Cytotoxicities of polysubstituted chlorodicarbonyl(cyclopentadienyl) and (indenyl)ruthenium complexes

Article abstract of DOI:10.1021/om400234p

Polysubstituted cyclopentadienyl and indenyl complexes of ruthenium were synthesized and investigated to elucidate their potential cytotoxic activities. In particular, substituted (indenyl)ruthenium complexes inhibited the proliferation of a panel of human adherent cancer cells with comparable activity to reference agent cisplatin. One of the active compounds exerted a concentration dependent inhibition of cell cycle at G1-S transiton as evidenced by flow cytometry.

Full text of DOI:10.1021/om400234p

Article
pubs.acs.org/Organometallics
Cytotoxicities of Polysubstituted Chlorodicarbonyl(cyclopentadienyl)
and (Indenyl)ruthenium Complexes
Denys Mavrynsky,† Jani Rahkila,† Daniel Bandarra,‡ Soraia Martins,‡ Margarida Meireles,‡
Maria Jose Calhorda,‡ Ida J. Kovacs,§ Istvan Zupko,§ Mikko M. Hanninen,∥ and Reko Leino*,†
́
́
́
́
̈
†Laboratory of Organic Chemistry, Åbo Akademi University, FI-20500 Åbo, Finland
‡Departamento de Química e Bioquímica, CQB, Faculdade de Cien
cias, Universidade de Lisboa, 1749-016 Lisboa, Portugal
̂
§Department of Pharmacodynamics and Biopharmacy, University of Szeged, H-6720 Szeged, Hungary
∥Department of Chemistry, University of Jyvaskyla, FI-40014 Jyvaskyla, Finland
̈
̈
̈
̈
S
* Supporting Information
ABSTRACT: Polysubstituted cyclopentadienyl and indenyl
complexes of ruthenium were synthesized and investigated to
elucidate their potential cytotoxic activities. In particular,
substituted (indenyl)ruthenium complexes inhibited the
proliferation of a panel of human adherent cancer cells with
comparable activity to reference agent cisplatin. One of the
active compounds exerted a concentration dependent
inhibition of cell cycle at G1−S transiton as evidenced by
flow cytometry.
chlorodicarbonyl complexes of ruthenium for use as racemization
catalysts in chemoenzymatic dynamic kinetic resolution of sec-
alcohols (Figure 2).6,7 As a spin-off from this work, and with the
INTRODUCTION
Cisplatin (1) (Figure 1), along with other Pt derivatives including
carboplatin and oxaliplatin, belongs to the most well-known drugs
■
Figure 1. Examples of organometallic compounds with anticancer
activity.
containing transition metals.1 Because of the generally high toxicities
and adverse side effects of platinum compounds in therapeutic
applications, and also to the potentially occurring and developing
drug resistance in rapidly mutating cancer cells, alternative
approaches toward other metal-based anticancer agents have been
under active investigation. For example, titanocene dichloride (2)
has been extensively tested as an anticancer drug although its low
hydrolytic stability sets limits for in vivo utilization and, so far, this
compound has not been approved for clinical use.2 Ruthenium
complexes in turn have gained significant attention due to their
lower toxicities toward normal cells and different spectrum of activity
compared to platinum compounds.3 Earlier research on π-complexes
of ruthenium has focused mainly on η6-arene complexes such as 3,4
whereas cytotoxicity studies on η5-(cyclopentadienyl)ruthenium
complexes have remained relatively scarce.5 In this context, the po-
tential cytotoxicities of chlorodicarbonyl ligated (cyclopentadienyl)
ruthenium complexes have remained virtually unexplored.
Figure 2. Structures of chlorodicarbonyl(cyclopentadienyl) and
(indenyl) complexes 4−6 investigated in the present study.
limited information available on the potential cytotoxicities of such
compounds in mind, we became interested in their potential use
as cytotoxic agents. In this paper, initial insights into their
pharmaceutical potential is presented and briefly discussed.
RESULTS AND DISCUSSION
■
The synthesis of 4a,7b 4b,7b 5a,7a and 5b7a has been described
earlier by us and that of 4c by Backvall and co-workers.8
̈
In recent work, we have prepared and investigated, as part of
structure optimization work, several cyclopentadienyl and indenyl
Received: March 19, 2013
© XXXX American Chemical Society
A
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The new polysubstituted indenyl complexes 6a and 6b were
prepared from the corresponding tribenzylindenes by a method
similar to that utilized earlier for 5a and 5b (Scheme 1).7a All
Table 1. Selected Bonding Distances in the Complexes 5a
and 6a
distances (Å)
5a
6a
Ru−C1
Ru−C2
Ru−C3
Ru−C4
Ru−C5
Ru−C37
Ru−C38A
Ru−C38B
Ru−Cl1A
2.244(2)
2.238(2)
2.241(2)
2.240(2)
2.238(2)
1.933(3)
1.879(4)
1.80(3)
2.322(2)
2.202(2)
2.237(2)
2.218(2)
2.323(2)
1.874(2)
1.93(1)
1.90(1)
2.398(2)
2.387(2)
1.899
Scheme 1. Preparation of Indenyl Complexes 6a and 6b
a
a
a
2.3194(9)
2.38(1)
a
Ru−Cl1B
b
Ru−Ct
1.875
a
The molecular structure shows a disorder in which the Cl ligand and
one of the two CO ligands have two orientations (rotamers) in about
b
9:1 and 1:1 ratio for 5a and 6a, respectively. Ct is the C5 ring
centroid.
