor activity, although other additional or
supplementary mechanisms are possible as well [31-38].
Investigations into binding of HInd trans- [RuCl4(ind)2]
and HIm trans- [RuCl4(im)2] towards calf-thymus DNA and
the synthetic double-stranded homopolymers poly(dG)
.poly(dC) and poly(dA).poly(dT) have been carried out using
UV/VIS- and ICP-AES [31, 32]. Both complexes bind
covalently to calf-thymus DNA and show a binding
preference for poly(dG) . poly(dC) compared to poly(dA) .
poly(dT).
Further investigations are necessary to obtain a deeper
insight into binding of the Ru-complexes to nucleobases and
possible DNA intra- or interstrand cross linking properties.
The use of NMR-techniques, helpful in the case of platinum
complexes, is limited because of paramagnetism of Ru(III)compounds. Also of great importance will be the knowledge
of redox reactions, taking place in the hypoxic milieu of the
tumor cell, as the resulting Ru(II)-species should exhibit
other ligand, nucleophile preferences than Ru(III)complexes. Thus, Ru(III)-complexes have to be seen as
Ruthenium Complexes as Anticancer Agents
prodrugs, being transformed into antitumor active species in
the body by hydrolysis and redox reactions and reactions
with biologically occurring nucleophiles.
Topoisomerase II Poisoning by Indazole and Imidazole
Complexes of Ruthenium
DNA is a very dynamic molecule and during the lifetime
of a cell, it constantly undergoes various topological changes
without affecting its genetic makeup. Numerous topological
problems like negative/positive supercoiling and catenation
arise in DNA during replication and transcription, which
causes intertwining of DNA [39]. This intertwining is
resolved by a class of enzymes called topoisomerases, which
thus play important roles in maintaining genome integrity
(Wang 1985 [40], 1991 [41], 1996 [42]; Watt and Hickson
1994 [43]; Pruss and Drlica 1986 [44]). These enzymes are
also involved in decatenation of DNA in the G2 phase of cell
division for separation of newly replicated chromatids
(Downes et al. 1994 [45]). In the M phase, they help in
chromosome condensation and segregation (Adachi et al.
1991 [46]). Of the two types of topoisomerases (type I and
type II) the type II enzymes are most important for cell cycle
progression and survival of dividing cells. The catalytic
cycle of topoisomerase II (topo II) typically involves
breaking both strands of a duplex DNA segment, passing
another duplex segment through the gate created by the
broken DNA strands and finally resealing the broken strands
(Berger et al. 1996 [47]). This strand passage reaction is
central to the various functions of topo II, as well as for
targeting the enzyme by anticancer chemotherapeutics called
topo II poisons (Froelich-Ammon and Osheroff 1995 [48]).
In order to examine whether coordination complexes of
ruthenium with similar ligand conformation are inert towards
topo II, two anticancer coordination complexes of
ruthenium, HInd trans- [RuCl4(ind)2] and HIm t r a n s [RuCl4(im)2], have been tested for topo II antagonism [39].
Though very effective on animal models, their clinical
development was hindered due to extreme toxic effects.
Histological and blood-chemical investigations showed
major liver and kidney damage, hyperplasia and
hyperkeratosis of gastric mucosa and anemia [22]. The study
on topoisomerase II poisoning by these two compounds
suggested that they may be promising candidates for further
development as topoisomerase II poisons. The topo II
antagonism studies showed that these complexes poison topo
II and HInd trans- [RuCl4(ind)2] was more potent compared
to HIm trans- [RuCl4(im)2]. The cleavage assay by both
complexes revealed that these compounds had the ability to
form the “cleavage complex” similar to other topo II
poisons. This is an important feature of topo II poisons
because in the presence of these drugs, the enzyme induces
permanent double stranded nicks in DNA. Accumulation of
sufficient double strand breaks in DNA brings about
numerous adverse genetic aberrations, which ultimately
force the affected cells to undergo apoptosis or necrosis [48].
As in the case of topo II antagonism, HInd t r a n s [RuCl4(ind)2] was also more potent than HIm t r a n s [RuCl4(im)2] in inhibiting the [3H]thymidine incorporation
by the two cancer cell lines. This data also corroborates with
the anticancer activity of the two drugs reported by Keppler
et al. [22]. Though the data presented in [39] does not give
any direct evidence between topo II antagonism and
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1091
anticancer activity, it does suggest that topo II antagonism
may partly account for the anticancer activity of these drugs,
in addition to inhibition of DNA synthesis. Most of the work
done on metal containing anticancer drugs suggests that
these molecules have multiple levels of complex interactions
with the cellular DNA and proteins. Many may have
preferential interactions with particular DNA sequences and
proteins, leading to inhibition of important cellular
pathways, eventually causing anticancer activity.
Identification of such interactions and the resulting
anticancer properties will immensely help in the continual
development of drug entities, which are high on therapeutic
specificity and low on toxicity. This is particularly important
because cancer cells regularly evolve mechanisms to resist
the cytostatic action of anticancer drugs. Topo II is an
important target for many DNA binding anticancer drugs.
Since most anticancer metal complexes of ruthenium, cobalt,
platinum and titanium primarily target DNA, it would be
worthwhile to search for DNA binding metal complexes that
poison topo II. The biochemical studies on metal complexes
and topo II antagonism [39, 49] give an insight into the
molecular interactions of these molecules with DNA and
topo II and the subsequent effect on cancer cell proliferation.
Understanding the molecular interactions leading to topo II
poisoning merits a deeper investigation for the development
of more potent topo II poisons of this class.
PROPERTIES OF RU-DMSO COMPLEXES
Because of the contrasting binding properties of the S
and O atoms, dimethyl sulfoxide can form S-DMSO and ODMSO linkage isomers, depending on the nature and
characteristics of the transition metal ions. The sulfynil
group provides a good acceptor site for -electron donor
species, such as low spin iron(II) and ruthenium(II) ions,
while the oxygen atom is the preferred site for hard metals,
such as the 3d trivalent cations, aluminum(III) and
lanthanides. While most of the Ru(II) complexes exhibit a
great affinity for sulfur ligands, the preference demonstrated
by the corresponding Ru(III) species is usually inverted,
favoring the binding of the O-donor sites.
Several Ru(II) and Ru(III) complexes with coordinated
dimethyl sulfoxide have been shown to possess good
antitumor and, above all, antimetastatic properties against
animal models. Among these compounds, a Ru(III) complex
called NAMI-A, [ImH][trans-RuCl4(S-DMSO)(Im)] (Im =
imidazole, DMSO = dimethylsulfoxide), Fig. 3, was selected
because of its very good antimetastatic activity. NAMI-A is
O
Cl
Cl
CH3
CH3
S
Cl
Ru Cl
N
NH
N
H
NH
Fig. (3). Chemical structure of NAMI-A, [ImH][trans-RuCl4(Sdmso)(Im)].
a Ru(III) complex that, after extensive preclinical
investigations that evidenced its remarkable and specific
activity against metastases, has recently and successfully
completed a Phase I trial (first ruthenium complex ever to
1092
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
reach clinical testing). The procedures of the synthesis of
NAMI-A were reported and its rights are protected by the
patent [50].
The review article [6], after a brief summary of the main
chemical and pharmacological aspects of NAMI-A, focused
on the development of new classes of ruthenium complexes
originated from the NAMI-A frame. In particular, the
chemical and biological features of the following classes of
compounds were treated: NAMI-A-type complexes, derived
from NAMI-A by changing the nature of the N-ligand;
dinuclear NAMI-A-type compounds containing heterocyclic
bridging N-N ligands; and new Ru-DMSO nitrosyls broadly
derived from NAMI-A-type complexes. Several of these new
compounds have been found to have antimetastatic activity
comparable to, or even better than, NAMI-A; however, the
nature of the target(s) responsible for the antimetastatic
activity remaind unclear. Common to any type of NAMI-Atype compound, both monomeric and dimeric, cell
cytotoxicity (which is generally very low) is not sufficient to
explain their potent and peculiar antitumor activity. All
active NAMI-A-type compounds share the capacity to
modify important parameters of metastasis such as tumor
invasion, matrix metallo-proteinases activity and cell cycle
progression.
Several NAMI-A-type monomeric and dimeric Ru(III)
complexes have been recently developed [35]. In such
compounds, the coordinative environment of each Ru(III)
nucleus was very similar of that of NAMI-A. Preliminary in
v i v o results have shown that some of them had an
antimetastatic activity comparable to that of NAMI-A at
dosages that were 3.5 times lower in terms of moles of Ru.
Ruthenium complexes originally were synthesized as
compounds selectively toxic for solid tumors, because of the
selective activation to cytotoxic species into these tissues
[51, 52]. A wide series of investigations, performed on
sulfoxide-ruthenium complexes, pointed out a more specific
activity of these compounds on solid-tumor metastases,
putting light on the pharmacological possibilities of such
drugs (Sava, 1994 [53]; Sava and Bergamo, 1997 [54]). The
property that renders ruthenium complexes unique among
anticancer agents is principally the lack of evident direct cell
cytotoxicity at doses that increase lifetime expectancy in
tumor-bearing hosts (Sava et al. 1994 [55]; 1995 [56] ;
Capozzi et al. 1998 [57]). Rather than being a limitation, the
lack of direct cell cytotoxicity was the leading aspect of these
complexes in that it indirectly means a low or absent bone
marrow or epithelial toxicity at active dosages (Giraldi et al.
1977 [58]; Sava et al. 1984 [59]; Gagliardi et al. 1994 [60]).
Although many studies point out the capacity of
ruthenium complexes to bind to DNA of isolated plasmids or
eukaryotic cells (Clarke and Stubbs, 1996 [61]), many others
seem to suggest a certain difficulty of these complexes to
penetrate cell membrane, preferring extracellular components
as binding sites (Ghosh et al. 1981 [62]; Deinum et al. 1985
[63]). These characteristics may contribute to the
understanding of the mechanism of antitumor activity in in
vivo systems, where ruthenium interactions may deprive
tumor cells of normal cell-cell and cell-matrix contacts,
which are essential for cell growth, division, and metastasis
formation (Fox et al. 1995 [64]; Schadendorf et al. 1995
[65]; Umansky et al. 1996 [66]).
Irena Kostova
A series of 18 ruthenium(III) complexes, structurally
related to the selective antimetastatic drug Na[transRuCl4(DMSO)Im], NAMI, Fig. 4, and characterized by the
presence of sulfoxide and nitrogen-donor ligands were tested
on TLX5 lymphoma and some of them on MCa mammary
carcinoma to evaluate the dependence of the degree of
cytotoxicity and of antimetastatic activity on the chemical
properties [56]. In vitro cytotoxicity was present only at high
concentrations (> 10-4 M), depended upon lipophilicity and
was markedly affected by the presence of 5% serum or
plasma samples in the culture medium. The comparison of
the effects on in vitro cytotoxicity with in vivo antitumor and
antimetastatic action pointed out that these compounds
reduce metastasis formation by a mechanism unrelated to a
direct tumor cell cytotoxicity. In particular, Na[transRuCl4(DMSO)Im] seemed to distinguish between artificially
induced metastases and spontaneous metastases and reduced
only the former by a cytotoxic mechanism. Out of all the
tested compounds Na[t r a n s-RuCl4(DMSO)Im] was
confirmed to be the most selective antimetastatic agent of the
group.
