Oxidative DNA cleavage by copper ternary complexes of 1,10-phenanthroline and ethylenediamine-sulfonamide derivatives

Article abstract of DOI:10.1016/j.poly.2009.05.017

Ternary copper(II) complexes (1-3) of 1,10-phenanthroline and ethylenediamine-R-sulfonamide derivatives (R = benzene, toluene and naphthalene rings) have been synthesized and characterized with the aid of X-ray diffraction and spectroscopic and electroche

Full text of DOI:10.1016/j.poly.2009.05.017

Polyhedron 28 (2009) 2537–2544
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Oxidative DNA cleavage by copper ternary complexes of 1,10-phenanthroline
and ethylenediamine-sulfonamide derivatives
Andreea Bodoki a, Adriana Hangan a, Luminita Oprean a, Gloria Alzuet b, Alfonso Castiñeiras c,
Joaquín Borrás b,
*
a Department of Inorganic Chemistry, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
b Department of Inorganic Chemistry, University of Valencia, Burjassot (Valencia), Spain
c Department of Inorganic Chemistry, University of Santiago de Compostela, Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Ternary copper(II) complexes (1–3) of 1,10-phenanthroline and ethylenediamine-R-sulfonamide deriva-
tives (R = benzene, toluene and naphthalene rings) have been synthesized and characterized with the aid
of X-ray diffraction and spectroscopic and electrochemical techniques. The crystal structures of the com-
plexes show that the coordination polyhedron around copper(II) is distorted square planar. Both 1,10-
phenanthroline and ethylenediamine-R-sulfonamide act as bidentate ligands. The three structures are
Received 31 March 2009
Accepted 5 May 2009
Available online 15 May 2009
Keywords:
Ternary Cu(II) complexes
Phenanthroline
Sulfonamide derivatives
Oxidative DNA cleavage
stabilized by p–p stacking interactions. The interaction of the complexes with calf thymus DNA has been
investigated by thermal denaturation studies which indicated that DNA was stabilized in the presence of
the compounds. The increase in DNA stability induced by the complexes follows the order: 3 > 2 > 1. All
three complexes were found to be very efficient agents of plasmid DNA cleavage in the presence of ascor-
bate as reducing agent. Mechanistic studies of the DNA cleavage process performed with radical scaveng-
ers show that the reactive oxygen species involved in the DNA damage are the hydroxyl radical, singlet
*
oxygen-like species, the superoxide and hydrogen peroxide.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ward a second target of biological action [9]. Such combined effects
may result in an important enhancement of the activity of the drug
The success of cisplatin and other platinum anticancer drugs
has stimulated a renaissance of inorganic medicinal chemistry
and the search for complexes of other transition metals with inter-
esting biological properties [1,2]. The continuous demand for new
anticancer drugs has addressed chemotherapeutic research based
on the use of essential metalloelements [3] since potential drugs
developed in this way may be less toxic and more prone to exhibit
antiproliferative activity against tumors. In this context, copper(II)
and its complexes play an important role as suitable species for
antiproliferative tests [4,5].
In recent years, there has been substantial interest in the ra-
tional design of novel transition metal complexes which bind and
cleave duplex DNA with high sequential or structural selectivity
[6–8]. An approach to the discovery of new metallodrugs involves
binding an organic compound of known therapeutic value to a me-
tal-containing fragment; this results in a metal–drug synergism in
which the metal acts as a carrier and stabilizer for the drug until it
reaches its target, while at the same time the organic drug carries
and protects the metal preventing side reactions in its transit to-
and the opening of new mechanism of action, such as when the
well-known breast cancer drug tamoxifen is coordinated to iron
(Ferrocifen) [10]. The use of transition metal complexes as artificial
nucleases is an area of extensive research due to the diverse struc-
tural features and reactivity of such complexes [11,12].
For the past several years, our group has worked on the synthesis
of copper–sulfonamide complexes in order to obtain novel antitu-
mor agents. These complexes are especially attractive as anticancer
agents because several substituted sulfonamides have been found
to arrest the cell cycle, causing apoptosis in the M phase [13]. In this
context, we have described the nuclease activity of several copper–
sulfonamide complexes [14–21]. In the present paper, we report
three new copper ternary complexes of substituted N-sulfonamides
derived of ethylenediamine (Chart 1) and 1,10-phenanthroline.
1,10-Phenanthroline is a versatile ligand able to chelate copper to
give mono-phen or bis-phen copper complexes at the oxidation
state I or II [22]. In the presence of hydrogen peroxide, the CuI(-
phen)2 complex efficiently cleaves DNA by oxidative attack on
deoxyribose units in the DNA minor groove [23,24]. The presence
of two organic ligands with biological properties and, on the other
hand, the known nuclease ability of copper(II) complexes in the
presence of reducing agents may lead to the development of new
products with remarkable antitumoral properties.
* Corresponding author. Fax: +34 963544960.
E-mail address: Joaquin.Borras@uv.es (J. Borrás).
0277-5387/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2009.05.017

2538
A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
85%. IR (KBr) (m
max/cmꢀ1): 3321, 3273 (N–H); 1324, 1157 (SO2).
