Synthesis, characterization, and in vitro evaluation of a potentially selective anticancer, mixed-metal [ruthenium(III)-platinum(II)] trinuclear complex

Article abstract of DOI:10.1021/ic062419h

A new type of mixed-metal trinuclear complex containing platinum(II) and ruthenium(III) fragments that resemble both cisplatin and NAMI-A has been synthesized and characterized by IR, ¹H NMR, elemental analysis, and X-ray crystallography. The water-soluble compound Na₂{trans,cis, trans-[Ru^IIICl₄(DMSO-S)(μ-pyz)]₂Pt ^IICl₂} (AH-197, pyz = pyrazine) was assessed for its effects on DNA mobility and toxicity against human cancer cell lines. When compared to cisplatin and KP-1019 (which structurally resembles NAMI-A), IC ₅₀ results showed that AH-197 had an intermediate toxicity. When this data was coupled with a subsequent COMPARE evaluation (standard COMPARE queries resulted in insignificant correlation coefficients (<0.70) while very low COMPARE correlation coefficients were found in the matrix queries as well), AH-197 yielded a correlation coefficient of 0.19 when compared to cisplatin and 0.25 when compared to KP1019 indicating that AH-197 has a unique behavior.

Full text of DOI:10.1021/ic062419h

Inorg. Chem. 2008, 47, 274−280
Synthesis, Characterization, and in Vitro Evaluation of a Potentially
Selective Anticancer, Mixed-Metal [Ruthenium(III)
Complex
−Platinum(II)] Trinuclear
Amnon Herman,† Joseph M. Tanski,‡ Michael F. Tibbetts,† and Craig M. Anderson*,†
Departments of Chemistry, Bard College, P.O. Box 5000, Annandale-on-Hudson,
New York 12504-5000, and Vassar College, Poughkeepsie, New York 12604
Received December 17, 2006
A new type of mixed-metal trinuclear complex containing platinum(II) and ruthenium(III) fragments that resemble
1
both cisplatin and NAMI-A has been synthesized and characterized by IR, H NMR, elemental analysis, and X-ray
crystallography. The water-soluble compound Na2
pyrazine) was assessed for its effects on DNA mobility and toxicity against human cancer cell lines. When
{ µ } (AH-197, pyz
trans,cis,trans-[RuIIICl4(DMSO-S)( -pyz)]2PtIICl2
)
compared to cisplatin and KP-1019 (which structurally resembles NAMI-A), IC50 results showed that AH-197 had
an intermediate toxicity. When this data was coupled with a subsequent COMPARE evaluation (standard COMPARE
queries resulted in insignificant correlation coefficients (<0.70) while very low COMPARE correlation coefficients
were found in the matrix queries as well), AH-197 yielded a correlation coefficient of 0.19 when compared to
cisplatin and 0.25 when compared to KP1019 indicating that AH-197 has a unique behavior.
Introduction
have been of initial interest and subsequent study.3,6,7,8
Although different platinum-containing complexes have been
successfully tested, most of them have been proven to have
the same or only slightly better efficacy than cisplatin.3
However, some platinum complexes, such as carboplatin and
oxaliplatin, have shown to be active against many types of
cancer.9,10
Multinuclear platinum complexes, particularly trinuclear,
are one specific class of compounds that have been recently
synthesized and have shown to bind to DNA in a manner
different from that of cisplatin.11-17 These compounds which
With the discovery of the anticancer properties of cis-
diamminedichloroplatinum(II), cisplatin (Figure 1),1,2 the
research of metallopharmaceuticals increased dramatically.
Cisplatin is still a widely used anticancer drug and is effective
in treating a variety of cancers.3 Cisplatin’s cytotoxicity is
achieved by forming, mainly, 1,2-intrastrand d(GpG) DNA
cross-links. The covalent cross-links cause significant distor-
tion of helical structure and result in inhibition of DNA
replication and transcription.4,5 The clinical success of
cisplatin has proved to be limited due to significant side
effects and resistance that cause relapse.3 Therefore, much
interest has focused on developing new chemotherapeutic
metal complexes with improved properties. Due to the
comprehensive studies of cisplatin and insights into its
mechanism of interaction with DNA, platinum complexes
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* To whom correspondence should be addressed. E-mail: canderso@
bard.edu. Phone: 845-758-7293.
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J. I.; Rose, P. G. Gynecol. Oncol. 2006, 103, 523-526.
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† Bard College.
‡ Vassar College.
