Synthesis, characterization, and bioactivities of copper complexes

with N-substituted Di(picolyl)amines

Lin-Yun Wang Qiu-Yun Chen Juan Huang
Kun Wang Chang-Jian Feng Zhi-Rong Gen
Received: 24 November 2008 / Accepted: 27 January 2009 / Published online: 24 February 2009
Springer Science+Business Media B.V. 2009
Abstract Three new Cu(II) complexes with ethyl bis(2-
pyridylmethyl)amino-2-propionate (Etdpa), or bis(2-pyri-
dylmethyl)amino-2-propionate (Adpa), were synthesized
and characterized by physico-chemical and spectroscopic
methods. The X-ray crystal structure of [(Adpa)CuCl] shows
that the copper(II) atomis coordinated by three Natoms, one
oxygen atomfromthe ligand (Adpa) and one chloride anion,
forming a trigonal bipyramidal geometry. The spectropho-
tometric and uorescence titration data indicate that the
interaction of square pyramidal [(Etdpa)CuCl
2
] with ct-DNA
is weak, but the trigonal bipyramidal complexes [(Adpa)
Cu(H
2
O)](ClO
4
) and [(Adpa)CuCl] interact with ct-DNA
with the mode of intercalation. The inhibition activities of the
three newcopper(II) complexes on the four cancer cells (Mcf-
7, Eca-109, A549, and Hela) are in the order: [(Adpa)Cu
(H
2
O)](ClO
4
) [[(Adpa)CuCl] [[(Etdpa)CuCl
2
], which
correlates with their DNA-binding properties. The results
showthat the substituents introduced on the secondary amino
nitrogen atom of dpa have great contribution to the antitumor
activities of these copper(II) complexes. It is also found that
the coordination of copper(II) ions with AdpaH can decrease
the toxicity of AdpaH.
Introduction
Copper plays a key role in biological processes. The great
majority of the copper proteins are involved mainly in
oxidation/reduction reactions as well as in the dioxygen
transport and activation [1]. The development of mimic
systems for copper metalloenzymes has provided important
compounds which allowed scientists to understand both the
physicalchemical properties of the active site of the
copper enzymes as well as the reactivity exhibited by the
metalloenzymes [2]. Some of these mimetic compounds
show similar activities to those of natural metalloenzymes
[3]. It has been demonstrated that copper can accumulate in
tumors due to the selective permeability of cancer cell
membranes to copper compounds [4]. Copper(II) com-
plexes are the preferred over platinum (II) complexes for
cancer inhibition [5]. Di(picolyl)amine and its derivatives
are used as neutral, nondeprotonated chelating ligands to
complex copper(II) atoms to mimic non-heme dioxygenase
[6]. The reaction of dpa with Cu(ClO
4
)
2
or CuCl
2
lead to
hexa-coordinated [Cu(dpa)
2
](ClO
4
)
2
[7] or the mononu-
clear complexes [Cu(dpa)Cl
2
] [8], respectively, in which
the geometry of reported coordinated Cu(II)-dpa com-
plexes is a distorted square pyramidal or trigonal
bipyramidal. The utility of these ligands is enhanced by the
ease with which substituents may be introduced on the
imino nitrogen atom, thus allowing the controlled modi-
cation of solubility and molecular conformation through
the non-bonding interactions [9]. Recently N-substituted
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-009-9200-5) contains supplementary
material, which is available to authorized users.
L.-Y. Wang Q.-Y. Chen (&) J. Huang K. Wang
School of Chemistry and Chemical Engineering, Jiangsu
University, 212013 Zhenjiang, Peoples Republic of China
e-mail: chenqy@ujs.edu.cn
C.-J. Feng
College of Pharmacy MSC09 5360, 1 University of New
