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Am(m)ines make the difference: organoruthenium am(m)ine complexes and their chemistry in anticancer drug development.
DOI: 10.1002/chem.201202657
Am(m)ines Make the Difference: Organoruthenium Am(m)ine Complexes
and Their Chemistry in Anticancer Drug Development
Maria V. Babak,[a] Samuel M. Meier,[a, b] Anton A. Legin,[a] Mahsa S. Adib Razavi,[a]
Alexander Roller,[a] Michael A. Jakupec ,[a, b] Bernhard K. Keppler,[a, b] and
Christian G. Hartinger *[a, b, c]
Abstract: With the aim of systematically studying fundamental structure–activity relationships as a basis for the development of RuII arene complexes
(arene = p-cymene or biphenyl) bearing
mono-, bi-, or tridentate am(m)ine ligands as anticancer agents, a series of
ammine, ethylenediamine, and diethACHTUNGREylenetriamine complexes were prepared by different synthetic routes. Especially the synthesis of mono-, di-,
and triammine complexes was found to
be highly dependent on the reaction
conditions, such as stoichiometry, temperature, and time. Hydrolysis and pro-
tein-binding studies were performed to
determine the reactivity of the compounds, and only those containing
chlorido ligands undergo aquation or
form protein adducts. These properties
correlate well with in vitro tumor-inhibiting potency of the compounds.
The complexes were found to be active
in anticancer assays when meeting the
following criteria: stability in aqueous
Keywords: arene ligands · cancer ·
N ligands · ruthenium · structure–
activity relationships
Introduction
The discovery of the anticancer activity of cisplatin by
Rosenberg et al.[1] has broadened the range of routinely applied chemotherapeutics from organic drugs to metal-based
compounds. Complexes based on titanium,[2] arsenic, and
ruthenium[2a, 3] succeeded cisplatin in clinical trials, and especially Ru anticancer agents show promising results.[4] The
unique properties of Ru compounds are thought to be related to an enhanced degree of selectivity compared to many
[a] M. V. Babak, S. M. Meier , A. A. Legin, M. S. Adib Razavi, A. Roller,
Dr. M. A. Jakupec , Prof. Dr. B. K. Keppler ,
Prof. Dr. C. G. Hartinger
Institute of Inorganic Chemistry
University of Vienna
Waehringer Strasse 42, 1090 Vienna (Austria)
[b] S. M. Meier , Dr. M. A. Jakupec , Prof. Dr. B. K. Keppler ,
Prof. Dr. C. G. Hartinger
Research Platform “Translational Cancer Therapy Research”
University of Vienna
Waehringer Strasse 42, 1090 Vienna (Austria)
[c] Prof. Dr. C. G. Hartinger
School of Chemical Sciences
The University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
Fax: (+ 64) 9-3737-599 ext. 87422
E-mail: c.hartinger@auckland.ac.nz
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202657.
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solution and low rates of hydrolysis
and binding to proteins. Therefore, the
complexes least reactive to proteins
were found to be the most cytotoxic in
cancer cells. In general, complexes with
biphenyl as arene ligand inhibited the
growth of tumor cells more effectively
than the cymene analogues, consistent
with the increase in lipophilicity. This
study highlights the importance of finding a proper balance between reactivity
and stability in the development of organometallic anticancer agents.
other metallodrugs due to binding to proteins in the blood
stream and activation by reduction (RuIII/II) once inside the
tumor.[4b, 5] As cisplatin consists of a PtII core with two
ammine and two chlorido ligands, researchers focused initially on analogous multichlorido ruthenium complexes with
ammine ligands. Two mixed-valent ruthenium complexes,
namely, [(NH3)5RuIIIORuIVACHTUNGRE(NH3)4ORuIIIACHTUNGRE(NH3)5]6 + (ruthenium red) and [ClACHTUNGRE(NH3)4RuIIIORuIVACHTUNGRE(NH3)4(OH)]3 + (Ru360),
which have been used as cytological stains,[6] were found to
be inhibitors of the mitochondrial uniporter (Ca2 + uptake)[7]
and, like other ruthenium compounds, such as [RuIII(py)ACHTUNGRE(NH3)5]3 + (py = pyridine), cis-[RuIIIACHTUNGRE(Him)2ACHTUNGRE(NH3)4] (Him =
imidazole), they show remarkable immunosuppressant activity.[3a, 8] The extensive work of Clarke and colleagues demonstrated that am(m)ine coordination complexes of RuIII
and RuII are active antitumor agents (e.g., cis-[RuIIICl2ACHTUNGRE(NH3)4] + ,[9] fac-[RuIIICl3ACHTUNGRE(NH3)3],[10] [RuIII(O2CCH2CH3)ACHTUNGRE(NH3)5]2 + ,[3a] [RuIIACHTUNGRE(H2O)ACHTUNGRE(NH3)5]2 + ,[11] etc.).[3a, 12] Other studies indicated the potential of mixed-ligand ruthenium(II)
complexes with ethylenediamine and its derivatives.[13] In
contrast to cisplatin, which is known to form preferably intrastrand cross-links between adjacent guanine residues of
DNA,[14] ruthenium am(m)ine complexes favor interstrand
cross-link formation, probably due to the steric hindrance of
the octahedrally configured ruthenium center as opposed to
the less crowded square-planar coordination geometry of
PtII.[3a] Although the complexes exhibited very good anticancer activity in primary tumors, low solubility prevented
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FULL PAPER
their further development. However, complexes with simple
nitrogen-containing ligands (NH3, 1,2-ethylenediamine, pyridine) other than indazole and imidazole proved to have potential in anticancer drug development.
More recently, RuII arene compounds made an appearance in medicinal chemistry, and their biological activity is
significantly dependent on the nature of the coligands.[3b, 15]
Complexes of the type [(h6-arene)Ru(en)X] + with bidentate
ethylenediamine (en) and chloride as a leaving group (X)
exhibited excellent cytotoxicity against primary tumors
which was in good correlation with their DNA binding ability.[16] Surprisingly, recently published neutral half-sandwich
ruthenium complexes [(h6-arene)RuACHTUNGRE(NH3)Cl2] (arene = pcymene or biphenyl) do not inhibit the growth of tumor
cells in an in vitro setting, probably due to their low aqueous
stability and high reactivity in cell culture medium.[17] Similar observations were also made with other RuACHTUNGRE(arene) complexes, the cytotoxicity of which greatly depends on their reactivity.[18] To control interactions with the wide variety of
biomolecules present in the cell, the choice of the type of ligands (chelating/nonchelating), arene (more/less lipophilic),
and the presence of a leaving group (inert/labile to aquation
as an activation step) is of high importance. With the aim of
weaving together all of the disparate threads running
through the development of half-sandwich RuII complexes
bearing various am(m)ine ligands, we present a systematic
investigation including their synthesis, spectroscopic properties, crystal structures, behavior in aqueous solution, and
studies on their antiproliferative activity in cancer cells and
interactions with biomolecules.
Experimental Section
Materials and methods: Materials from chemical suppliers were used as
received, and all reactions were carried out under argon atmosphere in
anhydrous solvents if not otherwise stated. RuCl3·3 H2O was purchased
from Johnson Matthey. [(h6-p-cymene)RuII(ethylenediamine)Cl]PF6 (7 a),
(7 b),[19]
[{(h6-p-cymene)[(h6-biphenyl)RuII(ethylenediamine)Cl]PF6
ACHTUNGRERuCl2}2] (A), and [{(h6-biphenyl)RuCl2}2] (B)[20] were prepared according
to literature procedures. Methanol was dried and distilled over Mg under
argon atmosphere. Ethylenediamine was distilled over Na prior to use or
was purchased from Aldrich (purified by redistillation, > 99.5 %).
NH4OH (25 % solution in water) and formic acid (98 %) were obtained
from Fluka; NH4PF6 (> 95 %), AgPF6 (98 %), diethylenetriamine (97 %)
from Aldrich; and ubiquitin (from bovine erythrocytes) and horse heart
cytochrome c from Sigma. Dimethyl sulfoxide was obtained from Acros,
and 9-ethylguanine (EtG) from Sigma. Products were isolated without
taking any special precautions, but anhydrous solvents were used for isolation of 2 a, 2 b, 5 a, 5 b, 6 a, 7 a, and 7 b.
Elemental analyses were performed by the Microanalytical Laboratory
of the Faculty of Chemistry of the University of Vienna. Electrospray
ionization mass spectrometry was carried out with a Bruker Esquire 3000
instrument (Bruker Daltonics, Bremen, Germany), MilliQ water
(18.2 MW; Millipore Synergy 185 UV Ultrapure Water System; Molsheim, France) and methanol (VWR Int., HiPerSolv, CHROMANORM)
were used as solvents for ESI-MS studies. The 1H and 31P NMR spectra
were recorded at 500.10 and 202.44 MHz on a Bruker FT NMR spectrometer Avance II 500 MHz. 1H NMR kinetic experiments were performed at 500.32 MHz on a Bruker DPX500 (Ultrashield Magnet).
