Synthesis, characterization, DNA interaction and cleavage, and in vitro cytotoxicity of copper(ii) mixed-ligand complexes with 2-phenyl-3-hydroxy-4(1H)- quinolinone

Article abstract of DOI:10.1039/c1dt10674k

A series of mixed-ligand complexes [Cu(qui)(L)]NO₃· xH₂O (1-6), where Hqui = 2-phenyl-3-hydroxy-4(1H)-quinolinone, L = 2,2′-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), bis(2-pyridyl)amine (ambpy) (3), 5-methyl-1,10-phenanthroline (mphen) (4), 5-nitro-1,10-phenanthroline (nphen) (5) and bathophenanthroline (bphen) (6), have been synthesized and fully characterized. The X-ray structures of [Cu(qui)(phen)]NO₃·H₂O (2) and [Cu(qui)(ambpy)] NO₃ (3a) show a slightly distorted square-planar geometry in the vicinity of the central copper(ii) atom. An in vitro cytotoxicity study of the complexes found significant activity against human osteosarcoma (HOS) and human breast adenocarcinoma (MCF7) cell lines, with the best results for complex 6, where IC₅₀ equals to 2.1 ± 0.2 μM, and 2.2 ± 0.4 μM, respectively. The strong interactions of the complexes with calf thymus DNA (CT-DNA) and high ability to cleave pUC19 DNA plasmid were found. A correlation has been found between the in vitro cytotoxicity and DNA cleavage studies of the complexes.

Full text of DOI:10.1039/c1dt10674k

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PAPER
Synthesis, characterization, DNA interaction and cleavage, and in vitro
cytotoxicity of copper(II) mixed-ligand complexes with
2-phenyl-3-hydroxy-4(1H)-quinolinone†
Roman Bucht´ık,a Zdeneˇk Tra´vn´ıcˇek,*b Ja´n Vancˇo,b Radovan Herchela and Zdeneˇk Dvorˇa´kc
Received 15th April 2011, Accepted 5th July 2011
DOI: 10.1039/c1dt10674k
A series of mixed-ligand complexes [Cu(qui)(L)]NO3·xH2O (1–6), where Hqui = 2-phenyl-3-hydroxy-
4(1H)-quinolinone, L = 2,2¢-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), bis(2-pyridyl)amine
(ambpy) (3), 5-methyl-1,10-phenanthroline (mphen) (4), 5-nitro-1,10-phenanthroline (nphen) (5) and
bathophenanthroline (bphen) (6), have been synthesized and fully characterized. The X-ray structures
of [Cu(qui)(phen)]NO3·H2O (2) and [Cu(qui)(ambpy)]NO3 (3a) show a slightly distorted square-planar
geometry in the vicinity of the central copper(II) atom. An in vitro cytotoxicity study of the complexes
found significant activity against human osteosarcoma (HOS) and human breast adenocarcinoma
(MCF7) cell lines, with the best results for complex 6, where IC50 equals to 2.1 0.2 mM, and 2.2
0.4 mM, respectively. The strong interactions of the complexes with calf thymus DNA (CT-DNA) and
high ability to cleave pUC19 DNA plasmid were found. A correlation has been found between the
in vitro cytotoxicity and DNA cleavage studies of the complexes.
DNA with high affinity and thus promotes DNA oxidation.7 It
Introduction
also causes apoptosis in cultured mammalian cells.8
The medicinal importance of cisplatin and other platinum-based
Back in 1965, Dwyer and co-workers first reported that a Cu(II)
complex of the composition of [Cu(tmphen)2]Cl2, where tmphen
of transition metal-based complexes that could be used in the
stands for 3,4,7,8-tetramethyl-1,10-phenanthroline, inhibits the
drugs1,2 has resulted in an extensive search for a broad spectrum
growth of Landschu¨tz ascites tumour.9 Since then, anticancer
properties of a wide range of copper complexes containing 1,10-
phenanthroline (phen) and related ligands have been intensively
investigated.10–13 However, the mechanism by which these com-
plexes enforce their biological activity is still unknown. It can
be supposed that the complexes bind directly to target molecules
within the cell or they participate in the redox system that produces
radicals consecutively damaging the molecules within the cell. The
next generation of the above-mentioned complexes is represented
by Casiope´ınas, a group of Cu(II) mixed-ligand antineoplastic
agents, containing 1,10-phenanthroline (phen), 2,2¢-bipyridine
(bpy) derivatives in combination with essential aminoacids.14–16
These compounds exhibit cytotoxicity, genotoxicity and antitu-
mour effects, however, the mechanism of their action has also not
been determined yet.
We have several reasons for choosing 2-phenyl-3-hydroxy-
4(1H)-quinolinone (Fig. 1) as the crucial ligand for the preparation
of the complexes (1–6). Firstly, generally 4-quinolinones, as aza-
analogues of flavones, are well known for their wide range of
diverse biological activities, such as antibacterial activity due to
the inhibition of DNA-gyrase (a class of enzymes known as topoi-
somerases) or antitumour activity, which is presumably caused
by mammalian topoisomerase II inhibition.17–19 Secondly the
advantage of a study of 2-phenyl-3-hydroxy-4(1H)-quinolinone
treatment of malignant tumours. Due to the generally high toxicity
and unwanted side-effects of platinum-based complexes, we have
focused our attention on the more favourable transition metal
for biological systems, i.e. copper. Copper is one of the metals
acting as an essential trace element involved in cellular respiration,
antioxidant defence, neurotransmission, connective tissue biosyn-
thesis and cellular iron metabolism. Several investigations provide
evidence that copper ions are capable of interacting directly with
nuclear proteins and DNA, causing site-specific damage.3,4 It has
been reported that copper compounds delay cell-cycle progression
and increase cell death in different cell cultures.5,6 Copper(II) ions
binding to specific sites can modify conformational structures of
proteins, polynucleotides, DNA or membranes. Cu(II) binds to
aDepartment of Inorganic Chemistry, Faculty of Science, Palacky´ University,
17. listopadu 12, CZ-771 46, Olomouc, Czech Republic
bRegional Centre of Advanced Technologies and Materials, Department
of Inorganic Chemistry, Faculty of Science, Palacky´ University, 17.
