New ternary bipyridine-terpyridine copper(ii) complexes as self-activating chemical nucleases

Article abstract of DOI:10.1039/c4ra12085j

Seeking self-activating chemical nucleases with potential applications as therapeutic agents, new ternary terpyridine-bipyridine-Cu(ii) complexes carrying pendant cyclic amines were developed. After detailed characterization, the nuclease activity of the synthesized compounds was evaluated by using circular plasmid DNA as substrate and analyzing the products by agarose-gel electrophoresis. The new complexes present an impressive plasmid DNA cleaving ability, which triggers double-strand DNA breaks in the absence of any exogenous agents, via an oxidative mechanism. The binding affinity towards duplex DNA was determined using UV-Vis and fluorescence spectroscopic titrations. These studies showed that the tested complexes bind moderately (in the order of 10⁴ M^-1) to duplex DNA. The copper complexes displayed high cytotoxicity against ovarian carcinoma A2780 cells (4-fold cisplatin activity), surpassing the resistance on the cisplatin-resistant cell line (A2780cisR) with lower resistance factors. Cellular uptake studies showed that the ternary complexes were able to enter the cell with a significant localization in the cytoskeleton. This journal is

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New ternary bipyridine–terpyridine copper(II)
complexes as self-activating chemical nucleases†
Cite this: RSC Adv., 2014, 4, 61363
a
a
a
b
b
Sofia Gama,* Ines Rodrigues, Fernanda Marques, Elisa Palma, Isabel Correia,
ˆ
b
b
c
c
M. Fernanda N. N. Carvalho, Joao Costa Pessoa, Andreia Cruz, Sonia Mendo,
Isabel C. Santos, Filipa Mendes, Isabel Santos and Antonio Paulo
˜
´
a
a
a
a
´
Seeking self-activating chemical nucleases with potential applications as therapeutic agents, new ternary
terpyridine–bipyridine–Cu(II) complexes carrying pendant cyclic amines were developed. After detailed
characterization, the nuclease activity of the synthesized compounds was evaluated by using circular
plasmid DNA as substrate and analyzing the products by agarose-gel electrophoresis. The new
complexes present an impressive plasmid DNA cleaving ability, which triggers double-strand DNA breaks
in the absence of any exogenous agents, via an oxidative mechanism. The binding affinity towards
duplex DNA was determined using UV-Vis and fluorescence spectroscopic titrations. These studies
4
ꢀ1
showed that the tested complexes bind moderately (in the order of 10
M
) to duplex DNA. The copper
Received 9th October 2014
Accepted 4th November 2014
complexes displayed high cytotoxicity against ovarian carcinoma A2780 cells (4-fold cisplatin activity),
surpassing the resistance on the cisplatin-resistant cell line (A2780cisR) with lower resistance factors.
Cellular uptake studies showed that the ternary complexes were able to enter the cell with a significant
localization in the cytoskeleton.
DOI: 10.1039/c4ra12085j
www.rsc.org/advances
host cell DNA aer viral infection, DNA repair, DNA recombi-
nation, DNA synthesis, DNA packaging in chromosomes and
1
Introduction
There has been considerable interest in recent years in the viral compartments and maturation of RNAs or RNA splicing.
development of DNA cleaving reagents due to their potential
Unsurprisingly, one of the challenges that has engaged many
1
application in biotechnology and medicine. DNA damage by chemists over the last years is the development of the so-called
exogenous molecules, mostly via oxidative or hydrolytic mech- “articial nucleases”. These are, or should be, synthetic systems
anisms, is an important process in cellular systems, because the able to reproduce the activity of hydrolytic enzymes that cleave
3
resulting lesions may have a signicant inuence on the phosphate esters. Chemists have been searching for low-
physiological/biological function of DNA. Importantly, in the molecular weight synthetic mimics of such enzymes, not only
absence of an efficient repair mechanism, DNA damage may to help to improve the fundamental understanding of mecha-
2
lead to mutagenesis, carcinogenesis, cell death or aging.
nistic aspects of enzyme action, but also to develop new
DNA cleavage induced by metal complexes recently attracted biotechnological tools (articial restriction enzymes and foot-
2
much attention, since the modes of action of these reagents printing agents) and nucleic acid-targeting therapeutics. The
2
resemble the ones employed by naturally occurring nucleases.
last few years have seen substantial progress as new approaches
Nucleases are ubiquitous enzymes indispensable for cellular have been described and the rst applications of articial
3
and viral development, acting at the DNA- (DNAses) and the hydrolytic agents to DNA manipulation have been reported.
RNA-level (RNAses). They are known to be involved on the
The knowledge that the active site of most nucleases contain
2
+
2+
2+
2+
control of several biological processes, namely protective divalent cations, such as Mg , Ca , Mn or Zn , prompted a
mechanisms against “foreign” (invading) DNA, degradation of massive amount of research aimed at discovering new metal
complexes capable of mimicking the hydrolytic activities of
metallo-nuclease enzymes. The diverse structural features and
a
2
Centro de Ci ˆe ncias e Tecnologias Nucleares (C TN), Instituto Superior T ´e cnico,
Universidade de Lisboa, Campus Tecnol ´o gico e Nuclear, Estrada Nacional 10 (km
versatility shown by transition metal-based compounds, such as
multiple/variable oxidation states and redox properties make
them good candidates to be exploited to discover novel articial
nucleases. Among the rst row transition elements, copper has
1
b
39,7), 2695-066, Bobadela LRS, Portugal. E-mail: scgama@ctn.ist.utl.pt
Centro de Qu ´ı mica Estrutural, Instituto Superior T ´e cnico, Universidade de Lisboa,
Avenida Rovisco Pais 1, 1049-001 Lisboa, Portugal
c
Departamento de Biologia & CESAM, Universidade de Aveiro, Campus de Santiago, elicited a special interest since the discovery of rst chemical
4
3
810-193 Aveiro, Portugal
nuclease by Sigman et al. Together with the possibility of
†
CCDC 1017524–1017525. For crystallographic data in CIF or other electronic
damaging DNA by hydrolytic and/or oxidative mechanisms,
format see DOI: 10.1039/c4ra12085j
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copper complexes may additionally induce efficient cleavage of
DNA due to their high nucleobase affinity and the relatively
strong Lewis acidity of Cu(II) ions.
For most copper complexes exhibiting strong DNA cleavage
2
Results and discussion
1,2
2.1. Synthesis, characterization, spectral and redox
properties
activity, the activity is mediated by reactive oxygen species (ROS) The bipyridine compounds were synthesized by reaction of
produced via the oxidation of Cu(II) to Cu(III) by the addition of chloro alkyl derivatives of the corresponding cyclic amine with
0
0
external oxidizing agents (e.g. dihydrogen peroxide, molecular the 2,2 -bipyridine-4,4 -diol precursor, in a 2 : 1 ratio, as depic-
oxygen) or the reduction of Cu(II) to Cu(I) by a reductive reagent ted in Scheme 1. The diol precursor was obtained by cleavage
5
,6
0
0
(
e.g. ascorbic acid, 3-mercaptopropionic acid). Transient- via hydrobromic acid of 4,4 -dimethoxy-2,2 -bipyridine, as
2
+
+
21
metal-bound species formulated as [CuOH] , [CuOOH] , and described in the literature.
CuO] have also been reported as responsible for DNA strand
scission. Additionally, copper complexes with appropriate pendant cyclic amines could be obtained in one step by reacting
+
2
4
[
The ternary Bpy–Tpy–Cu(II) complexes (C –C ) containing
5
ligand(s), can cleave DNA without any external agent, but oen stoichiometric amounts of the desired bipyridine derivative and
6
photo-induction is needed to initiate the cleavage process. For terpyridine with copper(II) triate, in acetonitrile at room
the above described reasons, the in vivo application of these temperature (Scheme 2). This approach was reported previously
1
reagents is limited since the addition of an oxidizing or for the synthesis of the parental complex C , which contains a
reducing agent or the use of photoactivation are not readily pentacoordinated metal atom bound to one tridentate terpyr-
22
1
feasible and attractive. Therefore, it would be of great interest to idine and one bidentate bipyridine ligand. Compound C was
nd self-activated systems not requiring any type of activation also synthesized in this work aiming to compare its nuclease
to generate reactive species for DNA cleavage activity.
activity with that exhibited by the congeners containing
Only a few reports on nuclease activity via self-activation pendant cyclic amines. The formation of complexes C –C was

1
4
5
–17
have been published
but recently copper complexes, pos- almost immediate as indicated by the sudden appearance of a
sessing inherently diverse structural and redox properties, blue color upon addition of ligands. The complexes precipitated
emerged as attractive self-activating agents for DNA from each reaction mixture as microcrystalline blue solids, aer
1,5,6,12–16,18
cleavage.
counterion exchange with hexauorophosphate and addition of
In the past few years, several copper complexes have been diethyl ether. Further purication was achieved by recrystalli-
proposed as potential anticancer metallodrugs, demonstrating zation, through diffusion of diethyl ether into acetonitrile
remarkable anticancer activity and showing toxicity generally solutions of the complexes.