Ru−C bond distances are observed for all carbon atoms of the
five-membered ring). In complex 6a, however, the Cp fragment
is slightly tilted producing marginally longer M−C1 and M−C5
bonds (2.322 and 2.323 Å, respectively) compared to the other
M−C(Cp) distances (ranging from 2.218 to 2.237 Å), even
though it is still best described as being η5-coordinated. The
observed distribution of M−C distances (three shorter and two
longer) is typical of the η5 coordination mode of indenyl, often
described as η3 + η2, even in the absence of steric constraints.
The fused ring systems around Cp rings of 5a and 6a are nearly
planar, while the other substituents have been rotated away
from the molecular plane. In 5a, the phenyl substituents are
canted about 56−72° from the cyclopentadienyl level while in
6a the benzyl substituents are arranged at the top of the ring
pointing at the opposite direction from the ruthenium cation.
The five M−C distances are very similar in this C5 ring,
reflecting the different nature of its π orbitals. The remaining
coordination sites of Ru are occupied by two carbonyl groups
and a chloride ion, producing a typical pseudo-octahedral three-
legged piano stool conformation. The carbonyl and chloride
ligands of both complexes are disordered in two orientations
(rotamers) with the main component 0.89 for 5a and a 1:1 ratio
in 6a, respectively (the minor or other component is presented
partly transparent in Figures 3 and 4). The formal oxidation state
of ruthenium in 5a and 6a can be assigned as +2, being further
supported by the relatively short Ru−carbonyl and Ru−C5
centroid distances (Table 1). All bond lengths around ruthenium
are in good accordance with previous studies.7a,b,8
1
new compounds were characterized by H and 13C NMR and
HRMS and in case of 6a also by X-ray crystallography. The
X-ray structures of 4a,7b 4b,7b and 4c7c have been described
earlier, while this is the first report on the X-ray structure of 5a.
The crystal structures of 5a and 6a are presented in Figures 3
and 4, respectively, with the selected bond distances reported in
Figure 3. The molecular structure of 5a. Thermal ellipsoids have been
drawn at 30% probability level, and hydrogen atoms are omitted for clarity.
The cytotoxic activity of these ruthenium complexes was
assayed against HeLa (cervical carcinoma), MCF-7 (breast
carcinoma), Caco-2 (colorectal adenocarcinoma), A2780 (ovarian
cancer), and A431 (skin epidermoid carcinoma) cell lines. The cells
were exposed to each of the compounds for a total of 48 or 72 h.
The IC50 values (final concentration ≤0.5% DMSO) were calcu-
lated from dose−response curves obtained by nonlinear regression
analysis from the colorimetric mitochondrial function-based
MTT viability assay. IC50 values are concentrations of drug required
to inhibit tumor cell proliferation by 50%. The results are
summarized in Table 2.
Figure 4. The molecular structure of 6a. Thermal ellipsoids have been
drawn at 30% probability level, and hydrogen atoms are omitted for clarity.
Overall, of the compounds studied here, the polysubstituted
cyclopentadienyl complexes 4a, 4b, and 4c and the 2-adamantano-
yloxy substituted cyclopenta[l]phenanthrenyl complex 5b
proved ineffective, failing to show 50% inhibition of cancer cell
Table 1. In both complexes, the modified cyclopentadienyl
(Cp) ring is η5-coordinated to the ruthenium metal (similar
B
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Table 2. Calculated IC50 Values for Selected Ruthenium
Complexes against HeLa, Caco-2, MCF7, A2780, and A431
Cell Lines
a
IC50 (μM)
b
b
complex
4a
HeLa
Caco-2
HeLa
MCF7
A431
A2780
46.0
>95
>30
>30
>30
14.3
>30
>30
>30
23.0
>30
>30
>30
>30
>30
>30
>30
19.1
4b
4c
>250
17.6
45.0
5.9
5a
81.7
5b
Figure 5. Effect of complex 6b on A2780 cell cycle distribution after
incubation for 24 h. * and ** indicate p < 0.05 and p < 0.01,
respectively, as compared with the control cells.
6a
5.2
2.4
4.7
7.8
5.7
6.3
5.1
8.0
8.0
5.6
8.8
3.9
4.3
0.9
6b
4.7
cisplatin (1)
a
compare very well with the binding constants of known
intercalators,11 such as doxorubicin and ethidium bromide,
determined under the same conditions to be 1.52 × 105 and
2.78 × 105 M−1, respectively. The plot in Figure 6 depicts the
Main value from two independent determinations, standard deviation
b
less than 15%. Results from experiments with 48 h of incubation. All
other data represent 72 h.
proliferation in up to 30 μM final concentrations. The larger range
of concentrations tested for these four complexes with 48 h
incubation showed high IC50 for 4a (46.0 μM) and 5b (45.0 μM),
and even higher for 4b and 4c. Owing to this low activity (IC50 >
95 or 250 μM), it was not considered relevant to determine a
precise value for these two complexes. Treatment with the
2-benzoyloxy substituted cyclopenta[l]phenanthrenyl complex 5a,
however, resulted in moderate inhibition of the cell growth,
whereas the activities of the two 1,2,3-tri(benzyl)substituted
indenyl complexes 6a and 6b were the most remarkable in all cell
lines tested and comparable with that of the clinically used
reference agent cisplatin 1.
To obtain information on the cell cycle distribution as a
function of the treatment of cancer cells with a test substance,
flow cytometric analysis was used. In short, diploid cells (cells
in “basic state”) contain a unit amount of DNA (population G1).
As the cell prepares for division, its DNA content gradually
increases, characterizing the synthetic (S) phase. Upon completion
of the synthesis, the G2 (G2/M) phase, with a DNA content
double from that of cells in the G1 phase, is reached. Next, a G2
cell undergoes division to produce two G1 cells. Cells containing
less-than-G1 amount of DNA (subG1 population) are considered
resulting from the organized self-decomposition (apoptosis),
typically elicited by harmful stimuli.
