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In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes.
J. Med. Chem. 2005, 48, 4161-4171
4161
In Vitro and in Vivo Evaluation of Ruthenium(II)-Arene PTA Complexes
Claudine Scolaro,† Alberta Bergamo,‡ Laura Brescacin,‡ Riccarda Delfino,‡ Moreno Cocchietto,‡
Gábor Laurenczy,† Tilmann J. Geldbach,† Gianni Sava,*,‡,§ and Paul J. Dyson*,†
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL),
CH-1015 Lausanne, Switzerland, Callerio Foundation Onlus, Via A. Fleming 22-31, 34127 Trieste, Italy, and
Dipartimento di Scienze Biomediche, Università di Trieste, Via L. Giorgieri 7-9, 34127 Trieste, Italy
J. Med. Chem. 2005.48:4161-4171.
Downloaded from pubs.acs.org by UNIV OF SASKATCHEWAN on 01/01/19. For personal use only.
Received January 7, 2005
The antitumor activity of the organometallic ruthenium(II)-arene complexes, RuCl2(η6-arene)(PTA), (arene ) p-cymene, toluene, benzene, benzo-15-crown-5, 1-ethylbenzene-2,3-dimethylimidazolium tetrafluoroborate, ethyl benzoate, hexamethylbenzene; PTA ) 1,3,5-triaza-7phosphaadamantane), abbreviated RAPTA, has been evaluated. In vitro biological experiments
demonstrate that these compounds are active toward the TS/A mouse adenocarcinoma cancer
cell line whereas cytotoxicity on the HBL-100 human mammary (nontumor) cell line was not
observed at concentrations up to 0.3 mM, which indicates selectivity of these ruthenium(II)arene complexes to cancer cells. Analogues of the RAPTA compounds, in which the PTA ligand
is methylated, have also been prepared, and these prove to be cytotoxic toward both cell lines.
RAPTA-C and the benzene analogue RAPTA-B were selected for in vivo experiments to evaluate
their anticancer and antimetastatic activity. The results show that these complexes can reduce
the growth of lung metastases in CBA mice bearing the MCa mammary carcinoma in the
absence of a corresponding action at the site of primary tumor growth. Pharmacokinetic studies
of RAPTA-C indicate that ruthenium is rapidly lost from the organs and the bloodstream.
Introduction
Current inorganic drugs such as cisplatin and related
compounds are successfully used in the treatment of
many cancer types with a high social incidence.1,2
However there are problems associated with their use,
in particular, cisplatin is highly toxic, leading to side
effects and limiting the dose that can be administrated.3
New developments in platinum drug design4,5 and drugdosing protocols6 have gone some way to reduce the
toxicity of these compounds. Complexes based on other
metals have been investigated for their possible application as antitumor drugs. One of the most promising
metals is ruthenium,7,8 and a number of ruthenium
complexes show high in vitro and in vivo antitumor
activity and some compounds are currently undergoing
clinical trials.9-11 Although the mechanism of action of
antitumor ruthenium compounds is not fully understood, it is thought that for certain species, similar to
the platinum drugs, the chloride ligands are substituted
by water in vivo,12,13 which subsequently undergo
substitution by the nucleobases of DNA.14
The ruthenium complex ImH[trans-RuCl4(DMSO)Im],
NAMI-A, has high selectivity for solid tumor metastases
and low host toxicity at pharmacologically active doses15
and was the first ruthenium complex to enter clinical
trials.16 Although this complex has a remarkably low
general toxicity,17,18 and shows marked efficacy against
metastases,19,20 it does not affect primary tumor
growth21-23 and does not exhibit cytotoxicity against
tumor cells in vitro. A related ruthenium(III) compound,
* Corresponding Author: Prof. Paul J. Dyson. E-mail: paul.dyson@
epfl.ch. Tel: +41 (0)21 693 98 54. Fax: +41 (0)21 693 98 85.
† Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique
Fédérale de Lausanne.
‡ Callerio Foundation Onlus.
§ Dipartimento di Scienze Biomediche, Università di Trieste.
KP1019,24 has also entered clinical trials, since it was
found to exhibit antiproliferative activity in vitro in
human colon carcinoma cell lines.25
The lower general toxicity of ruthenium complexes
compared to platinum drugs has been attributed to the
ability of ruthenium compounds to specifically accumulate in cancer tissues.26 The higher specificity of
these compounds for their targets may also be linked
to the selective uptake by the tumor compared with
healthy tissue27,28 and because of a selective activation
by reduction to cytotoxic species within the tumor.29
While the anticancer activity of ruthenium coordination complexes has been studied in detail for many
years, organoruthenium compounds, with direct ruthenium-carbon bonds, are now attracting interest. The
first organoruthenium compound to be evaluated as an
anticancer agent was Ru(η6-C6H6)Cl2(metronidazole)
(metronidazole ) 1-β-hydroxyethyl-2-methyl-5-nitroimidazole). While it was found that the complex had a
greater selective cytotoxicity than metronidazole itself
under hypoxic reducing conditions, as far as we are
aware, further studies were not forthcoming.30 Subsequently, several types of ruthenium(II)-arene complexes have shown high in vitro and/or in vivo antitumor
activity including those with phosphine, amine, and
sulfoxide co-ligands. Cationic [RuCl(η6-arene)(en)]+ (en
) ethylenediamine) complexes show in vitro inhibition
of cancer cell growth and in vivo antitumor activity.31,32
Although the interactions of the ruthenium(II)-arene
compounds have been most extensively studied with
DNA, RuCl2(η6-C6H6)(dimethylsulfoxide (DMSO) has
been shown to inhibit the DNA relaxation activity of
topoisomerase II.33 Another series of ruthenium(II)arene compounds with disulfoxide ligands have been
10.1021/jm050015d CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/21/2005
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Scheme 1. Protonation of the PTA Ligand in 1,
Postulated as Being Responsible for pH Dependant
Damage to DNA
tested for anticancer activity in vitro and showed
cytotoxicity against a human mammary cancer cell
line.34
Our attention has focused on ruthenium(II)-arene
complexes combined with the 1,3,5-triaza-7-phosphaadamantane (PTA) ligand. The compound RuCl2(η6C10H14)(PTA), named RAPTA-C, 1, was found to exhibit
pH-dependent DNA damage such that at the pH typical
of hypoxic tumor cells, DNA was damaged, whereas at
the pH characteristic of healthy cells, little or no damage
was detected.35,36 Such behavior was attributed to the
PTA ligand which can be protonated at low pH, and the
protonated form was considered to be the active agent.
