Synthesis, X-ray structure, electrochemical properties and cytotoxic effects of new arene ruthenium(II) complexes

Article abstract of DOI:10.1016/j.jorganchem.2013.07.020

A series of novel half-sandwich organoruthenium(II) complexes with the general formula [(η⁶-arene)Ru(L)Cl₂] (where L = flavone, chromone or benzofuranone derivatives) have been synthesized. All the ligands in the reaction with [(η⁶-p-cymene)RuCl) ₂(μ-Cl)₂] form monodentate compounds, which were fully characterized by elemental analysis, MS, UV-Vis, IR and NMR spectroscopy. The molecular structure of one of the complexes [(η⁶-p-cymene)Ru(7- amino-3H-benzofuran-1-one-κ¹-N)Cl₂] was determined by X-ray crystallography. The redox properties of the complexes were monitored by cyclic voltammetry. The cytotoxicity of all obtained compounds has also been evaluated on several melanoma and leukemic cell lines.

Full text of DOI:10.1016/j.jorganchem.2013.07.020

Journal of Organometallic Chemistry 745-746 (2013) 64e70
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, X-ray structure, electrochemical properties and cytotoxic
effects of new arene ruthenium(II) complexes
a
b
b
c
ꢀ
Adam Pastuszko , Karolina Niewinna , Malgorzata Czyz , Andrzej Józwiak ,
Magdalena Ma1ecka d, Elzbieta Budzisz a
Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Łódz, Muszynski 1 Street, 90-151 Łódz, Poland
Department of Molecular Biology of Cancer, Medical University of Łódz, Mazowiecka 6/8 Street, 92-215 Łódz, Poland
Department of Organic Chemistry, Faculty of Chemistry, University of Łódz, Tamka 12, 91-403 Łódz, Poland
,
*
a
ꢀ
ꢀ
b
c
ꢀ
ꢀ
ꢀ
ꢀ
d
ꢀ
ꢀ
Department of Structural Chemistry and Crystallography, University of Łódz, Pomorska 163/165, 90-236 Łódz, Poland
a r t i c l e i n f o
a b s t r a c t
A series of novel half-sandwich organoruthenium(II) complexes with the general formula [(h
6-arene)
Article history:
Received 27 May 2013
Received in revised form
4 July 2013
Ru(L)Cl2] (where L ¼ flavone, chromone or benzofuranone derivatives) have been synthesized. All the
ligands in the reaction with [( -Cl)2] form monodentate compounds, which were
6-p-cymene)RuCl)2(
fully characterized by elemental analysis, MS, UVeVis, IR and NMR spectroscopy. The molecular structure
of one of the complexes [(
6-p-cymene)Ru(7-amino-3H-benzofuran-1-one- 1-N)Cl2] was determined by
h
m
Accepted 5 July 2013
h
k
X-ray crystallography. The redox properties of the complexes were monitored by cyclic voltammetry. The
cytotoxicity of all obtained compounds has also been evaluated on several melanoma and leukemic
cell lines.
Keywords:
Synthesis
Arene ruthenium(II) complexes
X-ray structure
Ó 2013 Elsevier B.V. All rights reserved.
Cytotoxic effect
Cyclic voltammetry
1. Introduction
ligand cause the higher cytotoxicity. Moreover, complexes which
lack of NH groups on the coordinated ligand are often inactive [9].
Organometallic ruthenium(II) complexes have attracted
considerable interest as potential anticancer agents because of their
low toxicity, often good aqueous solubility [1] and their efficacy
against platinum drug resistant tumors [2e4]. Ruthenium(II) and
ruthenium(III) complexes have similar ligand-exchange kinetics to
those of platinum complexes. Ruthenium(II) complexes bearing a
In addition, the redox potential between the different accessible
oxidation states occupied by ruthenium complexes enables to
catalyze oxidation and reduction reactions, depending on physio-
logical environment.
On the other hand the ligands selected for the current experi-
ments belong to the well known group of the compounds in plants
e flavonoids. These derivatives are naturally occurring compounds
which are able to induce cytotoxic effect in various cell lines. Due to
their miscellaneous activities, such as anticancer [10], antioxidant,
antiproliferative [11], anti-HIV, anti-inflammatory [12], and many
other activities, they are used in several areas like chemistry,
biochemistry, genetics, cellular and molecular biology. Moreover,
flavonoids form metal ions complexes with important biological
activity [13]. Copper(II) complexes show higher anti-inflammatory
activity than free flavonoids [14]. From the above results the
approach to attach a bioactive ligand to arene ruthenium(II) moiety
is very promising and important in investigating of biological
properties.
