Ruthenium(II) complexes of thiosemicarbazones: The first water-soluble complex with pH-dependent antiproliferative activity

Article abstract of DOI:10.1002/ejic.200601196

Two ruthenium(II) complexes of 2-acetylpyridine N⁴,N ⁴-dimethylthiosemicarbazone (HL¹) and phenanthrenequinone thiosemicarbazone (HL²), namely [Ru^IICl(L ¹)(PPh₃)₂] (1)

Full text of DOI:10.1002/ejic.200601196

FULL PAPER
DOI: 10.1002/ejic.200601196
Ruthenium(II) Complexes of Thiosemicarbazones: The First Water-Soluble
Complex with pH-Dependent Antiproliferative Activity
Sanja Grguric-Sipka,[a][‡] Christian R. Kowol,[a] Seied-Mojtaba Valiahdi,[a] Rene Eichinger,[a]
Michael A. Jakupec,[a] Alexander Roller,[a] Sergiu Shova,[a][‡‡] Vladimir B. Arion,*[a] and
Bernhard K. Keppler*[a]
Keywords: Antitumour agents / Ruthenium / N,S ligands / X-ray diffraction
Two ruthenium(II) complexes of 2-acetylpyridine N4,N4-di-
methylthiosemicarbazone (HL1) and phenanthrenequinone
thiosemicarbazone (HL2), namely [RuIICl(L1)(PPh3)2] (1) and
[RuIICl(L2)(PPh3)2] (2), have been synthesised and character-
ised by IR, UV/Vis and NMR spectroscopy, electrospray mass
spectrometry, cyclic voltammetry and X-ray crystallography.
In addition, the X-ray crystal structure of [RuIIICl2(L2)-
PPh3]·dmso·1.25H2O (3·dmso·1.25H2O) is reported. The reac-
tion of [RuIICl2(dmso)4] with HL1 and 1,3,5-triaza-7-phos-
(S25°C Ն 250 mg/mL), which has been fully characterised.
Complex 4 shows strong antiproliferative effects in low
micromolar concentrations in the ovarian carcinoma cell line
41M (IC50 = 0.87 µM) and more moderate activity in the
breast cancer cell line SK-BR-3 (IC50 = 39 µM). The activity
of the compound is 6.5- and 5.4-times higher at pH = 6.0 than
at pH = 7.4 in the non-small cell lung cancer cell line A549
and the colon carcinoma cell line HT-29 (GI50 = 24 and 8.0 µ
M
at pH = 6.0 for A549 and HT-29, respectively).
phaadamantane (PTA) gives the highly water-soluble com- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
plex [RuIICl(L1)(HPTA)2]Cl2·C2H5OH·H2O (4·C2H5OH·H2O)
Germany, 2007)
azones are among the most potent known inhibitors of this
enzyme.[6,7] The inhibition of ribonucleotide reductase pre-
vents the conversion of ribonucleotides into deoxyribonu-
cleotides, which results in the impairment of DNA synthesis
and repair.[8] The applicability of thiosemicarbazones as
antitumour agents has been explored for over half a cen-
tury,[9] the first compound of this class to enter phase II
clinical trials as an antineoplastic agent being 3-aminopyr-
idine-2-carboxaldehyde thiosemicarbazone (3-AP, tri-
apine).[10,11] The effect of certain metals on the biological
activity of thiosemicarbazones has been receiving consider-
able attention recently.[12–14] Although a large number of
reports are available on the chemistry and biological ac-
tivity of copper(II), cobalt(III), gallium(III), iron(III), plati-
num(II) or palladium(II) thiosemicarbazonates,[15–18] there
are only a few reports on antitumour activity of the corre-
sponding ruthenium complexes.[19,20] Interest in ruthenium
was stimulated by phase I clinical trials of two ruthenium
complexes, namely (H2im)[trans-RuIIICl4(Him)(dmso)]
Introduction
Thiosemicarbazones form a class of mixed hard-soft oxy-
gen/nitrogen-sulfur chelating ligands that show a variety of
coordination modes in metal complexes. The thiosemicarb-
azone can act as a monodentate ligand that binds to the
metal ion through the sulfur atom[1] or as a bidentate ligand
that coordinates to the metal ion through the sulfur atom
and one of the nitrogen atoms of the hydrazine moiety to
form a four- or a five-membered metallacycle.[2,3] The coor-
dination capacity of thiosemicarbazones can be increased
by using for their preparation aldehydes or ketones contain-
ing additional functional group(s) in position(s) suitable for
chelation.[4] Beside their interesting coordination chemistry,
thiosemicarbazones have a wide pharmacological spectrum
of activity; their potential has been recognised to include
antitumour, antiviral, antibacterial, antifungal and antima-
larial properties.[5] The enzyme ribonucleotide reductase has
been identified as a principal target, and thiosemicarb-
(NAMI-A; Him
=
imidazole) and (H2ind)[trans-
RuIIICl4(Hind)2] (KP1019; Hind = indazole), the first as an
antimetastatic drug and the second as an anticancer agent
against primary tumours and metastases, and in particular
colon carcinomas.[21,22]
[a] Institute of Inorganic Chemistry, Faculty of Chemistry, Univer-
sity of Vienna,
Waehringer Str. 42, 1090 Vienna, Austria
Fax: +43-1427752680
E-mail: vladimir.arion@univie.ac.at
bernhard.keppler@univie.ac.at
The very high cytotoxicity of our recently synthesised
gallium(III) and iron(III) complexes with N4-substituted α-
N-heterocyclic thiosemicarbazones[23] in vitro together with
the above-mentioned interest in ruthenium chemistry
prompted us to synthesise novel ruthenium complexes of
[‡] On sabbatical leave from the Faculty of Chemistry of the Uni-
versity of Belgrade.
[‡‡]On sabbatical leave from the Department of Chemistry of the
Moldova State University.
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
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Ruthenium(II) Complexes of Thiosemicarbazones
FULL PAPER
thiosemicarbazones. We chose two ligands, 2-acetylpyridine and methanol and insoluble in water, whereas complex 4 is
N4,N4-dimethylthiosemicarbazone (HL1), which shows very very soluble in water (S25°C Ն 250 mg/mL), dmso, ethanol
high cytotoxicity in the low nanomolar range and the abil- and methanol.
ity to destroy the tyrosyl radical of mouse ribonucleotide
reductase R2 protein,[23] and phenanthrenequinone thio-
semicarbazone (HL2), which is known to have antiprolifera-
tive activity in the low micromolar range.[24] The presence
of the phenanthrene moiety in HL2 renders this ligand cap-
able of DNA intercalation in addition to its potential ribo-
nucleotide reductase inhibiting properties.
Ruthenium(II) complexes with two monoanionic triden-
tate thiosemicarbazones are neutral species that are spar-
ingly soluble or even insoluble in aqueous media.[25] Water
solubility is required to assay the cytotoxicity in vitro, there-
fore we focused on the synthesis of complexes with a 1:1
metal/ligand stoichiometry. A literature survey showed that
Figure 1. Structures of the ligands used.