O
S
H
N
H
N
O
S
FAB: m/z+ 341 [H2L1 + H]+.
H2L1
H2L2
H2L3
Anal. Calc. for C22H20N2S2O4 (H2L3) (440.09): C, 59.98; H, 4.58;
N, 6.36; S, 14.56. Found: C, 59.32; H, 4.55; N, 6.36; S, 14.15%. Yield:
O
O
75%. IR (KBr) (m
max/cmꢀ1): 3282 (N–H); 1336, 1156 (SO2). FAB: m/z+
441 [H2L3 + H]+.
2.2.2. N,N0-(Ethane-1,2-diyl)bis(4-methylbenzenesulfonamide) (H2L2)
This compound was obtained and characterized as previously
reported [25].
O
S
H
N
H
N
O
S
H3C
CH3
O
O
2.3. Synthesis of [Cu(L1)(phen)] (1), [Cu(L2)(phen)]ꢁH2O (2) and
[Cu(L3)(phen)](3)
0.5 mmoles of the ligand were dissolved in 30 mL of methanol
(H2L1 or H2L2) or in a mixture of 35 mL methanol and 10 mL
N,N0-dimethylformamide (H2L3). To this solution were then added
2 mL of a methanolic solution of KOH (0.5 M) and 0.25 mmol of
Cu(CH3COO)2ꢁH2O. Separately, another solution was prepared by
dissolving 0.5 mmol of 1,10-phenanthroline (phen) in 25 mL of
methanol and then added dropwise under continuous stirring to
the solution containing the ligand and the Cu(II) salt. The final
green reaction mixture was stirred for an hour at 30 °C and was
then left to stand at room temperature. Slow evaporation of this
resulting solution provided well-developed blue crystals of the
compounds that were suitable for X-ray diffraction.
O
S
H
N
H
N
O
S
O
O
Chart 1. Scheme of the ligands.
2. Experimental
2.1. Materials and physical measurements
Anal. Calc. for C26H22CuN4S2O4 (1) (582.15): C, 53.64; H, 3.81; N,
9.62; S, 11.02. Found: C, 53.18; H, 3.94; N, 9.12; S, 11.18%. Yield:
Reagents and solvents were commercially available (Sigma) in
high purity and used as received. Plasmid pUC18 (0.25 g/ L,
750 M in nucleotides) in TE (tris 10 mM and EDTA 1 mM, pH 8.0)
72%. IR (KBr) (
(kmax, nm): 396 (
DMF: 386 (
spectra in DMF: m/z+ 582.0 [1+H]+.
m
max/cmꢀ1): 1269, 1135
*
m(SO2). Diffuse reflectance
l
l
p ? p ), 590, 740sh (d–d). UV–Vis (kmax, nm) in
l
e e
= 1440 Mꢀ1 cmꢀ1), 627 ( = 305 Mꢀ1 cmꢀ1), ESI+ mass
was purchased from Roche Diagnostics, Germany. CT-DNA (type XV)
was from Sigma. Solutions of the metal complexes and other
reagents for strand scission experiments were prepared fresh daily.
Elemental analyses (C, N, H, S) were performed on a Carlo Erba
AAS instrument. Infrared spectra were recorded with a Mattson
Satellite FT-IR spectrophotometer from 4000 to 400 cmꢀ1 on KBr
disks. Diffuse reflectance spectra (nujol mulls) of the complexes
were performed on a Shimadzu 2101-PC UV–Vis spectrophotome-
ter. UV–Vis spectra of the complex solutions were recorded with an
HP 8453 instrument. Fast atom bombardment (FAB) mass spectra
were obtained with a VG Autospec spectrometer with 3-nitrobenzyl
alcohol as matrix. ESI mass (+ mode) analyses were performed on a
Bruker Esquire 3000 plus LC-MS system. Electronic Paramagnetic
Resonance (EPR) spectra of ground crystals were collected at the
X-band frequency at room temperature with a Bruker ELEXSYS
spectrometer. Cyclic voltammetry experiments were performed
in a single compartment cell with a three-electrode system on a
PAR 273A potentiostat/galvanostat. The working and auxiliary
electrodes were platinum, and the reference electrode was Ag/
AgCl. The supporting electrolyte was tetrabutylammonium per-
chlorate. Solutions of the complexes were deoxygenated by purg-
ing with nitrogen for 15 min prior to measurement.
Anal. Calc. for C28H28CuN4S2O5 (2) (628.22): C, 53.53; H, 4.49; N,
8.92; S, 10.21. Found: C, 53.92; H, 4.32; N, 8.89; S, 10.24%. Yield:
65%. IR (KBr) (
m
max/cmꢀ1): 1266, 1133
*
m(SO2). Diffuse reflectance
? p ), 590, 740sh (d–d). UV–Vis (kmax, nm) in
(kmax, nm): 400 (
p
DMF: 386 (e e
= 1680 Mꢀ1 cmꢀ1), 623 ( = 265 Mꢀ1 cmꢀ1), ESI+ mass
spectra in DMF: m/z+ 610.1 [2-H2O+H]+.
