Synthesis, characterization, and biological properties of N-phenolato-N′-(3-dimethylaminopropyl)oxamide and its binuclear copper(II) complexes

Article abstract of DOI:10.1007/s11243-012-9662-8

A new asymmetric N,N′-bis(substituent)oxamide ligand, N-phenolato-N′-(3-dimethylaminopropyl)oxamide (H₃pdmapo), and two of its binuclear Cu(II) complexes with different terminal ligands, namely [Cu₂(pdmapo)(phen)(H₂O)](ClO

Full text of DOI:10.1007/s11243-012-9662-8

Transition Met Chem (2013) 38:69–78
DOI 10.1007/s11243-012-9662-8
Synthesis, characterization, and biological properties
0
of N-phenolato-N -(3-dimethylaminopropyl)oxamide
and its binuclear copper(II) complexes
Wan-Ju Zhang
Zhi-Yong Wu
•
Fang Wang
•
Yan-Tuan Li
•
Received: 19 July 2012 / Accepted: 17 October 2012 / Published online: 2 November 2012
Ó Springer Science+Business Media Dordrecht 2012
0
Abstract A new asymmetric N,N -bis(substituent)oxam-
0
DNA-binding properties of H pdmapo and the two com-
3
ide ligand, N-phenolato-N -(3-dimethylaminopropyl)oxam-
ide (H pdmapo), and two of its binuclear Cu(II) complexes
plexes were studied. The experimental evidence suggests that
the ligand binds to DNA via a groove binding mode, while
the binuclear complexes bind intercalatively to DNA.
3
with different terminal ligands, namely [Cu (pdmapo)-
2
(
(
phen)(H O)](ClO ) (1) and [Cu (pdmapo)(bpy)(CH OH)]-
2 4 2 3
ClO ) (2), where phen = 1,10-phenanthroline and bpy =
4
0
2
,2 -bipyridine, have been synthesized and characterized.
Introduction
The crystal structures of both complexes have been deter-
mined by single-crystal X-ray diffraction. Both structures
Studies on DNA interactions of organic and inorganic
small molecules are important in understanding cellular
processes, such as mutagenesis and cancer. Some mole-
cules have been developed as cancer therapeutics or
molecular biology tools [1–5]. Among these compounds,
transition metals have attracted much interest. Their com-
plexes can bind to DNA in noncovalent modes such as
electrostatic, intercalative, and groove binding [6, 7]. Many
potential applications of such complexes require that the
complexes bind to DNA through an intercalative mode.
Cu(II) complexes have been extensively studied in this
context [8]. Copper is a physiologically important metal
that plays an important role in the endogenous oxidative
DNA damage associated with aging and cancer [9]. For the
past several decades, great effort has been devoted to
binding studies of copper complexes with DNA [10–12]. In
particular, many studies have been focused on binuclear
copper complexes due to their presence in metallopro-
teinases as well as their affinity for DNA [13–17].
3-
contain binuclear Cu(II) cationic complexes with pdmapo
3-
ligands. The asymmetric pdmapo
ligands bridge two
Cu(II) atoms in the cis conformation and the CuꢀꢀꢀCu sep-
arations through the oxamide bridge are 5.2046(18) and
˚
5
.207(2) A for complexes 1 and 2, respectively. The coor-
dination environments of the two Cu(II) atoms in each
binuclear complex are different. The copper occupying the
3-
inner site of the pdmapo ligand is four-coordinated in a
CuN O distorted square-planar environment, while the other
3
is five-coordinated in a square pyramid geometry. In com-
plex 1, O–HꢀꢀꢀO and C–HꢀꢀꢀO hydrogen bonds link the
complex into a one-dimensional chain. In complex 2,
O–HꢀꢀꢀO hydrogen bonds link the molecules to form a dimer,
together with two types of strong p–p interactions, giving a
two-dimensional network structure. The cytotoxicities and
W.-J. Zhang (&) ꢀ Y.-T. Li (&) ꢀ Z.-Y. Wu
Marine Drug and Food Institute, Ocean University of China,
5 Yushan Road, Qingdao 266003, People’s Republic of China
e-mail: wanjuzhang@yahoo.com.cn
0
It is well known that N,N -bis(substituent)oxamides are
good candidates for the formation of binuclear complexes,
since their coordinatingabilitytowardtransitionmetalscan be
Y.-T. Li
e-mail: yantuanli@ouc.edu.cn
modified by changing the nature of the amide substituents
0
[
18]. Symmetrical N,N -bis(substituent)oxamides have been
extensively studied. However, relatively few studies on
W.-J. Zhang ꢀ F. Wang
School of Chemical Engineering, Huanggang Normal
University, 146 Xingang Road, Huanggang 438000,
People’s Republic of China
0
asymmetric N,N -bis(substituent)oxamides have been repor-
ted because of the associated synthetic difficulties. In recent
123