Spectroscopic Characterisation
Complexes 1 and 4 show two well-resolved MLCT tran-
phosphorus-containing species and, in particular, tri- sitions in their electronic absorption spectra. The bands at
phenylphosphane often serve as ancillary ligands to com- 476 (1) and 450 nm (4) can be attributed to an
plete the coordination sphere of ruthenium upon coordina- Ru(4dπ)Ǟπ*(py) MLCT transition, whereas the bands at
tion of a thiosemicarbazone.[26–28] This prompted us to con- 379 (1) and 378 nm (4) can be assigned to an
sider 1,3,5-triaza-7-phosphaadamantane (PTA) as a co-li- Ru(4dπ)Ǟπ*(imine) transition.[25] The strong absorptions
gand as it is known to endow metal complexes with aque- at 597 and 470 nm in complex 2 are assignable to a combi-
ous solubility and solubility in polar organic sol- nation of MLCT bands and RuII d–d bands, and the high-
vents.[29a–29e,30] The (PTA)ruthenium compounds reported energy absorptions in the ultraviolet region for 1, 2 and 4
so far that show antiproliferative activity are organometallic can be attributed to intraligand π–π* transitions.
compounds derived from [Ru(η6-p-cymene)Cl2(PTA)],[29a]
some of which have been found to cause pH-dependent positive mode contains a strong peak at m/z = 847 attrib-
The ESI mass spectrum of complex 1 recorded in the
DNA damage.
uted to the [M – Cl]+ ion and a second remarkable signal
Herein we report on the synthesis, spectroscopic charac- at m/z = 585 assigned to [M – Cl – PPh3]+. Complex 2
terisation and X-ray diffraction structures of three novel shows a low intensity peak at m/z = 942 due to the [M +
complexes and the pH-dependent in vitro antiproliferative H]+ ion and more intense peaks at m/z = 964 and 980 attrib-
activity of the first thiosemicarbazone/PTA mixed rutheni- uted to [M + Na]+ and [M + K]+ ions, respectively. An
um(II) complex with high aqueous solubility.
intense signal at m/z = 906 corresponds to [M – Cl]+, while
that at m/z = 644 corresponds to [M – Cl – PPh3]+. The
ESI mass spectrum of complex 4 contains peaks at m/z =
673 and 695, which were assigned to [M – H]+ and [M – 2
H + Na]+, respectively. A signal at m/z = 637 is due to [M –
Results and Discussion
The reaction of [RuIICl2(PPh3)3] with either 2-acetylpyr- Cl]+, while that at m/z = 538 is due to [M – H – HPTA
idine N4,N4-dimethylthiosemicarbazone (HL1) in dry + Na]+. The observed isotopic patterns fit well with the
dichloromethane or phenanthrenequinone thiosemicarb- theoretical isotopic distributions.
1
azone (HL2) in absolute ethanol afforded the complexes
The H NMR spectrum of complex 1 in CDCl3 shows
trans-[RuIICl(L1)(PPh3)2] (1) and trans-[RuIICl(L2)(PPh3)2] signals attributable to the pyridine ring protons of coordi-
(2) in 41 and 80% yield, respectively. In one case the synthe- nated thiosemicarbazone at δ = 8.62, 7.36, 6.86 and
sis of 2 was accompanied by marginal formation of a side 6.47 ppm. The lack of any NH signal indicates the depro-
product, a few red crystals of which separated first on crys- tonation of the ligand. Proton resonances due to the PPh3
tallisation from dimethyl sulfoxide. An X-ray diffraction ligand are found between δ = 7.54 and 7.14 ppm. The 31P
study (see Supporting Information) showed that this minor NMR spectrum shows one resonance at δ = 30.42 ppm,
species has the composition [RuIIICl2(L2)PPh3] (3). Re- thus indicating the equivalence of the two PPh3 ligands in
peated crystallisation of 2 from dimethyl sulfoxide did not 1. This signal is shifted only slightly downfield from that
produce any significant amounts of 3, thereby preventing for trans-[RuCl(pabh)(PPh3)2] (Hpabh is a Schiff base de-
its full characterisation. The complex trans-[RuIICl- rived from 2-pyridinecarbaldehyde and benzoylhydrazine)
(L1)(HPTA)2]Cl2 (4) was prepared by the reaction of 2-ace- at δ = 27.62 ppm (CDCl3).[31] Complex 2 in [D6]dmso shows
tylpyridine N4,N4-dimethylthiosemicarbazone and PTA aromatic proton signals for the phenanthrenequinone moi-
with [RuIICl2(dmso)4] in absolute ethanol (Figure 1). Com- ety between δ = 7.19 and 8.89 ppm. The signal integrations
plex 4 appears to be the first reported ruthenium(II) com- and multiplicities are in agreement with the proposed for-
plex that contains a thiosemicarbazone and 1,3,5-triaza-7- mula, although exact assignment is not possible due to ex-
phosphaadamantane (PTA) as ligands. Complexes 1 and 2 tensive overlap with resonances originating from the tri-
are well soluble in CHCl3 but sparingly soluble in ethanol phenylphosphane ligands. A singlet that can be assigned to
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the NH2 group was found at δ = 8.13 ppm. The hydrazinic chlorido ligand. Two trans-positioned triphenylphosphane
NNH proton signal at δ = 14.42 ppm present in the 1H ligands in 1 complete the octahedron. A comparison of the
NMR spectrum of the metal-free thiosemicarbazone is ab- bond lengths in the coordination polyhedron of 1 with
sent in the spectrum of the complex, thus indicating the those in [Ru(HL)(PPh3)2Cl]+ [HL = 2-acetylpyridine 4-(4-
deprotonation of the thiosemicarbazone ligand upon com- tolyl)thiosemicarbazone][26] shows that the Ru–Cl bond in
plexation. As for 1, the 31P NMR spectrum of 2 is very 1 [2.4812(13) Å] is significantly longer than in [Ru(HL)-
simple, displaying one resonance at δ = 27.36 ppm due to (PPh3)2Cl]+ [2.459(2) Å]. The interatomic Ru–N1 and Ru–
the equivalence of the two PPh3 ligands.
N2 distances of 2.092(4) and 1.976(4) Å, respectively, are
Complex 4 in [D6]dmso shows a signal at δ = 9.87 ppm similar to those in [Ru(HL)(PPh3)2Cl]+ [2.085(5) and
that can be assigned to the protons of the two quaternary 1.984(5) Å, respectively], whereas the Ru–S and Ru–P
nitrogen atoms in the PTA ligands. As for 1, the absence of bonds are shorter in 1 {2.3725(13) and 2.3747(8) Å com-
a further NH resonance indicates the presence of a pared to 2.386(2) and 2.399(1) Å in [Ru(HL)(PPh3)2Cl]+}.
monoanionic thiosemicarbazone ligand in 4. The pyridine The distribution of electron density over the thiosemicarb-
ring protons occur as multiplets at δ = 8.77, 7.85, 7.67 and azide moiety in the compared complexes is different,
7.33 ppm. The singlet due to the N(CH3)2 group is found thereby indicating the prevailing double-bond character of
at δ = 3.33 ppm together with the water signal. The proton C8–S in [Ru(HL)(PPh3)2Cl]+ [1.707(6) Å vs. 1.751(5) Å in
resonances of the methylene groups bridging the nitrogen 1]. The N3–C8 bond length in 1 [1.335(7) Å] is equal within
atoms in PTA appear as a singlet at δ = 4.62 ppm, while 3σ to that in [Ru(HL)(PPh3)2Cl]+ [1.359(7) Å]. The mole-
the resonances due to the PCH2N protons are observed as cules of 1 interact weakly with each other through C–H···Cl
a multiplet at δ = 3.71 ppm. The 31P NMR spectrum of 4 contacts, as shown in Figure S1 (Supporting Information).
