Interaction with DNA and different effect on the nucleus of cancer cells for copper(ii) complexes of N-benzyl di(pyridylmethyl)amine

Article abstract of DOI:10.1039/c0dt01616k

Three new copper(ii) complexes of N-benzyl di(pyridylmethyl)amine (phdpa) were synthesized and characterized by spectroscopic methods. The interaction between CT-DNA and the complexes was studied by UV and fluorescence titration methods. It was found that the complex [(phdpa)Cu(H₂O)Ac)](Ac), with the non-planar aromatic heterocyclic ring ligand (phdpa), showed good anticancer properties and could cause the fragmentation of the nucleus, although its interaction with CT-DNA was weaker than that of 1,10-phenanthroline (phen) -based copper(ii) complexes. The anticancer activities of copper(ii) complexes with phdpa and phen based ligands are correlated to their binding constants with DNA, but phen-based copper(ii) complexes did not cause the nucleus fragmentation of HeLa cells. [(phdpa)Cu(H₂O)Ac)](Ac) can noticeably decrease the oxygen content of a culture solution and of HeLa cells, which make it a new nucleus and oxygen related anticancer copper(ii) complex. Information obtained here would be helpful in the design of new antitumor complexes in oxidative therapy.

Full text of DOI:10.1039/c0dt01616k

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PAPER
Interaction with DNA and different effect on the nucleus of cancer cells for
copper(II) complexes of N-benzyl di(pyridylmethyl)amine†
Qiu-Yun Chen,*a Hai-Jian Fu,a Wei-Hua Zhu,a Yan Qi,a Zheng-Ping Ma,a Kai-Di Zhaob and Jing Gaob
Received 21st November 2010, Accepted 11th February 2011
DOI: 10.1039/c0dt01616k
Three new copper(II) complexes of N-benzyl di(pyridylmethyl)amine (phdpa) were synthesized and
characterized by spectroscopic methods. The interaction between CT–DNA and the complexes was
studied by UV and fluorescence titration methods. It was found that the complex
[(phdpa)Cu(H2O)Ac)](Ac), with the non-planar aromatic heterocyclic ring ligand (phdpa), showed
good anticancer properties and could cause the fragmentation of the nucleus, although its interaction
with CT–DNA was weaker than that of 1,10-phenanthroline (phen) -based copper(II) complexes. The
anticancer activities of copper(II) complexes with phdpa and phen based ligands are correlated to their
binding constants with DNA, but phen-based copper(II) complexes did not cause the nucleus
fragmentation of HeLa cells. [(phdpa)Cu(H2O)Ac)](Ac) can noticeably decrease the oxygen content of
a culture solution and of HeLa cells, which make it a new nucleus and oxygen related anticancer
copper(II) complex. Information obtained here would be helpful in the design of new antitumor
complexes in oxidative therapy.
strength of DNA with metal complexes related to its antitumor
Introduction
activities? Whether the antitumor properties of copper(II) com-
Copper complexes have been used as metallopharmacons receiv-
plexes is related to their nucleus-binding capabilities, needs to be
ing special attention in relation to their action on DNA.1 Copper,
further examined. In an effort to throw some light on the structure–
as an essential element, may be less toxic than non-essential metals
activity relationship of this class of copper complexes, we report
such as platinum.2 A number of copper complexes have been
the synthesis, characterization, interaction with DNA, effect on
screened for anticancer activity, and some of them were found to be
the nucleus of cancer cells and antitumor activities of Cu(II)
active both in vitro and in vivo.3,4 The copper(II) complexes of 1,10-
complexes with N-benzyl bis(2-pyridylmethyl)amine (phdpa).