O
Cl
Cl
CH3
CH3
S
Cl
Ru Cl
N
Na+
NH
Fig. (4). Chemical structure of Na[trans-RuCl4(DMSO)Im].
In vitro and in vivo pharmacological experiments have
shown that NAMI-A and other active ruthenium compounds
were scarcely cytotoxic, suggesting that their mechanism of
action was very likely different from that of platinum drugs
and might be unrelated to interactions with DNA.
Modifications of natural DNA by the anticancer heterocyclic
ruthenium(III) compounds were studied by methods of
molecular biophysics [33]. These methods included DNA
binding studies using atomic absorption spectrophotometry,
inhibition of restriction endonucleases, mapping of DNA
adducts by transcription assay, interstrand cross-linking
employing gel electrophoresis under denaturing conditions,
DNA unwinding studied by gel electrophoresis, circular
dichroism analysis, and DNA melting curves measured by
absorption spectrophotometry. The results indicated that the
complexes HIm[trans-Cl4Im2RuIII], HInd[trans-Cl4Ind2Ru
III], and Na[trans-Cl4Im(DMSO)RuIII] (Im and Ind stand
for imidazole and indazole, respectively) coordinated irreversibly to DNA. Their DNA binding mode was, however,
different from that of cisplatin. Interestingly, Na[transCl4Im(DMSO)RuIII] bound to DNA considerably faster than
the other two ruthenium compounds and cisplatin. In
addition, when Na[trans-Cl4Im(DMSO)RuIII] bound to
DNA it exhibited an enhanced base sequence specificity in
comparison with the other two ruthenium complexes.
Na[trans-Cl4Im(DMSO)RuIII] also formed bifunctional
intrastrand adducts on double-helical DNA which were
capable of terminating RNA synthesis in vitro, while the
capability of the other two ruthenium compounds to form
such adducts was markedly lower. This observation has been
interpreted to mean that the bifunctional adducts of
HInd[trans-Cl4Ind2RuIII] and Na[trans-Cl4Im2RuIII] formed
Ruthenium Complexes as Anticancer Agents
on rigid double-helical DNA were sterically more crowded
by their octahedral geometry than those of Na[transCl4Im(DMSO)RuIII]. In addition, the adducts of all three
ruthenium compounds affected the conformation of DNA,
Na[trans-Cl4Im(DMSO)RuIII] being most effective. It has
been suggested that the altered DNA binding mode of
ruthenium compounds in comparison with cisplatin might be
an important factor responsible for the altered cytostatic
activity of this class of ruthenium compounds in tumor cells.
One of the possibilities for explaining the activity of
NAMI-A against disseminated tumors is that it interferes
with NO methabolism in vivo. Nitric oxide is known to play
an important role in many biological functions, and recently
it was demonstrated to be involved as mediator in one tumorinduced angiogenic process, which is a key step in the
formation of metastases. Increased levels of NO correlate
with tumour growth and spreading in different experimental
and human cancers. Drugs interfering with the nitric oxide
synthase (NOS) pathway may be useful in angiogenesisdependent tumours. NO is also known to interact in vivo
with iron proteins, thus ruthenium action might also occurr
through an iron-mimicking mechanism. Serli et al. [67, 68]
investigated the reactivity of basic Ru(II)- and Ru(III)chloride-DMSO complexes and of NAMI-A towards NO
with the goal of producing spectroscopically and structurally
well characterized models. The spectroscopic and X-ray
structural features for all the new complexes were consistent
with the formulation, in which a diamagnetic Ru(II) nucleus
bound to NO+. Electrochemical measurements on the Ru-NO
complexes showed that they were all redox active in DMF
solutions and the site of reduction was the NO+ moiety. The
reduced complexes were not stable and rapidly released the
NO radical. Moreover, spectroscopic studies indicated that in
physiological conditions the active Ru(III) compounds, both
monomers and dimers, are easily and quantitatively reduced
to Ru(II) species by stoichiometric amounts of biological
reducing agents, such as ascorbic acid, cysteine and
glutathion. This important feature suggests that NAMI-A and
the Ru(III)-DMSO compounds might indeed be reduced also
in vivo to generate Ru(II) active species.
Morbidelli et al. [37] characterised pharmacologically
certain ruthenium-based compounds, as potential NO
scavengers to be used as antiangiogenic antitumour agents.
NAMI-A and other ruthenium-based compounds were able
to bind tightly and inactivate free NO in solution. Formation
of ruthenium--NO adducts was documented by electronic
absorption, FT-IR spectroscopy and 1H-NMR. Pretreatment
of rabbit aorta rings with NAMI-A reduced endotheliumdependent vasorelaxation elicited by acetylcholine. The key
steps of angiogenesis, endothelial cell proliferation and
migration stimulated by vascular endothelial growth factor
(VEGF) or NO donor drugs, were blocked by NAMI-A the
compound being devoid of any cytotoxic activity. When
tested in vivo, NAMI-A inhibited angiogenesis induced by
VEGF. It was likely that the antitumour properties
previously observed for ruthenium-based NO scavengers,
such as NAMI-A, were related to their NO-related
antiangiogenic properties.
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1093
NAMI-A-Comparison with Cisplatin
Many of studies were conducted by comparing the effects
of NAMI-A with those of cis-dichlorodiammineplatinum(II)
(cisplatin), to which the ruthenium complex is often referred
because it contains a heavy metal of group VIII transition
metals. In this context, c i splatin represents a drug of
particular interest. It is myelosuppressive, emetic, and
nephrotoxic (Tognella, 1990 [69]; Rozenweig et al. 1977
[70]); nevertheless, it must be considered a unique agent,
and, since its introduction into clinical trials, it is a drug that
has completely changed the prognosis of some tumors and
significantly ameliorated that of others.
Typically, it is expected that a compound such as
cisplatin that shows antitumor action on experimental tumors
also would be cytotoxic against tumor cells in vitro.
Therefore, a compound like NAMI-A, which often is
compared with cisplatin because it similarly is based to a
heavy metal of the same group, is totally atypical if it shows
the same action as cisplatin (or even better) in vivo on solid
metastasizing tumor but is virtually devoid of cytotoxicity
against tumor cells in vitro. Data reported show this
discrepancy and show further that NAMI-A is much less
toxic than cisplatin for healthy tissues at equieffective doses.
In vivo doses are optimal for both compounds, considering
the schedule of administration used and the ratio between
host toxicity and antitumor effect (i.e., optimal doses).
Furthermore, atomic absorption spectroscopy studies of
tissue disposition showed that NAMI-A, at the in vivo dose
of 35 mg/kg given for 6 consecutive days, reaches a 100-M
order concentration in tumor, liver, and lungs. Therefore, in
vitro comparison of both drugs using a top dose of 100 M
appears to be appropriate. Cisplatin was as effective as
NAMI-A in mice bearing MCa mammary carcinoma, which
is slightly less effective than NAMI-A in mice bearing TS/A
adenocarcinoma and markedly less effective than NAMI-A
in mice bearing Lewis lung carcinoma. These data, added to
those related to the toxicity for healthy epithelia, stress the
more favorable therapeutic properties of NAMI-A versus
cisplatin.
Tumor cells, challenged in vitro with NAMI-A, do not
show a significant alteration of cell cycles. NAMI-A causes
a mild and transient arrest of cell cycle in the premitotic
phase, which, besides that reported for KB cells, also was
demonstrated with TS/A cells cultured in vitro, with the same
doses and timings. The correspondent mean increase of total
protein content, as determined by the SRB test, should be
regarded as a consequence of this effect. This effect is a
property of NAMI-A that is not shared with NAMI (sodium
trans-imidazole-dimethyl sulfoxide-tetrachlororuthenate, Na
[trans-RuCl4(DMSO)Im]), which was totally ineffective in
vitro on tumor cells, probably associated with the advantages
given by a molecule endowed with better pharmaceutical
characteristics (Mestroni et al. 1998 [71]). The relevance of
these mild effects of NAMI-A on in vitro tumor cells for the
selective action on in vivo metastases is not clear yet. It
could be speculated that NAMI-A has a receptor-mediated
effect, which might explain both its selectivity for tumor
metastases, which behave differently from the primary tumor
counterparts, and the reversibility of its in vitro as well
1094
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
as in vivo effects. Furthermore, such a mechanism might also
help to explain the effects of NAMI on mRNAs for
metalloproteinases and their inhibitors (Sava et al. 1996
[72]), the selection of tumor cell populations that NAMI
caused with vitro-vivo and vivo-vivo bioassays of treated
tumor cells, and, particularly, the irrelevance of intracellular
migration of NAMI for its effects on TLX5 lymphoma. That
in vitro treated tumor cells do not show a reduced rate of
growth as compared with in vivo treated lung metastases
might be attributed to the cross-talk interactions that in vivo
growing cells have with other healthy cells and with
extracellular matrix constituents that might accentuate
growth arrest or apoptosis.
Considering that the main target for antitumor
chemotherapy is represented very often by distant
metastases, which are present, although not always
diagnosable, at the time of eradication of the primary lesion
and invariably are responsible for the failure of most of the
available antitumor therapies (Poste, 1986 [73]; Fidler and
Balch, 1987 [74]), the studies present NAMI-A as a reliable
candidate. The ruthenium complex NAMI-A was found to be
particularly effective in inhibiting lung metastasis formation
and growth in animal models of solid metastasising tumors
[75-77]. At doses active on lung metastases, it seems to be
devoid of important organ toxicity and its pharmacological
effect is not attributable to a direct cytotoxicity for tumor
cells [51, 57, 78].
NAMI-A is devoid of any link with cisplatin, used as a
reference compound, because it is free of direct cytotoxicity
for tumor cells. The lack or low toxicity of NAMI-A for host
tissues, compared with that exhibited by active doses of
cisplatin, is a support for this new compound, which also
should be regarded as a novel and potent agent for the
treatment of solid tumor metastases when these tumor lesions
are already present and in an advanced stage of growth.
Ruthenium-Based NAMI-A Type Complexes
After the promising results obtained with NAMI-A, new
NAMI-A type complexes, that differ from the parental
compound only for the nature of the nitrogen ligand
coordinated trans- to DMSO, Fig. 5, were prepared by
Alessio et al. [79, 80]. The complexes were synthesised with
the aim of investigating the relevance of this part of the
molecule on the chemical behavior, on the pharmacological
properties and on the host toxicity.
NAMI-A was recognized as a novel antitumor agent
endowed with properties such as the capability of controlling
metastasis growth of solid tumors with a mechanism of
action comprising, among others, antiangiogenic properties
[81, 82] and inhibition of matrix metalloproteinases [72, 82].