Chemical shifts are given in parts per million (ppm) relative to the resid-
Chem. Eur. J. 2013, 19, 4308 – 4318
ual solvent peak. NaOD (40 % in D2O, Fluka) was used for H/D-exchange NMR experiments.
X-ray diffraction measurements were performed on a Bruker X8 APEX
II CCD diffractometer at 100 (2 b, 6 a), 150 (4 b), or 296 K (3 a). Single
crystals were positioned 40, 35, 35, and 35 mm from the detector, and
1468, 2295, 2780 and 823 frames were measured, each for 10, 30, 10 and
10 s over 18 scan width for 2 b, 3 a, 4 b, and 6 a, respectively. The data
were processed with SAINT software.[21] Crystal data, data collection parameters, and structure refinement details for 2 b, 3 a, 4 b, and 6 a are
given in Table S1 of the Supporting Information and key bond lengths
and angles in Table S2. The structures were solved by direct methods and
refined by full-matrix least-squares techniques. Non-hydrogen atoms
were refined with anisotropic displacement parameters. Hydrogen atoms
were placed at calculated positions and refined as riding atoms in the
subsequent least squares model refinements. The isotropic thermal parameters were estimated to be 1.2 times the values of the equivalent isotropic thermal parameters of the non-hydrogen atoms to which hydrogen
atoms are bonded. Severe disorder of the hexafluorophosphate anion in
3 a, modeled with two positions for P1, resulted in relatively high residual
electron density. The following computer programs, equipment and
tables were used: structure solution, SHELXS-97; refinement, SHELXL97;[22] molecular diagrams, Mercury 3.0.
Cell lines and culture conditions: The human cancer cell line CH1 (ovarian carcinoma) was provided by Lloyd R. Kelland (CRC Centre for
Cancer Therapeutics, Institute of Cancer Research, Sutton, UK). A549
(non-small cell lung cancer) and SW480 (colon carcinoma) cells were
supplied by Brigitte Marian (Institute of Cancer Research, Department
of Medicine I, Medical University of Vienna, Austria). Adherent cell cultures were grown in 75 cm2 culture flasks (Iwaki/Asahi Technoglass,
Gyouda, Japan) in complete medium [i.e., minimal essential medium
(MEM) supplemented with 10 % heat-inactivated fetal bovine serum,
1 mm sodium pyruvate, 4 mm l-glutamine, and 1 % v/v nonessential
amino acids from 100 ready-to-use stock (all purchased from Sigma-Aldrich, Austria)]. Cell cultures were incubated at 37 8C in a moist atmosphere containing 5 % CO2.
Cytotoxicity test in cancer cell lines: The cytotoxicity of the compounds
was determined by means of a colorimetric microculture assay (MTT
assay). The cells were harvested from culture flasks by trypsinization and
seeded into 96-well microculture plates (Iwaki/Asahi Technoglass,
Gyouda, Japan) in densities of 1 103 cells per well (for CH1), 2.5 103
cells per well (for SW480), and 3 103 cells per well (for A549). After the
cells were allowed to resume exponential growth for 24 h, the test compounds were dissolved in complete medium and 100 mL of serial dilution
was added per well. After exposure for 96 h, drug solutions were replaced with 100 mL of RPMI 1640 culture medium (supplemented with
10 % heat-inactivated fetal bovine serum and 2 mm of l-glutamine) plus
20 mL of MTT solution in phosphate-buffered saline (5 mg mL 1). After
incubation for 4 h, the RPMI/MTT mixtures were removed, and the formazan crystals formed in viable cells were dissolved in 150 mL of DMSO
per well. Optical densities were measured at 550 nm with a microplate
reader (Tecan Spectra Classic), by using a reference wavelength of
690 nm to correct for unspecific absorption. The quantity of viable cells
was expressed in terms of treated/control (T/C) values by comparison to
untreated control microcultures, and 50 % inhibitory concentrations
(IC50) were calculated from concentration–effect curves by interpolation.
Evaluation was based on means from at least three independent experiments, each comprising three replicates per concentration level.
Protein-binding studies: The metal compounds (400 mm) were dissolved
in 1 % aqueous dimethyl sulfoxide, and the proteins ubiquitin and cytochrome c (200 mm) in water. These stock solutions were mixed to obtain
2:1 metal-to-protein molar ratios and then kept at 37 8C in the dark.
Mass spectra of the incubation solutions were recorded after 0, 3, 6, 24,
and 48 h. Furthermore, the compounds were incubated under comparable
conditions with an ubiquitin–cytochrome c mixture to yield a molar ratio
of 1:1:1.
The samples were analyzed by using a MaXis ESI-Q-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) with the following parameters: capillary 4.5 kV, gas flow 8 psi, dry gas 6 L min 1, dry temperature
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C. G. Hartinger et al.
150 8C, 400 Vpp funnel RF, 4 eV quadrupole ion energy and 100 ms transfer time. The samples were diluted to 2 mm with water/methanol/formic
acid (50:50:0.2) and thereafter injected into the mass spectrometer by
direct infusion at a flow rate of 180 mL h 1. The spectra were recorded in
positive-ion mode over 0.5 min and averaged. The Data Analysis 4.0 software package (Bruker Daltonics, Bremen, Germany) was used for proACHTUNGREcessing, and maximum entropy deconvolution (automatic data point
spacing and 30 000 instrument resolving power) was applied.
Binding to the DNA model 9-ethylguanine: Compounds 2 b, 5 b, and 7 b
(400 mm) were dissolved in 1 % dimethyl sulfoxide aqueous solution. EtG
(800 mm) was dissolved in water. The compounds and EtG were incubated at 1:2 molar ratio at metal-to-complex concentrations of 50–100 mm
for 3, 6, 24, and 48 h at 37 8C. Samples were diluted with water/methanol
(1:1) to final concentrations of 5–10 mm and immediately introduced into
the mass spectrometer. ESI-IT mass spectra were recorded on an
AmaZon Ion Trap mass spectrometer (Bruker Daltonics, Bremen Germany) by direct infusion at a flow rate of 180 mL h 1. The following parameters were employed: capillary 2.5 kV, gas flow 9 psi, dry gas
6 L min 1, dry temperature 200 8C, and trap drive 55.1. The Data Analysis
4.0 software package (Bruker Daltonics, Bremen, Germany) was used for
processing the raw data.
Synthesis of complexes
[(h6-p-cymene)RuIIACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2 (1 a): Method 1: [{(h6-p-cymene)ACHTUNGRERuCl2}2] (0.3 mmol, 0.184 g) was stirred for 10 min in dry methanol
(10 mL). Ammonia gas was bubbled through the solution, which caused
significant warming of the reaction mixture and was accompanied by a
fast change of the color from red-orange to light yellow. After the solution had cooled down, the gas supply was stopped and the reaction mixture was stirred for 30 min. The yellow solution was concentrated by
rotary evaporation under reduced pressure to 1 mL, and a saturated solution of NH4PF6 in methanol (2–4 mL) was added. The resulting solution
was filtered and diethyl ether (15 mL) added. The light yellow precipitate
was collected by filtration, washed with diethyl ether (3 5 mL), and
dried in vacuo to yield 286 mg (90 %) of the target complex.
Method 2: Ammonia (2 mL, 25 % aqueous solution) was added to a suspension of [{(h6-p-cymene)RuCl2}2] (0.2 mmol, 0.122 g) in dry methanol
(10 mL). The reaction mixture was heated to reflux for 1 h at 85 8C. The
resulting yellow solution was concentrated by rotary evaporation under
reduced pressure to 1 mL and a saturated solution of NH4PF6 in methanol (2–4 mL) was added. The resulting solution was filtered and diethyl
ether (15 mL) was added. The yellow precipitate was collected by filtration, washed with diethyl ether (3 5 mL), and dried in vacuo to yield
152 mg (64 %) of the target complex.
Elemental analysis (%) calcd for C10H23RuN3P2F12·0.1 NH4PF6
(592.61 g mol 1): C 20.26, H 3.98, N 7.33; found: C 20.01, H 3.82, N
7.23 %; MS (ESI +): m/z 270.1 {[(h6-p-cymene)RuACHTUNGRE(NH3)2 H] + + [(h6-pcymene)RuACHTUNGRE(NH3)2] + }; found: 270.0; MS (ESI ): m/z 145.0 [PF6] ; found:
144.5; 1H NMR (500.10 MHz, [D6]DMSO): d = 1.19 (d, 6 H, 3JHH = 7.0 Hz,
CHACHTUNGRE(CH3)2), 2.12 (s, 3 H, C6H4ACHTUNGRE(CH3)), 2.81 (sept, 1 H, 3JHH = 6.9 Hz,
CHMe2), 3.41 (s, 9 H, NH3), 5.41 (d, 2 H, 3JHH = 6.0 Hz, CHcym), 5.70 ppm
(d, 2 H, 3JHH = 6.0 Hz, CHcym); 31P NMR (202.44 MHz; [D6]DMSO): d =
144.10 ppm (sept, 1 P, 1JPF = 727.7 Hz, PF6).