phenanthroline (phen) based ligands can inhibit DNA synthesis.5
Recently, a number of copper complexes, which were mimics of
non-heme dioxygenases, have been shown to induce apoptosis in
Results and discussion
cultured mammalian cells.6,7 Di(picolyl)amine (dpa) and its deriva-
General characterization
tives were used as neutral, nondeprotonated chelating ligands to
complex copper(II) atoms to mimic non-heme dioxygenase.8,9 N-
Substituted di(picolyl)amines and their copper(II) complexes were
found to be active against the proliferation of cancer cells.10 The
family of [(etdpa)Cu(phen)](ClO4)2 complexes are more active
against the proliferation of cancer cells than the [(dpa)CuCl2]
(etdpa = ethyl 2-[bis(2-pyridylmethyl)amino]propionate) due to
their different interactions with DNA.11 DNA is the primary target
molecule for most anticancer complexes. Thus, investigations on
the interactions of DNA with metal complexes are meaningful to
design new types of pharmaceutical molecules.4,12 Is the binding
Copper(II) complexes were prepared in good yields and char-
acterized by elemental analysis, IR and UV spectra. The re-
sults suggested that the composition of the complexes was
[(phdpa)Cu(H2O)(Ac)](Ac) (1), [(phdpa)Cu(bpy)(ClO4)]ClO4 (2)
and {[(phdpa)Cu(phen)(H2O)](ClO4)2}2(H2O) (3), respectively
(bpy = 4,4-dimethyl-2,2-bipyridine). The IR spectra of the free
ligand (phdpa) shows two pyridyl ring bands at approximately
1589 and 1569 cm-1 and the d(CH) vibration of the pyridyl ring at
~762 cm-1.13 These bands are all shifted in complexes (1–3). The
Cu(II) complexes (1), (2) and (3) showed the characteristic imine
nC band at 1609 cm-1, 1619 cm-1, and 1628 cm-1 respectively,
N
aSchool of Chemistry and Chemical Engineering, Jiangsu University, Zhen-
jiang, 212013, P. R. China. E-mail: chenqy@ujs.edu.cn
which shifted to lower frequencies due to metal coordination. The
pyridyl bands (d(CH)) were found at approximately 769, 772 and
776 cm-1, respectively. These shifts indicate that the nitrogen atom
of the pyridyl rings donates a pair of electrons to the central metal,
forming a coordinate bond. The strong broad peaks at 3428 cm-1
and 3502 cm-1 for complexes (1) and (3) indicate the existence of
bSchool of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China
† Electronic supplementary information (ESI) available: DSC figure, UV
data and crystal structure parameters. CCDC reference numbers 766526
and 782308. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c0dt01616k
4414 | Dalton Trans., 2011, 40, 4414–4420
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Table 1
the coordinated water molecules. The strong and wider band at
1578 cm-1 and the band at 1403 cm-1 for complex (1) belong to the
asymmetric stretching nas(COO-) frequencies and the symmetric
vibration nsym(COO-), respectively. The Dn(COO-) between the
observed asymmetric nas(COO-) and the symmetric ns(COO-)
bands could provide important information about the different
binding modes of the CH3COO- group.18 The difference between
nas(COO-) and nsym(COO-) is about 175 cm-1, suggesting that the
acetate groups coordinate to the metal atoms only as bidentate
ligands. The bands at 1095, 1094 cm-1 for (2) and (3) are assigned
◦
˚
(a) Selected bond lengths (A) and bond angles ( ) for (2)
Bond distances
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
2.314(4)
2.040(4)
2.016(4)
Cu(1)–N(4)
Cu(1)–N(5)
Cu(1)–O(1)
2.000(4)
2.006(4)
2.647(4)
Bond angles
N(4)–Cu(1)–N(5)
N(4)–Cu(1)–N(3)
N(5)–Cu(1)–N(3)
N(4)–Cu(1)–N(2)
N(5)–Cu(1)–N(2)
N(1)–Cu(1)–O(1)
(b) Selected bond lengths (A) and bond angles ( ) for (3)
Bond distances
Cu(1)–N(1)
Cu(1)–N(2)
80.79(17)
97.42(17)
177.44(17)
174.51(17)
94.26(17)
164.8(17)
N(3)– Cu(1)–N(2)
N(4)–Cu(1)–N(1)
N(5)–Cu(1)–N(1)
N(3)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
87.45(17)
103.98(16)
101.66(17)
80.53(17)
79.25(16)
-
to the free ClO4 ion. To assess the lipophilicity of the copper(II)
complexes, the partition coefficients (Pow) in an octanol–water
system were determined (Table S1†). The Pow values increase in
the order 1 < 2 < 3 with 0.12 for 1, 1.53 for 2 and 2.36 for 3.