One relatively unfavorable aspect of this compound was the
relatively slow dissociation of coordinated DMSO in
aqueous solution (slightly acidic pH, i.e. before
administration) since this might affect its pharmacological
activity, as shown by in vivo experiments in which tumorbearing mice where treated with aged-solution of NAMI-A.
It have been found a 25% decrease in the capability of
inhibiting metastasis formation for solution of NAMI-A in
which DMSO loss from the molecule reached up to 50%
[83].
Irena Kostova
Cl
Cl
SOMe2
Cl
Ru
Cl
N
H+
N
SOMe2
Cl
Cl
N
a
Cl
Cl
N
Cl
Cl
N
H+
b
N
SOMe2
Cl
Ru
Cl
N
Ru
H+
N
Cl
S
H+
N
SOMe2
Cl
Ru
N
Cl
HN
Cl
HN
S
c
d
Fig. (5). Chemical structures of Ru(III) complexes with pyrazine (a,
b), thiazole (c), pyrazole (d).
The chemical behavior of NAMI-A in aqueous solution
has been investigated and it is known that it undergoes a
series of hydrolytic processes whose nature and rates are
strongly pH-dependent [81]. At physiological pH NAMI-A
undergoes stepwise chloride hydrolysis, also accompanied
by the partial dissociation of the DMSO ligand and by a
progressive darkening of the solution, attributed to the
formation of oxobridge polymeric species [81]. In aqueous
solution (slightly acidic pH) NAMI-A is remarkably more
inert, nevertheless slow dissociation of DMSO occurs (ca.
2% per hour at 25°C). It had been established that the rates
of loss of DMSO from NAMI-A type complexes in aqueous
solutions were inversely related to the basicity of the
nitrogen ligand [80]. Thus, with the aim of increasing the
stability of the ruthenium complexes in solution, Alessio et
al. prepared new NAMI-A type complexes bearing a weakly
basic heterocyclic nitrogen ligand coordinated trans to
DMSO, Fig. 5: thiazole, pyrazole or pyrazine. Depending on
the synthetic procedure, the pyrazine complex could be
obtained either as pyrazinium salt, or as zwitterion. As
expected, it was found that in slightly acidic solution the new
complexes are more stable than NAMI-A, owing to a lower
loss of DMSO. Therefore they can be useful for determining
the importance of the integrity of the chemical structure for
the pharmacological activity, and also for assessing if the
nature of the nitrogen ligand plays a role in the antimetastatic
activity of ruthenium-dimethylsulfoxide complexes. Past
studies already evidenced that the presence of DMSO
molecule seems to be a prerequisite for the antimetastatic
activity of this class of ruthenium complexes [78]. In these
studies it has been found that the compound named ICR
(imidazolium trans-imidazoletetrachlororuthenate), which
has no DMSO molecule was not active on metastasis
inhibition as compared to NAMI-A at optimal dosages.
Alessio et al. [79] investigated the in vitro and in vivo
activity of these new complexes and compare their effects to
those of the parental compound NAMI-A. In fact, the better
stability in aqueous solutions of these compounds may be a
good prerequisite for clinical handling. The in vitro study
was carried out on human (MCF-7 mammary carcinoma),
and on murine (TS/A mammary adenocarcinoma and B16-
Ruthenium Complexes as Anticancer Agents
F10 highly metastatic variant of melanoma B16) cell lines.
For in vivo anti-metastasis effect determination, two
experimental tumors of the mouse (MCa mammary
carcinoma and Lewis lung carcinoma) were used.
The analogues of NAMI-A shared the same chemical
structure of NAMI-A, and differed from it in the nature of
the coordinated nitrogen ligand, such as pyrazole, thiazole
and pyrazine, which are less basic than imidazole. This
modification confered to the new NAMI-A type complexes a
better stability in aqueous solution compared to the parent
compound. The new complexes showed a pharmacological
activity very similar to that of the parental compound
NAMI-A: in vitro they were devoid of meaningful
cytotoxicity against tumor cells, and in vivo they inhibited
metastasis formation and growth approximately to the same
extent as NAMI-A.
The complexes, analogues of NAMI-A have been
presented in the patents [84, 85]. The invention [84]
concerned coordination complexes with Ru(III) as central
metal attached to a heterocyclic ligand in the apical position
trans to a sulfoxide and their pharmaceutical compositions
useful in antitumor therapy either alone or in combination
with platinum complexes. The inventors have found that the
contemporary presence of sulphoxide and basic nitrogen
ligands in the apex positions of the octahedric neutral and
anionic ruthenium (III) complexes produced compounds
endowed with considerable anti-tumour activity, as it has
been proved in some pharmacological tests usings the model
of Lewis lung carcinoma in rats. It was remarkable that the
co-administration of effective doses of the compounds of the
invention with an effective dose of a platinum(II) complex,
e.g. cisplatin, gave rise to increased cumulative effects on the
reduction of the primary tumor. The described complexes
could be administered to human patients in form of
pharmaceutical compositions for treating various kinds of
neoplasiae. The compounds of the invention could also be
used in experimental protocols of polychemotherapy in
combination with other anti-tumor compounds such as
anthracyclines, cyclophosphamide, bleomycine, vinblastine,
5-fluorouracyl and particularly with c i splatin or its
derivatives, in consideration of the surprising synergistic
effects. The invention further included compositions in form
of combinations with platinum complexes, in particular
cisplatin, for contemporary, sequential or separate use in
cytostatic and anti-tumor therapy.
The invention [85] related to new salts of anionic
complexes of Ru(III) with ammonium cations which were
particularly useful as antimetastatic and antineoplastic
agents. Despite the antimetastatic activity of alkaline and
alkaline earth salt complexes of Ru(III) with DMSO it was
found that they exhibited some serious inconveniences
which make the administration and formulation in adequate
therapeutical compositions extremely difficult. As a matter
of fact, these anionic complexes when isolated in the form of
sodium salts, always contained two solvent molecules of
crystallisation and cannot be isolated in a pure form, that is
without these crystallisation molecules. A further
disadvantage of the above mentioned compounds was due to
pharmacological negative effects caused by DMSO,
introduced in the organism. The inventors [85] have found
new salts of Ru(III) anionic complexes with ammonium
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1095
cations which exhibited a remarkable antimetastatic and
antineoplastic activity, which was significantly higher than
that of the corresponding sodium salts. The salts of the
invention did not exhibit the pharmacological drawbacks
which were due to the presence of DMSO crystallisation
molecules. A further object of the invention was the use of
the above salts in the treatment of neoplasms of a various
nature and in the prevention of the formation of metastases.
The above neoplasms were preferably solid spawning
tumors, such as the carcinoma of the gastrointestinal tract,
the mammary carcinoma, the lung tumors, the metastatic
carcinoma and the lung metastases of metastatic tumors. The
salts of the invention could be profitably administered by
parenteral, oral, topical or transdermal route. Moreover, the
ammonium salts could profitably be used in experimental
protocols of polychemotherapy in combination with other
antitumor drugs of common clinical use in the above
described pathologies, such as for example cisplatin, 5fluorouracil, vinblastin, cyclophosphamide, bleomycin,
anthracycline, taxol. The in vivo activity test of the salts on
mice affected by Lewis Lung Carcinoma outlined the fact
that the treatment with the presented complexes caused a
reduction both in the weight and in the number of metastases
which was higher than the one obtained with the reference
compound. The dosages chosen for the comparison of the
effects of the complexes and cisplatin on solid metastasizing
tumors were comparable. On tumor metastasis, the
compounds of the invention were as effective (Mca
mammary carcinoma) or even more effective (Lewis lung
carcinoma and TS/A Adenocarcinoma) than cisplatin.
The complexes on Fig. 5 were found to be active in
inhibiting in vitro spontaneous invasion in a modified
Boyden's chamber and they also inhibited gelatinase activity
with IC50 values comparable to or slightly better than that of
NAMI-A [75]. Thus, the change of the nitrogen donor ligand
from imidazole to pyrazole, pyrazine and thiazole does not
interfere with the effectiveness on two crucial steps of the
dissemination process, i.e. extra cellular matrix degradation
and migration [86-88]. Accordingly, they markedly reduce
metastasis weight of solid experimental tumors in vivo. Thus
the new NAMI-A type complexes retained the same potent
characteristic of NAMI-A to selectively interacted with solid
tumor metastases. However, compared to NAMI-A they did
not stop cell cycle progression at G2-M level and were more
active in preventing the spontaneous invasion of Matrigel by
tumor cells exposed for 1 h to 10-4 M concentration.
Globally, these complexes took advantage of the knowledge
on NAMI-A and appear particularly interesting for future
clinical handling and applications [75].
These novel ruthenium complexes behaved similarly to
NAMI-A; they were very effective on in vivo metastases
despite having no effects on primary tumor growth nor in
vitro cytotoxicity. They differed from NAMI-A because of
the complete lack of induction of cell cycle arrest in the G2M phase. This result may suggest that the effect on cell
distribution among cell cycle phases was dependent on the
nature of the heterocyclic nitrogen ligand coordinated to
ruthenium. Conversely these complexes, similarly to NAMIA, inhibited Matrigel invasion by tumor cells and the
gelatinase activity of MMP-2 and MMP-9. This latter effect
correlated with the reported inhibition of matrix
metalloproteinases [89-91] by compounds with thiadiazole
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Current Medicinal Chemistry, 2006, Vol. 13, No. 9
groups, because of their interaction with the catalytic zinc
center. Since the in vitro inhibition of ‘invasion’ correlated
with the in vivo inhibition of metastasis formation, it appears
that this in vitro test was suitable for determining the
‘antimetastatic’ efficacy of this type of ruthenium
complexes.
Taken globally, these data seemed to indicate an in vivo
effectiveness of Ru complexes with thiazole and pyrazine
slightly better than NAMI-A, therefore confirming that the
reinforcement of the axis DMSO Ru- N-donor ligand by
using N-containing heterocycles less basic than imidazole
reduced the loss of DMSO from the complex and increased
the antitumor action. To further stress this hypothesis,
Alessio et al. [92] investigated NAMI-A derivative with
imidazole replaced with N-methylimidazole which resulted
only poorly active on MCa mammary carcinoma in vivo at
three sub-toxic dosages. This compound differed from
NAMI-A by the higher basicity of N-methylimidazole which
encouraged a faster loss of DMSO from the molecule. Data
of this investigation provided new evidence on the metastasis
inhibiting properties of sulfoxide-ruthenium complexes and
showed that the new NAMI-A type complexes with
increased stability in solution retained the potent
characteristic of the parental compound to selectively
interact with solid tumor metastases.
Two ruthenium(III) complexes bearing the thiazole
ligand, namely, thiazolium (bisthiazole) tetrachlororuthenate
and thiazolium (thiazole, DMSO) tetrachlororuthenate were
prepared and characterized by Mura et al. [36]. The crystal
structures of both complexes were solved by X-ray
diffraction methods and found to match closely those of the
corresponding imidazole complexes. The behavior in
aqueous solution of both complexes was analyzed
spectroscopically. It was observed that replacement of
imidazole with thiazole, a less basic ligand, produced a
significant decrease of the ligand exchange rates in the case
of the NAMI-like compound. The main electrochemical
features of these ruthenium(III) thiazole complexes were
determined and compared to those of NAMI-A. Moreover,
some preliminary data were obtained on their biological
properties. Notably, both complexes exhibited higher
reactivity toward serum albumin than toward calf thymus
DNA; cytotoxicity was negligible in line with expectations.