Anal. Calc. for C34H26CuN4S2O4 (3) (682.28): C, 59.85; H, 3.84; N,
8.21; S, 9.40. Found: C, 59.74; H, 3.89; N, 8.19; S, 9.32%. Yield: 76%.
IR (KBr) (
nm): 400 (
m
max/cmꢀ1): 1279, 1122
*
m(SO2). Diffuse reflectance (kmax,
? p ), 630 (d–d). UV–Vis (kmax, nm) in DMF: 629
p
(e
= 215 Mꢀ1 cmꢀ1), ESI+ mass spectra in DMF: m/z+ 718.9
3+2H2O+H]+.
2.4. X-ray crystallography
A crystal of compound 1 (blue prismatic), compound 2 (blue
needle) or compound 3 (blue plate) was mounted on a glass fiber
and used for data collection. Crystal data were collected at
100.0(1) K, using a Bruker X8 Kappa APEXII diffractometer. Graph-
ite monochromated Mo Ka radiation (k = 0.71073 Å) was used
throughout. The data were processed with APEX2 [26] and corrected
for absorption using SADABS (transmissions factors: 1.000–0.099)
[27]. The structure was solved by direct methods using the pro-
gram SHELXS-97 [28] and refined by full-matrix least-squares tech-
niques against F2 using SHELXL-97 [29]. Positional and anisotropic
atomic displacement parameters were refined for all nonhydrogen
atoms. Hydrogen atoms were located in difference maps and in-
cluded as fixed contributions riding on attached atoms with isotro-
pic thermal parameters 1.2 times those of their carrier atoms.
Criteria of a satisfactory complete analysis were the ratios of rms
shift to standard deviation less than 0.001 and no significant fea-
tures in final difference maps. Atomic scattering factors were from
‘‘International Tables for Crystallography” [30]. Molecular graphics
were from PLATON [31] and SCHAKAL [32]. A summary of the crystal
2.2. Synthesis of the ligands
2.2.1. N,N0-(Ethane-1,2-diyl)dibenzenesulfonamide (H2L1) and
N,N0-(ethane-1,2-diyl)dinaphthalene-2-sulfonamide (H2L3)
A solution containing 1 g of ethylenediamine (16.6 mmol) and
34 mmol of benzenesulfonylchloride (6.0 g) (for H2L1) or naphtha-
lenesulfonylchloride (7.7 g) (for H2L3) in 6 mL of pyridine was
heated at reflux for 10 h. The mixture was added to 10 mL of cold
water and stirred for several minutes. The resulting solid was
recrystallized from ethanol.
Anal. Calc. for C14H16N2S2O4 (H2L1) (340.06): C, 49.40; H, 4.74;
N, 8.23; S, 18.84. Found: C, 49.35; H, 4.73; N, 8.27; S, 19.13%. Yield:

A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
2539
data, experimental details and refinement results for the three
complexes are listed in Table 1.
scavengers were DMSO (0.4 M), tert-butyl alcohol (0.4 M), sodium
formate (0.4 M), 1,4-diazabicyclo[2,2,2]octane (DABCO) (0.4 M), 4,
5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) (0.1 M) and cata-
2.5. DNA-melting experiments
lase (10
groove binder distamycin (8
green (3 M) or the copper(I) stabilizer neocuproine, at different
concentrations (12, 120 and 240 M) were also performed. Sam-
l
g/mL, 650 U/mL). Assays in the presence of the minor
l
M) and major groove binder methyl
DNA-melting experiments were carried out by monitoring the
absorption spectrum between 1000 and 200 nm of CT-DNA
l
l
(50
lM base pairs) at different temperatures in both the absence
ples were treated as described above.
and the presence of the ligands or the complexes in a ratio of
2.5:1 DNA to complex. Measurements were performed with an
Agilent 8453 UV–Vis spectrophotometer equipped with a Peltier
temperature controlled sample cell and driver (Agilent 89090A).
Solutions containing the ternary complexes (1–3) and CT-DNA in
borate buffer, pH 8 (1 mM borate, 2 mM NaCl) was stirred contin-
uously and heated with a temperature increase of 1 °C minꢀ1. The
temperature interval studied ranged from 25 to 90 °C. The melting
point was obtained with the first derivative using the Savitsky–Go-
lay algorithm.
3. Results and discussion
3.1. Crystal structures of the complexes 1–3
Figs. 1–3 show the molecular structure of complexes 1–3
respectively with the atomic labeling scheme adopted. The
relevant structural parameters for the compounds are listed in
Table 2. The crystal structures of compounds 1–3 consist of a dis-
crete monomeric copper(II) species surrounded by two N atoms
belonging to a phenanthroline molecule and two sulfonamido N
atoms of the dideprotonated N-sulfonamide ligands in a distorted
square-planar geometry.
2.6. pUC18 DNA cleavage
Both, the dideprotonated sulfonamide ligand (L12ꢀ, L22ꢀ or L32ꢀ
and the 1,10-phenanthroline, act as bidentate forming five-mem-
bered rings with the0 metal ion. The Cu–Nphen distances which
range from 1.992(5) ÅA to 2.024(5) ÅA are similar to those reported
for copper–phenanthroline complexes [33]. The Cu–Nsulfonamida-
to bond lengths lie in the range from 1.947(4) ÅA to 1977(5) ÅA and
are normal distances for copper–sulfonamidato complexes [25].