Mexico, Albuquerque, NM 87131-0001, USA
Z.-R. Gen
State Key Laboratory of Coordination Chemistry, Nanjing
University, 212093 Nanjing, Peoples Republic of China
1 3
Transition Met Chem (2009) 34:337345
DOI 10.1007/s11243-009-9200-5
di(picolyl)amines were found to be active against the
proliferation of cancer cells [10]. The mononuclear
copper(II) complex of 1-(bis-pyridin-2-ylmethyl-amino)-
3-chloropropane-2-ol) was reported to promote cell death
of THP-1 carcinogenic cells by apoptosis [11]. Here, we
report the synthesis, characterization, interaction with
DNA, and antitumor activities of Cu(II) complexes with
ethyl bis(2-pyridylmethyl)amino-2-propionate (Etdpa) and
bis(2-pyridylmethyl)amino-2- propionate (Adpa).
Experimental section
Chemical reagents, analysis, and physical
measurements
All chemicals used in the syntheses were of reagent grade
and were used without further purication (China). Calf
thymus DNA (ct-DNA), Tris(tris(hydroxymethyl)amino-
methane) (tris), ethidium bromide (EB), and di(picol-
yl)amine (dpa) were purchased from Sigma-Aldrich. The
complex [Cu(dpa)Cl
2
] was synthesized and characterized
as reported [8]. Water was puried with a Millipore Milli-
Q system. The C, H, and N microanalyses were performed
on Vario EL elemental analyzer. Metal ions were deter-
mined by Vista ICP-OES. The molar electrical
conductivities in DMF solution containing 10
-4
mol L
-1
complex were measured at 25.0 0.1 C using a BSA-A
conductometer. The electronic absorption spectrum was
recorded in the 900190 nm region using the VARIAN
CARY 50-BIO UVVIS spectrophotometer. Infrared
spectra were recorded on a Nicolet-470 spectrophotometer
in the spectral range 4,000400 cm pellets. The electro-
spray mass spectrum (ES-MS) was determined on a
Finnigan LCQ mass spectrograph, and the concentration of
the samples was about 1.0 lmol L
-1
. The mobile phase
was methanol. Electrochemical measurements were made
on a Princeton E.G and G-PARC model potentiostat. A
three electrode system comprising a glassy carbon working
electrode, a platinum wire auxiliary electrode, and a satu-
rated calomel reference (SCE) electrode were used for
voltammetric work. Cyclic voltammetry data were
obtained in water solution using 0.05 M NaClO
4
and 0.5 M
NaF as supporting electrolyte at 20.0 0.1 C. The solu-
tion was degassed with high purity N
2
prior to carrying out
the electrochemical measurements. The uorescence was
measured with Fp-750w Fluorometer.
Synthesis of ethyl bis(2-pyridylmethyl)amino-2-
propionate (Etdpa)
To a stirred solution of di(2-picolyl)amine (205.5 mg,
1 mmol), ethyl 2-bromide-propionate (182.9 mg, 1 mmol)
was added in 10 mL CH
2
Cl
2
. The resulting solution was
reuxed for 1 h. Then the solution was washed with water,
dried with sodiumsulfate, and concentrated in vacuo to give
colorless liquid after column chromatography on silica gel
(R
f
= 0.45, cyclohexane: ethyl acetate = 3:1) in 75%yield.
% Found: C, 68.33; H, 7.05; N, 13.92. % Calc: C, 68.20; H,
7.05; N, 14.04 for C
17
H
21
N
3
O
2
.
1
H NMR (400 MHz,
CDCl
3
): d = 1.261.40 (m, 6H, CH
3
); 3.65 (m, 1H, CH),
3.95 (s, 4H, CH
2
N); 4.20 (m, 2H, CH
2
); 7.147.72 (m, 8H,
H-py). IR (KBr) (cm
-1
): m(=CH) 3064 m, m(CH
2
)
2979 m, m(C=O) 1735 m, m(C=C, C=N) 1590 s, 1570 m,
d(CH, pyridine) 760 s. UVvis (H
2
O/nm) (e 9 10
-4
/
dm
3
mol
-1
cm
-1
): 203 (4.12), 260 (1.75).
Synthesis of bis(2-pyridylmethyl)amino-2-propionic
acid (AdpaH)
Etdpa (299.2 mg, 1 mmol) was stirred with 0.1 M NaOH
(10 mL, 1.0 mmol) for 2 h at 30 C. Then the solution was