◦
˚
2.014(4)
2.030(4)
2.008(4)
Cu(1)–N(4)
Cu(1)–N(5)
Cu(1)–O(9)
2.294(4)
2.015(4)
2.490(4)
Cu(1)–N(3)
Bond angles
Thermal analysis and electronic spectra
N(4)–Cu(1)–N(5)
N(4)–Cu(1)–N(3)
N(5)–Cu(1)–N(3)
N(4)–Cu(1)–N(2)
N(5)–Cu(1)–N(2)
N(4)–Cu(1)–O(9)
80.92(14)
80.10(16)
88.27(17)
100.34(14)
177.89(15)
172.94(17)
N(3)– Cu(1)–N(2)
N(4)–Cu(1)–N(1)
N(5)–Cu(1)–N(1)
N(3)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
93.60(17)
102.28(15)
96.02(16)
175.36(16)
82.07(16)
The existence of coordinated water molecules was further con-
firmed by the thermal analysis data for (1) (Fig. S1†). The thermal
decomposition of (1) proceeds with two decomposition steps. The
◦
first step falls in the range of 0–150 C, which is assigned to the
loss of one coordinated H2O molecule with a weight loss of 3.75%
(calcd 3.68%). The 71.63% weight loss in the range of 150–450 ◦C
is assigned to the loss of the ligand (phdpa) and one coordinated
CH3COO- group (calcd 71.16%). The thermal decomposition
data obtained supports the proposed structure of the complex
(1). The electronic spectra in the ultraviolet and visible ranges
for complexes (1), (2), and (3) were carried out in water solution
(Table S2†). The strong band at 254 nm for (1) and three bands
in the range of 206–308 nm for (2) and (3) are attributed to the
p–p* transition of ligands. Three copper complexes all exhibit
visible spectra with a single broad band in the range of 597–
660 nm, characteristic of a copper(II) d–d transition in a tetragonal
ligand field, in which the copper(II) has a distorted octahedral
coordination environment.
one oxygen atom O9 of a water molecule resulting in a distorted
octahedron geometry, which is similar to (2). The N1, N2, N3,
N5 and Cu1 form the equatorial plane with a deviation of 0.0092,
while the N4 and O9 occupy the apical positions. The copper(II)
atom is located nearly in the center of this plane. The bond
˚
distances of Cu1–N4 and Cu1–O9 are 2.294(4) A and 2.490(4)
˚
A, indicating that the O9 is weakly coordinated to Cu1. The
N1–Cu1–O9, N1–Cu1–N3, N2–Cu1–N5 angles are 172.94(17)◦,
175.36(16)◦, 177.89(15)◦, respectively.
Investigation of interaction with CT–DNA by electronic
spectroscopy
The binding ability of complexes (1), (2) and (3) with CT–DNA
was investigated by looking at the changes in absorbance and
shifts in the wavelength of their electronic spectra. The absorption
spectra of complexes (1) and (3) with increasing concentration
of CT–DNA are shown in Fig. 2 and Fig. S2.† It was observed
that the addition of CT–DNA to a solution of the complexes
induced remarkable bathochromic shifts and hypochromicities.
The absorption bands of (1–3) at 200 nm and 260 nm exhibited
bathochromic shifts of 12 nm, 14 nm, 17 nm and hypochromism of
2%, 3%, 5%, respectively. The bathochromic shift indicates that the
p* orbital of the phdpa couples with the p orbital of the base pairs,
thus reducing the p–p* transition energy. These results suggest the
existence of an association between these three complexes and CT–
DNA. So we primarily speculate that the complexes interacting
with CT–DNA have the same mode of electrostatic effect or
intercalation. Ligand (phdpa) in the three complexes containing a
benzyl group and several methylene groups, can bind to DNA by
van der Waals interactions.20
Crystal structure of complexes (2) and (3)
The molecular structure of [(phdpa)Cu(bpy)(ClO4)](ClO4) (2) with
the atomic labeling scheme is shown in Fig. 1(a) and selected bond
lengths and angles are listed in Table 1(a). The copper atom is
six coordinated by three N atoms of phdpa (N1, N2, N3), two
-
N atoms of bpy (N4, N5) and one oxygen atom (O1) of ClO4
resulting in a distorted octahedral geometry, which is different
from the trigonal bipyramidal complex [Cu(apme)(Cl)](BPh4)
(apme = tris(2-pyridylmethyl)amine).19 The N2, N3, N4 and N5
form the equatorial plane with a deviation of 0.0051, while the
N1 and O1 occupy the apical positions. The copper(II) atom is
˚
located nearly in the center of this plane (shifted by 0.038 A out of
the equatorial plane towards N3). The bond distances of Cu1–N1
˚
˚
and Cu1–O1 are 2.314(4) A and 2.647(4) A, indicating that the
O1 is weakly coordinated with Cu1. The N1–Cu1–O1, N2–Cu1–
N4, N3–Cu1–N5 angles are 168.48(17)◦, 174.51(17)◦, 177.44(17)◦,
respectively.