The N–Cu–N angles that describe the coordination polyhedron
)
A typical reaction for complexes was undertaken by mixing 7
of 0.05 M borate buffer (pH 8.0), 1 L of pUC18 (0.25 g/ L), 6
l
l
L
L
l
l l
of a solution of the tested complex at increasing concentrations
0
to obtain final concentrations between 3 and 12 M and 6 L of
l
l
ascorbate, in a 1000-fold molar excess relative to the concentration
of the complex in borate buffer (0.05 M, pH 8.0). The mixtures were
allowed to stand at 37 °C for 60 min, after which, 3 lL of a loading
buffer solution consisting of 0.25% bromophenol blue, 0.25% xylene
cyanole, and 30% glycerol was added. The solution was then sub-
jected to electrophoresis on a 0.8% agarose gel in 0.5 ꢂ TBE buffer
(0.045 M tris(hydroxymethyl)aminomethane (tris), 0.045 M boric
acid, and 1 mM EDTA) containing 2 lL/100 mL of a solution of ethi-
dium bromide (10 mg/mL) at 80V for about 2 h. The gel was pho-
0
0
[82.13(19)–104.67(19)° in 1, 81.99(15)–103.64(15)° in
2 and
81.67(18)–103.30(2)° in 3] deviate significantly from 90°. In all
complexes, the smaller angle corresponds to that formed by the
copper ion and the two phenanthroline nitrogen atoms, as a conse-
quence of the constrained geometry of the phen rings [34–36].
The distortion of the square-planar geometry (commonly
known as tetrahedrality) can be determined from the angle sub-
tended by two planes, each encompassing the copper and two
adjacent atoms. The tetrahedrality values of 46.46°, 34.0° and
tographed using a TDI Gel Printer Plus system.
To test for the presence of reactive oxygen species (ROS) gener-
ated during strand scission, reactive oxygen intermediate scaveng-
ers were added alternatively to the reaction mixtures. These
Table 1
Crystal and structure refinement data for complex 1, complex 2 and complex 3.
Complex 1
Complex 2
Complex 3
Empirical formula
Formula weight
C26H22CuN4O4S2
582.14
C28H28CuN4O5S2
628.20
C34H26CuN4O4S2
682.25
T (K)
100 (2)
100 (2)
100 (2)
Wavelength (Å)
Crystal system, space group
0.71073
monoclinic, P21/c (number 14)
7.2144(9)
30.913(4)
10.7325(15)
90.000
0.71073
triclinic, P1 (number 2)
7.4589(8)
10.7390(13)
17.7483(17)
106.147(7)
92.209(7)
0.71073
ꢀ
monoclinic, P2(1)/n (number 14)
10.1370(3)
11.1709(3)
25.9632(8)
90.000
a (Å)
b (Å)
c (Å)
a (°)
b (°)
94.576(7)
100.637(2)
c
(°)
90.000
2385.9(5)
4, 1.621
1.134
96.264(8)
1353.9(3)
2, 1.541
1.008
90.000
2889.54(15)
4, 1.568
0.950
1404
V (Å3)
Z, calculated density (Mg mꢀ3
Absorbtion coefficient (mm1)
F(000)
)
1196
650
Crystal size (mm)
h range for data collection (°)
Limiting indices
0.11 ꢂ 0.03 ꢂ 0.08
1.32–26.48
ꢀ9 6 h 6 8
06k638
0.37 ꢂ 0.07 ꢂ 0.04
1.99–25.68
ꢀ9 6 h 6 9
ꢀ13 6 k 6 12
0 6 l 6 21
0.22 ꢂ 0.20 ꢂ 0.10
1.60–27.88
ꢀ13 6 h 6 13
0 6 k 6 14
06l613
0 6 l 6 34
Reflections collected/unique
Data/restraints/parameters
45124/4893
[Rint = 0.1249]
4893/0/334
99.2
17555/5119
[Rint = 0.0812]
5119/0/361
99.9
36832/6896
[Rint = 0.0318]
6896/0/361
100
Completeness to h = 26.48 (%)
Final R indices [I > 2
R indices (all data)
r(I)]
R1 = 0.0756, wR2 = 0.1840
R1 = 0.1163, wR2 = 0.2038
R1 = 0.0694, wR2 = 0.1770
R1 = 0.0900, wR2 = 0.1879
R1 = 0.0887, wR2 = 0.2366
R1 = 0.1089, wR2 = 0.2514

2540
A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
Fig. 3. ORTEP view of [Cu(L3)(phen)] (3).
Complex 3 presents strong p–p stacking interactions involving
the phenanthroline molecules, the ‘‘Cu chelate” (the chelate ring
formed by the copper atom and the coordinated phenanthroline,
Cu1–N41–C53–C52–N51) and the naphthalene rings. Phen–phen
and phen–‘‘Cu chelate” interactions occur between neighboring
complex molecules. Additional phen–naphthalen interactions oc-
cur between the resulting pairs and adjacent complex molecules,
giving rise to 2D frameworks, parallel to ac plane (Fig. 5). The geo-
Fig. 1. ORTEP view of [Cu(L1)(phen)] (1).
metric parameters for these
p–p interactions are summarized in
Table 5. These interactions are similar to those reported for
p–p
other copper complexes of 1,10-phenanthroline [39,40].