19
2
4
lower than platinum compounds. In particular, mixed ligand
The formulation of compounds C –C as ternary bipyridine–
copper(II) complexes were found to exhibit prominent anti- terpyridine–copper(II) complexes was established on the basis of
cancer activity by inducing apoptosis, strongly binding and their characterization by elemental analysis, MALDI-TOF mass
20
cleaving DNA. Inspired by these ndings, we developed a new spectrometry, EPR and single crystal X-ray diffraction analysis
3
4
family of ternary bipyridine–terpyridine–copper(II) complexes (for C and C ). In particular, the MALDI-TOF spectra of all the
Bpy–Tpy–Cu(II)] containing pendant cyclic amine groups. The complexes showed peaks with m/z values corresponding to the
[
+
cyclic amines introduced at the pyridine ligands were expected respective [M ] molecular ion and displaying isotopic distribu-
to enhance the DNA binding affinity of the complexes by elec- tion patterns consistent with the presence of copper.
3
4
trostatic interactions between the protonated amines and the
X-ray quality crystals of C and C were obtained by recrys-
phosphodiester backbone of the nucleic acid. Herein, we report tallization from saturated solutions of the compounds in
3
on new bipyridine derivatives functionalized with different acetonitrile. Complex C crystallized in the triclinic system,
4
cyclic amine groups (pyrrolidine, piperazine and morpholine) space group P 1ꢀ , and C crystallized in the monoclinic system,
and on their ternary Bpy–Tpy–Cu(II) complexes. The action of space group P2 /n. The crystal data and nal renement details
1
3
4
these ternary complexes as self-activating DNA nucleases was for complexes C and C are given in Table 1. A selection of bond
evaluated and, additionally, their biological behavior in human lengths and angles are given in Table 2. The respective ORTEP
cancer cell lines was correlated to their biophysical properties diagrams are presented in Fig. 1.
and cellular uptake.
Scheme 1
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Scheme 2
3
4
3
The X-ray structural analysis of C and C conrmed that the coordinated bipyridine nitrogen atoms in the complexes C and
4
4
central Cu(II) metal ion in each of these complexes is penta- C , respectively. In the complex C , the atoms of both mor-
coordinated by tridentate terpyridine (tpy) and bidentate 2,2 - pholinyl containing arms (C26C27–C26aC27a and O2C32C33–
0
bipyridine (bpy) ligands. The coordination polyhedron is best O2aC32aC33a) are disordered over two sites (0.57–0.43 and
described as a distorted square-pyramidal with an N atom from 0.45–0.55) respectively.
3
4
the bipyridine ligand in the apical position. The angle between
In the crystal packing of C and C , hexauorophosphate
the basal plane dened by N1 N2 N3 N5 atoms and the Cu1–N4 counter-ions are involved as acceptors in several intermolecular
ꢁ
apical bond is 72.7(2) in both compounds; the Cu1–N4 apical C–H/F hydrogen-bonding interactions with both the bipyridyl
˚
˚
bond (2.160(4) A and 2.179(5) A) is longer than the Cu1–N5 and terpyridyl ligands, resulting in a three-dimensional supra-
˚
˚
equatorial bond distance (1.976(5) A and 1.986(4) A) for the molecular structure (Fig. 2). The same extensive network of
hydrogen bonds was found by Ulrich S. Schubert in the solid
1
22
state structure of the parental complex C . Dai-Zhi Kuang
23
et al. have also reported on related Cu(II) complexes containing
Table 1 Crystallographic data and structure refinement parameters
for compounds C and C
0
0
0
00
a tridentate 4 -ferrocenyl-2,2 :6 ,2 -terpyridine and a bidentate
3
4
0
2
,2 -bipyridine ligand, which crystallizes with formation of
3
4
similar three-dimensional supramolecular structures.
C
C
1
4
The EPR spectra of complexes C –C measured from DMSO
Crystal size (mm)
Crystal colour
and shape
0.60 ꢂ 0.30 ꢂ 0.04
0.18 ꢂ 0.10 ꢂ 0.03
solutions frozen at 77 K (Fig. 3) show axial symmetry with a
Green, plate
Blue, plate
more intense absorption at higher eld (g
one at lower eld (g ). The spin Hamiltonian parameters g
depend, among other factors, on the nature of the donor
t
) and a less intense
k
k
and
Temperature (K)
Empirical formula
Molecular mass
Crystal system
Space group
150(2)
150(2)
C H N O F P Cu
37 40 7 4 12 2
A
k
C
41
H
48
N
8
O
2
F
24
P
4
Cu
1328.29
Triclinic
P1ꢀ
8.2891(7)
17.7944(8)
19.6703(10)
78.996(2)
85.893(2)
89.038(2)
2840.7(3)
2, 1.553
0.618
1342
3.40 to 25.03
ꢀ5/9, ꢀ21/21, ꢀ23/23
1000.24
Monoclinic
P2 /n
atoms, and can be used to conrm the binding mode around
the metal ion. The simulation of the experimental spectra
resulted in good spectral t and reliable spin Hamiltonian
parameters that are in agreement with those published for
1
˚
a (A)
20.9700(7)
8.7014(4)
28.5460(10)
90
91.070(2)
90
5207.8(3)
4, 1.276
0.563
˚
b (A)
24,25
other terpyridine or bipyridine Cu(II) complexes.
The values
˚
c (A)
ꢁ
obtained for the g and A (Table 3) are in agreement with the
a ( )
k
k
ꢁ
26,27
b ( )
expected for coordination of copper to nitrogen atoms only.
No superhyperne coupling with the nitrogen donors could be
observed, due to the low bandwidth resolution, which is
common in solvents with high viscosity such as DMSO.
As the nuclease effect induced by most of the known met-
allonucleases oen proceeds via redox cycles between different
oxidation states of the metal ions, the redox behavior of the
ꢁ
g ( )
3
˚
V (A )
ꢀ3
Z, Dcalcd (Mg m
)
ꢀ1
m (mm
F(000)
)
2040
ꢁ
Theta range ( )
2.92 to 25.03
ꢀ24/24, ꢀ10/10,
33/33
Index range (h, k, l)
ꢀ
1
4
copper complexes C –C was studied by cyclic voltammetry.
Re. collected/unique
17 125/9746
32 880/9061
[Rint ¼ 0.0731]
0.9458/0.8673
9061/19/541
0.958
2
3
Representative cyclic voltammograms for C and C are dis-
[
R
int ¼ 0.04]
T max./min.
