Anthracene-tethered ruthenium(II) arene complexes as tools to visualize the cellular localization of putative organometallic anticancer compounds

Article abstract of DOI:10.1021/ic202530j

Anthracene derivatives of ruthenium(II) arene compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) or a sugar phosphite ligand, viz., 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-d-glucofuranoside, were prepared in order to evaluate their a

Full text of DOI:10.1021/ic202530j

Article
pubs.acs.org/IC
Anthracene-Tethered Ruthenium(II) Arene Complexes as Tools To
Visualize the Cellular Localization of Putative Organometallic
Anticancer Compounds
Alexey A. Nazarov,*,† Julie Risse,† Wee Han Ang,‡ Frederic Schmitt,§ Olivier Zava,† Albert Ruggi,†
Michael Groessl,† Rosario Scopelitti,† Lucienne Juillerat-Jeanneret,§ Christian G. Hartinger,⊥
and Paul J. Dyson*,†
†Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), 1015 Lausanne, Switzerland
‡Department of Chemistry, National University of Singapore, Singapore 117543, Singapore
§University Institute of Pathology Centre Hospitalier Universitaire Vaudois (CHUV), 1011 Lausanne, Switzerland
⊥School of Chemical Sciences, The University of Auckland, Auckland 1142, New Zealand
S
* Supporting Information
ABSTRACT: Anthracene derivatives of ruthenium(II) arene
compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane
(pta) or a sugar phosphite ligand, viz., 3,5,6-bicyclophosphite-
1,2-O-isopropylidene-α-D-glucofuranoside, were prepared in
order to evaluate their anticancer properties compared to the
parent compounds and to use them as models for intracellular
visualization by fluorescence microscopy. Similar IC50 values
were obtained in cell proliferation assays, and similar levels of
uptake and accumulation were also established. The X-ray
structure of [{Ru(η6-C6H5CH2NHCO-anthracene)Cl2(pta)] is
also reported.
tumor cells over healthy cells.10 The strategy of using sugar-
INTRODUCTION
Ruthenium-based antitumor drugs are being intensively
■
based ligands to preferentially deliver metal drugs to tumors has
been widely explored,21 but definitive experiments demonstrat-
studied, with two representatives currently undergoing clinical
trials.1−4 Many ruthenium compounds exhibit features that
ing preferential uptake are lacking. Several attempts have been
undertaken to localize metallodrugs in tumor cells, and
make them interesting for drug development including mild
different methods have been used.22−31 However, in the case
of ruthenium anticancer agents, the field is rather unex-
plored.32,33 We describe herein functionalization of RAPTA-
derived complexes with the anthracene fluorophore and an
investigation of the antineoplastic activity and intracellular
localization of the complexes with a pta ligand or a sugar-
derived phosphite, in order to establish the role of the latter
ligand in tumor uptake.
toxicity in vitro and high activity in in vivo models.5−7 These
biological effects may be attributed to a number of factors
including selective accumulation in the tumor environment
and/or tumor cells,8−10 inhibition of the antimetastatic
progression, and antiangiogenic properties.7,11−13 It has also
been shown that enzyme inhibition, as opposed to or in
addition to covalent binding to DNA, may be responsible for
these observed effects.14−17
Organoruthenium(II) arene compounds with 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane (pta) coligands, termed
RAPTA, and ethylene-1,2-diamine (en) ligands18 have been
studied extensively with respect to their tumor-inhibiting
properties. In particular, the RAPTA scaffold has been
functionalized with bioactive molecules.1,19,20 One such
modification entails substitution of the pta ligand with sugar-
derived phosphites that might be able to preferentially
accumulate in tumors because tumor cells have a high energy
demand and a limited oxygen supply, leading to higher glucose
uptake and glycolysis. Indeed, this modification resulted in
compounds with enhanced in vitro anticancer activity against a
panel of tumor cell lines and a high degree of selectivity for
RESULTS AND DISCUSSION
■
Fluorescent analogues of RAPTA complexes and their
carbohydrate derivatives were prepared by derivatization of
the arene ring with anthracene via an amide bond to the piano-
stool-configured ruthenium scaffold. Attachment of an
anthracene moiety is expected to change the biological
properties of the resulting derivative to a much lesser extent
than other widely used fluorescent tags.24,34−39 The synthetic
route was adapted from an earlier report that describes the
Received: November 23, 2011
Published: March 6, 2012
© 2012 American Chemical Society
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preparation of RAPTA compounds carrying the glutathione-S-
transferase inhibitor ethacrynic acid (EA).14 9-Anthracenecar-
boxylic acid was converted into its acid chloride by oxalyl
chloride/N,N-dimethylformamide (DMF) and reacted with
1,4-cyclohexadiene-1-methylamine or 1,4-cyclohexadiene-1-eth-
ylamine to yield N-(cyclohexa-1,4-dienylmethyl)anthracene-9-
carboxamide and N-(2-(cyclohexa-1,4-dienyl)ethyl)anthracene-
9-carboxamide, respectively. These cyclic dienes were reacted
with RuCl3 in ethanol under reflux to yield the respective
RuII(arene) dimers as brown solids. Finally, the dimers were
treated with 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-
glucofuranoside or pta to yield complexes 1−4 (Scheme 1).