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274 Inorganic Chemistry, Vol. 47, No. 1, 2008
10.1021/ic062419h CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/07/2007

A Potentially SelectiWe Anticancer Complex
Figure 1. Structures of platinum(II) and ruthenium(III) chemotherapeutic complexes.
contain two or more platinum centers can have both cis and/
or trans configurations. BBR3464 (Figure 1) is a representa-
tive compound of the polymetallic group which has entered
clinical trials and has shown to be potent against pancreatic,
lung, and melanoma cancers.18,19 It was also found that
BBR3464 binds to DNA by forming both intrastrand and
interstrand cross-links. Mechanistic studies have shown that
the interstrand cross-links, which account for 20% of the
cross-link adducts formed by BBR3464, are most important
to its anticancer behavior.3,20
levels at tumor sites, ruthenium’s “activation by reduction”
process makes it very selective as the metal complex may
accumulate in these hypoxic environments.24 Only a small
number of ruthenium(III) complexes have been found to be
effective with the most prominent examples being the
following: Na[trans-RuCl4(DMSO)(Im)],25 NAMI; its more
stable imidazolium analogue [ImH][trans-RuCl4(DMSO)-
(Im)],26 NAMI-A; the closely resembled indazole compound
[IndH][trans-RuCl4(Ind)2],27 KP1019 (Figure 1).
NAMI-A, new antitumor metastasis inhibitor-series A,
shows high selectivity for solid tumor metastases28,29 and low
toxicity at pharmacologically active doses and was found to
effectivelyinterferewithcellcycleregulationandangiogenesis.30-32
These properties have made NAMI-A the first ruthenium-
(III) compound to successfully complete phase I clinical
trials.33
For the past two decades much work has focused on the
synthesis and pharmacological evaluation of new compounds
containing metal centers different from platinum.21-23
A
major reason for this can be attributed to the reduced side
effects of these complexes due to their lower systemic
toxicity. Complexes with ruthenium(III) centers have been
synthesized and have demonstrated antitumor activity as well
as remarkable antimetastatic behavior.22,23 It is speculated
that ruthenium(III) complexes are reduced in vivo to the more
labile ruthenium(II), and this is a major reason for its
biological activity.21-23 Due to lower oxygen and lower pH
In this paper we report the successful modular synthesis
of heterotrinuclear complexes that combine the classes of
compounds described above. These compounds have a
platinum(II) center that resembles cisplatin as well as two
ruthenium(III) centers attached to the platinum by bridging
dinitrogen ligands that resemble NAMI. The design of these
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Inorganic Chemistry, Vol. 47, No. 1, 2008 275

Herman et al.
Complexes with the TPP (AH-166), TBA (AH-171), and
imidizolium (AH-235) cations were prepared similarly or by simple
metathesis reactions (see Supporting Information).
complexes has been done to achieve both efficacy and
selectivity with the objective of obtaining complexes with
properties of both types of metal complexes. The new type
of complex may then be potent against both neoplastic and
metastatic cancers. In addition, the ruthenium(III) centers are
susceptible to reduction to the more labile ruthenium(II)
which may weaken the ruthenium(II)-bridging ligand
bonds.34 These trinuclear species may also achieve better
selectivity due to the transferrin cycle mediated accumulation
of ruthenium(III) compounds in hypoxic environments and
subsequent “activation by reduction” of the ruthenium(III)
centers in these conditions.23
[TPP]2 {trans,trans,trans-[RuI I I Cl4 (DMSO-S)(µ-
pyz)]2PtIICl2}, AH-229. A solution of AH-166 was left in acetone
solution at 4 °C for 1 month. Dark red crystals appeared very
slowly. The crystals were then collected by filtration and washed
with ether. Alternatively, [TPP]Cl was mixed with 0.5 equiv of
AH-197 and AH-229 was allowed to slowly crystallize over a period
of several weeks at -15 °C. Selected IR (νmax/cm-1): 1707 m (pyz),
1106 s (Ru-DMSO-S), 521 vs, br (Pt-N), 421 m (Ru-S), 328 s,
307 sh (Pt/Ru-Cl). 1H NMR (CD3CN): -1.0 br (pyz H2,6), -12.9
br (DMSO-S).
The water-soluble trinuclear complex AH-197 was evalu-
ated for its in vitro activity with linearized plasmid DNA as
well as against the NCI’s 60 human tumor cell line screening
procedure and its algorithm, COMPARE.
DNA Migration Studies. The plasmid pBluescript SK+ was
purified from Escherichia coli using a QIAGEN plasmid midi kit
as described by the manufacturer (QIAGEN Inc.). Plasmid DNA
was linearized with the restriction endonuclease EcoRI and purified
from the reaction with a QIAquick PCR Purification Kit (QIAGEN
Inc.) eluted in deionized water. The procedure used in these studies
is a modification of that described by Stellwagen.37 Briefly, 0.5 µg
of linearized pBluescript SK+ was incubated with varying con-
centrations of the metal compounds in phosphate buffered saline,
PBS (3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 150 mM NaCl, pH
6.4), in a total volume of 50 µL at 37 °C for 24 h. Following
incubation, 20 µL samples were analyzed by agarose gel electro-
phoresis (0.9% agarose, 1X TBE (89 mM Tris base, 89 mM boric
acid, 3.1 mM EDTA)). Gels were stained with 5 µg/mL ethidium
bromide in 1X TBE for 45 min and photographed with UV
illumination.