extracted with CH
2
Cl
2
(10 mL), washed with water, and
concentrated to give white solid powder. White prism
crystals AdpaH (192 mg) obtained after recrystallizing
with methanol/H
2
O (1:1) in yield 60%. % Found: C, 66.40;
H, 6.32; N, 15.49. % Calc: C, 66.42; H, 6.31; N, 15.50 for
C
15
H
17
N
3
O
2
.
1
H NMR (400 MHz, CDCl
3
): d = 1.23
(d, 3H, CH
3
); 3.65 (m, 1H, CH), 3.95 (s, 4H, CH
2
N);
7.147.82 (m, 8H, H-py). 10.5(s, 1H, COOH). IR (KBr)
(cm
-1
): m(COOH) 3549 s, m(=CH) 3064 m, m(CH
2
)
2979 m, m(C=O) 1669 m, m(C=C, C=N) 1590 m, 1570 m,
d(CH, pyridine) 760 s. UVvis (H
2
O/nm) (e 9 10
-4
/
dm
3
mol
-1
cm
-1
): 203 (4.23), 260 (1.84).
Synthesis of [(Etdpa)CuCl
2
] (1)
To a stirred ethanol solution (5 ml) of ethyl bis(2-pyri-
dylmethyl)amino-2-propionate (160.8 mg, 0.54 mmol), a
solution of CuCl
2
2H
2
O (90.1 mg, 0.54 mmol) in ethanol
(10 mL) was added dropwise. The mixture was heated at
70 C for 3 h then the solution was evaporated by a rotary
evaporator under reduced pressure. The blue product
[(Etdpa)CuCl
2
] was obtained. Yield: 64%. % Found: C,
47.13; H, 4.91; N, 9.62; Cu, 8.51. % Calc: C, 47.07; H,
4.88; N, 9.69, Cu, 8.56 for C
17
H
21
Cl
2
CuN
3
O
2
. IR (KBr)
(cm
-1
): m(=CH) 3064 m, m(CH
2
) 2979 m, m(C=O)
1732 m, m(C=C, C=N) 1609 s, 1573 m, d(CH, pyridine)
774 s. UVvis (H
2
O/nm) (e 9 10
-4
/dm
3
mol
-1
cm
-1
):
254 (3.6), 691 (0.0088). k
m
(CH
3
OH): 15.6 S cm
2
mol
-1
.
Synthesis of [(Adpa)Cu(H
2
O)](ClO
4
) (2)
To a stirred ethanol solution (10 mL) of ethyl bis(2-pyri-
dylmethyl)amino-2-propionate (160.8 mg, 0.54 mmol), the
pH of the solution was adjusted to ca. 8 with NaOH, a
338 Transition Met Chem (2009) 34:337345
1 3
solution of Cu(ClO
4
)
2
6H
2
O (140.5 mg, 0.5 mmol) in
ethanol (5 mL) was added dropwise. After reaction for 2 h,
the light blue product was ltered off and dried under vac-
uum at room temperature. Yield: 67%. % Found: C, 39.91;
H, 4.15; N, 9.22, Cu, 14.16. % Calc: C, 39.92; H, 4.02; N,
9.31, Cu, 14.08 for C
15
H
18
ClCuN
3
O
7
. IR (KBr) (cm
-1
):
m(OH) 3443 m, m(=CH) 3084 m, m(CH
2
) 2986 m,
m(C=O) 1643 s, 1388 m, m(C=C, C=N) 1612 m, 1569 m,
d(CH, pyridine) 779 s, m(ClO
4
-
) 1120, 1108 s. UVvis
(H
2
O/nm) (e 9 10
-4
/dm
3
mol
-1
cm
-1
): 255 (3.89), 866
(0.0087). ^
M
(S cm
2
mol
-1
): 102.
Synthesis of [(Adpa)CuCl] (3)
To a stirred ethanol solution (10 mL) of ethyl bis(2-pyri-
dylmethyl)amino-2-propionate (160.8 mg, 0.54 mmol), the
pH of the solution was adjusted to ca. 8 with NaOH, a
solution of CuCl
2
2H
2
O (84.6 mg, 0.49 mmol) in ethanol
(7 mL) was added dropwise. Then the reaction solution
was reuxed for 4 h. The resulting solution was kept aside
for slow evaporation. The bright light green crystals suit-
able for X-ray diffraction were obtained after a few days.
Yield: 56%. % Found: C, 48.62; H, 4.57; N, 11.23; Cu,
17.17. % Calc: C, 48.78; H, 4.37; N, 11.38; Cu, 17.21 for
C
15
H
16
ClCuN
3
O
2
. IR (KBr) (cm
-1
): m(=CH) 3064 m,
m(CH
2
) 2979 m, m(C=O) 1643 s, 1388 m, m(C=C, C=N)
1612 s, 1569 m, d(CH, pyridine) 776 s. UVvis (H
2
O/nm)
(e 9 10
-4
/dm
3
mol
-1
cm
-1
): 255 (3.37), 863 (0.0081). k
m
(CH
3
OH): 18.1 S cm
2
mol
-1
.
X-ray data collections and crystal structure
determinations
Crystallographic data for the [(Adpa)CuCl] are listed in
Table 1. The blue prism crystals of the complex were
selected for lattice parameter determination and collection
of intensity data at 293 K on a SMART CCDC Area
Detector (Brukers, 2000) with monochromatized Mo Ka
radiation (k = 0.71073 A