To test this hypothesis a detailed investigation of
RAPTA compounds, as well as model Me-PTA analogues, has been undertaken, paying particular attention to their aqueous chemistry and in vitro cytotoxicity
on tumor cells.37,38 In parallel, in vivo experiments were
carried out to evaluate the anticancer and antimetastatic activity of these complexes and the distribution
of 1 in the organs and the blood. Herein, we describe
the outcome from these studies.
Scolaro et al.
Results and Discussion
We previously reported the synthesis, characterization, and effect of 1 on plasmid DNA.35 It was found
that at a pH >7 almost no damage to the DNA is
observed, whereas below pH 7 DNA damage is prevalent. Since healthy cells grow at pHs above 7, typically
pH 7.2, and (hypoxic) cancer cells have characteristically
lower pH values, typically pH 6.8, we proposed that the
compound might selectively target cancer cells. At the
time, we proposed that PTA could be protonated at
lower pH and in this form cause damage to the DNA as
illustrated in Scheme 1.
With this mechanism in mind, we proposed that the
methylated PTA derivative [RuCl2(η6-C10H14)(PTAMe)]+ (2) would display indiscriminate DNA damage
and show no specific selectivity toward cancer cells over
healthy cells. To test this hypothesis, and consolidate
the preliminary data obtained using plasmid DNA, cell
studies were undertaken. RAPTA compounds 1-9 (see
Chart 1) were prepared according to the method previously outlined for 1. Full synthetic details and spectroscopic characterization is provided in the Experimental
Section and in the Supporting Information.
Hydrolysis of 1 in Aqueous, Buffered, and Salt
Solutions. To delineate the differences in activity
between the RAPTA compounds and the methyl-PTA
derivatives 2 and 4 it is important to know the identity
of the compound that reaches the cell, and accordingly,
the aqueous chemistry of the complexes is very important. Hydrolysis of 1 was studied using UV-vis spectrophotometry under various conditions. The UV-vis
spectrum of 1 at 298 K immediately after dissolution
in pure water exhibits a strong absorption band at 342
nm which rapidly changes (over several minutes) to 326
nm (see Figure 1) indicating that rapid hydrolysis of the
complex takes place, although the exact nature of the
hydrolysis product was not characterized until later (see
below).
A similar approach was adopted to study the hydrolysis of 1 in phosphate buffered solutions at pH 2, 7, and
Chart 1. Structure of the Ruthenium(II)-arene-PTA (RAPTA) Compounds and the Me-PTA Derivatives of RAPTA-C
and RAPTA-T, Prepared as the Chloride Salts
Ruthenium(II)-Arene PTA complexes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4163
Figure 2. Determination of the free chloride concentration
in an aqueous solution of complex 1 by ionic chromatography
at 298 K.
Scheme 2. Proposed Hydrolysis of 1
Figure 1. (Top) Time evolution of the UV-vis absorption
spectra of 1 in water at 25 °C recorded at 1 min intervals over
a period of 20 min (no further change was observed after this
time). (Bottom) UV-vis absorption spectra of 1 in phosphate
solution at pH 7, recorded every minute at 25 °C.
12 and in NaCl solutions representative of blood plasma
(100 mM) and intracellular (4 mM) concentrations. In
phosphate buffer solution at pH 2, essentially the same
species is formed as that observed in water, as is
demonstrated by the isosbestic point at 346 nm. Surprisingly, at pH 7, the spectrum (see Figure 1) is
different from that observed in water and is similar to
that determined in phosphate buffer at pH 12, indicating that the phosphate interacts with the complex. The
initial absorption band at 342 nm, which corresponds
to the dichloro compound, decreases with time and a
species which absorbs at 300 nm is formed, and this
equilibrium is characterized by an isosbestic point at
324 nm.
The UV-vis spectrum of 1 at 298 K in 4 mM NaCl
solution is essentially the same as that observed in
water, whereas in a 100 mM NaCl solution hydrolysis
of 1 does not take place, that is, no change is observed
in the UV-vis spectrum. These latter results indicate
that the hydrolysis of 1 only takes place inside the cells
where the chloride concentration is approximately 4
mM, whereas hydrolysis is suppressed at an extracellular concentration of chloride. Moreover, the maximum
absorption band in the spectrum of 1 with 100 mM NaCl
is similar to the spectrum obtained in dichloromethane
which indicates that the compound is not hydrolyzed.
These results suggest that the compound is only activated by hydrolysis inside the cell by substitution of
chloride by water which then allows the compound to,
for example, react further with DNA.
The identity of the hydrolysis product of 1 could not
be ascertained from the UV-vis studies, and it is
feasible that one or both of the chloride ligands could
be replaced by water. Attempts to characterize the
hydrolysis product using 17O enriched water were
unsuccessful. Thus, ionic chromatography was used to
measure the concentration of chloride in aqueous solution to determine the extent of hydrolysis. A graph
showing the concentration of the complex 1 vs the free
chloride concentration measured in the solution is
shown in Figure 2.
Somewhat unexpectedly, it was found that if one
chloride is lost from 1, then the second is also lost
simultaneously, as indicated by the two equilibrium
constants K1 ) 0.03 mM and K2 ) 107 mM at 298 K
(see Scheme 2). However, loss of the chlorides is strongly
dependent on the concentration of chloride present in
solution. According to Scheme 2, an excess of free
chloride displaces the equilibria to the left. The hydrolysis of 1 takes place relatively rapidly, and afterward
there is no further change in the chloride concentration
for several days. The equilibrium constants K1 and K2
were calculated from the total complex and free chloride
concentrations using the least-squares fit method (the
sum of squares of the difference (measured [chloride]
- calculated [chloride]) were minimized).