Figure 1. Molecular structure of 3. The hydrogen-bonding network
[N3···N4′ 3.000(1) Å] and π−π-stacking interactions [the shortest
distance is 3.324(19) Å] are shown as solid and dashed lines,
respectively. The ellipsoids are drawn at the 50% probability level.
Scheme 1. Synthesis of 1−4 (a, 3,5,6-Bicyclophosphite-1,2-
O-isopropylidene-α-D-glucofuranoside; b, 1,3,5-Triaza-7-
phosphatricyclo[3.3.1.1]decane)
Table 1. Comparison of the Bond Lengths (Å) and Angles
(deg) of 3 with RAPTA-C and RAPTA-EA
3
RAPTA-C
RAPTA-EA
Ru−arenecentroid (Å)
Ru−P (Å)
Ru−Claver (Å)
1.691
1.701
1.697
2.2911(8)
2.415
2.296(2)
2.421
2.321(4)
2.429
Cl−Ru−Cl (deg)
P−Ru−Cl1 (deg)
P−Ru−Cl2 (deg)
88.84(3)
82.49(3)
86.31(3)
87.25(8)
83.42(8)
87.09(9)
89.32(11)
84.58(11)
82.09(12)
spectra suggest hydrolysis of the amide bond and the release of
anthracene under the conditions, and therefore it is not
unreasonable to assume that in in vitro studies the applied
compounds enter the cells intact. Hydrolysis can be remarkably
suppressed (>95%) by the addition of 100 mM NaCl.
The cytotoxicity of 1−4 was studied using MTT and
[3H]thymidine incorporation assays in 12 human cell lines
comprising SW480, HT29, and CaCo2 colon carcinoma, MDA-
MB231 and MCF-7 breast carcinoma, A549 lung carcinoma,
A2780 and A2780cisR ovarian carcinoma, LN18, LN229, and
LNZ308 glioblastoma, and HCEC cerebral endothelial cells
(Table 2). Only 1 exhibited IC50 values lower than 200 μM in
the MTT assay, whereas the other complexes are basically
nontoxic even after 72 h of drug exposure. Low in vitro
cytotoxicities have been observed previously for structurally
related compounds, and ruthenium compounds are, in general,
less cytotoxic than the clinically established platinum
compounds.10 The RuII(η6-p-cymene) compound with the
same phosphorus-based ligand as that of 1 and 2 exhibited IC50
values in most cell lines in the range 360−680 μM after 72 h of
exposure. Incubation of SW480 and A549 cells for 96 h with
the parent compound of 3 and 4, i.e., RAPTA-C, showed no
significant cytotoxicity with IC50 values of 170 and >640 μM,41
similar to the mentioned sugar derivative in SW480 (361
μM).10 Such behavior is often observed for ruthenium
anticancer agents, and high cytotoxicity is not a prerequisite
for antitumor activity in vivo or even in patients.44,45 Moreover,
carbohydrate complexes often exhibit low antineoproliferative
activity in cell culture, which is accompanied by improved
tolerability and only slightly lower antitumor activity in animal
experiments.21
The compounds were characterized by 1H, 13C{1H}, and
31P{1H} NMR spectroscopy, mass spectrometry, and elemental
analysis. 31P{1H} NMR spectroscopy was used to monitor the
formation of complexes 1−4. In these spectra, a shift from ca.
119 to 133 ppm and from −100 to −33 ppm was observed for
the phosphite and pta ligands, respectively, upon coordination.
1
In addition, characteristic shifts to lower frequency in both H
and 13C{1H} NMR spectra, as reported for similar types of
complexes, were observed, confirming the proposed structure
of the complexes.10
Single crystals of 3 suitable for X-ray diffraction analysis were
obtained by slow diffusion of pentane into a chloroform
solution containing the complex. Complex 3 features a piano-
stool geometry (Figure 1) with bond lengths and angles similar
to those of related RAPTA-C40 and RAPTA-EA14 complexes
(Table 1). In the crystal, a combination of hydrogen bonding
involving the pta ligands and π−π-stacking interactions
between the anthracene units of adjacent molecules connects
the molecules.