listopadu 12, CZ-771 46, Olomouc, Czech Republic. E-mail: zdenek.
travnicek@upol.cz; Fax: +420585 634 954; Tel: +420 585 634 352
cRegional Centre of Advanced Technologies and Materials, Department of
ˇ
Cell Biology and Genetics, Faculty of Science, Palacky´ University, Slechtitelu˚
11, CZ-783 71, Olomouc, Czech Republic
† Electronic supplementary information (ESI) available. CCDC reference
numbers 822513–822515. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c1dt10674k
9404 | Dalton Trans., 2011, 40, 9404–9412
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instructions. Cells were maintained at 37 ◦C and 5% CO2 in a
humidified incubator.
Human cancer cell lines were treated with the tested compounds
for 24 h and 48 h, using 96-well culture plates.22 The tested
compounds (cisplatin and 1–6) were applied to the cells at
concentrations of 0.1, 1, 10, 25 and 50 mM. In parallel, the cells
were treated with vehicle (DMF; 0.1% v/v) and Triton X-100 (1%
v/v) to assess the minimal (i.e. positive control) and maximal
(i.e. negative control) cell damage, respectively. The MTT as-
say [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] was measured at TECAN, Schoeller Instruments LLC
(540 nm). The data were expressed as the percentage of viability,
when 100% and 0% represent the treatments with DMF and Triton
X-100, respectively. The data from cancer cell lines were acquired
from three independent cell passages. The values of IC50 were
calculated, unless the concentration of the tested compound was
limited by its solubility.
Fig.
1
A representation of 2-phenyl-3-hydroxy-4(1H)-quinolinone
(Hqui), which was used as a ligand for the preparation of complexes 1–6.
complexes of suitable transition metals is the fact that 2-
phenyl-3-hydroxy-4(1H)-quinolinones themselves, as a specific
group of 4-quinolones, have already been studied for their
potential biological activity only recently due to their long
time synthetic unavailability.20 Finally complexes involving these
ligands are practically unknown, except for [Cu(qui)2] and
[Cu(tmeda)(qui)(N-baa)] complexes, where tmeda and N-baa
stand for N,N,N¢,N¢-tetramethyl-ethane-1,2-diamine, and 2-
(benzoylamino)benzoate, respectively.21
During this work, we strived not only to prepare and
characterize a novel group of copper(II) complexes and to
broaden a spectrum of Casiope´ınas-like complexes but also
to find out if the prepared complexes show in vitro cyto-
toxicity against selected human cancer cell lines. Thus, we
focused on studying Cu(II) mixed-ligand complexes of the
general composition [Cu(qui)(L)]NO3·xH2O, where qui = 2-
phenyl-3-hydroxy-4(1H)-quinolinonate anion and L = 2,2¢-
bipyridine (bpy), 1,10-phenanthroline (phen), 2,2¢-bipyridylamine
(ambpy), 5-methyl-1,10-phenanthroline (mphen), 5-nitro-1,10-
phenanthroline (nphen) or bathophenanthroline (bphen).
DNA interactions assessed by UV-Vis titration
The UV-Vis titration experiments were performed as a supple-
mental method for analyzing the efficiency and prediction of the
possible mechanism of complex-DNA interaction. The method is
based on measuring the absorption spectra of mixtures, containing
the same concentration of the tested complexes at 50 mM and vary-
ing concentrations of the nucleic acid. All experiments involving
the interactions of the tested compounds with CT-DNA were per-
formed in Tris/HCl buffer [tris(hydroxymethyl)aminomethane],
containing 5 mM Tris and 50 mM NaCl, adjusted to pH 7.2 by
the addition of HCl. The DNA samples were prepared by the
dilution of viscous saturated solutions of CT-DNA in Tris/HCl
buffer (approx. conc. 4 mg mL-1), which spectral analysis at
260 nm and 280 nm gave the ratios of ca 1.8–1.9, indicating that
CT-DNA was sufficiently free of protein.23 The absorption spec-
tra were analyzed for the nucleic acid concentration-dependent
changes, like bathochromic (red-shift), hypsochromic (blue-shift),
or hypochromic effects, usually considered as the evidence of DNA
interactions.24
Experimental
Complexes of the composition [Cu(qui)(bpy)]NO3·1/2H2O (1),
[Cu(qui)(phen)]NO3·H2O (2), [Cu(qui)(ambpy)]NO3·1/2H2O (3),
[Cu(qui)(mphen)]NO3·H2O (4), [Cu(qui)(nphen)]NO3·H2O (5)
and [Cu(qui)(bphen)]NO3·H2O (6), have been synthesized via
the reaction of 2-phenyl-3-hydroxy-4(1H)-quinolinone (Hqui) and
the corresponding bidentate N-donor ligand (L) with copper(II)
nitrate in an ethanol/water mixture (11 : 1, v/v) in the 1 : 1 : 1 molar
ratios, with average yields of 55–67%. They were characterized by
elemental analysis, UV-Vis, FTIR, Raman and EPR spectroscopy,
mass spectrometry, magnetic, conductivity and thermogravimetric
TG/DTA measurements, and single crystal X-ray analysis (for
detailed methodology used and the syntheses see ESI S1, S2†).