[(h6-biphenyl)RuIIACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2 (1 b): Method 1: [{(h6-biphenyl)RuCl2}2]
(0.2 mmol, 0.130 g) was stirred for 10 min in dry methanol (10 mL). Ammonia gas was bubbled through the solution, which caused significant
warming of the reaction mixture and changed the black-brown suspension to a light yellow solution. After the solution had cooled, the gas
supply was stopped and the reaction mixture was stirred for 1 h. The
yellow solution was filtered and concentrated by rotary evaporation
under reduced pressure to 1 mL, and saturated solution of NH4PF6 in
water (2–4 mL) was added. A light yellow precipitate formed immediately, which was collected by filtration, washed with water (2 2 mL) and diethyl ether (3 15 mL), and dried in vacuo to yield 175 mg (70 %) of the
target complex.
Method 2: Ammonia (2 mL of 25 % aqueous solution) was added to a
suspension of [{(h6-biphenyl)RuCl2}2] (0.2 mmol, 0.130 g) in dry methanol
(15 mL) and the reaction mixture was heated to reflux for 1 h at 85 8C.
Then it was filtered, the resulting light-orange solution concentrated by
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rotary evaporation under reduced pressure to 1 mL, and a saturated solution of NH4PF6 in water (2–4 mL) added. The orange-yellow precipitate
was collected by filtration, washed with water (1 2 mL) and diethyl
ether (3 15 mL), and dried in vacuo to give 160 mg (64 %) of the target
product.
Elemental analysis (%) calcd for C12H19RuN3P2F12·0.2 NH4PF6
(628.90 g mol 1): C 22.92, H 3.17, N 7.13; found: C 22.77, H 3.24, N
6.86 %; MS (ESI +): m/z 290.0 {[(h6-biphenyl)RuACHTUNGRE(NH3)2 H] + + [(h6biphenyl)RuACHTUNGRE(NH3)2] + }; found: 290.0; MS (ESI ): m/z 145.0 [PF6] ;
found: 144.5; 1H NMR (500.10 MHz; [D6]DMSO): d = 3.51 (s, 9 H, NH3),
5.87 (m, 3 H, CHphen), 6.17 (m, 2 H, CHphen), 7.57 (m, 3 H, RuCHphen),
7.78 ppm (m, 2 H, RuCHphen); 31P NMR (202.44 MHz; [D6]DMSO): d =
144.13 ppm (sept, 1 P, 1JPF = 720.5 Hz, PF6).
[(h6-p-cymene)RuIIACHTUNGRE(NH3)2Cl]PF6 (2 a): Ammonia (150 mL of 25 % aqueous solution) was added to a suspension of [{(h6-p-cymene)RuCl2}2]
(0.2 mmol, 0.122 g) in dry methanol (10 mL) and the reaction mixture
was heated to reflux for 1 h at 85 8C. The resulting orange solution was
concentrated by rotary evaporation under reduced pressure to 1 mL and
a saturated solution of NH4PF6 in methanol (2–4 mL) was added. The resulting bright orange solution was filtered and diethyl ether (15 mL) was
added. The yellow precipitate was collected by filtration, washed with diethyl ether (3 5 mL) and dried in vacuo to yield 88 mg (49 %) of the
target complex.
Elemental analysis (%) calcd for C10H20RuN2PF6Cl (449.77 g mol 1): C
26.70, H 4.48, N 6.23, found: C 26.68, H 4.33, N 6.04 %; MS (ESI +): m/z
288.0 [(h6-p-cymene)RuACHTUNGRE(NH3)Cl] + ; found: 288.4; MS (ESI ): m/z 145.0
[PF6] ; found: 144.9; 1H NMR (500.10 MHz; [D6]DMSO): d = 1.21 (d,
3
JHH = 7.0 Hz, 6 H, CHACHTUNGRE(CH3)2), 2.12 (s, 3 H, C6H4ACHTUNGRE(CH3)), 2.81 (sept, 1 H,
3
JHH = 6.9 Hz, CHMe2), 3.37 (s, 6 H, NH3), 5.37 (d, 2 H, 3JHH = 6.0 Hz,
CHcym), 5.61 ppm (d, 2 H, 3JHH = 6.1 Hz, CHcym); 31P NMR (202.44 MHz;
[D6]DMSO): d = 144.15 ppm (sept, 1 P, 1JPF = 702.7 Hz, PF6).
[(h6-biphenyl)RuIIACHTUNGRE(NH3)2Cl]PF6 (2 b): Ammonia (150 mL of 25 % aqueous
solution) was added to a suspension of [{(h6-biphenyl)RuCl2}2] (0.2 mmol,
0.130 g) in dry methanol (15 mL) and the reaction mixture was heated to
reflux for 1 h at 85 8C. Then it was filtered, the resulting orange solution
concentrated by rotary evaporation under reduced pressure to 1 mL, and
a saturated solution of NH4PF6 in methanol (2–4 mL) was added. The
yellow-ochre precipitate that formed immediately was collected by filtration, washed with water (1 2 mL) and diethyl ether (3 15 mL), and
dried in vacuo. The ochre solid was redissolved in the minimum amount
of methanol, and diethyl ether was added to precipitate 65 mg of the
target complex as a yellow powder. The orange aqueous supernatant was
left to stand at 0 8C for 8 h and orange microcrystals of the complex
formed (15 mg, overall yield 43 %). Crystals suitable for X-ray diffraction
analysis (orange needles) were grown by slow diffusion of diethyl ether
into a methanol solution.
Elemental analysis (%) calcd for C12H16RuN2PF6Cl (469.76 g mol 1): C
30.68, H 3.43, N 5.96; found: C 30.90, H 3.14, N 5.86 %; MS (ESI +): m/z
308.0 [(h6-biphenyl)RuACHTUNGRE(NH3)Cl] + ; found: 308.4; MS (ESI ): m/z 145.0
[PF6] ; found: 144.5; 1H NMR (500.10 MHz; [D6]DMSO): d = 3.49 (s,
6 H, NH3), 5.82 (m, 3 H, CHphen), 6.11 (m, 2 H, CHphen), 7.51 (m, 3 H,
RuCHphen), 7.80 ppm (m, 2 H, RuCHphen); 31P NMR (202.44 MHz;
[D6]DMSO): d = 144.24 ppm (sept, 1 P, 1JPF = 711.7 Hz, PF6).
[(h6-p-cymene)RuII(diethylenetriamine)]ClACHTUNGRE(PF6) (3 a): Diethylenetriamine
(27 mL, 0.25 mmol) was added to a suspension of [{(h6-p-cymene)RuCl2}2]
(0.09 mmol, 0.056 g) in dry methanol (15 mL). The reaction mixture immediately turned into a light yellow solution, which was stirred for 1.5 h,
filtered, and concentrated by rotary evaporation under reduced pressure
to 1 mL, after which a saturated solution of NH4PF6 in methanol (2–
4 mL) was added. The solution was filtered and diethyl ether (15 mL)
added. The mixture was allowed to stand at 0 8C for 8 h, and the yellow
precipitate was collected by filtration, washed with diethyl ether (3
15 mL), and dried in vacuo to yield 70 mg (75 %) of the target complex.
Crystals suitable for X-ray diffraction analysis were grown by slow diffusion of diethyl ether into a methanol solution.
Elemental analysis (%) calcd for C14H27RuN3PF6Cl (518.87 g mol 1): C
32.41, H 5.24, N 8.10; found: C 32.21, H 4.95, N 8.21 %; MS (ESI +): m/z
338.1 [(h6-p-cymene)Ru(diethylenetriamine) H] + ; found: 337.9; MS
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Chem. Eur. J. 2013, 19, 4308 – 4318
Ruthenium Complexes in Anticancer Drug Development
(ESI ): 145.0 [PF6] ; found: 144.8; 1H NMR (500.10 MHz; [D6]DMSO):
d = 1.21 (d, 6 H, 3JHH = 6.9 Hz, CHACHTUNGRE(CH3)2), 2.25 (s, 3 H, C6H4ACHTUNGRE(CH3)), 2.48–
2.55 (m, 4 H, C2H4), 2.61(m, 2 H, C2H4), 2.76 (m, 2 H, C2H4), 2.96 (sept,
1 H, 3JHH = 6.9 Hz, CHMe2), 5.14 (m, 2 H, NH2), 5.61 (d, 2 H, 3JHH =
6.2 Hz, CHcym), 5.73 (d, 2 H, 3JHH = 6.2 Hz, CHcym), 6.48 (m, 2 H, NH2),
7.92 ppm (m, 1 H, NH); 31P NMR (202.44 MHz; [D6]DMSO): d =
144.22 ppm (sept, 1 P, 1JPF = 720.6 Hz, PF6).