3.2. Spectroscopic properties
All the complexes present the same infrared spectrum pattern.
As for the sulfonamide group, the bands assigned to
m(SO2)as and
m(SO2)s vibrations are slightly shifted to lower frequencies with re-
spect to those of the uncoordinated ligands. In general, the IR spec-
tra are similar to those observed for other copper N-sulfonamide
derivatives [17,41] with the modifications noted above being
mainly due to ligand deprotonation at the sulfonamide group. In
addition, the spectrum of the complex exhibits new bands at
1428, 1254, and 850 cmꢀ1, which are assigned to the characteristic
vibrations of the coordinated phenanthroline molecules [42,43].
The diffuse reflectance spectra of complexes 1 and 2 exhibit a
d–d maximum at 590 nm and a shoulder at about 740 nm while
that of complex 3 shows a broad d–d maximum centered at about
630 nm. These bands correspond to ligand field transitions of dis-
torted square planar complexes with CuN4 chromophores [44].
The spectra of DMF solutions of complexes 1–3 show the same
maxima at 630 nm, thus suggesting that all three compounds con-
tain the same chromophore in solution. The shift of the d–d band
position with respect to that in the diffuse reflectance spectra is
probably a consequence of the coordination of solvent molecules.
These results are in agreement with those obtained from the ESI
experiments which show that the copper(II), a sulfonamidate li-
gand, and a phenanthroline species exist as a whole entity.
The EPR spectrum of the complex 1 (Fig. 6) is slightly rhombic.
The EPR parameters obtained by simulation [45] are: gx = 2.042,
gy = 2.063 and gz = 2.233. For systems with gz > gy > gx the ratio
R = (gy ꢀ gx)/(gz ꢀ gy) indicates the ground state of the complex
[46]. The R value of 0.12 for 2 suggests than the unpaired electron
of copper(II) is in the dx2–y2 orbital. The EPR spectra of the com-
plexes 2 and 3 are axial with the following EPR parameters
g|| = 2.218 and 2.210 and g\ = 2.057 and 2.050, respectively. The
g|| > g\ indicates a mainly copper(II) dx2–y2 ground state.
Fig. 2. ORTEP drawing of complex [Cu(L2)(phen)]ꢁH2O (2).
28.70° for 1, 2 and 3, respectively indicates that the copper ion
adopts a significant distorted square-planar geometry. This distor-
tion can be attributed to the presence of two bidentate ligands.
One remarkable aspect to be considered in the crystal
structures of the compounds is the presence of the
p–p stacking
interactions which are similar for complexes 1 and 2 and have dif-
ferent characteristics for complex 3. The structures of complexes 1
and 2 are stabilized by
p–p interactions brought on by intermolec-
ular parallel stacking between the phenanthroline rings
p–p
belonging to adjacent complex molecules (Fig. 4) [33,34,36,37].
The geometric parameters characterizing these interactions are
summarized in Tables 3 and 4. In addition, the structure of
complex 2 is stabilized by an intermolecular hydrogen bond of
moderate strength [38] formed between the O(1) of a water mole-
cule and the O(11) of the sulfonamide group [O(1)ꢁ ꢁ ꢁO(11),
2.841(7) Å, O(1)–H(10B)ꢁ ꢁ ꢁO(11), 168.1°].

A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
2541
Table 2
Selected bond lengths (Å) and angles (°) for complexes 1–3.
Complex 1
Complex 2
Complex 3
Cu(1)ꢀN(1)
1.952(5)
1.972(5)
1.992(5)
Cu(1)ꢀN(1)
1.950(4)
1.951(4)
1.997(4)
Cu(1)ꢀN(1)
1.947(4)
1.977(5)
2.005(5)
Cu(1)ꢀN(2)
Cu(1)ꢀN(2)
Cu(1)ꢀN(2)
Cu(1)ꢀN(31)
Cu(1)ꢀN(41)
Cu(1)ꢀN(41)
Cu(1)ꢀN(41)
2.024(5)
Cu(1)ꢀN(30)
2.012(4)
Cu(1)ꢀN(51)
2.015(4)
N(1)ꢀCu(1)ꢀN(2)
N(1)ꢀCu(1)ꢀN(31)
N(2)ꢀCu(1)ꢀN(31)
N(1)ꢀCu(1)ꢀN(41)
N(2)ꢀCu(1)ꢀN(41)
N(31)ꢀCu(1)ꢀN(41)
83.59(19)
157.43(19)
104.03(19)
104.67(19)
143.21(19)
82.13(19)
N(1)ꢀCu(1)ꢀN(2)
N(1)ꢀCu(1)ꢀN(41)
N(2)ꢀCu(1)ꢀN(41)
N(1)ꢀCu(1)ꢀN(30)
N(2)ꢀCu(1)ꢀN(30)
N(41)ꢀCu(1)ꢀN(30)
85.53(15)
160.56(16)
103.04(15)
97.93(15)
154.24(15)
81.99(15)
N(1)ꢀCu(1)ꢀN(2)
N(1)ꢀCu(1)ꢀN(41)
N(2)ꢀCu(1)ꢀN(41)
N(1)ꢀCu(1)ꢀN(51)
N(2)ꢀCu(1)ꢀN(51)
N(41)ꢀCu(1)ꢀN(51)
85.04(19)
96.28(19)
162.0(2)
159.36(19)
103.3(2)
81.67(18)
3.3. Cyclic voltammetry experiments
The electrochemical profiles of the complexes were studied in
DMF by cyclic voltammetry in the range of 1.0 to ꢀ1.0 V. In the
three cases, the voltammograms show irreversible redox processes
with one cathodic peak at ꢀ0.108 V, ꢀ0.144 V and ꢀ0.103 V for
1–3, respectively (versus NHE) (Fig. S1). All the redox processes
are clearly irreversible suggesting that the copper(I) species
decompose at higher potential. The reduction peaks indicate that
the stability of the copper(II) complex decreases as follows:
2 > 1 ꢃ 3.