DNA cleavage studies
Supercoiled plasmid DNA pUC19 was obtained by the standard
isolation process from Escherichia coli TOP10F cells using the
preparation kit QIAprep Spin Miniprep Kit (Qiagen, Hilden,
Germany), and its purity was checked by UV-Vis spectroscopy
at 230, 260, 280, and 320 nm. The native pUC19 plasmid was
solubilized in TE buffer (10 mM Tris, 1 mM EDTA, pH = 8.0,
final concentration 23.1 mM calculated as base-pairs). Supercoiled
plasmid (3–5 mL per reaction) was mixed with various concentra-
tions of the tested complexes (in different experiments, various
final concentrations of the Cu(II) complex to one base pair (bp) of
dsDNA were used, usually 1 : 1, 5 : 1, and 10 : 1 bp) in the presence
and absence of the reducing agent L-ascorbic acid in TE buffer.
Reaction mixtures were incubated at 37 ◦C for 1 h and then
analyzed by 0.8% agarose gel electrophoresis and detected with
ethidium bromide staining. The electrophoreogram was analyzed
by the software AlphaEaseFC version 4.0.0.34 (Alpha Innotech,
USA) and the relative percentages of the supercoiled circular
Cytotoxicity in vitro
Human cancer cell lines were purchased from a European Collec-
tion of Cell Cultures (ECACC). Human Caucasian osteosarcoma
cells (HOS; ECACC No.87070202) and human Caucasian breast
adenocarcinoma cells (MCF7; ECACC No.86012803) were used
in the study. Cells were cultured according to the manufacturer’s
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(CCC) form, single-strand nicked (OC) form and linear (L) form
were evaluated.
TG curves confirmed the presence of the water molecules of
crystallization in the complexes, the compounds 1 and 3 have
been determined as hemihydrates, while complexes 2 and 4–6 are
monohydrates. The thermal decomposition data are listed above
in preparation of complexes section, TG/DTA curves of 1–6 are
shown in ESI (Fig. S4†). The determined weight losses correlated
well with those calculated for the final product (CuO).
Cytological visualization of copper
The cytological visualization experiment was performed on a
culture of human acute monocyte leukemia cells (THP-1, ATCC).
The THP-1 cells were cultured (in RPMI-1640 supplemented with
10% FBS and 1% of mixture of penicillin and streptomycin) for
24 h with a 5mM DMSO solution of Cu(II) complex and the control
sample was cultivated with dimethylsulfoxide as vehicle. The
culture medium over the adherent THP-1 cells was removed from
the culture dish and the cells were washed with 6.7 mM phosphate
buffer solution (pH = 7.4). The cells were fixed for 30 min by a
70% ethanol solution at 4 ◦C. After fixation, 500 ml of rhodanine
working solution, prepared by adding 1 ml of saturated ethano-
lic solution of 5-(dimethylamino)benzylidenerhodanine (Sigma-
Aldrich) to 30 ml of distilled water, was pipetted into each culture
dish. The culture dishes were incubated for 2 h at 60 ◦C and then
the changes in colour were evaluated by light microscopy at 120¥
magnification.
X-ray structure of [Cu(qui)(phen)]NO3·H2O (2) and
[Cu(qui)(ambpy)]NO3 (3a)
The molecular structures of 2 and 3a were determined and are
shown in Fig. 2, and Fig. 3, respectively. The crystal data and
structure refinements are given in Table 1, the selected bond
lengths and angles are listed in Table 2. The geometry of the
CuN2O2 chromophore in both the complexes is distorted square-
planar. The Cu1–N2 and Cu1–N3, and Cu1–O1 and Cu1–O2
bond lengths of 2 and 3a (see Table 2) correlate well with the mean
˚
˚
value of 1.981 A (1.947–2.107 A interval; for Cu–N) and 1.923
˚
˚
A (1.869–2.182 A interval; Cu–O) determined for both discussed
bond types for eighteen Cu(II) complexes involving the square-
planar N2O2 donor set deposited in the Cambridge Structural
Database CSD to date.25 A degree of deformation in the vicinity of
the central Cu(II) atoms can be also seen from the deviations from
the least-square planes fitted through the CuN2O2 atoms, which
Interaction with human serum albumin
Deconvolution analysis of ESI-MS spectra of mixtures of Cu(II)
complex 2 (at 40 and 400 mM concentrations) with HSA (human
serum albumin, Sigma-Aldrich, at 40 mM) were carried out. The
HSA and complex 2 were both separately diluted in 2.5 mM
ammonium acetate dissolved in 2% acetic acid. The mass spectra
of the interacting systems were measured for a period of 30 min
starting with the addition of the corresponding complex to
the HSA solution, and the last spectra (in the 30th minute)
were evaluated by deconvolution analysis using the Promass for
Xcalibur, ver. 2.8, the automated ESI-deconvolution software
(Novatia LLC).
˚
were found to be (in A): Cu1 = -0.0008(3), O1 = -0.0185(2), O2 =
0.0297(2), N1 = -0.0409(2) and for N2 = 0.0613(2) for complex 2;
Cu1 = 0.0033 (3), O1 = -0.117 (3), O2 = -0.004 (3), N2 = -0.014
(3) and for N3 = -0.116 (3) for complex 3a.
Results and discussion
Characterization
The colour of the prepared crystalline products varied from
yellow-green (1–4), through greenish brown (5) to dark brown
(6). The compounds, excluding complex 5, have been found to be
very soluble in DMF and DMSO, while their solubility in ethanol,
acetone and water is very low. The solubility of complex 5 is low
even in DMF and DMSO, presumably due to the substitution on
1,10-phenanthroline, which increases its hydrophobic character
and makes the metal complexes soluble in a limited range extent
(up to 10-3 M), and moreover, the solubility of the complex
in water was even lower. Each of the complexes behaved as
an electrolyte 1 : 1 in DMF solutions, which corresponds to the
presumption of dissociation of the complexes in the solvent used
into the [Cu(qui)(L)]+ cation and nitrate anion. ESI-MS spectra
provided direct evidence about the formation of the [Cu(qui)(L)]+
complex cations, whose peaks have been observed. The MS2
spectra of the complexes afforded the same fragmentation, the
loss of the 2-phenyl-3-hydroxy-4(1H)-quinolinonate anion was
observed in all the cases with the [Cu(L)]+ peak as the product
of the fragmentation. The MS2 spectrum of complex 2, as a
representative example, is shown in ESI (Fig. S3†).
