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Selective, cytotoxic organoruthenium(II) full-sandwich complexes: a structural, computational and in vitro biological study.
FULL PAPERS
DOI: 10.1002/asia.201100637
Selective, Cytotoxic Organoruthenium(II) Full-Sandwich Complexes: A
Structural, Computational and In Vitro Biological Study
Bradley T. Loughrey,*[a] Benjamin V. Cunning,[b] Peter C. Healy,[a]
Christopher L. Brown,[b] Peter G. Parsons,[c] and Michael L. Williams[a]
tential, with the cationic charge of the
[RuCp*] + moiety completely delocalizing throughout the molecular structure
of each metallocene. In vitro cytotoxicity studies demonstrate these delocalized lipophilic cations to be potent
growth inhibitors of eleven unique tumorigenic cell lines, while exhibiting
Abstract: A structurally diverse range
of lipophilic, cationic h6-arene h5-cyclopentadienyl (h5-Cp*) full-sandwich
complexes of ruthenium(II) have been
prepared and structurally characterized
by Fourier-transform IR and NMR
spectroscopy, electrospray mass spectrometry, and elemental microanalyses.
Computational experiments incorporating the Hartree–Fock theory and the
second-order Møller–Plesset perturbation theory predict each complex to
possess a uniform d+ electrostatic po-
Keywords: arenes · cancer · cyclopentadienyl ligands · organometallic · ruthenium
Introduction
The significant involvement of metal ions and complexes in
biological processes and systems has, over recent times, led
to the realization that considerable opportunities exist for
the design of metal-based therapeutics.[1] The landmark antitumor properties of cisplatin proved to be the herald of this
new area of metallopharmaceutical research, with cisplatin
and its numerous derivatives now firmly established as some
of the most effective chemotherapeutic agents in clinical
use.[2] Despite their overall success and clinical popularity,
these platinum analogues suffer from disadvantages such as
high secondary toxicity and both intrinsic and acquired drug
resistance. Due to these limitations, it is of importance to
discover metallopharmaceuticals that possess higher efficacy
and activity while exerting fewer side-effects. In pursuit of
this goal, a diverse array of complexes containing various
[a] Dr. B. T. Loughrey, Prof. P. C. Healy, Dr. M. L. Williams
Eskitis Institute for Cell and Molecular Therapies
Griffith University
Brisbane (Australia)
Tel: (+ 61) 07-373-57728
E-mail: bradley.loughrey@griffithuni.edu.au
[b] B. V. Cunning, Prof. C. L. Brown
Queensland Micro- and Nanotechnology Facility
Griffith University
Brisbane (Australia)
[c] Prof. P. G. Parsons
Drug Discovery Group
Queensland Institute of Medical Research
Brisbane (Australia)
112
significantly lower levels of toxicity towards both a normal human fibroblast
and a mouse macrophage cell line.
Single-crystal X-ray structural determinations are additionally reported for
five
complexes,
[RuACHTUNGRE(h6-C6H5
5
ACHTUNGRE(CH2)2CH3)ACHTUNGRE(h -C5ACHTUNGRE(CH3)5)]BPh4,
[Ru(h6-C6H5CO2CH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BF4,
[RuACHTUNGRE(h6-C10H8)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4,
[Ru(h6-C14H10)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4, and [Ru(h6-C16H10)ACHTUNGRE(h5C5ACHTUNGRE(CH3)5)]BPh4.
transition metals have been evaluated, and elements such as
cobalt, gallium, gold, iron, osmium, rhodium, and ruthenium
have all produced promising libraries of novel anticancer
complexes.[3] Out of the assortment of metallodrugs that
contain metals other than platinum, ruthenium compounds
have proven to be the most promising ones, with two coordination complexes, KP1019 and NAMI-A, having successfully
completed phase I clinical trials.[4]
While the biological properties of inorganic coordination
complexes have been thoroughly explored over the past
decade, organometallic compounds have only been sparingly
investigated, with recent results suggesting that these systems hold the potential to find use as therapeutic agents.[5]
Essentially every class of metal–carbon bond has demonstrated some form of biological activity, including metal carbonyls, metal alkyls (found in naturally occurring systems
such as the B12 series of vitamers), metal carbenes, organometallic compounds comprising metal–metal bonds, and
metal–arene p-bond systems.[6] Ruthenium compounds comprising metal–arene bonds have seen particular success, with
a plethora of interesting biological results having already
been published for the RuII h6-arene half-sandwich (pianostool) complexes.[5] The most prevalent examples of these
molecules within the literature are the [RuACHTUNGRE(h6-arene)ACHTUNGRE(Y
Z)L] + (Y-Z = bidentate ligand, and L = monodentate anion)
and RAPTA [RuACHTUNGRE(h6-arene)ACHTUNGRE(Y-Z)PTA] (PTA = 1,3,5-triaza-7phospha-adamantane) series of complexes, which display
promising cytotoxic and antimetastatic activities, respectively.[7] Cationic organoruthenium full-sandwich complexes of
the structure [RuACHTUNGRE(h6-arene)ACHTUNGRE(h5-C5R5)] + , in which R = H (Cp)
or CH3 (Cp*), have also been demonstrated to possess inter-
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Chem. Asian J. 2012, 7, 112 – 121
esting in vitro biological activity.[8] The Cp derivatives, despite only exhibiting minimal cytotoxicity against human
promyelocytic leukemia cell line HL-60, were found to interact with plasmid pBR322 DNA when investigated by atomic
force microscopy, inducing significant supercoiling and kinking of the free DNA form. The Cp* derivatives, which are
investigated in our research group, were found to exhibit
potent cytotoxic activity in vitro, thereby inhibiting the
growth of tumorigenic cell lines at concentrations comparable to that of cisplatin. The promising antitumor effects displayed by these cationic organoruthenium molecules
prompted us to synthesize and biologically evaluate structurally diverse libraries of ruthenium(II)-based full-sandwich
complexes (Figure 1; in which R represents a series of sub-
chondrial membranes.[10] Once inside the mitochondrial organelle, they concentrate within the lipid boundary in response to the negative mitochondrial membrane potential,
ultimately triggering cell death through the mitochondrial
pathway.[10]
In the present study, we endeavored to gain greater insight into the cytotoxic profile of these lipophilic organoruthenium cations through the in vitro evaluation of a structurally diverse series of molecules against a panel of thirteen
different cell lines (including both tumorigenic and normal
cells). Computational and structural experiments were also
performed as a means of investigating how the cationic
charge of the [RuIICp*] + moiety is distributed throughout
the molecular structure of each metallocene.