0.9757/0.7080
9746/0/722
1.010
played in Fig. 4. Three distinct regions are observed for all
1
Data/restr/param
complexes, except C . The rst region (I) displays cathodic
2
G.O.F. on F
waves at relatively accessible reduction potentials (ꢀ0.20 to
R [I > 2s(I)]
R1 ¼ 0.0827,
wR2 ¼ 0.2160
1.29/ꢀ0.842
R1 ¼ 0.0996,
wR2 ¼ 0.2543
1.074/ꢀ0.582
I red
ꢀ
0.23 V) with quasi-reversible character ( E ), which are
1/2
ꢀ3
preceded by shoulder waves at slightly higher potential; the
Dr max/min [eA
]
II red
second region (II) shows irreversible waves ( E
p
) in the range
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Table 2 Selected bond lengths ( A˚ ) and angles ( ) for complex C and C
Paper
ꢁ
3
4
Bond lengths ( A˚ )
Bond angles ( )
ꢁ
3
4
3
4
C
C
C
C
Cu1–N1
Cu1–N2
Cu1–N3
Cu1–N4
Cu1–N5
2.047(5)
1.915(4)
2.065(5)
2.160(4)
1.976(5)
Cu1–N1
Cu1–N2
Cu1–N3
Cu1–N4
Cu1–N5
2.064(6)
1.908(5)
2.040(5)
2.179(5)
1.986(4)
N1–Cu1–N2
N1–Cu1–N3
N1–Cu1–N4
N1–Cu1–N5
N2–Cu1–N3
N2–Cu1–N4
N2–Cu1–N5
N3–Cu1–N4
N3–Cu1–N5
N4–Cu1–N5
80.44(19)
158.41(19)
106.68(18)
98.7(2)
N1–Cu1–N2
N1–Cu1–N3
N1–Cu1–N4
N1–Cu1–N5
N2–Cu1–N3
N2–Cu1–N4
N2–Cu1–N5
N3–Cu1–N4
N3–Cu1–N5
N4–Cu1–N5
80.8(2)
159.3(2)
95.51(19)
100.1(2)
79.3(2)
115.2(2)
166.5(2)
98.2(2)
80.4(2)
110.06(18)
170.13(19)
89.15(19)
98.5(2)
98.5(2)
78.30(19)
79.66(18)
3
4
Fig. 1 ORTEP diagram of C and C . Ellipsoids were drawn at the 40%
probability level.
ꢀ
1.09 to ꢀ1.16 V, which are associated with strong adsorption
4
waves on the reverse scan (except for C ); nally, a third region
1
(
III, not observed for C ) at potentials (ꢀ2.22 and ꢀ2.30 V) close
to the limit of the potential window (Table 4) is registered.
Region III is omitted for clarity in the cyclic voltammograms of
C and C presented on Fig. 4. These lowest potential reduction
2
3
III red
waves can be attributed to reduction of the ligands ( Ep ),
Fig. 3 EPR X-band spectra recorded at 77 K for 5 mM solutions of
1
4
C –C in DMSO.
based on the study of the redox properties of the free ligands
that has been performed under identical experimental condi-
tions (Table 4).
Overall, the electrochemical data obtained for complexes
1
4
C –C are consistent with Cu(II) / Cu(I) reductions (region I)
II red
followed by Cu(I) / Cu(0) reduction (region II)
E
. The
p
I
red
measured Cu(II)/Cu(I) reduction potentials E
compare well
/2
1
30,31
with those previously reported for related complexes.
Upon
Cu(I) / Cu(0) reduction, the observed adsorption wave gives
evidence for decomposition of the complexes with formation of
copper metal (Scheme 3).
1
4
In summary, the cyclic voltammetry studies of C –C have
shown that these Cu(II) complexes have very similar redox
potentials, despite displaying slight differences in their elec-
trochemical behavior due probably to the irreversible nature
4
Fig. 2 The crystal structure of C . View along the stacking axis a observed for some processes. These results can be explained by
emphasizing the short contacts.
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a
Table
3 Spin Hamiltonian parameters for the Cu(II) complexes Table 4 Cyclic voltammetry data obtained for the ligands and Cu(II)
28
obtained by simulation of the experimental spectra
complexes
I
red
p
II red
III red
ox
EPR parameters
E
E
p
E
p
E
p
4
ꢀ1
4
ꢀ1
b
g
k
g
t
A
k
ꢂ 10 [cm
]
A
t
ꢂ 10 [cm
]
Terpyridine
Bipyridine
—
—
—
—
—
—
—
—
—
ꢀ1.09
ꢀ1.18
ꢀ1.22
ꢀ1.16
—
ꢀ2.21
No
No
0.98
1.10
1.24
—
—
—
—
—
No
1
2
3
4
2
C
C
C
C
2.255
2.255
2.255
2.255
2.066
2.063
2.063
2.062
156.5
155.4
156.0
156.4
11.9
12.1
11.6
11.9
L
L
L
C
C
C
C
ꢀ2.33
ꢀ2.32
<ꢀ2.5
No
ꢀ2.26
ꢀ2.30
ꢀ2.22
—
3
4
—
1
c
e
e
f
d
ꢀ0.23
ꢀ0.19
ꢀ0.26
ꢀ0.20
2
3
4
2+
g
[
Cu(bpy)
2
]
ꢀ0.169
the structural similarity of the studied complexes, that maintain
the same coordination environment in solution, as conrmed
by the EPR studies discussed above.
a
[
NBu
4
][BF
4
]/CH
3
CN (0.1 M) as electrolyte. Potentials (ꢃ10 mV) quoted
5 0/+ ox
29
5 5 2
versus SCE, using [Fe(h -C
H
)
]
(E1/2 ¼ 0.382 V) as internal
red c I red
p
b
reference. Quasi-reversible wave E1/2 ¼ ꢀ2.16 V. Shoulder at E
¼
d
red
p
ꢀ
f
0.011 V. A new wave was formed at E
¼ ꢀ0.58 V during the
I red
p
e
electrochemical study of this complex. Shoulder at E
Shoulder at Ep ¼ ꢀ0.17 V. Ref. 30.
¼ ꢀ0.12 V.
I
red
g
2
.2. DNA interaction
It has been considered that the interaction of Cu(II) complexes
with DNA could play a vital role in their potential cytotoxicity
activity. The binding to DNA double helical structures is one of
the ways by which anticancer drugs exert its effect. It can be
divided in two main categories: covalent and non-covalent, such
as electrostatic interaction, intercalation between base pairs
and minor and major DNA groove binding interaction. Drugs
like cisplatin interact with DNA forming covalent adducts in an
irreversible way causing inhibition of DNA processes and
subsequent cell death. Although, when concerning drug
metabolism and toxicity, reversible non-covalent interactions
Scheme 3
3
12 nm and two weaker absorption bands at 326 and 339 nm are
also registered, corresponding to the metal-perturbed intra-
ligand p–p* transition.
A simple way to determine whether interactions between
DNA and the metal complex are present is to look for changes in
the UV-Vis spectrum of the complex upon addition of DNA. As
32
are typically preferred for anticancer drugs.
To evaluate such interactions several experimental tech-
niques were applied, based on the fact that interactions
between DNA and drugs/inorganic compounds can cause
chemical and conformational modications and, thus, varia-
3
exemplied for C in Fig. 5b, upon incremental additions of calf
thymus DNA (CT-DNA) to the complex solutions, small pertur-
bations in the ligand-centered bands, and a smaller effect in the
metal-perturbed intraligand bands are observed. Furthermore,
there is no change in the position of the absorption bands (no
red shi). This behavior was observed for all the complexes.
The UV-Vis titration experiments have shown that there is no
strong intercalative interaction between the complexes and CT-
DNA, which is usually characterized by an accentuated red shi
and hypochromism. In the case of a strong electrostatic
attraction between the compound and DNA, an hyperchromic
32
tions of the electrochemical properties of nucleobases.
.2.1. DNA binding. First, UV-Vis and uorimetric spec-
2
troscopic titrations were performed to evaluate the binding
affinity and gain some insight on the binding mode of
1
4
complexes C –C to DNA.
The electronic absorption spectra of complexes C –C
Fig. 5a), measured in PBS buffer (10 mM, pH 7.2) present three
1
4
(
strong absorption bands with maxima at 268, 277 and 285 nm,
which can be attributed to intraligand transitions. A shoulder at
2
3
3
Fig. 4 Representative cyclic voltammograms of (a) C and (b) C obtained in CH CN. (*) adsorption wave.