p-bonded arene ligand and other mono- or bidentate ligands
are considered to be promising candidates for anticancer treatment
[5]. The arene substituent is known to stabilize ruthenium in
its þ2 oxidation state and the arene ligand is relatively inert
toward displacement under physiological conditions [6]. All sub-
stituents, the monodentate ligand and the bidentate ligand as well
the arene substituent possess influent on the pharmacological
properties of areneeRu(II) complexes [7]. Substitution of chloride
by other halides, such as iodide has only a small effect on the
cytotoxicity of complexes with ethylenediamine as ligand [8]. The
size of the coordinated arene substituent appears to increase on
activity. More hydrophobic arene ligand and bidentate chelating
In this paper, we report the synthesis and characterization of
(h
6-p-cymene)Ru(II) complexes with aminochromone derivatives
and its analogs. The cytotoxic effect has been assayed on human
melanoma A375, DMBC11, DMBC12 and leukemic K562 cell lines.
* Corresponding author. Tel.: þ48 42 272 55 95; fax: þ48 42 678 83 98.
E-mail address: elzbieta.budzisz@umed.lodz.pl (E. Budzisz).
0022-328X/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2013.07.020

A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
65
2. Experimental
2.93 (m, 1H), 4.97 (d, 2H, 3JHH ¼ 5.9 Hz), 5.09 (d, 2H, 3JHH ¼ 6.2 Hz),
5.35 (d, 2H, 3JHH ¼ 6.2 Hz), 5.48 (d, 2H, 3JHH ¼ 5.9 Hz), 8.10e6.92 (m,
3H), 6,13 (s, 1H). MSeFAB (m/z): 481 (LRuCl2), 446 (LRuClþ), 410
(LRu2þ), 176 (L).
2.1. Material and methods
All substances were used without further purification. The p-
cymeneruthenium(II) dichloride, 3-nitrophthalic anhydride, 7-
amino-2-methylochromone, 7-aminoflavone and 6-aminoflavone
were purchased from Aldrich. Solvents for synthesis (dichoro-
methane, isopropanol, acetone, diethyl ether) were reagent grade
or better. The melting points were determined using an Electro-
thermal 1A9100 apparatus and they are uncorrected. The IR spectra
were recorded on an FTIRe8400S Shimadzu Spectrophotometer in
KBr. The 1H NMR spectra were registered at 300 MHz on a Varian
Mercury spectrometer. The MSeFAB were measured on Finnigan
Matt 95 mass spectrometer. Elemental analyses were performed by
using a Perkin Elmer PE 2400 CHNS analyzer. The ligands 1e3 were
commercially available. Ligands 4 and 5 were prepared according to
published methods.
2.1.4. Synthesis of [(
benzopyran-4-one-
1-N)Cl2] (4a)
h
6-p-cymene)Ru(5-amino-8-methyl-4H-1-
k
To a stirred solution of 5-amino-8-methyl-4H-benzopyran-4-
one (72.89 mg, 0.416 mmol) in dichloromethane (5 ml) a solution
of ruthenium p-cymene (124.20 mg, 0.208 mmol) in dichloro-
methane (5 ml) was added dropwise. The reaction mixture was
stirred at room temperature for 24 h. The resulting precipitate was
separated and dried under reduced pressure. Yield: 108.1 mg (54%).
M.p.: 190e191 ꢀC. Anal. Calc. for C20H23NO2RuCl2 (481.385 g/mol):
C, 49.89; H, 4.82; N, 2.91. Found: C, 49.34; H, 4.77; N, 2.84. IR (KBr,
cmꢁ1) (selected bands):
NMR (300 MHz, CDCl3)
n(CeNH2) 3544, 3477; n
(C]O) 1640. 1H
d
(ppm): 1.28 (d, 6H, 3JHH ¼ 6.9 Hz), 2.10 (s,
3H), 2.18 (s, 3H), 2.94 (m,1H), 5.00 (d, 2H, 3JHH ¼ 5.9 Hz), 5.05 (d, 2H,
3JHH ¼ 5.9 Hz), 5.36 (d, 2H, 3JHH ¼ 5.9 Hz), 5.49 (d, 2H, 3JHH ¼ 5.9 Hz),
7.98e6.27 (m, 4H). MSeFAB (m/z): 446 (LRuClþ), 410 (LRu2þ),
176 (L).