In the flow cytometric experiment, A2780 cells were
incubated for 24 h with 1 and 3 μM concentrations of complex
6b prior to analysis. Treatment with the tested complex
resulted in a substantially increased population of G1 cells. A
significant and concentration-dependent decrease in the ratio of
cells from S and G2/M phases was also observed (Figure 5).
On the basis of these experimental results, a cell cycle blockade
at G1−S transition could be suggested as a mechanism of
action of the detected antiproliferative property. Interestingly,
the subdiploid (subG1) cell population, which is generally
accepted as an apoptotic marker, exhibited no characteristic
change under the applied conditions.9
Figure 6. UV−vis absorption spectra of 6b (20 μM) in Tris buffer in
the presence of increasing amounts of ct DNA (0−200 μM). The inset
plot represents [DNA]/(εa − εf) (M2 cm) vs [DNA] (μM) for the
titration. The arrow indicates the absorbance changes monitored at
281 nm upon increasing DNA concentration.
UV−vis absorption spectra of complex 6b (20 μM) in Tris buffer
in the presence of increasing amounts of ct DNA (0−200 μM).
The arrow indicates the absorbance changes monitored at 281 nm
upon increasing DNA concentration. The inset plot represents
[DNA]/(εa − εf) (M2 cm) as a function of [DNA] (μM) for the
titration and allows the determination of Kb.
SUMMARY AND CONCLUSIONS
■
In this work, two new indenyl chlorodicarbonyl complexes of
ruthenium(II) were synthesized and characterized and together
with three earlier prepared chlorodicarbonyl ruthenium cyclo-
pentadienyl and two cyclopenta[l]phenanthrenyl complexes
screened against several cancer cell lines for investigation of
their potential cytotoxic activities. New crystal structures of two
of the complexes were determined, displaying the expected
piano stool coordination environment of the metal. In
both complexes, the five-membered rings of the ligands are
η5-coordinated to ruthenium, although in the indenyl complex the
The binding of complexes 5a, 6a, and 6b to DNA was also
studied by electronic absorption spectroscopy.10 The absorption
pattern of each complex was followed upon adding increasing
amounts of ct DNA (200 μM bp−1). The absorption maxima at
268 nm (5a), 281 nm (6a), and 290 nm (6b) were shifted to
higher wavelengths while the intensity decreased, suggesting that
the complexes may be acting as intercalators. The intrinsic binding
constants (Kb) of each compound were calculated as 1.05 × 105
(5a), 7.12 × 104 (6a), and 3.26 × 104 (6b) M−1. These values
C
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mixture was refluxed for 2 h and then cooled down to room
temperature and washed with 2 × 100 mL of a saturated solution of
NaHCO3 and 100 mL of brine. The organic layer was dried over
Na2SO4 and concentrated. The crude product was purified by filtration
two Ru−C bonds to the hinge carbon atoms are longer than
the other three.
Testing of the antiproliferative activity of these seven
complexes revealed five complexes as ineffective, inhibiting
less than 50% of cancer cell proliferation in up to 30 μM final
concentration. Two η5-indenyl ligated ruthenium complexes
containing 1,2,3-tribenzyl substituents in the five-membered
rings of the indenyl moieties displayed activities similar to that
of the reference metal complex cisplatin. Treatment with a
selected test agent resulted in a profound cell cycle disturbance
which could be the consequence of intercalation into the DNA.
Titration experiments showed that the binding constants to
DNA of the more active complexes were comparable to those
of known intercalators (doxorubicin and ethidium bromide).
On the basis of the results obtained in this study, it can be con-
cluded that chlorodicarbonyl indenyl complexes of ruthenium
are viable candidates for further search and optimization of
new lead compounds for anticancer therapies with promising
efficacies.
through silica using hexane as an eluent. Yield: 1.36 g (24% based on 7).
3
1H NMR (600.13 MHz, CDCl3): δ 7.12 (d, 2 H, J2‑Bn‑o‑H, 2‑Bn‑m‑H
=
8.2 Hz, 2-Bn-o-H), 7.10 (m, 2 H, 2-Bn-m-H), 6.94 (d, 1 H, 3J = 7.6 Hz,
H-6), 6.81 (d, 1 H, 3J = 7.6 Hz, H-5), 6.62 (m, 1 H, H-3), 3.78 (s, 2 H,
2-CH2Ph), 3.12 (s, 2 H, H-1), 2.35 (s, 3 H, 7-Me), 1.32 (s, 3 H,
2-p-MeBn). 13C NMR (150.90 MHz, CDCl3): δ 148.6 (C-2), 143.8
(C-4), 142.0 (C-3′), 137.3 (2-Bn-i-C), 135.7 (2-Bn-p-C), 130.0 (C-7′),
129.3 (2-Bn-m-C), 128.8 (2-Bn-o-C), 127.8 (C-6), 127.1 (C-7), 126.4
(C-3), 125.3 (C-5), 40.0 (C-1), 37.8 (2-CH2Ph), 21.2 (2-p-MeBn),
18.5 (4-Me), 14.4 (7-Me)
4,7-Dimethyl-2-(4-methoxybenzyl)-1H-indene (8b). Magnesi-
um turnings (7.1 g, 0.3 mol) and 100 mL of dry THF were placed into
a round-bottom flask under argon atmosphere, and then a single
crystal of iodine was added. Next, 4-methoxybenzyl chloride (12 mL,
0.09 mmol) was added slowly over 1.5 h. To the resulting mixture
containing the Grignard reagent, 4,7-dimethyl-2-indanone (7) (5 g,
32 mmol) was added slowly and the reaction mixture was stirred at
room temperature overnight. The reaction mixture was quenched with
saturated NH4Cl and filtered, and the residue was washed with TBME.