[(h6-biphenyl)RuII(diethylenetriamine)]ACHTUNGRE(PF6)2
(3 b):
[{(h6-biphenyl)RuCl2}2] (0.14 mmol, 0.091 g) was heated to reflux in water/methanol
(10:1) at 85 8C for 2 h and then cooled to 55 8C. Diethylenetriamine
(38 mL, 0.35 mmol) was added to this suspension, which turned from redbrown to yellow-greenish. This was then slowly heated to reflux again for
another 1.5 h and filtered while hot. The resulting yellow solution was
then concentrated by rotary evaporation under reduced pressure to
1 mL, and a saturated solution of NH4PF6 in water (2–4 mL) was added.
The flask was briefly shaken, and a light-yellow precipitate immediately
formed. The mixture was allowed to stand at 0 8C for 8 h, the precipitate
was collected by filtration, washed with water (2 2 mL) and diethyl
ether (3 15 mL), and dried in vacuo to yield 98 mg (54 %) of the target
complex.
Elemental analysis (%) calcd for C16H23RuN3P2F12 (648.37 g mol 1): C
29.64, H 3.58, N 6.48; found: C 29.54, H 3.20, N 6.25 %; MS (ESI +): m/z
358.1 [(h6-biphenyl)Ru(diethylenetriamine) H] + ; found: 357.9; MS
(ESI ): 145.0 [PF6] ; found: 144.8; 1H NMR (500.10 MHz; [D6]DMSO):
d = 2.41 2.60 (m, 4 H, C2H4), 2.72 (m, 4 H, C2H4), 4.31 (m, 2 H, NH2),
5.92 (t, 2 H, 3JHH = 6.0 Hz, CHphen), 6.04 (t, 1 H, 3JHH = 5.7 Hz, CHphen),
6.30 (d, 2 H, 3JHH = 6.0 Hz, CHphen), 5.92 (m, 2 H, NH2), 7.55 (m, 3 H,
RuCHphen), 7.80 (m, 2 H, RuCHphen), 8.32 ppm (m, 1 H, NH); 31P NMR
(202.44 MHz; [D6]DMSO): d = 144.19 ppm (sept, 1 P, 1JPF = 711.3 Hz,
PF6).
[(h6-biphenyl)RuII(ethylenediamine)ACHTUNGRE(NH3)]ACHTUNGRE(PF6)2 (4 b): A solution of
AgPF6 (0.11 mmol, 0.028 g) in methanol (2 mL) was added to a solution
of [(h6-biphenyl)Ru(ethylenediamine)Cl]PF6 (0.1 mmol, 0.050 g) in dry
methanol (7 mL) and the mixture stirred for 2 h. A white precipitate of
AgCl was quickly filtered off under aerobic conditions and the resulting
solution was flushed with Ar. Ammonia gas was bubbled through the solution, which caused significant warming of the reaction mixture with no
color change. After the clear solution had cooled the gas supply was stopped and the reaction mixture was stirred for 1 h. The yellow solution was
filtered, concentrated to 1 mL by rotary evaporation under reduced pressure, and a saturated solution of NH4PF6 in water (2–4 mL) was added.
A green-yellow precipitate formed immediately, which was collected by
filtration, washed with water (2 2 mL) and diethyl ether (3 15 mL) and
dried in vacuo to yield 21 mg (33 %) of the target complex. Crystals suitable for X-ray diffraction analysis were grown by slow diffusion of diethyl ether into a methanol solution.
Elemental analysis (%) calcd for C14H21RuN3P2F12·0.5 CH3OH
(638.36 g mol 1): C 27.28, H 3.63, N 6.58; found: C 27.59, H 3.89, N
6.59 %; MS (ESI +): m/z 315.0 [(h6-biphenyl)Ru(diethylenediACHTUNGREamine) H] + ; found: 315.1; MS (ESI ): m/z 145.0 [PF6] ; found: 144.9;
1
H NMR (500.10 MHz; [D6]DMSO): d = 2.18 (m, 2 H, CH2), 2.34 (m, 2 H,
CH2), 3.51 (s, 3 H, NH3), 4.30 (m, 2 H, NH2), 5.89 (t, 2 H, 3JHH = 6.0 Hz
CHphen), 5.97 (t, 1 H, 3JHH = 5.6 Hz, CHphen), 6.23 (d, 2 H, 3JHH = 6.1 Hz,
CHphen), 6.41 (m, 2 H, NH2), 7.57 (m, 3 H, RuCHphen), 7.76 ppm (m, 2 H,
RuCHphen); 31P NMR (202.44 MHz; [D6]DMSO): d = 143.18 ppm (sept,
1 P, 1JPF = 720.5 Hz, PF6).
[(h6-p-cymene)RuIIACHTUNGRE(NH3)Cl2]ACHTUNGRE[(dmbaH)ACHTUNGRE(PF6)] (5 a): The procedure of Betanzos-Lara et al.[17] was used after minor modification. In brief, N,N-dimethylbenzylamine (dmba, 0.098 mL, 0.65 mmol) and NH4PF6 (0.106 g,
0.65 mmol) were added to a suspension of [{(h6-p-cymene)RuCl2}2]
(0.32 g, 0.2 mmol) in dry methanol (15 mL) and the reaction mixture was
stirred for 18 h at room temperature. Then it was filtered and the resulting orange solution evaporated to dryness. The oily residue was redissolved in the minimum amount of CH2Cl2 and diethyl ether (30 mL)
added. The flask was briefly shaken, and a light yellow precipitate immediately formed. The mixture was allowed to stand at 0 8C for 8 h, and the
precipitate was collected by filtration, washed with diethyl ether (3
15 mL), and dried in vacuo to yield 155 mg (64 %) of the target complex.
Chem. Eur. J. 2013, 19, 4308 – 4318
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Elemental analysis (%) calcd for C19H31RuN2Cl2PF6 (604.40 g mol 1): C
37.75, H 5.17, N 4.63; found: C 37.58, H 5.46, N 4.96 %; MS (ESI +): m/z
289.0
{[(h6-p-cymene)RuACHTUNGRE(NH3)Cl] + + [(h6-p136.1
[dmba+H] + ,
+
cymene)RuACHTUNGRE(NH3)Cl+H] }; found: 136.6, 289.3; MS (ESI ): m/z 145.0
[PF6] ; found: 144.5; 1H NMR (500.10 MHz; CD3NO2): d = 1.31 (d, 6 H,
3
JHH = 7.0 Hz, CHACHTUNGRE(CH3)2), 2.10 (s, 3 H, C6H4ACHTUNGRE(CH3)), 2.90 (sept, 1 H, 3JHH =
7.0 Hz, CHMe2), 2.74 (s, 3 H, NH3), 2.94 (s, 6 H, NACHTUNGRE(CH3)2) 4.38 (s, 2 H,
CH2), 5.35 (d, 2 H, 3JHH = 6.0 Hz, CHcym), 5.56 (d, 2 H, 3JHH = 6.0 Hz,
CHcym), 7.53 (m, 3 H, CHdmba), 7.58 ppm (m, 2 H, CHdmba); 31P NMR
(202.44 MHz; CD3NO2): d = 146.53 ppm (sept, 1 P, 1JPF = 734.1 Hz, PF6).
[(h6-biphenyl)RuIIACHTUNGRE(NH3)Cl2]ACHTUNGRE[(dmbaH)ACHTUNGRE(PF6)] (5 b): [{(h6-biphenyl)RuCl2}2]
(0.3 mmol, 0.196 g) was heated to reflux in methanol (80 mL) at 85 8C for
2 h and then cooled to room temperature. N,N-Dimethylbenzylamine
(0.09 mL, 0.6 mmol) and NH4PF6 (0.98 g, 0.6 mmol) were added to the
brown solution and the reaction mixture was stirred for 18 h at ambient
temperature while no color change occurred. The solution was filtered
and the orange-brown filtrate was evaporated to dryness. The oily residue
was redissolved in a minimum amount of CH2Cl2 and 30 mL of diethyl
ether was added. The precipitate was collected by filtration, washed with
diethyl ether (3 15 mL), and dried in vacuo to yield 123 mg (33 %) of
the target complex.