2.1.1. Synthesis of [(
pyran-4-one-
1-N)Cl2] (1a)
h
6-p-cymene)Ru(6-amino-2-phenyl-4H-benzo
k
To a stirred solution of 6-aminoflavone (94.89 mg, 0.4 mmol) in
isopropanol (8 ml) and dichloromethane (4 ml) a solution of
ruthenium p-cymene (122.48 mg, 0.2 mmol) in dichloromethane
(4 ml) was added dropwise. The reaction mixture was stirred at
room temperature for 24 h. The resulting precipitate was separated
and dried under reduced pressure. Yield: 152.5 mg (70%). M.p.:
235e238 ꢀC. Anal. Calc. for C25H25NO2RuCl2 (543.433 g/mol): C,
55.25; H, 4.63; N, 2.58. Found: C, 55.20; H, 4.14, N, 2.75. IR (KBr,
2.1.5. Synthesis of [(
one-
1-N)Cl2] (5a)
To a solution of 7-aminophtalide (59.65 mg, 0.4 mmol) in
dichloromethane (5 ml) solution of ruthenium p-cymene
h
6-p-cymene)Ru(7-amino-3H-benzofuran-1-
k
a
(122.48 mg, 0.2 mmol) in dichloromethane (5 ml) was added
dropwise. The reaction mixture was stirred at room temperature
for 24 h. The resulting precipitate was separated and dried under
reduced pressure. Yield: 104.9 mg (59%). M.p.: 221e223 ꢀC. Anal.
Calc. for C18H21NO2RuCl2 (455.347 g/mol): C, 47.48; H, 4.65; N, 3.08.
Found: C, 47.15; H, 4.75, N, 2.97. IR (KBr, cmꢁ1) (selected bands):
cmꢁ1) (selected bands):
NMR (300 MHz, CDCl3)
n(CeNH2) 3228, 3191; n
(C]O) 1619. 1H
d
(ppm): 1.28 (d, 6H, 3JHH ¼ 7.0 Hz), 2.18 (s,
3
3H), 2.93 (m, 1H), 4.91 (s, 2H), 4.98 (d, 2H, JHH ¼ 5.9 Hz), 5.09 (d,
3
3
2H, JHH ¼ 5.9 Hz), 5.38 (d, 2H, JHH ¼ 5.9 Hz), 5.50 (d, 2H,
3JHH ¼ 5.9 Hz), 6.90 (s, 1H), 8.05e7.56 (m, 8H). MSeFAB (m/z): 543
(LRuCl2), 508 (LRuClþ), 472 (LRu2þ), 238 (L).
n
(CeNH2) 3264, 3181;
(ppm): 1.29 (d, 6H), 2.18 (s, 3H), 2.94 (m, 1H), 5.26 (s, 4H), 5.36 (d,
n
(C]O) 1741. 1H NMR (300 MHz, CDCl3)
d
3
3
2H, JHH ¼ 5.9 Hz), 5.49 (d, 2H, JHH ¼ 5.9 Hz), 7.62e7.21 (m, 3H).
MSeFAB (m/z): 460 (LRuCl2), 154 (L).
2.1.2. Synthesis of [(
pyran-4-one-
1-N)Cl2] (2a)
To a stirred solution of 7-aminoflavone (118.63 mg, 0.5 mmol) in
dichloromethane (8 ml) solution of ruthenium p-cymene
h
6-p-cymene)Ru(7-amino-2-phenyl-4H-benzo
k
2.2. Crystallographic analysis
a
Very small crystals were obtained by recrystallization from
dichloromethane/hexane by diffusion technique. An orange shaped
crystal of dimension 0.05 ꢂ 0.04 ꢂ 0.01 mm was used to collect data
(153.09 mg, 0.25 mmol) in dichloromethane (5 ml) was added
dropwise. The reaction mixture was stirred at room temperature
for 24 h. The resulting precipitate was separated and dried under
reduced pressure. Yield: 204.7 mg (71%). M.p.: 207e209 ꢀC. Anal.
Calc. for C25H25NO2RuCl2 (543.433 g/mol): C, 51.82; H, 5.04; N, 2.41.
Found: C, 51.99; H, 4.46; N, 2.36. IR (KBr, cmꢁ1) (selected bands):
in 100 K. 11,469 reflections were measured with omega scan mode
ꢁ
on SuperNova diffractometer using MoK
a
radiation (
l
¼ 0.7107 A).
The structure was solved by direct methods with the program
SHELXS-97 and refined by full-matrix least-squares method on F2
with SHELXL-97 [15]. The non-H atoms were refined anisotropi-
cally. All hydrogen position were placed geometrically and refined
using a riding model, with Uiso(H) ¼ x Ueq(C) where x ¼ 1.5 for
methyl groups and 1.2 for all others. CeH distances were fixed for
n
(CeNH2) 3345, 3213; n
(C]O) 1647. 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.28 (d, 6H, 3JHH ¼ 6.8 Hz), 2.17 (s, 3H), 2.93 (m, 1H), 4.98
3
3
(d, 2H, JHH ¼ 5.8 Hz), 5.12 (d, 2H, JHH ¼ 5.8 Hz), 5.35 (d, 2H,
3JHH ¼ 6.0 Hz), 5.48 (d, 2H, 3JHH ¼ 5.9 Hz), 6.78 (s, 1H), 8.13e7.54 (m,
8H). MSeFAB (m/z): 544 (LRuCl2 þ 1H), 508 (LRuClþ), 475 (LRu2þ),
238 (L).