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1
4
3
Fig. 5 UV-Vis absorption spectra of (a) Cu(II) complexes (C –C ) and (b) spectrophotometric titration of CT-DNA with complex C (50 mM), in
3
PBS buffer (10 mM, pH 7.2), at room temperature. The arrows indicate the changes of the bands upon addition of CT-DNA ([DNA]/[C ] ¼ 0–3.2).
effect should be observed, reecting the alterations in DNA were performed, taking advantage of the high sensitivity, large
conformation and structure aer the complex-DNA interaction linear concentration range and selectivity of molecular uo-
3
3
32
has occurred, which is also not the case. The observed small rescence. As an example, Fig. 6 shows the effect of the
change in the intensity of the intraligand spectral bands may be concentration of DNA on the uorescence emission spectra
2
indicative of a weak interaction, usually characterized by measured for solutions of complex C (5 mM). Upon addition of
hypochromic or hyperchromic effects without signicant increasing amounts of CT-DNA, a decrease of the emission
34
wavelength shis in the spectral proles.
band is clearly observed, as a result of the chromophore (Cu(II)
To quantify and compare the CT-DNA binding affinity of complex) quenching due to the interaction with CT-DNA. This
1
4
complexes C –C , their intrinsic binding constants K
b
were effect was observed with all complexes.
In the case of intercalating drugs, the molecules are inserted
determined by monitoring the changes in the absorbance of the
intraligand bands with increasing concentration of CT-DNA, into the base stack of the helix, and the rotation of the free
using a simple Scatchard model (see Experimental section). molecules favors the radiationless deactivation of the excited
4
ꢀ1
The calculated binding constant values (3.7–6.4 ꢂ 10 M ) are states. Also if the drugs are bound to DNA, the deactivation
comparable to those reported for other polypyridyl–copper(II) through uorescence emission is favored, and a signicant
4
38
complexes, such as copper(II)–tolyl-terpyridine (K ¼ 0.84 ꢂ 10
increase in the uorescence emission is normally observed.
ꢀ1
M
), copper(II)–tolyl-terpyridine-dimethylphenanthroline (K ¼ Nevertheless it is possible to observe a decrease in the uores-
5
ꢀ1 35,36
2.3–3.3 ꢂ 10
M
),
and copper(II)–terpyridine complexes cence intensity in the presence of DNA when groove binding,
functionalized with piperidine substituents (K ¼ 1.35–6.71 ꢂ electrostatic, hydrogen bonding or hydrophobic interactions
4
ꢀ1 37
1
0 M ).
are involved and the molecules are close to the sugar–phos-
To obtain additional information on the mode of interaction phate backbone. Considering the three widely accepted non-
of the complexes with DNA and their physical “localization” covalent interaction modes between metal complexes and
relative to the double helix, uorescence spectroscopy studies DNA (electrostatic interaction/”outside” binding, groove
39
binding and intercalation binding ) the UV-Vis and uori-
metric results indicate that intercalation is not the main process
1
4
involved in the interaction of C –C with CT-DNA.
This conclusion is further supported by DNA melting studies
(T , the temperature at which the double helix denatures into
m
single strand DNA) made in the absence and presence of the
ꢁ
complexes. In the absence of complexes, T
m
¼ 82.8 ꢃ 0.5 C for
1
4
CT-DNA. The addition of complexes C –C to DNA under identical
ꢁ
experimental conditions resulted in a DTm of ꢀ0.3 ꢃ 0.5 C, which
is within the experimental error. The intercalation of small
molecules into the DNA double helix is known to increase the
40
DNA melting temperature, while the interactions realized via
non-traditional intercalation, groove binding or electrostatic
41
forces are known to have slight effects on Tm. Therefore, the
quite low and small negative DT values obtained indicate that
there is no extra stabilization of the double-stranded nucleic acids
m
1
4
by C –C , giving further support to the conclusion that these
complexes and CT-DNA do not interact via intercalation.
These observations can be rationalized taking into consid-
2
4
2
Fig. 6 Fluorescence emission titration of complex C (5 mM) in PBS eration the structures of C –C . In each of these complexes, the
buffer (pH ¼ 7.4) with CT-DNA. The arrow indicate changes upon bipyridine co-ligand carries two protonable aliphatic amines.
2
addition of increasing amounts of CT-DNA ([DNA]/[C ] ¼ 0–11).
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According to Pearson's hard/so acid/base theory, these present the highest DNA cleavage activity since the linear DNA
positively-charged aliphatic amino groups are hard acid, and was formed even at very low concentration of these complexes
2
are most likely capable of binding to the negatively-charged (ca. 5 mM). In case of C , at 5 mM around 55% of the initial
42
phosphate groups, rather than to the DNA bases. Therefore, supercoiled DNA (Form I) was converted into nicked (53%) and
outside electrostatic interaction is the most plausible binding linear DNA form (2%). At 10 mM the cleavage of supercoiled DNA
2
4
mode between C –C complexes and duplex DNA.
was already ca. 90% and, for higher concentrations, no super-
3
2
.2.2. DNA cleavage activity. In order to evaluate the ability coiled form is observed. For C , the behavior is quite similar but
of the new ligands and corresponding Cu(II) complexes to act as the cleavage of supercoiled DNA to linear form is not so efficient
metallo-nucleases, DNA cleavage on plasmid fX174 phage DNA and a higher percentage of nicked DNA remains, even at high
was monitored by gel electrophoresis. Supercoiled fX174 phage concentrations. In the case of the morpholine complex deriva-
4
4
DNA was incubated with different concentrations of the tive (C ), the linear form was almost not observed (2% at [C ] ¼
ꢁ
compounds, in phosphate buffer (pH 7.2) for 18 h at 37 C. The 250 mM) indicating lower activity when compared with the other
naturally occurring supercoiled form (Form I), when nicked, two Cu(II) complexes bearing cyclic amine derivatives.
gives rise to an open circular relaxed form (Form II) and upon
2.2.3. Mechanistic investigation of the DNA cleavage reac-
further cleavage, results in the linear form (Form III). When tion. In general, metal-mediated DNA cleavage occurs through
subjected to gel electrophoresis, relatively fast migration is one of three different pathways, including photocleavage,
observed for Form I. Form II migrates slowly and Form III hydrolysis and oxidative cleavage. To get insight on the mech-
migrates between Forms I and II. The distribution of the three anism involved in the strong cleavage activity observed, the
DNA forms in the agarose gel electrophoresis provides a copper-mediated DNA cleavage was investigated under different
measure of the extent of DNA cleavage. Fig. 7 shows the elec- experimental conditions (see Fig. 8). Nevertheless, the results
1
4
trophoretic pattern of plasmid DNA treated with C –C , where it obtained without any additive indicate very high “self-acti-
can be observed that these Cu(II) complexes were able to induce vating” nuclease effect.
conformational changes in DNA and to promote
concentration-dependent DNA cleavage. Control experiments cleavage in the presence of redox agents, two additives were
revealed that no measurable DNA cleavage occurred when tested, namely ascorbic acid (AA) and H (Fig. 8a and b). AA
fX174 phage DNA was incubated with either 250 mM of the non- and H O are very important for the action of redox-active
a
As most of the copper(II) complexes only accomplish DNA
2 2
O
2
2
metallated ligands or Cu(CF SO ) , clearly indicating that the “chemical nucleases” that are effective cleavers of DNA,
3
3 2
observed cleavage was due to the metal complexes action.
From the gel electrophoresis analysis (Fig. 7) it is possible to reaction. The effect of adding AA (10 mM, lanes 2, 4 and 10) or
conclude that the cleavage activity is proportional to the (50 mM, lanes 3, 4 and 11) was evaluated and the result
because they are required to initiate and sustain the radical
2 2
H O
1
4
concentrations of C –C . All the complexes could convert the showed that both agents could dramatically enhance the
supercoiled DNA (Form I) to nicked DNA (Form II) and even to cleavage activity, converting supercoiled DNA to small frag-
2
3
linear DNA (Form III). From all the complexes, C and C
ments which could not be easily detected by gel electrophoresis
1
4
Fig. 7 (a) Agarose gel electrophoresis patterns for the cleavage of supercoiled fX174 phage DNA by C –C complexes after 18 h of incubation at
ꢁ
2
3
7 C in phosphates buffer (pH 7.2) and (b) percentage of plasmid relaxation for C . Forms I, II and III are supercoiled, nicked circular and linear
forms of DNA, respectively.
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2
3
1
4
Fig. 8 Cleavage of supercoiled fX174 DNA by Cu(II) complexes (a) C and C and (b) C and C in the presence of reducing (ascorbic acid, 10 mM),
1
4
oxidizing agents (H
scavenger (DMSO, 80 mM) and singlet oxygen species scavengers (NaN
2
O
2
, 50 mM), hydroxyl radical scavenger (DMSO, 5%) or in the dark (*), and (c) C –C in the presence of hydroxyl radical
ꢁ
3
and KI, 80 mM), after 18 h of incubation at 37 C in phosphate buffer (pH
7.2), DNA lin – linear DNA obtained by digestion with XhoI. Forms I, II and III are supercoiled, nicked circular and linear forms of DNA, respectively.