Results and Discussion
Synthesis, Characterization, and Structural Analysis
Figure 1. A focused library of organoruthenium full-sandwich complexes
previously evaluated for antitumor activity; R represents substituted
functional groups such as carboxylic acids, acid fluorides, esters, thioesters, ketones, alcohols, carbamates, amides, sulphonamides, and glycoconjugates.
stituted functional groups including carboxylic acids, acid
fluorides, esters, thioesters, ketones, alcohols, carbamates,
amides, sulphonamides, and glycoconjugates.[9] The results of
these studies demonstrated the cationic organoruthenium
complexes to possess potent and selective antiproliferative
activity towards a range of cancerous cell lines in vitro, including human skin carcinoma (MM96L) and two individual
phenotypes of breast cancer (MCF7 and MDA-MB-231),
with the degree of growth inhibition dependent on the size
and lipophilicity of the arene ligand.[9] Of particular interest,
however, was the relative inactivity of the neutral ruthenocenyl complexes when compared to their corresponding cationic derivatives. These mono-, 1,1’-di-, and pentasubstituted
ruthenocenyl molecules were on average over two orders of
magnitude less active than the respective cationic complexes, highlighting a relationship between positive charge
and cytotoxic activity.[9] This structural dependence on both
lipophilicity and a cationic charge, in addition to the notable
specificity displayed by these complexes towards cancerous
cells, thus suggests that these highly stable organoruthenium
molecules may be exerting their cytotoxic effect in a
manner similar to that of biologically active delocalized lipophilic cations (DLCs). As the name suggests, DLCs are lipophilic compounds that carry a positive charge delocalized
across the surface of their molecular structure. DLCs have
been shown through a number of studies to possess the capability to rapidly permeate cellular plasma and inner mito-
Chem. Asian J. 2012, 7, 112 – 121
Modern preparation of h6-arene ruthenium h5-pentamethylcyclopentadienyl complexes can be achieved through a plethora of multi-step synthetic routes, with the most popular
methods often involving the preparation of labile, dimeric
ruthenium starting materials (either [RuACHTUNGRE(h6-C6H6)Cl2]2 or
[Cp*RuCl2]2).[11] The former readily reacts with HCp* under
ultraviolet radiation in the presence of acetonitrile as solvent to form [(arene)RuACHTUNGRE(Cp*)] + complexes, while the latter
opens up a variety of additional synthetic mechanisms involving aromatic ligands and various reducing agents.[12] Reduction of [Cp*RuCl2]2 with LiBEt3H yields a ruthenium(II)
tetramer of the structure [Cp*RuACHTUNGRE(m3-Cl)]4, which undergoes
facile halide abstraction when exposed to AgCF3SO3 in acetonitrile. This forms the solvated, labile half-sandwich complex [Cp*RuACHTUNGRE(CH3CN)3]CF3SO3, which can further react in
the presence of aromatic ligands to produce [RuACHTUNGRE(arene)ACHTUNGRE(Cp*)] + complexes in good yield. The highly arenophilic
nature of [Cp*RuACHTUNGRE(m3-Cl)]4 can also be exploited in aqueous
media under microwave irradiation, with exposure of the
tetramer to aromatic ligands under these conditions, thus
forming a diverse range of water soluble [(Cp*)RuACHTUNGRE(arene)]Cl salts with no noticeable by-products. An alternate route involves the quantitative, two-step formation of
alkoxoruthenium(II) complexes [Cp*RuACHTUNGRE(O-alkyl)]2 through
the reaction between [Cp*RuCl2]2 and potassium carbonate
in various alcohol solvents. These alkoxo intermediates,
when in the presence of an aromatic ligand and suitable
proton donor, will readily facilitate arene metalation. A similar procedure achieving comparable yields has also been
undertaken using NaOCH3 as a direct replacement to potassium carbonate. Preparative routes to h6-arene ruthenium
h5-pentamethylcyclopentadienyl cations that did not involve
the use of ruthenium dimer complexes were originally pioneered by Kudinov et al.,[13] with the initial method involving the reaction between ruthenium trichloride hydrate, aromatic ligands, and HCp* in refluxing ethanol.
The prior described methodologies each possess their own
inherent advantages and disadvantages, with each procedure
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B. T. Loughrey et al.
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ultimately capable of preparing various libraries of [RuACHTUNGRE(arene)ACHTUNGRE(Cp*)] + full-sandwich complexes in good to moderate yields. For the purposes of our investigations, it was of
interest to incorporate a synthetic procedure that was facile,
convenient, and eligible for use with a wide variety of aromatic starting materials. As these organoruthenium complexes were to undergo in vitro cytotoxic evaluation, it was
also necessary to ensure that the chosen synthetic technique
produced analytically pure samples of the desired product.
To fulfill these requirements, we incorporated a modified
version of the aforementioned synthetic scheme developed
by Kudinov and co-workers. The devised method is a facile,
one-pot procedure that affords the user the capability of
preparing structurally diverse libraries of h6-arene ruthenium h5-pentamethylcyclopentadienyl cations by a combinatorial synthetic approach. This approach proceeds readily
without the use of ultraviolet or microwave irradiation, and
avoids the requirement of preparing highly reactive starting
materials (such as [Cp*RuACHTUNGRE(m3-Cl)]4 and [Cp*RuACHTUNGRE(CH3CN)3])
prior to use. Incorporation of this method (Scheme 1) af-
Scheme 1. Synthesis of ruthenium(II) arene Cp* full-sandwich complexes.
Aromatic protons are labeled as per their 1H NMR assignment; M designates the site of metal complexation.
forded the preparation of a series of cationic organoruthenium full-sandwich complexes (1–12) as air stable, crystalline
tetraphenylborate salts. The reaction is initiated through
heating rutheniumACHTUNGRE(III) trichloride hydrate in ethanol under
reflux conditions. The alcohol acts as a gentle reducing
agent, and this facilitates conversion of the rutheniumACHTUNGRE(III)
transition metal into its +2 oxidation state. The arene ligand
(Ar) and HCp* are then introduced, thereby prompting formation of the organoruthenium sandwich complex over a
period of 8–12 hours. Following aqueous workup, the complex is isolated as a solid, crystalline material through a
metathesis reaction involving an aqueous solution of sodium
tetraphenylborate of appropriate strength. Pure samples of
each complex are obtained after alumina filtration and recrystallization from acetone. Complexes were characterized
using 1H and 13C NMR spectroscopy, ESI-MS, FT-IR spectroscopy, and elemental microanalysis. Single-crystal X-ray
structural determinations of complexes 4, 5 a, and 10–12
were also performed.
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The p complexation of the [RuIICp*] + moiety to each aromatic ligand was confirmed post-synthesis by the characteristic upfield shift of the aromatic protons in the 1H NMR
spectra (Table 1). The obtained 13C NMR data were in good
agreement with the 1H NMR results, thus presenting the expected shielding of the h6-C6H6 carbons. The NMR data
were also of use in determining the ruthenium metal coordination site for complexes incorporating polycyclic aromatic
ligands (i.e., naphthalene (10), phenanthrene (11), and
pyrene (12)). For each multi-ring system the metal undergoes p complexation to a terminal ring, thus shifting the
1
H NMR signals for this phenyl group upfield, while the
NMR properties of the noncomplexed aromatic protons
remain relatively unchanged. An X-ray crystallographic
analysis of complexes (10–12) supports this result, with
ORTEP representations (Figure 2) clearly demonstrating
the ruthenium metal to favor p complexation to the terminal
ring of each polycyclic ligand. These observations are in accordance with Clars aromatic sextet theory, which predicts
the terminal ring of polycyclic aromatic systems to possess
the highest degree of aromaticity,[15] a property that is favorable for stable metal p-complexation.