exhibits one singlet resonance at δ = –38.13 ppm for the two
equivalent PTA ligands {cf. δ = –33.45 ppm for the two
equivalent phosphorus atoms in [RuCl(PTA)2([9]aneS3)]-
CF3SO3}.[29e]
The electrochemical properties of complexes 1, 2 and 4
have been studied by cyclic voltammetry in dmso solution
(0.2  [nBu4N][BF4]) using a glassy carbon working elec-
trode. The cyclic voltammograms of all complexes show a
single one-electron oxidation wave at E1/2 = 0.59, 0.71 and
0.73 V vs. NHE for 1, 2 and 4, respectively, which can be
assigned to RuII/RuIII electron transfer. The peak-to-peak
separation (∆Ep) of 70–90 mV in complexes 1 and 2 is al-
most independent of the scan rate. In addition, the equality
of the anodic peak current (ipa) and the cathodic peak cur- Figure 2. Perspective view of the molecule of [RuII(L1)(PPh3)2Cl]
rent (ipc) indicates a reversible electron-transfer. The RuII/
(1) with atom labelling scheme. Thermal ellipsoids are drawn at the
50% probability level, and the hydrogen atoms have been omitted
RuIII oxidation wave in complex 4 shows ∆Ep values of 110–
for clarity. Selected bond lengths [Å] and angles [°]: Ru–N1
130 mV in the range of scan rates from 50 to 1000 mVs–1
2.092(4), Ru–N2 1.976(4), Ru–S 2.3725(13), Ru–Cl 2.4812(13), Ru–
P1 2.3747(8), S–C8 1.751(5), C8–N3 1.335(7) Å; P1–Ru–P1i
and ipa/ipc ratios close to 1. Deviations from the ideal
Nernstian value of 59 mV are well documented for this type
of complexes.[26] Complex 2 shows two additional irrevers-
ible reduction waves with Ep/2 values of –0.78 and –1.27 V
i
175.08(6). Symmetry code: : –x, y + 1, z.
The ruthenium(II) ion in 2 is coordinated by the thio-
which can be ascribed to the reduction of the thiosemicarb- semicarbazone anion (L2)– through carbonyl oxygen atom
azone ligand. The more positive oxidation response O, imine nitrogen atom N1 and thiolate sulfur atom S and
(+140 mV) in 4 in comparison to that in 1 is in line with by the chlorido ligand in the equatorial plane. In addition,
the poorer electron-donating ability of PTA compared to two triphenylphosphane ligands are bound in axial posi-
PPh3.[32]
tions (Figure 3). Relevant bond lengths and angles are given
in the legend to Figure 3. A comparison of the bond lengths
of the thiosemicarbazone moiety in complex 2 with those
of the metal-free thiosemicarbazone[24] shows that the S–
C15 bond [1.707(4) Å] in 2 is longer than in HL2·CH3CN
Crystal Structures
The thiosemicarbazone ligand in 1 acts as a monoanion [1.681(2) Å], whereas the C15–N2 bond [1.348(9) Å] in 2 is
(L1)– and occupies a meridional plane about the ruthenium similar to that in HL2·CH3CN [1.365(3) Å]. The coordina-
ion, coordinating to it through pyridine nitrogen atom N1, tion of HL1 and HL2 (Figure 1) to the ruthenium(II) ion in
imine nitrogen atom N2 and the thiolate sulfur atom S (Fig- their deprotonated forms in 1 and 2, respectively, is also
ure 2). A Cl ligand completes the square plane around the indicated by the absence of counterions. The ligand in 2
ruthenium ion. The thiosemicarbazone ligand is planar. All has also undergone a configurational change from the (E)
non-hydrogen atoms of (L1)– lie in the crystallographic sym- configuration found in the metal-free state[24] to a (Z) con-
metry plane together with the central metal ion and the figuration relative to the C15–N2 bond. The phenanthrene
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Ruthenium(II) Complexes of Thiosemicarbazones
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moieties in the crystal structure of 2 are stacked pairwise in
an offset manner with an interplanar separation between
the benzene rings (C9–C14) and (C9–C14)Ј of around
3.32 Å; the distance between their centroids (X···XЈ) is
3.857 Å (Figure S2). The hydrogen bond between N3 as
proton donor and the Cl atom of the neighbouring mole-
cule as proton acceptor, with an N3···Cl contact of 3.490 Å,
is also worthy of note.
Figure 4. Perspective view of the complex [RuII(L1)(HPTA)2Cl]Cl2
(4) with atom numbering scheme. Thermal ellipsoids are drawn at
the 50% probability level, and the hydrogen atoms have been omit-
ted for clarity. Selected bond lengths [Å] and angles [°]: Ru–N1
2.081(5), Ru–N2 1.979(5), Ru–S 2.3707(17), Ru–Cl1 2.4571(16),
Ru–P1 2.3019(19), Ru–P2 2.2958(19), S–C8 1.771(6), C8–N3
1.303(8); P1–Ru–P2 166.05(5).
Determination of the pKa Value
The pH of a 0.005  aqueous solution of [RuII-
Cl(L1)(HPTA)2]Cl2 (4) is about 3. 31P NMR spectra of an
aqueous (D2O) solution of 4 were recorded at different pH*
(= pD) values (from 2.11 to 11.40). We suppose that the
complex exists predominantly in the monoprotonated form
(δ = –37.03 ppm) at pH* = 2.11, whereas the signal of the
neutral complex (with unprotonated PTA ligands) is found
at δ = –48.45 ppm at pH* = 11.40 (see Figure S4, Support-
Figure 3. Perspective view of the molecule of [RuII(L2)(PPh3)Cl] (2)
with atom numbering scheme. Thermal ellipsoids are drawn at the
30% probability level, and the hydrogen atoms have been omitted
for clarity. Selected bond lengths [Å] and angles [°]: Ru–O 2.096(2),
Ru–N1 1.936(3), Ru–S 2.3608(14), Ru–P1 2.3932(10), Ru–P2
2.3824(10), Ru–Cl 2.4528(9), S–C15 1.707(4), C15–N2 1.348(9) Å;
P1–Ru1–P2 170.36(3), O–Ru–S 160.96(6), N1–Ru–Cl 171.31(8).
The structure of complex 4 is shown in Figure 4. The ing Information). At pH = 3.82 the main signal is shifted
coordination polyhedron of the ruthenium ion can be de- to δ = –40.02 ppm and a second, minor (Ͻ 4%) 31P signal
scribed as a distorted octahedron. As in 1, the metal ion is emerges at δ = –41.30 ppm, which increases gradually in
coordinated in the equatorial plane by the thiosemicarb- intensity and shifts to higher frequency more sluggishly
azone monoanion through pyridine nitrogen atom N1, im- than the first peak (Figure S4). At the end of the titration
ine nitrogen atom N2 and the thiolate sulfur atom S, and (pH* = 11.40) the intensity of the second signal is 50%
by the chlorido ligand Cl1. Both axial positions are occu- of the main signal. Moreover, the intensity of this second
pied by two PTA ligands, which are bound to the ruthe- resonance increases with time. The appearance of this sec-
nium ion through phosphorus atoms P1 and P2. The Ru– ond 31P resonance can be suppressed by addition of an ex-
P(PTA) separations [Ru–P1 2.3019(19), Ru–P2 2.2958(19) Å] cess of NaCl to a solution of 4 in D2O. Repeated titration
are in the range expected for Ru–PTA complexes.[30,33] The of a 0.15  NaCl solution of 4 in D2O with NaOD from
Ru–Cl1 bond [2.4571(16) Å] is normal for Ru–PTA com- pH* = 3.29 to 10.30 showed that the second peak now ap-
plexes containing chlorido co-ligands.[34] In addition, the pears first at pH = 5.16 and remains of very low intensity
two PTA ligands are protonated at N5 and N8, respectively, (Ͻ 5%) until the end of the titration (pH = 10.30). These
and form hydrogen bonds to Cl2 and Cl3. The S–C8 bond results show that a solution of complex 4 in D2O in the pH
[1.771(6) Å] is longer than the C–S bond found in 2-acetyl- range indicated above undergoes both protolytic reactions
pyridine N-pyrrolidinylthiosemicarbazone [1.692(4) Å][35] and hydrolysis of the RuII–Cl bond.