) using a h - 2h scan mode. The

data was corrected for Lorenz and polarization effects
during data reduction. An empirical absorption correction
based on w scans was applied. The structure was solved by
the direct methods and rened on F
2
by full-matrix least-
squares methods using SHELXTL version 5.10 [12]. All
non-hydrogen atoms were rened anisotropically. Hydro-
gen atoms were introduced in their calculated positions. All
computations were carried out on a PC-586 computer using
the SHELXTL-PC program package.
DNA-binding studies
All experiments involving ct-DNA were performed in
buffer solution (50 mM NaCl, 5 mM TrisHCl, and pH 7.1)
at room temperature. The concentration of DNA was
determined by UV absorbance at 260 nm. The extinction
coefcient at 260 nm was taken as 6600 mol L
-1
cm
-1
[13]. Absorption titration experiments of copper(II) com-
plexes in buffer solution (50 mM NaCl, 5 mM TrisHCl,
pH 7.1) were performed by using a xed complex con-
centration to which increments of the DNA stock solutions
were added. For uorescence quenching experiments,
DNA (20 lM) was pretreated with ethidium bromide (EB,
0.68 lM) for 1 h at saturating binding level [14]. The
copper(II) complexes were then added to the mixture, and
their effect on the mission intensity (k
ex
= 532 nm, k
em
=
550.0700.0 nm) was measured at 20 C.
According to the SternVolmer equation, the quenching
nature of DNA-complex in the presence of EB can be
analyzed [15]
F
0
=F 1 K
sv
Q 1 K
q
s
0
Q ; 1
where F
0
and F are the uorescence intensities of DNA
EB complex in the absence and presence of complex, [Q]
the complex concentration, K
q
the DNAEB complex
quenching rate constant, s
0
the average lifetime of uoro-
phore in the absence of complex and its value is 10
-9
to
10
-7
s [16], and K
sv
is the SternVolmer dynamic
quenching constant.
Table 1 Crystal data and structure renement details for the com-
plex [(Adpa)CuCl]
Empirical formula C
15
H
16
ClCuN
3
O
2
Formular weight 369.30
Crystal system Monoclinic
Space group P2
1
/n
a (A