Single-crystal X-ray structure determinations for complexes 4, 5 a, and 10–12 demonstrate the ability of complexes
to crystallize as discrete [RuACHTUNGRE(h6-arene)ACHTUNGRE(h5-Cp*)] + cations in
the Pbca (4), P21/c (5 a and 12) and P21/n (10 and 11) space
groups, respectively. Representative views of each structure
are shown in Figure 2, while selected crystal data are available within Table 2. Geometric parameters and bond lengths
for each structure (4, 5 a, and 10–12) were found to be comparable with those previously published for complexes 1, 2,
and 8, respectively (Table 3).[8, 14] For each compound the
phenyl and Cp* rings are co-planar, with the angle formed
by the ring centroids and ruthenium atom equaling 180.028
(4), 179.968 (5 a), 176.448 (10), 177.778 (11), and 179.398
(12), respectively. The Cp*–arene interplanar separation is
comparable for each complex, with an average distance of
3.52 . Ruthenium–carbon distances to both the h5-Cp* and
h6-phenyl carbons (Table 2) are mostly comparable for each
complex, with average values of 2.17 and 2.21 , respectively. Bond lengths between the metal and fusion arene carbons of complexes 10–12, however, are observed to be significantly longer, with values ranging between 2.25 (12) and
2.30 (10). This increase in bond length indicates movement of the ruthenium atom away from the fusion carbons
of the polycyclic ligands, a trend previously observed for
[RuACHTUNGRE(arene-R)ACHTUNGRE(Cp*)] + complexes incorporating electronwithdrawing aromatic substituents.[9, 16]
Computational Studies
Computational experiments using the Hartree–Fock (HF)
theory and the second-order Møller–Plesset (MP2) perturbation theory were performed on each molecule as a means
of further examining both the structural and electrostatic
properties of complexes 1–12.[17] Geometries of complexes 3,
6, 7, and 9 were optimized at the HF level of theory with
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 112 – 121
Organoruthenium(II) Full-Sandwich Complexes
Table 1. 1H NMR chemical shift comparison between the prepared organoruthenium(II) full-sandwich complexes (1–12) and their noncomplexed aromatic ligands.[a]
Complex Complexed arene
1
2
3
4
5
6
7
8
9
10
11
12
Noncomplexed arene
5.87 (s, 6 H, C6H6)
5.76-5.79 (m, 5 H, C6H5)
Upfield Shift
7.27 (s, 6 H, C6H6)
7.08–7.13 (m, 3 H, meta, para), 7.19–7.22 (m, 2 H,
ortho)
1.40
1.43 (ortho)
1.33 (meta)
1.33 (para)
7.08–7.14 (m, 3 H, meta, para), 7.20–7.23 (m, 2 H,
1.38 (ortho)
5.83-5.85 (m, 5 H, C6H5)
ortho)
1.27 (meta)
1.27 (para)
7.08–7.13 (m, 3 H, meta, para), 7.20–7.24 (m, 2 H,
1.39 (ortho)
5.80-5.86 (m, 5 H, C6H5)
ortho)
1.28 (meta)
1.28 (para)
6.07–6.09 (m, 3 H, meta, para), 6.34–6.38 (m, 2 H, ortho)
7.48–7.53 (m, 2 H, meta), 7.61–7.66 (m, 1 H, para),
1.60 (ortho)
7.94–7.97 (m, 2 H, ortho)
1.43 (meta)
1.56 (para)
6.07–6.12 (m, 3 H, meta, para), 6.36–6.38 (m, 2 H, ortho)
7.49–7.52 (m, 2 H, meta), 7.61–7.65 (m, 1 H, para),
1.58 (ortho)
7.94–7.96 (m, 2 H, ortho)
1.41 (meta)
1.53 (para)
6.08–6.10 (m, 3 H, meta, para), 6.43–6.45 (m, 2 H, ortho)
7.43–7.47 (m, 2 H, meta), 7.53–7.58 (m, 1 H, para),
1.47 (ortho)
7.89–7.92 (m, 2 H, ortho)
1.36 (meta)
1.47 (para)
6.05–6.09 (m, 3 H, meta, para), 6.42–6.44 (m, 2 H, ortho)
7.43–7.47 (m, 2 H, meta), 7.53–7.58 (m, 1 H, para),
1.48 (ortho)
7.89–7.92 (m, 2 H, ortho)
1.38 (meta)
1.49 (para)
6.44-6.49 (m, 1 H, C6H5 para), 6.52-6.56 (m, 2 H, C6H5
1.34 (ortho)
5.20 (d, 2 H, C6H5 ortho), 5.42-5.44 (m, 1 H, C6H5 para), 5.56ortho), 6.96-7.01 (m, 2 H, C6H5 meta)
1.41 (meta)
5.59 (m, 2 H, C6H5 meta)
1.04 (para)
6.11-6.13 (m, 2 H, aromatic (B, C)), 6.67-6.69 (m, 2 H, aromatic 7.49-7.52 (m, 4 H, aromatic (B, C, F, G)), 7.89-7.92 (m, 1.23 (A) 0.23 (E)
(A, D)), 7.60-7.64 (m, 2 H, aromatic (F, G)), 7.66-7.70 (m, 2 H,
4 H, aromatic (A, D, E, H))
1.39 (B) -0.11 (F)
aromatic (E, H))
1.39 (C) -0.11 (G)
1.23 (D) 0.23 (H)
6.19-6.24 (m, 2 H, aromatic (B, C)), 6.62-6.64 (m, 1 H, aromatic 7.62-7.71 (m, 4 H, aromatic (B, C, H, I)), 7.83 (s, 2 H,
1.02 (A) -0.18 (F)
(D)), 7.39-7.42 (m, 2 H, aromatic (H, I)), 7.78-7.80 (m, 2 H, aro- aromatic (E, F)), 7.98 (d, 2 H, aromatic (D, G)), 8.81
1.45 (B) -0.03 (G)
matic (A, E)), 7.98-8.03 (m, 2 H, aromatic (F, G)), 8.57-8.59 (m, (d, 2 H, aromatic (A, J))
1.45 (C) 0.26 (H)
1 H, aromatic (J))
1.35 (D) 0.26 (I)
0.04 (E) 0.23 (J)
6.36-6.39 (m, 1 H, aromatic (B)), 6.75-6.77 (m, 2 H, aromatic
8.04-8.08 (t, 2 H, aromatic (B, G)), 8.17 (s, 4 H, aromat- 1.52 (A) 0.00 (F)
(A, C)), 7.72-7.74 (m, 2 H, aromatic (D, J)), 8.08-8.12 (m, 1 H,
ic (D, E, I, J)), 8.28 (d, 4 H, aromatic (A, C, F, H))
1.68 (B) -0.04 (G)
aromatic (G)), 8.26-8.29 (m, 4 H, aromatic (E, F, H, I))
1.52 (C) 0.00 (H)
0.44 (D) -0.11 (I)
-0.11 (E) 0.44 (J)
[a] dH in ppm. Coupling patterns, multiplicities, and assignments are shown in parentheses. All experiments were referenced against a deuterated DMSO
internal standard (reference peak: [D6]DMSO, 1H d = 2.49 ppm).
the LanL2DZ basis set, with diagrammatic representations
and selected bond parameters available as Figure 3 and
Table 4, respectively.[18] Optimized geometries of 3, 6, 7, and
9 are isostructural with those obtained through X-ray crystallographic analysis of complexes 1, 2, 4, 5 a, 8, and 10—12,
respectively (Table 3), with the only noticeable difference
observed to be the Cp*–arene interplanar distances. The
average computational interplanar separation for complexes
3, 6, 7, and 9 is 3.87 , with ruthenium–carbon distances to
both the h5-Cp* and h6-phenyl carbons (Table 4) possessing
average values of 2.25 and 2.43 , distances significantly
longer than the experimental averages discussed previously
(3.52, 2.17, and 2.21 , respectively).