This behavior is markedly different from that observed for cisplatin, which like 1 does not undergo
hydrolysis in the extracellular medium, but rapidly loses
one chloride for water once inside a cell, with the second
hydrolysis step being considerably slower.39 With ruthenium drugs, or drug candidates, hydrolysis also
appears to be an important part of their activation. For
example, the degradation of NAMI-A consists of stepwise hydrolysis of the chloride ligands in acidic media,
accompanied by the hydrolysis of the DMSO group,
followed by the formation of a poly-oxo species.40 The
coordinated water in [Ru(OH2)(η6-p-cymene)(en)]2+ deprotonated to the hydroxo species [Ru(OH)(η6-p-cymene)(en)]+, and the respective acid dissociation constants of
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Scolaro et al.
Figure 3. UV-vis absorption spectrum of 1 in water (b) and
in a solution of calf thymus DNA (2) overlaid with the calf
thymus DNA spectrum (9).
Figure 5. 31P chemical shift vs pD for PTA (top), 1 (middle
left), 3 (middle right), 5 (bottom left), and 9 (bottom right).
Table 1. pKa Values for the PTA Ligand and for Complex 1, 3,
5, and 9 at 298 K in 0.1 M NaCl Solution
Figure 4. DNA melting curves of calf thymus DNA alone (9)
and calf thymus DNA with 1 (1:1 ratio) (O). Buffer consisted
of 10 mM phosphate buffer and 50 mM NaClO4.
the aqua adducts were determined,41 whereas in [Ru(OH2)2(η6-arene)(PTA)]2+ no such transformation had
been observed under physiological pH values (see
below).
DNA Interactions. The absorption spectra of calf
thymus DNA, 1, and a mixture of the two (1:1 ratio)
are illustrated in Figure 3. Calf thymus DNA and 1 have
overlapped transitions below 300 nm. Consequently, any
metal complex spectral changes can be observed above
300 nm where calf thymus DNA has no absorbance.
Effectively, when 1 is incubated with calf thymus DNA
a change in the absorption spectra is observed, indicating that the compound binds to DNA. The melting of
calf thymus DNA was studied in the presence of complex
1. The increase in absorbance at 260 nm was recorded
from 50 to 90 °C, and the melting temperature, Tm, was
then determined from the denaturation curves shown
in Figure 4.
Complex 1 is able to modify in vitro calf thymus DNA
with the melting temperature increasing from 67 to 80
°C (∆Tm ) 13 °C) upon binding of the compound (see
Figure 4). These data indicate that complex 1 induces
a stabilization of the double helix with a corresponding
increase in melting temperature. The effect of 1 on calf
thymus denaturation can be compared to other ruthenium antitumor compounds. For example, the ruthenium(III) compounds, Na[trans-RuCl4(DMSO)(Im)]
(NAMI) and Ru(PDTA)Cl2 (PDTA ) propylenediamine-
complex
pKa
PTA
RuCl2(η6-C10H14)(PTA), 1
RuCl2(η6-C7H8)(PTA), 3
RuCl2(η6-C6H6)(PTA), 5
RuCl2(η6-C6Me6)(PTA), 9
5.63 ( 0.05
3.13 ( 0.02
3.31 ( 0.03
3.23 ( 0.06
2.99 ( 0.02
tetraacetate), both induce a slight stabilization of the
double helix (∆Tm ≈ +1-2 °C).42 The ruthenium(II)arene complex RuCl2(η6-C6H6)(DMSO) induces a stabilization of the double helix between 9 and 23 °C
depending on the concentration of the complex.33
Protonation of the Phosphine PTA Ligand (pKa).
The pKa of PTA, PTA-Me+, and complexes 1, 3, 5, and
9 was determined using 31P NMR spectroscopy in D2O
(see Experimental Section). Graphs showing the chemical shift of the 31P nucleus vs pD are shown in Figure
5, since no pKa was observed for the methylated PTA
compounds. The curves were fitted with the Henderson-Hasselbach equation. The pH titration curves were
carried out in D2O, and the pKa value is obtained from
the midpoint of the curve where 0.44 are subtracted.43
The pKa values of the free and coordinated ligand are
summarized in Table 1. The pKa of PTA has been
reported on more than one occasion with values of 6.044
and 5.7,45 which are in keeping with those estimated
herein. Upon coordination, the pKa of the PTA ligand
decreases, and it can be fine tuned by varying the arene
ligand. These values were determined in the presence
of chloride to ensure that the process being determined
is the protonation of the PTA ligand attached to the
complex. However, essentially identical values are also
obtained in the absence of chloride, and the formation
of hydroxo ligands from the hydrolyzed species was not
observed.
Ruthenium(II)-Arene PTA complexes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4165
Figure 6. Effects of 1 on TS/A (top left) and HBL-100 (top right) cell proliferation, 2 on TS/A (middle left) and HBL-100 (middle
right) cells, and 3 on TS/A (bottom left) and HBL-100 (bottom right). The cells were sown on day 0 and treated on day 1 with a
range of concentrations between 1 and 300 µM, for the TS/A cells and HBL-100, of compound dissolved in water for 24, 48, and
72 h. The cell number was evaluated using the MTT test at the end of the treatment. Standard error bars (always below 10% of
the mean values) are omitted for clarity.
These results indicate that a more complicated mechanism is required to rationalize the selective anticancer
activity of RAPTA compounds. It is possible that once
the complexes bind to DNA the pKa of the PTA ligand
increases to a value which is compatible with its
protonation in the hypoxic environment of a cancer cell,
which then exerts a secondary hydrogen bonding interaction much in the same way as cisplatin.46 Combined,
the two interactions may induce greater toxicity than
coordination alone, but further experiments are required
to prove such a hypothesis. However, protonation of the
PTA ligand represents only one possible reason for the
selectivity of the RAPTA compounds, and it is worth
noting that while DNA is viewed as the traditional
target for inorganic drugs, there is a great deal of
evidence to suggest that for ruthenium compounds other
non-DNA targets are important, and in the case of DNA
targets many different kinds of DNA adducts can be
formed with the anticancer agents. For example, the
most prevalent DNA adduct with cisplatin is a linking
of platinum to adjacent purine bases.47 When cisplatin
is allowed to react with DNA in vitro, the main adducts
formed are 1,2-intrastrand cross-links, monofunctional
adducts, or protein-DNA cross-links.48,49 The possibility
of drug interactions with proteins on the cell membrane
in the understanding of antitumor activity of some
anticancer compounds is a consideration that must be
taken into account in further investigations.