The NAMI-A-type Ru(III) complex (Hdmtp)[transRuCl4(DMSO-S)(dmtp)] (dmtp is 5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine), and the corresponding sodium
analogue, (Na)[trans-RuCl4(DMSO-S)(dmtp)], were synthesized by Velders et al. [35]. In vitro (Hdmtp)[transRuCl4(DMSO-S)(dmtp)] was not cytotoxic on tumor cells,
following challenges from 1 to 72 h and concentrations up to
100 microM, it inhibited matrigel invasion at 0.1 mM and
MMP-9 activity with an IC50 of about 1 mM, and it was
devoid of pronounced effects on cell distribution among cell
cycle phases. In vivo this complex, similar to NAMI-A,
significantly inhibited metastasis growth in mice bearing
advanced MCa mammary carcinoma tumors. In the lungs,
the same complex was significantly less concentrated than
NAMI-A, whereas no differences between these two
compounds were found in other organs such as tumor, liver,
and kidney. However, (Hdmtp)[trans- R u C l4(DMSOS)(dmtp)] caused edema and necrotic areas on liver
Irena Kostova
parenchyma that were more pronounced than those caused
by NAMI-A. Conversely, glomerular and tubular changes on
kidney were less extensive than with NAMI-A. In
conclusion, (Hdmtp)[t r a n s - R u C l4(DMSO-S)(dmtp)]
confirmed the excellent antimetastatic properties of this class
of NAMI-A-type compounds and qualified as an interesting
alternative to NAMI-A for treating human cancers.
Recently, NAMI-A has attracted significant attention and
several various reviews and research papers concerning the
use of NAMI-A in clinical and/or experimental treatment
have been published [93-122]. In summary, it is generally
appreciated that enormous progress has been made in the
understanding of the mode of action of this compound. The
success of NAMI-A against metastasis should stimulate
laboratory studies with appropriate experimental models to
predict clinical activity, since the use of experimental
conditions closely similar to those of human tumours should
help the identification of more active compounds.
Ru(II)/Ru(III) COMPLEXES
Octahedral ruthenium(III) and ruthenium(II) complexes
show antineoplastic properties on a number of experimental
tumors. Tetraammine-, pentaammine-, heterocycle-, and
dimethylsulfoxide-coordinated ruthenium complexes have
shown high affinity for nitrogen donor ligands in vitro and as
a result exhibit various degrees of biological activities
including antitumor action in vivo. The chemical behavior of
ruthenium(III) complexes indicates the possibility of opening
a window of selective toxicity, in practice lacking in the
chemotherapeutic approach to neoplastic diseases.
Ruthenium ions may accumulate in tumor tissues via a
mechanism mediated by transferrin transport. Moreover,
binding of ruthenium to DNA is several times higher in its
reduced ruthenium(II) form and the reduction from
ruthenium(III) prodrugs to the more toxic ruthenium(II)
compounds is particularly efficient in tumor hypoxic
environments [123]. Correspondingly, solid tumors appear to
be more susceptible than those of the lymphoproliferative
type. In particular, tumors of the colorectal region and lung
tumors (primary or metastatic), which are generally
associated with a bad prognosis, have given interesting
responses in experimental models, indicating these tumors as
preferential targets for the development of ruthenium
anticancer drugs.
Antineoplastic ruthenium(III) complexes are generally
regarded as prodrugs, being activated by reduction. Within a
homologous series of ruthenium(III) complexes, cytotoxic
potency is therefore expected to increase with increasing
ease of reduction. Complexes of the general formula
[Ru(III)Cl6-n(ind)n](3-n)- (n = 0-4; ind = indazole; counterions
= Hind+ or Cl- and the compound trans-[Ru(II)Cl2(ind)4]
have been prepared and characterized electrochemically
[124]. Lever's parametrization method predicted that a higher
indazole-to-chloride ratio resulted in a higher reduction
potential, which was confirmed by cyclic voltammetry. In
vitro antitumor potencies of these complexes in colon cancer
cells (SW480) and ovarian cancer cells (CH1) varied by
more than 2 orders of magnitude and increased in the
following rank order: [Ru(III)Cl6]3- < [Ru(III)Cl4(ind)2]- <
[Ru(III)Cl5(ind)]2- << [Ru(III)Cl3(ind)3] < [Ru(III)Cl2(ind)4]+
approximately [Ru(II)Cl2(ind)4]. Thus, the observed
Ruthenium Complexes as Anticancer Agents
differences in potency correlated with reduction potentials
largely, though not perfectly, pointing to the influence of
additional factors. Differences in the cellular uptake
(probably resulting from different lipophilicity) contributed
to this correlation but could not solely account for it.
The electrochemical behavior of [trans-RuCl4L(DMSO)]and [trans-RuCl4L2]- [L = imidazole (Him), 1,2,4-triazole
(Htrz), and indazole (Hind)] complexes has been studied in
DMF, DMSO, and aqueous media by cyclic voltammetry
and controlled potential electrolysis [125]. They exhibited
one single-electron Ru(III)/Ru(II) reduction involving, at a
sufficiently long time scale, metal dechlorination on
solvolysis, as well as, in organic media, one single-electron
reversible Ru(III)/Ru(IV) oxidation. The redox potential
values were interpreted on the basis of the Lever's
parametrization method. The kinetics of the reductively
induced stepwise replacement of chloride by DMF were
studied by digital simulation of the cyclic voltammograms,
and the obtained rate constants were shown to increase with
the net electron donor character of the neutral ligands
(DMSO < indazole < triazole < imidazole) and with the
basicity of the ligated azole, factors that destabilize the
Ru(II) relative to the Ru(III) form of the complexes.
The reduction of Cl(NH3)5Ru(III) and subsequent binding
of heterocyclic ligands by the resultant (H2O)(NH3)5Ru(II)
ion was shown to be catalyzed by components of rat-liver
cells [126]. The presence of air significantly decreased the
rate of heterocyclic ligand binding. In the case of microsome
and soluble component catalysis, this was probably due to
oxidation of the Ru(II) ion prior to complexation. Various
inhibitors of electron-transfer proteins were employed in an
effort to determine the preferred reducing species. These
results lent support to the hypothesis that the antitumor
activity of acido ruthenium(III) ammine complexes involved
activation by reduction in vivo prior to metal coordination to
nucleic acids. Anticancer drugs functioning by this
mechanism might be preferentially toxic to or might localize
in hypoxic areas of tumors.
Ru(II)/Ru(III) polypyridyl complexes containing 2,6-(2'benzimidazolyl)-pyridine or chalcone as co-ligands were
synthesized and characterized [127]. Their interaction with
aqueous buffered calf thymus DNA was measured and these
results prompted additional screening for anti-HIV (human
immunodeficiency virus) activity against DNA replication in
H9 lymphocytes and cytotoxic activity against eight tumor
cell lines. The most active compounds, especially selectively
against the 1A9 ovarian cancer cell line, were determined.
PROPERTIES OF Ru(II) COMPLEXES
An important activation mechanism for Ru(III) is thought
to be reduction to Ru(II). Tumors are often hypoxic (low in
O2) and contain reducing agents such as thiols (eg.
glutathione, E0 = -0.24 V). Reduction of Ru(III) to Ru(II)
weakens bonds to -donor ligands and increases ligand
substitution rates; –acceptors such as DMSO can raise the
redox potential.
Ruthenium (II) complexes have been presented in the
patents [128-130]. The invention [128] related to sitespecific chiral ruthenium (II) antitumor agents. It has been
discovered that certain bis-substituted metal complexes of
phenanthrolines were capable of binding covalently and
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1097
stereospecifically to DNAs. Such complexes were useful in
stereospecific labeling and cleavage of DNAs and were
further useful as antitumor agents. These compounds were
potentially very effective anti-tumor drugs. The advantages
such compounds provided over cisplatin included lower
heavy-metal toxicity, greater selectivity owing to
stereochemistry, greater site specificity given the organic
ligands and the possibility of linkage to monoclonal
antibodies, and easier and less expensive preparation. The
ruthenium (II) complexes with phenanthrolines were useful
in methods for labeling, nicking and cleaving DNA [129,
130]. The invention of Thorp et al. [131] related to the use of
oxoruthenium (IV) complexes for cleaving nucleic acids.
Diphenyl tris ruthenium (II) complexes of the inventions
[129, 130] were screened for cytotoxic activity against
mouse leukemia cells (L 1210 and P 815) and showed high
potency against leukemia cells.
Object of the inventions [132, 133] was a class of anionic
and neutral complexes of ruthenium (II) containing nitrogen
oxide (NO) and optionally also a nitrogen ligand. The
preparation process included the use of starting complexes of
ruthenium (III) which were reacted with suitable reagents so
as to obtain complexes containing NO coordinated to
ruthenium (II). Additional substitution reactions allowed the
introduction of new groups that coordinated to the ruthenium
atom, among which some nitrogen ligands. The above
described ruthenium complexes exerted their antitumour
activity by releasing one or more of their ligands and
covalently binding to biological targets. Therefore, most
ruthenium complexes endowed with antitumour activity
were reactive and selectively labile species and were
hydrolised in aqueous solution, in particular at physiological
pH. Although they were more inert, the ruthenium
complexes of the invention had a more pronounced
antiproliferative and cytotoxic activity compared with that of
the ruthenium complexes of the prior art and were suitable
for use in the treatment of hyperproliferative or tumoral
pathologies. The cytotoxicity of the ruthenium complexes
prepared in the invention was tested on two tumor cell lines:
MCF-7, a human hormone dependent mammary carcinoma
cell line and TS/A, a murine adenocarcinoma cell line. The
results obtained showed that the ruthenium complexes tested
exerted a cytotoxic and antiproliferative action. Although
murine cells were more sensitive towards the cytotoxic and
antiproliferative activity of the compounds tested for short
contact times, no difference between human and murine cells
was observed for prolonged contact times. The cytotoxic
activity of the ruthenium complexes tested was significantly
higher compared with that of similar compounds of the prior
art.
The invention [134] was related to ruthenium (II) derived
antitumoral compounds with high antimetastatic activity and
with low toxicity for the host. It described the use of
inorganic ruthenium complexes in the therapy of solid
tumors, in particular of those tumors characterized by high
metastatizing ability. Pharmaceutical compositions containing ruthenium (II) complexes in formulations suitable for the
administration and a kit for the preparation of such
complexes at the moment of use or immediately before use,
were moreover described. In vivo activity assays of the
compositions of the ruthenium(II) complexes according to
the present invention on mice affected by MCa mammary
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Current Medicinal Chemistry, 2006, Vol. 13, No. 9
Irena Kostova
carcinoma showed prevention activity on the formation of
lung metastases. As the result of all the activity and toxicity
tests performed, it was therefore possible to conclude that the
ruthenium(II) compounds of the invention, were markedly
more active than the corresponding ruthenium(III)
complexes and than the ruthenium(II) complexes belonging
to the state of the art. Moreover, unexpectedly, the
complexes according to the invention were endowed with
alower systemic toxicity contrarily to what expected from
the state of the art.