Solvent was removed in vacuo, providing 5 g (approximately 55%) of
oil crystallizing upon overnight storage at RT. This product was
dissolved in 100 mL of toluene, and 200 mg of p-toluenesulfonic acid
were added. The reaction mixture was refluxed for 1.5 h and then
cooled down to room temperature and washed with 2 × 100 mL of a
saturated solution of NaHCO3 and 100 mL of brine. The organic layer
was dried over Na2SO4 and concentrated. The crude product was
purified by filtration through silica (hexane → hexane:DCM 10:1 →
EXPERIMENTAL SECTION
■
General Considerations. All glassware utilized for organometallic
reactions was oven-dried at 150 °C overnight and cooled down in a
desiccator over phosphorus pentoxide. Solvents were dried according
to standard procedures. Starting material for the indenyl ligand
precursors, 4,7-dimethyl-2-indanone (7), was prepared according to a
literature procedure.12 The RPMI 1640 cell culture medium, fetal
bovine serum, tripsin, glutamine, and pen-step were purchased from
LONZA Co. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide), doxorubicin, and ethidium bromide were purchased
from Sigma Aldrich. A stock solution of calf thymus DNA (ct DNA)
from Sigma Aldrich was prepared by diluting in a buffer solution
(50 mM NaCl/5 mM Tris-HCl, pH 7.2) and stirring at 4 °C for two
days. The solution was stored at 4 °C. The ratio of UV absorbance at
260 and 280 nm (A260/A280) of 1.83−1.86 of this ct DNA stock
solution indicated that the DNA was sufficiently free of protein
contamination. The DNA concentration was determined by the UV
absorbance at 260 nm after 1:10 dilution using ε = 6600 M−1 cm−1.13
MTT was dissolved in RPMI 1640 medium (0.5 mg/mL). Ruthenium
complexes 4a,7b 4b,7b 4c,8 5a,7a and 5b7a were prepared as described
previously. For X-ray structure determination, 5a was recrystallized
from a DCM−heptane mixture. NMR spectra were recorded using a a
600 MHz NMR spectrometer equipped with a BBI-5 mm-Zgrad-ATM
probe or BBO-5 mm-Zgrad probe at 298 K operating at 600.13 MHz
1
hexane:DCM 4:1). Yield: 3.9 g (47% based on 7). H NMR (600.13
3
MHz, CDCl3): δ 7.16 (d, 2 H, J2‑Bn‑o‑H, 2‑Bn‑m‑H = 8.7 Hz, 2-Bn-o-H),
6.95 (d, 2 H, 3J = 7.7 Hz, H-6), 6.85 (m, 2 H, 2-Bn-m-H), 6.83 (d, 1 H,
3J = 7.7 Hz, H-5), 6.61 (m, 1 H, H-3), 3.80 (s, 3 H, 2-p-MeOBn), 3.77
(s, 2 H, 2-CH2Ph), 3.14 (s, 2 H, H-1), 2.36 (s, 3 H, 7-Me), 2.24 (s, 3
H, 4-Me). 13C NMR (150.90 MHz, CDCl3): δ 158.2 (2-Bn-p-C),
148.9 (C-2), 143.8 (C-7′), 142.0 (C-3′), 132.5 (2-Bn-i-C), 130.1 (C-4),
129.9 (2-Bn-o-C), 127.8 (C-6), 127.1 (C-7), 126.2 (C-3), 125.3 (C-5),
114.0 (2-Bn-m-C), 55.4 (2-p-MeBn), 40.0 (C-1), 37.4 (2-CH2Ph), 18.5
(4-Me), 18.4 (7-Me).
4,7-Dimethyl-1,2,3-tris-(4-methylbenzyl)-1H-indene (9a).
4,7-Dimethyl-2-(4-methylbenzyl)-indene (1.3 g, 5.2 mmol) was
dissolved in 100 mL of dry THF under argon atmosphere, and the
mixture was cooled down to −50 °C. Then a 1.6 M solution of n-BuLi
in hexane (3.6 mL, 5.8 mmol) was added slowly, keeping the
temperature below −50 °C. The reaction mixture was allowed to warm
up to 0 °C and then cooled down to −70 °C. Next, 4-methylbenzyl
chloride (0.76 mL, 5.8 mmol) was added slowly, keeping the
temperature at −70 °C. The reaction mixture was stirred at −70 °C
for 10 min and then allowed to warm up to room temperature. The
reaction mixture was then cooled down to −50 °C again, and another
3.6 mL (5.8 mmol) of a 1.6 M solution of n-BuLi in hexane were
added slowly and the reaction mixture was allowed to warm up to 0 °C
and then again cooled down to −70 °C. Another 0.76 mL (5.8 mmol)
of 4-methylbenzyl chloride was added slowly, and then the reaction
mixture was stirred at room temperature overnight. The reaction was
quenched by carefully adding 10 mL of a saturated solution of NH4Cl.