Experimental Section
cis-PtCl2(pyz)2 and [(DMSO)2H][trans-RuCl4(DMSO)2] were
synthesized by following literature preparations.35,25 The sodium,
tetrabutylammonium (TBA), and tetraphenylphosphonium (TPP)
salts of the [trans-RuCl4(DMSO)2]- anion were obtained from
simple metathesis reactions.36 Ethanol (200 proof) was purchased
from Pharmaco Products Inc. All other reagents and solvents were
purchased from Sigma Aldrich Inc. IR spectra were recorded in
the solid state on a Nicolet 4700 FTIR spectrometer between 4000
1
and 250 cm-1. H NMR spectra were measured in D2O, acetone-
d6, CDCl3, and CD3CN on a Varian Gemini 300 MHZ spectrometer.
The solvent peaks at 4.80, 2.05, 7.24, and 1.96 ppm for D2O, C2D6O
(acetone), CDCl3, and CD3CN, respectively, were used as internal
standards for the 1H NMR spectra. UV/vis were run on an Agilent
8453 diode array instrument. Plasmid’s miniprep extractions as well
as purification kits were purchased from Qiagen Inc. IC50 assays
were conducted as part of the in vitro anticancer screening offered
by the Developmental Therapeutic Program in the National Cancer
Institute (Bethesda, MD). Detailed experimental procedures for
these assays are found online (http://dtp.nci.nih.gov/branches/btb/
ivclsp.html). Spectral abbreviations used below: br (broad); m
(medium); s (strong); sh (shoulder); vbr (very broad); w (weak).
For NMR labels see Scheme 1.
Na2{trans,cis,trans-[RuIIICl4(DMSO-S)(µ-pyz)]2PtIICl2}, AH-
197. A 0.21 g amount of Na[trans-RuCl4(DMSO)2] (0.49 mmol)
was partially dissolved in 30 mL of acetone, and 0.10 g of cis-
PtCl2(pyz)2 (0.23 mmol) was added in situ to the orange suspension.
The mixture was then refluxed for 1 h, during which all of the
reactants dissolved and the solution turned dark red. The solution
was cooled to room temperature. An orange precipitate was afforded
by the addition of 10 mL of diethyl ether that was collected by
filtration, washed with diethyl ether, and vacuum-dried. A yield of
0.17 g obtained (65%). Selected IR (νmax/cm-1): 1699 m, 1614 m,
br (pyz), 1077 s (Ru-DMSO-S), 515 m (Pt-N), 429 m (Ru-S),
331 s (Pt/Ru-Cl). 1H NMR (D2O): -14.0 br (DMSO-S), -2.2 br
(pyz H2,6). Anal. Calcd for C12H20Cl10N4Na2O2S2Ru2Pt‚1/2CH3-
COCH3: C, 14.1; H, 2.01; N 4.89. Found: C, 13.7; H, 2.08; N,
4.37. Selected UV/vis (PBS): λmax/nm 393 (ꢀ/L mol-1 cm-1 11
900); immediately after reduction 478 (11 200).
NCI’s 60 Cell in Vitro Cancer Screenings. AH-197 was
screened against the 60 human cancer cell lines (NCI-60) used by
the National Cancer Institute’s Developmental Therapeutics Pro-
gram (detailed procedure found at http://dtp.nci.nih.gov/branches/
btb/ivclsp.html). However, AH-197 was dissolved in water, not
DMSO, prior to dilution. The pH of the medium used in the
procedure was 7.2. Additional evaluation of data from screenings
of AH-197 (NSC: 742192) was conducted using the pattern-
recognition algorithm COMPARE (http://itbwork.nci.nih.gov).38
Standard COMPARE queries were performed against all synthetic
compounds in the NCI’s Developmental Therapeutic Program
Database (http://dtp.nci.nih.gov/docs/dtp_search.html) to obtain
correlation coefficients. Furthermore, matrix COMPARE of our
compound was performed against screening data from cisplatin
(NSC: 119875) and KP1019 (NSC: 666158).
X-ray Structure Determinations. X-ray diffraction data were
collected on a Bruker SMART APEX 2 CCD platform diffracto-
meter (Mo KR (λ ) 0.710 73 Å)). Suitable crystals were mounted
in a nylon loop with Paratone-N cryoprotectant oil. The structures
were solved using direct methods and standard difference map
techniques and were refined by full-matrix least-squares procedures
on F2 with SHELXTL (version 6.14).39 All non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were included in
calculated positions and were refined using a riding model. Crystal
data and refinement details are presented in Table 1.