and also longer than in HL1 [1.720(2) Å].[36] The lengthen-
A pKa value of 4.46 for one of the axially coordinated
ing of this bond in 4, along with the N2–N3 and N3–C8 PTA ligands in 4 was obtained from a 31P NMR pH* ti-
bonds, indicates a significant electron delocalisation over tration curve in D2O (Figure 5). This value is higher than
the thiosemicarbazide moiety.[37] The Ru–N2 distance that (3.13) found from 31P NMR titration of the coordi-
[1.979(5) Å] is slightly shorter than the Ru–N1 distance nated PTA in [RuCl2(PTA)(η6-p-cymene)],[38] but lower
[2.081(5) Å], presumably because of steric reasons. The as- than those reported for metal-free PTA (5.63[29c] and
sociation of a proton with N5 of one PTA and of another 6.0[39]).
one with N8 of the second PTA ligand makes the corre-
The evolution of the absorption spectrum of 4 in 0.15 
sponding atoms good proton donors, and they are conse- NaCl aqueous solution on increasing the pH from 2.88 to
quently involved in hydrogen bonding to the two chloride 9.30 is given as Supporting Information (Figure S5) and is
anions Cl2 and Cl3, respectively (see Figure 4).
characterised by an isosbestic point at 445 nm.
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Figure 5. Plot of δ(31P) vs. pD of 4.
Antiproliferative Activity in Cancer Cell Lines
The antiproliferative effects of compound 4 were first ex-
amined in the human cancer cell lines 41M and SK-BR-3
by means of a colourimetric microculture assay (MTT as-
say) with continuous exposure at pH = 7.4 for 96 h. Con-
centration-effect curves are presented in Figure 6A. The
compound exerts strong antiproliferative effects in low
micromolar concentrations in the ovarian carcinoma cell
line 41M (IC50 = 0.87 µ), while the breast cancer cell line
SK-BR-3 is much less sensitive (IC50 = 39 µ). The IC50
values obtained in the non-small cell lung cancer cell line
A549 and the colon carcinoma cell line HT-29 (Table 1)
suggest that these cells are rather insensitive, but a direct
comparison with the other two cell lines is not appropriate
because of the different exposure times. However, experi-
ments on the pH dependency of the antiproliferative effects,
with exposure at either pH = 6.0 or 7.4 for 24 h, followed
Figure 6. Antiproliferative effects of compound 4, as determined
by an MTT assay. A) Concentration-effect curves in 41M and SK-
BR-3 cells (continuous exposure for 96 h). B) Concentration-effect
curves (corrected for differences in growth kinetics of the corre-
sponding controls) in A549 and HT-29 cells at pH = 7.4 and 6.0
(24 h exposure), indicating enhanced cytostatic effects in a slightly
acidic medium.
Table 1. Antiproliferative effects of compound 4 in four human
cancer cell lines and the pH dependency in two of these cell lines.
Cell line
Exposure IC50 [µ][a]
GI50 [µ][b]
GI50 ratio
time [h]
pH = 7.4 pH = 7.4 pH = 6.0
41M (ovary)
SK-BR-3 (breast)
A549 (lung)
96
96
24
24
0.87Ϯ0.09
39Ϯ5
186Ϯ16
47Ϯ4
156Ϯ18
43Ϯ4
24Ϯ1
8.0Ϯ0.4
6.5
5.4
HT29 (colon)
by incubation in a drug-free medium (pH = 7.4) for a fur- [a] 50% inhibitory concentrations in the MTT assay. [b] 50% growth
inhibition (corrected for differences in growth kinetics of the corre-
ther 72 h revealed that the activity of the compound is 6.5-
sponding controls) after exposure for 24 h in the MTT assay. All val-
and 5.4-times higher under slightly acidic conditions in
ues are the means with standard deviations obtained from at least
A549 and HT-29 cells, respectively, with IC50 values shifted
three independent experiments.
towards lower micromolar concentrations (Figure 6B,
Table 1). The extent of this activation clearly exceeds that
of cisplatin (3.2- and 2.2-fold in A549 and HT-29 cells, may therefore contribute to the reduced chemosensitivity of
respectively), while the effects of oxaliplatin are indepen- acidic tumours.[43–45] On the other hand, weakly acidic
dent of the pH under the same experimental conditions.[40] drugs such as chlorambucil are more cytotoxic when the
This pronounced pH dependency suggests that com- extracellular pH is slightly acidic.[44,46] These observations
pound 4 may be activated preferentially in a slightly acidic are consistent with ion trapping, according to which weak
environment. Such acidic conditions occur in the extracellu- electrolytes that are not transported actively concentrate in
lar fluid of solid tumours as a consequence of constitutive the compartment where their ionised fraction is larger. Ion
upregulation of glycolysis (independent of oxygen supply), trapping has also been discussed as a possible explanation
which is a hallmark of malignant progression that confers for the pH-dependent cellular uptake and cytotoxicity of
a growth advantage to tumour cells under the conditions of the metal complex cisplatin, the hydrolysis products of
intermittent hypoxia resulting from hypovascularis- which are in a pH-dependent equilibrium.[47] Therefore, the
ation.[41,42] The altered pH gradients across cell membranes importance of appropriate pKa values for the design of anti-
in acidic tumour regions affect the cellular drug accumu- tumour drugs has been emphasised.[48] Furthermore, the
lation and cytotoxicity of weakly basic chemotherapeutics development of prodrugs that are selectively activated in an
such as anthracyclines, vinca alkaloids or mitoxantrone and acidic environment is being pursued as a strategy to in-
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Ruthenium(II) Complexes of Thiosemicarbazones
FULL PAPER
thiosemicarbazone,[24] 2-acetylpyridine N4,N4-dimethylthiosemi-
carbazone,[51] 1,3,5-triaza-7-phosphaadamantane (PTA)[52] and cis-
[RuCl2(dmso)4][53] were prepared according to published pro-
cedures. Elemental analyses were carried out at the Institute of
Physical Chemistry of the University of Vienna with a Carlo Erba
microanalyser. Infrared spectra were recorded with a Bruker Vertex
70 FTIR spectrometer as KBr pellets (4000–400 cm–1). Electronic
spectra were obtained with a Perkin–Elmer Lambda 650 UV/Vis
spectrophotometer. Electrospray ionisation mass spectrometry was
carried out in methanol with a Bruker Esquire 3000 instrument
crease the therapeutic index by effectively killing tumour
cells without impairing normal tissues.[49]
Some (PTA)ruthenium(II) complexes related to the com-
pounds presented here have been found to induce DNA
damage in isolated bacterial plasmids only at pH values less
than about 7.0[50] and to exert a stronger cytotoxicity in
breast cancer cells than in a non-malignant mammary
gland cell line, while analogues containing methylated PTA,
which is not protonated in acidic solution, do not show this
kind of selectivity.[38] PTA has therefore attracted attention (Bruker Daltonics, Bremen, Germany). Expected and experimental
isotope distributions were compared. The NMR spectra were re-
corded with a Bruker DPX 400 instrument using standard Bruker
pulse programs. Chemical shifts for 1H NMR spectra were refer-
enced to the residual 1H present in deuterated dmso and CHCl3,
respectively, and 31P NMR to external H331PO4 in D2O. Cyclic vol-
tammograms were measured in a three-electrode cell using a 0.2-
mm-diameter glassy carbon working electrode, a platinum auxiliary
electrode and an Ag|Ag+ reference electrode containing 0.1 
AgNO3, the potential of which was corrected using an internal
standard redox couple of ferrocenium/ferrocene. Measurements
as a ligand that apparently confers tumour selectivity to
(arene)ruthenium(II) compounds, which has been tenta-
tively attributed to activation by protonation (assuming that
the protonated form is actually the active species). While it
has not been reported whether the cytotoxicity of arene–
RuII–PTA complexes is pH-dependent, our findings with
compound 4, which exerts stronger antiproliferative effects
at pH values encountered in solid tumours (6.0) than at pH
values prevailing in normal tissues (7.4), support this no-
tion. The presence of two PTA ligands in compound 4 may were performed at room temperature using an EG & G PARC
273A potentiostat/galvanostat. Deaeration of solutions was ac-
complished by passing a stream of argon through the solution for
5 min prior to the measurements and then maintaining a blanket
of argon over the solution during the measurement. The potentials
were measured in 0.2  [nBu4N][BF4]/dmso, with [Fe(η5-C2H5)2]
(E1/2ox = +0.68 V vs. NHE)[54] as internal standard, and are quoted
relative to NHE.
