Anticancer activity of self-assembled molecular rectangles via arene-ruthenium acceptors and a new unsymmetrical amide ligand

Article abstract of DOI:10.1021/om2012826

Two new and large molecular rectangles 4 and 5 were synthesized from two different arene-ruthenium [Ru₂(μ-η⁴-C ₂O₄)(MeOH)₂(η⁶-p-Pr ⁱC₆H₄Me)₂][O₃SCF ₃]₂ (2) and [Ru₂(p-cymene)₂(donq) (OH₂)₂][O₃SCF₃]₂ (donq = 5,8-dioxydo-1,4-naphthaquinonato) (3) acceptors and a new unsymmetrical N-(4-(pyridin-4-ylethynyl)phenyl) isonicotinamide (1) donor ligand. X-ray crystallography of 4 confirmed a molecular rectangle. The ¹H NMR spectra of both rectangles 4 and 5 showed a mixture of two structural, head-to-tail (HTT) and head-to-head (HTH), type isomers in a 1:1 ratio. The cytotoxicities of both rectangles have been established against Colo320 (colorectal cancer), A549 (lung cancer), MCF-7 (breast cancer), and H1299 (lung cancer) human cancer cell lines. The cytotoxicity of rectangle 5 was found to be considerably stronger against all cancer cell lines than that of the reference drug cisplatin.

Full text of DOI:10.1021/om2012826

Article
pubs.acs.org/Organometallics
Anticancer Activity of Self-Assembled Molecular Rectangles via
Arene−Ruthenium Acceptors and a New Unsymmetrical Amide
Ligand
Anurag Mishra,† Hyunji Jung,† Jeong Woo Park,‡ Hong Kyeung Kim,‡ Hyunuk Kim,§ Peter J. Stang,∥
and Ki-Whan Chi*,†
†Department of Chemistry, University of Ulsan, Ulsan 680-749, Republic of Korea
‡School of Biological Sciences, University of Ulsan, Ulsan 680-749, Republic of Korea
§Energy Materials and Convergence Research Department, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea
∥Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
S
* Supporting Information
ABSTRACT: Two new and large molecular rectangles 4 and 5 were synthesized
from two different arene−ruthenium [Ru2(μ-η4-C2O4)(MeOH)2(η6-p-
PriC6H4Me)2][O3SCF3]2 (2) and [Ru2(p-cymene)2(donq)(OH2)2][O3SCF3]2
(donq = 5,8-dioxydo-1,4-naphthaquinonato) (3) acceptors and a new unsym-
metrical N-(4-(pyridin-4-ylethynyl)phenyl) isonicotinamide (1) donor ligand. X-
ray crystallography of 4 confirmed a molecular rectangle. The 1H NMR spectra of
both rectangles 4 and 5 showed a mixture of two structural, head-to-tail (HTT) and
head-to-head (HTH), type isomers in a 1:1 ratio. The cytotoxicities of both
rectangles have been established against Colo320 (colorectal cancer), A549 (lung
cancer), MCF-7 (breast cancer), and H1299 (lung cancer) human cancer cell lines.
The cytotoxicity of rectangle 5 was found to be considerably stronger against all
cancer cell lines than that of the reference drug cisplatin.