ꢁ
ꢁ
ꢁ
methyl group at 0.96 A, for aromatic at 0.93 A, for methine 0.98 A,
ꢁ
ꢁ
for methylene 0.97 A and for NH2 group 0.9 A. Final results are:
R1 ¼ 3.47% and 3.04% for all and observed reflections, respectively.
3
ꢁ
2.1.3. Synthesis of [(
h
6-p-cymene)Ru(7-amino-2-methyl-4H-benzo
The largest difference peak and hole are: 0.52 and ꢁ0.56 e/A .
Atomic coordinates and relevant data have been deposited at the
Cambridge Crystallographic Data Centre as supplementary publi-
cation number CCDC 936546. These data can be obtained free of
charge from CCDC via http://www.ccdc.cam.ac.uk/products/csd/
request/.
pyran-4-one-
k
1-N)Cl2] (3a)
To a stirred solution of 7-amino-2-methylochromone (87.60 mg,
0.5 mmol) in dichloromethane (8 ml) a solution of ruthenium p-
cymene (153.10 mg, 0.25 mmol) in dichloromethane (5 ml) was
added dropwise. The reaction mixture was stirred at room tem-
perature for 24 h. The resulting precipitate was separated and dried
under reduced pressure. Yield: 129.0 mg (51%). M.p.: 211e213 ꢀC.
Anal. Calc. for C20H23NO2RuCl2 (481.385 g/mol): C, 47.25; H, 5.15; N,
2.75. Found: C, 47.23; H, 4.71; N, 2.73. IR (KBr, cmꢁ1) (selected
2.3. Biological evaluation
Three melanoma cell lines were used in the current study.
Leukemia represented by K562 cell line was maintained in
RPMI1640 medium supplemented with 10% FBS and antibiotics.
bands):
CDCl3)
n(CeNH2) 3202, 3101; n
(C]O) 1618. 1H NMR (300 MHz,
d
(ppm): 1.27 (d, 6H, 3JHH ¼ 7.0 Hz), 2.16 (s, 3H), 2.37 (s, 3H),

66
A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
CH3
CH3
CH3
H2N
O
CH3
O
O
H2N
O
CH3
Cl
Cl
H3C
H2N
Ru
CH3
CH3
Ru
H3C
Cl
H3C
Cl
O
O
Ru
Cl
Cl
L(2)
L(1)
L(3)
NH2
CH3
NH2
L-NH2
O
L
CH2Cl2
O
1a - 5a
1 - 5
O
O
NH2
L(5)
L(4)
Scheme 2. Synthesis of complexes 1ae5a.
Fig. 1. Selected ligands 1e5.
Standard three electrodes were used: glassy carbon electrode
(working), silver wire (pseudo reference) and platinum wire
(auxiliary).
A375 human melanoma cell line with high metastatic potential (a
gift from Prof. Piotr Leidler, Jagiellonian University, Poland) was also
maintained in RPMI1640 medium supplemented with 10% FBS and
antibiotics. DMBC11 and DMBC12 cell lines, derived from patients
with nodular melanoma (NM), were established and maintained in
our laboratory as described before [16].
3. Results and discussion
3.1. Chemistry
To this work we have selected five ligands, the derivatives of
flavone (1, 2), chromone (3, 4), and benzofuranone (5), see Fig. 1. In
our previous papers we reported that ligand 3 form monodentate
Zn(II) complex via oxygen atom in carbonyl group [17]. While
ligand 4 can form monodentate Zn(II) complex via nitrogen atom
in amino group in C-7 position [18], also bidentate coordination
with Cu(ClO4)2 ꢂ 6H2O via nitrogen and oxygen atoms has been
reported [19].
2.3.1. Measurement of cell number
Cancer cells were counted after staining with Trypan blue
(SigmaeAldrich) and plated at a density of 2e4 ꢂ 103 viable cells
per well in 96-well plates. Cells were cultured for 2 days with tested
compounds at indicated concentrations. An acid phosphatase ac-
tivity (APA) assay was used to measure viable cell number in the
culture. Briefly, the plates were centrifuged, the medium was dis-
carded and replaced with 100 ml assay buffer containing 0.1 M so-
Ligands 1e3 were commercially available. The ligand 4 was
synthesized according to procedure described previously [17].