[RuL1Cl(PPh3)2] (1): A suspension of 2-acetylpyridine N4,N4-di-
methylthiosemicarbazone (22.3 mg, 0.1 mmol) in dry dichloro-
methane (5 mL) was added to a solution of [RuCl2(PPh3)3]
(95.8 mg, 0.1 mmol) in dry dichloromethane (5 mL) and the re-
sulting mixture was refluxed under argon for 4 h. The solution was
concentrated to about 5 mL, and allowed to stand at +4 °C over-
night. The product was obtained as green-brown crystals, which
be favourable from this point of view and may constitute a
basis for further attempts to increase tumour-selective ac-
tivity. However, the pronounced decrease of pKa (from 5.6
to 3.0–3.3) resulting from the coordination of PTA to ru-
thenium(II) in arene–RuII–PTA complexes renders the pos-
sibility that ion trapping accounts for any pH-dependent
cytotoxicity of this type of compounds rather unlikely[38]
and raises the need for other explanations that might be
completely different from previous assumptions.
In contrast to arene–RuII–PTA complexes, the presence
of the biologically active thiosemicarbazone ligand may
play a role in the activity of complex 4 even though this
ligand is tightly bound to the metal centre in a tridentate
manner. However, even a partial release of 2-acetylpyridine were filtered off, washed with n-hexane and dried in vacuo. Yield:
N4,N4-dimethylthiosemicarbazone should result in a tre-
36 mg (41%). C46H43ClN4P2RuS·0.5CH2Cl2 (924.86): calcd. C
60.39, H 4.80, N 6.06, S 3.47; found C 60.61, H 4.80, N 6.30, S
mendous increase in cytotoxicity because its IC50 values of
3.57. ESI-MS: m/z = 847 [M – Cl–]+, 585 [M – Cl– – PPh ]+. IR: ν
0.22 and 2.7 n in 41M and SK-BR-3 cells, respectively,[23]
are three to four orders of magnitude below those of com-
plex 4. If a release of the thiosemicarbazone ligand ac-
counts for the effects of compound 4, it must be assumed
to occur only to a very small degree.
˜
3
= 1522 [w, ν(C=N)], 1482 (m), 1391 (s), 1314 (s), 907 (w), 742 (m),
695 (s) and 516 (s, ν[Ru(PPh ) ]) cm–1. 1H NMR (400.13 MHz.
3
2
3
CDCl3): δ = 8.62 (d, JH,H = 5.1 Hz, 1 H, py), 7.54–7.51 (m, 12 H,
PPh3), 7.36 (m, 1 H, py), 7.24–7.21 (m, 6 H, PPh3), 7.18–7.14 (m,
3
3
12 H, PPh3), 6.86 (d, JH,H = 8.1 Hz, 1 H, py), 6.47 (t, JH,H
=
6.2 Hz, 1 H, py), 2.70 [s, 6 H, N(CH3)2], 1.82 [s, 3 H, C(CH3)] ppm.
31P{1H} (162.00 MHz; CHCl3): δ = 30.42 and 26.19 [equal intensity
(PPh3)2] ppm. UV/Vis (CH2Cl2): λmax (ε) = 599 sh (1620), 546 sh
(2280), 476 (7500), 397 sh (31760), 379 (39880), 274
(47120 –1 cm–1) nm. Single crystals suitable for an X-ray diffrac-
tion study were selected directly from the reaction vessel.
Conclusions
Following a known approach based on using P-contain-
ing derivatives as co-ligands, a novel highly water-soluble
ruthenium(II) thiosemicarbazonate complex, namely [RuII-
(L1)(HPTA)2Cl]Cl2, has been prepared. This, to the best of
our knowledge, is the first ruthenium(II) complex with
mixed thiosemicarbazone/PTA ligands to be isolated and
characterised by X-ray crystallography. The complex shows
pH-dependent antiproliferative activity in vitro in micromo-
lar concentrations against the non-small cell lung cancer
cell line A549 and the colon carcinoma cell line HT29.
[RuL2Cl(PPh3)2]·C2H5OH (2). Method a): A suspension of phenan-
threnequinone thiosemicarbazone (28.2 mg, 0.1 mmol) in dry etha-
nol (5 mL) was added to a suspension of [RuCl2(PPh3)3] (95.8 mg,
0.1 mmol) in dry ethanol (5 mL), and the resulting mixture was
refluxed under argon for 4 h. The solution was concentrated to
about 5 mL and allowed to stand at +4 °C overnight. The solid
residue was stirred in n-hexane to remove the excess of ligand and
triphenylphosphane, filtered off and dried in vacuo. Yield: 61 mg
(65%). C51H40ClN3OP2RuS·C2H5OH (987.49): calcd. C 64.46, H
4.70, N 4.26, S 3.25; found C 64.46, H 4.36, N 4.55, S 3.34. ESI-
MS: m/z = 942 [M + H+]+, 964 [M + Na+]+, 980 [M + K+]+, 906
Experimental Section
General: All chemicals were obtained from commercial suppliers
and were used without further purification. Phenanthrenequinone
[M – Cl–]+, 644 [M – Cl– – PPh ]+. IR: ν = 1600 [s, ν(C=N)], 1590
˜
3
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2875

V. B. Arion, B. K. Keppler et al.
FULL PAPER
(s), 1434 (s), 1190 (s), 743 (m), 694 (s) and 519 (s, ν[Ru(PPh ) ]) cm–1.
by Krezel and Bal,[57] (with pKa* as the pKa value determined in
D2O) for conversion of protonation constants, measured in D2O
3
1H NMR (400.13 MHz, [D6]dmso): δ = 8.88 (dd, 3JH,H = 8.23, 4JH,H
3
4
= 1.8 Hz, 1 H, phq), 8.43 (dd, JH,H = 7.8, JH,H = 1.0 Hz, 1 H, into constants valid for H2O solutions. The error is estimated as
3
phq), 8.35 (d, JH,H = 8.6 Hz, 1 H, phq), 8.26 (m, 1 H, phq), 8.13
(s, 2 H, NH2), 7.65 (m, 1 H, phq), 7.52–7.49 (m, 12 H, PPh3), 7.26–
7.19 (m, 9 H, PPh3 and phq), 7.14–7.11 (m, 12 H, PPh3) ppm.
31P{1H} (162.00 MHz, [D6]dmso): δ = 27.36 [s, 2 P, (PPh3)2]. UV/
Vis (CH2Cl2): λmax (ε) = 597 nm (9080) , 470 (14580), 320 (13600),
268 (49330 –1 cm–1) nm. Method b). [RuL2Cl(PPh3)2]: Phenan-
threnequinone thiosemicarbazone (28.2 mg, 0.1 mmol) in dry etha-
nol (5 mL) and triphenylphosphane 76.0 mg (0.29 mmol) in dry
ethanol (5 mL) were added to [RuCl2(dmso)4] (48.0 mg, 0.1 mmol)
in dry ethanol (5 mL) and the mixture refluxed under argon for
5 h. The black solid was filtered off, washed with absolute ethanol
and n-hexane and dried in vacuo. Yield: 79 mg (80%).