Fig. 2 Molecular structure of complex 2 together with the atom
numbering scheme. The water molecule of crystallization was omitted
for clarity. The non-H atoms are drawn as thermal ellipsoids at the 50%
probability level.
The two bidentate chelating ligands qui (excluding the phenyl
group of qui) and L are almost coplanar with the dihedral angle
being 2.65(4)◦ (for 2) and 3.34(6)◦ (for 3a). The dihedral angle
between the phenyl ring and a quinoline moiety in qui was found
to be 26.72(5)◦ (for 2) and 36.38(8)◦ (for 3a).
In the crystal structure of 2, the isolated [Cu(qui)(phen)]+
cations, nitrate anions and crystal water molecules are held
together via the N–H ◊ ◊ ◊ O and O–H ◊ ◊ ◊ O hydrogen bonds and
9406 | Dalton Trans., 2011, 40, 9404–9412
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Table 1 Crystal data and structure refinements for [Cu(qui)(phen)]NO3·H2O (2) and [Cu(qui)(ambpy)]NO3 (3a)
Compound
2
3a
Empirical formula
Formula weight
T/K
C27H20CuN4O6
560.02
100(2)
C25H19CuN5O5
532.99
100(2)
Crystal system, space group
Unit cell dimensions
Monoclinic, P21/n
Monoclinic, Cc
˚
a/A
17.5992(5)
7.1785(2)
19.0423(5)
90
111.443(3)
90
2239.19(10)
2, 1.660
1.032
14.0853(7)
22.5202(11)
6.8273(4)
90
95.373(5)
90
2156.1(2)
4, 1.642
1.065
˚
b/A
˚
c/A
a (◦)
b (◦)
g (◦)
3
˚
V/A
Z, Dc/g cm-3
Absorption coefficient (mm-1)
Crystal size/mm
0.35 ¥ 0.10 ¥ 0.10
1146
0.20 ¥ 0.10 ¥ 0.10
1092
F(000)
q range for data collection (◦)
Index ranges (h, k, l)
3.06 £ q £ 25.00
-20 £ h £ 20
-8 £ k £ 8
-22 £ l £ 22
18461/3939(0.0450)
0.9038 and 0.7140
3939/2/346
0.952
2.91 £ q £ 25.00
-13 £ h £ 16
-26 £ k £ 26
-8 £ l £ 8
9658/3083 (0.0302)
0.9010 and 0.8152
3083/2/325
1.042
Reflections collected/unique (Rint
Max. and min. transmission
Data/restraints/parameters
Goodness–of–fit on F2
)
Final R indices [I > 2s(I)]
R1 = 0.0331, wR2 = 0.0793
R1 = 0.0503, wR2 = 0.0826
0.539, -0.307
R1 = 0.0296, wR2 = 0.0750
R1 = 0.0316, wR2 = 0.0759
0.675, -0.470
R indices (all data)
-3
˚
Largest peak and hole/e A
◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for [Cu(qui)(phen)]NO3·H2O (2) and [Cu(qui)(ambpy)]NO3 (3a)
Bond Lengths
2
3a
Bond Angles
2
3a
Cu1–O1
Cu1–O2
Cu1–N2
Cu1–N3
N2–C1
N2–C5
N3–C10
N3–C6
N1–C15
N1–C16
O2–C13
O1–C14
1.892(2)
1.916(2)
1.978(2)
1.988(2)
1.334(3)
1.356(3)
1.334(3)
1.366(3)
1.372(3)
1.375(3)
1.296(3)
1.328(3)
1.918(2)
1.956(2)
1.995(3)
1.988(3)
1.360(5)
1.350(5)
1.353(4)
1.351(4)
1.354(5)
1.373(5)
1.312(5)
1.317(5)
O2–Cu1–N2
O1–Cu1–N3
O1–Cu1–O2
N3–Cu1–N2
O2–Cu1–N3
O1–Cu1–N2
C1–N2–Cu1
C5–N2–Cu1
C10–N3–Cu1
C6–N3–Cu1
C13–O2–Cu1
C14–O1–Cu1
177.87(8)
176.22(8)
86.81(7)
82.92(9)
95.84(7)
173.90(11)
172.26(11)
83.70(10)
93.03(12)
92.97(10)
90.20(12)
116.9(2)
124.5(3)
117.1(2)
125.0(2)
110.1(2)
110.6(2)
94.51(7)
128.82(17)
112.68(15)
129.73(17)
112.10(15)
109.55(14)
109.79(14)
C–H ◊ ◊ ◊ C, C–H ◊ ◊ ◊ O, N–H ◊ ◊ ◊ O, C ◊ ◊ ◊ C and C ◊ ◊ ◊ O non-covalent
contacts (for interatomic parameters of non-covalent contacts
see ESI Table S3†). The shortest Cu1 ◊ ◊ ◊ N4 distance between
Cu(II) atom and N4 atom of nitrate anion equals 5.791(3) A.