The reaction mixture was then extracted with 3 × 100 mL EtOAc,
washed with 100 mL brine, dried over Na2SO4, and concentrated. The
crude product was purified by column chromatography using hexane:DCM
5:1 as an eluent. Yield: 1.14 g (48%). 1H NMR (600.13 MHz, CDCl3): δ
7.07 (m, 2 H, 2-Bn-m-H), 7.03 (d, 2 H, 3J = 7.9 Hz, 2-Bn-o-H), 6.89 (m,
2 H, 1-Bn-m-H), 6.85,(d, 1 H, 3J = 8.0 Hz, H-6), 6.84 (m, 2 H, 3-Bn-m-H),
6.83 (d, 1 H, 3J = 8.0 Hz, H-5), 6.67 (m, 2 H, 2-Bn-m-H), 6.40 (d, 2 H,
3J = 7.9 Hz, 3-Bn-o-H), 4.02 (d, 1 H, 2J = 17.1 Hz, 3-CH2Ph), 3.92 (d, 1 H,
2J = −17.1 Hz, 3-CH2Ph), 3.86 (d, 1 H, 2J = −15.6 Hz, 2-CH2Ph), 3.56 (d,
1 H, 2J = −15.6 Hz, 2-CH2Ph), 3.62 (dd, 1 H, 3J = 4.7 Hz, 3J = 4.6 Hz, H-
1), 3.46 (dd, 1 H, 2J = −14.1 Hz, 3J = 4.6, 1-CH2Ph), 3.16 (dd, 1 H, 2J =
−14.1 Hz, 3J = 4.7 1-CH2Ph), 2.47 (s, 3 H, 4-Me), 2.32 (s, 3 H, p-MeBn),
1
for H and 150.92 MHz for 13C. Numbering of the indene moiety, in
accordance with the IUPAC rules, used for assignment of the NMR
spectra but deviating from the crystallographic numbering used in
Figures 3 and 4 and Table 1 is shown in Figure 7.
Figure 7. Numbering of the indene moiety in NMR spectra.
4,7-Dimethyl-2-(4-methylbenzyl)-1H-indene (8a). A solution
of 4,7-dimethyl-2-indanone (7) (3.6 g, 23 mmol) in THF was slowly
added to 50 mL of a 0.5 M solution of 4-methylbenzylmagnesium
chloride in THF over 4 h. The reaction mixture was stirred at room
temperature overnight. The reaction was quenched by slowly adding
20 mL of a saturated solution of NH4Cl upon which a white
precipitate formed. Next, 15 mL of water were added to dissolve the
precipitate. The organic layer was separated and dried over Na2SO4
and concentrated, providing 4.91 g of the crude product. Part of this
product (2 g, approximately 7.5 mmol) was dissolved in 80 mL of
toluene, and 130 mg of p-toluenesulfonic acid was added. The reaction
D
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1
2.31 (s, 3 H, p-MeBn), 2.26 (s, 3 H, p-MeBn), 2.11 (s, 3 H, 7-Me). 13C
NMR (150.90 MHz, CDCl3): δ 130.0 (C-5), 129.1 (2-Bn-o-C), 129.0 (1-
Bn-o-C), 128.9 (3-Bn-m-C), 128.6 (1-Bn-m-C), 128.5 (2-Bn-m-C), 127.8
(3-Bn-o-C), 126.2 (C-6), 49.3 (C-1), 33.3 (1-CH2Ph), 32.3 (2-CH2Ph),
31.9 (3-CH2Ph), 21.0 and 21.1 (1-p-MeBn, 2-p-MeBn, 3-p-MeBn), 19.6 (4-
Me), 19.0 (7-Me). The quaternary carbons were omitted due to
overlapping and complex coupling patterns caused by the highly conjugated
system.
(11%). H NMR (600.13 MHz, CDCl3): δ 7.01 (s, 2 H, H-5, H-6),
6.75 (d, 4 H, 3J = 8.6 Hz, 1-Bn-o-H, 3-Bn-o-H), 6.66 (m, 4 H, 1-Bn-m-
H, 3-Bn-m-H), 6.34 (d, 2 H, 3J = 8.7 Hz, 2-Bn-o-H), 6.16 (m, 2 H, 2-
Bn-m-H), 4.54 (d, 2 H, 2J = −17.5 Hz, 1-CH2Ph, 3-CH2Ph), 4.02 (d, 2
2
H, J = −17.5 Hz, 1-CH2Ph, 3-CH2Ph), 3.77 (s, 2 H, 2-CH2Ph), 3.74
(s, 6 H, 1-p-MeOBn, 3-p-MeOBn), 3.54 (s, 3 H, 2-p-MeOBn), 2.44
(s, 6 H, 4-Me, 7-Me). 13C NMR (150.90 MHz, CDCl3): δ 197.2 (Ru-
CO), 158.1 (1-Bn-p-C, 2-Bn-p-C, 3-Bn-p-C), 132.9 (C-4, C-7), 131.1
(C-5, C-6), 131.0 (1-Bn-i-C, 3-Bn-i-C), 129.9 (2-Bn-o-C), 128.7
(2-Bn-i-C), 128.2 (1-Bn-o-C, 3-Bn-o-C), 116.2 (C-2), 113.9 (1-Bn-m-
C, 3-Bn-m-C), 113.2 (2-Bn-o-C), 110.4 (C-3′, C-7′), 89.1 (C-1, C-3),
55.2 (1-p-MeOBn, 3-p-MeOBn), 55.0 (2-p-MeOBn), 31.7 (1-CH2Ph,
3-CH2Ph), 31.3 (1-CH2Ph), 20.6 (4-Me, 7-Me). MS: exact mass calcd
for C37H35102RuO5 (M+-Cl) 661.1528, found 661.1528; 633.14 (M+-
Cl-CO); 605.15 (M+-Cl-2CO). Anal. Calcd for C37H35ClO5Ru: C,
63.56; H, 5.34. Found: C, 63.00; H, 5.06. Because of the small amount
of sample (>2 mg) remaining for elemental analysis, also contributing
to the accuracy of analysis, this run could not be duplicated. While the
carbon analysis for this compound is slightly greater than the 0.4%
range viewed as establishing analytical purity, it is provided to illustrate
the best value obtained. For spectroscopic analytical data (NMR, MS),
see Supporting Information.
4,7-Dimethyl-1,2,3-tris-(4-methoxybenzyl)-1H-indene (9b).