AH-197. Single crystals of AH-197 were grown from acetone
at 4 °C. Several crystals were screened and found to be multiply
twinned. X-ray diffraction data were collected at 115 K on a crystal
(34) Clarke, M. J.; Zhu, F.; Frasca, D. R. Chem. ReV. 1999, 99, 2511-
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(35) Foulds, G.; Thoronton, D. Spectrochim. Acta. 1981, 37, 917-921.
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Geremia, S.; Calligaris, M. Inorg. Chem. 1995, 34, 4716. (b) Anderson,
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pp 1-53.
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(39) Sheldrick, G. M. SHELXTL, An integrated system for solVing, refining
and displaying crystal structures from diffraction data; University of
Go¨ttingen: Go¨ttingen, Germany, 1981.
276 Inorganic Chemistry, Vol. 47, No. 1, 2008

A Potentially SelectiWe Anticancer Complex
Table 2. Selected Bond Lengths (Å) and Angles (deg)a for AH-197
Pt-N(1)
Pt-N(3)
2.01(1)
2.03(1)
2.290(3)
2.293(3)
2.16(2)
2.305(5)
2.2(3)
2.12(1)
2.281(4)
2.347(7)
N(1)-Pt-N(3)
N(1)-Pt-Cl(5)
89.8(4)
90.2(3)
86.4(3)
93.7(1)
175.0(4)
88(6)
92(5)
178.0(3)
Pt-Cl(5)
N(3)-Pt-Cl(6)
Pt-Cl(6)
Cl(5)-Pt-Cl(6)
Ru(1)-N(2)
Ru(1)-S(1)
Ru(1)-Cl av
Ru(2)-N(4)
Ru(2)-S(2)
Ru(2)-Cl av
N(2)-Ru(1)-S(1)
N(2)-Ru(1)-Cl av
S(1)-Ru(1)-Cl av
N(4)-Ru(2)-S(2)
N(4)-Ru(2)-Cl av
S(2)-Ru(2)-Cl av
89(1)
91(1)
a Estimated standard deviations in the least significant figure are given
in parentheses.
Figure 2. ORTEP diagram of the asymmetric unit of cis-AH-197 (20%
ellipsoids; hydrogen atoms removed for clarity).
Table 1. Crystal Data, Data Collection, and Refinement Parameters for
AH-197 and AH-229
param
formula
cis-AH-197
trans-AH-229
C12H20Cl10N4O2Na2
S2Ru2Pt‚C3H6O
plate, orange
0.13 × 0.10 × 0.03
triclinic
C60H60Cl10N4O2
P2S2Ru2Pt‚2C3H6O
needle, yellow
0.32 × 0.07 × 0.04
triclinic
habit, color
size, mm
lattice type
space group
a, Å
P1h
P1h
12.526(2)
12.667(2)
17.570(3)
106.517(2)
95.159(2)
14.6756(7)
15.3198(7)
17.0886(8)
76.981(1)
73.257(1)
84.799(1)
b, Å
c, Å
Figure 3. Molecular structures of the two independent molecules of trans-
AH-229 (20% ellipsoids; hydrogen atoms removed for clarity).
R, deg
â, deg
γ, deg
115.745(2)
2332.9(7)
Table 3. Selected Bond Lengths (Å) and Angles (deg)a for AH-229
V, Å3
3583.2(3)
Z
fw
2
2
Pt(1)-N(1)
2.013(3)
2.300(1)
2.101(3)
2.289(1)
2.349(4)
2.024(3)
2.289(1)
N(1)-Pt(1)-Cl(1)
89.09(9)
90.91(9)
178.52(9)
88.8(3)
91(1)
90.95(9)
89.05(9)
178.51(9)
89(1)
1172.23
1.669
4.331
1116
1.68-28.17
-16 e h e 16, -16
k e 16, -23
e l e 22
1863.07
1.727
2.889
1852
1.45-28.52
-19 e h e 19, -19
e k e 19, -22
e l e 22
Pt(1)-Cl(1)
Ru(1)-N(2)
Ru(1)-S(1)
Ru(1)-Cl av
Pt(1A)-N(1A)
Pt(1A)-Cl(1A)
N(1#2)-Pt(1)-Cl(1)
N(2)-Ru(1)-S(1)
Dc, g cm-3
µ, mm-1
F(000)
θ range, deg
index ranges
N(2)-Ru(1)-Cl av
S(1)-Ru(1)-Cl av
N(1A)-Pt(1A)-Cl(1A)
N(1A#2)-Pt(1A)-Cl(1A)
N(2A)-Ru(1A)-S(1A)
N(2A)-Ru(1A)-Cl av
Ru(1A)-N(2A) 2.103(3)
Ru(1A)-S(1A)
Ru(1A)-Cl av
2.289(1)
reflcns collcd
unique reflcns
completeness
abs corr
50 545
42 414
2.352(17) S(1A)-Ru(1A)-Cl av
91(1)
10 350 (Rint ) 0.0928)
90.4 (to Φ ) 28.17°)
empirical
16 490 (Rint ) 0.0497)
90.6 (to Φ ) 28.52°)
empirical
a Estimated standard deviations in the least significant figure are given
in parentheses.