3.4. Thermal denaturation studies
Since DNA is the primary pharmacological target of many antitu-
mor compounds [47], the interaction between DNA and metal com-
plexes is of paramount importance in understanding the
mechanism of tumor inhibition for the treatment of cancer. Thus,
the binding of the ligands and the complexes 1–3 to CT-DNA was
studied by measuring changes in the melting temperature (Tm) of
DNA that characterizes the transition from a double-stranded nu-
cleic acid to a single stranded one [48]. The melting curves for com-
plex 1 and the CT DNA are shown in Fig. 7. The three compounds
produced
a large increase in Tm (Tm = 50.8 °C for CT-DNA;
Tm = 62.7, 64.6 and 69.9 °C for 1, 2 and 3, respectively) when the
concentration ratio of DNA to complex is 2.5:1, which indicates a
strong metal complex–DNA interaction that stabilizes the native
DNA conformation [49]. The considerable increase of the melting
temperature of DNA produced by the compounds is also indicative
of an intercalative binding mode involving the phenanthroline mol-
ecule. The stronger interaction of complex 3 may be the result of the
presence of a naphthalene ring in the sulfonamide ligand, which
reinforces its DNA-binding propensity by stacking interaction.
Moreover, our results reveal that the sulfonamide ligands have
not significant effect on Tm of CT-DNA, suggesting that the free sul-
fonamides do not interact with the nucleic acid (data not shown).
Fig. 4. Crystal packing of complex 1 showing the
p
–
p
interactions.
Table 3
Geometric parameters for
p–p stacking (Å/°) in complex 1.
p
–
p
stacking
dc–c
a
b
c
d\[Cg(I)–P(J)]
d\[Cg(J)–P(I)]
Phen–phen
Cg(4)–Cg(4)a
Cg(4)–Cg(4)b
3.7505
3.6682
0.00
0.00
29.52
27.56
29.52
27.56
3.264
3.252
3.264
3.252
dC–C: distance between ring centroids; a: dihedral angle between the rings I and J; b,
c
: slipping angles; d\[Cg(I)–P(J)] and d\[Cg(J)–P(I)]: perpendicular distance of Cg(4)
on the ring Cg(4)a or Cg(4)a.
a
1 ꢀ x, ꢀy, 2 ꢀ z.
3.5. Cleavage of pUC18 DNA
b
2 ꢀ x, ꢀy, 2 ꢀ z. Cg(4): N41–C40–C39–C38–C36–C41.
The DNA endonucleolytic cleavage, activated by metal ions, has
been of interest to researchers [50]. Transition metal-mediated rad-
ical production may result in an efficient DNA cleavage. DNA cleav-
age is controlled by relaxation of the supercoiled circular form of
pUC18 into the nicked circular and linear forms. When circular
plasmid DNA is run in horizontal gel using electrophoresis, the fast-
est migration will be observed for the supercoiled form (Form I). If
one strand is cleaved, the supercoils will relax to produce a slower-
moving open circular form (Form II). If both strands are cleaved, a
linear form (Form III) that migrates in between will be generated.
The ability of complexes 1–3 to cleave DNA was investigated by
means of gel electrophoresis with supercoiled pUC18 in 0.05 M
borate buffer (pH 8.0) and with ascorbate activation. Control
Table 4
Geometric parameters for
p–p stacking (Å/°) in complex 2.
p–
p
stacking
dc–c
a
b
c
d\[Cg(I)–P(J)]
d\[Cg(J)–P(I)]
Phen–phen
Cg(3)–Cg(3)a
Cg(3)–Cg(3)b
3.7923
3.6765
0.00
0.00
29.27
25.35
29.27
25.35
3.308
3.323
3.308
3.323
dC–C: distance between ring centroids; a: dihedral angle between the rings I and J; b,
c
: slipping angles; d\[Cg(I)–P(J)] and d\[Cg(J)–P(I)]: perpendicular distance of Cg(3)
on the ring Cg(3)a or Cg(3)b.
a
2 ꢀ x, 1 ꢀ y, 1 ꢀ z.
b
1 ꢀ x, 1 ꢀ y, 1ꢀz. Cg(3): N30–C31–C32–C33–C34–C43.

