← Back
New cytotoxic and water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes.
J. Med. Chem. 2003, 46, 1743-1750
1743
New Cytotoxic and Water-Soluble Bis(2-phenylazopyridine)ruthenium(II)
Complexes
Anna C. G. Hotze,† Marina Bacac,† Aldrik H. Velders,†,‡ Bart A. J. Jansen,† Huub Kooijman,§ Anthony L. Spek,§
Jaap G. Haasnoot,† and Jan Reedijk*,†
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands, and Bijvoet Center for Biomolecular Research,
Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
Received December 10, 2002
New water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes, all derivatives of the
highly cytotoxic R-[Ru(azpy)2Cl2] (R denoting the coordinating pairs Cl, N(py), and N(azo) as
cis, trans, cis, respectively) have been developed. The compounds 1,1-cyclobutanedicarboxylatobis(2-phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(cbdca-O,O′)] (1), oxalatobis(2-phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(ox)] (2), and malonatobis(2-phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(mal)] (3), have been synthesized and fully characterized. X-ray analyses
of 1 and 2 are reported, and compound 1 is the first example in which the cbdca ligand is
coordinated to a ruthenium center. The cytotoxicity of this series of water-soluble bis(2phenylazopyridine) 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 R-[Ru(azpy)2Cl2], R-[Ru(azpy)2(NO3)2], β-[Ru(azpy)2Cl2] (β indicating
the coordinating pairs Cl, N(py), and N(azo) as cis, cis, cis, respectively), and β-[Ru(azpy)2(NO3)2] have been determined in this cell line. All the bis(2-phenylazopyridine)ruthenium(II)
compounds display a promising cytotoxicity in the A2780 cell line (IC50 ) 0.9-10 µM), 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 β isomeric
compounds) are similar in the cisplatin-resistant A2780cisR cell line compared to the normal
cell line A2780, suggesting that the activity of these compounds might not be influenced by
the multifactorial resistance mechanism that affect platinum anticancer agents.
Introduction
Cisplatin is one of the most widely used antitumor
drugs, especially for the treatment of testicular and
ovarian cancer.1,2 However, cisplatin has some major
drawbacks, like severe toxic side effects, a limited
applicability to a relatively small range of tumors, and
often occurring resistance. This resistance is either
developed or intrinsic.2 The second-generation platinum
drug diammine(1,1-cyclobutanedicarboxylato)platinum(II), carboplatin, is also in wide clinical use. It has less
toxic side effects and can be administered in a significant higher dose than cisplatin.2 In the search for other
antitumor-active metal complexes, several ruthenium
complexes have been reported to be promising as
anticancer drugs.3 For example, complexes such as
trans-Hind[RuIIICl4(ind)2] (Hind ) indazole), mer-[Ru(terpy)Cl3] (terpy ) 2,2′:6′,6′′-terpyridine), and [RuIV(chd-H2)Cl2] (chd ) 1,2-cyclohexanediamine tetraacetate) have been reported to be highly antitumoractive.4-6 Recently, some Ru(II) arene complexes have
also been reported, which show inhibition of cancer cell
growth.7,8 Currently, the most promising antimetastatic
agent appears to be trans-(H2im)[RuCl4(dmso)(Him)]
* To whom correspondence should be addressed. Fax: +31715274671. E-mail: reedijk@chem.leidenuniv.nl. Phone: 31-71 5274459.
† Leiden University.
‡ Current address: Molteni Farmaceutici, S.S. 67 Loc. Granatieri,
I-50018, Scandicci, Florence, Italy.
§ Utrecht University.
(nicknamed NAMI-A) (Him ) imidazole), which has
successfully undergone the preclinical stage of testing9,10
and has recently finished phase I clinical trials as an
antimetastatic drug.11 So at present, a number of
antitumor-active ruthenium complexes are known, but
no structure-activity relationships (SARs) have been
established as yet.
The isomeric dichlorobis(2-phenylazopyridine)ruthenium(II) complexes exhibit quite different cytotoxicities
against a series of tumor cell lines.12 The R-isomer
R-[Ru(azpy)2Cl2] (R-Cl), in which R indicates the coordinating pyridines (trans), azo nitrogens (cis), and
chlorides (cis) (Figure 1), has been reported to show a
remarkably high cytotoxicity, even more pronounced
than cisplatin in most of the tested cell lines (i.e., MCF7, EVSA-T, WIDR, IGROV, M19, A498, and H266).12
Noteworthy is the difference in cytotoxicity between the
two cis isomers (Figure 1) R and β (β indicating the
coordinating pairs Cl, N(py), and N(azo) as cis, cis, cis,
respectively) of which the latter shows a cytotoxicity of
a factor 10 lower than the R isomer in the panel of used
cell lines.12 Because the dichlorobis(2-phenylazopyridine)ruthenium(II) complexes are not water-soluble,
the water-soluble compounds R-[Ru(azpy)2(NO3)2] (RNO3) and β-[Ru(azpy)2(NO3)2] (β-NO3) (Figure 1) have
been developed in our group.13,14
The present paper reports the structural characterization of a new series of water-soluble compounds: 1,1cyclobutanedicarboxylatobis(2-phenylazopyridine)ru-
10.1021/jm021110e CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/25/2003
1744
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hotze et al.
Figure 1. Schematic structures of R-[Ru(azpy)2X2] (left) and
β-[Ru(azpy)2X2] (right) (X ) Cl or NO3).
Figure 3. Atomic displacement ellipsoid plot35 of 1 drawn at
the 50% probability level. Hydrogen atoms are drawn as
spheres of arbitrary radius.
Figure 2. Schematic structures of R-[Ru(azpy)2(cbdca-O,O′)]
(1), R-[Ru(azpy)2(ox)] (2), and R-[Ru(azpy)2(mal)] (3).
thenium(II), R-[Ru(azpy)2(cbdca-O,O′) (1), oxalatobis(2phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(ox)] (2),
and malonatobis(2-phenylazopyridine)ruthenium(II),
R-[Ru(azpy)2(mal)] (3) (Figure 2). The single-crystal
X-ray diffraction studies of both 1 and 2 are reported.