max, min transm
data, restraints,
params
R1, wR2 (I > 2σ(I))
R1, wR2 (all data)
goodness-of-fit
(on F2)
0.8810, 0.6029
33 000/33/350
0.9056, 0.4583
16 490/0/793
two of which have been located and refined and two of which are
disordered. The atoms of the disordered acetone molecules were
included in the refinement as a diffuse contribution to the scattering
using the program SQUEEZE in the PLATON suite of programs.40
ORTEP diagrams of the two independent molecules are shown in
Figure 3. Selected bond length and angles are given in Table 3.
0.1045, 0.2570
0.1776, 0.3003
1.012
0.0377, 0.0620
0.0592, 0.0671
1.009
largest diff peak,
8.659, -5.649
1.300, -0.793
hole, e Å-3
Results and Discussion
that was determined with CELL_NOW to contain two twin
domains. Both domains were integrated with SAINT using the two-
component orientation matrix produced by CELL_NOW. The data
were scaled and absorption corrected with TWINABS. The initial
solution was refined with single component data for the stronger
domain before final refinement with HKLF 5 format data for both
twin domains as produced by TWINABS. An ORTEP diagram of
the molecule is shown in Figure 2. Selected bond length and angles
are given in Table 2.
AH-229. Suitable X-ray-quality single crystals of AH-229 were
grown from acetone at 4 °C. X-ray diffraction data were collected
at 125 K. The asymmetric unit contains two unique half-molecules,
one from each of the independent trinuclear molecules of AH-229,
each platinum atom residing on an inversion center. The unit cell
contains four molecules of acetone solvent of crystallization,
Synthesis and Characterization of the Complexes.
Ruthenium(III) precursors were synthesized by modifications
of those reported by Allesio et al.25 [(DMSO)2H][trans-
RuCl4(DMSO)2], 1, was the initial ruthenium(III) compound
from which the other ruthenium(III) compounds were
derived. Complexes with different cations (Na, 2; TPP, 3;
TBA, 4) resulted from simple metathesis reactions of 1 with
the chloride salts of the appropriate cations (NaCl, TPP-Cl,
TBA-Cl) in appropriate molar ratios. The platinium(II)
precursor was synthesized according to an earlier report by
Foulds et al.35
(40) Spek, A. L. Acta Crystallogr. 1990, A46, C34.
Inorganic Chemistry, Vol. 47, No. 1, 2008 277

Herman et al.
Scheme 1. Synthesis of the Complexes
Synthesis of the trinuclear complex AH-197 was achieved
by refluxing a mixture of the ruthenium and platinum
precursors listed above, in a 2.1:1 ratio, respectively. The
initial insoluble nature of the platinum precursor was used
as indication for the extent of the reaction; as the platinum
complex dissolved and the solution reached homogeneity,
the reaction was assumed to be complete. This resulted in
the bridging of the pyrazine ligand between the platinum
center and the ruthenium center as a result of a substitution
of the pyrazine for the DMSO ligand on the ruthenium. The
general route is outlined in Scheme 1. The trans isomer of
the trinuclear species was obtained from aged solutions of
AH-197 in acetone with the addition of the tetraphenylphos-
phonium cation or from aged solutions of the cis-TPP isomer,
AH-166. Cis chloro/pyridine platinum complexes are known
to slowly isomerize to the trans configuration.41,42 In addition,
computation studies using Gaussian ’0343 show the trans
isomer of the dianion to be slightly lower in energy than the
cis dianion (see Supporting Information).
The vibrational spectra for cis-PtCl2(pyz)2 and [(DMSO)2H]-
[trans-RuCl4(DMSO)2] have been reported,35,25 and the
assignments on the platinum(II) and ruthenium(III) fragments
of the new complexes are based on these studies. Infrared
spectroscopy is diagnostic of the binding mode (terminal or
bridging) of coordinated pyrazines.44 Monocoordinated pyra-
zine compounds (terminal) are characterized by a “breathing
mode” which gives a sharp peak at about 1590 cm-1.45
That peak is absent if pyrazine acts as a bridging ligand
as in complex AH-197. Two new peaks at 1699 and 1615
cm-1 are present in the spectrum of AH-197. The trans
complex shows only one peak for the pyrazine mode at 1707
cm-1.