Complexes 1−4 undergo aquation in water via substitution
of a chlorido ligand by an aqua group, as indicated by 31P{1H}
NMR spectroscopy and consistent with studies on related
compounds.10 This transformation is characterized by a shift of
the P atom resonance from approximately 133 to 140 ppm in
the carbohydrate−phosphite complexes and from −33 to −28
ppm in the pta compounds. In addition, 1 and 2 undergo a P−
O cleavage of the sugar phosphite carrier ligand in aqueous
solution, as observed for structurally related ruthenium and
osmium complexes.10,43 Notably, neither 1H nor 31P{1H} NMR
Tethering the anthracene moiety to the arene ligand did not
significantly influence the in vitro antitumor activity, whereas
the incorporation of conjugate π systems results in enhanced
antitumor activity because of their intercalating properties and
higher lipophilicities resulting in increased cellular uptake.46
Because anthracene derivatives are known to exhibit intrinsic
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Table 2. IC50 Values As Determined by the MTT Assay after 24, 48, and 72 h of Exposure of the Cells to 1−4 and RAPTA-C and
by the [3H]Thymidine Assay (Values in Parentheses As Measured after 24 h of Incubation with the Test Compound)
IC50/μM
MDA-
MB231
compound
cell line
LN18
LN229
LNZ308
SW480
>200
HT29
CaCo2
A549
MCF-7
HCEC
A2780
A2780cisR
1
MTT, 24 h
MTT, 48 h
MTT, 72 h
[3H]thym
>200
90
110
165
>200
100
95
>200
>200
>200
(12)
>200
120
>200
>200
>200
(11)
>200
>200
>200
(14)
>200
>200
>200
(14)
>200
>200
>200
(25)
>200
60
120
>200
40
40
140
70
40
35
50
70
200
80
120
125
110
50
30
>200
(12)
(25)
>200
>200
>200
(25)
>200
>200
>200
(35)
>200
>200
>200
(>200)
(10)
>200
100
(15)
>200
>200
>200
(50)
>200
>200
>200
(13)
>200
>200
>200
(>200)
(35)
(25)
(24)
>200
>200
>200
(16)
>200
>200
>200
(20)
>200
>200
>200
(50)
(21)
(20)
>200
>200
>200
(52)
>200
>200
>200
(18)
>200
>200
>200
(>200)
(10)
>200
>200
>200
(50)
>200
>200
>200
(35)
>200
>200
>200
(>200)
2
3
4
MTT, 24 h
MTT, 48 h
MTT, 72 h
[3H]thym
>200
>200
>200
(75)
>200
>200
>200
(150)
>200
>200
>200
(>200)
>200
>200
>200
(>200)
>200
>200
>200
(14)
>200
>200
>200
(50)
>200
>200
>200
(50)
100
(14)
>200
>200
>200
(13)
>200
>200
>200
(>200)
MTT, 24 h
MTT, 48 h
MTT, 72 h
[3H]thym
>200
>200
>200
(>200)
>200
>200
>200
(>200)
170 60
>200
>200
>200
(13)
>200
>200
>200
(>200)
>200
>200
>200
(>200)
>200
>200
>200
(>200)
>200
>200
>200
(>200)
MTT, 24 h
MTT, 48 h
MTT, 72 h
[3H]thym
>200
>200
>200
(>200)
a
b
b
b
RAPTA-C MTT
436
>1000
>1000
a
b
96 h (taken from ref 41). 72 h (taken from ref 42). LN18, LN229, and LNZ308: human glioblastoma cells. SW480, HT29, and CaCo2: human
colon carcinoma cells. A549: human lung carcinoma cells. MDA-MB231 and MCF-7: human breast carcinoma cells. HCEC: human endothelial cells.
A2780 and A2780cisR: human ovarian carcinoma cells, cisplatin-sensitive and resistant, respectively.
fluorescence, the compounds were thought to be suitable
models for cellular uptake studies using fluorescence micros-
copy. Consequently, fluorescence emission spectra were
recorded for 1 and 3. Because of the relatively quick aquation
of the compounds in aqueous solutions, the spectra were
recorded in DMF. In both cases, the emission maxima were
very similar at 412 and 411 nm, respectively (Figure 2).
Figure 3). Cells were fixed with a 4% buffered paraformalde-
hyde solution on a glass slide, and images were taken after
excitation at 365 nm. Intense fluorescence was observed in the
case of 1 with the sugar phosphite ligand, whereas only
moderate fluorescence was detected for the pta complex 3 (ca.
three times higher for 1, resembling approximately the
quantum yield ratio for both compounds), which is most
Figure 2. Fluorescence spectra of 1 and 3 (11 μM) after excitation at
λex = 365 nm.
However, the fluorescence intensity of 1 was much higher than
that of 3, demonstrating the influence of the phosphorus-
derived ligands. This fact was also shown by determining the
quantum yields of 0.126 and 0.036 for 1 and 3.
Fluorescence microscopy was used to study the localization
of the anthracene-functionalized compounds in A549 lung
carcinoma cells in order to delineate the influence of the
phosphorus ligand on the cellular visualization of the
complexes. A549 lung carcinoma cells were incubated with 1
and 3 for 24 h at 50 μM (well below the cytotoxicity IC50;
Figure 3. Fluorescence microscopy of A549 lung carcinoma cells
treated with (a) 1 and (b) 3 (50 μM each) for 24 h (λex 365 nm). (c
and d) 6-fold-magnified zoom images of cells shown in a) and b),
respectively.
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probably related to the, in general, lower fluorescence emission
of 3. It appears that neither 1 nor 3 accumulate in the nucleus,
as was observed by other methods with structurally related
complexes.33
Table 3. Crystal Data and Structure Refinement for 3
empirical formula
fw
C28H29Cl2N4OPRu
640.49
temperature (K)
wavelength (Å)
cryst syst
140(2)
In order to elucidate the influence of the phosphorus-
containing ligands on the cellular accumulation of anthracene-
tethered RuII(arene) complexes, the ruthenium concentration
was measured after incubation of A2780 cells with 1 and 3.