Moreover, the strong intermolecular interactions are present
between aromatic rings of phen and qui with the distance of
UV-Vis, FTIR and Raman spectra
There were three absorption maxima observed in each solid
state spectrum in the measured region. The position of the first
maximum of all the complexes varies from 404 nm (1) to 422 nm
(6) and due to its very high intensity may be assigned to the
charge-transfer (CT) transition. Next two maxima located around
˚
˚
3.338(2) A (see Fig. S5†). The O1 atom from qui makes the nearest
580 and 700 nm may be assigned to the d–d transitions B1g
→
intermolecular contact to the central copper atom at the distance
˚
Eg and B1g → B2g in quasi D4h point group symmetry of square
planar chromophore (Table 3).26 The electronic spectra were also
measured in DMF solutions, in which the CT transition was
observed in similar region close to 400 nm. On the contrary, the
pattern of the d–d transitions was significantly altered and the
only one absorption maximum was observed in the range of 590–
620 nm (emax ranging from 51 to 154 mol-1 dm3 cm-1), which might
be explained by the coordination of the solvent to the metal centre
resulting in the changing of configuration from square-planar to
of 4.38(2) A.
As for the crystal structure of 3a, the nitrate anion is situated
outside the inner coordination sphere with the shortest Cu1 ◊ ◊ ◊ N5
-
˚
distance of 6.846(3) A. The NO3 anions are also included in the
N–H ◊ ◊ ◊ O hydrogen bonds towards the N–H groups of ambpy and
qui, which results in formation of a one-dimensional polymeric
chain. Except of these hydrogen bonds, the crystal structure of
3a is stabilized by the non-covalent contacts of the C–H ◊ ◊ ◊ C,
C–H ◊ ◊ ◊ O, N–H ◊ ◊ ◊ O and C ◊ ◊ ◊ C type (see ESI Fig. S5†).
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Table 5 The spin Hamiltonian parameters derived from simulating
frozen solution EPR spectra of complexes 1–6 in DMF measured at liquid
nitrogen temperature
gxy
gz
Axy (MHz)
Az (MHz)
1
2
3
4
5
6
2.05
2.06
2.06
2.06
2.06
2.06
2.24
2.25
2.25
2.26
2.26
2.26
38
30
31
32
30
35
569
551
549
560
562
560
while other very strong bands at 493–496 could originate in n(Cu–
O), (lit.27 500–515 cm-1).
The Raman spectra measured in the 150–3750 cm-1 range
yielded practically the same information as the spectra measured
in FTIR excluding complex 3, which was not measurable due to
its burning in the laser beam.
Fig. 3 Molecular structure of complex 3a together with the atom
numbering scheme. The non-H atoms are drawn as thermal ellipsoids
at the 50% probability level.
Magnetic and EPR data
Table 3 Electronic solid state and 10-3 DMF solution spectral data for
complexes 1–6
The X-band EPR spectroscopy was performed at liquid nitrogen
temperature both for the powder samples 1–6 as well as for their
DMF frozen solutions. Based on the general pattern of the powder
spectra and obtained results, the compounds 1–6 can be divided
into two groups. As for the first group, the isotropic signals were
observed with geff = 2.10; 2.07 and 2.10 for compounds 2, 3, and 4,
respectively. The EPR spectrum of 2, as a representative example,
is shown ESI Fig. S8†. The powder spectra of the second group
Solid (CT)
Solid (d–d)
l1 (nm)
DMF (10-3 M)
l2 (nm) l (nm)
e (mol-1 dm3 cm-1)
l (nm)
1
2
3
4
5
6
404
416
405
413
415
422
583
584
595
514
603
588
692
707
760
709
709
706
601
608
607
615
620
590
84
85
51
82
83
154
(compounds 1, 5 and 6) are more complicated. Three DMS =
1
transitions were observed in the 270–380 mT region accompanied
by DMS = 2 transitions around 150 mT (for the sample spectrum
of 1 see ESI Fig. S9†). Such transitions indicate the formation of
the quasi-dimers in the solid state due to the weak intermolecular
interactions. On the contrary, the frozen solution spectra in DMF
exhibited the same pattern for all the compounds 1–6. The main
features of the experimental spectra were simulated using the
EasySpin package including the hyperfine interactions due to
nuclear spin of copper I = 3/2 with parameters listed in Table 5.28
The spectrum of 1 is shown in Fig. 4. The g-values were found
to be 2.05–2.06 and 2.14–2.26 in the perpendicular, and parallel
direction, respectively. The hyperfine splitting tensor’s components
have values of Axy = 30–35 and Az = 549–569 MHz. Also, the super-
hyperfine splitting was observed corresponding to two nitrogen
atoms of the N-donor based ligands L. To conclude, the analysis
of the EPR solution spectra comply well with elongated octahedral
copper(II) complexes, whose basal plane is formed by [Cu(qui)(L)]+
motif and the axial positions are the most likely occupied by two
O-coordinated DMF molecules.29
The magnetic measurements were performed on compounds 1
and 2 as representatives of both groups of compounds determined
by solid state EPR. The magnetic susceptibility of 1, calculated
from magnetization measured at B = 1 T, is monotonously increas-
ing on temperature lowering up to Tmax = 3.4 K and then starts
to decrease. This feature is characteristic for antiferromagnetically
coupled Cu(II) dimers or one-dimensional polymeric species. Also,
the isothermal magnetizations do not resemble the Brillouin
function for an isolated paramagnetic monomer with S = 1/2
(Fig. 5). Firstly, the susceptibility above T = 10 K was successfully
fitted to the Curie–Weiss law with g = 2.14 and H = -3.3 K (see ESI
Table 4 Selected FTIR spectral data (cm-1) for complexes 1–6
Nujol (150–600 cm-1) KBr (400–4000 cm-1)
n(Cu–O)
n(Cu–N)
n1(NO3)
n(C Car)
n(C O)
1
2
3
4
5
6
494
493
495
496
496
494
544
542
543
543
546
545
1309
1317
1325
1317
1327
1318
1562
1563
1568
1566
1564
1562
1627
1629
1631
1629
1631
1628
a distorted octahedral (see also EPR spectroscopy discussion for
more details).