Electrostatic potential surfaces for complexes 2–12 were
calculated and mapped on electron density surfaces by using
structural parameters obtained through both computational
structure optimization and X-ray structural analyses of each
metallocene. The cc-pVDZ basis set was used for all atoms,
Chem. Asian J. 2012, 7, 112 – 121
with ruthenium parameters adjusted for the inclusion of a
pseudo potential.[19] Predicted atomic charges for each complex were generated using Mulliken population analyses,
with the electrostatic potential surfaces represented in
Figure 4. The calculated electrostatic potential maps predict
that the net positive charge of the [RuCp*] + moiety is completely delocalized throughout the molecular structure of
each metallocene, such that the complexes possess a uniform
(slightly positive d+) electrostatic potential. As expected,
these calculations therefore suggest that [(arene)RuACHTUNGRE(Cp*)] +
complexes exist as delocalized lipophilic cations, a property
which would impact their behavior within biological systems.[10]
In Vitro Cytotoxic Evaluation
After verifying their purity, complexes 1–12 were subjected
to cytotoxic evaluation using a sulforhodamine B (SRB) col-
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B. T. Loughrey et al.
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Figure 2. X-ray crystal structures with atom numbering schemes for (A) [RuACHTUNGRE(h6-C6H5ACHTUNGRE(CH2)2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (4), (B) [Ru(h6-C6H5CO2CH2CH3)
ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BF4 (5 a), (C) [RuACHTUNGRE(h6-C10H8)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (10), (D) [Ru(h6-C14H10)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (11), and (E) [Ru(h6-C16H10)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (12). Thermal ellipsoids are drawn at 25 % probability; counterions are omitted for clarity.
Table 2. Crystal data for [RuACHTUNGRE(h6-arene)ACHTUNGRE(h5-Cp*)] + complexes (4, 5 a, 10–12).
Complex
4
5a
10
11
12
Formula
Mol. Weight
Crystal System
Space Group
a []
b []
c []
a [8]
b [8]
g [8]
V [3]
Z
dcalcd [g cm 3]
m [mm 1]
Crystal Size [mm]
T min,max
2q max [8]
N (total)
N (unique)
No (I > 2s(I))
Rint
R1
wRF2 (all data)
C43H47BRu
675.7
Orthorhombic
Pbca
18.506(3)
16.730(2)
22.325(3)
90
90
90
6911.6(2)
8
1.30
0.48
0.65 0.58 0.54
0.74, 0.78
55.0
68 312
7927
6424
0.025
0.028
0.072
C19H25O2BF4Ru
473.3
Monoclinic
P21/c
15.9234(2)
16.5056(2)
15.2162(2)
90
92.180(1)
90
3996.31(9)
8
1.57
0.83
0.45 0.40 0.36
0.71, 0.75
55.0
21 387
9159
7920
0.016
0.030
0.080
C44H43BRu
683.7
Monoclinic
P21/n
10.2731(2)
31.0155(9)
11.0446(3)
90
93.812(2)
90
3511.3(2)
4
1.29
0.48
0.28 0.18 0.09
0.88, 0.96
50.0
15 564
5903
4520
0.038
0.058
0.140
C48H45BRu
733.7
Monoclinic
P21/n
31.402(9)
10.681(2)
11.216(3)
90
97.560(2)
90
3729.2(16)
4
1.31
0.45
0.40 0.20 0.15
0.84, 0.94
50.0
7217
6566
3377
0.051
0.058
0.185
C50H45BRu
757.7
Monoclinic
P21/c
10.5574(5)
14.8108(7)
24.2992(10)
90
90.438(3)
90
3799.4(3)
4
1.33
0.45
0.34 0.21 0.18
0.86, 0.92
50.0
13 913
6401
5198
0.019
0.031
0.084
orimetric assay for cell density determination following drug
treatment in microtiter wells for 6 days.[20] A diverse panel
of both tumorigenic and normal cell lines were chosen for
this study: A549 (lung cancer), B16 (murine melanoma),
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CI80-13S (ovarian cancer), DU145 (prostate cancer grade
II), HT29 (colon cancer), MCF7 (hormone-dependent
breast cancer), MDA-MB-231 (hormone-independent breast
cancer), MM418c5 (human melanoma), MM96L (human
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Chem. Asian J. 2012, 7, 112 – 121
Organoruthenium(II) Full-Sandwich Complexes
Table 3. Selected geometric parameters () for organoruthenium(II) full-sandwich complexes, with atoms numbered as per Figure 2.
Complex
1[a]
2[a]
4
5 (1)[b]
5 (2)[b]
8[a]
10
11
12
Ru C1
Ru C2
Ru C3
Ru C4
Ru C5
Ru C11
Ru C12
Ru C13
Ru C13a
Ru C14
Ru C14a
Ru C15
Ru C16
Ru C18a
Ru C20a
Ru C20b
Ru C21
C11 C17
C17 O1
C17 O2
2.170(8)
2.170(8)
2.183(5)
2.170(6)
2.183(5)
2.208(6)
2.186(8)
2.181(8)
2.147(8)
2.178(7)
2.167(7)
2.149(8)
2.167(7)
2.208(8)
2.215(8)
2.219(8)
2.192(2)
2.182(2)
2.170(2)
2.183(2)
2.175(2)
2.223(2)
2.199(2)
2.204(2)
2.183(2)
2.179(2)
2.179(2)
2.178(3)
2.191(3)
2.205(2)
2.214(2)
2.221(2)
2.195(3)
2.177(2)
2.177(3)
2.180(3)
2.188(3)
2.210(2)
2.219(2)
2.224(3)
2.192(2)
2.175(2)
2.175(2)
2.164(2)
2.173(2)
2.191(2)
2.210(3)
2.217(3)
2.168(4)
2.180(4)
2.194(4)
2.166(4)
2.177(4)
2.242(8)
2.201(7)
2.200(6)
2.128(7)
2.112(12)
2.134(11)
2.151(9)
2.156(7)
2.221(7)
2.190(9)
2.197(9)
2.172(3)
2.153(3)
2.162(2)
2.173(2)
2.186(2)
2.229(3)
2.229(3)
2.226(3)
2.263(3)
2.208(6)
2.210(8)
2.209(2)
2.218(2)
2.220(3)
2.206(3)
2.190(6)
2.252(4)
2.196(8)
2.254(7)
2.186(6)
2.181(8)
2.213(8)
2.182(7)
2.209(2)
2.207(2)
2.226(3)
2.216(3)
2.222(3)
2.213(2)
2.206(3)
2.184(3)
2.303(6)
2.250(2)
2.293(6)
2.249(2)
1.487(14)
1.518(4)
1.501(3)
1.323(3)
1.203(3)
1.504(3)
1.321(4)
1.193(3)
1.507(4)
1.204(3)
[a] Structural parameters obtained from Gemel et al. (1), Navarro Clemente et al. (2) and Loughrey et al. (8), respectively.[8, 14] [b] Structure of 5 a with two
molecules in the asymmetric unit. X-ray data obtained using the BF4 counterion as crystals produced using the BPh4 counterion (5) provided data unsuitable for publication.
melanoma), PC3 (prostate cancer grade IV), T47-D (hormone-dependent breast cancer), RAW264 (a murine macrophage cell line), and NFF (neonatal foreskin fibroblasts).