(
lanes 2–4 and 10–12). This suggests that the redox process is such as terephthalic acid (TPA) and 1,3-diphenyl-isobensofuran
highly enhanced by the use of reducing/oxidant external agents. (DPBF) (results not shown).
To identify the reactive oxygen intermediates which might be The DNA cleavage could be inhibited when NaN
formed in the DNA cleavage process, experiments in the pres- added to the system (Fig. 8c, lanes 3 –4 , 7 –8 , 12 –13 , 15 –16 ),
3
or KI were
0
0
0
0
0
0
0
0
2
3
ence of a variety of radical scavengers were also carried out particularly in case of C and C . These results suggested the
Fig. 8). DMSO was found to have little effect on the DNA participation of singlet oxygen as active species. Moreover, the
(
0
0
0
0
cleavage reaction (lanes 7, 15 and 2 , 6 , 10 , 14 ), indicating that dramatic cleavage activity enhancement observed by the addi-
hydroxyl radicals are not involved. Additionally, this conclusion tion of H O and AA on the reaction catalyzed by C –C further
1
4
2
2
was corroborated by uorescent methods using different dyes, supported the oxidative mechanism of action.
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1
4
1
2
Fig. 9
k
obs for the cleavage of supercoiled fX174 DNA by Cu(II) complexes (a) C –C at 40 mM and (b) C and C at indicated concentration, after
ꢁ
incubation over time at 37 C, in phosphate buffer (pH 7.2).
The photochemical cleavage pathway was excluded by per- positive amine groups. This may also explain the lower activity
4
forming control experiments in the absence of light. Compar- observed for C complex, where the presence of a oxygen atom
ison of lines 1 and 9 with 8* and 16* respectively, in Fig. 8a and in the morpholine ring may delocalize the positive charge of the
b, conrms that the process is not photochemically activated amine and decrease its ability to interact with phosphodiester
since the DNA cleavage extend is the same in the presence (1 negatively charged backbone.
and 9) or in absence (8* and 16*) of light.
To quantify the nuclease activity of the complexes, the
2
.3. Cell-based studies
reaction that leads to formation of nicked circular DNA (Form
II) from the supercoiled (Sc, Form I) over various concentrations
of complexes (20–60 mM) and constant DNA concentration was
followed over time (0–22 h). The results of gel electrophoresis
were subjected to densitometric quantication and the kinetic
parameters were analyzed by assuming a simple pseudo rst-
order process for conversion of Form I to Form II. The rate
constants (kobs) were determined from the plot of ln(% Sc DNA)
versus time at different concentrations, and in Fig. 9a the kobs
obtained for 40 mM of complex are presented. The higher kobs
The high DNA cleavage ability displayed by the complexes
encouraged us to evaluate their cellular uptake and cytotoxicity.
The cytotoxic activity of all ligands, Cu(II) complexes and CuCl
2
was assayed in the A2780/A2780cisR ovarian cisplatin sensitive/
resistant cancer cells by a colorimetric method (MTT assay).
Comparison between the activity of the reference drug cisplatin
and the activity of these compounds was performed in this cell
pair. Dose–response curves aer 24 and 72 h exposure were
obtained by using an appropriate range of concentrations. IC50
values were calculated and are presented in Table 5.
Aer 72 h of incubation, C –C presented high cytotoxic
activity, surpassing cisplatin in both cell lines. Moreover, all
complexes were able to overcame resistance in the A2780cisR
2
values for C are clearly indicative of the best nuclease activity
2
3
4
1
1
4
within this family, which follows the order C > C > C > C .
This effect is even more visible if we represent the kobs vs.
concentration of complexes (Fig. 9b).
2
4
1
Overall, the DNA cleavage studies of C –C complexes
cells when compared to cisplatin. Bipyridine (L ) and its deriv-
2
4
showed their remarkable self-activated nuclease activity. For the
atives (L –L ) displayed lower cytotoxic activity than the corre-
sponding complexes, with IC50 values that ranged from 27–94
mM in the A2780 cell line and 44–200 mM in the A2780cisR cell
2
3
most active complexes (C and C ), the activity seems to involve
reactive oxygen species as showed by the moderate inhibitory
effects caused by the presence of the radical scavengers NaN
3
and KI. Nevertheless, the hydrolytic pathway can not be
completely ruled out, as these complexes still showed strong
Table 5 IC50 values for A2780 ovarian carcinoma cells and the
nuclease activity even in the presence of the radical scavengers. cisplatin-resistant variant A2780cisR after 24 h and 72 h of continuous
1
4
treatment with terpyridine, and the mixed Cu(II) complexes (C –C ),
If we consider the cyclic amine ring as a centroid, the
distances between the two centroids in the same Cu(II) molecule
CuCl
2
and the reference drug cisplatin
˚
are about 12.1 A, twice the distance between adjacent phos-
IC50 (mM)
phorus atoms of the phosphodiester in the double helix DNA
˚
43
24 h
72 h
backbone (2 ꢂ ca. 6 A). This suggests that the two protonated
amines can interact with alternate phosphodiester groups in a
DNA strand. This interaction between neighboring phosphoryl
oxygen atoms and the protonated amine may lead to more
favorable electrostatic interactions, allowing the DNA to be
Compounds
Terpyridine
A2780
A2780
A2780cisR
16.3 ꢃ 5.2
20.9 ꢃ 3.4
14.6 ꢃ 2.3
20.5 ꢃ 5.1
17.9 ꢃ 5.0
—
0.93 ꢃ 0.3
0.89 ꢃ 0.3
0.47 ꢃ 0.2
0.27 ꢃ 0.1
0.26 ꢃ 0.1
>100
4.19 ꢃ 1.3
2.05 ꢃ 1.2
1.15 ꢃ 0.6
0.99 ꢃ 0.6
0.88 ꢃ 0.4
[200
1
C
2
2
3
C
cleaved more readily. Therefore, the higher activity of C and C
3
C
can be attributed to their structure matching the phospho-
diester backbone of the nucleic acids and cooperative interac-
tion from the highly active Bpy–Tpy–Cu(II) moiety and two
4
C
CuCl
Cisplatin
2
16.9 ꢃ 2.4
1.30 ꢃ 0.5
16.05 ꢃ 1.2
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1
3
4
6
Fig. 10 The subcellular distribution (copper content) of C , C and C into the A2780 cells expressed as: (a) nmol of Cu per 10 cells; (b)
percentage of total copper uptake by the cells. Cells were incubated with the compounds at a concentration equivalent to the IC50 values found
at 24 h challenge (20 mM). The cytosol, membrane/particulate, nuclear and cytoskeletal fractions were extracted and their copper content
determined by ICP-MS.
line. Complexes containing cyclic amine derived bipyridines onset of metastases, and hence a potential target of interest in
4
4
were much more active (two to three fold) than the parental numerous therapies.
complex C that contains the non substituted bipyridine.
1
A small part of complexes (5–10%) is localized in the nuclear
Cellular uptake studies were performed to evaluate cell fraction, and consequently could damage DNA. Therefore, a
permeability in A2780 cells. Furthermore, and to ascertain if the comet assay was performed to evaluate DNA damage of A2780
cellular toxicity is related to DNA cleavage, sub-cellular frac- cells aer exposure to the different Cu(II) complexes. This assay
tioning were also performed. Cells were incubated for 24 h with allows the detection of early nuclear changes in the cells, as well
1
3
4
C , C and C at 20 mM, a concentration equivalent to the IC
as changes on chromatin organization within a single cell. As
5
0
found for these compounds (Table 5). The amount of copper in seen in Fig. 11, the Cu(II) complexes induced DNA strand breaks
1
2
4
the cytosolic, membrane/particulate, nuclear and cytoskeletal in A2780 cells, particularly in the case of C , C and C . This
fractions of the A2780 cancer cells was quantied by ICP-MS. result further demonstrates the ability of these Cu(II) complexes
1
3
Results in Fig. 10 show that the three tested complexes (C , C
to induce DNA damage.