Very similar amounts of ruthenium were found after treatment
with 1 and 3 at equimolar concentrations (332 16 and 305
48 pmol/106 cells of ruthenium, respectively). This is in the
same dimension as that observed for the ruthenium compounds
KP1019 and NAMI-A.33 These data sets reveal only a minor
influence of the phosphorus ligand on the uptake, which,
however, might be leveled by the high lipophilicity related to
the anthracene substituent.
0.710 73
monoclinic
space group
unit cell dimens
P21/c
a = 17.1875(4) Å, α = 90°
b = 12.9255(3) Å, β = 93.548(2)°
c = 13.7320(4) Å, γ = 90°
3044.80(13)
volume (Å3)
Z
4
density (calcd) (mg/m3)
1.397
abs coeff (mm−1
)
0.769
F(000)
cryst size (mm3)
1304
0.34 × 0.26 × 0.21
In addition to the MTT assay, [3H]thymidine incorporation
was studied as a measure of the DNA synthesis as a function of
the compound concentration. In this experimental setup, 1 was
found to be the most inhibitory of the test compounds,
followed by 2 and 3. In contrast, 4 showed no activity against
the majority of the human tumor cell lines, with the exception
of the MCF-7 and CaCo2 cell lines. Compound 1 was similarly
potent in all cell lines, with IC50 values in the range 10−35 μM
and LN229 and MCF-7 representing the most sensitive cell
lines. Interestingly, the MCF-7 cell line showed no sensitivity to
the test compounds under the MTT assay conditions but was
the most sensitive cell line in the [3H]thymidine incorporation
experiments.
θ range for data collection (deg) 2.85−28.41
index ranges
−22 ≤ h ≤ 22, −17 ≤ k ≤ 17, −16 ≤ l ≤
16
reflns collected
21233
indep reflns
6641 [R(int) = 0.0273]
95.2%
completeness to θ = 25.00°
abs corrn
semiempirical from equivalents
1.00000 and 0.87796
full-matrix least squares on F2
6641/0/334
max and min transmn
refinement method
data/restraints/param
GOF on F2
1.068
final R indices [I > 2σ(I)]
R indices (all data)
R1 = 0.0380, wR2 = 0.1062
R1 = 0.0512, wR2 = 0.1122
largest diff peak and hole (e/Å3) 0.845 and −0.587
CONCLUSIONS
■
washed with toluene (3 × 5 mL), toluene was removed under reduced
pressure, and the crude product was purified by column chromatog-
raphy [eluent: n-hexane/EtAc (3:1)]. Yield: 3.31 g (54%). Mp: 152−
153 °C. Elem anal. Calcd for C22H19NO: C, 84.31; H, 6.11; N, 4.47.
Found: C, 84.22; H, 5.97; N, 4.23. 1H NMR (400.13 MHz, CDCl3): δ
8.50 (s, 1H, Hant), 8.11 (d, J = 8.2 Hz, 2H, Hant), 8.03 (d, J = 8.2 Hz,
2H, Hant), 7.53 (m, 4H, Hant), 6.06 (br s, 1H, NH), 5.76 (m, 3H, C
CH, CHCH), 4.27 (d, J = 5.6 Hz, 2H, CH2NH), 2.84 (m, 2H,
CH2), 2.75 (m, 2H, CH2). 13C{1H} NMR (100.63 MHz, CDCl3): δ
169.7 (CO), 131.9 (Cdiene), 131.4 (Cant), 130.9 (Cant), 128.4 (Cant),
128.1 (Cant), 127.9 (Cant), 126.5 (Cant), 126.4 (Cant), 125.0 (Cant),
124.0 (Cdiene), 123.7 (Cdiene), 121.4 (Cdiene), 45.5 (NHCH2), 27.5
(Cdiene), 26.5 (Cdiene). MS (ESI+): m/z 314 ([M + H]+).
In conclusion, we have prepared and characterized a series of
fluorescent RuII(arene) complexes tethered with an anthracene
moiety bearing pta or a sugar phosphite ligand with the aiming
of assessing the relative cellular uptake of compounds. Cells
incubated with the complex carrying the carbohydrate−
phosphite ligand exhibited higher fluorescence intensities than
those with pta because of the differences in the quantum yields
of the complexes and, importantly, reveal that the complexes do
not accumulate in the cell nucleus. No influence of the P-ligand
on the cellular accumulation was observed, which might be
related to the lipophilicity of the molecules.