Each of these cell lines is susceptible to a variety of applied
chemotherapeutics and also displays differing mechanisms
of cross-resistance to drug treatment.
Results of this assay are
listed (Table 5) and demonstrate these complexes to be
cytotoxic and relatively selective growth inhibitors of all
eleven tumorigenic cell lines,
achieving IC50 values (concentration at which 50 % of cell
growth is inhibited) in the low
micromolar
range
(0.03–
37.6 mm). The average antiproliferative effect of these cationic organoruthenium complexes
follows the sequence 12 > 11 >
10 > 4 > 6 8 3 > 2 > 1 > 5 >
7 > 9, with cytotoxic activity
again found to be dependent
upon both the size and lipophilicity of the complexed h6arene ligand. Complexes 10–
12, which incorporate the
large, hydrophobic polycyclic
aromatic ligands naphthalene
(10), phenanthrene (11), and
pyrene (12) achieve the highest levels of growth inhibition
recorded for complexes of this
type, thereby registering IC50
values
in the nanomolar range
6
Figure 3. Computationally optimized structures with atom numbering schemes for (A) [Ru(h -C6H5CH2CH3)
5
+
6
5
+
6
5
(0.03–0.35
mm).
ACHTUNGRE(h -C5ACHTUNGRE(CH3)5)] (3), (B) [Ru(h -C6H5CO2ACHTUNGRE(CH2)2CH3)ACHTUNGRE(h -C5ACHTUNGRE(CH3)5)] (6), (C) [Ru(h -C6H5COCH2CH3)ACHTUNGRE(h -C5ACHTUNGRE(CH3)5)] + (7), and (D) [Ru(h6-C6H5NH2)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)] + (9).
Chem. Asian J. 2012, 7, 112 – 121
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117
B. T. Loughrey et al.
FULL PAPERS
Table 4. Selected calculated bond lengths () for [Ru(h6-C6H5CH2CH3)
ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)] + (3), [Ru(h6-C6H5CO2ACHTUNGRE(CH2)2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)] + (6),
[Ru(h6-C6H5COCH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)] + (7), and [Ru(h6-C6H5NH2)ACHTUNGRE(h5C5ACHTUNGRE(CH3)5)] + (9).
Complex
3
6
7
9
Ru C1
Ru C2
Ru C3
Ru C4
Ru C5
Ru C11
Ru C12
Ru C13
Ru C14
Ru C15
Ru C16
C11 C17
C11 N1
C17 O1
C17 O2
2.260
2.257
2.255
2.254
2.256
2.460
2.415
2.411
2.415
2.410
2.421
1.520
2.251
2.252
2.252
2.252
2.249
2.398
2.429
2.435
2.433
2.427
2.413
1.495
2.253
2.254
2.252
2.251
2.249
2.412
2.429
2.433
2.433
2.424
2.409
1.513
2.256
2.256
2.253
2.260
2.253
2.521
2.431
2.406
2.405
2.406
2.431
1.366
1.331
1.217
1.221
Four human cancer cell lines were found to be particularly susceptible to growth inhibition under in vitro conditions,
these being the rapidly dividing human melanoma cell line
(MM96L), both hormone-dependent phenotypes of breast
cancer (T47-D and MCF7), and the cisplatin-resistant ovarian cancer cell line (CI80-13S). Complexes 1–12 inhibit the
growth of these cell lines at potencies 18.5-, 15.8-, 11.1-, and
10.9-fold greater than that displayed towards the control
human fibroblast cell line (NFF). The cell lines A549,
MDA-MB-231, HT29, PC3, and DU145 (which each exhibit
a slow in vitro proliferation rate) were found to be more tolerant, however, with the complexes inhibiting these particular cell lines at potencies 2.9-, 4.9-, 5.8-, 6.0-, and 7.0-fold
greater than that exhibited towards NFF.
Conclusions
A structurally diverse series of organoruthenium(II) fullsandwich complexes have been prepared in good yield and
purity through a simple one-pot reaction of RuCl3·xH2O,
HCp*, and aromatic ligands in refluxing alcohol. Computational experiments incorporating the Hartree–Fock method
and the second-order Møller–Plesset perturbation theory
predict each complex to possess a uniform d+ electrostatic
potential, with the cationic charge of the [RuCp*] + moiety
completely delocalizing throughout the molecular structure
of each metallocene. Cytotoxic evaluation of these delocalized lipophilic cations show them to be potent antitumor
agents against a wide range of cancerous cell lines, while displaying significantly lower toxicity towards both the normal
human fibroblast and mouse macrophage controls. Complexes are found to favor inhibition of rapidly dividing
cancer cells including human melanoma (MM96L), two hormone-dependent phenotypes of breast cancer (T47-D and
MCF7), and the cisplatin-resistant ovarian cancer cell line
(CI80-13S). Cytotoxic activity
is found to increase with size
and lipophilicity of the attached aromatic ligand, with
complexes incorporating the
polycyclic aromatic groups
naphthalene,
phenanthrene,
and pyrene registering IC50
values in the nanomolar range.
Experimental Section
General Procedures
Figure 4. Electrostatic potential surfaces of organoruthenium(II) full-sandwich complexes 2–12. The electrostatic potential is represented with a color scale ranging from red (0.00 au) to blue (+ 0.12 au).
118
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2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
All reactions were conducted under
argon using standard Schlenk techniques unless stated otherwise. [RuACHTUNGRE(h6-C6H5R)ACHTUNGRE(Cp*)] + (where R = CH3
(2),
CO2ACHTUNGRE(CH2)2CH3
(6),
COACHTUNGRE(CH2)2CH3 (8), and NH2 (9)) compounds were prepared and isolated
using literature methods.[9] The identification and purity of these compounds was assured through comparison of experimentally attained characterization data with prior published
literature values. Starting materials
and solvents were obtained from a
commercial supplier (Aldrich) and
used as received. Fourier transform
infrared spectroscopy (FT-IR) was
Chem. Asian J. 2012, 7, 112 – 121
Organoruthenium(II) Full-Sandwich Complexes
Table 5. Inhibitory concentration that limits proliferation by 50 % (IC50) for the organoruthenium(II) full-sandwich complexes 1–12 against a range of cancer
cell lines and two non-tumorigenic cell lines (RAW264 and NFF). Cisplatin (Csp) was incorporated for the purposes of a positive control.