A lot of Ru(II) complexes with anticancer activity have
been synthesized [135-143] with derivatives of pyridine,
pyrazine, pyrimidine, 2,2'-bipyridine, 4,4'-bipyridine, 1,10phenanthroline, and N-substituted thiosemicarbazide: N-methyl-isatin-3-thiosemicarbazone, isatin-3-(4-Cl-phenyl)thiosemicarbazone) and acetazolamide, (Fig. 6).
N
N
N
N
2
1
N
3
N
N
N
N
5
4
RNH
NHNH2
NNHC(S)NH2
S
7
N
N
N
O
CH3
8
6
NNHC(S)NH(C6H4Cl)
N
N
H
9
O
H
SO2NH2
NHCOCH3
S
10
Fig. (6). Chemical structures of the ligands for the preparation of
Ru(II) complexes: pyridine (1), pyrazine (2), pyrimidine (3), 2,2'bipyridine (4), 4,4'-bipyridine (5), 1,10-phenanthroline (6), Nsubstituted thiosemicarbazide (7), N-methyl-isatin-3-thiosemicarbazone (8), isatin-3-(4-Cl-phenyl)thiosemicarbazone) (9) and acetazolamide (10).
In search of potential anticancer drug candidates in
ruthenium complexes, a series of mononuclear ruthenium(II)
complexes of the type [Ru(phen)2(nmit)]Cl2, [Ru(bpy)2
(nmit)]Cl2, [Ru(phen)2(icpl)]Cl2, Ru(bpy)2(icpl)]Cl2 (phen=
1,10-phenanthroline; bpy=2,2'-bipyridine; nmit=N-methylisatin-3-thiosemicarbazone, icpl=isatin-3-(4-Cl-phenyl)thiosemicarbazone) and [Ru(phen)2(aze)]Cl2, [Ru(bpy)2(aze)]Cl2
(aze=acetazolamide) and [Ru(phen)2(R-tsc)](ClO4)2 (R=methyl, ethyl, cyclohexyl, 4-Cl-phenyl, 4-Br-phenyl, and 4EtO-phenyl, tsc=thiosemicarbazone) have been prepared and
characterized by elemental analysis, FTIR, 1H-NMR and
FAB-MS [135]. Effect of these complexes on the growth of
a transplantable murine tumor cell line (Ehrlich Ascites
Carcinoma) was studied. The effect of hematological profile
of the tumor hosts has also been studied. Some of the
complexes have remarkably decreased the tumor volume.
Treatment with the ruthenium complexes prolonged the
lifespan of Ehrlich Ascites Carcinoma (EAC) bearing mice.
Tumor inhibition by the ruthenium chelates was followed by
improvements in hemoglobin, RBC and WBC values. Thus,
the results suggest that these ruthenium complexes have
significant antitumor property. The results also reflect that
the drug does not adversely affect the hematological profiles
as compared to that of cisplatin on the host.
Antineoplastic and antibacterial activity of some
mononuclear Ru(II) complexes has been investigated by
Mazumder et al. [136]. The ligands show a bidentate
behavior, forming octahedral ruthenium complexes [135,
136]. The complexes were subjected to in-vivo anticancer
activity tests against a transplantable murine tumor cell line,
Ehrlich's Ascitic Carcinoma (EAC) and in-vitro antibacterial
activity against several Gram positive and Gram negative
bacterial strains. [Ru(bpy)2(ihqs)]Cl2 and [Ru(bpy)2(hc)]Cl2
(where bpy = 2,2'-bipyridine, ihqs = 7-iodo-8hydroxy
quinoline-5-sulphonic acid and hc = 3-hydroxycoumarin)
showed promising antitumor activity. Treatment with these
complexes prolonged the life span of EAC bearing mice as
well as decreased their tumor volume and viable ascitic cell
count.
A series of compounds were synthesized from ruthenium
trichloride, and their i.p. LD50s were determined in mice:
chloronitrobis(2,2'-dipyridyl)ruthenium(II), 55;dichlorobis
(2,2'-dipyridyl)ruthenium(II), 63; ruthenium trichloride, 108;
and potassium pentachloronitrosylruthenate(II), 127 mg/kg
[137]. The two bis-bipyridyl complexes produced death in
convulsions within minutes, whereas the remaining
compounds resulted in long, debilitating courses with death
occurring in 4-7d. When given in massive overdoses,
however, the compounds with inorganic ligands also
produced rapid convulsive death in mice, and when given i.v.
to anesthetized cats, they produced respiratory arrest. The
major toxic effects of all the complexes appeared to be due
to the metal and not to its associated ligands. No compound
tested was as potent as cisplatin in antitumor activity. In
minimally lethal doses, the complexes with inorganic ligands
may affect a variety of contractile tissues, perhaps by a
general mechanism involving Ca. These complexes were apt
to be generally cytotoxic as well.
Ru(II) polypyridyl complexes containing 3hydroxyflavone derivatives as coligands were screened for
cytotoxic activity against eleven tumor cell lines [138]. In
order to check the effect of flavones containing Ru(II)
complexes in vivo on a mammal, a representative complex
Ru(L)2(DMSO)2.5H2O (LH-3-Hdroxy-4'-benzyloxyflavone)
was orally administered to adult male mice. Its effects on
protein content and LDH were studied in different tissues of
the animal. The compound got absorbed and retained in the
blood between 1-3 hr after feeding. As compared to the
normal and DMSO control sets, tissue specific significant
reversible changes in the protein content as well as in LDH
activity were observed between 1-4 hr of treatment.
However, on polyacrylamide gel electrophoresis, except
some tissue specific transitory alterations, expression
patterns of five LDH isozymes were unchanged after feeding
the compound. The present results suggested that in addition
to its potent cytotoxic effect on cell lines in vitro,
Ruthenium Complexes as Anticancer Agents
Ru(L)2(DMSO)2.5H2O inhibited LDH activity, but reversibly
with a little effect on biosynthetic status of the enzyme in
mice.
A group of four ruthenium chelates of the mixed
hard/soft N-S donor ligands 2-formylpyridine (4-H/4phenyl)thiosemicarbazone has been studied in the
experimental models of MCa mammary carcinoma and
TLX5 lymphoma in the CBA mouse [139]. Although all the
tested complexes, reduced the formation of lung metastases
at the same extent only one of the compounds caused parallel
inhibition of the growth of the primary tumor. The chemical
nature of the tested compounds seems to determine the
nature of the antitumor effects and the bis-chelates were
found to be endowed with greater cytotoxic properties
towards primary tumor than the monochelates. This opens up
a very interesting point, whether it is the presence of two
chelate rings around the ruthenium(II)/(III) acceptor centre
or the increase in the number of the soft S donor centers that
generates greater cytotoxic properties in the corresponding
ruthenium complexes. As far as the reduction of the
metastasis formation is concerned, it appears that among the
four ruthenium chelates tested, it is possible to identify
structures capable of controlling the spread of tumor to the
lungs in the absence of significant cytotoxicity for tumor
cells. This finding appears of importance in that it indicates
the possibility of a specific mechanism of interaction with
cells of the metastatic tumor. In this context it appears
necessary to investigate other congeners of this "family"
with more sulfur donor sites and particularly those with
better water solubility.
Complexes of the type [Ru(II)Cl2(DMSO)2L], where L
are 5-nitrofurylsemicarbazone derivatives, were prepared in
an effort to combine the potential anti-tumor activity of the
metal and the free ligands [140]. The new complexes were
excellent DNA binding agents for calf thymus DNA. Their
in vitro anti-tumor activity was tested in cellular models and
the complexes were found to be non-cytotoxic on the tumor
cell lines assayed, neither in aerobic conditions nor in the
bio-reductive assay performed. Redox behavior, lipophilicity
and stability were studied in order to explain the lack of
cellular cytotoxic effects. The complexes resulted 10-100
times more hydrophilic than the parent ligands thus the bioactivity of these compounds would be compromised by their
inadequate lipophilic properties.
The reaction of trans-[RuCl2(PPh3)3] (Ph = C6H5) with 2thio-1,3-pyrimidine and 6-thiopurines produced mainly
crystalline solid complexes [141]. Selected ruthenium(II)thiobase complexes were studied for their structural,
reactivity, spectroscopic, redox, and cytotoxic properties.
The results suggested that thiopyrimidinato anions chelated
to the metal center via N and S. Cytotoxic activity
measurements for the complexes were performed against
ovarian cancer cells A2780/S.
The dichlorobis(2-phenylazopyridine)ruthenium(II) complexes, [Ru(azpy)2Cl2], are under renewed investigation due
to their potential anticancer activity, Fig. 7. New watersoluble bis(2-phenylazopyridine)ruthenium(II) complexes,
all derivatives of the highly cytotoxic -[Ru(azpy)2Cl2]
(alpha denoting the coordinating pairs Cl, N(py), and N(azo)
as cis, trans, cis, respectively) have been developed by Hotze
et al. [142]. The compounds 1,1-cyclobutanedicarboxy-
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1099
latobis(2-phenylazopyridine)ruthenium(II), -[Ru(azpy)2
(cbdca-O,O')], oxalatobis(2-phenylazopyridine)ruthenium
(II), -[Ru(azpy)2(ox)], and malonatobis(2-phenylazopyridine)ruthenium(II), -[Ru(azpy)2(mal)], have been synthesized and fully characterized. The cytotoxicity of this series
of water-soluble bis(2-phenylazopyridine) complexes has
been determined in A2780 human ovarian carcinoma and
A2780cisR, the corresponding cisplatin-resistant cell line.
For comparison reasons, the cytotoxicity of the complexes
-[Ru(azpy)2Cl2], -[Ru(azpy)2(NO3)2], -[Ru(azpy)2Cl2]
(beta indicating the coordinating pairs Cl, N(py), and N(azo)
as cis, cis, cis, respectively), and -[Ru(azpy)2(NO3)2] have
been determined in this cell line. All the bis(2-phenylazopyridine)ruthenium(II) compounds displayed a promising
cytotoxicity in the A2780 cell line (IC50 = 0.9-10 M), with
an activity comparable to that of cisplatin and even higher
than the activity of carboplatin. Interestingly, the IC50 values
of this series of ruthenium compounds (except the beta
isomeric compounds) were similar in the cisplatin-resistant
A2780cisR cell line compared to the normal cell line A2780,
suggesting that the activity of these compounds might not
have been influenced by the multifactorial resistance
mechanism that affect platinum anticancer agents.
N
N
N
Cl
Ru
N
Cl
N
N
Fig. (7). Chemical structure of dichlorobis(2-phenylazopyridine)
ruthenium(II) complexes, [Ru(azpy)2Cl2].