Cytotoxic Effects on TS/A Adenocarcinoma Cells
and on HBL-100 Mammary Cells. The biological
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test which measures mitochondrial dehydrogenase activity as an indication of cell viability was
carried out with all nine of the RAPTA compounds (see
Chart 1) using two different cell lines, tumor mouse
TS/A cells and normal human HBL-100 cells. The effects
of ruthenium(II)-arene compounds 1-9 on the viability
of these cells were evaluated by measuring the variations after 24, 48, and 72 h of treatment. The cell
viability after these times was determined using the
MTT assay, and the results from these studies are
summarized in Figure 6 for RAPTA-C, its Me-PTA
derivative 2, and the toluene analogue 3. The experiments were repeated twice for all of the compounds, and
the IC50 values resulting from an average over the two
experiments are listed in Table 2.
From Table 2 it is apparent that the RAPTA compounds 3 and 7 are clearly more cytotoxic for the cancer
cells (TS/A) than for the normal cells (HBL-100) which
suggest that they may be selective toward cancer cells
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Scolaro et al.
Table 2. IC50 Values of the RAPTA Compounds 1, 3, 5, 6, 7, 8,
and 9 and Their Methyl-PTA Analogues 2 and 4 on the TS/A
and HBL-100 Cell Lines after 72 h of Incubation
Table 3. Ruthenium Uptake after Treatment for 24 h in TS/A
Cells in Vitro with 100 µM of RAPTA Compounds
compound
RuCl2(η6-C10H14)(PTA), 1
[RuCl2(η6-C10H14)(Me-PTA)]+, 2
RuCl2(η6-C7H8)(PTA), 3
[RuCl2(η6-C7H8)(Me-PTA)]+, 4
RuCl2(η6-C6H6)(PTA), 5
RuCl2(η6-benzo-15-crown-5)(PTA), 6
[RuCl2(η6-C6H5(CH2)2Im)(PTA)][BF4], 7
RuCl2(η6-C6H5CO2Et)(PTA), 8
RuCl2(η6-C12H18)(PTA), 9
IC50 (TS/A) IC50 (HBL-100)
72 h (µM)
72 h (µM)
>300
>300
74
110
231
159
66
103
199
>300
246
>300
77
>300
>300
>300
>300
>300
in vivo, possibly leading to low toxicity. Most of the IC50
values could not be determined because the values are
out of the concentration range used for the experiments
and would be higher than 300 µM. The only two
compounds which showed a measurable cytotoxicity for
the normal cells are the methyl-PTA derivatives of
RAPTA-C and RAPTA-T, both being slightly more toxic
to the healthy cells. All of the other compounds can be
considered as nontoxic toward the HBL-100 cells. Even
the functionalized compounds 6 and 7 exhibit selective
toxicity toward the cancer cells.
The most striking difference in cytotoxicity between
the two cells lines is observed for 3 and 7 which exhibit
IC50 values of 74 and 66 µM after 72 h of incubation
with the TS/A adenocarcinoma cells but are essentially
nontoxic to the HBL-100 normal cells under analogous
conditions. Moreover, the cytotoxicity of RAPTA-T 3
shows activity dependence with the time of exposure
(see Figure 6). A similar pattern is observed for the
p-cymene analogue 1, but it is less effective toward the
cancer cells than the toluene analogue. The selective
cytotoxicity of the RAPTA compounds toward the cancer
cells could potentially lead to a drug with lower side
effects compared to other metal-based anticancer drugs.
However, it is worth noting that all of the series of
RAPTA compounds are less active than cisplatin where
the MTT assay using the same cell line (TS/A) gave a
corresponding IC50 of 0.53 µM, although cisplatin displays a very high general cytotoxicity.50,51 Other ruthenium(II)-arene compounds have been tested on the
human mammary cancer cell line (MDA-MB-435s) and
the human ovarian cancer cell lines and exhibit IC50
values between 55 and 360 and 6-200 µM,34 respectively, which is in keeping with the compounds described herein and characteristic of ruthenium compounds which are generally less cytotoxic than platinum
compounds. Another series of ruthenium(II)-arene
complexes have been evaluated for activity in vitro
against the platinum-sensitive human ovarian cancer
cell line A2780 which showed high cytotoxicity with IC50
values between 0.5 µM and 100 µM, some being comparable to carboplatin and cisplatin antitumor agents.31,32
These complexes seem to act in the same way as the
cytotoxic agent, cisplatin, that is, via DNA binding.
However, the antimetastatic NAMI-A complex does not
show direct cytotoxicity for tumor cells in vitro, but it
is highly effective in vivo against metastasis cells. Its
mode of action is consistent with antiangiogenic properties, inhibition of matrix metalloproteinase, and modulation of tumor-tissue interactions.28,52
Determination of Intracellular Ruthenium Concentration. After TS/A cells were exposed to 100 µM
compounds
intracellular RAPTA
(µg/106 cells)
intracellular RAPTA
(×10-4 M)
RAPTA-C, 1
RAPTA-B, 5
RAPTA-H, 9
RAPTA-T, 3
RAPTA-BI, 7
RAPTA-BC, 6
RAPTAMe+-T, 4
RAPTA-CO2Et, 8
0.12 ( 0.02a
0.13 ( 0.02
0.15 ( 0.01
0.16 ( 0.02
0.19 ( 0.04
0.28 ( 0.02
0.33 ( 0.08
3.40 ( 0.41
2.55 ( 0.06
3.26 ( 0.04
3.16 ( 0.27
2.9 ( 0.3
3.03 ( 0.67
4.63 ( 0.35
5.9 ( 1.4
54.67 ( 6.66
a Each number is the mean ( SE of an experiment made in
triplicate.
of RAPTA compounds for 24 h, their uptake was
determined by flameless atomic absorption spectroscopy
(AAS), and the results are reported in Table 3.