C51H40ClN3OP2RuS (941.42): calcd. C 65.07, H 4.28, N 4.46;
found C 64.78, H 4.43, N 4.38. Single crystals suitable for X-ray
data collection were obtained from a chloroform solution of 2 satu-
rated with n-hexane.
Ϯ0.04 pK units for two 31P resonances, and the mean value was
taken.
X-ray Crystallography: X-ray diffraction measurements were per-
formed with an X8 APEXII CCD diffractometer. Single crystals of
1–4 were positioned at 40, 40, 40 and 37.5 mm from the detector,
respectively, and 1187, 1438, 1328 and 1320 frames were measured
each for 70, 120, 50 and 10 s with a 1° scan-width. The data were
processed using the SAINT software package.[58] The structures
were solved by direct methods and refined by full-matrix least-
squares techniques. Non-hydrogen atoms were refined with aniso-
tropic displacement parameters. H atoms were inserted in geometri-
cally calculated positions and refined with a riding model. The fol-
lowing computer programs were used: structure solution:
SHELXS-97;[59] refinement: SHELXL-97;[60] molecular diagrams:
ORTEP;[61] computer CPU: Pentium IV; scattering factors were
taken from the literature.[62] Crystal data, data collection param-
eters, and structure refinement details are given in Table 2. CCDC-
630689 (1·3CH2Cl2), -630686 (2), -630687 (3·dmso·1.25H2O) and
-630688 (4·2H2O) contain the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
[RuL1Cl(HPTA)2]Cl2·C2H5OH·H2O (4·C2H5OH·H2O): A solution
of 2-acetylpyridine N4,N4-dimethylthiosemicarbazone (22.3 mg,
0.1 mmol) in dry ethanol (5 mL) was added to a suspension of cis-
[RuCl2(dmso)4] (44.8 mg, 0.1 mmol) in dry ethanol (5 mL), and the
resulting mixture was heated at 65 °C under argon for 1 h to yield
a red solution. PTA (31.6 mg, 0.2 mmol) was then added and heat-
ing was continued for a further 4 h. The green-yellow side product
was separated by filtration and the red solution concentrated
to about 1 mL and allowed to stand at +4 °C overnight. The
red crystals formed were filtered off, washed with cold dry
ethanol and dried in vacuo. Yield: 7 mg (10%).
C22H39Cl3N10P2RuS·C2H5OH·H2O (809.14): calcd. C 35.63, H
5.85, N 17.31, S 3.96; found C 35.09, H 5.40, N 17.49, S 4.07. ESI-
MS: m/z = 673 [M – H+]+, 695 [M – 2 H+ + Na+]+, 637 [M – 2
Cell Lines and Culture Conditions: Human 41M (ovarian carci-
noma) and SK-BR-3 (mammary carcinoma) cells were kindly pro-
vided by Lloyd R. Kelland (CRC Centre for Cancer Therapeutics,
Institute of Cancer Research, Sutton, UK) and Evelyn Dittrich
(General Hospital, Medical University of Vienna, Austria), respec-
tively. Human A549 (non-small cell lung cancer) and HT29 (colon
carcinoma) cells were kindly provided by Brigitte Marian (Institute
of Cancer Research, Medical University of Vienna, Austria). Cells
were grown in 75-cm2 culture flasks (Iwaki/Asahi Technoglass, Gy-
ouda, Japan) as adherent monolayer cultures in complete culture
medium [Minimal Essential Medium (MEM) supplemented with
10% heat-inactivated fetal bovine serum, 1 m sodium pyruvate,
4 m -glutamine and 1% non-essential amino acids (100ϫ) (all
purchased from Sigma–Aldrich, Vienna, Austria)]. Cultures were
maintained at 37 °C in a humidified atmosphere containing 5%
CO2.
H+ – Cl–]+, 538 [M – 2 H+ – PTA + Na+]+. IR: ν = 1531 [w, ν
˜
(C=N)
], 1503 (w), 1397 (s), 1315 (s), 768 (m), 566 (m) cm–1. 1H NMR
3
(400.13 MHz, [D6]dmso): δ = 9.87 (s, 2 H, NH), 8.77 (d, JH,H
=
3
5.3 Hz, 1 H, py), 7.85 (m, 1 H, py), 7.67 (d, JH,H = 8.1 Hz, 1 H,
py), 7.33 (m, 1 H, py), 4.62 (s, 12 H, NCH2N), 3.78–3.67 (m, 3JH,P
= 10.6 Hz, 12 H, NCH2P), 3.33 [s, 6 H, N(CH3)3, below the water
signal], 2.42 (s, 3 H, CH3) ppm. 31P{1H} (162.00 MHz, [D6]dmso):
δ = –38.13 [s, 2 P, (HPTA)2]. UV/Vis (H2O): λmax (ε) = 522 sh
(870), 450 (5460), 378 sh (18320), 355 (25910), 280 sh (9220), 222
sh (38930 –1 cm–1) nm. Red crystals of composition 4·2H2O of
X-ray diffraction quality were obtained by recrystallisation from
ethanol.
Antiproliferative Activity in Cancer Cell Lines: Antiproliferative ef-
fects were determined by means of a colourimetric microculture
assay [MTT assay; MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-di-
phenyl-2H-tetrazolium bromide]. Cells were harvested from the
culture flasks by trypsinisation and seeded into 96-well microcul-
ture plates (Iwaki/Asahi Technoglass, Gyouda, Japan) in cell densi-
ties of 4ϫ103 (except HT-29: 3ϫ103 cells/well), in order to ensure
exponential growth throughout drug exposure. After pre-incu-
bation for 24 h, 41M and SK-BR-3 cells were exposed to serial
dilutions of the test compound in 200 µL/well complete culture me-
dium for 96 h under an atmosphere containing 5% CO2. For pH-
dependency experiments in A549 and HT-29 cells, the test com-
pound was dissolved in Dulbecco’s Modified Eagles Medium
(DMEM) (w/o sodium hydrogencarbonate) supplemented with
10% heat-inactivated fetal bovine serum, 1 m sodium pyruvate,
4 m -glutamine, 1% non-essential amino acids (100ϫ) and so-
dium hydrogencarbonate in either 8.8 m (pH = 6.0) or 39.6 m
(pH = 7.4) concentration (all purchased from Sigma–Aldrich, Vi-
enna, Austria), and cells were exposed under an atmosphere con-
taining 8% CO2 for 24 h, followed by incubation in 200 µL/well
drug-free DMEM (pH = 7.4) for a further 72 h. At the end of
Determination of the pKa Values: The pH* titration experiment was
carried out in D2O at 298 K using an Ecoscan pH 6 (Eutech) pH
meter equipped with a micro-combination pH electrode (Orion) for
NMR tubes calibrated with buffer solution at pH = 4, 7 and 10.
The pH* values were adjusted with diluted DCl and NaOD. The
pKa value of the coordinated PTAs in 4 was determined by plotting
the change in chemical shift of the 31P signal against pH* and fit-
ting the curve to the modified Henderson–Hasselbalch equation[55]
(δobs, δHA and δA are the chemical shifts for the observed, proton-
ated and deprotonated species, respectively), by using the program
KALEIDAGRAPH,[56] with the assumption that the observed val-
ues are weighted averages according to the populations of the pro-
tonated and deprotonated species.