Elemental analysis (%) calcd for C21H27RuN2Cl2PF6 (624.39 g mol 1): C
40.40, H 4.36, N 4.49; found: C 40.22, H 4.46, N 4.50 %; MS (ESI +): m/z
136.1 [dmba+H] + , 308.0 [[(h6-biphenyl)RuACHTUNGRE(NH3)Cl] + ; found: 136.3,
308.3; MS (ESI ): m/z 145.0 [PF6] ; found: 144.6; 1H NMR
(500.10 MHz; CD3NO2): d = 2.18 (br, 3 H, NH3), 2.88 (s, 6 H, NACHTUNGRE(CH3)2)
4.14 (s, 2 H, CH2), 5.97 (m, 2 H, CHphen), 5.76 (m, 2 H, CHphen), 7.51 (m,
3 H, RuCHphen), 7.58 (m, 5 H, CHdmba), 7.79 ppm (m, 2 H, RuCHphen);
31
P NMR (202.44 MHz; [D6]DMSO): d = 144.15 ppm (sept, 1 P, 1JPF =
746.7 Hz, PF6).
[(h6-p-cymene)RuIIACHTUNGRE(NH3)Cl2]ACHTUNGRE[(Et3NH)ACHTUNGRE(PF6)]
(6 a):
Triethylamine
(0.090 mL, 0.65 mmol) and NH4PF6 (0.106 g, 0.65 mmol) were added to a
suspension of [{(h6-p-cymene)RuCl2}2] (0.32 g, 0.2 mmol) in dry methanol
(15 mL) and the reaction mixture was stirred for 18 h at room temperature. Then it was filtered and the resulting orange solution was concentrated to 2 mL, whereby it turned dark brown. The solution was filtered
again and crystals suitable for X-ray diffraction analysis (long brown needles) were grown by slow diffusion of diethyl ether into a dichloromethane solution. The crystals were manually separated from the brown residue and washed with diethyl ether (5 15 mL) to give 80 mg (35 %) of
the target product.
Elemental analysis (%) calcd for C16H33RuN2Cl2PF6 (570.39 g mol 1): C
33.69, H 5.83, N 4.91; found: C 33.40, H 6.20, N 4.86 %; MS (ESI +): m/z
102.2 [Et3N+H] + , 270.7 [(h6-p-cymene)RuCl] + , 289.0 {[(h6-cymene)RuACHTUNGRE(NH3)Cl] + + [(h6-p-cymene)RuACHTUNGRE(NH3)Cl+H] + }; found: 102.6, 271.1, 289.1;
MS (ESI ): m/z 145.0 [PF6] ; found: 144.5; 1H NMR (500.10 MHz;
CD3NO2): d = 1.30 (d, 6 H, 3JHH = 7.0 Hz, CHACHTUNGRE(CH3)2), 1.37 (t, 9 H, 3JHH =
7.3 Hz, CH3), 2.19 (s, 3 H, C6H4ACHTUNGRE(CH3)), 2.88 (sept, 1 H, 3JHH = 6.9 Hz,
CHMe2), 2.71 (s, 3 H, NH3), 3.34 (m, 6 H, CH2), 5.31 (d, 2 H, 3JHH =
5.9 Hz, CHcym), 5.53 ppm (d, 2 H, 3JHH = 5.9 Hz, CHcym); 31P NMR
(202.44 MHz; CD3NO2): d = 146.53 ppm (sept, 1 P, 1JPF = 737.0 Hz, PF6).
Results and Discussion
Synthesis of organoruthenium(II) am(m)ine complexes: Numerous studies on RuII h6-arene complexes with different
numbers of coordinated ammine ligands have been published[17, 23] since the first report of the synthesis of [(h6benzene)RuACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6)3·NH4PF6 in 1978[23b] until the
most recent publications.[17, 23g] Despite the variety of synthetic pathways, some of them are contradictory.[23a,c,d] We
found that the type of products, that is, with two ammine ligands in [(h6-arene)RuACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) or three in [(h6arene)RuACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2, and their yields strictly depend on
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4311
C. G. Hartinger et al.
tion but with an equimolar amount of NH3 did not lead to
the reaction conditions, such as temperature and reaction
time, concentration of the reactants, and quality of the solthe target products 5 a and 5 b featuring one ammonia and
vents.
two chlorido ligands. These complexes were successfully preAll of the complexes were synthesized from the ruthenipared by in situ generation of NH3 from stoichiometric
um precursors [{(h6-arene)RuCl2}2] (A: arene = p-cymene;
amounts of base (e.g, dimethylbenzylamine or triethylACHTUNGREamine) and NH4PF6 and were always isolated as adducts of
B: arene = biphenyl). Dimeric complex B has low solubility
in most commonly used solvents. Therefore, in general, reacthe complexes with the base.
tions involving the h6-biphenyl fragment require longer reacComplexes 7 a and 7 b with bidentate ethylenediamine
(en) ligand were synthesized according to a literature procetion times and additional purification steps and resulted in
dure.[19] Complex 4 b can be obtained in a single step from
lower yields in comparison to h6-p-cymene complexes. The
metal species formed during the reaction are highly reactive
7 b by removal of the chlorido ligand with silver salts and
and can be stabilized by coordination of solvent molecules
subsequent reaction with ammonia gas bubbled through the
which are quickly substituted by nitrogen ligands with
solution (yield: 33 %). We also investigated the reactions of
higher affinity to ruthenium. It was reported that solvents of
A and B with various tridentate ligands. Although osmium
medium polarity, such as methanol and nitromethane, yield
complexes with tridentate macrocycles such as 1,4,7-trimeththe target complexes in higher yields,[17] a fact also observed
yl-1,4,7-triazacyclononane and 1,4,7-triazacyclononane are
known,[23f, 24] similar reactions with the ruthenium precursors
in our studies. The generated compounds with labile chlorido ligands can be easily hydrolyzed, and therefore synthesis
resulted in fast darkening of the solution and decomposiand isolation were performed in absolute solvents.
tion. However, we succeeded in the synthesis of ruthenium
It was recently reported that the reaction of A and B with
complexes with the tridentate ligand diethylenetriamine
aqueous ammonia results in a mixture of products.[23g] How(dien), which readily forms a complex with dimer A on stirring for 1.5 h with an excess of the ligand (yield: 75 %). The
ever, by treating A and B with a large excess of 25 % aquereaction of biphenyl precursor B under the same conditions
ous ammonia in refluxing methanol we obtained ruthenium
resulted in the formation of 3 b in low yield. However, 3 b
complexes 1 a and 1 b with three ammonia ligands, which
was obtained in 54 % yield when B was heated to reflux for
were unambiguously characterized by NMR spectroscopy
2 h in water/methanol before an excess of dien was added,
and mass spectrometry. The reaction time does not affect
followed by stirring for 1.5 h at 55 8C.
the type of product, but the yield of the reaction. Indeed, a
series of experiments was performed with refluxing or stirAll complexes were characterized by 1H and 31P NMR
ring the reagents for 1 or 24 h, and it was found that the
spectroscopy as well as ESI-MS and elemental analysis. The
maximum yield can be obtained after 1 h of reflux (64 %) or
NH3 protons of [(h6-p-cymene)RuACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2 (1 a), [(h624 h of stirring (60 %), while the yields after 1 h of stirring
biphenyl)RuACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2
(1 b),
[(h6-p-cymene)Ru6
and 24 h of reflux were unsatisfactory (37 and 22 %). Low
ACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 a), and [(h -biphenyl)RuACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6)
or moderate yields of the reaction prompted the use of alternative sources of ammonia.
Commercially available solutions of NH3 in dioxane or
ethanol were found to be inappropriate for preparing the desired compounds. However, reaction with ammonia gas bubbled through the reaction mixture yielded the target complexes in good yields (90 and
70 % for 1 a and 1 b, respectively).
When ruthenium precursors
A and B were heated to reflux
with a slight excess of 25 %
aqueous ammonia for one hour,
complexes with two ammonia
and one chlorido ligand were
formed in moderate yields (49
and 43 % for 2 a and 2 b;
Scheme 1). The optimal reac- Scheme 1. Synthesis of RuII arene complexes with am(m)ine ligands. i) MeOH, NH , NH PF /MeOH or 25 %
3,g
4
6
tion time was found to be 1– NH4OH excess, NH4PF6 ; ii) MeOH, 25 % NH4OH, NH4PF6 ; iii) MeOH, dien, NH4PF6 ; iv) MeOH, en, NH3,g,
3 h. However, the same reac- AgPF6, NH4PF6 ; v) MeOH, dmba, NH4PF6 ; vi) MeOH, Et3N, NH4PF6 ; vii) MeOH, en, NH4PF6.[19]
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Chem. Eur. J. 2013, 19, 4308 – 4318
Ruthenium Complexes in Anticancer Drug Development
(2 b) appear as a broad singlet. The NH3 proton resonances
of cymene complexes are detected at a higher field
(3.36 ppm) than those of biphenyl derivatives (3.49 ppm) because of the higher electron density at the RuII centers
caused by the weaker p-accepting and stronger p-donating
capability of p-cymene. The NH3 signal has approximately
the same chemical shift in [(h6-biphenyl)RuACHTUNGRE(NH3)3]ACHTUNGRE(PF6)2
(1 b), [(h6-biphenyl)RuACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 b), and [(h6biphenyl)Ru(ethylenediamine)ACHTUNGRE(NH3)]ACHTUNGRE(PF6)2 (4 b).