The three most common isomers -, - and -[RuL2Cl2]
with L= o-tolylazopyridine (tazpy) and 4-methyl-2phenylazopyridine (mazpy) (alpha indicating the
coordinating Cl, N(pyridine) and Nazo atoms in mutual cis,
trans, cis positions, beta indicating the coordinating Cl,
N(pyridine) and Nazo atoms in mutual cis, cis, cis positions,
and gamma indicating the coordinating Cl, N(pyridine) and
Nazo atoms in mutual trans, c i s , cis positions) were
synthesized and characterized by NMR spectroscopy [143].
The molecular structures of -[Ru(tazpy)2Cl2] and [Ru(mazpy)2Cl2] were determined by X-ray diffraction
analysis. The IC50 values of the geometrically isomeric
[Ru(tazpy)2Cl2] and [Ru(mazpy)2Cl2] complexes compared
with those of the parent [Ru(azpy)2Cl2] complexes were
determined in a series of human tumour cell lines (MCF-7,
EVSA-T, WIDR, IGROV, M19, A498 and H266). These
data unambiguously showed for all complexes the following
trend: the -isomer showed a very high cytotoxicity,
whereas the - isomer was a factor 10 less cytotoxic. The isomers of [Ru(tazpy)2Cl2] and [Ru(mazpy)2Cl2] displayed a
very high cytotoxicity comparable to that of the - isomer of
the parent compound [Ru(azpy)2Cl2] and to that of the isomer. These biological data were of the utmost importance
for a better understanding of the structure-activity
relationships for the isomeric [RuL2Cl2] complexes.
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Current Medicinal Chemistry, 2006, Vol. 13, No. 9
Irena Kostova
Ketoconazole (KTZ), Fig. 8 has been used as a secondline agent in hormone-refractory cancer therapy. Since
transition metal complexes including those of Ru(III) and
Ru(II), show important anticancer activity with limited
toxicity, Strasberg et al. investigated the potential antitumor
efficacy of Ru(II) complexed to KTZ or clotrimazole (CTZ)
compared to Ru(II) alone or uncomplexed azoles [144].
RuCl2(KTZ)2 exerted greater apoptosis- associated caspase-3
activation than RuCl2(CTZ)2, KTZ, CTZ or RuCl2(MeCN)4
against several human tumor cell monolayers. Treatment of
WM164 melanoma monolayers with 25 M of cisplatin or
RuCl2(KTZ)2 showed that the latter was more effective than
cisplatin. Such results suggested that the Ru(II) and Pt(II)
metal complexes were unequally effective and acted through
alternative signaling pathways.
N
N
CH2
O
O
H3C
C
N
N
OCH2
Cl
O
Cl
Fig. (8). Chemical structure of Ketoconazole (KTZ).
RUTHENIUM(II)-ARENE COMPLEXES
Ru(II) is stabilized in organometallic Ru(II) arene
complexes [( 6-arene)Ru(en)Cl]PF6 (where en is
ethylenediamine) which have the characteristic ‘piano-stool’
structure, Fig. 9 and exhibit anticancer activity. They contain
a reactive Ru-Cl bond and undergo hydrolysis in aqueous
solution. They bind strongly to G through N7 coordination
together with arene-purine base stacking when the arene is
large enough (e.g. biphenyl or tetrehydroanthracene). Such
coordinated arenes can therefore intercalate into DNA.
Polyhaloaromatic ruthenium complexes have been
described in the invention [145]. Novel ruthenium arene
complexes could react with phenoxides or thiophenoxides to
form polyfunctional ruthenium complexes. Such complexes
were useful as crosslinking agents in polymerizations.
The recently published inventions [146-155] related to
the use in medicine, particularly for the treatment and/or
prevention of cancer, and to a process for the preparation of
ruthenium (II) arene complexes. The compounds of the
inventions could be used directly against a tumour.
Alternatively or additionally, the compounds might be used
to prevent or inhibit metastasis and/or to kill secondary
tumours. Compounds of the inventions might be effective in
treating and/or preventing tumours caused by cells that are
resistant to other cytotoxic drugs, such as cisplatin, for
example. Certain compounds of the inventions had the
surprising advantage that they did not bind selectively to
guanine bases but showed roughly equal affinity for binding
to guanine and adenine. This effect was unexpected, given
that the presence of an N-H group available for hydrogen
bonding was thought to be a requirement for bonding to
guanine. Furthermore, binding to guanine and adenine gives
the compounds a potentially greater ability to be less
susceptible to drug resistance in tumour cells. In one
embodiment, the compounds could be used in a method of
binding non-selectively to guanine bases, preferably a
method of binding to guanine and adenine bases with
roughly equal affinity. The compounds and compositions of
the inventions could be administered alone or in combination
with other compounds. A number of compounds of the
inventions were tested on A2780 ovarian cancer cell line.
The tested compounds had an IC50 of less than 50 M. The
experiments were repeated to investigate the effect of the
compounds of the inventions on drag-resistant variants of the
A2780 cell line. The compounds of the inventions had
cytotoxicity against cancer cells that were resistant to
treatment by other drugs.
Organometallic ruthenium(II)-arene complexes are
currently attracting increasing interest as anticancer
compounds with the potential to overcome drawbacks of
traditional drugs like cisplatin with respect to resistance,
selectivity, and toxicity. Rational design of new potential
pharmaceutical compounds requires a detailed understanding
of structure–property relationships at an atomic level.
Gossens et al. performed in vacuo density functional theory
(DFT) calculations, classical MD, and mixed QM/MM CarParrinello MD explicit solvent simulations to rationalize the
binding mode of two series of anticancer ruthenium(II) arene
complexes to double-stranded DNA (dsDNA) [156]. Binding
energies between the metal centers and the surrounding
ligands as well as proton affinities were calculated using
DFT. The results supported a pH-dependent mechanism for
the activity of the ruthenium(II)-arene complexes. Adducts
of these compounds with the DNA sequence
d(CCTCTG*G*TCTCC)/d(GGAGACCAGAGG), where G*
are guanosine bases that bind to the ruthenium compounds
through their N(7) atom, have been investigated. The
resulting binding sites were characterized in QM/MM
molecular dynamics simulations showing that DNA can
easily adapt to accommodate the ruthenium compounds.
Organometallic ruthenium(II) arene anticancer complexes of
the type [( 6-arene)Ru(en)Cl]PF6 specifically target guanine
bases of DNA oligomers and form monofunctional adducts.
Chen et al. have determined the structures of monofunctional
adducts of the "piano-stool" complexes [( 6-Bip)Ru(II)(en)
Cl][PF6] (Bip = biphenyl) Fig. 9, [( 6-THA)Ru(II)(en)Cl]
[PF6] (THA = 5,8,9,10-tetrahydroanthracene), and [6-DHA)
Ru(II)(en)Cl][PF6] (DHA = 9,10-dihydroanthracene) with
guanine derivatives, in the solid state by X-ray crystallography, and in solution using NMR methods [157]. Strong arene-nucleobase stacking was present in the crystal
structures of [( 6-C14H14)Ru(en)(9EtG-N7)][PF6]2.(MeOH)
and [( 6-C14H12)Ru(en)(9EtG-N7)][PF6]2.2(MeOH) (9EtG =
9-ethylguanine). In the crystal structure of [( 6-biphenyl)
Ru(en)(9EtG-N7)][PF6]2.(MeOH), there was intermolecular
stacking between the pendant phenyl ring and the purine sixmembered ring. This stacking stabilized a cyclic tetramer
structure in the unit cell. The guanosine (Guo) adduct [( 6biphenyl)Ru(en)(Guo-N7)][PF6]2.3H2O exhibited intramolecular stacking of the pendant phenyl ring with the purine
five-membered ring and intermolecular stacking of the
purine six-membered ring with an adjacent pendant phenyl
ring. These occured alternately giving a columnar-type
structure. There were significant reorientations and
conformational changes of the arene ligands in [( 6-arene)
Ru(II)(en)(G-N7)] complexes in the solid state, with respect
to those of the parent chloro-complexes [(6-
Ruthenium Complexes as Anticancer Agents
arene)Ru(II)(en)Cl]+. The arene ligands had flexibility
through rotation around the arene-Ru -bonds, propeller
twisting for Bip, and hinge-bending for THA and DHA.
Thus propeller twisting of Bip decreased by ca. 10 degrees
so as to maximize intra- or intermolecular stacking with the
purine ring, and stacking of THA and DHA with the purine
was optimized when their tricyclic ring systems were bent by
ca. 30 degrees, which involved increased bending of THA
and a flattening of DHA. This flexibility made simultaneous
arene-base stacking and N7-covalent binding compatible.
These studies suggested that simultaneous covalent
coordination, intercalation, and stereospecific H-bonding
could be incorporated into Ru(II) arene complexes to
optimize their DNA recognition behavior, and as potential
drug design features.
Cl
Ru
NH2
H 2N
Fig. (9). Chemical structure of [(6-arene)Ru(en)Cl]+ complex of
biphenyl.
Chen et al. have investigated the recognition of nucleic
acid derivatives by organometallic ruthenium(II) arene
anticancer complexes of the type [( 6-arene)Ru(II)(en)X]
where en = ethylenediamine, arene = biphenyl (Bip),
tetrahydroanthracene (THA), dihydroanthracene (DHA), pcymene (Cym) or benzene (Ben), X = Cl- or H2O using
NMR spectroscopy [158]. For mononucleosides, [( 6Bip)Ru(en)]2+ bound only to N7 of guanosine, to N7 and N1
of inosine, and to N3 of thymidine. Binding to N3 of
cytidine was weak, and almost no binding to adenosine was
observed. The reactivity of the various binding sites of
nucleobases toward Ru at neutral pH decreased in the order
G(N7) > I(N7) > I(N1), T(N3) > C(N3) > A(N7), A(N1).
Therefore, pseudo-octahedral diamino Ru(II) arene
complexes were much more highly discriminatory between
G and A bases than square-planar Pt(II) complexes.
Reactions with nucleotides proceeded via aquation of [(6arene)Ru(en)Cl]+, followed by rapid binding to the 5'phosphate, and then rearrangement to give N7, N1, or N3bound products. Small amounts of the dinuclear species were
also detected. Ru-H2O species were more reactive than RuOH species. The presence of Cl- or phosphate in neutral
solution significantly decreased the rates of Ru-N7 binding
through competitive coordination to Ru. In kinetic studies
(pH 7.0, 298 K, 100 mM NaClO4), the rates of reaction of
cGMP with [(6-arene)Ru(II)(en)X]n+ (X = Cl- or H2O)
decreased in the order: THA > Bip > DHA >> Cym > Ben,
suggesting that N7-binding is promoted by favorable arenepurine hydrophobic interactions in the associative transition
state. These findings have revealed that the diamine NH2
groups, the hydrophobic arene, and the chloride leaving
group have important roles in the novel mechanism of
recognition of nucleic acids by Ru-arene complexes, and will
aid the design of more effective anticancer complexes, as
well as new site-specific DNA reagents.