The FTIR spectra measured in the 150–4000 cm-1 region
indirectly confirmed the coordination of heterocyclic ligands to
the Cu(II) atom (Table 4). The characteristic band of the carbonyl
groups of the coordinated qui appears at 1628–1631 cm-1, which
surprisingly does not differ from those of free Hqui found
at 1631 cm-1. The characteristic vibration of n(CCar) in the
aromatic system present in the uncoordinated bidentate N-donor
heterocycles (L) appeared in the range of 1562–1568 cm-1. The
bands of a very strong intensity in the region of 1450–1560 cm-1
are assignable to the vibrations of n(CCar) of qui. The very strong
bands of the uncoordinated nitrate group n1(NO3) were observed
in the region of 1309–1327 cm-1. FTIR spectra of the complexes
showed new bands in comparison to those of free organic ligands
in the region between 150 and 600 cm-1. The very strong bands at
542–546 cm-1 might be assigned to n(Cu–N), (lit.27 530–560 cm-1),
9408 | Dalton Trans., 2011, 40, 9404–9412
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Fig. 6 The magnetic data for complex 2 scaled per one Cu(II). Left:
the temperature dependence of the effective magnetic moment and molar
magnetization measured at B = 1 T. Right: the isothermal magnetizations
measured at T = 2.0 and 4.6 K. Empty circles – experimental data, full
lines – the best fit of experimental data using eqn (3), with J = -0.23 cm-1
and g = 2.13.
Fig. 4 The frozen solution X-band EPR spectrum of complex 1 in
DMF at liquid nitrogen temperature. The experimental data (full line)
and calculated data (dashed line) with the parameters given in Table 5.
state through the intermolecular interactions as already supported
by solid state EPR.
The magnetic data of compound 2 are different (Fig. 6). In
this case, the molar susceptibility is monotonically increasing on
cooling and no maximum is observed. The inverse susceptibility
was fitted successfully in the whole temperature range to the Curie–
Weiss law with g = 2.13 and H = -0.1 K (see ESI Fig. S11†). The
small and negative value of the Weiss constant is in agreement
with decreasing of the effective magnetic moment below 12 K
from 1.84 mB to 1.72 mB at T = 2 K. Firstly, we tried to simulate the
magnetic behaviour with a monomeric model including molecular
field correction zj, but it was unsuccessful. By inspecting the
X-ray crystal structure of 2, we can see forming of the quasi
one-dimensional chain through p–p stacking (see ESI Fig. S5†).
That is why, the susceptibility was fitted to spin Hamiltonian for
antiferromagnetically coupled uniform 1D chain with Si = 1/2
using the Johnston et al. equation31 with J = -0.30 cm-1 and
g = 2.13 (see ESI Fig. S12†). In order to fit also the isothermal
magnetization data and to confirm our presumption, the magnetic
data were fitted to finite-size closed ring model according to spin
Hamiltonian
Fig. 5 The magnetic data for complex 1 scaled per one Cu(II). Left:
the temperature dependence of the effective magnetic moment and molar
magnetization measured at B = 1 T. Right: the isothermal magnetizations
measured at T = 2.0 and 4.6 K. Empty circles – experimental data, full
lines – the best fit of experimental data using eqn (1), with J = -3.9 cm-1
and g = 2.10.
16
17
⎧
⎫
⎪
⎪
⎪
⎪
⎪
⎪
ˆ
(3)
H = −J
Si ⋅Si+1 + S ⋅S + m
B⋅gi ⋅Si
(
)
⎬
⎪
⎨
⎪
⎪
Fig. S10†). The negative value of the Weiss constant points out
to the presence of antiferromagnetic interactions. Magnetic data
for 1 were successfully fitted to the following spin Hamiltonian for
dimer
17
1
B
∑
∑
⎪
⎪
⎭
i=1
i=1
⎪
⎩
where number of 17 centres is sufficient to mimic infinite chain
properties for such small expected antiferromagnetic exchange as
was already proved in lit.32 The fitting procedure resulted in J =
-0.23 cm-1 and g = 2.13 (Fig. 6).
2
ˆ
(1)
H = −J(S1⋅S2) + mB
B⋅gi ⋅Si
∑
i=1
where J-parameter represents the isotropic exchange between two
local spins SA = SB = 1/2. The simple formula for the molar
magnetization of such dimer was used
In vitro cytotoxicity
All the prepared Cu(II) complexes have been tested for their in
vitro cytotoxicity against human osteosarcoma (HOS) and human
breast adenocarcinoma (MCF7) cancer cell lines. Results are
expressed as the IC50 values and are summarized in Table 6.
The tested complexes achieved the dose-dependent cytotoxicity
in both cancer cell lines. The in vitro cytotoxicity of these
compounds was in almost all cases considerably higher than
Mmol = mBgNA[exp((J + x)/kT) - exp((J - x)/kT)]/[1 +
(2)
exp((J + x)/kT) + exp(J/kT) + exp((J - x)/kT)]
where x = mBgB and the fitting procedure resulted in J = -3.9 cm-1
and g = 2.10 (Fig. 4).30 Such relatively small antiferromagnetic
exchange can be understood by forming quasi-dimers in the solid
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Table 6 In vitro cytotoxic activity data together with their standard
deviations of complexes 1–6 and cisplatin against the HOS and MCF7
human cell lines, expressed as the IC50 values
complexes involving low-molecular weight ligands derived from
phenanthroline have been identified in many publications as
prominent minor-groove binding agents.34,35 Furthermore, cells
undergoing oxidative stress, which is speculated to be the cause or
manifestation of more than 100 pathological processes, including
some types of cancer and different inflammatory processes, have
been shown to undergo oxidative DNA cleavage when exposed to
transition metal complexes that bind in the minor groove and are
able to participate in the Fenton’s reaction.36–38
HOSa
MCF7b
1
2
3
4
21.4 5.2
4.3 0.1
22.0 5.1
3.7 0.1
> 1.0
27.2 0.8
7.3 2.3
18.9 4.1
3.9 0.4
> 1.0
5
6
2.1 0.2
37.7 5.4
2.2 0.4
24.5 6.1
cisplatin
DNA cleavage
a human osteosarcoma cell line, b human breast adenocarcinoma cell line
DNA cleavage experiments were performed on a model system
involving a mixture of circular plasmid pUC19 and the Cu(II)
complexes in different concentrations in a state of oxidative
stress (i.e. Fenton’s reaction with hydrogen peroxide either in
the presence or absence of a reducing agent, ascorbic acid).