) 9.358 (5)
b (A

) 12.664 (10)
c (A

) 12.770 (10)
a () 90.00
b () 98.047 (13)
c () 90.00
V (A

3
)/Z 1499 (3)/4
Density (calc) (Mg/m
3
) 1.637
Absorption coefcient (mm
-1
) 1.645
F(000) 756
Index ranges -11 B h B 10; -15 B k B 15;
-10 B l B 15
H Range () 2.2825.99
Reection collected 6935
Independent reections [R
int
] 2890
Data/restraints/parameters 2890/0/200
Goodness-of-t on F
2
1.054
Final R indices [I [2r(I)] R
1
= 0.0361; wR
2
= 0.0990
R indices (all data) R
1
= 0.03870; wR
2
= 0.1004
Transition Met Chem (2009) 34:337345 339
1 3
Cytotoxicity testing
The cytotoxicity assay was in four kinds of cell lines (human
breast carcinoma cells Mcf-7, human esophageal cancer
cells Eca-109, human cervical cancer Hela cells, and human
lung adenocarcinoma A549 cells). Cells were cultured in
RMPI 1640 medium containing 4.8 g/L of Hepes, 2.2 g/L
NaHCO
3
and supplemented with penicillin/streptomy-
cin(1000 units/mL), and 10% calf serum. Hela, Mcf-7 cells
were cultured in DMEM medium containing 10% fetal
bovine serum. All cells were grown at 37 C in a humidied
atmosphere in the presence of 5%CO
2
. Eca-109, A549, Mcf-
7, Hela cells were seeded at a density of 4 9 10
4
cells/mL
into sterile 96 well plates and grown in 5% CO
2
at 37 C.
Test compounds were dissolved in H
2
O and diluted with
culture media. After 24 h, compounds were added and
treated for 48 h. Cell viability was determined by the 3-[4,5-
Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide
(MTT) assay by measuring the absorbance at 570 nm with
ELISA reader. IC
50
was calculated by the software provided
by Nanjing University. Each test was performed in triplicate.
Results and discussion
Synthesis and spectroscopic data
The synthesis route of the copper(II) complexes is shown in
Scheme 1. The reaction of the ligand ethyl bis(2-pyridyl-
methyl)amino-2-propionate (Etdpa) with Cu(ClO
4
)
2
and
CuCl
2
in the presence of sodium hydroxide produced the
new complexes [(Adpa)Cu(H
2
O)](ClO
4
) (2) and [(Adpa)-
CuCl] (3), respectively. The molar conductivities of the
complexes (1) and (3) in methanol are 15.6 and 18.1 S
cm
2
mol
-1
, respectively, indicating that the complexes are
non-electrolytes. The molar conductivity (102 S cm
2
mol
-1
)
of the complex (2) indicates this complex is a 1:1 electrolyte.
The IR spectra of the ligands show that there are two
pyridyl ring vibration bands at *1570 and 1590 cm
-1
and
d(CH) vibration of pyridyl ring at *760 cm
-1
[10]. These
vibrations in the copper complexes are all shifted. The
pyridyl ring vibrations bands were *1609 and 1573 cm
-1
for [(Etdpa)CuCl
2
] (1) and 1612 and 1569 cm
-1
for (2) and
(3). The d(CH) vibration bands of pyridyl ring for all of the
copper complexes were found at *774, 779, and
776 cm
-1
, respectively. These shifts indicate the pyridine
nitrogen atoms of the ligands donate a pair of electrons
each to the central metal forming coordinate bonds [17].
The m(C=O) band of the Etdpa and the complex (1) appears
at 1729 cm
-1
indicating that the existence of ester group of
Etdpa. The infrared spectra of the complexes (2) and (3)
show m
as
(COO) stretching frequencies at 1643 cm
-1
and
m
sym
(COO) at 1388 cm
-1
, respectively. The difference
between m
as
(COO) and m
sym
(COO) are about 255 cm
-1
,
suggesting that the carboxylate groups coordinate to the
copper(II) atoms only as monodentate ligands [18].
The Cu-pyridine charge transfer bands at ca. 254 nm
dominated the UV spectra for the three complexes. The
copper atom may adopt geometries ranging from typical
trigonal bipyramidal to distorted square pyramidal depend-
ing on the nature of the ligands. The [(Etdpa)CuCl
2
] (1)
exhibits visible spectra with single broad bands at 650
700 nm, characteristic of a copper(II) d
zx
, d
yz
? d
x2-y2
(
2
B
1
?
2
E) [19] transition in a tetragonal ligand eld, in
which the copper(II) ion has a distorted square-pyramidal
coordination environment. Because the dd transition bands
of [(Adpa)Cu(H
2
O)](ClO
4
) (2) and [(Adpa)CuCl] (3) in
aqueous solution were 866 and 863 nm, respectively, rather
than 650 nm, we conclude that the copper(II) atoms in the
complexes (2) and (3) mainly adopt a trigonal bipyramidal
rather than a distorted square-pyramidal geometry [20].
Crystal structure of [(Adpa)CuCl] (3)
The molecular structure of [(Adpa)CuCl] (3) with the atomic
labeling scheme is shown in Fig. 1, and the selected bond
lengths and angles are listed in Table 2. The monodepro-
nated Adpa acts as a tetradentate ligand toward a copper(II)
ion. The copper atom is coordinated by three N atoms (N1,
N2, N3, one oxygen atom (O2) of the (Adpa) and one chlo-
ride anion (Cl2), resulting a ve-coordinated mononuclear
copper(II) complex, which is different fromthe reported syn-
anticarboxylate bridged polymeric one-dimensional chain
copper(II) complex {[Cu(l-pmea)](ClO
4
) H
2
O} (pmea =
bis(2-pyridylmethyl)amino-2-ethanoic acid) [19]. The ve-
coordinated copper(II) complex [(Adpa)CuCl] forms a
trigonal bipyramidal, similar to the geometry of reported
[Cu(apme)(Cl)](BPh
4
) (apme = tris(2-pyridylmethyl) amine)
[21]. The N1, N2, and O2 form the equatorial trigonal plane,
while the N3 and Cl2 occupy the apical positions. The
N
N
H
3
CH
2
COOC
N
Cu
Cl
Cl
N
N
N
Cu
Cl
N
N
COOCH
2
CH
3
N
Etdpa
N
N
COO
N
Cu
CuCl
2
Cu(ClO
4
)
2
CuCl
2
OH
2
2
+
NaOH
NaOH
1
3
O
O
Scheme 1 Synthesis route of copper(II) complexes [(Etdpa)CuCl
2
]
(1), [(Adpa)Cu(H
2
O)](ClO
4
) (2), and [(Adpa)CuCl] (3)
340 Transition Met Chem (2009) 34:337345
1 3
copper(II) atomis shiftedby0.326 A

out of the equatorial plane

toward the tertiary amino ligand. The bond distances of Cu1
N3 and Cu1O2 are 2.012(2) and 2.028(2) A

. The N3Cu1
Cl2 angle is 178.29(6). Intermolecular hydrogen bonds
involving the carbon atoms, oxygen atoms (O1, O2), and the
chloride atom(Cl2) result in networks in 3. The Cl2 is linked to
the hydrogen atoms H15 (Cl(2)H15 [-1/2 ? x, 1.5 - y,
-1/2 ? z] of 2.653 A

) of the neighboring molecule. The car-

boxyl oxygen atoms (O1, O2) bond to hydrogen atoms from
carbon atoms of aromatic rings C11 [1 ? x, y, z] and C9
[1/2 ? x, 1.5 - y, -1/2 ? z] with interatomic distances
O1H11 of 2.709 (4) A