2542
A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
Fig. 5. Portion of the crystal packing of complex 3 showing the p–p interactions and the scheme of numeration of the rings.
experiments with CuCl2 were also carried out under the same
experimental conditions. All the complexes were found to exhibit
nuclease activity (Fig. 8) and were more effective at higher concen-
[Cu(phen)2]2+ induces the conversion of DNA into small linear frag-
ments and a considerable smear appears (lane 20).
trations. At 9
lM the complexes were able to transform almost
3.6. DNA cleavage mechanism
completely Form I into Form II. At 12
l
M the compounds induced
complete degradation of the supercoiled Form I to yield open circu-
lar (Form II) and linear DNA (Form III) (lanes 10, 11 for complex 1,
lanes 14, 15 for complex 2 and lanes 18, 19 for complex 3).
Neither the copper salt with ascorbate (lane 4) nor the copper
complexes without ascorbate at the maximum concentration as-
sayed (lanes 5, 6 and 7, respectively) yields significant strand scis-
sion, proving that both the copper complex and the ascorbate are
required for the efficient DNA cleavage. Even though active, the
three complexes exhibit lower nucleolytic efficiency as compared
We used different inhibiting reagents to study various aspects
of the mechanism of pUC18 DNA cleavage induced by the com-
plexes with ascorbate activation (Figs. 9 and 10).
The reduction of the copper(II) complexes during the DNA
cleavage process was evaluated by adding neocuproine, a ligand
that strongly chelates copper(I), to the reaction mixtures. The
to that of the bis(o-phenanthroline)copper(II) complex,
a
well-known and very efficient chemical nuclease. Thus, at 12
lM
Table 5
Geometric parameters for
p–p stacking (Å/°) in complex 3.
Experimental
Simulated
p–
p
stacking
dC–C
a
b
c
d\[Cg(I)–P(J)]
d\[Cg(J)–P(I)]
Phen–phen
Cg(4)–Cg(10)a
Cg(5)–Cg(4)a
3.5623
3.7297
0.42
1.01
15.95
23.69
16.17
22.73
3.421
3.440
3.425
3.415
Phen–‘‘Cu chelate [Cg(3)]”
Cg(3)–Cg(4)a
Cg(3)–Cg(10)a
3.9296
3.6940
2.86
2.66
31.04
21.74
28.29
20.79
3.460
3.454
3.367
3.431
Phen–naphthalene
Cg(5)–Cg(6)b
Cg(5)–Cg(7)b
Cg(6)–Cg(10)b
3.8765
3.5505
3.5820
2.39
0.32
1.60
29.06
19.49
19.03
31.31
19.65
18.83
3.313
3.344
3.390
3.389
3.347
3.386
dC–C: distance between ring centroids; a: dihedral angle between the rings I and J; b,
c
: slipping angles; d\[Cg(I)–P(J)] and d\[Cg(J)–P(I)]: perpendicular distance of Cg(I)
on the ring Cg(J)a or Cg(J)b.
1.05 104
1.1 104
1.15 104
1.2 104
1.25 104
a
1 ꢀ x, 1 ꢀ y, ꢀz.
b
1 ꢀ x, 1 ꢀ y, ꢀz. Cg(3): Cu1–N41–C53–C52–N51; Cg(4): N41–C41–C42–C43–
H gauss
C44–C53; Cg(5): N51–C50–C49–C48–C47–C52; Cg(6): C11–C12–C17–C18–C19–
C20; Cg(7): C12–C13–C14–C15–C16–C17; Cg(10): C44–C45–C46–C47–C52–C53.
Fig. 6. X-band polycrystalline powder EPR spectrum of complex 1.

A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
2543
1.15
1.1
Complex 1
DNA
1.05
1
Fig. 9. Agarose gel electrophoresis of pUC18 plasmid treated with 12
lM complex 2
and 12 mM ascorbate in the presence of increasing concentrations of neocuproine.
Incubation time: 1 h (37 °C). Lane 1: supercoiled DNA; lane 2: supercoiled
DNA + ascorbate; lane 3: 2 without inhibitor; lane 4: 2 + neocuproine (12
lM);
0.95
0.9
lane 5: 2 + neocuproine (120 M); lane 6: 2 + neocuproine (240 M).
l
l
0.85
20
30
40
50
60
70
80
90
100
Temperature ( ºC )
Fig. 7. Thermal denaturation curves of complex 1 and calf thymus DNA.