Complex
IC50 values [mM][a]
1
2
3
4
5
6
7
8
9
10
11
12
A549
B16
CI80-13S
DU145
HT29
MCF7
MDA-MB-231
MM418c5
MM96L
PC3
T47-D
RAW264
NFF
12.2
11.8
4.05
7.86
8.32
6.71
11.6
5.98
6.03
7.66
6.32
20.7
37.9
12.7
12.5
3.86
8.17
9.84
4.99
5.20
6.32
2.28
8.80
5.17
16.6
92.2
8.14
8.25
3.21
6.73
6.86
2.89
5.65
4.51
1.70
5.82
1.81
12.4
30.7
7.05
3.63
2.88
3.96
3.83
2.24
5.74
2.44
1.26
3.41
1.12
6.76
18.1
24.1
18.5
3.78
7.75
7.40
5.57
19.7
7.96
2.73
11.5
3.93
31.2
48.5
10.1
8.04
2.72
4.98
4.25
2.33
3.36
3.77
2.54
8.14
2.86
9.42
10.6
32.2
14.1
13.0
8.38
8.23
4.31
24.7
12.2
4.17
7.68
4.18
27.2
101
9.72
8.58
2.92
4.27
3.95
3.00
9.14
5.21
2.85
3.93
1.83
30.2
32.7
36.1
36.5
4.45
12.2
37.6
4.73
12.0
10.3
3.50
13.5
9.10
26.8
98.2
1.31
1.24
0.44
0.54
0.41
0.34
1.42
0.36
0.16
0.89
0.15
2.70
2.38
1.31
1.24
0.44
0.54
0.41
0.34
1.42
0.36
0.16
0.89
0.15
2.70
2.38
0.36
0.94
0.10
0.11
0.48
0.25
0.80
0.17
0.04
0.24
0.03
1.83
1.02
Csp
3.20
1.78
1.80
0.80
1.70
3.30
[a] Errors are within the range of 5–10 % of the reported value. Results are the average of three separate experiments.
conducted on a Thermo Nicolet Nexus FT-IR spectrometer with all samples prepared as KBr discs. The following abbreviations apply to the intensity of peaks found within the spectra: vs = very strong; s = strong;
m = medium; and w = weak. Electrospray ionization mass spectrometry
experiments were conducted on a Waters ZQ 4000 mass spectrometer by
direct injection. All data were processed using the Mass Lynx Version IV
(IBM) software. 1H and 13C NMR spectra were obtained on a 400 MHz
Varian Gemini NMR spectrometer with each sample being prepared in a
solution of [D6]DMSO. Peaks obtained for the deuterated solvent were
used as internal reference points for the spectra (reference peaks:
[D6]DMSO, 1H, d = 2.45 ppm, 13C, d = 39.5 ppm). All signals were recorded using their appropriate chemical shift (d in ppm), multiplicity, integral
ratio, and coupling constants (Hz). The following abbreviations apply to
the signal multiplicity of peaks within the spectra: s = singlet, d = doublet,
t = triplet, and m = multiplet. Signals for the aromatic protons of complexes 10–12 were assigned according to the labeling system shown in
Scheme 1. All deuterated solvents were supplied by Cambridge Isotope
Laboratories and were used without further purification. Microanalyses
were performed at the Microanalytical Unit of the University of Queensland.
Synthetic Procedure for the Preparation of Complexes 1, 3–5, 5 a, 7, and
10–12
Ruthenium trichloride hydrate (0.20 g, 0.76 mmol) was transferred into
the reaction vessel using ethanol (20 mL). This mixture was refluxed
until all starting material had dissolved. Pentamethylcyclopentadiene
(0.24 mL, 1.52 mmol) and the arene ligand (1.52 mmol) were added to
the reaction mixture, and the resulting solution heated at reflux for a further 10 h. The solvent was concentrated in vacuo and the remaining residue dissolved in a water/diethyl ether partition mixture (20 mL/20 mL).
The aqueous portion was retained and washed with diethyl ether (20 mL
x3). The aqueous layer was then mixed slowly with 5 mL of a 0.30 m
aqueous solution of sodium tetraphenylborate (1, 3–5, 7, 10–12) or ammonium tetrafluoroborate (5 a), respectively. The resulting precipitate
was filtered off and dried in vacuo. Complex 5 a, which incorporated the
BF4 counter ion, required no further purification. The remaining tetraphenylborate complexes were redissolved in a minimum quantity of acetone and filtered through a short alumina column (neutral, 150 mesh)
using acetone as the eluent. Analytically pure samples of the complexes
were obtained after concentration of the acetone solvent in vacuo.
[RuACHTUNGRE(h6-C6H6)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (1)
White powder, yield = 0.395 g, 82 %; IR: ñ = 3060 (C H aromatic, w, br),
2968 (C H aliphatic, w, br), 2922 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 1.94 (s, 15 H, C5ACHTUNGRE(C5H15)), 5.87 (s, 6 H, C6H6), 6.76–6.80
(m, 4 H, BACHTUNGRE(C6H5)4 para), 6.90–6.93 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.15–
7.20 ppm (m, 8 H, BACHTUNGRE(C6H5)4 ortho); 13C ([D6]DMSO): d = 10.3 (C5ACHTUNGRE(C5H15)), 87.1 (C6H6), 95.9 (C5ACHTUNGRE(C5H15), 121.5 (4CH, BACHTUNGRE(C6H5)4), 125.3
Chem. Asian J. 2012, 7, 112 – 121
(8CH, BACHTUNGRE(C6H5)4), 135.5 (8CH, BACHTUNGRE(C6H5)4), 162.4, 163.0, 163.7, 164.3 ppm
(4CH, BACHTUNGRE(C6H5)4, signals split by 11B); ESMS (m/z): positive ion, calcd m/
z for [(h5-C5ACHTUNGRE(CH3)5)RuACHTUNGRE(h6-C6H6) + ]: 314.44, found: 315 (100 %), negative
ion, calcd m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental analysis, calcd (%) for C40H41BRu: C 75.8, H 6.53; found: C 75.9, H 6.69.
[Ru(h6-C6H5CH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (3)
White powder, yield = 0.403 g, 77 %; IR: ñ = 3052 (C H aromatic, m, br),
2974 (C H aliphatic, m, br), 2922 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 1.15 (t, 3 H, CH2CH3), 1.89 (s, 15 H, C5ACHTUNGRE(C5H15)), 2.33 (q,
2 H, CH2CH3), 5.83–5.85 (m, 5 H, C6H5), 6.76–6.81 (m, 4 H, BACHTUNGRE(C6H5)4
para), 6.89–6.94 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.15–7.20 ppm (m, 8 H, BACHTUNGRE(C6H5)4 ortho); 13C ([D6]DMSO): d = 10.0 (C5ACHTUNGRE(C5H15)), 15.3 (CH2CH3),
25.3 (CH2CH3), 86.5 (aromatic), 86.9 (2CH, aromatic), 87.2 (2CH, aromatic), 95.3 (C5ACHTUNGRE(C5H15), 105.1 (C-CH2CH3), 121.5 (4CH, BACHTUNGRE(C6H5)4), 125.3
(8CH, BACHTUNGRE(C6H5)4), 135.5 (8CH, BACHTUNGRE(C6H5)4), 162.3, 163.0, 163.7, 164.3 ppm
(4CH, BACHTUNGRE(C6H5)4, signals split by 11B); ESMS (m/z): positive ion, calcd m/
z for [(h5-C5ACHTUNGRE(CH3)5)Ru(h6-C6H5CH2CH3) + ]: 342.50, found: 343 (100 %),
negative ion, calcd m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental analysis, calcd (%) for C42H45BRu: C 76.2, H 6.87; found: C 76.1,
H 6.82.