Compound 8a (3.9 g, 15 mmol) was dissolved in 100 mL of dry
THF under argon atmosphere, and the mixture was cooled down
to −50 °C. Then a 1.6 M solution of n-BuLi in hexane (11 mL, 18 mmol)
was added slowly, keeping the temperature below −50 °C. The reaction
mixture was allowed to warm up to 0 °C and then cooled down to −70 °C.
Next, 4-methoxybenzyl chloride (2.2 mL, 16 mmol) was added slowly,
keeping the temperature at −70 °C. The reaction mixture was stirred at
−70 °C for 10 min and then allowed to warm up to room temperature.
The reaction mixture was then cooled down to −50 °C again, and another
11 mL (16 mmol) of a 1.6 M solution of n-BuLi in hexane wasadded
slowly and the reaction mixture was allowed to warm up to 0 °C and
then again cooled down to −70 °C. Another 2.2 mL (16 mmol) of
4-methoxybenzyl chloride was added slowly, and then the reaction mixture
was stirred at room temperature overnight. The reaction was quenched by
careful addition of 10 mL of a saturated solution of NH4Cl. The reaction
mixture was then extracted with 3 × 100 mL EtOAc, washed with 100 mL
of brine, dried over Na2SO4, and concentrated. The crude product was
purified by column chromatography using hexane:DCM 2:1 as an eluent.
Yield: 4.33 g (58%). The product was still unpure after the purification, and
was used as such in the following reactions.
X-ray Structure Determination. For crystal data and other experi-
mental details for compounds 5a and 6a, see Supporting Information.
The data collection was performed with Agilent SuperNova dual
wavelength diffractometer equipped with Atlas CCD area detector using
Cu Kα radiation and CrysAlisPro program package.14 The empirical
(5a) absorption correction with SCALE3 ABSPACK scaling algorithm
or analytical numeric one using multifaceted crystal (6a) was performed
as implemented in CrysAlisPro program.14 The structures were solved
by direct methods by using the SHELXS-9715 program or the SIR-9716
program, and the full-matrix least-squares refinements on F2 were
performed using the SHELXL-9715 program. Figures were drawn with
Diamond 3.17 For all complexes, the heavy atoms were refined
anisotropically. The CH hydrogen atoms were included at the calculated
distances with fixed displacement parameters from their host atoms (1.2
or 1.5 times of the host atom).
The site occupations of the groups involved in the rotational
disorder of compound 5a were refined to be 0.89 and 0.11. The atoms
of the main component could be refined anisotropically (ISOR
restraint for atoms O2A, C37, and C38A was used), while the atoms of
the minor component had to be refined with isotropic displacement
parameters and the C38B−O2B distance had to be fixed to 1.15 Å. For
6a, the site occupation factors were initially refined, but as they
converged to values very close to 0.5, the parameters were fixed (to 0.5
for both parts). Atoms of the both groups were refined anisotropically,
but the ISOR restraint for atoms C38A, O1, O2a, and O2b had to be
used.
Cytotoxicity Studies. HeLa (cervical adenocarcinoma) and Caco-
2 (colorectal adenocarcinoma) cell lines were maintained in RPMI
1640 supplemented with 10% fetal bovine serum (FBS), 200 U/mL
penicillin, 100 μg/mL streptomycin, and 0.3 g/mL L-glutamine. MCF7
(breast adenocarcinoma), A2780 (ovarian cancer), and A431 (skin
epidermoid carcinoma) cell lines were maintained in MEM supplemented
with 10% fetal bovine serum, 1% nonessential amino acids, and an
antibiotic antimycotic mixture (AAM). All cell lines were purchased from
the European Collection of Cell Culture (Salisbury, UK) and cultivated in
a humidified atmosphere of 95% air/5% CO2 at 37 °C.
For the first screening of the complexes, exponentially growing cells
were seeded at a density of approximately 5000 cells/well, in 96-well
flat-bottomed microplates, and were treated with the complexes after
48 h. The complexes were dissolved in DMSO and tested in
concentrations ranging from 1 to 200 μM. Each experiment included
10 replicates for each concentration of complexes, and the results
usually represent at least two independent experiments. The MTT
assay was used to determine the cell viability or the cytotoxicity of test
compounds.18 The optical density was measured at 570 nm using a
96-well multiscanner autoreader. The IC50 were calculated by nonlinear
regression analysis (GraphPad Prism 4.0, GraphPad Software; San
Diego, CA, USA). Selected complexes were reevaluated similarly but
Chlorodicarbonyl(η5-4,7-dimethyl-1,2,3-tris-(4-methylben-
zyl)-indenyl)ruthenium(II) (6a). Compound 9a (540 mg, 1.18
mmol) was dissolved in 50 mL of dry THF under argon atmosphere,
and the reaction mixture was cooled down to −60 °C. Then a 1.6 M
solution of n-BuLi in hexane (0.81 mL, 1.30 mmol) was added slowly,
keeping the temperature at −60 °C. The reaction mixture was allowed
to return to room temperature and stirred for 30 min, after which
302 mg of [Ru(CO)3Cl2]2 dissolved in 5 mL of dry THF were added.
The reaction mixture was stirred at room temperature for 70 h. The
reaction was quenched by adding a few drops of H2O, and then the
solvent was evaporated. The crude product was purified by column
chromatography (hexane:DCM 5:1 → hexane:DCM 2:1 → DCM).