The products and intermediates of the reaction (the reactive
species CuO+ and Cu(OH)2+ or the highly reactive hydroxyl
radical)39–41 cause single-strand breaks of the supercoiled double-
strand DNA (CCC-form), which are manifested by the formation
of open circular forms (OC-forms), or alternatively, double-strand
breaks, which are evident from the formation of linear forms
(L-forms). The identification and distribution of different forms
were determined by agarose gel electrophoresis. Additionally, the
above mentioned reactive species can cleave the DNA into smaller
fragments (shown in electrophoreogram as a smear) or even yield
complete DNA cleavage.
those of the commercially used antineoplastic platinum-based
drug cisplatin (HOS IC50 = 37.7 5.4, MCF7 24.5 6.1 mM)
in both human cancer cell lines, and moreover, complex 6 was
found to be the most potent one (HOS 2.1 0.2 mM; MCF7
2.2 0.4 mM). The cytotoxicity of compound 5 in all the tested
cell types could not be assessed due to its low solubility in an
aqueous milieu.
Interaction with CT-DNA assessed by UV-Vis titration
In order to determine a possible mechanism of DNA-copper(II)
complex interaction, the spectrophotometric titration of the
complexes with highly polymerized CT-DNA was performed.
There exist three major modes for binding of metal complexes
to DNA: (a) intercalation inside a groove (stabilized by the
p–p interactions between the aromatic systems of ligands and
nucleobases), (b) binding outside a groove and (c) binding outside
the helix (the weakest, mostly electrostatic interaction).33 Effective
intercalation interactions exhibit themselves in UV-Vis spectra
through charge-transfer bands by significant hypochromicity and
red-shifting of the band maxima (bathochromic effect). The high-
est hypochromicity was found for complexes 1 and 2 (Table 7, for
UV-Vis spectra of the compounds see ESI Fig. S13†), suggesting
that these compounds intercalate with DNA more than the others.
On the other hand, the complexes 4 and 5, involving more
hydrophobic ligands 5-methyl-1,10-phenanthroline, and 5-nitro-
1,10-phenanthroline, respectively, provided a significantly lower
hypochromic effect, which could mean that although these com-
plexes form non-covalent interaction with DNA, the intercalation
is just partial, i.e. the steric hindrance of their substituents
prevent them from penetrating further into the minor groove
of DNA and p–p interactions between the aromatic systems
of ligands and nucleobases are much less effective. Copper(II)
Reaction without the reducing agent. In the absence of a reduc-
ing agent, Fenton’s reaction proceeds very slowly. Iron(II) sulphate,
used as a reference compound, showed low reactivity with the
model circular plasmid molecule. At a molar ratio of FeSO4:1bp
(pair of bases in DNA) of 10 : 1 (200 mM concentration), the
average DNA cleavage was only 19.1 1.92% (for a detailed report,
see ESI Table S4†). Analogous results were obtained for complexes
1 and 3 (see ESI Table S5 and S6†). On the other hand, complexes
2, 4, 5, and 6 cleaved DNA with significantly higher effectiveness
at lower concentrations than the reference compound FeSO4 (see
ESI Table S7–S10†), suggesting that these complexes either employ
stronger interaction with DNA or create more efficient production
system in the Fenton’s reaction yielding oxidatively damaged
polynucleotide chain, especiallyinthe C1¢, C4¢ andC5¢ positions of
the deoxyribose residue, leading to subsequent fragmentation.42,43
Reaction with the reducing agent. The presence of a reducing
agent in high excess in the reaction system strongly increases DNA
Table 7 Spectral shift analysis for complexes 1–6 during DNA titration, showing the maxima of charge-transfer bands at the given ratios of CT-DNA
vs. complex
lmax CT-band (unbound form; nm)
Hypochromicity maximum at Ra (%)
Blue(-) or red(+) shift (nm)
1
2
3
4
5
6
382
356; 400
408
394
390
29 (0.71)
17.1 (0.10)
n/a
4.7 (0.53)
3.9 (2.41)
n/a
-8
+2
+2
0
+8
0
422
a R stands for a ratio of CT-DNA vs. complex concentrations (the concentration of DNA is converted to the molar concentrations of its single bases); the
R values are given in parentheses; n/a = not detected
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cleavage efficiency. The increase in efficiency is probably due to the
formation of a cyclic production system, in which ascorbic acid
reduces Cu(II) to Cu(I) and then copper is re-oxidized by Fenton’s
reagent, with hydroxyl radical formation. The whole cycle can thus
run until complete depletion of the reducing agent or stabilization
of the copper oxidation state, e.g. by coordination with albumin.44
The results of DNA cleavage studies have been found to be in
good correlation with in vitro antitumour activity of the tested
Cu(II) complexes. This may indicate that the interaction with
DNA and mainly the ability of copper(II) complexes to act as
chemical nucleases are important in the mechanism of their action
in biological systems.
The cells with increased levels of intracellular copper were stained
red-brown. The intensity of red-brown colour of cells cultured with
complex 2 (see ESI Fig. S15†) compared to the control sample
cultured with vehicle (see ESI Fig. S16†), leads to the conclusion
that complex 2 penetrates into living cells of THP-1.