In fact, the X-ray structure of 1 is the first example of
this carboxylato ligand coordinated to ruthenium. The
choice of the carboxylato ligands was inspired by the
compound carboplatin, which is known to be less toxic
than cisplatin while being equally active as cisplatin.15
In the present paper, the cytotoxic activity of these new
water-soluble bis(2-phenylazopyridine)ruthenium(II) carboxylato complexes and of the water-soluble R-NO3 and
β-NO3 is described and compared to the cytotoxicity of
the parent compounds R-Cl and β-Cl. The cytotoxicity
of the compounds has been tested in A2780 (human
ovarian carcinoma) and A2780cisR, the corresponding
cisplatin-resistant cell line. A2780cisR is endowed with
multifactorial resistance mechanisms such as decreased
uptake, increased glutathion levels, and increased DNA
repair15 and thus represents a good model for the
screening of new anticancer metal-based agents.
Results and Discussion
X-ray Analyses. The bis(2-phenylazopyridine)ruthenium(II) complexes with carboxylato ligands have been
synthesized using the water-soluble starting compound
R-[Ru(azpy)2(NO3)2] and the particular carboxylato acid
based on the synthesis of carboplatin.16
A projection of the X-ray structures of R-[Ru(azpy)2(cbdca-O,O′)], 1, and R-[Ru(azpy)2(ox)], 2, is shown in
Figures 3 and 4, respectively. If the coordination pairs
O, N(py), and N(azo) are considered in that order, the
configuration of 1 and 2 is cis, trans, cis (ctc), so the
Figure 4. Atomic displacement ellipsoid plot35 of 2 drawn at
the 50% probability level. Hydrogen atoms are drawn as
spheres of arbitrary radius.
original R configuration is retained during the reaction.
The molecular structures clearly show the didentate
O,O′-coordinated dicarboxylato ligands. The Ru-N(py)
and Ru-N(azo) distances are comparable to those in
similar structures.17 The Ru-O distances of 2.044(2)
and 2.051(2) Å in 1 and 2.056(4) and 2.054(5) Å in 2
are consistent with common single bonding, although
slightly shorter than, for example, the average Ru-Oox
distance18 in [(tpy)(C2O4)RuIIIORuIII(tpy)(C2O4)]‚8H2O.
The distance C46-O4 of 1.223(3) Å (and C45-O3 ) 1.224(4) Å) in 1 indicates the double-bonded keto functionality, whereas the distances C46-O1 and C45-O2 of
1.296(3) and 1.303(3) Å, respectively, indicate single
bonding. These distances are similar to those found in
compound 2. The O-Ru-O angle in 1 is 88.43(8)° and
80.47(19)° in 2 and are comparable to the Cl-Ru-Cl
angle17 in R-[Ru(azpy)2Cl2] (89.4°) and the O-Ru-O
Ruthenium(II) Complexes
Figure 5. 1H NMR spectrum of 1 in CDCl3 showing the
aromatic region (top) and high-field region (bottom). For the
proton assignment, the numbering scheme of the X-ray
structure is used.
angle14 in R-[Ru(azpy)2(NO3)2] (77.9°). The X-ray structure of 1 shows the C42 and C44 atoms to be inequivalent
with Ru-C distances of 4.946(3) and 3.990(3) Å. This
inequivalence of the cyclobutane ring carbon atoms in
the solid-state structure was also observed in the case
of [Pt(en)(cbdca-O,O′)].19 In solution (vide infra) this
inequivalence is apparently removed because the two
azpy ligands are identical in NMR. Also, carboplatin
shows inequivalence of the analogous CR and Cβ atoms
in the solid state.20 The malonate-like ring from the
cbdca ligand coordinated to a metal ion may adopt three
conformations: boat, chair, and planar.21 In 1, the
conformation was found to be close to half-boat which
is different from the boat conformation found for carboplatin.20,22 In the complex [Pt(en)(cbdca-O,O′)] the Ptcbdca chelate ring adapts a flattened boat conformation,19 which seems comparable to the half-boat conformation found in 1. In the X-ray structure of 1, as well
as in solution (vide infra), it appears that the H42A and
H42B protons (and the H44A/H44B and H43A/H43B protons)
are not equivalent. This is in contrast to the (in solid
state and in solution) observed equivalence of these
protons in carboplatin.20,23
Characterization and Stability Properties Studied by NMR Spectroscopy. The 1H NMR spectra of
the complexes 1, 2, and 3 in CDCl3 (for 1H NMR of 1,
see Figure 5) show one set of azpy signals, indicating
two equivalent azpy ligands, so the complexes still
possess a C2 symmetry. The assignment of the aromatic
signals has been made by the use of 2D correlation NMR
spectroscopy, and H6 and H3 have been assigned on the
basis of the 3J coupling constants, which are known to
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1745
be larger for H3. The same order of signals accounts
for 1, 2, and 3, which proves that the carboxylato ligands
are chelating and that the R-configuration for the azpy
ligands is retained, in agreement with X-ray data (vide
supra). In the 600 MHz 1H NMR spectrum of 1 (Figure
5) in CDCl3, the cyclobutane signals are observed at
high field. Three multiplet signals are observed at 2.22,
1.93, and 1.70 ppm all having an intensity of 2 and
assigned to the cyclobutane ring. Because three signals
appear, one would expect that C42 and C44 are inequivalent. However, on the other hand, the two azpy ligands
are identical from NMR spectra. This might suggest
some dynamic motion of the six-membered ring part of
the cbdca ligand, which gives the C2 axis of the
compound. To solve this contradiction, a comparison
with the NMR data of carboplatin is drawn. The NMR
spectrum of carboplatin shows only two signals for the
cyclobutane ring (a triplet and quartet), and it has been
stated that the simplicity of the spectrum of carboplatin
may arise from a rapid inversion at the oxygen atoms.20,24 The 13C{1H} NMR spectrum of an analogue of
carboplatin, i.e., [Pt(en)(cbdca-O,O′)], shows only three
resonances for the four-membered ring of cbdca, so there
is also rapid ring inversion in solution.19 Ring flexibility
seems to be confirmed by crystal structure studies of
other malonato metal complexes. Both boat and chair
conformations have been observed.21 Moreover, because
the cyclobutane ring is planar in carboplatin, protons
are similar on either side of the cyclobutane ring.20,24
Combined with the X-ray data of 1 (vide supra), it seems
likely that rapid (on the NMR time scale) chelate ring
flipping of the six-membered ring occurs in solution to
account for the C2 symmetry, which makes the azpy
ligands equivalent. This flipping in a sense would also
make the carbon atoms C42 and C44 equivalent. However, as mentioned before, three signals appear for the
cbdca part of the compound. Combined with the complicated multiplet pattern, this may indicate the inequivalence of H42A and H42B (and also H44A/H44B and
H43A/H43B). The three facts, i.e., the azpy ligands being
equivalent, the assumed rapid ring motion, and the
inequivalence of the hydrogens on either side of the
cyclobutane ring, would result in one multiplet signal
assignable as H44B/H42B, the other as H44A/H42A, and the
final mulitplet at 1.70 ppm as H43A/H43B (which appears
most upfield corresponding to hydrogens that are the
most far away from the ruthenium metal ion). The exact
assignment of the cyclobutane signals has not been
attempted.