The NMR spectrum of AH-197 (Supporting Information)
shows peaks that are characteristic of complexes containing
paramagnetic ruthenium(III).46,47 Protons close to the para-
magnetic center have large line widths (often some peaks
are too wide to be observable) and are shifted greatly upfield.
The peak at -14.0 ppm integrating to 12 protons is assigned
to the coordinated DMSO while the peak at -2.2 ppm
integrating to 4 protons is assigned to the protons on the
pyrazine ring closest to the platinum center, thus furthest
from the paramagnetic ruthenium(III) center. The protons
on the pyrazine ring closest to the ruthenium center are not
observed (for labeling, see Scheme 1). The proton NMR of
the trans complex, AH-229, recorded in CD3CN showed
peaks at -13.2 ppm for the DMSO methyl protons and -1.0
ppm for the pyrazine protons furthest from the ruthenium
as well as the phenyl peaks associated with the TPP cation.
The trinuclear complex AH-197 has its ruthenium(III)
centers reduced to ruthenium(II) upon the addition of 2 equiv
of the biological reductant, ascorbic acid. This was witnessed
in both NMR and UV/vis experiments. Upon addition of
ascorbic acid to a solution of AH-197, an immediate reaction
is seen as the color changes from orange to red. In the UV/
vis spectrum, the lowest energy peak at 393 nm shifted to
480 nm upon addition of ascorbic acid as is characteristic
of reduction of ruthenium(III) to ruthenium(II).29,45 In the
NMR spectrum, upon addition of ascorbic acid, there is an
immediate and complete disappearance of the peaks associ-
ated with the paramagnetic ruthenium(III) species, AH-197,
indicating a quantitative reduction of the ruthenium(III). The
disappearance of the paramagnetic upfield peaks is ac-
companied with the appearance of several new, overlapping
downfield peaks between 8.6 and 9.6 ppm and 3.4 and 3.6
ppm characteristic of a mixture of a number of possible
species including ruthenium(II)/pyrazine(bridging or terminal)/
DMSO complexes.29,45
(41) Rochon, F. D.; Kong, P. C. Can. J. Chem. 1979, 57, 526-529.
(42) Rochon, F. D.; Kong, P. C.; Melanson, R. Can. J. Chem. 1981, 59,
195-198.
(43) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin,
K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone,
V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G.
A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,
H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.
E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J.
W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari,
K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian ‘03; Gaussian, Inc.: Wallingford CT, 2004.
The structures of the cis trinuclear complex and of the
trans trinuclear complex were determined. The bond lengths
(45) Allesio, E.; Mestroni, G.; Geremia, S.; Calligaris, M.; Iengo, E. Dalton
Trans. 1999, 19, 3361-3371.
(46) Anderson, C.; Beauchamp, A. L. Inorg. Chem. 1995, 34, 6065-6073.
(47) Anderson, C. Can. J. Chem. 2001, 79, 1477-1482.
(44) Collman, J. P.; McDevitt, J. T.; Leidner C. R.; Yee, G. T. Torrance,
J. B.; Little, W. A. J. Am. Chem. Soc. 1987, 109, 4606-4614.
278 Inorganic Chemistry, Vol. 47, No. 1, 2008

A Potentially SelectiWe Anticancer Complex
Table 4. IC50 Values (µM) for AH-197, Cisplatin, and KP1019 for
Selected Cell Lines
cell line
AH-197
cisplatin
KP1019
CCRF-CEM (leukemia)
HOP-62 (NSC lung)
BT-549 (breast)
HT29 (colon)
COLO 205 (colon)
4.3
5.0
14.4
18.8
5.5
4.8
4.5
14.8
27.4
56.0
24.9
100
59.8
17.4
53.6
no effect on the electrophoretic mobility of the plasmid
(Figure 4 lanes 12-15). Similar ruthenium(III)/DMSO-
containing compounds are hypothesized to act on tumor cells
through a different mechanism than cisplatin, not necessarily
involving DNA binding.32,53
Figure 4. Concentration-dependent interactions of metal complexes with
DNA. A total of 0.5 µg of linearized plasmid DNA was incubated with
varying concentrations of cisplatin, AH-197, or Na[trans-RuCl4(DMSO)-
(pyz)], at 37 °C for 24 h. A 1 kb plus molecular weight standard (Invitrogen)
was included for reference. Lanes 1, 6, and 11: 1 kb plus DNA ladder.
Lanes 2-5: 0, 0.1, 0.5, 1.0 mM cisplatin, respectively. Lanes 7-10: 0,
0.1, 0.5, 1.0 mM AH-197, respectively. Lanes 12-15: 0, 0.1, 0.5, 1.0 mM
Na[trans-RuCl4(DMSO)(pyz)], respectively.