[RuACHTUNGRE(h6-C6H5ACHTUNGRE(CH2)2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (4)
White powder, yield = 0.395 g, 74 %; IR: ñ = 3050 (C H aromatic, m, br),
2979 (C H aliphatic, m, br), 2929 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 0.90 (t, 3 H, (CH2)2CH3), 1.54 (m, 2 H, CH2CH2CH3),
1.89 (s, 15 H, C5ACHTUNGRE(C5H15)), 2.25 (t, 2 H, CH2CH2CH3), 5.80–5.86 (m, 5 H,
C6H5), 6.76–6.80 (m, 4 H, BACHTUNGRE(C6H5)4 para), 6.90–6.93 (m, 8 H, BACHTUNGRE(C6H5)4
meta), 7.16–7.19 ppm (m, 8 H, BACHTUNGRE(C6H5)4 ortho); 13C ([D6]DMSO): d = 10.0
(C5ACHTUNGRE(C5H15)), 13.2 (CH2)2CH3), 24.1 (CH2CH2CH3), 33.8 (CH2CH2CH3),
86.5 (aromatic), 87.2 (2CH, aromatic), 87.4 (2CH, aromatic), 95.3 (C5ACHTUNGRE(C5H15), 103.5 (C (CH2)2CH3), 121.5 (4CH, BACHTUNGRE(C6H5)4), 125.3 (8CH, BACHTUNGRE(C6H5)4), 135.5 (8CH, BACHTUNGRE(C6H5)4), 162.3, 163.0, 163.7, 164.3 ppm (4CH, BACHTUNGRE(C6H5)4, signals split by 11B); ESMS (m/z): positive ion, calcd m/z for
[(h5-C5ACHTUNGRE(CH3)5)RuACHTUNGRE(h6-C6H5ACHTUNGRE(CH2)2CH3) + ]: 356.53, found: 357 (100 %), negative ion, calcd m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental
analysis, calcd (%) for C43H47BRu: C 76.4, H 7.02; found: C 76.3, H 7.01.
[Ru(h6-C6H5CO2CH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (5)
White powder, yield = 0.277 g, 52 %; IR: ñ = 1727 (C=O stretch, m),
1274 cm 1 (C O stretch, m); NMR: 1H ([D6]DMSO): d = 1.31 (t, 3 H,
CH2CH3) 1.82 (s, 15 H, C5ACHTUNGRE(C5H15)), 4.33 (q, 2 H, CH2CH3), 6.07–6.09 (m,
3 H, C6H5 meta and para), 6.34–6.38 (m, 2 H, C6H5 ortho), 6.70–6.77 (m,
4 H, BACHTUNGRE(C6H5)4 para), 6.83–6.91 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.11–7.13 ppm
(m, 8 H, BACHTUNGRE(C6H5)4 ortho); 13C ([D6]DMSO): d = 9.7 (C5ACHTUNGRE(C5H15)), 14.1
(CH2CH3), 62.4 (CH2CH3), 86.5 (C CO2CH2CH3), 86.9 (2CH, aromatic),
88.3 (2CH, aromatic), 89.0 (aromatic), 97.1 (C5ACHTUNGRE(C5H15)), 121.5 (4CH, BACHTUNGRE(C6H5)4), 125.3 (8CH, BACHTUNGRE(C6H5)4), 135.5 (8CH, BACHTUNGRE(C6H5)4), 162.6, 163.1,
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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119
B. T. Loughrey et al.
FULL PAPERS
163.6, 164.1 ppm (4CH, BACHTUNGRE(C6H5)4, signals split by 11B), 164.3 (C=O);
ESMS (m/z): positive ion, calcd m/z for [(h5- C5ACHTUNGRE(CH3)5)Ru(h6C6H5CO2CH2CH3) + ]: 386.51, found: 387 (100 %), negative ion, calcd m/z
for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental analysis, calcd (%)
for C43H45O2BRu: C 73.2, H 6.40; found: C 73.4, H 6.47.
[Ru(h6-C6H5CO2CH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BF4 (5a)
Bronze powder, yield = 0.235 g, 67 %; IR: ñ = 1727 (C=O stretch, s),
1295 cm 1 (C O stretch, s); NMR: 1H ([D6]DMSO): d = 1.31 (t, 3 H,
CH2CH3) 1.83 (s, 15 H, C5ACHTUNGRE(C5H15)), 4.34 (q, 2 H, CH2CH3), 6.09–6.12 (m,
3 H, C6H5 meta and para), 6.36–6.40 ppm (m, 2 H, C6H5 ortho); 13C
([D6]DMSO): d = 9.7 (C5ACHTUNGRE(C5H15)), 14.1 (CH2CH3), 62.5 (CH2CH3), 86.5
(C-CO2CH2CH3), 86.9 (2CH, aromatic), 88.3 (2CH, aromatic), 89.1 (aromatic), 97.1 (C5ACHTUNGRE(C5H15)), 164.3 ppm (C=O); ESMS (m/z): positive ion,
calcd m/z for [(h5-C5ACHTUNGRE(CH3)5)Ru(h6-C6H5CO2CH2CH3) + ]: 386.51, found:
387 (100 %), negative ion, calcd m/z for BF4 : 86.81, found: 87 (100 %);
elemental analysis, calcd (%) for C19H25O2BF4Ru: C 48.2, H 5.30; found:
C 48.4, H 5.13.
[Ru(h6-C6H5COCH2CH3)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (7)
White powder, yield = 0.358 g, 70 %; IR: ñ = 1702 cm 1 (C=O stretch, s);
NMR: 1H ([D6]DMSO): d = 1.04 (t, 3 H, CH2CH3) 1.80 (s, 15 H, C5ACHTUNGRE(C5H15)), 2.89 (q, 2 H, CH2CH3), 6.08–6.10 (m, 3 H, C6H5 meta and para),
6.43–6.45 (m, 2 H, C6H5 ortho) 6.69–6.77 (m, 4 H, BACHTUNGRE(C6H5)4 para), 6.83–
6.91 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.09–7.16 ppm (m, 8 H, BACHTUNGRE(C6H5)4 ortho);
13
C ([D6]DMSO): d = 7.2 (CH2CH3) 9.8 (C5ACHTUNGRE(C5H15)), 32.1 (CH2CH3), 85.8
(2CH, aromatic), 88.2 (2CH, aromatic), 89.1 (aromatic), 91.4 (CCOCH2CH3), 96.9 (C5ACHTUNGRE(C5H15)), 121.5 (4CH, BACHTUNGRE(C6H5)4), 125.3 (8CH, BACHTUNGRE(C6H5)4), 135.5 (8CH, BACHTUNGRE(C6H5)4), 162.6, 163.1, 163.6, 164.1 ppm (4CH, BACHTUNGRE(C6H5)4, signals split by 11B), 199.3 (C=O); ESMS (m/z): positive ion,
calcd m/z for [(h5-C5ACHTUNGRE(CH3)5)Ru(h6-C6H5COCH2CH3) + ]: 370.51, found:
371 (100 %), negative ion, calcd m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319
(100 %); elemental analysis, calcd (%) for C43H45OBRu: C 74.9, H 6.59;
found: C 74.54, H 6.41.
[RuACHTUNGRE(h6-C10H8)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (10)
Yellow crystals, yield = 0.403 g, 75 %; IR: ñ = 3050 (C H aromatic, m, br),
2975 (C H aliphatic, m, br), 2928 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 1.59 (s, 15 H, C5ACHTUNGRE(C5H15)), 6.11–6.13 (m, 2 H, aromatic
(B & C)), 6.67–6.69 (m, 2 H, aromatic (A & D)), 6.75–6.80 (m, 4 H, BACHTUNGRE(C6H5)4 para), 6.89–6.94 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.14–7.20 (m, 8 H, BACHTUNGRE(C6H5)4 ortho), 7.60–7.64 (m, 2 H, aromatic (E & G)), 7.66–7.70 ppm (m,
2 H, aromatic (E & H)); ESMS (m/z): positive ion, calcd m/z for [(h5-C5ACHTUNGRE(CH3)5)RuACHTUNGRE(h6-C10H8) + ]: 364.50, found: 365 (100 %), negative ion, calcd
m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental analysis, calcd
(%) for C44H43BRu: C 77.3, H 6.35; found: C 77.1, H 6.38.