Based on the literature [20,21] we synthesized ligand 7-
aminophtalide L(5) using as materials 3-nitrophthalic anhydride
which is commercially available (Scheme 1).
dium acetate (pH ¼ 5), 0.1% Triton X-100 and 5 mM p-nitrophenyl
phosphate, pNPP (SigmaeAldrich) and incubated for additional 2 h
at 37 ꢀC. The reaction was stopped with 10
ml/well of 1 M NaOH, and
the absorbance values were measured at the wavelength of 405 nm
using a microplate reader (Infinite M200Pro, Tecan, Austria).
All complexes 1ae5a were prepared at room temperature in
dichloromethane by mixing equimolar amounts of ruthenium p-
cymene compound and corresponding or in solution of dichloro-
methane and isopropanol ligands L(1e5), respectively. All ligands
behaves as a strong monodentate chelating agents forming new
Ru(II) complexes through coordination of the amino nitrogen atom
(Scheme 2).
2.3.2. Flow cytometric analysis of cancer cell death
Detection of cell death was carried out by staining with propi-
dium iodide (PI). Cancer cells were seeded into 12-well plate and
treated for 46 h with ligands 1e5 and complexes 1ae5a at indi-
cated concentrations. After treatment, cells were collected, centri-
fuged at 400 ꢂ g for 5 min and stained with PI for 15 min at room
temperature in the dark. 15,000 events were analyzed for each
sample by flow cytometer FACSVerse (Becton Dickinson), and re-
sults were processed by using FACSuite software (Becton
Dickinson).
3.2. IR spectra
The most important IR spectral data for ligands 1e5 and their
complexes are listed in Table 1. The IR spectra of all compounds
show characteristic stretching vibrations at w3200e3400 cmꢁ1
2.4. Cyclic voltammetry
which correspond to the asymmetric and symmetric n(NH2). These
bands are negatively shifted in complexes 1ae5a in comparison to
free ligands (by 114 and 47 cmꢁ1 for compound 1a, 115 and 80 cmꢁ1
for compound 2a, 229 and 221 cmꢁ1 for compound 3a, 8 and
The compounds were measured electrochemically by cyclic
voltammetry. Complexes were dissolved in tetrabutylammonium
tetrafluoroborate. All measurements were carried out at room
temperature. Cyclic voltammograms were obtained on computer-
controlled electrochemical system under argon atmosphere.
Table 1
IR data (n
, cmꢁ1) for ligands 1e5 and complexes 1ae5a.
Compound
n(NH2)
n(C]O)
naromat
1
1a
2
2a
3
3a
4
4a
5
5a
3342, 3238
3228, 3191
3460, 3293
3345, 3213
3431, 3322
3202, 3101
3432, 3324
3424, 3264
3480, 3376
3264, 3181
1642
1619
1630
1647
1655
1618
1648
1640
1748
1741
1581, 1565, 1487
1600, 1579, 1470
1600, 1586, 1498
1607, 1456
NO2
NO2
O
O
NH2
O
O
CH3OH
i, ii, iii, iv
OCH3
OH
O
O
1584
O
1593, 1542, 1493
1625, 1588, 1569
1618, 1538, 1486
1642, 1600
i: ClCOOCH2CH3, N(CH2CH3)3; ii: NaBH4; iii: HCl; iv: H2, Pd/C, acetic acid
1608, 1567
Scheme 1. Synthesis of 7-aminophtalide (5).

A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
67
60 cmꢁ1 for compound 4a, 216 and 195 cmꢁ1 for compound 5a).
These bands shift to lower energy confirm that coordination of the
amino group has occurred. The (C]O) bands are presented for
each compounds at 1748e1618 cmꢁ1. Vibrations from aromatic
carbons are assigned for bands in the 1642e1456 cmꢁ1
n
.
3.3. 1H NMR spectra
1H NMR spectra data of complexes 1ae5a were recorded in
CDCl3 and are presented in the experimental section. Position and
intensity of the signals correspond to ligands used in synthesis.
Methyl protons of the isopropyl group give signals at
d 1.28 ppm as
doublets and methylene proton at 2.94 ppm as multiplet. The
d
aromatic protons from p-cymene in the complexes are presented as
four doublets at 4.98, 5.09, 5.38 and 5.45 ppm. This is probably due
to the long range coupling of diastereotopic methyl protons of
isopropyl group and aromatic protons of the p-cymene [22,23]. The
methyl protons from ligands are displayed as singlets at 2.37 for
complex 4a and 2.10 ppm for complex 5a.
3.4. UVeVis spectra
Fig. 2. An ORTEP view of complex 5a with atom numbering scheme.
The UVeVis absorption spectra of complexes 1ae5a were
recorded in dichloromethane at room temperature. All the studied
complexes showed intense absorption bands in the range 243e
325 nm attributed to intraligand charge transfer. In addition, a low
energy absorption bands were presented in the range 450e500 nm.