The 1H NMR spectrum of the free dien ligand was recorded in a variety of solvents (see Table S3 in the Supporting
Information). The methylene protons gave rise to two sharp
multiplets in all solvents, whereas NH and NH2 protons appeared as sharp singlets at d = 1.06 and 4.83 ppm in CDCl3
and CD3OD, and as a broad singlet at d = 1.44 ppm in
[D6]DMSO. In D2O, NH/NH2 signals were not observed due
to H/D exchange. On coordination to the metal center, the
proton spectra undergo drastic changes, and the NH2/NH
protons are detected as three significantly downshifted signals (see Table S2 in the Supporting Information), indicating
electron donation by the diethylenetriamine ligand to the
RuII center. The NMR spectra of [(h6-p-cymene)RuACHTUNGRE(dien)]-
FULL PAPER
ACHTUNGRE(PF6)2 (3 a) and [(h6-biphenyl)RuACHTUNGRE(dien)]ACHTUNGRE(PF6)2 (3 b) were
also recorded in various solvents, but no significant differences were found for the resonances of the arene rings. Methyl
and methylene proton resonances were also insensitive to
the nature of the solvent, unlike those of the amino groups.
In both complexes, the NH/NH2 proton resonances of coordinated diethylenetriamine recorded in [D6]DMSO lie
downfield (Dd = 1.06 and 0.57 ppm for 3 a and 3 b, respectively) with respect to those recorded in CD3OD and D2O.
The NH/NH2 protons of the diethylenetriamine ligand in 3 a
and 3 b undergo slow H/D exchange in protic solvents
ACHTUNGRE(>48 h), but it can be accelerated by addition of NaOD to
be complete within several minutes. The protons of the
ammine ligands in 1 a, 1 b, 2 a, 2 b, and 4 b undergo fast H/D
exchange (see Figure S1 in the Supporting Information),
which results in the disappearance of the corresponding signals in the 1H NMR spectra within several hours. Therefore,
coordination of chelating triethylenetriamine results in a
more pronounced reduction of the rate of H/D exchange in
protic solvent compared to monodentate ammine ligands.
X-ray structure determination: The molecular structures of
[(h6-biphenyl)RuIIACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 b), [(h6-p-cymene)RuIIACHTUNGRE(dien)]ACHTUNGRE(PF6)(Cl) (3 a), [(h6biphenyl)RuII(en)ACHTUNGRE(NH3)]ACHTUNGRE(PF6)2
(4 b), and [(h6-p-cymene)RuIIACHTUNGRE(NH3)Cl2]ACHTUNGRE[(Et3NH)ACHTUNGRE(PF6)] (6 a)
were determined by X-ray diffraction analysis (Figures 1 and
2, see Tables S1 and S2 in the
Supporting Information for
crystallographic data, bond
lengths, and bond angles).
Single crystals of the complexes
Figure 1. Molecular structure of [(h6-biphenyl)RuIIACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 b, left) and space-filling model (right)
were grown by slow diffusion of
showing p–p stacking interactions between the phenyl rings of two molecules. The PF6 anion has been omitdiethyl ether into saturated
ted for clarity. Selected bond lengths [] and angles [8]: Ru1 CACHTUNGRE(1–6)av 2.1826(35), Ru1 Cl1 2.4125(4), Ru1 N1
2.1417(14), Ru1 N2 2.1324(14); N1-Ru1-Cl1 83.53(4), N2-Ru-Cl1 83.36(4), N1-Ru-N2 82.88(6).
methanol solutions at 277 K.
Figure 2. Molecular structures of dicationic [(h6-p-cymene)RuIIACHTUNGRE(dien)]ACHTUNGRE(PF6)2 (3 a, left) and [(h6-biphenyl)RuII(en)ACHTUNGRE(NH3)]ACHTUNGRE(PF6)2 (4 b, center) and neutral
[(h6-p-cymene)RuIIACHTUNGRE(NH3)Cl2]ACHTUNGRE[(Et3NH)ACHTUNGRE(PF6)] (6 a, right). The PF6 anions and Et3NH + were omitted for clarity. Selected bond lengths [] and angles [8]
for 3 a: Ru1 CACHTUNGRE(1–6)av 2.190(1), Ru1 N1 2.120(5), Ru1 N2 2.127(5), Ru1 N3 2.130(5); N1-Ru1-N2 79.4(2), N3-Ru-N1 88.39(19), N2-Ru-N3 77.61(19).
For 4 b: Ru1 CACHTUNGRE(1–6)av 2.1937(52), Ru1 N1 2.1515(17), Ru1 N2 2.1324(17), Ru1 N3 2.1388(17), N1-Ru1-N2 84.79(7), N3-Ru-N1 88.35(7), N2-Ru-N3
79.39(7). For 6 a: The complex cation contains a reflection plane passing through Ru1, N1, C1, C4, C5, and C6; Ru1 CACHTUNGRE(1–6)av 2.170(8), Ru1 Cl1
2.4157(6), Ru1 Cl2 2.4157(6), Ru1 N1 2.130(3); N1-Ru1-Cl1 83.26(6), N1-Ru1-Cl2 83.26(6), Cl1-Ru1-Cl2 85.10.
Chem. Eur. J. 2013, 19, 4308 – 4318
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4313
C. G. Hartinger et al.
The pseudo-octahedral coordination environment of the
RuII center consists of Cl (2 b, 6 a), and NH3 (2 b, 4 b, 6 a) or
chelating ligands (3 a, 4 b) and the h6-arene ring. The unit
cell of 6 a also contains a triethylammonium hexafluorophosphate ion pair.
In general, the geometrical parameters of all complexes
are very similar. The Ru Cl bond length does not vary significantly with the coordinated arene [2.4125(4) for [(h6biphenyl)RuIIACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 b) and 2.4146(4) for [(h6cymene)RuIIACHTUNGRE(NH3)2Cl]ACHTUNGRE(PF6) (2 a)],[23g] which is in accordance
with observations for the mono-ammine complexes [(h6arene)RuIIACHTUNGRE(NH3)Cl2] (5 a,b) (2.421(2), 2.427(2) and
2.4246(9), 2.4284(8) for cymene/biphenyl respectively).[17] In
contrast, the Ru Cl bond in the ethylenediamine complexes
[(h6-arene)RuII(en)Cl]PF6 (7 a,b) is remarkably longer in the
cymene complex [2.4418(8) (7 a) vs. 2.4080(15) (7 b)].[19]
The Ru N bond lengths in 2 b, 3 a, 4 b, and 6 a are in the
range 2.120–2.152 , and N-Ru-N angles vary between 79
and 888. These values are similar to those reported previously for related RuIIACHTUNGRE(arene) complexes.[19, 23g] The Ru Phcentroid
distance in 2 b (1.662 ) is slightly longer than that of its
cymene analogue 2 a (1.659 ), which is again in accordance
with mono-ammine complexes, although less pronounced
(1.670 for 5 b vs. 1.657 for 5 a).[17] Furthermore, the
slightly longer bond lengths for Ru biphenyl correlate with
the higher p acidity compared to p-cymene, which leads to a
partial filling of the antibonding orbitals of the Ru arene
bonds. All structures feature longer Ru Csubstituted than Ru
CH bonds. In the X-ray structure of 2 b, but not of 4 b, intermolecular p stacking was observed in a parallel offset fashion (Figure 1) with a shortest interatomic distance of
3.347 and a Phcentroid···Phcentroid distance of 3.823 . In contrast to structurally related ruthenium(II) half-sandwich
complexes bearing biphenyl moieties, in which one phenyl
ring is twisted out of the plane by around 23.3(9)8 (7 b)[19] or
39.5(5)8 (5 b),[17] the biphenyl ligand in 2 b is surprisingly
almost planar (0.3(3)8). In 4 b, the phenyl rings of the biphenyl ligand are tilted from coplanarity by 28.6(3)8.
Hydrolysis and stability studies: Hydrolysis and stability
tests were carried out for the complexes prior to investigation of their reactivity toward biomolecules. The stability of
the complexes was investigated in water and in 0.1 m (simulating blood plasma conditions) and 1 m aqueous NaCl solutions by means of 1H NMR spectroscopy. It is known that
2 a,b,[23g] 5 a,b[19] and 7 a,b[17] form mono- and diaqua complexes in water through substitution of chlorido ligands with
water molecules. While aquation of the ammine complexes
2 a,b, 5 a,b and 6 a was not suppressed even in 1 m NaCl solution, the ethylenediamine species 7 a,b remained intact for
24 h in 0.1 m NaCl. The complexes without labile chlorido ligands (i.e., 1 a,b, 3 a,b and 4 b) are inert toward hydrolysis in
aqueous solution. In long-term stability studies (up to 90 d),
a second set of peaks was observed in the NMR spectra
after about 10 d (shifted upfield by ca. 0.3 ppm and at 6 %
relative intensity, see Figure S1 in the Supporting Information). The intensity of this set of peaks remained constant
4314
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over the following 80 d, which is indicative of the establishment of an equilibrium. Off-line electrospray ionization
mass spectrometry (ESI-MS) measurements of the NMR
samples containing 1 b and 3 b showed identical species as
observed after 24 h (see below). However, additional hydroxido- and methoxido-bridged dimers were detected in
the mass spectrum of 1 b. This suggests that the above-mentioned hydrolysis products may be formed after ammine
ligand cleavage.