The molecular structure of {[(phdpa)Cu(phen)(H2O)]-
(ClO4)2}2(H2O) (3) with the atomic labeling scheme is shown in
Fig 1(b) and the selected bond lengths and angles are listed in
Table 1(b). The copper atom is six coordinated by three N atoms
of phdpa (N3, N4, N5), two N atoms of phen (N1, N2) and
Since complex (1) contains a non-planar ligand and possesses a
smaller p system than phenanthroline, its classical intercalation is
weak.21 Electrostatic binding and van der Waals interactions may
be the main binding mode between (1) and DNA.22 The complex
(2) shows weak binding to the DNA due to less extended planarity
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Fig. 2 Electronic spectra of complex (1) (20 mM) in the presence of
increasing amounts of CT–DNA (b–f); DNA concentrations are 0, 10,
20, 30, 40 and 50 mM for spectra (a–f), respectively. The arrow indicates
the absorbance changes upon increasing DNA concentration.
Fig. 3 Fluorescence spectra of DNA–EB in the presence of complex (1)
at 0◦. The total concentrations of the complexes are (a) 0, (b) 10.0, (c) 20.0,
(d) 30.0, (e) 40.0, (f) 50.0 mM. The EB and DNA concentrations are 0.72
and 20 mM respectively.
Fig. 1 (a) Crystal structure of [(phdpa)Cu(bpy)(ClO4)]+ (2). Thermal
ellipsoids are drawn at 30% probability. Hydrogen atoms are omitted. (b)
Crystal structure of [(phdpa)Cu(phen)(H2O)]2+ (3). Thermal ellipsoids are
drawn at 30% probability. Hydrogen atoms are omitted.
log((F0 - F)/F) = nlogKA + nlog([Dt] - n[Nt](F0 - F)/F0) (1)
where [Nt] and [Dt] are the total concentrations of the DNA–EB
complex and the complex, respectively. On the assumption that
n in the bracket is equal to 1, the curve log(F0 - F)/F versus
log([Dt] - n[Nt](F0 - F)/F0) was drawn and fitted linearly, giving
the slope n. If n was not equal to 1, then it was substituted into the
bracket and the curve log(F0 - F)/F versus log([Dt] - n[Nt](F0 -
F)/F0) was drawn again. Further iterations were used to obtain
a constant value of n. Based on the intercept and n, KA can
be obtained. The binding constants are listed in Table 2. These
KA values followed the order: (3) > (2) > (1). This suggests that
complexes (2) and (3) have a greater affinity for CT–DNA than
does (1). The binding constants of the complex (1) increase greatly
with temperature suggesting that its binding reaction with DNA is
endothermic.
compared to phen, which is consistent with the observed trend in
hypochromism. The obvious hypochromism in complexes (2) and
(3) is possibly caused by the intercalation binding mode between
the complexes and CT–DNA. Intercalation involves the partial
insertion, between aromatic heterocyclic rings of the ligands,
of the DNA base pair.23 The higher binding propensity of the
complex (3) in comparison to the complex (2) may be due to
the presence of extended planar aromatic rings facilitating non-
covalent interactions with the DNA molecule. In order to test if
complexes could bind to DNA by intercalation, ethidium bromide
(EB) was employed.24 The fluorescence intensity of the DNA–EB
system decreased upon the addition of complexes (1), (2) and (3)
(Fig. 3, Fig. S3† and Fig. S4†). The emission of the DNA–EB
system increased with increasing amounts of the three complexes.