, O2H9 of 2.446 (4) A

. The addi-
tional interactions are p-pstackinginteractions betweenthetwo
adjacent pyring rings (N1B/C1B-C5B) [-1/2 ? x, 1.5 - y,
-1/2 ? z] and (N1AA/C1AA-C5AA) [1/2 ? X, 1.5 - y,
-1/2 ? z] or the (N1/C1-C5) [x, y, z] and (N1A/C1A-C5A)
[1 ? x, y, z], with the interplannar distance of ca. 3.687 A
(Fig. S1). The molecules are linked through intermolecular
hydrogenbonds of CHO, CHCl andpackedthroughp-p
stacking interaction forming network structures (Fig. S2).
Electrochemistry
Cyclic voltammograms for the copper(II) complexes at a
glassy carbon electrode in 0.05 M NaClO
4
and 0.05 M NaF
were shown in Fig. 2. The electrochemical behavior of the
[(Adpa)CuCl] and [(Etdpa)CuCl
2
] at a glassy carbon elec-
trode in 0.05 M NaClO
4
and 0.05 M NaF was characteristic
of quasi-reversible one-electron Cu(II)/Cu(I) redox pro-
cesses and a adsorptive stripping peak from deposition of
copper on the electrode, which is scan rate dependent. The
[(Adpa)Cu(H
2
O)](ClO
4
) exhibits a reversible one-electron
redox process with the half-wave potential of -0.403 V
involving the Cu
II
/Cu
I
couple (E
pc
= -0. 448 V, E
pa
=
-0.358 V, the ratio of anodic and cathodic peak currents
I
pa
/I
pc
are *1) and a irreversible one-electron Cu
II
/Cu
III
oxidation processes with E
PC
= -0.001 V. The one-elec-
tron Cu
II
/Cu
I
oxidation and reduction half-wave potentials
(E
1/2
) for the three complexes are in the range of -0.403 to
0.445 mVat 25 mV s
-1
, which are more negative than those
of the square-pyramidal complex [Cu(dpa)Cl
2
] [8]. The
ranking of the Cu
II
/Cu
I
potentials for the [(Adpa)
Cu(H
2
O)](ClO
4
) is near to that observed for the trigonal
bipyramidal Cu(II) complex of dpa (E
1/2
= -0.39 V) [22].
Fig. 1 Crystal structure
for the complex [(Adpa)CuCl].
Thermal ellipsoids are
drawn at 50% probability
Table 2 Selected bond lengths (A

) and bond angles () for the

complex [(Adpa)Cu(Cl)]
Bond distances
Cu(1)N(3) 2.012(2) Cu(1)N(2) 2.082(3)
Cu(1)N(1) 2.026(2) Cl(2)Cu(1) 2.2184(15)
Cu(1)O(2) 2.028(2)
Bond angles
N(3)Cu(1)N(1) 82.10(10) N(2)Cu(1)Cl(2) 99.86(8)
N(3)Cu(1)O(2) 80.35(10) N(3)Cu(1)N(2) 81.80(9)
N(1)Cu(1)O(2) 124.25(8) N(1)Cu(1)N(2) 121.12(9)
N(3)Cu(1)Cl(2) 178.29(6) O(2)Cu(1)N(2) 108.01(9)
N(1)Cu(1)Cl(2) 96.65(9) O(2)Cu(1)Cl(2) 99.45(9)
Transition Met Chem (2009) 34:337345 341
1 3
Because a positive shift in the half-wave potential reects
a less stable Cu(II) complex [23], the stability of the
Cu(II) complexes can be ranked as follows: [(Adpa)
CuCl] [[(Etdpa)CuCl
2
] [[(Adpa)Cu(H
2
O)](ClO
4
).
Binding characteristics of complex with DNA
The spectrophotometric titration spectra of the [(Adpa)-
Cu(H
2
O)](ClO
4
) (2), [(Adpa)CuCl] (3) are shown in Fig. 3a
and b, respectively. It is observed that the absorption bands
of (2) and (3) at 255 nm exhibited hypochromism of 17.2
and 13.1%, and bathochromism shift of about 5 nm when
the ct-DNA was added to the solution of the complexes.
Figure 3a and b shows that the absorption spectra of com-