4
and C ) present similar proles, i.e., low retention in the cytosol
and membranes and a high amount in the cytoskeleton,
1
4
3
particularly for C and C . Although C shows a lower total
3
Conclusions
uptake, its higher accumulation in the nucleus, membranes
and cytosol could probably contribute to confer a high cytotoxic New mixed Bpy–Tpy–Cu(II) complexes were isolated and char-
activity.
acterized aer reaction of equimolar amounts of Cu(II) with
0
Given the role of the cytoskeleton in cell transformation and terpyridine and 2,2 -bipyridine derivatives with different cyclic
in biological processes such as cell division and cell migration it amines. All new complexes show high antitumor activity in
is pertinent to consider that the cytoskeleton could be a target ovarian carcinoma A2780 cells, being three to ve times more
for these new Cu(II) complexes. In fact the dysfunction of the cytotoxic than cisplatin, at 72 h of exposure. Additionally,
1
4
cytoskeleton is oen associated with pathologies such as the complexes C –C show good activity in the cisplatin-resistant
cells A2780cisR, with lower resistance factors than cisplatin.
The four complexes tested exhibit similar cellular uptake and
distribution proles with preferential localization in the
cytoskeleton.
These new complexes present an impressive plasmid DNA
cleaving ability, triggering double-strand DNA breaks in the
absence of any exogenous oxidant or reducing agent. Aer
binding to DNA, the complexes cleave the DNA strands by a self-
activating mechanism. The cleavage may be carried out via an
oxidative pathway, but no reductant or oxidant is needed.
However, one cannot exclude the possible involvement of
hydrolytic reactions catalyzed by the complexes. The enhanced
2
3
cleavage activity observed for C and C complexes can be
attributed to electrostatic interaction between the positive
protonated cyclic amines of the complexes and the negative
1
4
Fig. 11 DNA damage of A7280 cells. Cells were exposed to C –C at
0 mM for 24 h; Control: non-exposed cells. Data are presented as
4
phosphodiester moiety in the DNA backbone. In C , delocal-
2
ization of the positive charge by the presence of the oxygen in
the amine ring may lead to a lower electrostatic interaction.
average ꢃ standard deviation. (*) stands for statistical significant
differences (p < 0.05). A.U. stands for arbitrary units.
61372 | RSC Adv., 2014, 4, 61363–61377
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1
6
3.0 N, 67.6 C, 9.2 H; calculated for C24
H
34
N
4
O
2
$H
2
O 13.1 N,
4
Experimental section
7.3 C, 8.8 H.
4.1. General procedures
0
0
4
4
.2.2.3. 4,4 -Bis(2-morpholinoethoxy)-2,2 -bipyridine
(L ).
1
All chemicals used were p.a. grade. The chemical reactions were Yield 56%. H NMR (300 MHz, CDCl ): d 8.45 (2H, dd, CHbpy),
3
1
13
followed by TLC. H and C NMR spectra were recorded on a 7.96 ppm (2H, t, CHbpy), 6.84 (2H, dt, CHbpy), 4.27 (4H, td, CH
Varian Unity 300 MHz spectrometer, and chemical shis are 3.72 (8H, dd, CH ca.), 3.46 (1H, d, CH ca.), 2.84 (4H, dt, CH
given in ppm, referenced with the residual solvent resonances 2.58 (8H, d, CH ca.). C RMN (75 MHz, CDCl
2
),
),
2
2
2
1
3
2
3
): d 165.87,
relative to SiMe . IR spectra were recorded in KBr pellets on a 157.88, 150.29, 111.44, 106.88, 67.00, 65.97, 57.43, 54.18. ESI-MS
4
+
Bruker Tensor 27 spectrometer. C, H and N analyses were per- (MeOH): (m/z) ¼ 415.3 [M + H] ; CHN (%): experimental 13.0 N,
formed on an EA 110 CE Instruments automatic analyzer. The 61.6 C, 7.9 H; calculated for C22
EPR spectra were recorded at 77 K (on glasses prepared by 7.5 H.
freezing solutions in liquid nitrogen) with a Bruker ESP 300E X-
H N O $H O 13.0 N, 61.1 C,
30 4 4 2
band spectrometer coupled to a Bruker ER041 X-band frequency 4.3. Synthesis of the copper(II) complexes
meter (9.45 GHz) operating at 100 KHz. The cyclic voltammetry
4
.3.1. General procedure for the synthesis of bipyridine–
studies were performed under dinitrogen using a potentiostat/
galvanostat Radiometer Analytical Voltammetry PST050
VoltaLab® equipment.
terpyridine–copper(II) complexes. To a solution of bipyridine
1
4
ligands L –L (0.100 g, 0.24 mmol) and terpyridine (0.056 g, 0.24
mmol) in acetonitrile (6 mL), copper(II) triate was added (0.24
mmol). The mixture was stirred at room temperature for 2 h.
The solution was then concentrated, and diethyl ether and
4 6
NH PF were added to help the complex precipitation. The
4.2. Synthesis of the ligands
recovered precipitates were dried under vacuum and formu-
0
0
21
4
.2.1. Synthesis of 2,2 -bipyridine-4,4 -diol. To a solution
1
4
lated as C –C .
.3.1.1. Bipyridine–terpyridine–copper(II) complex (C ). m ¼
.270 g; 87% yield. CHN (%): experimental 8.62 N, 36.50 C, 3.13
0
0
of 4,4 -dimethoxy-2,2 -bipyridine (1 g; 4.6 mmol) in acetic acid
60 mL) was added bromhidric acid (16 mL, 92 mmol). The
reaction mixture was le under reux for approximately 72 h
and then the solvent was removed under vacuum. The solid
residue was dissolved in water and neutralized with ammonium
hydroxide. The white precipitate which formed was ltered and
OD): d 8.18 (2H, s,
Haromatic), 7.24 (2H, s, Haromatic), 6.74 (2H, s, Haromatic); ESI-MS
1
4
(
0
2
+
ꢀ
H; calculated for C H CuN $2PF6 $4H O 8.59 N, 36.84 C,
2
5
19
5
2
+
3.34 H. MALDI-TOF-MS (DHB matrix): 452.09 [M ].
0
0
4
.3.1.2. 4,4 -Bis(2-(pyrrolidin-1-yl)ethoxy)–2,2 -bipyridine–ter-
2
pyridine–copper(II) complex (C ). m ¼ 0.249 g; 72% yield; CHN
1
dried. Yield 99%. H NMR (300 MHz, CD
3
(%): experimental 8.89 N, 36.11 C, 3.93 H; calculated for C37
-
4+
ꢀ
H
43CuN
7
O
2
$4PF
6
$1.5MeCN 9.00 N, 36.32 C, 3.62 H. MALDI-
ꢀ
(
MeOH): (m/z) ¼ 187.0 [M ꢀ H] .
+
TOF-MS (DHB matrix): 678.26 [M ].
4
.2.2. General procedure for the synthesis of derivatized
0
0
4
.3.1.3. 4,4 -Bis(2-(piperidin-1-yl)ethoxy)–2,2 -bipyridine–ter-
1
4
0
0
bipyridine ligands (L –L ). To a solution of 2,2 -bipyridine-4,4 -
diol (0.800 g, 4.3 mmol), potassium carbonate (3.526 g, 25.5
mmol) and potassium iodide (catalytic amount) in anhydrous
DMF (20 mL) was added 12.8 mmol of the cyclic amine (1-(2-
chloroethyl)pyrrolidine, 1-(2-chloroethyl)piperidine or 4-(2-
3
pyridine–copper(II) complex (C ). m ¼ 0.240 g; 76% Yield; CHN
(%): experimental 7.99 N, 36.75 C, 3.96H; calculated for C39
-
4
2
+
ꢀ
6
H
47CuN
7
O
$4PF
$0.5MeCN 8.02 N, 36.68 C, 3.73 H. MALDI-
+
TOF-MS (DHB matrix): 706.25 [M ].