Fig. 10. Agarose gel electrophoresis of pUC18 plasmid treated with 12 lM complex
3 and 12 mM ascorbate in the presence of potential inhibitors. Incubation time: 1 h
(37 °C). Lane 1: supercoiled DNA; lane 2: supercoiled DNA + ascorbate; lane 3: 3
without inhibitor; lane 4: 3 + DMSO (0.4 M); lane 5: 3 + tert-butyl alcohol (0.4 M);
lane 6: 3 + sodium formiate (0.4 M); lane 7: 3 + DABCO (0.4 M); lane 8: 3 + Tiron
effect of neocuproine on the DNA cleavage induced by the com-
plexes was tested at different concentrations. Results for complex
2 are shown in Fig. 9. The addition of neocuproine at a concentra-
(0.1 M); lane 9: 3 + distamycin (8
3 + catalase (6.5 units).
lM); lane 10: 3 + methyl green (3 lM); lane 11:
tion equivalent to that of the compound (12
attenuate the DNA cleavage; however higher concentrations of
neocuproine (120 and 240 M) (lanes 5 and 6) clearly inhibit the
lM) (lane 4) did not
l
DNA strand scission mediated by 2 which indicates that Cu(I) is
formed in the DNA damage reaction. Complexes 1 and 3 exhibit
a similar behavior (data not shown). Generation of a Cu(I) complex
induced in the presence of ascorbate was expected to lead to the
activation of dioxygen that, in turn, could produce the intermedi-
ate or intermediates responsible for plasmid DNA strand scission.
If the reaction follows a Fenton-like mechanism, several possible
intermediates could be formed, depending on the number of elec-
trons transferred from the complex to O2. The possible intermedi-
is one of the intermediates involved in the DNA scission process.
The addition of DABCO decreased the cleavage efficiency of the
compound (lane 7), which suggests the involvement of singlet oxy-
gen-like entities in the DNA damage. The DNA cleavage was atten-
uated in the presence of Tiron (lane 8) as a consequence of the
involvement of superoxide in the DNA scission. The significant
reduction in the ability of the complex to cleave DNA in the pres-
ence of catalase (lane 11) implies that hydrogen peroxide is neces-
sary for the breakdown of DNA. The cleavage is oxidative in nature
as the enzyme catalase, which lowers solution concentrations of
hydrogen peroxide, inhibited the cleavage reaction. From these re-
sults we concluded that a Cu(I) complex, the hydroxyl radical, sin-
glet oxygen-like species, the superoxide, and hydrogen peroxide
are the active species involved in the DNA cleavage induced by 3.
ates are superoxide anion ðOꢀÞ, hydrogen peroxide (H2O2) or
2
hydroxyl radical (ꢀOH). The three-electron reduced species, hydro-
xyl radical, is a well-accepted oxidant of DNA both free in solution
and coordinated to a metal complex [50]. The presence of free dif-
fusible radicals can be ascertained through their competitive
reduction due to additional hydrogen-atom donors in the reaction
mixture. Thus, we used several radical scavengers that might inhi-
bit the DNA scission induced in the presence of the title Cu(II) com-
plexes. Data for complex 3 are shown in Fig. 10. Complexes 1 and 2
presented the same behavior. The DNA breakdown mediated by
complex 3 was clearly inhibited in the presence of the hydroxyl
radical scavengers DMSO, tert-butyl alcohol and sodium formiate
(lanes 4–6), indicating that the freely diffusible hydroxyl radical
A
metal bound species similar to that proposed for the
+
[Cu(phen)2]2+, often formulated as [CuOH]2 or [CuO]+, remains a
possibility [12,50]. A cupric superoxo (CuII–O2ꢀ) or cupric hydro-
peroxo (CuII–OOH) such as that suggested for peptidylglycine mon-
oxygenase and dopamine b-hydroxylase may also serve as the
active O2-derived species [51–53].
In the presence of the minor groove binder distamycin and
major groove binder methyl green (lanes 9 and 10, Fig. 10), no
Fig. 8. Agarose gel electrophoresis of pUC18 plasmid DNA, in the presence of the complexes 1–3 or CuCl2, with or without ascorbate activation. Incubation time: 1 h (37 °C).
Lane 1: kDNA/EcoRI + Hind III Marker; lane 2: supercoiled DNA; lane 3: supercoiled DNA + ascorbate 12 mM; lane 4: 12 M CuCl2 + 12 mM ascorbate; lane 5: 12 M 1
without ascorbate; lane 6: 12 M 2 without ascorbate; lane 7: 12 M 3 without ascorbate; lane 8: 3 M 1 + 3mM ascorbate; lane 9: 6 M 1 + 6 mM ascorbate; lane 10: 9
1 + 9 mM ascorbate; lane 11: 12 M 1 + 12 mM ascorbate; lane 12: 3 M 2 + 3 mM ascorbate; lane 13: 6 M 2 + 6mM ascorbate; lane 14: 9 M 2 + 9 mM ascorbate; lane 15:
12 M 2 + 12 mM ascorbate; lane 16: 3 M 3 + 3mM ascorbate; lane 17: 6 M 3 + 6 mM ascorbate; lane 18: 9 M 3 + 9 mM ascorbate; lane 19: 12 M 3 + 12 mM ascorbate;
lane 20: 12
M [Cu(phen)2]2+ + 12 mM ascorbate.
l
l
l
l
l
l
lM
l
l
l
l
l
l
l
l
l
l

2544
A. Bodoki et al. / Polyhedron 28 (2009) 2537–2544
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Their interaction with DNA has been studied with the aid of Tm
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Acknowledgements
G.A., J.B. acknowledge financial support from the Spanish Com-
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A.C. thanks CICYT (CTQ2006-15329-C02/BQU) for financial
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