plexes increase on increasing the concentration of ct-DNA.
This is a typical hyperchromic effect, which was caused
possibly by the intercalation binding mode between the
complexes and ct-DNA. Hypochromic effect indicates the
complexes (2) and (3) induce the change of DNA double-
helix structure [24]. Bathochromism shift indicates the p*
orbital of intercalated ligand couple with the p orbital of the
base pairs, thus reducing the pp* transition energy [25].
Therefore, we speculate that complexes (2) and (3) inter-
acting with ct-DNA have the mode of intercalation.
However, there is no obvious bathochromic shifts and
hypochromicities when the ligand Etdpa and the [(Etdpa)
CuCl
2
] (1) were used in the same condition. The complex
(1) shows weak binding to the ct-DNA, which is similar
to the reported square-pyramidal ternary(L-leucine)-bpy
(2,2
0
-bipyridine) copper(II) complex [26].
It is well known that EB can intercalate nonspecically
into DNA. Competitive binding of other drugs to DNA and
EB will result in displacement of bound EB and a decrease
in the uorescence intensity [27, 28]. When [(Etdpa)CuCl
2
]
(1) was added into the solution of DNAEB complex,
respectively, the change of the uorescence intensity of
DNAEB complex was small and ruleless, which indicates
that there is nearly no intercalated interaction between the
complex (1) with DNA. However, when the complexes (2)
and (3) were added into the solution of DNAEB complex,
the uorescence intensity of DNAEB complex decreased
with the increasing concentration of (2) and (3). The
uorescence spectra for the complexes [(Adpa)Cu(H
2
O)]
(ClO
4
) (2) and [(Adpa)CuCl] (3) were shown in Fig. 4a and
b. Since intercalated EB is the only uorescent species, the
1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5
d
a
[(Adpa)CuCl]
[(Adpa)Cu(H
2
O](ClO
4
)
[(Etdpa)CuCl
2
]
Potential/V
Fig. 2 Cyclic voltammogram of [(Etdpa)CuCl
2
] (1), [(Adpa)
Cu(H
2
O)](ClO
4
) (2), and [(Adpa)CuCl] (3) in water containing
50 mM NaClO
4
and 50 mM NaF. Scan rate: 25 mV s
-1
(a),
50 mV s
-1
(b), 75 mV s
-1
(c), 100 mV s
-1
(d)
200 250 300 350 400
200 250 300 350 400
0.5
1.0
0.0
1.5
f
a
A
b
s
o
r
b
a
n
c
e
/nm
0.2
0.4
0.6
0.8
0.0
1.0
f
a
A
b
s
o
r
b
a
n
c
e
/nm
(a)
(b)
Fig. 3 a Electronic spectra of [(Adpa)Cu(H
2
O)](ClO
4
) (2) (50 lM)
in the presence of increasing amounts of ct-DNA (af); DNA
concentrations are 0, 23.8, 71.5, 95.3, 119, and 142.9 lM for spectra
(af), respectively. b Electronic spectra of [(Adpa)CuCl] (3) (50 lM)
in the presence of increasing amounts of ct-DNA (af); DNA
concentrations are 0, 23.8, 71.5, 95.3, 119, and 142.9 lM for spectra
(af), respectively
342 Transition Met Chem (2009) 34:337345
1 3
observed uorescence decrease indicates that the com-
plexes (2) and (3) can replace EB inside the DNA cavities.
Such a characteristic change is often observed in the
intercalative DNA interaction [29].
The binding constants K
A
of (2) and (3) with DNA in the
presence of EB were determined using the following
relationship [30]
log F
0
F =F n log K
A
n log