EXPERIMENTAL SECTION
N-[2-(Cyclohexa-1,4-dienyl)ethyl]anthracene-9-carboxa-
mide. N-[2-(Cyclohexa-1,4-dienyl)ethyl]anthracene-9-carboxamide
was obtained from anthracene-9-carbonyl chloride (5.53 g, 23.0
mmol), 1,4-cyclohexadiene-1-ethylamine50 (3 mL, 23.6 mmol), and
triethylamine (4.1 mL, 29.5 mmol) using the procedure described for
N-(cyclohexa-1,4-dienylmethyl)anthracene-9-carboxamide. Yield: 3.17
g (42%). Mp: 173−176 °C. Elem anal. Calcd for C23H21NO: C, 84.37;
H, 6.46; N, 4.28. Found: C, 84.30; H, 6.13; N, 4.41. 1H NMR (400.13
MHz, CDCl3): δ 8.49 (s, 1H, Hant), 8.09 (d, J = 8.2 Hz, 2H, Hant), 8.02
(d, J = 8.2 Hz, 2H, Hant), 7.50 (m, 4H, Hant), 6.04 (br s, 1H, NH), 5.75
(m, 2H, CHCH), 5.56 (s, 1H, CCH), 3.87 (m, 2H, NHCH2),
2.75 (m, 2H, CH2), 2.68 (m, 2H, CH2), 2.43 (t, J = 6.2 Hz, 2H,
NHCH2CH2). 13C{1H} NMR (100.63 MHz, CDCl3): δ 169.5 (CO),
132.0 (Cdiene), 131.6 (Cant), 131.1 (Cant), 128.3 (Cant), 128.1 (Cant),
128.0 (Cant), 126.7 (Cant), 125.5 (Cant), 125.0 (Cant), 124.1
(cyclohexadiene), 124.0 (Cdiene), 121.6 (Cdiene), 37.4 (NHCH2CH2),
37.3 (NHCH2CH2), 28.4 (CH2), 26.7 (CH2). MS (ESI+): m/z 328
([M + H]+).
■
All solvents were purified and degassed prior to use.47 1H, 13C{1H},
and 31P{1H} NMR spectra were recorded on a Bruker Avance II 400
spectrometer at room temperature and were referenced to the 1H
signal of the NMR solvent or to H3PO4 as an external reference for
31P{1H} NMR spectroscopy. Electrospray ionization mass spectra
(ESI-MS) of the compounds were obtained in MeOH on a
ThermoFinnigan LCQ Deca XP Plus quadrupole ion-trap instrument
operated in positive-ion mode over a mass range of m/z 150−1000.
The ionization energy was set at 3.5 kV and the capillary temperature
at 150 °C. Melting points were determined with a Stuart Scientific
SMP3 apparatus and are uncorrected. The flash chromatography
system Varian 971-FP was used for purification. Elemental analyses
were carried out by the microanalytical laboratory at EPFL (Table 3).
N-(Cyclohexa-1,4-dienylmethyl)anthracene-9-carboxamide.
A solution of anthracene-9-carbonyl chloride48 (5.53 g, 23.0 mmol) in
CH2Cl2 (60 mL) was added dropwise to a stirred solution of 1,4-
cyclohexadiene-1-methanamine49 (2.7 mL, 23.6 mmol) and triethyl-
amine (4.1 mL, 29.5 mmol) in CH2Cl2 (120 mL). The reaction
mixture was stirred for 12 h at room temperature. The solvent was
evaporated, and the semicrystalline solid was suspended in toluene (25
mL). Triethylamine hydrochloride was removed by filtration and
Bis[dichlorido(η6-N-benzylanthracene-9-carboxamide)-
ruthenium(II)]. A solution of N-(cyclohexa-1,4-dienylmethyl)-
anthracene-9-carboxamide (2.0 g, 6.3 mmol) in degassed ethanol
(150 mL) was added to ruthenium(III) chloride hydrate (0.55 g, 2.1
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mmol). The reaction mixture was refluxed at 90 °C for 10 h under a
nitrogen atmosphere. Upon completion, a brown solid has formed,
which was filtered, washed with ethanol (3 × 5 mL) and diethyl ether
(2 × 5 mL), and dried in vacuo. Yield: 1.0 g (97%). Mp: >200 °C
(dec). Elem anal. Calcd for C44H34N2O2Ru2Cl4: C, 54.67; H, 3.55; N,
Hz, 1H, H6), 4.25 (s, 1H, H4), 4.15 (m, 1H, H6′), 4.09 (m, 2H,
NHCH2CH2), 3.07 (t, J = 6.7 Hz, 2H, NHCH2CH2), 1.49 (s, 3H,
CH3), 1.33 (s, 3H, CH3). 13C{1H} NMR (100.63 MHz, CDCl3, 25
°C): δ 169.7 (CO), 131.4 (Cant), 131.0 (Cant), 128.5 (Cant), 128.3
Cant), 127.9 (Cant), 126.8 (Cant), 125.5 Cant), 125.0 (Cant), 112.5
(C(CH3)2), 110.6 (CAr), 105.6 (C1), 91.4 (CAr), 90.1 (CAr), 89.6
(CAr), 83.6 (C2, CAr), 77.2 (C4), 74.7 (C5), 69.4 (C6), 38.7
(NHCH2CH2), 33.1 (NHCH2CH), 26.8 (CH3), 26.2 (CH3).