Stability of Complexes 1, 2, and 3 in Aqueous
Solution. The stability of complexes 1, 2, and 3 in
solution has been followed in time in D2O by 1H NMR
spectroscopy. The aromatic resonances of the compounds 2 and 3 were not found to change over 4 weeks
at room temperature, and no new signals appeared,
indicating that these compounds are inert to hydrolysis
in D2O under these conditions. Compound 1 is less inert
to hydrolysis in comparison to 2 and 3. Following the
resonances of 1 in D2O in time shows the appearance
of one new set of aromatic signals, probably from R-[Ru(azpy)2(D2O)2]2+ and free cbdca2-, and after approximately 8 days the ratio is 1:1 between intact 1 and the
new signals in the aromatic region. The ratio of 1
relative to the new aromatic signals and the occurring
1746
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hotze et al.
free cbdca remained unchanged for the rest of the
observation period. This reactivity deviates from carboplatin in water, where no hydrolysis has been observed (k < 5 × 10-9 s-1).23-25
To investigate whether the complexes are stable
under physiological conditions (pH 7.4 and 0.05 M
phosphate, 0.1 M NaCl), samples of 1, 2, and 3 (1 mg/
mL) have been followed by 1H NMR spectroscopy for 1
month at room temperature. The spectra of 2 and 3 did
not show any changes, proving that the compounds also
remain intact for 1 month under physiological conditions. Compound 1, however, appeared to be less stable
under physiological conditions, and hydrolysis of the
cbdca ligand does occur. After 5 days, two hydrolysis
species are visible, but unfortunately, it was not possible
to assign unambiguously the appearing resonances
because of overlapping signals. Likely candidates are
R-[Ru(azpy)2(cbdca-O)(X)] (with X ) D2O, Cl, or HPO4)
and R-[Ru(azpy)2(X)2]2+ (with X ) D2O or HPO4). The
approximate rate constant for the decrease in the
concentration of 1 was determined to be k ) 1.3 × 10-6
s-1 (assuming first-order decay). The rate of hydrolysis
of 1 under physiological conditions is comparable to the
observed hydrolysis rates of carboplatin. Different hydrolysis studies of carboplatin have been performed, and
apparently, added nucleophiles or acids offer some
assistance to the lability of the coordinated cbdca. For
example, in phosphate buffer (310 K, 1 mM carboplatin,
0.1 M Na2HPO4), a rate constant of 7.2 × 10-7 s-1 has
been reported.25 A rate constant of 1.2 × 10-6 s-1 has
been observed for an aqueous solution containing phosphate and chloride (20 mM carboplatin, 140 mM Cl, 0.1
M Na2HPO4).23 In correlation to the cytotoxicity tests,
the stability of the carboxylato complexes was also
tested by dissolving compounds 1, 2, and 3 in DMSOd6 to which D2O was added in the same ratio as that
used in the cytotoxity tests. The NMR data show that
DMSO does not coordinate to the ruthenium azpy
moiety.
Cytotoxicity Tests. The IC50 values represent the
concentration of a drug that is required for 50% reduction of cellular growth. Because R-Cl and β-Cl are poorly
water-soluble, a DMSO stock solution was used for all
compounds to perform a proper comparison between the
compounds. NMR data, mass spectrometry, and conductivity measurements of R-Cl and R-NO3 in aqueous
solutions (0.1 mg/mL and 1% DMSO) show that DMSO
does not coordinate, just like the carboxylate complexes
(vide supra).26 In the case of R-Cl, the chloride ligands
remain coordinated, whereas upon dissolution of R-NO3,
the nitrate ligands are replaced by water ligands. The
IC50 values of R-Cl and β-Cl, R-NO3 and β-NO3, 1-3,
carboplatin, and cisplatin are listed in Table 1. The
order of cytotoxicities in A2780 is R-Cl > β-Cl > 2 ∼ 1
∼ 3 ∼ R-NO3 > β-NO3. The cytotoxic activity of the
complexes R-NO3 (and to a lesser extent β-NO3) and the
complexes 1, 2, and 3, which all show the improved
water solubility, is very interesting because they show
an appreciable cytotoxicity of only a factor 10 less than
the extremely cytotoxic but water-insoluble parent
complex R-Cl. Remarkable is the influence of the ciscoordinated ligands (i.e., Cl, NO3, ox, mal, and cbdca)
on the cytotoxicity and solubility. The cytotoxicity data
of R-NO3 , 1, 2, and 3 compared to the parent compound
Table 1. IC50 Values in A2780 and A2780cisR Cell Lines of Rand β-[Ru(azpy)2Cl2], R- and β-[Ru(azpy)2(NO3)2],
R-[Ru(azpy)2(cbdca-O,O′)], 1, R-[Ru(azpy)2(ox)], 2, and
R-[Ru(azpy)2(mal)], 3, Cisplatin, and Carboplatin
R-[Ru(azpy)2Cl2]
R-[Ru(azpy)2(NO3)2]
β-[Ru(azpy)2Cl2]‚CHCl3
β-[Ru(azpy)2(NO3)2]‚CHCl3
R-[Ru(azpy)2(ox)], 2
R-[Ru(azpy)2(mal)], 3
R-[Ru(azpy)2(cbdca-O,O′)], 1
cisplatin
carboplatin
IC50 in
A2780 (µM)
IC50 in
A2780cisR (µM)
0.85
8.5
1.6
9.7
6.3
7.9
7.2
2.3
8.2
0.98
6.3
7.3
12.1
6.3
6.2
4.9
7.8
41.6
R-Cl show that changing the Cl ligands with the other
ligands results in a decrease of activity but a parallel
increase in water solubility.