Fernandez et al. showed that the chelating ligand XY in
Ru(II) anticancer complexes of the type [Ru(6-
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
1101
arene)(XY)Cl]n+ had a major influence on the rate and extent
of aquation, the pKa of the aqua adduct, and the rate and
selectivity of binding to nucleobases [159]. Replacement of
neutral ethylenediamine (en) by anionic acetylacetonate
(acac) as the chelating ligand increased the rate and extent of
hydrolysis, the pKa of the aqua complex (from 8.25 to 9.41
for arene-p-cymene), and changed the nucleobase specificity.
For the complexes containing the hydrogen-bond donor en,
there was exclusive binding to N7 of guanine in competitive
nucleobase reactions, and in the absence of guanine, binding
to cytosine or thymine but not to adenine. In contrast, when
XY was the hydrogen-bond acceptor acac, the overall
affinity for adenosine (N7 and N1 binding) was comparable
to that for guanosine, but there was little binding to cytidine
or thymidine.
Modifications of natural DNA in a cell-free medium by
antitumor monodentate Ru(II) arene compounds of the
general formula [( 6-arene)Ru(en)Cl]+ (arene = biphenyl,
dihydroanthracene, tetrahydroanthracene, p-cymene, or
benzene; en = ethylenediamine) were studied by atomic
absorption, melting behavior, transcription mapping, circular
and linear dichroism, plasmid unwinding, competitive
ethidium displacement, and differential pulse polarography
[160]. The results indicated that these complexes bind
preferentially to guanine residues in double-helical DNA.
The data were consistent with DNA binding of the
complexes containing biphenyl, dihydroanthracene, or
tetrahydroanthracene ligands that involved combined
coordination to G N7 and noncovalent, hydrophobic
interactions between the arene ligand and DNA, which may
include arene intercalation and minor groove binding. In
contrast, the single hydrocarbon rings in the p-cymene and
benzene ruthenium complexes could not interact with
double-helical DNA by intercalation. Interestingly, the
adducts of the complex containing p-cymene ligand, which
had methyl and isopropyl substituents, distorted the
conformation and thermally destabilized double-helical
DNA distinctly more than the adducts of the three multiring
ruthenium arene compounds. It has been suggested that the
different character of conformational alterations induced in
DNA, and the resulting thermal destabilization, may affect
differently further "downstream" effects of damaged DNA
and consequently may result in different biological effects of
this new class of metal-based antitumor compounds. The
results pointed to a unique profile of DNA binding for Ru(II)
arene compounds, suggesting that a search for new
anticancer compounds based on this class of complexes may
also lead to an altered profile of biological activity in
comparison with that of metal-based antitumor drugs already
used in the clinic or currently on clinical trials.
The aqua adducts of the anticancer complexes [( 6X)Ru(en)Cl][PF6] (X=biphenyl (Bip), X=5,8,9,10-tetrahydroanthracene (THA), X=9,10-dihydroanthracene (DHA);
en=ethylenediamime) were separated by HPLC and
characterised by mass spectrometry as the products of
hydrolysis in water [161]. The X-ray structures of the aqua
complexes were reported. The rates of aquation of the
complexes determined by UV/VIS spectroscopy at various
ionic strengths and temperatures were >20x faster than that
of cisplatin. The reverse, anation reactions were very rapid
on addition of 100 mM NaCl (a similar concentration to that
in blood plasma). The aquation and anation reactions were
1102
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
about two times faster for the DHA and THA complexes
compared to the biphenyl complex. The hydrolysis reactions
appear to occur by an associative pathway. The pKa values
of the aqua adducts were determined by 1H NMR
spectroscopy. At physiologically-relevant concentrations
(0.5-5 M) and temperature (310 K), the complexes would
exist in blood plasma as >89 % chloro complex, whereas in
the cell nucleus significant amounts (45-65 %) of the more
reactive aqua adducts would be formed together with smaller
amounts of the hydroxo complexes (9-25 %, pH 7.4).
Inhibition of the growth of the human ovarian cancer cell
line A2780 by organometallic ruthenium(II) complexes of
the type [(6-arene)Ru(X)(Y)(Z)], where arene is benzene or
substituted benzene, X, Y, and Z are halide, acetonitrile, or
isonicotinamide, or X,Y is ethylenediamine (en) or Nethylethylenediamine, has been investigated [162]. The Xray crystal structures of the complexes [(6-p-cymene)
Ru(en)Cl]PF6, [( 6-p-cymene)RuCl2(isonicotinamide)], and
[(6-biphenyl)Ru(en)Cl]PF6 were reported. They had "piano
stool" geometries with 6 coordination of the arene ligand.
Complexes with X,Y as a chelated en ligand and Z as a
monofunctional leaving group had the highest activity.
Hydrolysis of the reactive Ru-Cl bond in complexes was
detected by HPLC but was suppressed by the addition of
chloride ions. The complexes bound strongly and selectively
to G bases on DNA oligonucleotides to form monofunctional
adducts. No inhibition of topoisomerase I or II by complexes
was detected. These chelated Ru(II) arene complexes have
potential as novel metal-based anticancer agents with a
mechanism of action different from that of the Ru(III)
complex currently on clinical trial.
Thirteen novel ruthenium(II) organometallic arene
complexes have been evaluated for activity (in vitro and in
vivo) in models of human ovarian cancer, and crossresistance profiles established in cisplatin and multi-drugresistant variants [163]. A broad range of IC50 values was
obtained (0.5 to >100 M) in A2780 parental cells. Stable
bidentate chelating ligands (ethylenediamine), a more
hydrophobic arene ligand (tetrahydroanthracene) and a
single ligand exchange centre (chloride) were associated
with increased activity. None of the six active ruthenium(II)
compounds were cross-resistant in the A2780cis cell line,
demonstrated to be 10-fold resistant to cisplatin/carboplatin.
Varying degrees of cross-resistance were observed in the P170 glycoprotein overexpressing multi-drug-resistant cell
line 2780AD that could be reversed by co-treatment with
verapamil. In vivo activity was established with RM175 in
the A2780 xenograft together with non-cross-resistance in
the A2780cis xenograft and a lack of activity in the 2780AD
xenograft. High activity coupled to non cross-resistance in
cisplatin resistant models merit further development of this
novel group of anticancer compounds.
Ruthenium(II) complexes with other derivatives of
ethylenediamine have been tested for antitumor properties
[164-166]. A new potential antitumour soluble drug
K[Ru(eddp)Cl2].3H2O, (eddp=ethylenediamine-N,N'-di-3propionate) has been isolated and characterized [167]. The
testing of the cytotoxic activity of this complex against
several human cancer cell lines evidenced that
K[Ru(eddp)Cl2] complex had a remarkable and selective
antiproliferative effect against the cervix carcinoma HeLa
Irena Kostova
and colon adenocarcinoma HT-29, behaving in these two
cases as an antineoplastic drug.
DINUCLEAR RUTHENIUM COMPLEXES
One important advancement in platinum pharmacology
was the introduction of multinuclear compounds which
highlight the real possibility to overcome the problem of
resistance, probably because of the increased interchain
DNA binding which is more refractory to cell repair systems.
The search for novel antitumour compounds based on the
ruthenium metal has brought to characterisation a series of
dinuclear complexes. These complexes, with a structure that,
considering each metal centre, mimics the antimetastasis
ruthenium(III) complex NAMI-A, show the capacity to
interact with tumour cells in vitro quite similarly to NAMI-A
and therefore we might expect that they exhibit also
interesting properties in vivo.
New ruthenium dimeric complexes, either anionic or
neutral, either symmetrical or asymmetrical, with high
antimetastatic and antitumour activity and remarkable
chemical stability were described in the invention [168]. The
two ruthenium atoms of said complexes had an oxidation
state (III) and were bound by means of a nitrogen
heterocyclic ligand containing at least two nitrogen
heteroatoms. The complexes object of the invention showed
a remarkable chemical stability and a high antimetastatic
activity. They could be used in the prevention and therapy of
tumour, especially those with an elevated degree of
metastatic diffusion, such as the digestive tract carcinomas,
mammary carcinomas, and lung carcinomas. Because of
their high stability it was possible to overcome the
inconveniences related to the processing of labile products.
This facilitated not only the administration but also the
preparation of suitable pharmaceutical formulations with
appropriate dosage and administration of the active form of
the compound. The complexes of the invention could be
profitably used in the treatment of various kinds of
neoplastic diseases. The compounds could be used in
combination with other antitumoural drugs, such as for
example
c i s platin, 5-fluorouracyl, vinblastine,
cyclophosphamide, bleomycin, anthracyclin, taxole. As
shown in the experimental results, the in vivo models related
to the treatment of a metastatizing solid tumour, such as the
MCa mammary carcinoma, with the compounds of the
invention, showed a marked and statistically significant
reduction in metastases formation. This reduction was
observed either when the treatment was performed before
surgical removal of the primary tumor or after it. In vitro
cytotoxicity of complexes of the invention was carried out
on murine TS/A adenocarcinoma cells. The data showed that
the dimeric complexes of the invention showed scarce
antiproliferative effect when they were tested in vitro on
tumour cells also when high doses were applied. In fact, the
inhibition of cell proliferation was not evident at doses lower
than 10-4 M. Under this condition the complexes did not
cause any cytotoxic effect. These data showed that the
antimetastatic activity of the complexes of the invention
could not be ascribed only to an in vitro cytotoxic effect.
Bergamo et al. have examined the biological and
antitumor activity of a series of dinuclear ruthenium
complexes [169]. The aim of this study was to compare the
Ruthenium Complexes as Anticancer Agents
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
in vitro effects of these new compounds, Fig. 10 on cell
proliferation, cell distribution among cell cycle phases, and
the expression of some proteins involved in cell cycle
regulation. Results obtained showed a mild cytotoxic activity
against human and murine cell lines, more evident after
prolonged exposure of cell challenge. These dinuclear
complexes modified cell cycle distribution similarly to
NAMI-A. Correlating the induction of cell cycle
modifications with ruthenium uptake by tumor cells and with
the modulation of proteins regulating cell cycle, the authors
stressed that the induction of G(2)-M cell cycle arrest was
related to the achievement of a threshold concentration of
ruthenium inside the cells, which was dependent on the cell
line being used, and that only cyclin B, among cell cycle
regulating proteins examined by immunoblotting assays,
appears to be significantly modified. This in vitro study
showed that dinuclear ruthenium complexes could have a
behavior similar to that of the monomer NAMI-A. These
results encouraged the future experimentation of their
pharmacological properties in in vivo models.
Ru
Cl
Cl
L
Cl
Ru
Cl
S
O
N
CH3
O
CH3
Cl
S
Cl
2-
CH3
O
S
Cl
2X+
Ru
Cl
Cl
Cl
Cl
CH3
Cl
L
Ru
NH4+
CH3
Cl
Cl
S
O
CH3
CH3
Cl
CH3
S
O
CH3
-
CH3
2
1
N
N
N
N
b
N
a
c
N
N
e
d
1103
C6H4SO3-, were tested for cytotoxicity against HeLa and
multidrug resistant CoLo 320DM human cancer cell lines.