31P{1H} NMR (161.98 MHz, CDCl3, 25 °C): δ 133.9. MS (ESI+):
1
2.90. Found: C, 54.64; H, 3.74; N, 3.11. H NMR (400.13 MHz,
DMSO-d6): δ 9.40 (t, J = 5.6 Hz, 1H, NH), 8.69 (s, 1H, Hant), 8.14
(m, 2H, Hant), 7.88 (m, 2H, Hant), 7.55 (m, 4H, Hant), 6.19 (m, 2H,
HAr), 6.07 (m, 2H, HAr), 5.91 (m, 1H, HAr), 4.53 (d, J = 5.6 Hz, 2H,
NHCH2). 13C{1H} NMR (100.63 MHz, DMSO-d6): δ 169.3 (CO),
132.6 (Cant), 131.0 (Cant), 128.9 (Cant), 128.1 (Cant), 127.8 (Cant),
127.2 (Cant), 126.1 (Cant), 125.4 (Cant), 102.2 (CAr), 89.2 (CAr), 86.7
(CAr), 85.2 (CAr), 41.8 (NHCH2). MS (ESI+): m/z 990 ([M + Na]+).
Bis[dichlorido(η6-N-phenethylanthracene-9-carboxamide)-
ruthenium(II)]. This compound was obtained from N-(2-(cyclohexa-
1,4-dienyl)ethyl)anthracene-9-carboxamide (1.0 g, 3.1 mmol) and
ruthenium(III) chloride hydrate (0.27 g, 1.0 mmol) using the
procedure described for bis[dichlorido(η6-N-benzylanthracene-9-
carboxamide)ruthenium(II)]. Yield: 0.57 g (96%). Mp: >200 °C
(dec). Elem anal. Calcd for C46H38N2O2Ru2Cl4: C, 55.54; H, 3.85; N,
m/z 711 ([M − Cl]+), 768 ([M + Na]+).
[Dichlorido(η6-N-benzylanthracene-9-carboxamide)(1,3,5-
triaza-7-phosphaadamantane)ruthenium(II)] (3). Complex 3 was
obtained from 1,3,5-triaza-7-phosphaadamantane (32 mg, 0.2 mmol)
and [{Ru(η6-C6H5CH2NHCO-anthracene)Cl2}2] (100 mg, 0.1 mmol)
using the procedure described for 1. Yield: 60 mg (44.7%). Mp: >220
°C (dec). Elem anal. Calcd for C28H29N4OPRuCl2·0.8CH2Cl2: C,
1
48.83; H, 4.35; N, 7.90. Found: C, 48.55; H, 4.45; N, 7.73. H NMR
(400.13 MHz, DMF-d7): δ 8.89 (1H, Hant), 8.34 (m, 2H, Hant), 8.21
(m, 2H, Hant), 7.75 (m, 4H, Hant), 6.24 (s, 4H, HAr), 5.71 (s, 1H, HAr),
4.79 (d, J = 5.8 Hz, 2H, NHCH2), 4.73 (s, 6H, NCH2N), 4.58 (s, 6H,
PCH2N). 13C{1H} NMR (100.63 MHz, DMF-d7): δ 169.4 (CO),
133.0 (Cant), 131.4 (Cant), 128.7 (Cant), 128.0 (Cant), 127.9 (Cant),
126.8 (Cant), 125.8 (Cant), 125.4 (Cant), 103.7 (CAr), 87.9 (CAr), 87.1
(CAr), 79.3 (CAr), 72.8 (J = 6.6 Hz, NCH2N), 52.6 (J = 17.8 Hz,
PCH2), 41.8 (CH2NH). 31P{1H} NMR (161.98 MHz, DMF-d7): δ
−32.1. MS (ESI+): m/z 663 ([M + Na]+).
1
2.81. Found: C, 55.76; H, 4.07; N, 2.92. H NMR (400.13 MHz,
DMSO-d6): δ 8.94 (t, J = 5.2 Hz, 1H, NH), 8.65 (s, 1H, Hant), 8.12
(m, 2H, Hant), 7.74 (m, 2H, Hant), 7.54 (m, 4H, Hant), 6.09 (m, 2H,
HAr), 5.92 (d, J = 5.9 Hz, 2H, HAr), 5.86 (m, 1H, HAr), 3.89 (m, 2H,
NHCH2CH2), 2.82 (t, J = 6.3 Hz, 2H, NHCH2CH2). 13C{1H} NMR
(100.63 MHz, DMSO-d6): δ 168.6 (CO), 133.5 (Cant), 131.1 (Cant),
128.8 (Cant), 127.7 (Cant), 127.6 (Cant), 126.9 (Cant), 126.0 (Cant),
125.7 (Cant), 105.0 (CAr), 88.9 (CAr), 86.6 (CAr), 84.4 (CAr), 39.1
(NHCH2CH2), 33.0 (NHCH2CH2). MS (ESI+): m/z 462 ([M −
2Cl]2+).