The pKa* value determined from the titration curve in Figure 5 was
corrected by using the equation pKa = 0.929pKa* + 0.42 suggested
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Eur. J. Inorg. Chem. 2007, 2870–2878

Ruthenium(II) Complexes of Thiosemicarbazones
FULL PAPER
Table 2. Crystal data and details of data collection for 1–4.
1·3CH2Cl2
2
3·dmso·1.25H2O
4·2H2O
Empirical formula
Formula mass
Space group
a [Å]
b [Å]
c [Å]
C49H49Cl7N4P2RuS
1137.14
Pmn21
18.4882(7)
9.3773(3)
14.7549(6)
C51H40ClN3OP2RuS
941.38
P1
C35H33.5Cl2N3O3.25PRuS2
814.70
P21/c
9.8926(3)
17.8078(6)
21.7962(7)
C22H43Cl3N10O2P2RuS
781.08
C2/c
31.404(4)
9.0952(9)
¯
9.4527(4)
11.8536(7)
20.2796(11)
81.177(4)
84.411(3)
72.261(3)
3822.1(2)
2
25.571(3)
α [°]
β [°]
95.493(2)
106.939(12)
γ [°]
V [Å3]
2558.05(16)
2
3822.1(2)
4
6986.8(14)
8
Z
λ [Å]
0.71073
1.476
0.18ϫ0.16ϫ0.14
100
0.71073
1.464
0.16ϫ0.07ϫ0.05
296
0.71073
1.417
0.34ϫ0.20ϫ0.18
100
0.71073
1.485
0.06ϫ0.06ϫ0.06
100
ρcalcd. [gcm–3]
Crystal size [mm]
T [K]
µ [cm–1]
Flack parameter
81.4
59.7
73.9
86.6
–0.01(3)
0.0359
0.0891
1.051
[a]
R1
0.0413
0.1025
0.997
0.0539
0.1631
1.093
0.0522
0.1388
1.058
[b]
wR2
GOF[c]
2 2
[a] R1 = Σ||Fo| – |Fc||/Σ|Fo|. [b] wR2 = {Σ[w(Fo2 – Fc2)2]/Σ[w(Fo ) ]}1/2. [c] GOF = {Σ[w(Fo2 – Fc2)2]/(n – p)}1/2, where n is the number of reflections
and p is the total number of parameters refined.
incubation, all media were replaced by 150 µL/well RPMI 1640 me-
dium (supplemented with 10% heat-inactivated fetal bovine serum
and 2 m -glutamine) plus 20 µL/well MTT solution in phos-
phate-buffered saline (5 mg/mL). After incubation for 4 h, the me-
dium/MTT mixtures were removed and the formazan crystals
formed by the mitochondrial dehydrogenase activity of vital cells
were dissolved in 150 µL of dmso per well. Optical densities at
550 nm were measured with a microplate reader (Tecan Spectra
Classic), using a reference wavelength of 690 nm to correct for un-
specific absorption. The quantity of vital cells was expressed in
terms of T/C values by comparison with untreated control micro-
cultures, and 50% inhibitory concentrations (IC50) were calculated
from concentration-effect curves by interpolation. In order to cor-
rect for differences in growth kinetics in the pH-dependency experi-
ments, concentration-effect curves were transformed by taking the
optical densities of separate MTT-stained control microcultures at
the beginning of drug exposure as zero values, and GI50 concentra-
tions were calculated instead of IC50 values. Evaluation is based on
the mean of at least three independent experiments, each compris-
ing at least six microcultures per concentration level.
[1]
[2]
[3]
[4]
T. S. Lobana, S. Khanna, R. J. Butcher, A. D. Hunter, M.
Zeller, Polyhedron 2006, 25, 2755–2763.
F. Basuli, M. Ruf, C. G. Pierpont, S. Bhattacharya, Inorg.
Chem. 1998, 37, 6113–6116.
G. Dessy, V. Fares, L. Scaramuzza, A. A. G. Tomlinson, G.
De Munno, J. Chem. Soc., Dalton Trans. 1978, 1549–1554.
D. Kovala-Demertzi, P. N. Yadav, J. Wiecek, S. Skoulika, T.
Varadinova, M. A. Demertzis, J. Inorg. Biochem. 2006, 100,
1558–1567.
D. X. West, S. B. Padhye, P. B. Sonawane, Struct. Bonding 1991,
76, 1–50.
E. C. Moore, A. C. Sartorelli, Pharm. Ther. 1984, 24, 439–447.
E. C. Moore, A. C. Sartorelli, in Inhibitors of Ribonucleoside
Diphosphate Reductase Activity (Eds.: J. G. Cory, A. H. Cory),
Pergamon Press, Oxford, 1989, pp. 203–215.
E. C. Moore, A. C. Sartorelli, Pharm. Ther. 1984, 24, 439–447.
R. W. Brockman, J. R. Thomson, M. J. Bell, H. E. Skipper,
Cancer Res. 1956, 16, 167–170.
L. Feun, M. Modiano, K. Lee, J. Mao, A. Marini, N. Savaraj,
P. Plezia, B. Almassian, E. Colacino, J. Fischer, S. MacDonald,
Cancer Chemother. Pharmacol. 2002, 50, 223–229.
J. Murren, M. Modiano, C. Clairmont, P. Lambert, N. Savaraj,
T. Doyle, M. Sznol, Clin. Cancer Res. 2003, 9, 4092–4100.
A. Gómez Quiroga, C. Navarro Raninger, Coord. Chem. Rev.
2004, 248, 119–133.
N. Farrell, Coord. Chem. Rev. 2002, 232, 1–4.
U. Abram, K. Ortner, R. Gust, K. Sommer, J. Chem. Soc.,
Dalton Trans. 2000, 735–744.
M. Belicchi-Ferrari, F. Bisceglie, C. Casoli, S. Durot, I. Mor-
genstern-Badara, G. Pelosi, E. Pilotti, S. Pinelli, P. Tarasconi,
J. Med. Chem. 2005, 48, 1671–1675.
V. B. Arion, M. A. Jakupec, M. Galanski, P. Unfried, B. K.
Keppler, J. Inorg. Biochem. 2002, 91, 298–305.
J. Patole, S. Padhye, M. S. Moodbidri, N. Shirsat, Eur. J. Med.
Chem. 2005, 40, 1052–1055.
L. Otero, M. Vieites, L. Boiani, A. Denicola, C. Rigol, L.
Opazo, C. Olea-Azar, J. D. Maya, A. Morello, L. Krauth-Sie-
gel, O. E. Piro, E. Castellano, M. González, D. Gambino, H.
Cerecetto, J. Med. Chem. 2006, 49, 3322–3331.
[5]
[6]
[7]
[8]
[9]
[10]
Supporting Information (see footnote on the first page of this arti-
cle): Crystal packing of complexes 1 and 2, the molecular structure
of 3, 31P NMR spectra of 4, UV/Vis spectra of 4 at different pH
values and IR spectra of 1, 2 and 4.
[11]
[12]
[13]
[14]
Acknowledgments
[15]
The authors would like to thank the Austrian Science Fund
(FWF), the Austrian Council for Research and Technology Devel-
opment, the Austrian Research Promotion Agency (FFG), the
European Cooperation in the Field of Scientific and Technical Re-
search (COST) and the Faustus Forschung Austria Translational
Drug Development AG, Vienna, Austria for financial support. We
also thank Dr. Hanspeter Kählig for NMR titrations and Mag.
Raffael Schuecker for helpful discussions, as well as Mag. Michael
Grössl for ESI-MS measurements.
[16]
[17]
[18]
Eur. J. Inorg. Chem. 2007, 2870–2878
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2877

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[19]
[38] C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchi-
etto, G. Laurenczy, T. J. Geldbach, G. Sava, P. J. Dyson, J.