Furthermore, the stability of the biphenyl compounds in
aqueous solution was assayed by ESI-MS, which largely confirmed the observations made by NMR spectroscopy. However, labile monodentate ligands, such as ammine or chlorido, attached to a ruthenium center may be cleaved to a considerable extent even during the soft electrospray process.
Such cleavage is observed at a dry gas temperature as low
as 80 8C and leads to the formation of dinuclear hydrolysis
products with bridging solvent molecules, which are usually
regarded as an inactive form of RuII arene anticancer
agents.[25] Accordingly, the most abundant signal in the mass
spectrum of 2 b was assigned to a dinuclear hydrolysis product after cleavage of the monodentate ligands, namely,
[(bip)2Ru2ACHTUNGRE(m-OCH3)3] + (m/z 604.87 0.1, mex = 605.02; bip =
h6-biphenyl), observed throughout the entire incubation
period. Similar dinuclear methoxido-containing hydrolysis
products were also observed in the mass spectra of 5 b. The
methoxide ligands probably stem from the dilution process
with water:methanol (1:1) prior to ESI-MS measurements.
In case of 1 b, the most abundant signal was found at m/z
288.92 0.01, corresponding to a [(bip)RuACHTUNGRE(NH3)ACHTUNGRE(NH2)] +
fragment (mex = 289.03). The isotope pattern suggests an
RuI/RuII redox couple in form of [(bip)RuACHTUNGRE(NH3)2] + and
[(bip)RuACHTUNGRE(NH3) H] + and a ratio of 1:0.75, while dimeric hydrolysis products were not observed. Due to the lability of
the investigated monodentate ligands under the conditions
applied in the MS experiments, unequivocal conclusions
could not be drawn on the stability of the complexes in
aqueous solution. In contrast, complexes containing chelating di- or triamines (e.g., 3 b, 4 b, and 7 b) exhibited higher
stability during the spraying process. Their mass spectra remained constant over the entire incubation period, and
therefore these compounds are believed to be stable for at
least 48 h in aqueous solution. The most abundant signals in
the mass spectra of 7 b were assigned to [(bip)Ru(en)Cl] +
(m/z 350.91 0.01, mex = 351.02) and [(bip)Ru(en) H] + (m/z
314.93 0.01, mex = 315.04); the latter was also observed in
the spectra of 4 b. This provides further proof of the lability
of monodentate ammine ligands during the electrospray
process, since no further assignable signals were detected for
4 b. The mass spectrum recorded for dicationic 3 b showed
solely the doubly charged [M]2 + ion (m/z 179.44 0.01,
mex = 179.55).
Reactivity toward proteins: Understanding the interaction
of novel metallodrugs with proteins is of particular interest,
since anticancer drugs are mostly administered intravenously into the blood stream, where they are exposed to plasma
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 4308 – 4318
Ruthenium Complexes in Anticancer Drug Development
proteins. Electrospray ionization mass spectrometry (ESIMS) has proven to be a very suitable tool for the investigation and monitoring of such interactions. In this study, incubations were carried out at 2:1 compound-to-protein molar
ratio in aqueous solution. The reactivity of 2 b, 3 b, and 7 b
was investigated toward ubiquitin (ub), cytochrome c (cyt),
and a mixture containing equimolar amounts of ub and cyt.
The MS studies were carried out in high-resolution time-offlight (ToF) mode. Mass spectra of the incubation mixtures
were recorded under denaturing conditions by adding 50 %
methanol and 0.2 % formic acid prior to injection to ensure
proper unfolding and protonation of the protein. Because of
the absence of labile ligands, 3 b was used as a negative control, and accordingly no protein adducts were observed
when it was incubated with ub, cyt, or the ub–cyt mixture
for 48 h.
Compound 2 b reacted readily with ub, and after 48 h extensive depletion of free ub (3 %) in favor of mono- (45 %)
and bis-adducts (52 %) was observed (Figure 3). The
detected masses correspond to a mono-adduct of the type
Figure 3. Deconvoluted mass spectra recorded for the incubation mixtures containing ub and 3 b (a), 7 b (b), or 2 b (c) after 48 h. The reaction
mixtures were incubated at a compound-to-protein ratio of 2:1 at 37 8C
in the dark.
[ub + (bip)Ru] + (8818.6031 Da,
mex = 8818.6051 Da, 0.2 ppm),
while free ub was found at
8564.6299 Da
(mex =
8564.6304 Da, 0.1 ppm) in the
deconvoluted mass spectrum
(Figure S2 of the Supporting Information), and a bis-adduct
with two (bip)Ru moieties was
detected at 9071.5659 Da (mex =
9071.5800 Da, 1.6 ppm). In case
of 2 b the interaction with pro-
Chem. Eur. J. 2013, 19, 4308 – 4318
FULL PAPER
teins is accompanied by cleavage of the ammine ligands, as
observed previously with pyridonato complexes,[18d] although
in-source ligand cleavage cannot be completely excluded
(see above). The interaction between 2 b and cyt resulted in
extensive metallation of the protein including higher order
adducts at low abundance relative to cyt (12358.4023 Da,
mex = 12358.3405 Da, 5 ppm). In particular, adducts corresponding to [cyt + RuACHTUNGRE(HCOO)] + (12504.3070 Da, mex =
12504.2310 Da,
6 ppm,
15 %),
[cyt + (bip)Ru] +
(12613.3526 Da, mex = 12613.3244 Da, 2 ppm, 26 %), and
[cyt + (bip)Ru + RuACHTUNGRE(HCOO)] +
(12757.2472 Da,
mex =
12757.1985 Da, 4 ppm, 18 %) were detected after 48 h. Similar to the stability studies, no adducts bearing monodentate
ammine ligands were observed. Of interest is the observation of the signal at 12504.33 Da. A previous study attributed this signal to [cyt + PF6 ] (12 505 Da).[23f] However, in the
present case, the high-resolution mass spectrometric data
and isotopic distribution suggest that it rather corresponds
to a [cyt + RuACHTUNGRE(HCOO)] + adduct (12504.23 Da), in which all
of the ligands including the arene were cleaved (see Figure S2 in the Supporting Information). This may be related
to oxidation of ruthenium to an RuIII species. This difference
in adduct formation between the two data sets may be attributed to differences in experimental conditions. Low
metal (and therefore PF6 ) concentrations and small metalto-protein molar ratios thereby seem to favor formation of
the [cyt + RuACHTUNGRE(HCOO)] + adduct. Additionally, no comparable adduct was observed in the reaction with ub. The reaction of 2 b with a 1:1 ub:cyt mixture yielded similar results
to the single-protein incubations. Complex 2 b seems to bind
preferentially to ub forming mainly [ub + (bip)Ru] + monoadducts, whereas mono-adduct formation with cyt was only
observed at low relative abundance (Table 1).
Compound 7 b formed monofunctional adducts with ub of
the type [ub + (bip)Ru(en)] +
(8878.6821 Da, mex =
8878.6676 Da, 2 ppm, 42 %), as reported for the undecapeptide substance P.[26] Retention of the ethylenediamine ligand
is related to the stability of the coordinative bond between
the N,N-bidentate ligand and the Ru center. In addition, the
bis-adduct [ub + 2ACHTUNGRE(bip)Ru(en)] + (9191.7175 Da, mex =
9191.7038 Da, 2 ppm, 15 %) was detected after 48 h. Incubation of 7 b with cyt yielded several metal–protein adducts
within 48 h, and both mono- and bis-adducts were observed
in considerable quantities. Similar to ub, these adducts corresponded to the [(bip)Ru(en)] + moiety attached to the pro-
Table 1. List of the detected metallodrug–protein adducts and their associated relative intensities during 2:1
incubation of 2 b, 3 b, or 7 b with ubiquitin (ub), cytochrome c (cyt), or an ub–cyt mixture after 48 h.
Compound
ub
adduct type
I [%]
adduct type
cyt
I [%]
2b
+ (bip)Ru
+ 2 (bip)Ru
45
52
26
15
18
3b
7b
–
+ (bip)Ru(en)
+ 2 (bip)Ru(en)
42
15
+ (bip)Ru
+ RuACHTUNGRE(HCOO)
+ (bip)Ru + RuACHTUNGRE(HCOO)
–
+ (bip)Ru(en)
+ 2 (bip)Ru(en)
+ RuACHTUNGRE(HCOO)
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
30
6
8
ub–cyt mixture
adduct type
I [%]
ub + (bip)Ru
ub + 2ACHTUNGRE(bip)Ru
cyt + (bip)Ru
–
ub + (bip)Ru(en)
cyt + (bip)Ru(en)
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84
5
9
22
13
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C. G. Hartinger et al.
tein, that is, [cyt + (bip)Ru(en)] + (12672.4068 Da, mex =
12672.3790 Da, 2 ppm, 30 %) and [cyt + 2ACHTUNGRE(bip)Ru(en)] +
(12986.4451 Da, mex = 12986.4169 Da, 2 ppm, 8 %). Like for
2 b, the [cyt + RuACHTUNGRE(HCOO)] + adduct was observed, albeit at
low abundance (Table 1). When 7 b was incubated with the
ub–cyt mixture, it reacted primarily with ub to form [ub +
(bip)Ru(en)] + , similarly to 2 b, whereas the analogous cyt
adducts are much less pronounced.