0
0
4
.3.1.4. 4,4 -Bis(2-morpholinoethoxy)–2,2 -bipyridine–terpyr-
ꢁ
chloroethyl)morpholine). The mixture was stirred at 75 C for 72
h under N . Then the solvent was removed and the residue was
4
idine–copper(II) complex (C ). m ¼ 0.252 g; 78% yield. CHN (%):
2
experimental 8.69 N, 36.07 C, 3.85H; calculated for C H -
3
7
43
puried by column chromatography (100% CH
2 2
Cl to 49/49/2
4
+
ꢀ
CuN O $4PF6 $1.5MeCN 8.79 N, 35.46 C, 3.53 H. MALDI-TOF-
MS (DHB matrix): 710.25 [M ].
7
4
CH Cl /MeOH/NH OH). The three compounds were obtained
2
2
4
+
as bright-white solids.
4
0
0
2
.2.2.1. 4,4 -Bis(2-(pyrrolidin-1-yl)ethoxy)-2,2 -bipyridine (L ).
1
4.4. X-ray diffraction analysis
Yield 51%. H NMR (300 MHz, CD
3
OD): d 8.48 (2H, d, CHbpy),
.89 ppm (2H, s, CHbpy), 7.07 (2H, dd, CHbpy), 4.38 (4H, t, CH
.18 (4H, t, CH ), 2.90 (8H, m, CH ca.), 1.92 (8H, m, CH ca.); were obtained from a acetonitrile solution of the complexes in a
3
4
7
3
2
), Blue crystals of C and C suitable for X-ray diffraction studies
2
2
2
1
3
C RMN (75 MHz, CD OD): d 167.42, 158.72, 151.60, 112.20, saturated chamber of diethyl ether, aer standing for some
3
1
09.00, 67.31, 55.63, 55.42, 24.16. ESI-MS (MeOH): (m/z) ¼ 383.3 days. The crystals were mounted on a loop with protective oil. X-
+
[M + H] ; CHN (%): experimental 13.9 N, 65.6 C, 8.4 H; calcu- ray data were collected at 150 K in the u and 4 scans mode on a
lated for C22
H
30
0
N
4
O
2
$H
2
O 14.0 N, 65.9 C, 8.1H.
Bruker APEX II CCD diffractometer using graphite mono-
0
3
˚
4
.2.2.2. 4,4 -Bis(2-(piperidin-1-yl)ethoxy)-2,2 -bipyridine (L ). chromated Mo Ka radiation (0.71073 A) and operating at 50 kV
1
3
Yield 50%. H NMR (300 MHz, CDCl ): d 8.45 (2H, d, CHbpy), and 30 mA. Cell parameters were retrieved using Bruker
45
45
7
2
1
1
.95 ppm (2H, d, CHbpy), 6.85 (2H, dd, CHbpy), 4.31 (4H, t, CH
.86 (4H, t, CH ), 2.59 (8H, m, CH ca.), 1.66 (8H, m, CH ca.), observed reections. A semi-empirical absorption corrections
.54 (4H, m, CH2 ca.). C RMN (75 MHz, CDCl ): d 165.89, were applied using SADABS. Structure solution and rene-
57.82, 150.12, 111.26, 106.88, 66.07, 57.60, 55.02, 25.94, 24.15. ment were performed using direct methods with program
2
), SMART soware and rened using Bruker SAINT on all
2
2
2
1
3
46
3
+
47
48
ESI-MS (MeOH): (m/z) ¼ 411.4 [M + H] ; CHN (%): experimental SIR97 and SHELXL97, both included in the package of
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4
9
ꢁ
programs WINGX-Version 2013.3. A full-matrix least-squares Peltier temperature-controlling programmer ETC-717 (ꢃ0.1 C)
renement was used for the non-hydrogen atoms with aniso- in phosphate buffer at various temperatures in the presence and
tropic thermal parameters, except for disordered atoms that absence of the complexes. UV melting proles were obtained by
ꢁ
were rened isotropically. All hydrogen atoms were inserted in scanning A260 absorbance monitored at a heating rate of 0.5 C
idealized positions and allowed to rene riding in the parent min for solutions of CT-DNA (100 mM) in the absence and
carbon atom. Molecular graphics were prepared using presence of different concentrations of copper(II) complexes
ꢀ
1
50
ꢁ
ORTEP3. A summary of the crystal data, structure solution and (10–40 mM) from 30 to 90 C with the use of the thermal melting
renement parameters are given in Table 1 ESI.†
.5. Cyclic voltammetry studies
program. The melting temperature Tm which is dened as the
temperature where half of the total base pairs is unbound was
determined from the midpoint of the melting curves.
4
4.6.2. Fluorescence spectroscopy studies. Fluorescence
The redox properties of the complexes and ligands were
studied, under nitrogen, by cyclic voltammetry using a
potentiostat/galvanostat Radiometer Analytical Voltammetry
spectra were recorded in a Perkin Elmer LS 55 uorescence
spectrometer using a quartz cuvette of 1 cm. Aer each addition
of CT-DNA in phosphate buffer, the solution was allowed to
equilibrate and the absorption at the excitation wavelength was
recorded. The uorescence spectra were then recorded until
there were no further changes in the uorescence intensity.
PST050 VoltaLab® equipment interfaced with
compartment cell provided with a Pt wire working electrode.
NBu ][BF ]/0.1 M THF solution was used as electrolyte. The
a three
[
4
4
solvent (CH CN Sigma-Aldrich) was puried by conventional
3
1
4
Complexes C –C were excited at 323 nm and the emission
spectra were recorded from l ¼ 335–550 nm, with a scan speed
techniques and distilled immediately before use. The potential
ꢀ
1
5
0/+
values were measured at 200 mV s using [Fe(h –C
5
H
5
)
2
]
as
ꢀ1
of 300 nm min . Emission and excitation slits were chosen in
internal reference.
order to maximize the uorescence intensity.
4.6. DNA binding studies
4.7. DNA cleavage activity
Calf thymus DNA (CT-DNA) sodium salt was purchased from
Sigma and was used without further purication. The DNA The plasmid DNA used for gel electrophoresis experiments was
concentration per nucleotide of stock solutions in phosphate fX174 (Promega). Linear DNA was obtained by digestion with
buffer (10 mM, pH 7.2) were determined by absorption spec- the single-cutter restriction enzyme XhoI and used as a refer-
troscopy at 260 nm, aer adequate dilution with the buffer and ence in agarose gel electrophoresis. DNA cleavage activity was
ꢀ
1
ꢀ1 51
using the reported molar absorptivity of 6600 M cm
.
All evaluated by monitoring the conversion of supercoiled plasmid
measurements involving DNA and the different tested DNA (Sc – form I) to nicked circular DNA (Nck – form II) and
compounds were carried out in phosphate buffer (10 mM, pH linear DNA (Lin –form III).
7
.2). The absorption and uorescence titrations were performed
by keeping the concentrations of the complex constant, while 2 mL (200 ng) of supercoiled DNA, 2 mL of 100 mM stock
varying the concentrations of DNA. To obtain the intrinsic Na HPO /HCl pH 7.2 buffer solution and 10 mL of the aqueous
binding constant (K ) all calculations were done considering the solution of the complex. The nal reaction volume was 20 mL,
DNA concentration in base pairs, and the data were corrected the nal buffer concentration was 10 mM and the nal metal
for volume changes. concentration varied from 1 to 500 mM. When indicated, the
.6.1. Absorption spectroscopy studies. UV-Vis absorption reaction was carried out in the same buffer but in the presence
spectra were recorded on a Perkin Elmer Lambda 35 spec- of ascorbic acid (10 mM), H O (50 mM), DMSO (5% or 80 mM),
Each reaction mixture was prepared by adding 6 mL of water,
2
4
b
4
2
2
trometer using 1 cm path-length quartz cells. In order to elim- NaN
3
(80 mM) and KI (80 mM) or in the dark. Samples were
ꢁ
inate any interference of the DNA absorbance in the region of typically incubated for 18 h at 37 C, except in the case of the
absorbance of the chromophores an equal amount of CT-DNA kinetics study where times of 1, 2, 4, 6, 8 and 22 h were used.