with structural complexity and functionalities by simple
reactions. In these endeavors, ruthenium complexes are an
attractive alternative to platinum-based anticancer agents
because of their rich synthetic chemistry, variable oxidation
states, accessibility under physiological conditions, selective
antimetastatic properties, multiple mechanisms of action
distinct from platinum-based drugs, and low systemic toxicity.11
Two Ru(III) complexes, NAMI-A and KP1019, have
successfully entered clinical trials for the treatment of metastatic
solid tumors.12 Therrien and co-workers have reported a half-
sandwich ruthenium-based coordination cage that encapsulates
the [M(acac)2] (M = Pt2+, Pd2+) complexes with a synergic
enhancement of their antitumor activity.13 Recently, we have
reported a new set of molecular rectangles constructed by the
coordination-driven self-assembly approach and that were
evaluated for in vitro anticancer activity.14 The results suggested
that larger rectangles show higher activity over small rectangles,
in agreement with the hypothesis that large macromolecules are
more retained inside cancer cells. Although a number of
metallarectangles have been reported for host−guest chemistry
or other properties,15 only a few have been explored for their
antitumor properties.14,16
INTRODUCTION
■
In the last two decades, the metal-directed self-assembly of
supramolecular architectures has attracted much attention, due
to their promise for various applications, including molecular
recognition,1 separation materials,2 catalysis,3 host−guest
chemistry,4 and biology.5 In particular, the coordination
bonding motif of pyridyl-based ligands with metal acceptors
has proven very useful for constructing a wide array of
molecular architectures.6 Most assemblies prepared to this date
are highly symmetrical in nature as they are built from simple
homodi- and tridentate pyridine-containing donors and metal
acceptors.7 A literature survey reveals that only a few
unsymmetrical and ambidentate ligands have been successfully
incorporated with discrete metallasupramolecules.8 Among the
several kinds of functionalities, the one that incorporates an
ethynyl spacer in the backbone serves as an excellent substrate
that endows interesting optical properties and rigidity to
supramolecules.9 Its rigid and π-conjugated structure makes the
acetylene group an outstanding candidate for the construction
of unsaturated molecular scaffolds.
The identification of new anticancer metal complexes is
presently receiving a surge of interest due to their structural
diversity and inimitable biological and medicinal properties
arising from either covalent or noncovalent bindings/
interactions with biomolecular cellular targets.10 Metal−ligand
coordination is a useful means to create bioactive molecules
Received: December 27, 2011
Published: April 13, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of Unsymmetrical Amide Ligand 1
Scheme 2. Synthesis of Molecular Rectangles 4 and 5
In this paper, we report the synthesis and characterization of
two new cationic molecular rectangles incorporating [Ru2(μ-η4-
C2O4)(MeOH)2(η6-p-PriC6H4Me)2][O3SCF3]2 (2) and
[Ru2(p-cymene)2(donq)(OH2)2][O3SCF3]2 (donq = 5,8-diox-
ydo-1,4-naphthaquinonato) (3) building blocks with a new N-
(4-(pyridin-4-ylethynyl)phenyl) isonicotinamide (1) donor
ligand and their cytotoxic activity against various cancer cell
lines.
carbon peak was observed at 86 and 89 ppm, and the CO
peak was at 165 ppm.
Synthesis and Characterization of Molecular Rectan-
gles. Ligand 1 was stirred with the [Ru2(μ-η4-C2O4)-
(MeOH)2(η6-p-PriC6H4Me)2][O3SCF3]2 (2) and [Ru2(p-
cymene)2(donq)(OH2)2][O3SCF3]2 (donq = 5,8-dioxydo-1,4-
naphthaquinonato) (3) acceptors, respectively, in acetone at
room temperature for 6 h to generate the [2 + 2] systems.
Precipitation with diethyl ether furnishes 4 and 5 as analytically
pure solids. Two isomeric molecular rectangles are possible, as
depicted in Scheme 2.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Ligand. The starting
ligand precursor (4-iodophenyl)-pyridine carbox-amide (PyI)
was synthesized by coupling 4-iodo-aniline and isonicotinoyl
chloride hydrochloride in CH2Cl2 in the presence of triethyl-
amine, followed by recrystallization from methanol/H2O (yield
74%). The 1H NMR showed one sharp singlet for the NH at δ
10.67 in d6-DMSO, and four aromatic resonances were found,
indicating the presence of eight protons (Figure S1, Supporting
Information). The ligand 1 was synthesized via Sonogashira
coupling of (4-iodophenyl)pyridine carboxamide (PyI) and 4-
ethynylpyridine in DMF solution (Scheme 1). In the IR
spectrum of 1, the NH and CO vibration stretching peaks for
the amide linkage were observed at 3345 and 1650 cm−1,
respectively, while the CC vibration stretching peak was
The evidence for the formation of two structural isomeric
1
products came from the H NMR spectra, which showed two
1
types of proton nuclei. In the H NMR spectrum of 4, two
different signals of amidic NH protons (δ 10.19 and 9.96 ppm)
and two sets of aromatic protons were observed in the ratio of
1:1, as shown in Figure 1. Two sets of doublets corresponding
to the pyridyl-H, (highlighted in Figure 1) may be assigned to
the two isomers, generally referred to as head-to-tail (HTT) and
head-to-head (HTH) (Scheme 2), and the relative stabilities of
these isomers were consistent with previous observations in
related systems.17 Moreover, we observed the 0.5 ppm upfield
shift of Ha and Hf protons as compared to ligand 1 (Figure 1).