According to the linear Stern–Volmer equation, the Ksv values
were obtained (Table 2). The binding constants of complexes (1–
3) with DNA in the presence of EB were determined using the
following relationship:25
Inhibition on the proliferation of cancer cells
Copper(II) complexes were reported to inhibit the proliferation
of the cancer cells A549, Hela and HepG-2.6,26 Complexes (1),
(2) and (3) were studied for their antitumor activity in vitro by
determining the inhibitory percentage against growth of the cancer
4416 | Dalton Trans., 2011, 40, 4414–4420
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Table 2 The quenching constant, binding constant and binding site of
the complexes (1–3) with DNA at various temperatures
Complex
Temperature (◦)
Ksv (M-1)
KA (M-1)
n
(1)
0
890
9.85 ¥ 103
1.21 ¥ 104
1.36 ¥ 104
3.08 ¥ 104
3.69 ¥ 104
5.06 ¥ 104
6.41 ¥ 104
8.20 ¥ 104
10.98 ¥ 104
1.11
1.39
1.36
1.25
1.37
1.41
0.97
1.27
1.32
15
35
0
15
35
0
1.18 ¥ 103
1.27 ¥ 103
2.78 ¥ 104
3.12 ¥ 104
4.35 ¥ 104
5.5 ¥ 104
8.78 ¥ 104
14.6 ¥ 104
(2)
(3)
15
35
Fig. 4 The change in nucleus morphology of HeLa cells. H2B-labelled
HeLa cells were exposed to 10 mM of the complexes for 24 h; change in
the nuclei was observed.
Table 3 IC50 of the complexes on four cancer cell lines (mM)
Effect on the O2 consumption of HeLa cells
Copper(II) complexes of di(picolyl)amine derivatives have been
widely used as models of non-heme dioxygenase.8,9 The effect on
the oxygen content of a culture solution (without HeLa cells) for
complexes (1) and (3) in the 120–600 s range is shown in Fig. 5.
Experimental results show that the oxygen content decreased at a
rate of 3.142 nmol mL-1 min-1 when complex (1) (20mM) was added
in 120–600 s, but the oxygen content change of the culture solution
was very similar to that of the blank for the complex (3) (20mM).
This indicates that complex (1) could bind with oxygen or catalyze
an oxygenation reaction leading to the obvious decease of oxygen
in solution. The effect of the O2 consumption for complexes (1)
and (3) on the HeLa cells is shown in Fig. 6. Experimental data
show that the complex (1) (20 mM) can obviously decrease the
O2 consumption of HeLa cells in 6 h. Although the complex (3)
exhibits almost no effect on the oxygen content of the culture
solution, it did decrease the oxygen consumption of HeLa cells.
This was possibly due to the fact that complex (3) could act on
the HeLa cells leading to a change in the cell respiration or in
an oxygen involved metabolite. The oxygen consumption rate of
HeLa in the presence of (3) was smaller than that in the presence
of complex (1).
Complex
HepG-2
A549
HeLa
(1)
13.4
17.4
3.1
13.1
14.2
3.1
3.3
15.1
3.3
32
(2)
(3)
5-Fluorouracil
32
32
*IC50 is the average data of a triplicate assay. SD is 0.07.
cells A549, Hela, and HepG-2 using the 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenpyltetrazolium bromide reduction method (MTT
method). The IC50 data for complexes (1–3) are shown in Table 3.
Complex (3) was active against the three cancer cells, with IC50
values in the range of 3.1–3.3 mM, which was similar to the
reported [Cu(CH3COO)2(phen)] (IC50, 1.8 mM and 1.19 mM to
A549 and Hela cells).26 The IC50 values (14.2–17.4 mM) of complex
(2) are a little smaller than that of complex (3), which corresponds
to the DNA binding force of the complexes. This indicates that
phen may contribute greatly to the antitumor activities of the
phen-based copper(II) complexes (2) and (3), which is similar to
the previously reported [(etdpa)Cu(phen)](ClO4)2.11 The complex
(1) also shows good inhibition on the proliferation of the three
cancer cells although its DNA binding force is smaller than those
of complexes (2) and (3). Experimental results indicate that the
antitumor activities of the complex (1) do not correlate well with
its DNA binding properties.
The morphology of drug-treated cells was used to determine
the extent of the cytological effects. After incubating for 24 h
with the complexes, HeLa cells were stained with DAPI, which
is specific for the visualisation of nuclear morphology and
detection of DNA condensation. It was interesting to find that
the complex (1) could cause the fragmentation of the nucleus
in HeLa cells, but only a few condensed nuclei in the HeLa
cells were observed when complex (3) was added (Fig. 4).
These results indicate that the anticancer properties of copper(II)
complex (1), with ligands containing non-planar rings, are nucleus
relevant, just like those of cisplatin.27 Copper complexes with dpa
derivatives [Cu(HPClNOL)Cl]Cl [HPClNOL = 1-(bis-pyridine-2-
ylmethylamino)-3-chloropropan-2-ol] were found to promote cell
death of THP-1 cells by apoptosis.5 Nucleus condensation or
fragmentation is a characteristic of apoptosis. Obvious nucleus
fragmentation was induced by complex (1) indicating that this
complex could possibly promote the cell death of HeLa cells by
apoptosis. The detailed anticancer mechanisms of complexes (1)
and (3) need further investigation.