NCI’s 60 Cell in Vitro Cancer Screenings. AH-197 was
accepted to the NCI’s Developmental Therapeutics Program
in vitro screening. A general comparison of the IC50 values
for the NCI-60 cell lines for AH-197, cisplatin, and KP1019
revealed that AH-197 has an intermediate cytotoxicity
compared to the other two compounds (Supporting Informa-
tion). IC50 values for selected cell lines that show interesting
results are presented in Table 4. As shown, AH-197 and
cisplatin were almost equally effective in CCRF-CEM
leukemia cells (IC50 ) 4.3 and 4.8 µM, respectively), HOP-
62 NSC lung cells (IC50 ) 5.0 and 4.5 µM, respectively),
and BT-549 breast cells (IC50 ) 14.4 and 14.8 µM,
respectively). The IC50 results for KP1019 in HT29 colorectal
cells, where efficacy of this compound exceeded cisplatin,
led KP1019 to clinical trials as a colorectal chemotherapeutic
drug.54 The IC50 value for AH-197 in HT29 colorectal cells
was similar to that for KP1019. Most interesting was the
dissimilar result shown for another colon cancer cell line,
COLO 205, for which AH-197 was found to be much more
potent in inhibiting cell growth in this cell line than either
cisplatin or KP1019.
and angles for the metal-ligand bonds in the coordination
sphere (Tables 2 and 3) are reasonable when compared to
similar monomeric and pyrazine-bridged ruthenium(III) and
platinum(II) complexes previously reported.41,45,48 ORTEP
diagrams can be seen in Figures 2 and 3. In the cis complex
(AH-197), the angle between the PtCl2pyz2 square plane and
the plane of the pyz ring is 65.7(5)° for the N1/N2 pyz and
68.8(4)° for the N3/N4 pyz. The trans complex, as stated in
the Experimental Section, crystallizes with two independent
trinuclear units. In this complex, the angle between the PtCl2-
pyz2 square plane and the plane of the pyz ring is 52.1(1)°
for the Pt1 molecule and 40.9(1)° for the Pt1A molecule.
This is the principal difference between the two independent
molecules in this structure. The larger values for the angles
on the cis complex are most probably due to steric hindrance
between the pyrazine groups.49 Also, when considering the
N-Ru-Cl and S-Ru-Cl angles (Tables 2 and 3), it appears
that the chlorides cant slightly toward the pyrazine ring and
away from the DMSO ligand in each of the cis and the trans
complexes.
DNA Migration Studies. Cisplatin is known to form
intrastrand cross-links with guanine bases and bend DNA
thereby reducing its electrophoretic mobility.4,50,51,52 The
concentration dependent inhibition of DNA migration due
to incubation with AH-197 is shown in Figure 4, suggesting
it tightly binds to DNA. To provide a basis for comparison,
incubation of DNA with cisplatin and Na[trans-RuCl4-
(DMSO)(pyz)] was also performed using the same concen-
trations and conditions. Interestingly, AH-197 appears to
have a greater effect on mobility than cisplatin at comparable
concentrations (compare Figure 4 lanes 3-5 with lanes
8-10) perhaps reflecting the greater size of AH-197. Not
surprisingly, Na[trans-RuCl4(DMSO)(pyz)] appears to show
The computerized, pattern-recognition algorithm, COM-
PARE, that was developed at the NCI was shown to correlate
biochemical mechanisms of action on the basis of the profile
of cell sensitivities produced from the screenings55,56 and
enables the evaluation and exploitation of data from all NCI
in vitro screenings. The profile for AH-197 was compared
to all synthetic compounds (standard COMPARE) as well
as to both cisplatin and KP1019 (matrix COMPARE).
Standard COMPARE queries resulted in insignificant cor-
relation coefficients (<0.70, data not shown). Additionally,
very low COMPARE correlation coefficients were found in
the matrix queries; AH-197 yielded a correlation coefficient
of 0.19 when compared to cisplatin and 0.25 when compared
to KP1019 (0.0-1.0 range). The low COMPARE
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Lackinger, L.; Keppler, B. K.; Marian, B. J. Cancer Res. Clin. Oncol.
2005, 131, 101-110.
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BC Decker, Inc.: Philadelphia, PA, 1993; pp 11-22.
(56) Paull, K. D.; Hamel, E.; Malspeis, L. In Cancer Chemotherapeutic
Agents; Foye, W. O., Ed.; American Chemical Society: Washington,
DC, 1995.
(48) Tessier, C.; Rochon, F. D. Inorg. Chim. Acta 1999, 295, 25-38.
(49) Rochon, F. D.; Priqueler, J. R. L. Can. J. Chem. 2004, 82, 649-658.