This shift was most likely due to the enhanced electron density
at the metal center and was more pronounced for the Ha and
1
obtained at 2245 cm−1. The H NMR showed one broad
1
Hf protons than for the other N-pyridine protons. The H
singlet for the NH at δ 9.97 in acetone-d6, and six aromatic
resonances were found, indicating the presence of 12 protons
(Figure S2, Supporting Information). The 13C NMR spectrum
of the ligand was also simple, and all carbon centers were
located easily (Figure S3, Supporting Information). The CC
NMR spectrum of complex 5 was very similar to that of 4
(Figure S4, Supporting Information).
Electrospray ionization mass spectrometry (ESI-MS) of
complexes 4 and 5 were obtained to provide additional
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1
Figure 1. Comparison of representative H NMR spectra showing the aromatic portion of (a) amide ligand 1, (b) Ru acceptor 2, and (c) the self-
assembled [2 + 2] rectangle 4 in acetone-d6.
evidence for the formation of the molecular rectangles. The
charged states for 4 at m/z = 1007.1 ([M − 2OTf]2+) and
621.8 ([M − 3OTf] 3+) (Figure 2), and 5 at m/z = 1106.8 ([M
− 2OTf]2+) and 688.4 ([M − 3OTf]3+) were clearly observed
and isotopically resolved (Figure 2). These peaks matched well
with the theoretical distributions.
λabs = 335 and 337, 454 nm for 4 and 5 (Figure 4). The high-
energy bands observed in both the rectangles 4 and 5 were also
present in the spectra of the free ligand 1. These bands are
likely due to π → π* transitions of the ethynyl backbone, which
are preserved upon self-assembly. The dinuclear arene−Ru
acceptors exhibited energy absorption bands at 263, 319 and
320, 450 nm for 2 and 3, respectively. These bands are likely a
combination of intra/intermolecular π → π* transitions mixed
with metal-to ligand charge-transfer transitions. As with the
bands of the pyridyl donors, these arene−Ru-based bands are
also preserved upon self-assembly, giving rise to strong
absorption for 4 and 5.6e The rectangle 4 gives a strong
absorption band with respect to acceptor 2 red shifted by ∼15
nm. Similar red shifts (∼17 nm) are observed for the bands in
5, which correspond to the absorption in donor 3.
In Vitro Anticancer Activity. Metal-based drugs are widely
used in clinical applications. Currently, organometallic arene−
ruthenium(II) compounds are attracting considerable attention
as antitumor agents.18−20 To explore the potential biological
effects of the interaction of these systems with biomolecules, we
have evaluated ligand 1, acceptors 2 and 3, and metal-
larectangles 4 and 5 for their cytotoxicity against Colo320
(colorectal cancer), A549 (lung cancer), MCF-7 (breast
cancer), and H1299 (lung cancer) cancer cell lines. All cancer
cells were exposed for 24 h to increasing concentrations of the
compounds, and their proliferation was determined using the 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay.