On account of position and intensity these transitions are believed
to be metal to ligand charge transfer (MLCT) [24].
structure with atom numbering scheme is presented at Fig. 2, while
selected geometric parameters are shown in Table 3. The complex
has an essentially octahedral coordination geometry comprising
the p-cymene ring carbons occupying one face of the octahedron
leaving the other three sites to be coordinated by two chloride
atoms and monodentate (7-amino-3H-benzofuran-1-one) ligand.
3.5. MSeFAB
ꢁ
The RueC(arene) distances vary considerably from 2.160(3) A to
ꢁ
2.203(3) A. The molecules of 5a complex symmetrical by inversion
Mass spectrometry was carried out for all newly synthesized
complexes. Parent peaks have been found in FABeMS spectra at (m/
z) 543 for complex 1a, at (m/z) 544 for complex 2a and at (m/z) 481
for complex 3a. Ion peaks corresponding to the ligands have been
observed at (m/z) 238 for compounds 1a, 2a, at (m/z) 176 for
compounds 3a, 4a and at (m/z) 154 for compound 5a. For more
detailed mass spectra analysis see experimental section.
form dimers via N1eH1B/Cl1 interaction (see Fig. 3). In the crystal
lattice another weak CeH/Cl and CeH/O intermolecular
Table 3
ꢀ
ꢀ
ꢁ
ꢁ
Selected bond lengths (A), angles ( ) and hydrogen bond geometry ( , A), symmetry
codes (i): 2 ꢁ x, 1 ꢁ y, ꢁz, (ii): 1 ꢁ x, 1 ꢁ y, ꢁz, (iii): 5/2 ꢁ x, ½ þ y, ½ ꢁ z.
ꢁ
Bond lengths (A)
3.6. Crystal structure of complex 5a
Ru1 C1
Ru1 C2
Ru1 C3
Ru1 C4
Ru1 C5
2.165(4)
2.197(3)
2.160(3)
2.176(3)
2.203(3)
Ru1 C6
Ru1 N1
Ru1 Cl2
Ru1 Cl1
2.180(4)
2.183(3)
2.4063(8)
2.4075(8)
The structure of complex 5a has been confirmed by X-ray
analysis, for crystallographic data see Table 2. The molecular
Bond angles (ꢀ)
C1 Ru1 N1
C2 Ru1 N1
C3 Ru1 N1
C4 Ru1 N1
C5 Ru1 N1
C6 Ru1 N1
C1 Ru1 Cl1
C2 Ru1 Cl1
C3 Ru1 Cl1
C4 Ru1 Cl1
C5 Ru1 Cl1
C6 Ru1 Cl1
C1 Ru1 Cl2
C2 Ru1 Cl2
Table 2
154.27(12)
117.31(12)
94.79(13)
98.10(12)
124.61(12)
162.59(11)
89.48(10)
91.99(9)
120.35(9)
158.92(10)
151.71(10)
113.89(9)
122.93(10)
161.10(10)
C3 Ru1 Cl2
C4 Ru1 Cl2
C5 Ru1 Cl2
C6 Ru1 Cl2
C1 Ru1 N1
C2 Ru1 N1
C3 Ru1 N1
C4 Ru1 N1
C5 Ru1 N1
C6 Ru1 N1
Cl2 Ru1 Cl1.
N1 Ru1 Cl1
N1 Ru1 Cl2
150.78(9)
112.92(10)
89.31(10)
93.65(10)
154.27(12)
117.31(12)
94.79(13)
98.10(12)
124.61(12)
162.59(11)
88.09(3)
Crystallographic data for compound 5a.
Complex 5a
Molecular formula
Molecular weight
Crystal description/size (mm)
Crystal system
C18H21Cl2NO2Ru
455.33
Orange block/0.05 ꢂ 0.04 ꢂ 0.01
Monoclinic
P21/n
8.6021(3)
8.0777(2)
24.9788(9)
90.160(3)
1735.65(2)
4/1.742
920
455.33
100
25.35
11,469/3179/4.37%
0.0305/1.040
0.523/ꢁ0.558
Space group
ꢁ
a [A]
ꢁ
b [A]
ꢁ
c [A]
82.78(7)
81.45(8)
b
[ꢀ]
3
ꢁ
V [A ]
Z/dx [g cmꢁ3
F(000)
]
ꢀ
ꢁ
Hydrogen bond geometry ( , A)
m
[cmꢁ1
]
DeH
H/A
D/A
<DeH/A
T [K]
max
q
[ꢀ]
N1eH1B/Cl1i
C1eH1/Cl2ii
0.90
0.93
0.97
0.97
2.48
2.82
2.54
2.57
3.290(3)
150
128
151
137
No. of measured/unique reflections/Rint
R1/wR2/S (goodness of fit)
3.471(4)
3.417(4)
3.340(4)
C19eH19B/O1iii
C19eH19B/O2iii
Difference peak/hole [eÅꢁ3
]

68
A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
Fig. 4. Cyclic voltammogram of compound 3a.