Under the conditions used, the intracellular concentration of ruthenium is 2- to 6-fold higher for all of the
RAPTA compounds when compared to the concentration
in the culture medium. The amount of ruthenium in the
tumor cells is between 0.12 and 0.33 µg with the
exception of 8 where the intracellular concentration is
54.67 × 10-4 M corresponding to 3.40 µg of ruthenium
per million cells. A reasonable hypothesis for explaining
the high amount of 8 in the tumor cells after only 24 h
of incubation may be the presence of esterases which
break the ester function such that the resulting charged
species remain inside the cell.53 The RAPTA compounds
may enter the tumor cells either by passive diffusion
or by active transport, or even by an association of these
two processes, but without further experiments it is not
possible to say explicitly which mechanism is involved.
Effects on Tumor Growth and Metastases Formation. In vivo experiments were carried out with 1
and 5 to evaluate the effects of these compounds on
primary tumor growth and lung metastasis formation,
using the MCa mammary carcinoma in CBA mice, with
ip (intraperitoneal) treatment started 5 days after tumor
implantation. The control group consisted of nine mice.
The tested groups with 1 contained five mice each.
Different doses of compound were used for each group
of five mice to establish the presence of a dose-effect
relationship. The first group received a single dose of
200 mg/kg on day 5 after tumor implantation. This
injection was also performed on another group with a
second injection on day 9. Another group received a dose
of 100 mg/kg on days 5, 7, 9, and 11. The total dose of
400 mg/kg was also divided into a daily injection of 40
mg/kg from days 5 to 14. The last treated group was
given a single injection of 400 mg/kg. The experiment
ended on day 20 after which tumor growth and lung
metastases were counted. The effects of 1 on the
primary tumor and lung metastasis formation are
reported in Table 4.
For RAPTA-B 5, the experiment comprised two treated
groups of mice and the control group. The first group
received a daily injection of 40 mg/kg from days 5 to 14
and the second group a dose of 100 mg/kg on days 5, 7,
9, and 11. The experiment ended on day 19 and lung
metastases were counted. The effects of 5 on the volume
of the primary tumor and lung metastases formation
are shown in Table 5.
The in vivo experiment where 1 was administrated
shows diverse behaviors depending on the schedule of
Ruthenium(II)-Arene PTA complexes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4167
Table 4. Effects of 1 on the Tumor Growth and Metastasis
Formation of MCa Mammary Carcinoma in CBA Mice
Table 6. Pharmacokinetic Dataa for a Monocompartment
Distribution
lung metastasesb
RAPTA-C 1
compd
treatment
group
primary tumora
(mg)
no.
weight
controls
200 mg/kg
200 mg/kg/day (×2)
100 mg/kg/day (×4)
40 mg/kg/day (×10)
400 mg/kg/day
3780 ( 164
3498 ( 233
3663 ( 160
3472 ( 161
4173 ( 215
3442 ( 164
14.6 ( 4.5
13.3 ( 9.9
6.3 ( 3.5
7.3 (2.1
12.8 ( 4.3
19.7 ( 8.1
29.5 ( 8.8
19.6 ( 18.9
21.4 ( 9.6
18.7 ( 6.3
21.1 ( 8.6
34.9 (12.5
treatment
groups
RAPTA-C 4 × 100 mg/kg/day
2 × 200 mg/kg/day
1 × 200 mg/kg
NAMI-Ab 1 × 200 mg/kg
T1/2
(h)
Vd
Cltot
AUC
(mL) (mL/h) (mg‚h/L)
10.39
12.21
11.47
12.00
100
163
153
110
6.5
9.2
9.0
7.33
615
873
891
689
a Data were obtained by determination of 1 plasma concentrations. b Pharmacokinetics parameters of NAMI-A compound from
ref 67.
a Measured on day 19. b Measured on day 20.
Table 5. Effects of 5 on the Tumor Growth and Metastasis
Formation of MCa Mammary Carcinoma in CBA Mice
lung metastasesa
RAPTA-B 5
treatment
group
primary
tumora (mg)
no.
weight
controls
40 mg/kg/day (×10)
100 mg/kg/day (×4)
3776 ( 352
3038 ( 398
3376 ( 194
15.8 ( 3.7
11.6 ( 2.7
11.5 ( 2.1
4.7 ( 1.8
2.8 ( 1.0
3.3 ( 1.4
a Measured on day 19.
administration. RAPTA-C 1 was found to reduce the
number of lung metastases when given at 2 × 200 and
4 × 100 mg/kg/day schedules. The single dose of 400
mg is totally devoid of effects (see Table 4). The dose of
200 mg/kg repeated twice appears to be the most
efficient in reducing lung metastases formation. It is
important to note that at the end of the experiment
when the metastases were counted, two mice (out of five)
in this treated group did not have any metastasis. From
these observations, it seems that the single dose of 400
mg/kg is too large, but if the injection is divided into 2
doses of 200 mg/kg the effect on lung metastases
formation is more significant. Effectively, the results
obtained with the other dose (4 × 100 mg/kg/day)
administrated with higher frequency also show similar
effects on lung metastases formation.
RAPTA-B 5 was also studied in the same mouse
model administered with a total dose of 400 mg/kg at
two different frequencies, and the effects of this complex
on the primary tumor growth and on lung metastases
formation are reported in Table 5. The two different
doses of 5 administrated to the mice have the same
slight, statistically nonsignificant, effect on the metastases number. Moreover, these two doses do not
influence the evolution of the primary tumor.
NAMI-A, given ip at 35 mg/kg/day for 6 consecutive
days, to mice bearing MCa mammary carcinoma, Lewis
lung carcinoma, and H460M2 human lung cancer,
reduces lung metastasis weight by 71-90% and metastasis number by 40-60% in the absence of a corresponding action at the site of primary tumor growth.54,55
The removal of only the metastatic cells from the
primary tumor may explain the modest activity of
NAMI-A at the primary tumor level, where these cells
often represent a small fraction.56
Pharmacokinetic Study with 1. A pharmacokinetic
study in healthy Swiss CD-1 mice was carried out to
determine the fate of 1 after ip administration. The
content of ruthenium in the blood, plasma, and in some
organs (liver, kidney, spleen, and lung) was examined
to define the fundamental pharmacokinetic parameters
of 1. The amount of ruthenium was measured, in three
separate mice per group per time, by AAS after the
different ip treatment schedule of 1. Two groups received a total dose of 400 mg/kg administrated with a
different frequency: the first group received 4 doses of
100 mg/kg every 2 days (days 1, 3, 5, and 7) and the
other one 2 doses of 200 mg/kg with 4 days of interval
(days 3 and 7). The last treated group received a single
ip treatment of 200 mg/kg.