In analogy to the proposed inverse correlation between
extent of protein binding of a metallodrug and cytotoxic activity,[18g] 2 b is expected to be only active to a limited extent
in in vitro assays due to pronounced binding to proteins and
ligand cleavage. In contrast, 7 b displays a reduced rate of
binding to proteins and also retains the ligand, which is anticipated to result in elevated anticancer activity. Compound
3 b does not react at all with proteins and would in principle
be expected to show increased cytotoxicity. However, the
absence of a leaving group does not allow conclusions on
antitumor properties to be made in this case.
Interaction with 9-ethylguanine as a model for DNA binding: DNA binding is responsible for the antitumor activity
of platinum anticancer agents.[14b] To evaluate the binding
capability of representative complexes to DNA, 2 b, 5 b, and
7 b were treated with the DNA model 9-ethylguanine (EtG)
and the reaction mixtures were analyzed by ESI-MS. In contrast to 5 b and 2 b, for which no interaction with EtG was
observable, 7 b interacts specifically with the model purine.
Mono-adducts are believed to form through hydrolysis of
the chlorido leaving group and subsequent coordination of
EtG to the RuII center. ESI-IT mass spectra featured peaks
assignable to [(bip)Ru(en)ACHTUNGRE(EtG)PF6] + (m/z 639.93 0.01,
mex = 640.10), [(bip)Ru(en)ACHTUNGRE(EtG)] + (m/z 493.97 0.01, mex =
494.12), and [(bip)Ru(en)ACHTUNGRE(EtG)]2 + (m/z 247.45 0.01, mex =
247.57), as well as free EtG (m/z 180.01 0.01). CID
tandem mass spectrometric experiments on the parent signal
at m/z 494 gave peaks at m/z 180.01 and 314.93 for EtG and
[(bip)Ru(en)] + , respectively, confirming adduct formation
of 7 b with EtG. The total percentage of signals attributable
to 7 b–EtG adducts increased from 12 % after 3 h to 78 %
after 48 h compared to all signals assigned to free 7 b
(Table S4 in the Supporting Information). The stability of
the 7 b–DNA adducts may be related to additional C6=
O···HN hydrogen-bond formation between guanine and the
ethylenediamine ligand.[27]
Inhibition of cancer cell growth: The in vitro anticancer activity of the Ru complexes was determined in ovarian
(CH1), colon (SW480), and non-small cell lung carcinoma
(A549) cells by means of the colorimetric MTT assay with
an exposure time of 96 h (see Table 2 for IC50 values; concentration–effect curves are shown in Figure S3 in the Supporting Information). CH1 cells are significantly more chemosensitive to the complexes under investigation than
SW480 and A549 cells. In general, the biphenyl complexes
are more cytotoxic than their p-cymene counterparts,[16b] and
only in case of 7 a and 7 b were both compounds approxi-
4316
www.chemeurj.org
Table 2. Cytotoxicity of ruthenium complexes 1–7 and cisplatin given as
50 % inhibitory concentrations (IC50) in CH1 (ovarian carcinoma), A549
(non-small cell lung cancer), and SW480 (colon carcinoma) cells, determined by means of the MTT assay. Values are means plus/minus standard
deviations obtained from at least three independent experiments with exposure times of 96 h.
Compound
1a
1b
2a
2b
3a
3b
4b
5a
5b
6a
7a
7b
cisplatin[28]
IC50 [mm]
CH1
404 29
91 17
400 20
3.3 0.3
> 640
54 5
30 3
258 21
85 13
343 50
7.3 0.1
6.2 0.5
0.14 0.03
SW480
474 9
272 20
586 15
16 2
> 640
204 16
78 8
360 26
298 21
454 19
5.9 0.7
12 2
3.3 0.4
A549
550 16
494 54
> 640
68 11
> 640
376 20
258 12
> 640
566 8
580 8
8.8 0.4
9.2 1.9
1.3 0.4
mately equally potent in all cell lines, with IC50 values
mostly lower than 10 mm. This might be related to improved
accumulation of more lipophilic complexes in cells, facilitated by diffusion of such compounds across membrane barriers. Furthermore, the arene ligand influences the reaction
with biological targets. Biphenyl complexes may undergo p–
p stacking interactions with nucleobases, leading to intercalation into DNA. However, it seems that the cytotoxicity of
the compounds is strongly related to their ability to form covalent bonds, and indeed complexes with labile chlorido ligands were found to undergo quick aquation and yield the
lowest IC50 values in the in vitro assays (2 b, 7 a, 7 b), whereas 1 a, 1 b, 3 a, 3 b, and 4 b with three monodentate ammine
or a tridentate amine ligand are virtually noncytotoxic,
which may be attributed to the absence of a leaving group.
Interestingly, 4 b shows medium in vitro activity, but seems
drastically more active in CH1 and SW480 cell lines than
1 a,b and 3 a,b. This may be due to the monodentate ammine
ligand, which under cellular conditions may nonetheless be
cleaved. The resulting complex is then identical to hydrolyzed 7 b. However, the doubly charged complexes are expected to show less efficient cellular uptake compared to 2 b
and 7 a,b and therefore reduced cytotoxicity. These results
also confirmed the predictions from protein binding assays.
On the contrary, the low activity of 2 a, 5 a, 5 b, and 6 a may
be attributed to their instability in organic and especially in
aqueous media, in which hydrolysis is not suppressible even
by addition of 1 m NaCl, and side reactions may occur already in the cell culture medium prior to contact with the
cells. This may also explain why the presence of two labile
chlorido ligands (5 a, 5 b, and 6 a) does not seem to be more
advantageous than the presence of only one (2 a, 2 b) despite
the charge of the latter complexes, although the presence of
two labile ligands is one of the prerequisites for the strong
cytotoxicity of cisplatin (Table 2). Remarkably, neutral
ruthenium complexes 5 a and 6 a, which contain noncova-
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 4308 – 4318
Ruthenium Complexes in Anticancer Drug Development
lently bound base–HPF6 ion pairs, show a difference in cytotoxicity, which may depend on the base strength, whereby
the former is slightly more active.
Conclusion
Ruthenium arene complexes of the bidentate ligand ethylACHTUNGREenediamine are potent anticancer agents and have the potential to overcome resistance of tumors to cisplatin. In a
systematic study by varying the am(m)ine and arene ligands
of Ru half-sandwich compounds, several important parameters for structure–activity relationships could be derived
when evaluating the in vitro anticancer activity of the compounds in human tumor cell lines (CH1, A549, and SW480).
The cytotoxicity of the complexes strongly depends on the
denticity of the ligand, and IC50 values varying by several
orders of magnitude were observed. The activity of the complexes appears to be related to their aqueous stability. It is
known that classic metal-based drugs serve as prodrugs and
are activated by aquation to undergo interactions with biomolecules and exert antiproliferative activity. Inside the cell,
activation can be achieved by slow hydrolysis of anionic ligands such as chloride in environments of low chloride concentration, while outside the cell the higher chloride ion
concentration shifts the equilibrium to the chlorido complex,
preventing formation of the active species. Compounds
which are quickly hydrolyzed in aqueous (NaCl-containing)
solution were found to be inactive in the anticancer assays,
as were compounds which are too reactive towards proteins.
These properties are of relevance to incubation with tumor
cells in cell culture medium. These observations are in line
with previous reports on organoruthenium complexes with
three monodentate ligands such as acetonitrile or isonicotinACHTUNGREamide,[19] and also with some more weakly bonding bidentate ligands.[18a, c, g] Furthermore, compounds lacking leaving
groups were less active due to their too high stability. Overall, these facts indicate the necessity of covalent bond formation of the anticancer agent with the intracellular target
for exerting anticancer activity.
Acknowledgements
This work was supported by the Platform Austria for Chemical Biology
of the Genome Research Program Austria (GEN-AU, BMWF-70.081/
0018-II/1a/2008), COST D39, and CM0902. We gratefully acknowledge
Prof. Vladimir Arion for the refinement of X-ray diffraction data and
Prof. Markus Galanski for recording the 2D NMR spectra.
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Received: July 26, 2012
Revised: December 12, 2012
Published online: January 22, 2013
Chem. Eur. J. 2013, 19, 4308 – 4318