employed. Those melanoma cells were grown in anchorage-
independent manner as spheroids [16]. Such as multicellular
structures are considered to portray the original tumor more
accurately than two-dimensional serum-driven monolayer cell
cultures and they could be considered as a good in vitro model for
anticancer drug screening. We have recently used this model to
study the activities of different drugs and drug combinations [25e
27]. In the current study cytostatic effects were assessed based on
changes in the activity of acid phosphatase (APA assay). The results
are shown in Fig. 6.
Fig. 3. Dimers of complex 5a formed by N1eH1B/Cl1 interaction. Hydrogen atoms
not involved in hydrogen bond are omitted for clarity, symmetry code (i): 2 ꢁ x,
1 ꢁ y, ꢁz.
interactions which stabilized crystal packing are observed (Table 3).
Additionally, the furan ring (O2/C17/C16/C15/C19) in the molecule
at x, y, z is linked by pep interactions with p-cymene ring (C1eC6)
ꢁ
at 1 þ x, y, z: the interplanar spacing is 3.406(2) A, while the ring-
Changes in viability were measured by flow cytometry after
labeling cells with propidium iodide. Cells that remained PI-
negative are considered as viable. Viability expressed as % of con-
trol is shown in Fig. 7.
ꢁ
centroid separation is 3.669(2) A.
3.7. Cyclic voltammetry
In general, ligand 3 and its complex 3a showed the highest ac-
tivity against both melanoma and leukemic cell lines. These two
compounds were much more effective against heterogenous mel-
anoma cell lines DMBC11 and DMBC12 than against established
A375 melanoma cell line. In case of A375 cells it was mainly the
cytostatic not cytotoxic activity as cell membrane did not become
permeable to propidium iodide. Similar effects were obtained for
A375 cells treated with cisplatin indicating that the mode of action
might be similar for cisplatin and arene ruthenium(II) complexes
[28]. Compound 1 and its derivative 1a represented moderate ac-
tivity against melanoma A375, DMBC11, DMBC12 cell lines. How-
ever, there is hardly any influence on leukemic K562 cell line. Other
compounds turned to be less active in the performed tests. It was
already shown that ruthenium complexes display promising anti-
cancer activities [29,30]. Some of them, NAMI-A (ImH[trans-
RuC14(DMSO)Im]) and KP1019 (indazolium trans-[tetrachloro
bis(1H-indazole)ruthenate(III)]), have entered clinical trials. NAMI-
A was shown to possess the ability to inhibit neoangiogenesis
The measurements were performed at glassy carbon electrode
(working), silver wire (pseudo reference) and platinum wire
(auxiliary) electrodes in tetrabutylammonium tetrafluoroborate.
The cyclic voltammograms were obtained by scanning the potential
between ꢁ2 and þ2 V versus Ag/AgCl at a scan rate of 0.20 V/s. The
potential data are given in Table 4.
On the forward scan for the negative to positive potential all
complexes exhibited oxidation associated to the Ru(II)/Ru(III) pro-
cess. On the return scan reduction process of the same redox couple
was observed. Analysis of cathodic and anodic peaks established
one metal centered voltammetric response with E1/2 values in the
range of ꢁ0.662 to ꢁ0.942 V assigned to (Ru(III)/Ru(II)) redox
couple. The peak-to-peak separation value DEp ranging from 536 to
970 mV suggest an irreversible one electron-transfer process. A
representative voltammograms have been depicted in Figs. 4 and 5.
3.8. Biological activity
Ligands 1e5 and their complexes 1ae5a were tested for their
biological activity against human melanoma A375 cell line and
leukemic K562 cell line. In addition, two melanoma cell lines,
DMBC11 and DMBC12, derived from surgical specimens were
Table 4
Cyclic voltammetric data for complexes 1ae5a.
Compound
Epc (V)
Epa (V)
E1/2 (V)
DEp (mV)
1a
2a
3a
4a
5a
ꢁ0.930
ꢁ1.119
ꢁ1.362
ꢁ1.214
ꢁ1.172
ꢁ0.394
ꢁ0.313
ꢁ0.523
ꢁ0.250
ꢁ0.202
ꢁ0.662
ꢁ0.716
ꢁ0.942
ꢁ0.732
ꢁ0.687
536
808
839
964
970
D
Ep ¼ Epa ꢁ Epc, where Epa and Epc are anodic and cathodic potentials, respectively;
E1/2 ¼ 0.5(Epa þ Epc).