The determination of 1 in plasma was used to
calculate T1/2, Vd, Cltot, and AUC with a monocompartment model, and the results are listed in Table 6. The
half-life time (T1/2) varies between 10.39 and 12.21 h
as a function of the treatment schedule used. The
distribution volumes (Vd) are between 100 and 163 mL
depending on the dose. The total clearance (Cltot), that
is, the volume of blood cleared of the drug by the various
elimination processes (metabolism and excretion) per
unit time, lies between 6.5 and 9.2 mL/h. The area
under plasma concentration vs the time curve (AUC),
an estimation of bioavailability and total clearance of
the drugs, is between 615 and 891 mg‚h/L. The results
obtained with 1 seem to be influenced mostly by the last
administrated dose rather than the cumulative doses.
Effectively, if the values obtained with the single and
repeated dose of 200 mg/kg are compared, it can be seen
that the parameters are similar for these two different
schedules of treatment. RAPTA-C 1 pharmacokinetic
parameters can be also compared to the antimetastatic
agent, NAMI-A given iv at 200 mg/kg;57 these results
are similar to those obtained with 1 given at 4 × 100
mg/kg/day. If the comparison is made between 200 mg/
kg of NAMI-A and 200 mg/kg (last administration) of
1, the results indicate that the volume of distribution
and the total clearance are higher for 1. This indicates
that 1 has a better tissue penetration and a higher blood
clearing than NAMI-A.
The concentration of 1 in the different organs as a
function of the time after the last administration is
shown in Figure 7. Ruthenium retained by the body (as
the sum of the quantities found in the tested organs and
the blood) 1 h after the last injection represents about
8-14% of the administrated dose depending on the dose
of the treatment. It might be hypothesized that the
biggest part of the administrated dose was eliminated
by urine while a minor part is probably present in other
body districts that were not analyzed. These percentages
of ruthenium recovered in the body can be compared to
the retention of the antimetastatic NAMI-A compound.
When this latter compound is administrated in a single
iv treatment of 200 mg/kg, the amount of recovered
ruthenium 1 h after the last treatment is around 20%
of the administrated dose and drops to 10% after 24 h.57
The retention of NAMI-A is slightly higher than 1, but
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Scolaro et al.
To conclude, it is worth making a general comparison
of the RAPTA compounds with NAMI-A, since NAMI-A
is showing promise in clinical trials. From a structural
and chemical viewpoint the compounds are very different, albeit that they are based on ruthenium. Their
oxidation states differ as do their ligand sets, RAPTA
being organometallic and NAMI-A a traditional coordination compound. However, in vitro cell studies show
distinct similarities, and on the basis of these similarities in vivo experiments were performed. Neither types
of compounds are active against primary tumors, but
both reduce the number and weight of metastasis cells,
with NAMI-A being slightly more effective. However,
the clearance rate of 1 is slightly superior to NAMI-A
which on balance suggests that the RAPTA compounds
are viable for entry into clinical trials, and experiments
continue.
Experimental Section
Figure 7. Concentration of 1 in the organs of mice treated ip
with 4 × 100 mg/kg/day (top), 2 × 200 mg/kg/day (middle),
and 1 × 200 mg/kg/day (bottom). The mice were killed by a
sublethal dose of ethyl uretane at t ) 1, 2, 4, 8, and 24 h. Data
are expressed as the mean ( SE of individual samples obtained
from three independent mice.
Table 7. T1/2 of Elimination of 1 from Liver, Kidney, Spleen,
and Lung of Swiss CD1-mice
treatment
groups
liver
T1/2a (h)
kidneys
spleen
lung
4 × 100 mg/kg/day
2 × 200 mg/kg/day
1 × 200 mg/kg
26.09
21.38
32.41
25.10
22.00
14.19
18.28
18.48
15.09
83.29
19.37
61.19
a T
1/2 was calculated according to the formula 0.693/Ke, where
Ke is the constant of elimination and obtained by linear interpolation of the ln of RAPTA-C concentration vs time.
it is interesting to look separately at the tested organs
to compare the amount found in the different parts.
The measurement of the elimination rate of 1 by the
organs was determined also by AAS, and the values are
reported in Table 7. Complex 1 is eliminated from the
lungs at a rate faster than that from the other organs
tested (see Table 7). NAMI-A complex stays longer in
the lung, and the slower release of this antimetastatic
agent can be attributed to its binding to extracellular
matrix collagen.52 Unexpectedly, the results indicate a
slow rate of elimination of 1 from the spleen.
Synthesis and Chemical Characterization. The PTA
ligand, its methyl derivative,58 [RuCl2(arene)]2,59-61 [RuCl2(C6H5(CH2)2Im)]2,62 and [RuCl2(η6-C10H14)(PTA)]35 1 were prepared as described previously. All of the other chemicals were
obtained from Sigma unless indicated otherwise. UV-vis
spectra were obtained on a JASCO V-550 or SpectraCount
Packard spectrophotometer. 1H and 31P NMR spectra were
recorded at 400 MHz on a Bruker Avance DPX spectrometer
at room temperature in DMSO or CDCl3. Electrospray ionization mass spectra were obtained on a Thermofinigan LCQ Deca
XP Plus quadrupole ion trap instrument set in positive mode
(solvent, methanol; flow rate, 5 µL/min; spray voltage, 5 kV;
capillary temperature, 100 °C; capillary voltage, 20 V) using
a literature method.63 Chloride ion concentrations were determined with a Dionex ICS 90 ion chromatograph, using two
columns (3 mm × 30 mm) IonPac AG14A and (3 mm × 150
mm) IonPac AS14A, with a suppressor MMS III, and a
conductometric detector DS5. All of the solutions were prepared in double distilled water (R >12 MΩ). Calibration was
conducted with 1-10 mM NaCl solutions. Elution: 0.5 mL/
min, 8.0 mM Na2CO3/1.0 mM NaHCO3.