For more insight into the structure-activity relationships for the azpy compounds in this study, the other
cis isomer, i.e., the β isomer, has been taken into account
also. Regarding the cytotoxicity data of the β-Cl and
β-NO3 complexes, it is concluded that the β isomer
shows lower activity in A2780 and A2780cisR than the
analogous R isomeric complexes but still shows considerable activity. Also, of these β isomeric compounds, the
β-NO3 shows less activity than the β-Cl complex but
increased water solubility.
Most interestingly, for compounds 1-3 and R-NO3,
the IC50 values for the resistant cell line are slightly
lower than or the same as for the normal cell line, in
contrast to the platinum compounds. In A2780cisR, the
multifactorial resistance mechanisms such as decreased
uptake, increased levels of GSH (γ-glutamate-cysteineglycine), and increased DNA repair cause a smaller
activity of the platinum compounds compared to that
for the normal cell line.
It is known that ruthenium complexes can bind to
DNA,3 and in particular, the bis(2-phenylazopyridine)ruthenium(II) complexes have also been studied for
their binding to DNA model bases.13,14,27,28 In general,
the binding mode of ruthenium complexes is thought
to be different from that of cisplatin.3 Therefore, it is
proposed that the here-reported series of ruthenium
complexes might be able to avoid DNA repair mechanisms that are present in cisplatin-resistant cells or be
unaffected by the higher levels of GSH.
Comparison of the cytotoxicity of the whole series of
azpy compounds with cisplatin and carboplatin shows
that R-Cl is the only compound far more active than
cisplatin. The other compounds show an activity slightly
below that of cisplatin, except for β-Cl, which is as active
as cisplatin. All compounds show a higher activity than
carboplatin and even β-NO3 shows comparable activity,
confirming the large potential of this series of compounds. Compound 1 shows a lower activity than the
parent compound R-Cl in both cell lines. Nevertheless,
compound 1 shows an activity in the A2780 cell line
similar to that of carboplatin, and 1 is even far more
active in the resistant cell line than carboplatin, showing an IC50 value of 4.9 µM compared to the value of
41.6 µM for carboplatin. This confirms once more that
the activity of 1 is not influenced by the resistance
mechanisms in the A2780CisR cell line (vide supra).
Ruthenium(II) Complexes
Together with the water solubity and stability, 1 appears to be a compound as interesting as carboplatin
itself.
The cytotoxicity data of the here-tested complexes are
better than other ruthenium polypyridyl complexes such
as the antitumor-active [RuIII(terpy)Cl3] with an IC50
value of 11.0 and 32.5 µM in A2780 and A2780cisR (data
not shown), respectively. The IC50 values of the azpy
compounds are in the same range as the reported8
cytotoxicity in the A2780 cell line of the ruthenium(II)
arene complexes. It is also interesting to see the huge
difference in activity between the bis(2-phenylazopyridine)ruthenium complexes and a related noncytotoxic5
complex, cis-[Ru(bpy)2Cl2], which exhibits IC50 values
exceeding the value of 130 µM in a series of human
tumor cell lines.29 In conclusion, the cytotoxicity data
of the water-soluble ruthenium azpy complexes are
promising in comparison to several other antitumoractive ruthenium and platinum compounds.
Activity after a Short Exposure Time To Investigate Influence of Uptake. In the cytotoxicity tests
described above, the cells are exposed to the compounds
for a relatively long time (3 days). This implies that all
compounds are eventually able to reach DNA, or other
biological targets, and act on this level. To investigate
whether the activity of the compounds can be correlated
to their ability to enter the cells, a short-exposure
experiment has been performed in which the cells have
been exposed to the drug for only 1 h in PBS. In this
way, it may be possible to determine if the different
ligands X in the R-[Ru(azpy)2X] complexes influence the
ability of the compounds to enter the cells. In this
experiment, only the R isomeric complexes have been
investigated.
The concentrations used, i.e., 500, 50, and 5 µM, are
higher than their IC50 values but are justified in this
experiment by the short exposure time of only 1 h.
Figure 6 shows the reduction of cell proliferation evaluated by the MTT test at 24 h after treatment at 50 and
5 µM. At the highest concentration, i.e., 500 µM (data
not shown), all compounds show a statistically significant reduction of cell proliferation in normal and
resistant cell lines when compared to the control group
((///) p < 0.001, Student-Newman-Keuls test). The
compounds R-Cl and R-NO3 maintain their significant
activity even at 50 and 5 µM in the resistant (data not
shown) and normal cell lines. At 50 µM, it is interesting
to see that cisplatin and compound 3 are not active
while 1, 2, R-Cl, and R-NO3 still show a considerable
reduction of cell proliferation in the A2780 cell line
(Figure 6). With a decrease in the concentration to as
low as 5 µM, the ruthenium carboxylato compounds and
cisplatin are inactive while R-Cl and R-NO3 still remain
very active in both A2780 and A2780cisR (Figure 6). The
activity shown in these graphs might thus be a combination of fast cell entering and rapid interaction with
biological targets, probably DNA, leading to cell death.