[Dichlorido(η6-N-phenethylanthracene-9-carboxamide)-
(1,3,5-triaza-7-phosphaadamantane)ruthenium(II)] (4). Com-
plex 4 was obtained from 1,3,5-triaza-7-phosphaadamantane (33 mg,
0.2 mmol) and [{Ru(η6-C6H5CH2CH2NHCO-anthracene)Cl2}2]
(100 mg, 0.1 mmol) using the procedure described for 1. Yield: 65
mg (48.8%). Mp: >220 °C (dec). Elem anal. Calcd for
C29H31N4OPRuCl2·0.25CH2Cl2: C, 51.99; H, 4.70; N, 8.29. Found:
C, 51.65; H, 4.47; N, 8.45. 1H NMR (400.13 MHz, DMSO-d6): δ 8.92
(t, J = 5.1 Hz, 1H, NH), 8.65 (s, 1H, Hant), 8.12 (d, J = 7.9 Hz, 2H,
Hant), 7.74 (d, J = 7.9 Hz, 2H, Hant), 7.53 (m, 4H, Hant), 5.88 (m, 2H,
HAr), 5.75 (m, 2H, HAr), 5.44 (m, 1H, HAr), 4.44 (s, 6H, NCH2N),
4.21 (s, 6H, PCH2N), 3.85 (m, 2H, NHCH2CH2), 2.67 (t, J = 6.4 Hz,
2H, NHCH2CH2). 13C{1H} NMR (100.63 MHz, DMSO-d6): δ 168.6
(CO), 133.6 (Cant), 131.1 (Cant), 128.5 (Cant), 127.7 (Cant), 127.4
(Cant), 126.4 (Cant), 125.6 (Cant), 106.7 (CAr), 88.2 (CAr), 86.3 (CAr),
78.5 (CAr), 72.7 (J = 6.6 Hz, NCH2N), 52.4 (J = 17.8 Hz, PCH2N),
39.1 (NHCH2CH2), 33.1 (NHCH2CH2). 31P{1H} NMR (161.98
MHz, DMSO-d6): δ −31.4. MS (ESI+): m/z 619 ([M − Cl]+).
Hydrolysis. Samples were dissolved in H2O/D2O/DMSO (8:1:1)
at 25 °C. For investigation on the effect of the chloride ion
concentration on the ligand exchange, sodium chloride (100 mM) was
added to the solution and samples were analyzed by 31P{1H} NMR
spectroscopy.
[Dichlorido(η6-N-benzylanthracene-9-carboxamide)(3,5,6-
bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside)-
ruthenium(II)] (1). A solution of 3,5,6-bicyclophosphite-1,2-O-
isopropylidene-α-D-glucofuranoside (51 mg, 0.2 mmol) in dichloro-
methane (20 mL) was added to a suspension of [{Ru(η6-
C6H5CH2NHCO-anthracene)Cl2}2] (100 mg, 0.1 mmol) in dichloro-
methane (20 mL), under nitrogen. The reaction mixture was stirred
for 12 h at room temperature, and the progress was monitored by
31P{1H} NMR specroscopy. The reaction mixture was concentrated
undervacuum, and diethyl ether was added to precipitate the product,
which was isolated by filtration and washed with diethyl ether (3 × 5
mL). The crude product was purified by short column chromatog-
raphy [CH2Cl2/MeOH (10:1)]. Yield: 93 mg (62%). Mp: >220 °C
(dec). Elem anal. Calcd for C31H30NO7PRuCl2·0.5CH2Cl2: C, 48.88;
H, 4.04; N, 1.81. Found: C, 49.14; H, 4.11; N, 2.01. 1H NMR (400.13
MHz, CDCl3): δ 8.53 (s, Hant), 8.08 (d, J = 8.4 Hz, 2H, Hant), 8.05 (d,
J = 8.4 Hz, 2H, Hant), 7.56 (m, 4H, Hant), 7.20 (br, 1H, NH), 5.98 (m,
5H, H-1, HAr), 5.53 (br s, 1H, HAr), 4.96 (m, 2H, NHCH2), 4.77 (m,
1H, H5), 4.48 (s, 1H, H3), 4.39 (s, 1H, H2), 4.06 (m, 2H, H-4, H6),
3.86 (br, 1H, H6′), 1.46 (s, 3H, CH3), 1.30 (s, 3H, CH3). 13C{1H}
NMR (100.63 MHz, CDCl3): δ 170.2 (CO), 130.9 (Cant), 130.5
(Cant), 128.8 (Cant), 128.7 (Cant), 128.0 (Cant), 127.2 (Cant), 125.6
(Cant), 124.7 (Cant), 112.4 (C(CH3)2), 106.8 (CAr), 105.5 (C1), 92.3
(CAr), 91.9 (CAr), 89.2 (CAr), 88.2 (CAr), 84.1 (CAr), 83.4 (C-3), 79.1
(C2), 77.2 (C4), 74.6 (C5), 69.3 (C6), 40.7 (NHCH2), 26.8 (CH3),
26.2 (CH3). 31P{1H} NMR (161.98 MHz, CDCl3): δ 133.2. MS
(ESI+): m/z 696 ([M − Cl]+).
Cellular Accumulation of Ruthenium. Cells were seeded in six-
well plates and incubated with the corresponding complex at a
concentration of 50 μM for 5 h. At the end of the incubation period,
the cells were rinsed twice with 2.0 mL of PBS, detached by adding 0.5
mL of an enzyme-free cell dissociation solution (Millipore, Switzer-
land), and collected by centrifugation. All samples were digested in
ICP-MS-grade concentrated nitric acid (Sigma Aldrich, Switzerland)
for 3 h at room temperature and filled to a total volume of 8.0 mL with
ultrapure water. Indium was added as an internal standard at a
concentration of 0.5 ppb. The metal contents were determined using
an Elan DRC II ICP-MS (Perkin-Elmer, Switzerland) equipped with a
Meinhard nebulizer and a cyclonic spray chamber. The ICP mass
spectrometer was tuned daily using a solution provided by the
manufacturer containing 1 ppb of each magnesium, indium, cesium,
barium, tin, and uranium. External standards were prepared gravi-
metrically from single element standards (CPI International,
Amsterdam, The Netherlands), using identical acid and internal
standard concentrations.