Cisplatin, a chemotherapeutic drug, was used as a control. On
the basis of the results of the MTS assay, only one of the cell
X-ray crystallography unambiguously established the nano-
scopic rectangular shape of the product 4. Single crystals of 4
suitable for X-ray diffraction were grown at ambient temper-
ature by vapor diffusion of diethyl ether into a concentrated
acetone solution of the complex. The molecular structure of 4
is shown in Figure 3. However, only the head-to-tail isomer
diffracted successfully. We were unable to crystallize the head-
to-head isomer even after repeated recrystallization. X-ray
analysis revealed that the two pyridine units bridge with two
[(Ru2(μ-η4-C2O4)(η6-p-PriC6H4Me)2]4+ building blocks to
form an M4L2 rectangle. In the difference Fourier map, two
independent structures were observed with similar geometries
(Figure S6, Supporting Information). The bond distances of
Ru1−Npyridine and Ru2−Npyridine are 2.13(2) and 2.10(1) Å, and
the Ru3−Npyridine and Ru4−Npyridine distances are 2.12(1) and
2.06 (2) Å, respectively. The observed Ru−N and Ru−O
distances are comparable to those found in oxalato-bridged
tetracationic rectangles.14,15a Despite the head-to-tail fashion of
ligand 1 binding to the Ru centers, the amide group carbonyl
(−CO−) and NH moieties aligned in the opposite direction.
Electronic absorption spectra of 4 and 5, along with their
corresponding metal acceptors 2 and 3 and donor 1, were also
investigated. The absorption spectra of 4 and 5 in methanol
solutions at 1 × 10−5 M concentration exhibit intense bands at
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Figure 2. Theoretical (top) and experimental (bottom) ESI-MS results for self-assembled [2 + 2] rectangles 4 and 5.
Figure 4. UV−visible spectra of 1−5 in methanol (1 × 10−5 M)
solution.
Interestingly, all four cell lines used in this study were highly
sensitive to the rectangle 5, and their growths were effectively
inhibited at a very low concentration (Table 1 and Figure 5). In
particular, the rectangle 5 was found to inhibit the proliferation
of H1299 cells with an IC50 of 3.62 μM, whereas the rectangle
4, donor 1, and both arene−ruthenium acceptors 2 and 3 did
not have any cytotoxic effect, as shown in Figure 5 and Table 1.
To determine the ability of rectangle 5 to induce apoptosis in
cancer cells, H1299 cells were treated with 10 μM rectangle 5
and cisplatin, respectively, for 12 h, after which they were
stained with TUNEL and examined by FACS analysis. Whereas
cisplatin caused apoptosis in 25% of H1299 cells, rectangle 5
Figure 3. (a) X-ray crystal structure of 4 showing the two Ru2+ ions
(green) coordinated with four pyridine N atoms in head-to-tail mode.
(b) and (c) side views are shown by the CPK model.
lines, Colo320, was found to be sensitive to cisplatin and the
other three cell lines, such as A549, MCF-7, and H1299, were
resistant to it. The comparative cytotoxicity of the ligand 1 and
arene−ruthenium acceptors 2 and 3 with tetranuclear
molecular rectangles 4 and 5 are presented in Table 1.
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various times before adding them to H1299 cell cultures. As a
control, the rectangles were preincubated in DMSO at 37 °C
for the same times. As shown in Figure 7, a 50% loss in growth-
Table 1. Cytotoxicity of the Compounds 1−5 in Human
Cancer Cells
a
IC50 (μM)
compound
Colo320
A549
H1299
MCF7
cisplatin
38.6
>100
70.98
40.94
18.05
38.86
10.18
>100
>100
>100
>100
>100
3.62
>100
>100
>100
63.97
80.91
<0.1
1
2
3
4
5
>100
51.01
12.95
>100
0.33
a
IC50: drug concentration necessary for 50% inhibition of cell
viability.
Figure 7. Loss of growth-inhibitory activity of the rectangles
preincubated in culture medium. Rectangles 4 and 5 at 20 μM were
preincubated in a cell culture medium containing 10% FBS and 100 μg
of penicillin−streptomycin (DMEM-10) at 37 °C for indicated times
before being added to cultures of H1299 cells. DMSO was used as a
control.