Cell survival was measured by means of the colorometric 3(4,5dimethylthiazol-2-yl)-diphenyltetrazodium bromide
assay. The antineoplastic activity of the highly water-soluble
m-sulpho derivative was the highest, while the poorly watersoluble imidazole derivatives did not exhibit any cytotoxic
properties. The CoLo 320DM cancer cells were 5 times more
prone to drug-induced cell death than the HeLa cells.
Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal
compounds having the generalized formula MaNH2(CH)4
NH2Mb were shown to form specific DNA lesions which can
efficiently cross-link proteins to DNA [171]. In this study,
the homodinuclear case was represented by Ma = Mb = [cisPtCl2(NH3)] and the heterodinuclear case was represented by
Ma = [cis- R u C l2(DMSO)3] and Mb = [cis- P t C l2(NH3)].
Native and denaturing polyacrylamide gel electrophoresis
was used to show the formation of ternary coordination
complexes between the metal-treated 49-bp DNA fragment
and the Escherichia coli UvrA and UvrB DNA repair
proteins. Treatment with proteinase K resulted in loss of the
DNA-protein cross-links. DNA-protein cross-links formed
between UvrA and DNA previously modified with the
dinuclear metal compounds were reversible with the
reducing agent beta-mercaptoethanol. The DNA lesion
responsible for efficient DNA-protein cross-linking was
most probably a DNA-DNA interstrand cross-link in which
each metal atom was coordinated with one strand of the
DNA helix. The formation of DNA repair protein associated
DNA cross-links, potential "suicide adducts", suggested a
novel action mechanism for these anticancer compounds. In
addition, these dinuclear metal compounds should be very
useful agents for the investigation of a wide range of proteinDNA interactions.
Considering the possibility that dinuclear complexes
might release the monomeric units in vivo or, alternatively,
that they might allow a different binding to their target, these
studies should allow to better understand the role of
ruthenium complexes for the development of selective
anticancer drugs.
RUTHENIUM COMPLEXES AS INHIBITORS OF
PROTEIN KINASES
N
N
f
Fig. (10). Chemical structures of dinuclear ruthenium complexes (1,
2) and their ligands: pyrazine (a), pyrimidine (b), 4,4'-bipyridine
(c), 1,2-bis-(4,4'-pyridil)ethane (d), 1,2-bis-(4,4'-pyridil)ethylene (e)
and 1,3-bis-(4,4'-pyridil)propane (f).
Mixed-valent diruthenium tetracarboxylate complexes
were shown to have slight antineoplastic activity against
P388 leukemia cell lines [170]. However these complexes
suffered from poor water-solubility, which may have
detrimentally affected their activity. Mixed-valent
diruthenium tetracarboxylates of the type [Ru2(O2CR)4(L)2]
(PF6) with L = imidazole, 1-methylimidazole and H2O when
R = CH3, L = ethanol when R = Fc (ferrocenyl) or FcCH=CH- and of the type M3[Ru2(O2CR)4(H2O)2]4H2O, M =
Na+ when R = m-C6H4SO3- and M = K+ when R = p-
Protein kinases are a large superfamily of homologous
proteins with 518 members in the human genome [172].
Protein kinases regulate most aspects of cellular life and are
one of the main drug targets. The microbial alkaloid
staurosporine is a very potent but relatively nonspecific
inhibitor of many protein kinases. Many staurosporine
derivatives and related organic compounds with modulated
specificities have been developed, and several are in clinical
trials as anticancer drugs. For this class of inhibitors,
specificity for a particular protein kinase can be achieved by
the moiety that is attached to the indole nitrogen atoms.
Meggers et al. [173-178] showed that by replacing the
indolocarbazole alkaloid scaffold with metal complex,
elaborate structures could be assembled in an efficient
manner by variation of the ligands. The authors are
interested in exploring organometallic and inorganic
compounds as structural scaffolds for enzyme inhibition.
Such metal-ligand assemblies allow convergent synthetic
1104
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
approaches and give access to structural motifs that differ
from purely organic molecules. Meggers’s group has made
progress with investigating the scope of using
organometallic compounds as structural scaffolds for the
design of bioactive molecules. In contrast to conventional
metal-containing bioactive compounds or imaging agents, in
these molecules the metal center plays a solely structural role
by organizing the organic ligands in the three-dimensional
receptor space. Several ruthenium complexes, this group has
designed to mimic the shape of staurosporine, have proven to
be potent and selective inhibitors of protein kinases,
including glycogen synthase kinase 3. Their efforts are
focused on ruthenium complex scaffolds because ruthenium
offers a hexavalent coordination sphere that cannot be easily
obtained by any organic element. In addition, ruthenium
tends to form kinetically very inert coordinative bonds,
making it possible to obtain compounds that display
stabilities that are comparable to purely organic molecules.
Meggers et al. [173-178] introduced a strategy that uses a
class of natural products as a lead for a ruthenium complex
scaffold. These were the first reports of ruthenium
complexes used as protein kinase inhibitors. Ruthenium
complexes were more potent than staurosporine. Such
properties of organometallic compounds indicates that the
strategy of using kinetically inert metal centers as rigid
structural scaffolds for the design of enzyme inhibitors leads
to new opportunities for creating highly potent molecular
probes and ultimately drugs.
CONCLUSION
Inorganic compounds have been used in medicine for
many centuries but often only in an empirical fashion with
little attempt to design the compounds used and with little or
no understanding of the molecular basis of their mechanisms
of action. The interaction of transition metal ions with
biological molecules provides one of the most fascinating
areas of coordination chemistry. The application of this field
to biomedical uses deals with the use of metal complexes as
drugs and chemotherapeutic agents. Anticancer agent
therapy is gradually changing and a variety of novel research
avenues is available with the assistance of which large
amount of new compounds were produced possessing strong
antitumour activity on a broad spectrum of preclinical
tumour models. Some of them display an activity and safety
profile completely different from the standard cytotoxic
agents. This fact raises the hope that we are experiencing the
advent of a new period in which the combination of both
cellular /classic/ and molecular chemotherapy approaches
will be highly successful in the treatment of cancer. In
summary this review certainly is a comprehensive overview
of the applications of coordination chemistry of ruthenium in
medicinal and biomedical applications. Since the middle
seventies, many studies have been published, showing in a
convincible and repetitive manner, the possible advantages
of ruthenium as a base for new competitive drugs in cancer
therapy. Ruthenium complexes offer the potential of reduced
toxicity, a novel mechanism of action, non-cross resistance
and a different spectrum of activity compared to platinum
containing compounds. An important activation mechanism
for Ru(III) is thought to be reduction to Ru(II). Both Ru(II)
and Ru(III) bind strongly to DNA bases with a strong
preference for G-N7. Ruthenium organometallic complexes
Irena Kostova
form monofunctional adducts with guanine in DNA in vitro
and have a cytotoxic anticancer activity spectrum in
preclinical models suggesting lack of cross-resistance with
cisplatin. The primary cytotoxic lesion remains to be
identified but the downstream mechanism of action is
nevertheless of interest. Protein binding may also play an
important part in the mechanism of action of Ru complexes.
The uptake of Ru(III) by cells appears to be mediated by
transferrin. Ru-transferrin complexes themselves exhibit
anticancer activity. Enzyme targets for Ru may also include
topoisomerase and matrix metalloproteinases. Metastasis of
solid tumours represent the target of election for the
pharmacological treatment of cancer. Nevertheless,
commonly used treatments do not represent any selective
approach, provided that drugs are mostly unspecific
cytotoxics. Today many strategies adopted to interfere with
metastasis growth concern the interaction with biological
signals of the metastatic cells or of the host. Many ruthenium
agents active on newly identified molecular targets are more
effective in preventing metastasis formation than in
inhibiting their growth and it seems to provide optimism for
the future.
ABBREVIATIONS
Cisplatin
= Cis-diamminedichloroplatinium(II)
MAC
15A
= Colon tumour cell line
B16
= Melanoma cell line
Gua
= Guanine
CD
= Circular dichroism
ICP
(AES)
= Inductively coupled
emission spectroscopy
Hind
= Indazole
Him
= Imidazole
topo II
= Type II of topoisomerase
plasma
atomic
topo I
= Type I of topoisomerase
NAMI
= Sodium trans-imidazole-dimethyl
ide-tetrachlororuthenate
sulfox-
NAMI-A
= Imidazolium trans-imidazole-dimethyl sulfoxide-tetrachlororuthenate
DMSO
= Dimethyl sulfoxide
NO
= Nitric oxide
VEGF
= Vascular endothelial growth factor
MCa
= Mammary carcinoma cell line
TS/A
= Mammary adenocarcinoma cell line
TLX5
= Lymphoma cell line
ICR
= Imidazolium trans-imidazoletetrachlororuthenate
MCF-7
= Mammary carcinoma cell line
B16-F10
= Highly metastatic variant of melanoma B16
cell lines
IC50
= Concentration of inhibitor that affords 50%
of inhibition
Ruthenium Complexes as Anticancer Agents
Current Medicinal Chemistry, 2006, Vol. 13, No. 9
[4]
[5]
MTT
= Microculture 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide reduction
test
dmtp
= 5,7-dimethyl[1,2,4]triazolo[1,5a]pyrimidine
[7]
SW480
= Colon cancer cells
[8]
CH1
= Ovarian cancer cells
[9]
Htrz
= 1,2,4-triazole
[10]
1A9
= Ovarian cancer cell line
phen
= 1,10-phenanthroline
bpy
= 2,2'-bipyridine
[11]
[12]
[13]
[14]
nmit
= N-methyl-isatin-3-thiosemicarbazone
[15]
icpl
= Isatin-3-(4-Cl-phenyl)thiosemicarbazone)
[16]
aze
= Acetazolamide
tsc
= Thiosemicarbazone
[17]
[18]
EAC
= Ehrlich Ascites Carcinoma
[19]
ihqs
= 7-iodo-8hydroxy quinoline-5-sulphonic acid
hc
= 3-hydroxycoumarin
[20]
[21]
[22]
TLX5
= Lymphoma cell line
A2780/S
= Ovarian cancer cells
A2780cisR = Cisplatin-resistant human carcinoma cell
line
[6]
[23]
[24]
[25]
[26]
tazpy
= o-tolylazopyridine
mazpy
= 4-methyl-2-phenylazopyridine
KTZ
= Ketoconazole
CTZ
= Clotrimazole
DFT
= Density functional theory
THA
= 5,8,9,10-tetrahydroanthracene
DHA
= 9,10-dihydroanthracene
9EtG
= 9-ethylguanine
[32]
Guo
= Guanosine
[33]
Cym
= P-cymene
[34]
Ben
= Benzene
en
= Ethylenediamine
eddp
= Ethylenediamine-N,N'-di-3-propionate
HeLa
= Cervix carcinoma cell line
HT-29
= Colon adenocarcinoma cell line
P388
= Leukemia cell lines
CoLo
320DM
= Human cancer cell line
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