Synthesis of RuCl2(η6-arene)(PTA). Compounds 3, 5, 8,
and 9 were prepared as follows: A methanolic solution (70 mL)
of [RuCl2(η6-arene)]2 (0.64 mmol) and PTA (1.28 mmol) was
heated to reflux for 24 h under nitrogen. The solution was
allowed to cool to room temperature and filtered, and then the
solvent was removed under vacuum to obtain the red-brown
product (85-95%). Spectroscopic and analytical data are
provided in the Supporting Information.
RuCl2(η6-benzo-15-crown-5)(PTA) 6. A mixture of [RuCl2(η6-benzo-15-crown-5)]2 (140 mg, 0.16 mmol) and PTA (50 mg,
0.32 mmol) was dissolved in chloroform and stirred at room
temperature for 15 min. The solution was concentrated, and
then diethyl ether was added to afford 6 as an orange solid.
Yield: 165 mg (86%). Spectroscopic and analytical data is
provided in the Supporting Information.
[RuCl2(η6-C6H5(CH2)2Im)(PTA)][BF4] 7. A mixture of
[RuCl2(η6-C6H5(CH2)2Im)]2[BF4]2 (150 mg, 0.16 mmol) and PTA
(51 mg, 0.32 mmol) in N,N-dimethylformamide (8 mL) was
heated to 50 °C for 10 min and then allowed to cool to room
temperature while stirring. The solution was concentrated and
diethyl ether was added which resulted in the precipitation
of the product, which was washed with CH2Cl2 to afford 7 as
an orange solid. Yield: 185 mg (92%). Spectroscopic and
analytical data are provided in the Supporting Information.
Synthesis of [RuCl2(η6-arene)(Me-PTA)]Cl. Compounds
2 and 4 were prepared as follows: A mixture of [RuCl2(η6arene)]2 (0.163 mmol) and [PTA-Me]Cl (0.326 mmol) was
heated to reflux under nitrogen in MeOH (25 mL) for 4 h. After
evaporation of the solvent under vacuum, the residue was
washed with ether (2 × 5 mL) and recrystallized from hot
methanol. The product was obtained as orange crystals (80-
Ruthenium(II)-Arene PTA complexes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4169
90%). Spectroscopic and analytical data are provided in the
Supporting Information.
Thermal Denaturation. Thermal denaturation experiments were performed in quartz cuvettes. Calf thymus DNA
(purchased from Sigma, St. Louis, MO) was dissolved in 10
mM phosphate buffer containing 50 mM NaClO4. The ruthenium compound was added to DNA at a concentration which
gave a drug to DNA ratio of 1:1. The melting curves of DNA
were recorded by measuring the increase in absorbance at 260
nm from 50 to 90 °C.
Determination of pKa Values. The pH values of NMR
samples in D2O were measured at 298 K, directly in the NMR
tube, using a 713 pH meter (Metrohm) equipped with an
electrode calibrated with buffer solutions at pH values of 4, 7,
and 9. The pH values were adjusted with dilute HCl and
NaOH. The pH titration curves were fitted to the HendersonHasselbalch equation using the program Micromath Scientist
(Micromath Scientifc Software Inc.) with the assumption that
the observed chemical shifts are weighted averages according
to the populations of the protonated and deprotonated species.
The resonance frequencies change smoothly with pH between
the chemical shifts of the charged form HA+, stable in acidic
solution, and those of the neutral, deprotonated form A, which
is present at a high pH. At any pH, the observed chemical
shift is a weighted average of the two extreme values δ(HA+)
and δ(A)
using the procedure of Tamura and Arai with slight modifications.67 For each complex tested, a six-well plate was prepared
by seeding 125 000 TS/A cells in 3 mL of complete medium
with 5% FBS to each experimental and control well. The plate
was incubated for 24 h at 37 °C. Control wells were then
washed with PBS 3 times. Control wells were filled with 3 mL
of complete medium and experimental wells with 3 mL of a
100 µM solution of RAPTA compounds prepared in complete
medium. The plate was incubated for 24 h at 37 °C. The wells
were then washed with PBS 3 times, and the cells were
collected and counted with the trypan blue exclusion test, and
the intracellular concentration of ruthenium was determined.
After this treatment, the cells were dried in Nalgene cryogenic
vials (a first drying step was performed overnight at 80 °C
and a second step at 105 °C until the samples reached a
constant weight). The dried cells were decomposed by the
addition of an aliquot of tetramethylammonium hydroxide
(25% in water) (Aldrich Chimica, Gallarate, Milano, Italy) and
of milliQ water at a ratio 1:1 directly in each vial, at room
temperature, and under shaking. Final volumes were adjusted
to 1 mL with milliQ water. The concentration of ruthenium
in the TS/A tumor cell line was measured in triplicate by
flameless AAS using a Zeeman graphite tube atomizer, model
SpectrAA-300, supplied with a specific ruthenium emission
lamp (hollow cathode lamp P/N 56-101447-00, Varian, Mulgrave, Victoria, Australia). Quantification of ruthenium was
carried out in 10 µL samples at 349.9 nm with an atomizing
temperature of 2500 °C, using argon as carrier gas at a flow
rate of 3.0 L/min (for further details concerning the furnace
parameter settings, see ref 19). Before each analysis, a fivepoint calibration curve was obtained to check the range of
linearity using ruthenium custom-grade standard 998 mg/mL
(Inorganic Ventures, Lakewood, NJ).
In Vivo Tests. Animal studies were carried out according
to guidelines enforced in Italy (DDL 116 of 21/2/1992) and in
compliance with the Guide for the Care and Use of Laboratory
Animals (Department of Health and Human Services Publ.
No. 86-23, Bethesda, MD, NIH, 1985).