Fig. 5 Effect of complexes on the oxygen content of a culture solution in
120–600 s, a) complex 1 (20 mM); b) blank; c) complex 3 (20 mM)).
Oxygen scarceness limits the growth of tumors, because cancer
cells—like most other cells in the organism—utilize oxygen to
generate energy (at least in part) and as a substrate for a number
of fundamental biochemical reactions including the synthesis of
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The C, H and N microanalyses were performed on Vario EL
elemental analyzer. Metal ions were determined using a Vista
ICP-OES instrument. Electronic absorption spectra were recorded
in the 900–190 nm region using a Varian Cary 50-BIO UV-VIS
spectrophotometer. IR spectra were recorded on a Nicolet-470
spectrophotometer in the range 4000–400 cm-1. Fluorescence was
measured with a Fp-750w Fluorometer.
Synthesis of [(phdpa)Cu(H2O)Ac)](Ac) (1)
A solution of CuAc2 (199 mg, 1 mmol) in ethanol (10 cm3) was
added to a stirred ethanol solution (10 cm3) of phdpa (289.0 mg,
1 mmol). The mixture was heated at 75 ◦C for 1.5 h, then cooled
to room temperature. To the resulting mixture was added 20.0 cm3
ethyl ether and it was then filtered. A blue solid was obtained.
Yield: 69%. Analytically calcd (%) for C23H27CuN3O5: C, 56.49;
H, 5.57; N, 8.59, Cu, 12.99; found: C, 56.58; H, 5.51; N, 8.65, Cu,
12.87. IR (KBr pellet, cm-1): 3428(s), 3074(m), 3027(m), 2923(m),
1609(s), 1578(s), 1478(s), 1441(s), 1403(s), 1336(m), 769(s).
Fig. 6 Effect on the O2 consumption of HeLa cells of complexes (1) and
(3), A: control; B: cccp, 20 mM, 6 h; C: (1), 20 mM, 6 h; D: (1), 20 mM, 6 h;
E: (3), 20 mM, 12 h; F: (3), 40 mM, 12 h.
macromolecules.28,29 So we deduced that strong interference in the
oxygenation of HeLa cells was a factor for the antitumor activity
of complex (1). Oxidative therapy is a relatively new anticancer
strategy.30 Information obtained here indicates that complex (1)
may be a potential anticancer complex for oxidative therapy in the
future.
Synthesis of [(phdpa)Cu(bpy)(ClO4)](ClO4) (2)
A green solution of Cu(ClO4)2·6H2O (370.5 mg, 1 mmol) in
ethanol (10 cm3) was added to a stirred ethanol solution (10 cm3) of
phdpa (289.0 mg, 1 mmol). The mixture was heated at 80 ◦C for 1 h,
then the ethanol solution (10 cm3) of bpy (184.1 mg, 1 mmol) was
added and the resulting mixture was stirred at 80 ◦C for another
1 h. On cooling the solution to room temperature, the mixture was
filtered and the filtrate on concentration yielded a blue crystalline
solid. Yield: 55%. Analytically calcd (%) for C31H31Cl2CuN5O8:
C, 50.58; H, 4.25; N, 9.51, Cu, 8.63; found: C, 50.64; H, 4.19; N,
9.60, Cu, 8.54. IR (KBr pellet, cm-1): 3066(m), 3037(m), 2911(m),
1619(s), 1481(s), 1442(s),1356(m), 1095(s), 772(s).
Conclusion
Copper(II) complex {[(phdpa)Cu(phen)(H2O)](ClO4)2}2(H2O) (3)
with phdpa and phen based ligands shows strong binding
with DNA and good anticancer activities, but it does not
cause the nucleus fragmentation of HeLa cells. The complex
[(phdpa)Cu(H2O)Ac)](Ac) (1), with non-planar aromatic hetero-
cyclic ring-containing ligand phdpa, also shows good anticancer
properties and can cause the fragmentation of the nucleus
although its binding constant with CT–DNA is weaker than that
of the phen-based copper(II) complexes (2) and (3). Hence, we
conclude that the inhibition of (1), with non-planar aromatic
heterocyclic rings, of the cancer cells is not related to its strength
of binding to DNA. The complex (1) could bind with oxygen or
catalyze an oxygenation reaction leading to an obvious decease of
oxygen in solution. Experimental results show that the complex
(1) is a new nucleus and oxygen related anticancer copper(II)
complex, different from phen-based copper(II) complexes (2) and
(3). Oxidative therapy is a relatively new anticancer strategy.