Figure 5. Comparative cytotoxic effects of 2, 3, 4, and 5 with cisplatin
on the H1299 cancer cell line.
inhibitory activity of the rectangle 5 was observed around 24 h
of preincubation in the cell culture medium. On the contrary, in
DMSO, its growth-inhibitory activity was stable until 72 h of
preincubation. In the case of the rectangle 4, consistent with
the data shown in Figure 5, its growth-inhibitory activity was
very low and we could not determine whether the
preincubation affected its activity. It would be expected that
the loss of growth-inhibitory activity parallels the loss of the
rectangles from the medium, suggesting that the rectangle 5
may be stable for 24 h of preincubation in the cell culture
medium. In addition, for stability studies, molecular rectangle 5
led to apoptosis in 84% of the cell population (Figure 6). This
result suggested that the inhibitory effect of rectangle 5 on the
1
was dissolved in DMSO, and the sample was analyzed by H
NMR spectroscopy immediately after dissolution and after 48
h. No change was observed even after 48 h, thus attesting to the
stability of the molecular rectangles in DMSO (Figure S5,
Supporting Information).
Stability of Rectangle 5 in the Presence of Thiol
(GSH). Previous work has shown that some complexes were
reactive with glutathione, and other small molecular weight
thiols.21 To gain insight into the potential reactivity of the thiol
group with 5, its stability in the presence of thiol was
investigated by using H NMR spectroscopy. The H NMR
spectrum of 5 was unaffected by the addition of reduced
glutathione (GSH) up to 24 h in a DMSO/H2O mixed solvent.
There was no change in the characteristic chemical shifts of 5,
and the resonance peaks remained sharp, even in the presence
of excess GSH. This result suggested that the thiol did not bind
to the complex or displace the coordination sphere, thus
attesting to the stability of the molecular rectangles in the
presence of 10 equiv of thiol (Figure S5, Supporting
Information).
Figure 6. Analysis of rectangle-induced apoptosis of H1299 cells by
TUNEL assay. DMSO and cisplatin were used as controls. Data
represent means SD of three independent experiments.
1
1
growth of cancer cells was obtained from the induction of
apoptosis.
This result indicated that rectangle 5 was much more
effective than cisplatin in the inhibition of cancer cell growth
and might be a candidate for the development of a
chemotherapeutic drug against cisplatin-resistant cancer cells.
Although both rectangles have a mixture of two structural
isomers, rectangle 5 was more cytotoxic than rectangle 4 in all
the cancer cell lines. The reason for the enhanced cytotoxicity
of 5 is not clear, but it can be related to the presence of
extended aromaticity in the dhnq ligand.
Our next goal was to determine the stability of the growth-
inhibitory activity of the rectangles in the cell culture medium.
This was tested by preincubating 20 μM rectangle 4 or 5 in the
culture medium with 10% FBS (DMEM-10) at 37 °C for
CONCLUSION
■
In conclusion, we report the synthesis of two new large
molecular rectangles via self-assembly of a new unsymmetrical
amide ligand and two different lengths of arene−ruthenium
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were plated in 60 mm dishes at 2 × 105cells/mL DMEM. The
following day, the cells were treated with 10 μM cisplatin or 10 μM
rectangle 5, harvested, and fixed with 2% paraformaldehyde solution
and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate.
After washing twice with PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM
Na2HPO4·7H20; 1.4 mM KH2PO4; pH 7.2), cells were incubated in a
TUNEL reaction mixture containing terminal deoxynucleotidyl
transferase and tetramethyl-rhodamine-dUTP. Cells were analyzed
for fluorescence intensity using a FACS flow cytometer (Becton
Dickinson, Inc.).
1
acceptors. Both rectangles 4 and 5 were characterized by H
NMR and ESI-MS analysis. The solid-state structure of 4 was
determined by a single-crystal X-ray diffraction study,
confirming the rectangular structure assigned to these
1
complexes. The H NMR spectra of both rectangles showed
a mixture of two structural isomers in a 1:1 ratio. The new self-
assembled rectangles were further screened for in vitro
anticancer activity, and the antitumor efficacy of rectangle 5
was found to be considerably stronger against all cell lines than
that of cisplatin.