Preliminary atomic absorption data showing the amount
of ruthenium inside the cells after 1 h of drug challenge
show indeed that the activity in these kinds of short
exposure experiments is correlated to the amount of
ruthenium inside the cell.30 The further mechanisms
underlying the different activity of the R-Cl and R-NO3
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1747
Figure 6. Reduction of proliferation in the A2780 cell line
after the 1 h of PBS treatment at 50 µM (top) and 5 µM
(bottom).
compounds compared to 1, 2, and 3 under these conditions are thus a subject of further research.31
Concluding Remarks
The synthesis of the carboxylatobis(2-phenylazopyridine)ruthenium(II) complexes has resulted in 1,1cyclobutanedicarboxylatobis(2-phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(cbdca-O,O′)] (1), oxalatobis(2phenylazopyridine)ruthenium(II), R-[Ru(azpy)2(ox)] (2),
and malonatobis(2-phenylazopyridine)ruthenium(II),
R-[Ru(azpy)2(mal)] (3). The X-ray structures of 1 and 2
have unambiguously proven the chelating coordination
of the carboxylato ligands. Its molecular structure shows
1 to be the first ruthenium compound in which the 1,1cyclobutanedicarboxylato ligand is coordinated to ruthenium. Just like carboplatin versus cisplatin in 1, the
cis chloride ligands in the parent compound R-[Ru(azpy)2Cl2] have been replaced by the carboxylato group
to generate a water-soluble compound, which might be
equally biologically interesting as carboplatin.
The here-reported complexes are water-soluble, but
2 and 3 are also inert to hydrolysis under physiological
conditions. Compound 1 shows slow hydrolysis in both
D2O and phosphate buffer. The water-soluble R-NO3 and
carboxylato complexes 1, 2, and 3 display a cytotoxicity
of a factor 10 less than the insoluble parent complex
R-Cl in the A2780 and A2780cisR cell lines. Regarding
the β isomeric complexes, β-Cl and β-NO3 also show
considerable activity in the same order as the other azpy
complexes in this series, although β-Cl and β-NO3 are
less active than their R counterparts (i.e., R-Cl and
1748
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hotze et al.
R-NO3). In comparison to cisplatin and carboplatin, R-Cl
is far more active than cisplatin. The water-soluble
compounds display a slightly lower activity than cisplatin but a higher or comparable activity relative to
carboplatin. In particular, it is interesting to focus on
compound 1, which is less active than the parent
compound R-Cl (note also that carboplatin is less active
than cisplatin). But interestingly, 1 displays in the
A2780 cell line an activity comparable to that of carboplatin, and in the resistant cell line, 1 is even 10-fold
more active than carboplatin. The cytotoxicity data of
1, together with the water solubility and stability (only
showing relatively slow hydrolysis), show that 1 might
be interesting for further in vitro and in vivo studies.
The cytotoxicity data of this series of compounds
indicate that structural differences such as isomeric
structures (R and β backbones) and variation of the
counterions influence the activity. However, the reason
behind all factors influencing the activity of these
compounds is yet far from understood, and more in vitro
and in vivo studies are needed in the search for
stucture-activity relationships for these kind of complexes.
values were obtained by GraphPad Prism software, version
3.05, 2000. Statistical analysis was done with the StudentNewman-Keuls test, Instat2.
Short Exposure Time Experiment by 1 h of PBS
Treatment. For the 1 h of incubation in PBS, 10 000 cells/
well were seeded in 100 µL of complete medium in 96-multiwell
flat-bottom microtiter plates (Corning Costar). The plates were
preincubated for 48 h to allow cell adhesion. The tested
concentrations were 500, 50, and 5 µM.
The stock solution of all compounds (50 mM in DMSO) was
freshly prepared. The first concentration (i.e., 500 µM) was
obtained by a 1:100 dilution in PBS from the stock solution.
Other concentrations were obtained by a sequence of two 1:10
dilutions in PBS of the 500 µM solution.
Before treatment, the complete medium was removed and
the cells were washed with 100 µL of PBS. For treatment, 100
µL of the desired concentration was added to the wells, each
concentration in eight wells. The control group consisted of
PBS added with 1% of DMSO (as for the 500 µM solution).
The plates were incubated for 1 h at 37 °C under 5% CO2. At
the end of the incubation time, the cells were washed twice
with PBS and o grew for an additional 24 h in 100 µL of
complete medium. The MTT test was performed at 24 h after
treatment (see above). Statistical analysis was done with the
Student-Newman-Keuls test, Instat2.
Crystal Structure Determination and Refinement of
1 and 2. Diffraction data were collected with a Nonius Kappa
CCD diffractometer on a rotating anode (Mo KR radiation, λ
) 0.710 73 Å). Structures were solved with direct methods33
and refined34 on F2. Non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were
included in calculated positions in riding mode. Final data for
1: C28H24N6O4Ru, Mr ) 609.60, black, needle-shaped crystal
(0.02 mm × 0.03 mm × 0.20 mm), monoclinic, space group
P21/c (No. 14) with a ) 9.6537(10) Å, b ) 18.397(2) Å, c )
16.492(2) Å, β ) 119.875(9)°, V ) 2539.7(5) Å3, Z ) 4, Dc )
1.594 g cm-3, 49 729 reflections measured, 5830 independent,
1.6° < θ < 27.5°, T ) 150 K, 352 parameters, wR2 ) 0.0725,
R1 ) 0.0365 (for 5830 reflections with I > 2σ(I)), S ) 1.003,
-0.48 < ∆F < 0.79. Final data for 2: C24H18N6O4Ru‚solvent,
Mr ) 555.51, purple, needle-shaped crystal (0.05 mm × 0.05
mm × 0.35 mm), monoclinic, space group C2/c (No. 15) with a
) 24.073(3) Å, b ) 17.195(2) Å, c ) 16.479(3) Å, β ) 129.536(12)°, V ) 5260.7(12) Å3, Z ) 8, Dc ) 1.403 g cm-3, 43 692
reflections measured, 4770 independent, 1.6° < θ < 25.3°, T
) 250 K, 317 parameters, wR2 ) 0.1396, R1 ) 0.0675 (for 2904
reflections with I > 2σ(I)), S ) 1.041, -0.68 < ∆F < 0.56. The
unit cells contains four symmetry-related voids of 240 Å3 each,
filled with disordered solvent (probably diethyl ether, 45.5e
per void) and incorporated in the final model via the squeeze
procedure.35
A CIF file for compounds 1 and 2 is available from the
authors.