[Ru(h6-C14H10)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (11)
Cream crystals, yield = 0.414 g, 64 %; IR: ñ = 3053 (C H aromatic, m, br),
2983 (C H aliphatic, w, br), 2930 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 1.45 (s, 15 H, C5ACHTUNGRE(C5H15)), 6.19–6.24 (m, 2 H, aromatic
(B & C)), 6.62–6.64 (m, 1 H, aromatic (D)), 6.76–6.80 (m, 4 H, BACHTUNGRE(C6H5)4
para), 6.89–6.93 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.12–7.17 (m, 8 H, BACHTUNGRE(C6H5)4
ortho), 7.39–7.42 (m, 2 H, aromatic (H & I)), 7.78–7.80 (m, 2 H, aromatic
(A & E)), 7.98–8.03 (m, 2 H, aromatic (F & G)), 8.57–8.59 ppm (m, 1 H,
aromatic (J)); ESMS (m/z): positive ion, calcd m/z for [(h5-C5ACHTUNGRE(CH3)5)Ru(h6-C14H10) + ]: 414.56, found: 415 (100 %), negative ion, calcd
m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319 (100 %); elemental analysis, calcd
(%) for C48H45BRu: C 78.6, H 6.19; found: C 78.6, H 6.10.
[Ru(h6-C16H10)ACHTUNGRE(h5-C5ACHTUNGRE(CH3)5)]BPh4 (12)
Yellow crystals, yield = 0.182 g, 31 %; IR: ñ = 3051 (C H aromatic, m, br),
2980 (C H aliphatic, w, br), 2934 cm 1 (C H aliphatic, w, br); NMR: 1H
([D6]DMSO): d = 1.26 (s, 15 H, C5ACHTUNGRE(C5H15)), 6.36–6.39 (m, 1 H, aromatic
(B)), 6.75–6.77 (m, 2 H, aromatic (A & C)), 6.75–6.79 (m, 4 H, BACHTUNGRE(C6H5)4
para), 6.89–6.93 (m, 8 H, BACHTUNGRE(C6H5)4 meta), 7.13–7.19 (m, 8 H, BACHTUNGRE(C6H5)4
ortho), 7.72–7.74 (m, 2 H, aromatic (D & J)), 8.08–8.12 (m, 1 H, aromatic
(7)), 8.26–8.29 ppm (m, 4 H, aromatic (E, F, H & J)); ESMS (m/z): posi-
120
www.chemasianj.org
tive ion, calcd m/z for [(h5-C5ACHTUNGRE(CH3)5)Ru(h6-C16H10) + ]: 438.58, found: 439
(100 %), negative ion, calcd m/z for BACHTUNGRE(C6H5)4 : 319.25, found: 319
(100 %); elemental analysis, calcd (%) for C50H45BRu: C 79.3, H 6.00;
found: C 79.1, H 5.95.
Crystallography
Unique data sets were collected on an Oxford Diffraction GEMINI S
Ultra CCD diffractometer (compounds 4, 5 a, 10, and 12) and a Rigaku
AFC 7R four-circle diffractometer (compound 11) utilizing Mo Ka radiation. The structures were solved by direct methods and refined by fullmatrix least-squares refinement on F2 after application of semiempirical
absorption corrections. Anisotropic thermal parameters were refined for
non-hydrogen atoms; (x, y, z, Uiso)H were included and constrained at estimated values. Conventional residuals at convergence are quoted; statistical weights were employed. Computation was done using the CrysAlis,
teXsan, SHELX97, and ORTEP-3 program and software systems.[21]
CCDC 821740, CCDC 821741, CCDC 821742, CCDC 821743, and
CCDC 821744 contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Computational Calculations
The Gaussian 03 computer package[22] was employed for all calculations
incorporating the Hartree–Fock theory in the case of the geometry optimizations and the second-order Møller–Plesset perturbation theory
(MP2) for single point calculations.[17] Geometry optimization calculations for complexes 3, 6, 7, and 9 were performed in the gas phase using
the LanL2DZ basis set.[18] Electrostatic potential surfaces for complexes
1–12 were calculated from the density matrices generated by single point
calculations at the MP2 level using the cc-pVDZ basis set[19] for all
atoms, with Ru parameters adjusted for the inclusion of a pseudo potential. The magnitude of the electrostatic potential is represented using a
color scale ranging from red (0.00 au) to blue (+0.12 au).
Cell Culture and Cytotoxic Evaluation
Each cell line was cultured at 37 8C in RPMI 1640 medium supplemented
with heat-inactivated fetal calf serum (10 %, CSL, Australia), 3 mm
HEPES buffer (pH 7.3–7.4), penicillin (100 U mL 1), and streptomycin
(100 mg mL 1) at 5 % CO2 and 99 % humidity. Primary human fibroblasts
were obtained from neonatal foreskin cultured in the above medium.
Culture media were replaced every three days, and cell monolayers were
split at 70–80 % confluency. Routine mycoplasma tests were performed
using Hoechst stains and were always negative.
Stock solutions of each complex were prepared by dissolving the compounds (ca. 10 mg) in ethanol (1 mL). These stock solutions were diluted
as necessary for testing. Cells were seeded in 96-well microtiter plates at
approximately 5000 cells per 100 mL (NFF), 3000 cells per 100 mL
(MCF7, MDA-MB-231, CI80-13S, T47-D, DU145, PC3, HT29, A549,
MM418c5, B16, RAW264), and 1000 cells per 100 mL (MM96L). Seven
dilutions of each drug were added to triplicate wells. The plates were incubated for 6 days prior to incorporation of the SRB staining method.[20]
The culture medium was removed from the plates and each plate washed
with phosphate-buffered saline (PBS). The plates were fixed with methylated spirit for 15 min and then washed with water. SRB solution (50 mL,
0.4 % SRB dye (w/v) in 1 % (v/v) acetic acid) was added to each well and
left at room temperature for 45 min. The SRB solution was removed and
the plates washed quickly, once with water and twice with 1 % (v/v)
acetic acid solution. For the NFF cell assay, the plates were washed
thrice with 1 % (v/v) acetic acid solution. Tris base (100 mL, 10 mm, unbuffered, pH > 9) was added to each well to solubilize the protein-bound
dye. After incubation for 5 min, the absorbance was measured at 564 nm
on a multiwell plate reader. The percentage of surviving cells was calculated from the absorbance of untreated control cells. The IC50 values for
the inhibition of cell viability were determined by fitting the plots of the
percentage of surviving cells against drug concentration using a sigmoidal
function.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 112 – 121
Organoruthenium(II) Full-Sandwich Complexes
Acknowledgements
We thank the Eskitis Institute for Cell and Molecular Therapies, the
Queensland Micro- and Nanotechnology Facility, and the Queensland Institute of Medical Research for their support of this project.
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2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: July 22, 2011
Published online: November 17, 2011
www.chemasianj.org
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