The in vivo experiments with the RAPTA compounds were
carried out with the MCa mammary carcinoma grown in CBA
mice obtained from a local breeding colony grown according
to the standard procedures for inbred strains. The tumor graft
was carried out by injecting 106 cells of a cell suspension
prepared from mincing with scissors the primary tumor
masses obtained from donors similarly implanted 2 weeks
before. The minced tissue was filtered through a double layer
of sterile gauze, centrifuged at 200g for 10 min, and resuspended in an equal volume of PBS; viable cells were counted
by the trypan blue exclusion test. These tumor cells were
injected im into the calf of the left hind leg of experimental
groups of mice.
Evaluation of the Primary Tumor Growth and Lung
Metastases. The primary tumor growth was determined by
calliper measurements, by determining two orthogonal axes,
and the tumor volume was calculated with the formula: (π/
6)a2b, where a is the shorter axis and b the longer, assuming
tumor density is equal to 1. The evaluation of the number and
the weight of lung metastasis are performed by examining the
surface of the lung immediately after sacrificing the animals
by cervical dislocation. Lungs were dissected into five lobes,
washed with PBS and examined under a low power microscope
equipped with a calibrated grid. The weight of each metastasis
was calculated by applying the same formula used for primary
tumors and the sum of each individual weight gave the total
weight of metastatic tumor per animal.
Pharmacokinetic Study: Determination of Ruthenium Content in the Organs, Entire Blood, and Plasma.
(A). The experiment was carried out with healthy Swiss CD-1
mice purchased from Harlan Nossan (San Pietro al Natisone,
Italy). Blood was obtained by intracardiac puncture after
induction of anaesthesia with 1.5 g/kg ethyl uretane; an aliquot
of entire blood was collected in Nalgene cryovials, and a second
aliquot was centrifuged 10 min to separate the cellular fraction
from the plasma. A piece of organ (liver, kidneys, lungs, spleen)
δav )
δ(HA+)[HA+] + δ(A)[A]
[HA+] + [A]
The midpoint of the titration occurs when the concentrations
of the acid and its conjugate base are equal: [HA+] ) [A], that
is, when the pH equals the pKa of the compound. The pH at
the midpoint of the curve is corrected by subtracting 0.44 to
the pD values since the measurements were made in D2O.43
In Vitro Tests. TS/A murine adenocarcinoma cell line,
initially obtained from Dr. G. Forni (CNR, Centro di Immunogenetica ed Oncologia Sperimentale, Torino, Italy) belong
to the tumor cell panel of the Callerio Foundation and are
stored in liquid nitrogen. Cells were cultured according to a
standard procedure64 and maintained in RPMI-1640 medium
(EuroClone, Wetherby, U.K.) supplemented with 10% fetal
bovine serum (FBS, Invitrogen, Milano, Italy), 2 mM Lglutamine, (EuroClone, Wetherby, U.K.) and 50 µg/mL gentamycin sulfate solution (EuroClone, Wetherby, U.K.). The cell
line was kept in a CO2 incubator with 5% CO2 and 100%
relative humidity at 37 °C. Cells from a confluent monolayer
were removed from flasks by a trypsin-EDTA solution (EuroClone, Wetherby, U.K.).
HBL-100, nontumorigenic human breast cells, obtained from
the American Type Culture Collection, was maintained in
McCoy’s 5A medium (SIGMA, St. Louis, MO) supplemented
with 10% FBS, 2 mM L-glutamine, 100 UI/mL penicillin, and
100 µg/mL streptomycin (EuroClone, Whetherby, U.K.) in a
humidified atmosphere with 5% CO2 at 37 °C.
Cell viability was determined by the trypan blue dye
exclusion test. For experimental purposes, the cells were sown
in multiwell cell culture plastic plates (Corning Costar Italia,
Milano, Italy). Cell growth was determined by the MTT
viability test.65 Cells were sown on 96 well plates and after 24
h were incubated with the appropriate compound at a concentration of 1-300 µM, prepared by dissolving in a medium
containing 5% of serum for 24, 48, and 72 h. Analysis was
performed at the end of the incubation time. Briefly, MTT
dissolved in phosphate buffered saline (PBS) (5 mg/mL) was
added (10 µL per 100 µL of medium) to all wells, and the plates
were then incubated at 37 °C with 5% CO2 and 100% relative
humidity for 4 h. After this time, the medium was discarded
and 100 µL of DMSO (SIGMA, St. Louis, MO) was added to
each well according to the method of Alley et al.66 Optical
density was measured at 570 nm on a SpectraCount Packard
(Meriden, CT) instrument.
Determination of Intracellular Ruthenium. Ruthenium
cell uptake was determined by AAS on samples processed
4170
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Scolaro et al.
was carefully weighed and collected in Nalgene cryovials.
Blood sample and organs for ruthenium determination were
collected at 1, 2, 4, 8, and 24 h after the last administration.
(B) Blood and Plasma Analysis. Samples were decomposed by the addition of an aliquot of [Me4N][OH] (25% in
water) (Aldrich Chimica, Gallarate, Milano, Italy) and milliQ
water at a ratio of 1:1 directly in each vial, at room temperature while shaking. The samples were left at room temperature for 72 h in closed Nalgene vials until complete digestion,
according to a procedure adapted from Tamura et al.67 Final
volumes were adjusted to 1 mL with milliQ water.
(C) Organ Analysis. A fragment of each organ specimen
was carefully weighed and dried at 105 °C until reaching a
constant dried weight in Nalgene vials. Weights were taken
continuously, considering that the fragment had completely
dried when no further change in weight occurred. The decomposition of the dried organs was obtained by adding an aliquot
of [Me4N][OH] (25% in water) and milliQ water at a ratio of
1:1 directly in each vial, at room temperature while shaking.
MilliQ water was added to give a total volume of 1 mL.
Ruthenium was then determined by AAS (see above for
details).
Statistical Analysis. Results were subjected to computerassisted statistical analysis using ANOVA, Tukey-Kramer
analysis of variance, and Dunnett’s multiple comparison test.
Differences of P < 0.05 were considered to be significantly
different from the controls.
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Acknowledgment. We thank the EPFL and COST
(Switzerland) for financial support. The present work
was carried out with contributions from the Laboratory
for the Identification of New Antimetastasis Drugs and
developed under the COST D20 action.
Supporting Information Available: Spectroscopic and
analytical data for compounds 2-9. This material is available
free of charge via Internet at http://pubs.acs.org.
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