Information obtained here will be helpful in the search for new
antitumor complexes for oxidative therapy.
Synthesis of {[(phdpa)Cu(phen)(H2O)](ClO4)2}2(H2O) (3)
A green solution of Cu(ClO4)2· 6H2O (370.5 mg, 1 mmol) in
ethanol (10 cm3) was added to a stirred ethanol solution (10 cm3)
◦
of phdpa (289.0 mg, 1 mmol). The mixture was heated at 80 C
for 1 h, then the ethanol solution (10 cm3) of phen (182.2 mg,
1 mmol) was added, and the resulting mixture was refluxed for
another 1 h. Cooling the solution to room temperature, a blue
solid product was obtained. Yield: 68%. Analytically calcd (%) for
C62H60Cl4Cu2N10O19: C, 49.05; H, 3.98; N, 9.23, Cu, 8.37; found: C,
49.42; H, 3.85; N, 9.27; Cu, 8.48. IR (KBr pellet, cm-1): 3502(s, br),
3091(m), 3067(m), 2925(m), 1628(s), 1521(s), 1431(s, br), 1344(m),
1094(s), 776(s).
X-Ray crystallography
Experimental
Crystallographic data for (2) and (3) are listed in Table S3.†
The blue prism crystals of the complexes were selected for lattice
parameter determination and collection of intensity data at 298 K
on a Rigaku Mercury2 CCD Area Detector with monochroma-
Materials and methods
All products were obtained from commercial sources and
were used without further purification. N-benzyl di(pyridyl-
methyl)amine (phdpa) was synthesized according to reported
procedures.13 4,4-Dimethyl-2,2-bipyridine (bpy) was purchased
from Sigma Aldrich. Tris(tris(hydroxymethyl)aminomethane)
(tris), CT–DNA, and ethidium bromide (EB) were obtained from
Sigma (USA). Water was purified with a Millipore Milli-Q system.
˚
tized Mo Ka radiation (l = 0.71073 A). The data was corrected
for Lorenz and polarization effects during data reduction. A
semi-empirical absorption correction from equivalents based on
multi- scans was applied. The structure was solved by direct
methods and refined on F2 by full-matrix least-squares methods
using SHELXTL version 5.10.14 All non-hydrogen atoms were
4418 | Dalton Trans., 2011, 40, 4414–4420
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©

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refined anisotropically. Hydrogen atoms were introduced in their
calculated positions. All computations were carried out using the
SHELXTL-PC program package.
dissolved in H2O or DMSO and diluted with culture media. After
24 h, the compounds were added, and the samples kept for 48 h.
Cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenpyltetrazolium bromide (MTT) assay by measuring the
absorbance at 570 nm with an ELISA reader. IC50 values were
calculated using software provided by Nanjing university. Each
test was performed in triplicate.
Partition coefficients of complexes in 1-octanol–water
Partition coefficients were measured at 25 ◦C. Briefly, water
(5 cm3) saturated with 1-octanol was added to each vial containing
weighed amounts of a copper(II) complex. Then 1 cm3 of each of
these preparative solutions was pipetted into three cuvettes. To
each cuvette, 1-octanol (9 cm3) saturated with water was added.
These solutions were then stoppered and shaken for 3 h, set aside
Nuclei observation on H2B-GFP-labelled HeLa cells
H2B-labelled HeLa cells were plated in a 24-well plate at a density
of 2.4 ¥ 104 cells mL-1. After incubating for 24 h with the
complexes, the cells were stained with DAPI, and nuclei change
was visualized under a fluorescence microscope (Nikon TE2000
inverted microscope).