Reaction of 5 with L-Glutathione Reduced (GSH). Aliquots of a
solution of 5 in DMSO were added to solutions containing 2 or 10
equiv of GSH in D2O to give final millimolar concentrations in
DMSO/D2O (1:1, v/v). 1H NMR spectra were recorded at l and 24 h
of reaction time.
Synthesis of (4-Iodophenyl)pyridine Carboxamide (PyI). A
solution of isonicotinoyl chloride hydrochloride (1.00 g, 5.60 mmol)
and triethylamine (1.5 mL) in 20 mL of CH2Cl2 was prepared and
chilled at 0 °C in an ice bath for 5 min; then, 4-iodo aniline (1.35 g,
6.16 mmol) was added slowly to the cold solution over a period of 15
min. The reaction mixture was stirred at room temperature overnight.
The resulting precipitate was collected on a frit, recrystallized with
MeOH/H2O, and dried in a vacuum oven to afford a dark powder.
Yield: ca. 78% (1.45 g). Anal. Calcd for C12H9N2O: C, 44.4; H,
2.80; N, 8.64; O, 4.94. Found: C, 44.35; H, 2.70; N, 8.63%; O, 5.11.
1H NMR (d6-DMSO, 300 MHz, δ, ppm): 10.58 (s, 1H, CONH), 8.78
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals used in this work were
purchased from commercial sources and used without further
purification. The starting acceptors [Ru2(μ-η4-C2O4)(MeOH)2(η6-p-
PriC6H4Me)2][O3SCF3]2 (2)15a and [Ru2(p-cymene)2(donq)-
(OH2)2][O3SCF3]2 (3)20 were prepared by reported procedures.
1
The H and 13C NMR spectra were recorded in a Bruker 300 MHz
1
spectrometer. The chemical shifts (δ) in the H NMR spectra are
reported in parts per million relative to tetramethylsilane (Me4Si) as
an internal standard (0.0 ppm). The infrared spectra (either as KBr
pellets or as a mull in mineral oil) were recorded using a Varian 2000
FTIR spectrometer. Mass spectra were recorded on a Micromass
Quattro II triple-quadrupole mass spectrometer using electrospray
ionization (ESI) with a MassLynx operating system. Elemental
analyses were performed by using the Elemental GmbH Vario EL-3
instrument.
(d, 2H, J = 4.8 Hz, Ha), 7.84 (d, 2H, J = 5.1 Hz, Hb), 7.72 (d, 2H, J =
8.7 Hz, Hc), 7.62 (d, 2H, J = 8.7 Hz, Hd).
Synthesis of N-(4-(Pyridin-4-ylethynyl)phenyl)-
isonicotinamide(HL) 1. In a flame-dried Schlenk flask, 4-
ethynylpyridine hydrochloride (0.388 g, 2.77 mmol) and triethylamine
(3 mL) were vigorously stirred for 20 min. During this time, a white
precipitate formed. Thereafter, DMF (15 mL), Pd(PPh3)2Cl2 (0.063 g,
0.09 mmol), CuI (0.012 g, 0.062 mmol), PPh3 (0.016 g, 0.062 mmol),
and (4-iodophenyl)pyridine-carboxamide (0.600 g, 1.851 mmol) were
successively added. The Schlenk flask was wrapped in aluminum foil to
keep the reaction mixture in the dark, and the mixture was refluxed for
24 h. The resulting brown precipitate was collected on a frit, washed
several times with water, and was recrystallized with CH3OH/H2O
(1:1). The yellow compound was filtered and dried under vacuum.
Yield: ca. 72% (0.4 g). Anal. Calcd for C19H13N3O: C, 76.24; H,
4.38%; N, 14.04; O, 5.35. Found: C, 76.35; H, 4.40; N, 14.13; O, 5.31.