D
t
n N
t

F
0
F =F
0

;
2
where [N
t
] and [D
t
] are the total concentration of DNAEB
complex and the complex, respectively. The plots of
log(F
0
- F)/F versus log([D
t
] - n[N
t
](F
0
- F)/F
0
) for
DNAEB complex in the presence of complexes are shown
in Fig. 5 at 8 C, and the binding constants are listed in
Table 3. The binding constants K
A
of (2) and (3) are
3.03 9 10
4
and 1.39 9 10
3
, which indicate that the
interaction of [(Adpa)Cu(H
2
O)](ClO
4
) (2) to ct-DNA is
stronger than that of the [(Adpa)CuCl] (3). This order is
well consistent with the results of spectrophotometric
titration. The different DNA-binding constants for com-
plexes (2) and (3) may due to their different total charge
[30].
Inhibition on the proliferation of cancer cells
Three copper(II) complexes (1)(3) and the reported com-
plex [Cu(dpa)Cl
2
] [8] were studied for their antitumor
activity in vitro by determining the inhibitory percentage
against growth of cancer cells Mcf-7, A549, Hela, and Eca-
109 using the method of 3-[4,5-Dimethylthiazol-2-yl]-2,
5-diphenpyltetrazolium bromide reduction (MTT method).
The IC
50
data of the copper(II) complexes (1)(3), [Cu(dpa)
Cl
2
], the ligand Etdpa, and AdpaH were shown in Table 4.
The complexes (1)(3) can inhibit the proliferation of the
Mcf-7 cell with IC
50
in the range of 37.1224.13 lM, which
is smaller than that (96.23 lM) of [Cu(dpa)Cl
2
]. This may be
due to the solubility, and molecular conformation of com-
plexes (1)(3) were different from [Cu(dpa)Cl
2
]. The
complexes (2) and (3) were more active against the cancer
cell Eca-109 than [(Etdpa)CuCl
2
] and [Cu(dpa)Cl
2
] with
IC
50
in the range 23.2131.09 lM (Table 4). These data
indicate that the substituents introduced on the secondary
amino nitrogen atom of dpa have great contribution to the
antitumor activities of these copper(II) complexes. It is also
found that the AdpaH was more active against the
560 580 600 620 640 660 680 700
560 580 600 620 640 660 680 700
0
50
100
150
200
250
300
f
a
F
/nm
0
50
100
150
200
250
300
f
a
F
/nm
(a)
(b)
Fig. 4 a Fluorescence spectra of DNAEB in the presence of
[(Adpa)Cu(H
2
O)](ClO
4
) at 8 C. The total concentrations of [(Adpa)-
Cu(H
2
O)](ClO
4
) are (a) 0, (b) 10.0, (c) 20.0, (d) 30.0, (e) 40.0,
(f) 50.0 lmol L
-1
. EB and DNA concentration are 0.68 and
20 lmol L
-1
. b Fluorescence spectra of DNAEB in the presence
of [(Adpa)CuCl] at 8 C. The total concentrations of [(Adpa)CuCl]
are (a) 0, (b) 10.0, (c) 20.0, (d) 30.0, (e) 40.0, (f) 50.0 lmol L
-1
. EB
and DNA concentration are 0.68 and 20 lmol L
-1
-5.1 -5.0 -4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2
-2.0
-1.8
-1.6
-1.4
-1.2
-1.0
-0.8
l
o
g
(
(
F
0
-
F
)
/
F
)
log([D
t
]) - n[N
t
](F
0
- F)/F
0
[(Adpa)Cu(H
2
O)](ClO
4
)
[(Adpa)CuCl]
Fig. 5 The plots of log((F
0
- F)/F) versus log([D
t
] - n[N
t
]
(F
0
- F)/F
0
) for [(Adpa)Cu(H
2
O)](ClO
4
) and [(Adpa)CuCl] at 8 C
Table 3 The binding constants and binding site of the complexes (2)
and (3) with DNA at 8 C
Complex K
A
(L mol
-1
) n r
[(Adpa)Cu(H
2
O)](ClO
4
) (2) 3.03 9 10
4
1.25 0.992
[(Adpa)CuCl] (3) 1.39 9 10
3
1.01 0.997
Transition Met Chem (2009) 34:337345 343
1 3
proliferation of the four cancer cells than the copper(II)
complexes (2) and (3), indicating the coordination of
copper(II) with AdpaH can decrease the toxicity of AdpaH.
Conclusion
Three new Cu(II) complexes with Etdpa or Adpa were syn-
thesized and characterized. The X-ray crystal structure of
[(Adpa)CuCl] (3) shows that the copper(II) atom is coordi-
nated by three N atoms, one oxygen atom of the (Adpa) and
one chloride anion (Cl(1)), forming a trigonal bipyramidal
geometry. The spectrophotometric and uorescence titra-
tion data indicate that the interaction of square pyramidal
[(Etdpa)CuCl
2
] (1) with ct-DNA is weak, but the complexes
trigonal bipyramidal [(Adpa)Cu(H
2
O)](ClO
4
) (2) and
[(Adpa)CuCl] (3) interact with ct-DNA with the mode of
intercalation. The complexes (1)(3) are more active against
the proliferation of four cancer cells (Mcf-7, Eca-109, A549,
and Hela) than [Cu(dpa)Cl
2
] due to their different solubility
and molecular conformation. The inhibition activities of the
three new copper(II) complexes are in the order: (2) [(3)
[(1), which is in correlation with their DNA-binding
properties. It is also found that the coordination of copper(II)
ions with AdpaH can decrease the toxicity of AdpaH. The
detailed mechanism of inhibition is under further investi-
gation. Information obtained from this study may be helpful
to the development of therapeutic agents.
Acknowledgements Financial support from National Science
Foundation of China (20777029B0702), distinguished scholar science
foundation of Jiangsu University (06JDG050), and the foundation of
state key Laboratory of Coordination Chemistry, Nanjing University.
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Table 4 Inhibition on the proliferation of human cancer cells for copper(II) complexes and ligands
Tested complex IC
a
50Mcf7
lM SD
b
IC
a
50HeLa
lM SD
b
IC
a
50A549
lM SD
b
IC
a
50Eca109
lM SD
b
[(Cu(dpa)Cl
2
] (0) 96.23 0.03 [100 [100 [100
[(Etdpa)CuCl
2
] (1) 37.12 0.02 85.46 0.02 90.08 0.02 82.45 0.02
[(Adpa)Cu(H
2
O)](ClO
4
) (2) 36.09 0.02 84.32 0.02 60.34 0.02 23.21 0.02
[(Adpa)CuCl] (3) 24.13 0.02 74.12 0.02 60.25 0.02 31.09 0.02
Etdpa 25.14 0.02 10.14 0.02 12.36 0.02 8.41 0.02
AdpaH 3.12 0.02 7.12 0.02 25.24 0.02 6.65 0.02
a
IC
50
was average data of triplicate assay
b
SD means standard deviation
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