Yield: 310 mg (40%). 1H NMR (600.13 MHz, CDCl3): δ 7.00 (s, 2 H,
3
H-5, H-6), 6.91 (m, 4 H, 1-Bn-m-H, 3-Bn-m-H), 6.71 (d, 4 H, J =
8.0 Hz, 1-Bn-o-H, 3-Bn-o-H), 6.41 (m, 2 H, 2-Bn-m-H), 4.55 (d, 2 H,
2
2J = −17.6 Hz, 1-CH2Ph, 3-CH2Ph), 4.02 (d, 2 H, J = −17.6 Hz,
1-CH2Ph, 3-CH2Ph), 3.78 (s, 2 H, 2-CH2Ph), 2.43 (s, 6 H, 4-Me, 7-Me),
2.27 (s, 6 H, 1-p-MeBn, 3-p-MeBn), 2.01 (s, 3 H, 2-p-MeBn). 13C
NMR (150.90 MHz, CDCl3): δ 197.3 (Ru-CO), 136.0 (2-Bn-p-C),
135.8 (1-Bn-i-C, 3-Bn-i-C). 135.7 (1-Bn-p-C, 3-Bn-p-C), 133.6 (2-Bn-
i-C), 132.9 (4-C, 7-C), 131.1 (5-C, 6-C), 129.1 (1-Bn-m-C, 3-Bn-m-C),
128.7 (2-Bn-m-C), 128.4 (2-Bn-o-C), 127.1 (1-Bn-o-C, 3-Bn-o-C),
116.3 (2-C), 110.3 (3′-C, 7′-C), 89.3 (1-C, 3-C), 32.1 (1-CH2Ph, 3-
CH2Ph), 31.8 (2-CH2Ph), 21.0 (4-Me, 7-Me), 20.7 (1-p-MeBn, 2-
p-MeBn, 3-p-MeBn). MS: exact mass calcd for C37H35102RuO2 (M+-Cl)
613.1681, found 613.1681; 585.16 (M+-Cl-CO), 557.16 (M+-Cl-
2CO). Anal. Calcd for C37H35ClO2Ru: C, 68.56; H, 5.44. Found: C,
68.38; H, 5.32 (average of two runs).
Chlorodicarbonyl(η5-4,7-dimethyl-1,2,3-tris-(4-methoxyben-
zyl)-indenyl)ruthenium(II) (6b). Compound 9b (395 mg, 0.78
mmol) was dissolved in 50 mL of dry THF under argon atmosphere,
and the reaction mixture was cooled down to −60 °C. Then 0.54 mL
(0.86 mmol) of a 1.6 M solution of n-BuLi in hexane was added
slowly, keeping the temperature at −60 °C. The reaction mixture was
allowed to warm up to room temperature and stirred for 30 min, after
which 200 mg of [Ru(CO)3Cl2]2 dissolved in 5 mL of dry THF were
added. The reaction mixture was stirred at room temperature for 70 h.
The reaction was quenched by adding a few drops of H2O, and then
the solvent was evaporated. The crude product was purified by column
chromatography (hexane:DCM 2:1 → DCM). The obtained product
was recrystallized from 10 mL of heptane:DCM (4:1). Yield: 62 mg
E
dx.doi.org/10.1021/om400234p | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
after 72 h of incubation. Near-confluent cells were seeded in 96-well
microplates at a density of 5000 cells/well and allowed to stand
overnight, after which the medium containing the tested compound
was added. Cisplatin, a clinically used anticancer complex, was used as
reference compound.
Flow Cytometric Studies. Flow cytometric analysis was
performed in order to characterize the cellular DNA content of
treated A2780 cells as described earlier.19 Briefly, after treatment for
24 h, the cells (200000/condition) were trypsinized (Gibco BRL,
Paisley, U.K.), washed with phosphate-buffered saline (PBS), and fixed
in 1.0 mL of cold 70% ethanol overnight at −20 °C. After fixation, the
cells were collected and resuspended in PBS containing 100 μg/mL
propidium iodide, 20 μg/mL RNA-ase, and 0.3% Triton-X-100 and
then incubated at room temperature for 1 h. The cells were evaluated
by flow cytometry using Partec CyFlow ML instrument (Partec
D. K. Onkologie 2000, 23, 60−62. For a general review on
bioinorganic chemistry of titanium, see also: (d) Buettner, K. M.;
Valentine, A. M. Chem. Rev. 2012, 112, 1863−1881.
(3) (a) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast,
B.; Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 891−904.
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(b) Nagy, E. M.; Pettenuzzo, A.; Boscutti, G.; Marchio, L.; Dalla Via,
L.; Fregona, D. Chem.Eur. J. 2012, 18, 14464−14472.
(4) For reviews, see: (a) Yan, Y. K.; Melchart, M.; Habtemariam, A.;
Sadler, P. J. Chem. Commun. 2005, 4764−4776. (b) Melchart, M.;
Sadler, P. J. Ruthenium Arene Anticancer Complexes. In Bioorgano-
metallicsBiomolecules, Labeling, Medicine; Jaouen, G., Ed.; Wiley-
VCH: Weinheim, 2006; pp 39−64. (c) Hartinger, C. G.; Metzler-
Nolte, N.; Dyson, P. J. Organometallics 2012, 31, 5677−5685.
(5) (a) Akbayeva, D. N.; Gonsalvi, L.; Oberhauser, W.; Peruzzini, M.;
Vizza, F.; Bruggeller, P.; Romerosa, A.; Sava, G.; Bergamo, A. Chem.
̈
GmbH, Munster, Germany). In each analysis, 20000 events were
̈
Commun. 2003, 264−265. (b) Dutta, B.; Scolaro, C.; Scopelliti, R.;
Dyson, P. J.; Severin, K. Organometallics 2008, 27, 1355−1357.