(50) Flores, A.; Pe´rez, J. M. Toxicol. Appl. Pharmacol. 2001, 161, 75-
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Inorganic Chemistry, Vol. 47, No. 1, 2008 279

Herman et al.
coefficients suggest that AH-197 does indeed have a unique
profile of tumor cell sensitivities and therefore it is likely
that AH-197 has a novel biological mechanism of action.56
Like NAMI-A, AH-197 failed the usual in vitro screenings
such as the NCI’s due to low overall toxicity to the cancer
cell lines. It is well-established that ruthenium(III) com-
pounds are not toxic compared to platinum complexes and
hence do not yield the required IC50 values.57 One reason
for the low values may be the failure of the in vitro
screenings to mimic the reduction potential that is found in
the in vivo cancer environments. Nevertheless, both NAMI-A
and KP1019 showed good results in treating cancer in vivo
and this led them to clinical trials.27,33 AH-197 generally
showed intermediate IC50 values between those of KP1019
and cisplatin. Given the above and our knowledge of
NAMI-A and KP1019, leads us to conclude that, generally,
the cytotoxicity of AH-197 is due to the activity of the
platinum center. Tumor environment has been found to be
acidic and be favorable for the reduction of ruthenium(III)
to ruthenium(II).58 Under such conditions KP1019 and
NAMI-A were found to respond differently; a chloride ligand
is the first ligand of KP1019 to hydrolyze upon reduction,
whereas, for NAMI-A, the imidazole ligand trans to DMSO
is the first to hydrolyze.58,59 Therefore, upon reduction, AH-
197 might fragment into one platinum(II) and two ruthenium-
(II) centers, which could result in better inhibition of cell
growth exerted by the cisplatin-like moiety while the two
ruthenium(II) centers could follow NAMI-A-like behavior
and reduce metastasis. Another possible advantage of com-
bining the ruthenium(III) and platinum(II) moieties stems
from data of in vitro and in vivo reactions of these fragments
with serum proteins such as albumin and transferrin.60,61 In
these studies, ruthenium and platinum compounds were found
to compete with iron for the binding sites of transferrin. The
high demand for iron in cancer cells, which results in high
expression of transferrin receptors in these cells, may lead
to accumulation of ruthenium and platinum compounds in
the tumor environment.61-63 Hence, the different reactivity
of cisplatin, NAMI-A, and KP1019 with these proteins are
important when evaluating a modular compound such as AH-
197. Large proportions of cisplatin has been found to bind
irreversibly to albumin and transferrin and thereby lose much
of its antitumor activity while NAMI-A and KP1019 were
found to bind reversibly to these proteins and retain their
heterocyclic ligands.23,60,61 Therefore, AH-197, which may
have restricted binding properties for its platinum center due
to the attached ruthenium moieties, could well achieve better
efficacy in its delivery to the tumor site. Recent combination
therapy results using both cisplatin and NAMI-A on lung
metastasis mouse models revealed an exceptional activity;
more than 60% of the animals were cured of the cancer.57,60
Concluding Remarks. AH-197 showed interesting be-
havior under our experimental conditions. AH-197 is quickly
and quantitatively reduced by ascorbic acid; therefore, the
compound may be activated by reduction as expected for
ruthenium(III) compounds. The hybrid (cisplatin-NAMI)
nature of our compound allows for the potential of combined
efficiency of mechanisms and, perhaps, higher selectivity
against both neoplastic tumors and metastatic cancer. Al-
though AH-197 was not chosen for continued studies by the
NCI due only to its lack of overall cytotoxicity, this is not
a grave concern. It has been very recently reported, for
several reasons, that the existing screening procedures are
in need of updating as many of the most promising new
pharmaceuticals have “failed” the NCI screening which does
not account for the possible in vivo activity of weakly
cytotoxic drugs.57,64 We look forward to further testing of
our new class of compounds and the related analogues that
we are preparing.
Acknowledgment. We thank Bard College, Vassar Col-
lege, and The National Science Foundation (Grant NSF
0521237 to J.M.T. (PI) and C.M.A.) for generous financial
support. We thank the Developmental Therapeutic Program
in the National Cancer Institute for accepting our compound
for cancer in vitro screenings. We thank Skylar Ferrara for
his valuable discussions. We thank James Morris for help
with the computations studies.
Supporting Information Available: Tables of IC50 values,
syntheses of analogous complexes, CIF files for each structure, and
calculated optimized molecular geometries of AH-197 and AH-
229 as well as total and zero point energies. This material is
available free of charge via the Internet at http://pubs.acs.org. CCDC
662891 for AH-197 and CCDC 662892 for AH-229 are available
at www.ccdc.cam.ac.uk/data_request/cif.
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IC062419H
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