Interaction with human serum albumin
ESI-MS analysis of a mixture of complex 2 with HSA was
performed in order to determine whether the complexes are able
to interact with biomolecules.46
Comparison of deconvoluted MS spectra of pure HSA with the
mixtures of complex 2 vs. HSA in molar ratios 1 : 1 and 10 : 1
revealed that the interaction of complex 2 with the HSA was
observed only in the 10 : 1 case. The interaction is probably caused
by the ligand exchange of qui for the appropriate binding site at
HSA. At these conditions, we identified that the coordination
species Cu(phen)2+ bind to the protein in a 1 : 1 ratio. The
presence of the complex [protein–Cu(phen)(H2O)] was confirmed
by the appearance of a deconvoluted peak at 66 123 Da, which
represents a 258 Da (see ESI Fig. S17†) difference from the mean
deconvoluted mass of intact HSA (65 865 Da) (see ESI Fig. S18†),
corresponding to the species of [Cu(phen)(H2O)]2+.
The stability of the complex 2 during the ESI-MS analysis was
monitored as well. In each experimental step in which complex
2 was involved, we were able to observe the specific masses
characteristic for the intact complex (479.16 corresponding to (M–
NO3)+ and 302.08 corresponding to Cu(phen)(acetate)+) in the
range of 80–600 m/z. This observation confirms that even in the
presence of a strong chelating agent, complex 2 is stable enough to
be able to provide a pharmacologically active coordination species
with the ability to bind to the structure of a transport protein such
as HSA.
Correlation between cytotoxicity and DNA cleavage
In addition to DNA interaction and DNA cleavage studies, we
strived to find the correlation between the in vitro cytotoxicity
and nuclease activity. We found very good correlation between
these two properties, which can be expressed in the form of
the correlation coefficients, with R = 0.999 (for the correlation
between the cytotoxicity to the HOS cell line vs. the percentage of
DNA cleavage at 20 mM in the experiment without the addition
of ascorbate, as seen in Fig. 7), R = 0.986 (for the correlation
between the cytotoxicity to the HOS cell line vs. the percentage
of DNA cleavage at 20 mM in the experiment with the addition
of ascorbate), R = 0.922 (for the MCF-7 cell line without the
addition of ascorbate), and R = 0.930 (for the MCF-7 cell line
with the addition of ascorbate as seen in ESI Fig. S14†). Based on
these findings, we may conclude that there exists a relationship
between the in vitro cytotoxicity and nuclease activity of the
studied complexes. Moreover, it could serve as an effective tool
for the prediction and evaluation of in vitro cytotoxicity of newly
prepared compounds of similar composition and stereochemistry
after the model extension and validation in future.
Conclusions
We prepared and fully characterized a series of mixed-ligand Cu(II)
complexes with the 2-phenyl-3-hydroxy-4(1H)-quinolinonate an-
ion (qui) of the formulas [Cu(qui)(bpy)]NO3·1/2H2O (1),
[Cu(qui)(phen)]NO3·H2O (2), [Cu(qui)(ambpy)]NO3·1/2H2O (3),
[Cu(qui)(mphen)]NO3·H2O (4), [Cu(qui)(nphen)]NO3·H2O (5) or
[Cu(qui)(bphen)]NO3·H2O (6). The X-ray molecular structures of
the complexes 2 and 3a exhibited Cu(II) in the distorted square-
planar geometry.
The results of in vitro cytotoxicity obtained for complexes 1–
4 and 6 against the HOS and MCF7 human cancer cell lines
are significantly better than those of cisplatin, a commercially
used antineoplastic drug, for the same cancer cell lines. Relatively
strong intercalating ability of the complexes to DNA showed the
possible mechanism of the cytotoxic effect of the prepared Cu(II)
complexes against the above-mentioned human cancer cell lines.
It can be seen from the obtained results that we not only prepared
and characterized novel type of Cu(II) complexes involving the
2-phenyl-3-hydroxy-4(1H)-quinolinonate anion but also found
that the obtained results regarding anticancer activity are very
promising and thus, the biological properties of such systems
should be studied in greater details in future. Based on these
findings, we may also conclude that there exists a good correlation
between the in vitro cytotoxicity and nuclease activity, which could
Fig. 7 The correlation between the cytotoxicity to HOS cell line vs. the
percentage of DNA cleavage at 20 mM in experiment without the addition
of ascorbate for complexes 1–4.
Cytological visualization of copper
In order to confirm the ability of complexes 1–6 to penetrate living
cells, a method called as a rhodanine method routinely used for
cytological diagnostic of the Wilson’s disease was carried out.45
This method is based on the formation of brown-red coloured
copper(II) complex with 5-(dimethylamino)benzylidenerhodanine.
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serve (after the model extension and validation) as an effective tool
for the prediction of cytotoxicity of newly prepared compounds
of similar composition and stereochemistry in future.
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Azuara, J. Inorg. Biochem., 2009, 103, 299.
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The authors gratefully thank the Ministry of Education, Youth
and Sports of the Czech Republic (MSM6198959218), the Opera-
tional Program Research and Development for Innovations - Eu-
ropean Regional Development Fund (CZ.1.05/2.1.00/03.0058),
the Operational Program Education for Competitiveness - Eu-
ropean Social Fund (project CZ.1.07/2.3.00/20.0017) of the
Ministry of Education, Youth and Sports of the Czech Republic,
and GACR P207/11/0841 for financial support, Dr Pavel Starha
for performing TG/DTA experiments, Ms. Radka Novotna´ for
measuring infrared and Raman spectra, Ms. Alena Klanicova´ for
mass spectra measurements, Dr Jan Hosˇek for technical assistance
with cytological visualization of intracellular copper, and Dr Peter
Baran for useful discussions.
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9412 | Dalton Trans., 2011, 40, 9404–9412
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©