(c) Garcia, M. H.; Morais, T. S.; Florindo, P.; Piedade, M. F. M.;
Moreno, V.; Ciudad, C.; Noe, V. J. Inorg. Biochem. 2009, 103, 354−
361. (d) Moreno, V.; Lorenzo, J.; Aviles, F. X.; Garcia, M. H.; Ribeiro,
J. P.; Morais, T. S.; Florindo, P.; Robalo, M. P. Bioinorg. Chem. Appl.
2010, 1−11. (e) Moreno, V.; Font-Bardia, M.; Calvet, T.; Lorenzo, J.;
recorded, and the percentages of the cells in the different cell-cycle
phases (subG1, G1, S, and G2/M) were calculated by using ModFit
LT (Verity Software House, Topsham, ME, USA).
DNA Binding Studies. Calf thymus DNA (ct DNA) solutions of
various concentrations (0−100 μM) were added to 20 μM buffered
solutions (5 mM Tris, 50 mM NaCl, pH 7.2) of the metal complexes,
and the same amount of ctDNA solution was added to the reference
cell. Absorption spectra were recorded after equilibration at 37.0 °C
for 10 min. The intrinsic binding constant, K, was determined from a
plot of D/Δεap vs D according to eq 1.20 D is the concentration of
DNA in base pairs, Δεap = |εA − εF|, εA = Aobs/[complex], and Δε =
|εB − εF|, with εB and εF corresponding to the extinction coefficient of
the DNA-bound complex and unbound complex, respectively.
́
Aviles, F. X.; Garcia, M. H.; Morais, T. S.; Valente, A.; Robalo, M. P. J.
Inorg. Biochem. 2011, 105, 241−249. (f) Morais, T. S.; Silva, T. J. L.;
Marques, F.; Robaldo, M. P.; Avecilla, F.; Amorim Madeira, P. J.;
Mendes, P. J. G.; Santos, I.; Garcia, M. H. J. Inorg. Biochem. 2012, 114,
65−74.
(6) For recent reviews on DKR of sec-alcohols using transition metal
racemization catalysts and enzymes, see, for example: (a) Martín-
Matute, B.; Backvall, J.-E. Curr. Opin. Chem. Biol. 2007, 11, 226−232.
̈
D/Δεap = D/Δε + 1/(Δε × K)
(1)
(b) Lee, J. H.; Han, K.; Kim, M.-J.; Park, J. Eur. J. Org. Chem. 2010,
999−1015.
(7) (a) Mavrynsky, D.; Sillanpaa, R.; Leino, R. Organometallics 2009,
̈
̈
ASSOCIATED CONTENT
■
28, 598−605. (b) Mavrynsky, D.; Paivio, M.; Lundell, K.; Sillanpaa, R.;
̈
̈
̈
̈
S
* Supporting Information
Kanerva, L. T.; Leino, R. Eur. J. Org. Chem. 2009, 1317−1320. See
also: (c) Mavrynsky, D.; Kanerva, L. T.; Sillanpaa, R.; Leino, R. Pure
Appl. Chem. 2011, 83, 479−487. (d) Paivio, M.; Mavrynsky, D.; Leino,
1
Copies of H and 13C NMR for all new ligands and metal
̈
̈
complexes, HRMS for new complexes. X-ray crystallographic
data and data for all complexes in CIF format [CDCC 929859
(5a) and CCDC 929860 (6a)]. This material is available free of
charge via the Internet at http://pubs.acs.org.
̈
̈
R.; Kanerva, L. T. Eur. J. Org. Chem. 2011, 1452−1457.
(8) (a) Csjernyik, G.; Bogar, K.; Backvall, J.-E. Tetrahedron Lett.
2004, 45, 6799−6802. See also: (b) Martín-Matute, B.; Edin, M.;
Bogar, K.; Kaynak, B.; Backvall, J.-E. J. Am. Chem. Soc. 2005, 127,
́
̈
́
̈
8817−8825.
AUTHOR INFORMATION
Corresponding Author
*Phone: +358-(0)2-2154132 (office); +358-(0)400-707195
(mobile). E-mail: reko.leino@abo.fi.
Notes
■
(9) Vermes, I.; Haanen, C.; Reutelingsperger, C. J. Immunol. Methods
2000, 243, 167−190.
(10) Kelly, J. M.; Murphy, M. J.; McConnell, D. J.; O′Huigin, C.
Nucl. Acids Res. 1985, 13, 167−184.
(11) Martínez, R.; Chacon
127−151.
(12) Leino, R.; Luttikhedde, H. J. G.; Lehtonen, A.; Sillanpaa, R.;
́
-García, L. Curr. Med. Chem. 2005, 12,
The authors declare no competing financial interest.
̈
̈
Penninkangas, A.; Stranden
Organomet. Chem. 1998, 558, 171−179.
(13) Quintal, S.; Matos, J.; Fonseca, I.; Fel
́
, J.; Mattinen, J.; Nasman, J. H. J.
̈
ACKNOWLEDGMENTS
■
Financial support from the Finnish Funding Agency for
Technology and Innovation (TEKES) (Technology Pro-
gramme SYMBIO project no. 40168/07: Developing New
Chemoenzymatic Methods and Biocatalysts) is gratefully acknowl-
edged. M.J.C., D.B., S.M., and M.M. acknowledge FCT, POCI,
and FEDER (PEst-OE/QUI/UI0612/2011) for financial
support.
́
ix, V.; Drew, M. G. B.;
Trindade, N.; Meireles, M.; Calhorda, M. J. Inorg. Chim. Acta 2008,
361, 1584−1596.
(14) CrysAlisPro; Agilent Technologies Ltd: Yarnton, England, 2011.
(15) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr.
2008, 64, 112−122.
(16) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.;
Spagna, R. J. Appl. Crystallogr. 1999, 32, 115−119.
(17) DIAMOND. J. Appl. Crystallogr. 1999, 32, 1028−1029.
(18) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.
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dx.doi.org/10.1021/om400234p | Organometallics XXXX, XXX, XXX−XXX