Syntheses. Oxalic acid dihydrate (Merck) (H2ox(H2O)2),
malonic acid (Merck-Schuchardt) (H2mal), and 1,1-cyclobutanedicarboxylic acid (Janssen Chimica) (H2cbdca) were obtained commercially and used without purification. 2-(Phenylazo)pyridine36 and the complexes37,38 R-[Ru(azpy)2Cl2] and
β-[Ru(azpy)2Cl2]‚CHCl3 were synthesized according to published methods. The compounds R-[Ru(azpy)2(NO3)2] and
β-[Ru(azpy)2(NO3)2]‚CHCl3 were prepared as described before.13,14
R-[Ru(azpy)2(cbdca-O,O′)] (1). 1,1-Cyclobutanedicarboxylic acid (0.027 g, 0.19 mmol) was added to a solution of R-[Ru(azpy)2(NO3)2] (0.100 g, 0.17 mmol) in 30 mL of acetone. A KOH
solution (3.4 mL, 0.1 M) was added, and the mixture was
stirred at 40 °C for 4 days. The blue-purple solution was
filtered and evaporated to dryness by rotary evaporation. The
solid was dissolved in 30 mL of absolute ethanol, and the
solution was then filtered. The solution was evaporated and
dissolved in 30 mL of acetone. Dropwise addition of ca. 40 mL
of diethyl ether resulted, after 24 h, in crystals suitable for
X-ray analysis. Yield: 0.040 g (39%). Anal. Calcd for
RuC28H24N6O4: C, 55.2; H, 3.97; N, 13.8. Found: C, 54.8; H,
Experimental Section
Instrumental Methods. 1H NMR spectra were obtained
at 300.13 MHz on a Bruker 300 DPX spectrometer and at
600.13 MHz on a Bruker 600 DMX where indicated. Spectra
were recorded in CDCl3 and D2O and calibrated on the residual
solvent peaks. All spectra were obtained at 25 °C. Elemental
analyses were performed with the use of a Perkin-Elmer
analyzer.
Cell Lines. A2780 (human ovarian carcinoma) and A2780
cisplatin-resistant cell lines were maintained in continuous
logarithmic culture in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco BRL, Invitrogen Corporation, The Netherlands) supplemented with 10% fetal calf serum (Perbio Science,
Belgium), penicillin G sodium (100 units/mL, Duchefa Biochemie BV, The Netherlands), streptomycin (100 µg/mL,
Duchefa Biochemie BV, The Netherlands), and Glutammax
100x (Gibco BRL, The Netherlands). The cells were harvested
from confluent monolayers.
Cytotoxicity Assay. For the cytotoxicity evaluation, 2000
cells/well were seeded in 100 µL of complete medium in 96multiwell flat-bottom microtiter plates (Corning Costar). The
plates were incubated at 37 °C in 5% CO2 for 48 h prior to
drug testing to allow cell adhesion.
The stock solutions (1 mg/mL DMSO) of all tested compounds were freshly prepared and directly used for the
dillutions. Because R-Cl and β-Cl are poorly water-soluble but
are needed in the comparison with the water-soluble compounds, a DMSO solution was chosen. The dilutions (six-step
dilutions except for the R-Cl for which two more dilutions were
used) were prepared in complete medium, and the final tested
concentrations were 0.01, 0.005, 0.001, 0.0005, and 0.0001 mg/
mL (and in the case of R-Cl, the concentrations were also
0.00005 and 0.00001). Each concentration was tested in
quadruplicate using 100 µL/well added to the 100 µL of
complete medium. In the control group, only 100 µL of
complete medium containing 1% of DMSO was added. The
range of concentrations used ended up with a concentration
from 1% to 0.01% DMSO (R-Cl up to 0.001%). Parallel
experiments showed that no difference in cell proliferation was
observed in control groups with or without 1% DMSO. The
plates were incubated for 72 h, and the evaluation of cell
proliferation was performed by the MTT colorimetric assay.32
A 50 µL MTT solution (5 mg/mL in PBS, Sigma Chemical Co.)
was added to each well and incubated for 1 h. Formazan
crystals were solubilized with 100 µL of DMSO. Optical density
was measured by microplate reader (Bio Rad) at 590 nm. IC50
Ruthenium(II) Complexes
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1749
4.49; N, 13.8. ESI-MS m/z: 611 [M + H]. 1H NMR (300 MHz,
chloroform-d): δ 8.64 (d, 2H), 8.45 (d, 2H), 8.05 (t, 2H), 7.41
(t, 2H), 7.31 (t, 2H), 7.11 (t, 4H), 6.72 (d, 4H), 2,22 (m, 2H),
1.93 (m, 2H), 1.70 (m, 2H).
R-[Ru(azpy)2(ox)] (2). To a solution of R-[Ru(azpy)2(NO3)2]
(0.100 g, 0.17 mmol) in 30 mL of acetone was added oxalic
acid dihydrate (0.017 g, 0.013 mmol). The procedure used
was the same as that for 1, and recrystallization from absolute ethanol and ether resulted in crystals suitable for
X-ray analysis. Yield: 0.080 g (85%). Anal. Calcd for
RuC24H18N6O4: C, 51.9; H, 3.27; N, 15.1. Found: C, 49.1; H,
2.99; N, 14.3. ESI-MS m/z: 556 [M + H]. 1H NMR (300 MHz,
chloroform-d): δ 8.68 (d, 2H), 8.33 (d, 2H), 8.10 (t, 2H), 7.48
(t, 2H), 7.34 (t, 2H), 7.10 (t, 4H), 6.75 (d, 4H).
R-[Ru(azpy)2(mal)] (3). The synthesis was analogous to the
synthesis of 1 and 2. Yield: 0.026 g (54%). Anal. Calcd for
RuC25H20N6O4: C, 52.7; H, 3.45; N, 14.8. Found: C, 51.1; H,
3.38; N, 14.4. ESI-MS m/z: 570 [M + H]. 1H NMR (300 MHz,
chloroform-d): δ 8.68 (d, 2H), 8.47 (d, 2H), 8.10 (t, 2H), δ 7.46
(t, 2H), 7.30 (t, 2H), 7.09 (t, 4H), 6.72 (d, 4H), 3.01 (s, 3H).