FT-IR spectrum (KBr, ν, selected peaks): 3345 (NH), 2250 (CC),
X-ray Diffraction Data for 4. Single crystals suitable for X-ray
diffraction study of 4 were obtained by the vapor diffusion of diethyl
ether to a acetone solution of the compound. A single crystal of
rectangle 4 was mounted on a loop, and data were collected at 100 K
on an ADSC Quantum 210 CCD diffractometer with a synchrotron
radiation (λ = 1.00000 Å) at Macromolecular Crystallography
Beamline 6B1, Pohang Accelerator Laboratory (PAL), Pohang,
Korea. The raw data were processed and scaled using the program
HKL2000. The structure was solved by direct methods, and the
refinements were carried out with full-matrix least-squares on F2 with
appropriate software implemented in the SHELXTL program package.
The contributions of the disordered solvent molecules were removed
from the diffraction data using the SQUEEZE routine of PLATON
software (c.a. 876 e/u.c), and then final refinements were carried out.
X-ray data for 4: C83H82F3N6O13Ru4S, M = 1864.89, monoclinic, P21/c
(No. 14), a = 34.156(7) Å, b = 10.371(2) Å, c = 27.318(6) Å, β =
98.05(3)°, V = 9581(3) Å3, Z = 4, T = 90 K, μ(synchrotron) = 1.736
mm−1, ρcalc = 1.293 g·cm−3, 18 743 reflections measured, 5913 unique
(Rint = 0.0443), R1 = 0.0946 (I > 2σ(I)), wR2 = 0.2883 (all data), GOF
= 1.190, 992 parameters and 852 restraints.
Cancer Cell Growth Inhibition Assay (MTS Assay). Human
cancer cell lines, Colo320, A549, H1299, and MCF7, were purchased
from the Korean Cell Line Bank (KCLB-Seoul, Korea). The cells were
cultured in Dulbecco’s Modified Eagle Medium (DMEM) supple-
mented with 10% heat inactivated fetal bovine serum (FBS) and 100
μg of penicillin−streptomycin at 37 °C in a humidified atmosphere of
5% CO2. For the MTS cell proliferation assay, cells were plated in
triplicate at 5.0 × 104 cells/well in 96-well culture plates in RPMI 1640
medium. Compounds 1−5 were preincubated in DMEM-10 or
DMSO for the indicated times and added to the cells. After 24 h of
incubation, MTS (CellTiter 96 Aqueous One Solution reagent,
Promega, USA) was added to each well according to the
manufacturer’s instructions. Absorbance at 490 nm was determined
for each well using a Victor 1420 multilabel counter (EG&GWallac,
Turku, Finland). The percentage of surviving cells was calculated from
the ratio of absorbance of treated to untreated cells. The IC50 values
were obtained by fitting values to sigmoidal dose−response curves
using the routines provided in GraphPad Prism.
1
1650 (CO) cm−1. H NMR (d6-acetone, 300 MHz, δ, ppm): 9.97
(s, 1H, CONH), 8.78 (d, 2H, J = 4.2 Hz, Ha), 8.61(d, 2H, J = 4.2 Hz,
Hf), 7.92 (d, 2H, J = 8.7 Hz, Hb), 7.88 (d, 2H, J = 8.7 Hz, Hc), 7.62 (d,
2H, J = 4.2 Hz, He), 7.46 (d, 2H, J = 4.2 Hz, Hd). 13C NMR (DMSO-
d6, 300 MHz, δ, ppm): 164.23 (CO, C4), 150.33 (C13), 149.36 (C1),
142.23 (C3), 140.10 (C5), 132.51 (C7), 130.26 (C11), 125.32 (C12),
122.10 (C6), 120.28 (C2), 116.60 (C8), 94.05 (C9), 86.56 (C10).