Fig. 5. Cyclic voltammogram of compound 4a.

A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
69
Fig. 6. The influence of ligands 1e5 and complexes 1ae5a on viable cell numbers in melanoma and leukemic cell cultures. Melanoma cells A375 grown as the monolayer were more
sensitive to tested compounds than heterogenous populations of anchorage-independent melanoma cells. Viability was assessed by acid phosphatase activity assay. Absorbance
given by control cells was taken as 100%. Data represent the average ꢃ SD.
Fig. 7. Induction of cell death by ligands 1e5 and complexes 1ae5a in melanoma and leukemic cell cultures. Cells were treated with tested compounds at concentrations of 10 and
100
m
M for 44 h. Cells with PI-permeable membrane were assessed by flow cytometry. Percentages of viable cells (PI-negative) are shown as bars. The data are mean ꢃ SD.
induced by vascular endothelial growth factor (VEGF) [31] and
metastatic activity [32].
characterized by standard analytical techniques. The molecular
structure of complex 5a was determined by X-ray crystallography.
The complex has an essentially octahedral coordination geometry
comprising the p-cymene ring carbons occupying one face of the
octahedron leaving the other three sites to be coordinated by two
chloride atoms and monodentate (7-amino-3H-benzofuran-1-one)
ligand. The molecules of the complex form dimers in the crystal
lattice. Electron-transfer properties of all complexes have been
4. Conclusions
Arene ruthenium(II) complexes with flavone, chromone and
benzofuranone derivatives as ligands were prepared. The com-
plexes coordinated in
a monodentate manner and were

70
A. Pastuszko et al. / Journal of Organometallic Chemistry 745-746 (2013) 64e70
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studied by cyclic voltammetry. The electrochemical analysis
confirmed an irreversible one electron-transfer process occurring
to all compounds which probably results in increased electro-
chemical stability of the compounds in vivo. In viability assays
ligand 3 and its complex 3a showed the highest activity against
both melanoma and leukemic cell lines. Heterogenous patient-
derived melanoma cell lines, DMBC11 and DMBC12, were more
sensitive than established melanoma cell line, A375. Also com-
pounds 1 and 1a exerted some activity against melanoma cell lines
A375, DMBC11 and DMBC12. In general, the biological study has
revealed that leukemic cells were less susceptible to tested ligands
1e5 and complexes 1ae5a than melanoma cells. This is in agree-
ment with our previous results obtained for oximine rhenium(I)
complexes for which higher anticancer activity was also induced
against melanoma cells [33]. To our knowledge, the current report
is the first study showing the biological activity of Ru(h
6-p-cymene)
complexes with flavone and chromone against melanoma and
leukemic cells. Further studies are necessary to resolve the mech-
anism(s) of the cellular responses evoked by these compounds and
to assess their effectiveness in in vivo models.
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Acknowledgments
[19] M. Grazul, A. Kufelnicki, M. Wozniczka, I.-P. Lorenz, P. Mayer, A. Jozwiak,
M. Czyz, E. Budzisz, Polyhedron 31 (2012) 150e158.
Financial support from Medical University of Lodz (grant No.
503/3-066-02/503-01 to E. Budzisz and grant No. 503/1-156-01/
503-01 to M. Czyz), grants No. 502-03/3-066-02/502-34-025 and
503/3-066-02/503-06-300 to A. Pastuszko and the DAAD fellow-
ship No A/11/13479 are gratefully acknowledged. The authors wish
to thank I. Dobinska for her support in biological experiments.
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(2010) 1144e1150.
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695 (2010) 567e573.
[25] M. Czyz, K. Koprowska, M. Sztiller-Sikorska, Cancer Biol. Ther. 14 (2013)
135e145.
[26] K. Koprowska, M.L. Hartman, M. Sztiller-Sikorska, M. Czyz, Anti-Cancer Drugs
(2013), http://dx.doi.org/10.1097/CAD.0b013e3283635a04.
[27] M. Wozniak, A. Szulawska-Mroczek, M.L. Hartman, D. Nejc, M. Czyz, Anti-
cancer Res. 33 (2013) 3205e3212.
[28] M. Czyz, K. Lesiak-Mieczkowska, K. Koprowska, A. Szulawska-Mroczek,
M. Wozniak, Br. J. Pharmacol. 160 (2010) 1144e1157.
[29] O. Letzen, C. Moucher, A. Kirsch-De Mesmaker, John Wily & Sons, Ltd., West
Sussex, 2005, pp. 359e378.
Appendix A. Supplementary material
CCDC 936546 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
[30] T. Chen, Y. Liu, W.J. Zheng, J. Liu, Y.S. Wong, Inorg. Chem. 49 (2012) 6366e
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