(13) Velders, A. H. Ph.D. Thesis, Leiden University, Leiden, The
Netherlands, 2000.
(14) Hotze, A. C. G.; Velders, A. H.; Ugozzoli, F.; Biagini-Cingi, M.;
Manotti-Lanfredi, A. M.; Haasnoot, J. G.; Reedijk, J. Synthesis,
characterization, and crystal structure of R-[Ru(azpy)2(NO3)2]
(azpy ) 2-(phenylazo)pyridine) and the products of its reactions
with guanine derivatives. Inorg. Chem. 2000, 39, 3838-3844.
(15) Kelland, L. R.; Sharp, S. Y.; O’Neill, C. F.; Raynaud, F. I.; Beale,
P. J.; Judson, I. R. Mini-review: discovery and development of
platinum complexes designed to circumvent cisplatin resistance.
J. Inorg. Biochem. 1999, 77, 111-115.
(16) Pasini, A.; Caldirola, C. A New Synthetic Method for Diaminomalonatoplatinum Type Complexes and the Unexpected Behaviour of [PtCl2(trans-dach)]. Inorg. Chim. Acta 1988, 151, 1920.
(17) Seal, A.; Ray, S. Structures of two isomers of dichlorobis(2phenylazopyridine)ruthenium(II), [RuCl2(C11H9N3)]. Acta Crystallogr. 1984, C40, 932.
(18) Lebeau, E. L.; Adeyemi, A.; Meyer, T. J. Water Oxidation by
[(tpy)(H2O)2RuIIIORu(III)(H2O)2(tpy)]4+. Inorg. Chem. 1998, 37,
6476-6484.
(19) Guo, Z.; Habtemariam, A.; Sadler, P. J.; Palmer, R.; Potter, B.
S. Conformational flexibility within the chelate rings of [Pt(en)(CBDCA-O,O′)], an analogue of the antitumour drug carboplatin: X-ray crystallographic and solid-state NMR studies. New
J. Chem. 1998, 11-14.
(20) Neidle, S.; Ismail, I. M.; Sadler, P. J. The Structure of the
Antitumor Complex cis-(Diammino) (1,1-cyclobutanedicarboxylato)Pt(II): X-ray and NMR Studies. J. Inorg. Biochem. 1980,
13, 205-212.
(21) Deng, Y.; Khokhar, A. R. Crystallographic characterization of
trans-bis(acetato)(1,1-cyclobutanedicarboxylato)ethylendiamineplatinum(IV) trihydrate. Inorg. Chim. Acta 1993, 204,
35-38.
(22) Beagly, B.; Cruickshank, D. W. J.; McAuliffe, C. A.; Pritchard,
R. G.; Zaki, A. M. The crystal and molecular structure of cisdiammine-1,1-cyclobutanedicarboxoplatinum(II) [cis-Pt(NH3)2(CBDCA)]. Dynamic puckering of the cyclobutane ring. J. Mol.
Struct. 1985, 130, 97-102.
(23) Frey, U.; Ranford, J. D.; Sadler, P. J. Ring-Opening Reactions
of the Anticancer Drug Carboplatin: NMR Characterization of
cis-[Pt(NH3)(CBDCA-O)(5′-GMP-N7)] in Solution. Inorg. Chem.
1993, 32, 1333-1340.
(24) Canovese, L.; Cattalini, L.; Chessa, G. Kinetics of the Displacement of Cyclobutane-1,1-dicarboxylate from Diammine(cyclobutane-1,1-dicarboxylato)platinum(II) in Aqueous Solution. J.
Chem. Soc., Dalton Trans. 1988, 2135-2140.
(25) Knox, R. J.; Friedlos, F.; Lydall, D. A.; Robers, J. J. Mechanism
of Cytotoxicity of Anticancer Platinum Drugs: Evidence That
cis-Diamminedichloroplatinum(II) and cis-Diammine(1,1-cyclobutanedicarboxylato)platinum(II) Differ Only in the Kinetics of
Their Interaction with DNA. Cancer Res. 1986, 46, 1972-1979.
(26) The 1H NMR spectrum of R-[Ru(azpy)2Cl2] in an aqueous solution
(0.1 mg/mL, 1% DMSO) shows one set of signals and an H6
signal at relatively low field, suggesting the presence of both Cl
ligands. The 1H NMR spectrum of R-[Ru(azpy)2(NO3)2] in an
aqueous solution (0.1 mg/mL, 1% DMSO) is the same as the
spectrum of R-[Ru(azpy)2(NO3)2] in pure D2O. In fact, this is the
spectrum of R-[Ru(azpy)2(D2O)2]2+ in which the NO3 ligands are
replaced by D2O ligands upon dissolution. Mass spectra (MALDITOF) of R-[Ru(azpy)2Cl2] in an aqueous solution (0.1 mg/mL, 1%
DMSO) show, as main signals, m/z 503.0 for R-[Ru(azpy)2Cl]+
and m/z 467.1 for R-[Ru(azpy)2]H+. Also present in the spectrum
is a small signal at m/z 538.0for R-[Ru(azpy)2Cl2]. These data
agree with the retained coordination of the Cl ligands. Conductivity measurements of R-[Ru(azpy)2Cl2] (done at a higher
concentration of 1 mg/mL, 10% DMSO) show that this complex
does not display any conductivity. In contrast, conductivity
measurements of R-[Ru(azpy)2(NO3)2] in an aqueous solution (1
mg/mL, 10% DMSO) show a higher conductivity, i.e., in the
range of that for R-[Ru(azpy)2(H2O)2]2+ in H2O. This proves that
in the case of R-[Ru(azpy)2Cl2], a neutral complex is present,
which has to be the dichloro complex. The higher conductivity
of the R-[Ru(azpy)2(NO3)2] related to the dichloro complex is due
to the replacement of the NO3 ligands with H2O ligands (as
shown by NMR spectroscopy), resulting in a charged complex.