in phosphate buffer was added to the sample and reference Aer incubation, 5 mL of DNA loading buffer (0.25% bromo-
cells. Aer each addition of CT-DNA, the solution was allowed to phenol blue, 0.25% xylene cyanol, 30% glycerol in water,
equilibrate and the absorption spectrum was recorded until Applichem) were added to each tube and the sample was loaded
there were no further changes in the absorbance. The K was onto a 0.8% agarose gel in TBE buffer (89 mM Tris–borate, 1
b
ꢀ
1
then calculated by tting the data to a reciprocal plot of D/D3ap mM EDTA pH 8.3) containing ethidium bromide (0.5 mg mL ).
versus D by using the following equation: D/D3ap ¼ D/D3 + 1/ Controls of non-incubated and of linearized plasmid were
(
ꢀD3 ꢂ K
b
), in which the concentration of DNA (D) is expressed loaded on each gel electrophoresis. The electrophoresis was
in terms of base pairs, the apparent molar extinction coefficient carried out for 2.5 h at 100 V. Bands were visualised under UV
ap ¼ Aobserved/[complex], D3ap ¼ [3 ꢀ 3 ] and D3 ¼ [3 ꢀ 3 ], in light and images captured using an AlphaImagerEP (Alpha
which 3 is the extinction coefficient of the DNA bound complex, Innotech). Peak areas were measured by denstiometry using
and 3 is the extinction coefficient of the free complex (Scatchard AlphaView Soware (Alpha Innotech). The photos chosen for
model).
3
a
f
b
f
b
f
52
this publication were rearranged to show only the relevant
DNA melting experiments were carried out by monitoring samples. All samples in each gure were obtained from the
the absorption at 260 nm of CT-DNA (100 mM) in the same same run. Peak areas were used to calculate the percentage (%)
Perkin-Elmer Lambda 35 spectrophotometer equipped with a of each form (Sc, Nck and Lin), with a correction factor of 1.47
61374 | RSC Adv., 2014, 4, 61363–61377
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ꢁ
for the Sc form to account for its lower staining by ethidium pH 10) at 4 C for 120 min in the dark. The slides were then
53
bromide. The decrease of Form I over time was tted as a placed on a horizontal electrophoresis tray previously lled with
pseudo-rst order kinetics, aer logarithmic linearization of the freshly prepared cold alkaline buffer and le for 15 min to allow
equation y ¼ (y0 ꢀ a)exp(ꢀkobst) + a, where y0 is the initial DNA unwinding. Electrophoresis was performed at 43 V, 300
percentage of a form of DNA, y is the percentage of a specic mA for 10 min in alkaline buffer (0.3 M NaOH and 1 mM EDTA,
form of DNA at time t, a is the percentage of uncleaved DNA, pH 13). Then, slides were neutralized with ice cold 0.4 M Tris–
and kobs is the apparent rate constant.
HCl (pH 7.5). Just before analysis cells were stained with 100 mL
of ethidium bromide solution (20 mg mL ).
ꢀ1
Cells were counted under inverted uorescence microscopy.
Visual scoring of cellular DNA on each slide was based on the
categorization of 100 cells randomly selected. The comet-like
formations were visually graded into 5 classes, depending on
DNA damage, and scored as described by Garc ´ı a et al. Positive
controls were always included, and consisted of cells previously
4
.8. Cell viability in human tumoral cell lines
The tumoral cell lines A2780 and A2780cisR were cultured in
RPMI 1640 culture medium (Gibco) supplemented with 10%
FBS and 1% penicillin/streptomycin at 37 C in a humidied
ꢁ
55
2
atmosphere of 95% of air and 5% CO (Heraeus, Germany). The
cells were adherent in monolayers and, when conuent, were
harvested by digestion with 0.05% trypsin–EDTA (Gibco) and
seeded farther apart.
Cell viability was evaluated by using a colorimetric method
based on the tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2-yl)-
2 2
exposed to 200 mM of H O , for 1 h. Statistical analyses were
performed with GraphPad Prism 6 (GraphPad soware, Inc.,
USA). One-way univariate analysis of variance model (ANOVA)
was used. A value of p < 0.05 was considered signicant.
2,5-diphenyltetrazolium bromide]), which is reduced by living
cells to yield purple formazan crystals. Cells were seeded in 96-
4
.10. Cellular uptake
4
well plates at a density of 1–1.5 ꢂ 10 cells per well in 200 mL of
To explore the uptake of the compounds and its intracellular
distribution, A2780 cells (10 cells in 5 mL medium) were
exposed to C , C and C complexes at 20 mM for 24 h, a
concentration equivalent to the IC50 values found for a 24 h
incubation. Aer that period, cells were washed with cold PBS
and centrifuged to obtain a cellular pellet following a previously
culture medium and le to incubate overnight for optimal
adherence. Aer careful removal of the medium, 200 mL of a
6
1
3
4
dilution series of the compounds in fresh medium were added
ꢁ
and incubation was performed at 37 C/5% CO for 24 h and 72
2
h. The percentage of DMSO in cell culture medium did not
exceed 1%. Cisplatin was rst solubilized in saline and then
added at the same concentrations used for the other
compounds. At the end of the incubation period, the
compounds were removed and the cells were incubated with
56
described procedure. Cytosol, membrane/particulate, nuclear
and cytoskeletal fractions were obtained using a commercial kit
PREP
(Fraction
cell fractionation kit from BioVision) following
ꢀ1
ꢁ
the manufacturer's recommendations.
2
00 mL of MTT solution (500 mg mL ). Aer 3–4 h at 37 C/5%
The copper content in the different fractions was measured,
aer digestion, by a Thermo XSERIES quadrupole ICP-MS
instrument (Thermo Scientic). Briey, samples were digested
with ultrapure HNO , H O , and HCl in a closed pressurized
microwave digestion unit (Mars5, CEM) with medium-pressure
HP500 vessels and then diluted in ultrapure water to obtain a
CO , the medium was removed and the purple formazan crys-
tals were dissolved in 200 mL of DMSO by shaking. The cell
viability was evaluated by measurement of the absorbance at
2
3
2 2
570 nm using a plate spectrophotometer (Power Wave Xs, Bio-
Tek). The cell viability was calculated dividing the absorbance
of each well by that of the control wells (cells treated with
medium containing 1% DMSO). Each experiment was repeated
at least three times and each point was determined in at least six
replicates. Statistical analysis was done with GraphPad Prism
soware.
2.0% (v/v) acid solution. The instrument was tuned using a
multielement ICP-MS 71 C standard solution (Inorganic
ꢀ1
Venture). Indium-115 at 10 mg L was used as an internal
standard.
4
.9. Comet assay
Acknowledgements
The comet assay was performed in A2780 cells to detect the
presence of DNA strand breaks. Five different conditions were The authors would like to acknowledge FCT for nancial
dened: untreated cells (negative control) and cells treated with support (PTDC/QUI-QUI/114139/2009, PEst-OE/QUI/UI0100/2013,
1
4
C –C at 20 mM. The assay was performed aer 24 h exposure, SFRH/BPD/29564/2006 grant to S. Gama, SFRH/BPD/80758/
under yellow light, to prevent UV-induced DNA damage and 2011 grant to E Palma and FCT Investigator contract to F.
with slight modications of the protocol described by Nogueira Mendes and I. Correia). A. Cruz was funded by the post-doctoral
54
et al. Briey, exposed and non-exposed A2780 cells were grant (BPD/UI88/2886/2013), from the project “Sustainable Use
4
adjusted to a concentration of 1 ꢂ 10 cells per mL, mixed with of Marine Resources” – MARES (CENTRO-07-ST24-FEDER-
0
.5% low melting point agarose, at a ratio of 1 : 10 (v/v) and 002033), funded by QREN, Mais Centro – Programa Oper-
placed on top of 1% (w/v) normal melting point agarose pre- acional Regional do Centro e Uni ˜a o Europeia/Fundo Europeu
coated microscope slides. The slides with the embedded cells de Desenvolvimento Regional. Dr Eug ´e nio Soares from Labo-
were immersed into precooled lysing solution (2.5 M NaCl, 100 rat ´o rio Central de An ´a lises, Universidade de Aveiro is
mM EDTA, 10 mM Tris base and 1% Triton X-100, 10% DMSO, acknowledged for the ICP-MS analyses. The IST-UTL Centers of
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5 D. Sanna, P. Buglyo, A. I. Tomaz, J. C. Pessoa, S. Borovic,
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