[Dichlorido(η6-N-phenethylanthracene-9-carboxamide)-
(3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D -
glucofuranoside)ruthenium(II)] (2). Complex 2 was obtained from
3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside (50
mg, 0.2 mmol) and [{Ru(η6-C6H5CH2CH2NHCO-anthracene)Cl2}2]
(100 mg, 0.1 mmol) using the procedure described for 1. Yield: 100
mg (67%). Mp: >220 °C (dec). Elem anal. Calcd for
C32H32NO7PRuCl2·0.25CH2Cl2: C, 50.52; H, 4.27; N, 1.83. Found:
C, 50.25; H, 4.14; N, 1.73. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ
8.46 (s, 1H, Hant), 7.99 (d, J = 8.3 Hz, 2H, Hant), 7.94 (d, J = 8.3 Hz,
2H, Hant), 7.49 (m, 4H, Hant), 6.69 (br s, 1H, NH), 6.17 (d, J = 3.4 Hz,
1H, H1), 5.82 (m, 4H, HAr), 5.60 (m, 1H, HAr), 4.99 (m, 1H, H5), 4.75
(m, 1H, H3), 4.72 (d, J = 3.4 Hz, 1H, H2), 4.36 (dd, J = 12.4 and 9.6
Fluorescence Spectroscopy. Fluorescence measurements were
performed on a Varian Cary Eclipse spectrofluorimeter at room
temperature. Solutions 1 and 3 were prepared in DMF (11 μM). The
3637
dx.doi.org/10.1021/ic202530j | Inorg. Chem. 2012, 51, 3633−3639

Inorganic Chemistry
Article
fluorescence spectra of 1 and 3 were recorded 30 min after sample
preparation (λex = 365 nm; slit width of 5 nm). Quantum yields (Φ)
were determined by comparing the wavelength-integrated intensities (I
or IR) of isoabsorptive diluted solutions (Abs < 0.1) with reference to
anthracene (ΦR = 1.27 in ethanol) by the equation51
ASSOCIATED CONTENT
* Supporting Information
■
S
Crystallographic data for the structural analysis of 3. This
material is available free of charge via the Internet at http://
pubs.acs.org. The atomic coordinates for this structure have
also been deposited with the Cambridge Crystallographic Data
Centre as CCDC 854958. The coordinates can be obtained, on
request, from the Director, Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.
2
n I
Φ = Φ
R
2
n
I
R
R
where n and nR are the refractive index of the sample and reference
solvent, respectively.
AUTHOR INFORMATION
Corresponding Author
*E-mail: alexey.nazarov@epfl.ch (A.A.N.), paul.dyson@epfl.ch
(P.J.D.). Tel: +41-21-6939854. Fax: +41-21-6939780.
Cell Lines and Culture Conditions. Human A2780 and
A2780cisR ovarian carcinoma cell lines were obtained from the
European Centre of Cell Cultures (ECACC, Porton Down, Salisbury,
U.K.). Human SW480, HT29, and CaCo2 colon carcinoma, MDA-
MB231 and MCF-7 breast carcinoma, and A549 lung carcinoma cells
were obtained from the American Type Culture Collection, human
cerebral endothelial cells (HCEC) were a kind gift of D. Staminirovic
and A. Muruganandam, Ottawa, Canada, LN18, LN229, and LNZ308
glioblastoma cells were obtained from AC Diserens Neurosurgery
Service, CHUV, Lausanne, Switzerland. These cells are routinely
grown in Dulbecco’s Modified Eagle Medium containing 4.5 g/L
glucose and glutamax and were supplemented with 10% heat-
inactivated fetal bovine serum and antibiotics. Cultures were
maintained at 37 °C in a humidified atmosphere containing 6%
CO2. All cell culture reagents were purchased from Gibco-BRL, Basel,
Switzerland.
Determination of the Cell Viability and DNA Synthesis. For
these experiments, cells were grown in 48-well plates (Costar, Integra
Biosciences, Cambridge, MA) as monolayers for 24 h in a complete
medium with 10% fetal calf serum (FCS) to reach subconfluence.
Then a fresh complete medium with 5% FCS was added together with
the drugs, and the culture was continued for another 72 h. The
compounds were predissolved at 20 mM in DMSO and then added to
the cell culture medium at the required concentration with a maximum
DMSO content of 0.5% (v/v) to be incubated for 24, 48, or 72 h. At
these concentrations, DMSO has no effect on the cell viability and
synthesis of DNA.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the Swiss National Science
Foundation, National Centre of Competence in Research
“Chemical Biology−Visualisation and Control of Biological
Processes Using Chemistry”, COST D39, and EPFL for
financial support. This research was supported by a Marie
Curie Intra European Fellowship within the 7th European
Community Framework Programme (Project 220890-SuRuCo
to A.A.N.).
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