(27) Hotze, A. C. G.; Broekhuisen, M. E. T.; Velders, A. H.; Kooijman,
H.; Spek, A. L.; Haasnoot, J. G.; Reedijk, J. Crystallographic and
NMR evidence of the unusual N6,N7-didentate chelation of
3-methyladenine coordinated to the cytotoxic R-dichlorobis(2phenylazopyridine)ruthenium(II) complex. J. Chem. Soc., Dalton
Trans. 2002, 2809-2810.
(28) Hotze, A. C. G.; Broekhuisen, M. E. T.; van der Schilden, K.;
Velders, A. H.; Haasnoot, J. G.; Reedijk, J. Unusual Coordination
of the Rare Neutral Imine Tautomer of 9-Methyladenine Chelating in the N6,N7-Mode to Ruthenium(II) Complexes. Eur. J.
Inorg. Chem. 2002, 369-376.
(29) Velders, A. H.; de Vos, D. Unpublished results.
Acknowledgment. This work was supported in part
(A.L.S.) by the Council for Chemical Sciences of The
Netherlands Organization for Scientific Research (Grant
CWS-NWO). We thank Johnson Matthey Chemicals
(Reading, U.K.) for a generous loan of RuCl3·3H2O.
Additional support from COST Action D20 allowing
regular research exchanges to partner laboratories
inside EU countries is gratefully acknowledged.
Supporting Information Available: Tables of atomic
coordinates, bond lengths and angles, and displacement
parameters for complexes 1 and 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Reedijk, J. Improved understanding in platinum antitumour
chemistry. Chem. Commun. 1996, 801-806.
(2) Wong, E.; Giandomenico, C. M. Current Status of PlatinumBased Antitumor Drugs. Chem. Rev. 1999, 99, 2451-2466.
(3) Clarke, M. J.; Zhu, F.; Frasca, D. R. Non-Platinum Chemotherapeutic Metallopharmaceuticals. Chem. Rev. 1999, 99, 25112533.
(4) Vilaplana, R. A.; Gonzalez-Vilchez, F.; Guteirrez-Puebla, E.;
Ruiz-Valero, C. The first isolated antineoplastic Ru(IV) complex: synthesis and structure of [Cl2(1,2-cyclohexanediaminotetraacetate)Ru]‚2H2O. Inorg. Chim. Acta 1994, 224, 15.
(5) Nováková, O.; Kaspárková, J.; Vrána, O.; vanVliet, P. M.;
Reedijk, J.; Brabec, V. Correlation between cytotoxicity and DNA
binding of polypyridyl ruthenium complexes. Biochemistry 1995,
34, 12369-12378.
(6) Keppler, B. K.; Henn, M.; Juhl, U. M.; Berger, M. R.; Niebl, R.;
Wagner, F. E. New ruthenium complexes for the treatment of
cancer. Prog. Clin. Biochem. Med. 1989, 10, 41-69.
(7) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris, R.
E.; Chen, H.; Sadler, P. J.; Jodrell, D. I. In vitro and in vivo
activity and cross resistance profiles of novel ruthenium(II)
organometallic arene complexes in human ovarian cancer. Br.
J. Cancer 2002, 86, 1652-1657.
(8) Morris, R. E.; Aird, R. E.; Del Socorro Murdoch, P.; Chen, H.;
Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.;
Jodrell, D. I.; Sadler, P. J. Inhibition of cancer cell growth by
ruthenium(II) arene complexes. J. Med. Chem. 2001, 44, 36163621.
(9) Sava, G.; Gagliardi, R.; Bergamo, A.; Alessio, E.; Mestroni, G.
Treatment of metastases of solid mouse tumours by NAMI-A:
comparison with cisplatin, cyclophosphamide and dacarbazine.
Anticancer Res. 1999, 19, 969-972.
(10) Sava, G.; Bergamo, A.; Zorzet, S.; Gava, B.; Casarsa, C.;
Coccietto, M.; Furlani, A.; Scarcia, V.; Serli, B.; Iengo, E.; Alessio,
E.; Mestroni, G. Influence of chemical stability on the activity
of the antimetastasis ruthenium compound NAMI-A. Eur. J.
Cancer 2002, 38, 427-435.
(11) Sava, G. Personal communication, 2002.
(12) Velders, A. H.; Kooijman, H.; Spek, A. L.; Haasnoot, J. G.; de
Vos, D.; Reedijk, J. Strong differences in the in vitro cytotoxicity
of three isomeric dichlorobis(2-phenylazopyridine)ruthenium(II)
complexes. Inorg. Chem. 2000, 39, 2966-2967.
1750
Journal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hotze et al.
(30) Hotze, A. C. G. Unpublished results.
(31) Hotze, A. C. G. Results to be published.
(32) Tada, H.; Shiho, O.; Kuroshima, K.; Koyama, M.; Tsukamoto,
K. An improved colorimetric assay for interleukin 2. J. Immunol.
Methods 1986, 93, 156-165.
(33) Sheldrick, G. M. Program for Crystal Structure Determination;
University of Göttingen: Götingen, Germany, 1986.
(34) Sheldrick, G. M. SHELXL-97-2, Program for Crystal Structure
Refinement; University of Göttingen: Göttingen, Germany, 1997.
(35) Spek, A. L. PLATON, a Multi-Purpose Crystallographic Tool;
UtrechtUniversity: TheNetherlands(http://www.cryst.chem.uu.nl/
platon/).
(36) Krause, R. A.; Krause, K. Chemistry of bipyridyl-like ligands.
Isomeric complexes of ruthenium(II) with 2-(phenylazo)pyridine.
Inorg. Chem. 1980, 19, 2600-2603.
(37) Bao, T.; Krause, K.; Krause, R. A. Hydroxide-assisted stereospecific isomerization of a trans-dichloro bis chelate of ruthenium(II). Inorg. Chem. 1988, 27, 759-761.
(38) Goswami, S.; Chakravarty, A. R.; Chakravorty, A. Chemistry of
ruthenium. 2. Synthesis, Structure and Redox Properties of
2-(arylazo)pyridine Complexes. Inorg. Chem. 1981, 29, 2246-2250.
JM021110E