◦
to allow the two phases to separate at 25 C. Concentrations of
copper(II) complexes in the aqueous phase before (C0) and after
partitioning (Cw) were evaluated by spectrophotometry using the
pyridyl ring bands (259 nm) of ligands. Based on the concentration
ratio of each complex between the 1-octanol and water phases, the
partition coefficients were calculated as:
Oxygen detection
The polarographic signal from a standard Clark oxytherm
(Hansatech Ltd.) separated from the sample solution by a 12.7 mm
thick Teflon membrane was recorded for the entire duration of the
experiments at 1 s intervals. Air saturated water solutions ([O2] =
253 mM) were used for the calibration of the electrode. The volume
of the solution at the beginning of each experiment was 1 mL,
thermostated inside the double walled cell at 37 ◦C. Complexes
were freshly dissolved before each experiment in water (or mixtures
of DMSO–H2O (1 : 1) for complex 3). The final concentration of
the complexes was 20 mM in each Clark cell. After an equilibration
time of 2 min (120 s), oxygen concentration was monitored.
Oxygen change were determined by a linear fit of the Clark cell
data in the 120–600 s range after the complex was added.
Pow = (C0V0 - CwVw)/CwVw
(2)
DNA binding experiments
The application of electronic absorption spectroscopy in DNA-
binding studies is one of the most useful techniques. All the
experiments involving the interaction of complexes with CT–DNA
were conducted in buffer solution (50 mM NaCl, 5 mM Tris-HCl,
pH 7.1) at room temperature. A solution of CT–DNA in Tris-HCl
buffer solution gave ratios of UV absorbance at 260 and 280 nm
of about 1.9, indicating that the DNA was sufficiently free of
protein.15 The DNA concentration was determined by absorption
spectroscopy using the molar absorption coefficient (6600 M cm-1)
at 260 nm.16 Absorption titration measurements were done by
varying the concentration of CT–DNA–buffer solution, keeping
the fixed complex concentration constant in Tris–HCl buffer
(pH 7.11). For fluorescence quenching experiments, initially the
CT–DNA was pretreated with ethidium bromide (EB) for 0.5 h
and the complexes were added to this mixture and their effect
on the emission intensity was measured at different temperatures.
According to the Stern–Volmer equation, the quenching nature of
the DNA–complex in the presence of EB can be analyzed17 as:
Oxygen consumption of HeLa
HeLa cells were seeded at a density of 1 ¥ 105 cells mL-1 into
sterile 96 well plates and grown in 5% CO2 at 37 ◦C for 24 h. Test
compounds were dissolved in H2O or DMSO and diluted with
culture media. After 24 h, the compounds were added. Samples
were transferred into Clark cells. After an equilibration time of
2 min (120 s), oxygen concentration was monitored by Clark
oxytherm. The oxygen consumption rate was determined based on
counting the cell numbers and density (nmol (ml-1 min-1 105cells)).
Each test was performed in triplicate.
F0/F = 1 + Ksv[Q]
(3)
where, F0 and F are the fluorescence intensities of the DNA–
EB complex in the absence and presence of complex, [Q] is
the complex concentration, Ksv is the Stern–Volmer dynamic
quenching constant.
Supplementary material
Crystallographic data for the structural analysis have been de-
posited with the Cambridge Crystallographic Data Centre, CCDC
No. 766526, 782308 for complexes (2) and (3). The data can be
obtained free of charge via http://www.ccdc.cam.ac.uk, or from
the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB21EZ, UK; fax: (+ 44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
Cytotoxicity testing
The cytotoxicity assay used three kinds of cell lines (hepatocellular
carcinoma HepG-2, human cervical cancer HeLa cells and human
lung adenocarcinoma A549 cells). Cells were cultured in RMPI
1640 medium containing 4.8 g L-1 of Hepes, 2.2 g L-1 NaHCO3
and supplemented with penicillin/streptomycin (1000 units cm-3),
and 10% calf serum. HeLa cells were cultured in DMEM medium
containing 10% fetal bovine serum. All cells were grown at 37 ◦C
in a humidified atmosphere in the presence of 5% CO2. A549 and
HeLa cells were seeded at a density of 4 ¥ 104 cells mL-1 into sterile
96 well plates and grown in 5% CO2 at 37 ◦C. Test compounds were
Acknowledgements
Financial support from National Science Foundation of China
(20971059) and distinguished scholar science foundation of
Jiangsu University (06JDG050) and the foundation of state key
Laboratory of Coordination Chemistry, Nanjing University.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 4414–4420 | 4419
©

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16 J. K. Barton, A. T. Danishefsky and J. M. Golderg,, J. Am. Chem. Soc.,
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4420 | Dalton Trans., 2011, 40, 4414–4420
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©