Med. Chem. 2005, 48, 4161–4171.
U. K. Mazumder, M. Gupta, S. S. Karki, S. Bhattacharya, S.
Rathinasamy, S. Thangavel, Chem. Pharm. Bull. 2004, 52, 178–
185.
[39] K. J. Fisher, E. C. Alyea, N. A. Shahnazarian, Phosphorus, Sul-
fur, Silicon Relat. Elem. 1990, 48, 37–40.
[40] S-M. Valiahdi, M. A. Jakupec, B. K. Keppler, unpublished re-
sults.
[20]
[21]
[22]
[23]
F. Bregant, S. Pacor, S. Ghosh, S. K. Chattopadhyah, G. Sava,
Anticancer Res. 1993, 13, 1011–1017.
G. Sava, R. Gagliardi, A. Bergamo, E. Alessio, G. Mestroni,
Anticancer Res. 1999, 19, 969–972.
M. Galanski, V. B. Arion, M. A. Jakupec, B. K. Keppler, Curr.
Pharm. Des. 2003, 9, 2078–2089.
C. R. Kowol, R. Berger, R. Eichinger, A. Roller, M. A. Jaku-
pec, P. P. Schmidt, V. B. Arion, B. K. Keppler, J. Med. Chem.
2007, 50, 1254–1265.
Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. New-
ton, C. E. Anson, A. K. Powell, J. Inorg. Biochem. 2003, 95,
306–314.
M. Maji, S. Ghosh, S. K. Chattopadhyay, T. C. W. Mak, Inorg.
Chem. 1997, 36, 2938–2943.
M. Maji, M. Chatterjee, S. Ghosh, S. Kumar Chattopadhyay,
B. M. Wu, T. C. W. Mak, J. Chem. Soc., Dalton Trans. 1999,
135–140.
F. Basuli, S. M. Peng, S. Bhattacharya, Inorg. Chem. 2000, 39,
1120–1127.
[41] M. Stubbs, P. M. J. McSheehy, J. R. Griffiths, C. L. Bashford,
Mol. Med. Today 2000, 6, 15–19.
[42] R. A. Gattenby, R. J. Gillies, Nat. Rev. Cancer 2004, 4, 891–
899.
[43] L. E. Gerweck, S. V. Kozin, S. J. Stocks, Br. J. Cancer 1999, 79,
838–842.
[44] B. P. Mahoney, N. Raghunand, B. Baggett, R. J. Gillies, Bi-
ochem. Pharmacol. 2003, 66, 1207–1218.
[45] L. E. Gerweck, S. Vijayappa, S. Kozin, Mol. Cancer Ther. 2006,
5, 1275–1279.
[24]
[25]
[26]
[46] S. V. Kozin, P. Shkarin, L. E. Gerweck, Cancer Res. 2001, 61,
4740–4743.
[47] C. M. Laurencot, K. A. Kennedy, Oncol. Res. 1995, 7, 371–
379.
[27]
[48] L. E. Gerweck, K. Seetharaman, Cancer Res. 1996, 56, 1194–
1198.
[28] F. Basuli, S. M. Peng, S. Bhattacharya, Inorg. Chem. 2001, 40,
[49] L. F. Tietze, T. Feuerstein, Curr. Pharm. Des. 2003, 9, 2155–
2175.
1126–1133.
[29] a) W. H. Ang, P. J. Dyson, Eur. J. Inorg. Chem. 2006, 4003– [50] C. S. Allardyce, P. J. Dyson, D. J. Ellis, S. L. Heath, Chem.
4018; b) A. D. Phillips, L. Gonsalvi, A. Romerosa, F. Vizza,
M. Peruzzini, Coord. Chem. Rev. 2004, 248, 955–993; c) W. H.
Ang, E. Daldini, C. Scolaro, R. Scopelliti, L. Juillerat-
Jeannerat, P. J. Dyson, Inorg. Chem. 2006, 45, 9006–9013; d) C.
Scolaro, T. J. Geldbach, S. Rochat, A. Dorcier, C. Gossens, A.
Bergamo, M. Cocchietto, I. Tavernelli, G. Sava, U. Rothlis-
berger, P. J. Dyson, Organometallics 2006, 25, 756–765; e) B.
Serli, E. Zangrando, T. Gianferrata, C. Scolaro, P. J. Dyson, A.
Bergamo, E. Alessio, Eur. J. Inorg. Chem. 2005, 3423–3434; f)
D. N. Akbayeva, L. Gonsalvi, W. Oberhauser, M. Peruzzini, F.
Vizza, P. Brügeller, A. Ramerosa, G. Sava, A. Bergamo, Chem.
Commun. 2003, 264–265.
Commun. 2001, 1396–1397.
[51] D. L. Klayman, J. P. Scovill, J. F. Bartosevich, C. J. Mason, J.
Med. Chem. 1979, 22, 1367–1373.
[52] D. J. Daigle, Inorg. Synth. 1998, 32, 40–45.
[53] I. P. Evans, A. Spencer, G. Wilkinson, J. Chem. Soc., Dalton
Trans. 1973, 204–209.
[54] W. C. Barette Jr, H. W. Johnson Jr, D. T. Sawyer, Anal. Chem.
1984, 56, 1890–1898.
[55] Z. Szakacs, G. Hägele, R. Tyka, Anal. Chim. Acta 2004, 522,
247–258.
[56] KALEIDAGRAPH, version 4.03, Synergy Software, Reading,
PA, 2006.
[30] D. J. Darensbourg, F. Joo, M. Kannisto, A. Katho, J. H. Rei- [57] A. Krezel, W. Bal, J. Inorg. Biochem. 2004, 98, 161–166.
benspies, D. J. Daigle, Inorg. Chem. 1994, 33, 200–208.
[31] R. Raveendran, S. Pal, Polyhedron 2005, 24, 57–63.
[32] B. J. Frost, S. B. Miller, K. O. Rove, D. M. Pearson, J. D. Kori-
nek, J. L. Harkreader, C. A. Mebi, J. Shearer, Inorg. Chim. Acta
2006, 359, 283–288.
[33] A. Romerosa, T. Campos-Malpartida, C. Lidrissi, M. Saoud,
M. Serrano-Ruiz, M. Peruzzini, J. A. Garrido-Cárdenas, F.
García-Maroto, Inorg. Chem. 2006, 45, 1289–1298.
[34] B. J. Frost, C. A. Mebi, Organometallics 2004, 23, 5317–5323.
[35] A. Sreekanth, H.-K. Fun, M. R. P. Kurup, J. Mol. Struct. 2005,
737, 61–67.
[58] M. R. Pressprich, J. Chambers, SAINT + Integration Engine,
Program for Crystal Structure Integration, Bruker Analytical
X-ray systems, Madison, USA, 2004.
[59] G. M. Sheldrick, SHELXS-97, Program for Crystal Structure
Solution, University of Göttingen, Germany, 1997.
[60] G. M. Sheldrick, SHELXL-97, Program for Crystal Structure
Refinement, University of Göttingen, Germany, 1997.
[61] C. K. Johnson, Rep. ORLN-5138, Oak Ridge National Labo-
ratory, Oak Ridge, TN, 1976.
[62] International Tables for X-ray Crystallography (Ed.: A. J. C.
Wilson), Kluwer Academic Press, Dordrecht, The Netherlands,
1992, vol. C, Tables 4.2.6.8 and 6.1.1.4.
[36] C. R. Kowol, V. B. Arion, B. K. Keppler, unpublished results.
[37] A. K. Nandi, S. Chaudhuri, S. K. Mazumdar, S. Ghosh, J.
Chem. Soc., Perkin Trans. 2 1984, 1729–1733.
Received: December 18, 2006
Published Online: May 22, 2007
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