Synthesis of Molecular Rectangle 4. The ligand 1 (0.006 g,
0.020 mmol) and solid [Ru2(μ-η4-C2O4)(MeOH)2(η6-p-
PriC6H4Me)2][O3SCF3]2 (2) (0.018 g, 0.020 mmol) were suspended
in CH2Cl2/CH3OH (1:1, 1 mL), and the resulting mixture was then
stirred for 6 h at room temperature. The reaction mixture was filtered,
followed by the removal of the solvent under reduced pressure, and
the product was washed with diethyl ether. The crude product thus
obtained was redissolved in acetone and subjected to vapor diffusion of
diethyl ether. This resulted in the highly crystalline product within a
day. The product was filtered and dried under vacuum. Yield: ca. 92%
(0.022 g). Anal. Calcd for C86H86F12N6O22Ru4S4: C, 44.60; H, 3.74; N,
3.63. Found: C, 44.80; H, 3.72; N, 3.60. FT-IR spectrum (KBr, ν,
selected peaks): 3340 (NH), 2245 (CC), 1620 (CO) cm−1. MS
(ESI) calcd for [M − 2OTf]2+ m/z 1007.1, found 1007.1; calcd for [M
1
− 3OTf]3+ m/z 622.1, found 622.1. H NMR (d6-acetone, 300 MHz,
δ, ppm): 10.18 (s, H, CONH), 9.94 (s, H, CONH), 8.30−8.20 (m,
8H, Ha/Ha’), 8.13−8.08 (m, 8H, Hf/Hf’), 8.02−7.89 (m, 8H, Hb/Hb,),
7.70−7.62 (m, 8H, Hc/Hc’), 7.47−7.39 (m, 8H, He/He,), 7.26−7.23
(m, 2H, Hd/d,), 6.10 (m, 16H; −C6H4), 5.94 (m, 16H, −C6H4), 2.86
TUNEL Staining. TUNEL staining was conducted using an in situ
cell death detection kit, TMR Red, according to the protocol supplied
by the manufacturer (Roche Molecular Biochemicals). Briefly, cells
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Organometallics
Article
(m, 8H, −CH(CH3)2), 2.28−2.27 (m, 24H; −CH3), 1.39−1.36 (m,
48H; −CH(CH3)2).
Synthesis of Rectangle 5. The synthesis was performed as
described for rectangle 4 except that the [Ru2(p-cymene)2(donq)-
(OH2)2][O3SCF3]2 (donq = 5,8-dioxydo-1,4-naphthaquinonato) (3)
acceptor was used, and the product was isolated as a green crystalline
solid.
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Yield: ca. 91%. Anal. Calcd for C86H86F12N6O22Ru4S4: C, 48.76; H,
3.61; N, 3.35. Found: C, 48.80; H, 3.72; N, 3.40. FT-IR spectrum
(KBr, ν, selected peaks): 3342 (NH), 2247 (CC), 1633 (CO)
1
cm−1. MS (ESI) calcd for [M − 3OTf]3+ m/z 755.4, found 755.6. H
NMR (d6-acetone, 300 MHz,δ, ppm): 10.04 (s, H, CONH), 10.01 (s,
H, CONH), 8.72−8.68 (m, 8H, Ha/Ha’), 8.55−8.53 (m, 8H, Hf/Hf’),
7.93−7.85 (m, 8H, Hb/Hb’), 7.83−7.78 (m, 8H, Hc/Hc’), 7.51−7.48
(m, 8H, He/He’), 7.43−7.40 (m, 8H, Hd/Hd’), 7.31−7.30 (d, 16H,
Har), 6.02 (m, 16H; −C6H4), 5.82 (m, 16H, −C6H4), 3.08 (m, 8H,
−CH(CH3)2), 2.28 (m, 24H; −CH3), 1.49−1.35 (m, 48H; −CH-
(CH3)2).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Supporting Information available: H NMR spectra of starting
PyI and ligand 1, 13C NMR spectrum of ligand 1, H NMR
1
spectrum of 5 in acetone-d6, comparative 1H NMR spectrum of
5 with and without GSH, X-ray crystal structure of rectangle 4
with two independent molecules, and table of bond lengths and
angles for 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: kwchi@ulsan.ac.kr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.-W. C. gratefully acknowledges generous financial support
from the World Class University (WCU) program (R33-2008-
000-10003) and Priority Research Centres program (2009-
0093818) through the National Research Foundation of Korea
(NRF) and the X-ray diffraction experiments performed at the
Pohang Accelerator Laboratory in Korea. P.J.S. thanks the
National Institutes of Health (NIH), USA (Grant No. GM-
057052), for financial support.
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