7
0
Transition Met Chem (2013) 38:69–78
years, this area has received more attention since complexes
0
ethyl oxalyl chloride (10 mmol) at 5 °C. After stirring for an
additional 1 h at 25 °C, the solid was filtered off. Recrys-
tallization from ethyl acetate gave ethyl N-(2-hydroxy-
phenyl)oxalamate as a beige solid (Yield: 1.70 g, 81 %).
bridged by asymmetric N,N -bis(substituent)oxamides have
displayed interesting properties in many fields [19–22]. We
became interested in designing complexes of asymmetric
0
N,N -bis(substituent)oxamides and exploring their biological
H pdmapo was prepared in the second step as follows: A
3
activities. Many complexes which contain an aromatic het-
erocycle can bind to DNA through intercalative modes. The
extent of binding is expected to depend on the size and elec-
tron density of the aromatic rings, as well as on the combined
effect of hydrophobic and hydrophilic interactions [23]. To
understand the effects of the terminal ligands on the DNA-
binding behavior of Cu(II) complexes, the selection of inter-
calating ligand is very important [24].
solution of N,N-dimethyl-1,3-propanediamine (5 mmol) in
ethanol (20 ml) was added to a solution of ethyl N-(2-
hydroxyphenyl)oxalamate (5 mmol) in ethanol (20 ml). The
mixture was stirred for 3 h and then concentrated under
vacuum to give a precipitate. Recrystallization from ethanol
gave H pdmapo as a white powder. Yield: 1.12 g, 85 %.
3
-
1
Mp: 160.6–161.2 °C. IR: mmax (KBr)/cm 3319vs, 1,668 s.
-
1
-1
UV–visible (in CH OH): k (nm) [emax (M cm )]: 297
3
max
1
Based on these considerations, we synthesized a new
0
(26,000). H NMR (800 MHz, CDCl , 25 °C, TMS): d/ppm
3
asymmetric N,N -bis(substituent)oxamide ligand, N-pheno-
0
1.63 [m, 2H], 2.12 [s, 6H], 2.23 [t, 2H, J = 6.87 Hz], 3.22
[m, 2H], 6.82 [s, 1H], 6.91 [s, 1H], 6.97 [s, 1H], 8.12 [s, 1H],
lato-N -(3-dimethylaminopropyl)oxamide (H pdmapo), and
3
?
9.18 [s, 1H], 9.78 [s, 1H], 10.50 [s, 1H]. m/z (ES ) 266.1
two of its binuclear Cu(II) complexes with different terminal
?
[M ? H] . Anal Calcd for C H N O : C, 58.9; H, 7.2; N,
ligands, [Cu (pdmapo)(phen)(H O)](ClO ) (1) and [Cu -
2
2
4
2
13 19 3 3
(
pdmapo)(bpy)(CH OH)](ClO ) (2) (phen = 1,10-phenan-
3
15.8 %. Found: C, 58.8; H, 7.3; N, 16.0 %.
Synthesis of the complexes
4
0
throline, bpy = 2,2 -bipyridine), in order to study the
influence of the terminal ligands on the biological properties
of these binuclear complexes. The results of this study are
reported in this paper.
A solution of copper(II) perchlorate hexahydrate (0.0742 g,
0.2 mmol) in methanol (10 ml) was added dropwise to a
solution of H pdmapo (0.0265 g, 0.1 mmol) and piperidine
3
Experimental
(0.0128 g, 0.15 mmol) in methanol (10 ml). Then, a solu-
tion of phen (0.0198 g, 0.1 mmol) in methanol (10 ml) was
added dropwise over 0.5 h. The mixture was continuously
stirred at 60 °C for 5 h, whereupon a green precipitate
formed. The precipitate was filtered off, washed sequen-
tially with methanol and ethyl ether several times, and then
dissolved in a water/methanol (1:3 v/v) mixture. Green
crystals of the complex suitable for X-ray analysis were
obtained from this solution by slow evaporation at room
Materials and methods
All reagents were commercially available and of analytical
grade. Carbon, hydrogen, and nitrogen elemental analyses
were performed with a Perkin-Elmer elemental analyzer
Model 240. The metal contents were determined on an ICP-
4
300 isoionic emission spectrophotometer. IR spectra were
-
1
recorded with samples as KBr pellets in a Nicolet model
Impact 470 FTIR spectrophotometer. UV–Vis spectra were
obtained on a Varian Cary-300 type UV–Vis spectropho-
tometer equipped with quartz cuvettes of 1 cm path length at
temperature. Yield: 0.058 g (84 %). IR: mmax (KBr)/cm
1,639 s, 1,470 m, 1,428 m, 1102vs, 621 m. UV–visible (in
-
1
-1
CH OH): k
(nm) [emax (M cm )]: 640 (200), 272
(65,000), 225 (75,100). Anal. Calcd for C H N O Cu Cl:
3
max
2
5
26
5
8
2
1
room temperature. H NMR spectra were recorded on a
C, 43.7; H, 3.8; N, 10.2; Cu, 18.5 %. Found: C, 43.7; H,
3.9; N, 10.3; Cu, 18.5 %. Complex 2 was prepared in the
same way as complex 1, but replacing phen with bpy. IR:
JEOL-ECP 600 spectrometer with tetramethylsilane (TMS)
as internal standard, and chemical shifts are reported as d
values. Mass spectra were recorded with a Q-TOF Global
mass spectrometer. Fluorescence spectra were measured
with an Fp-750w Fluorometer.
-
1
mmax (KBr)/cm
1,638 s, 1,471 m, 1,430 m, 1120vs,
623 m. UV–visible (in CH OH): kmax (nm) [emax
3
-
1
-1
(M cm )]: 645 (220), 300 (48,400), 234 (45,000). Anal.
Calcd for C H ClN O Cu : C, 42.6; H, 4.2; N, 10.3; Cu,
2
4
28
5
8
2
0
Synthesis of N-phenolato-N -(3-
dimethylaminopropyl)oxamide
18.8 %. Found: C, 42.6; H, 4.3; N, 10.4; Cu, 18.7 %.
The synthesis is summarized in Scheme 1.
The synthesis of H pdmapo was carried out in two steps. The
3
Crystal structure determination and refinement
first step consisted of preparing the ethyl N-(2-hydroxy-
phenyl)oxalamate according to the reported method [25];
thus, a solution of 2-aminophenol (10 mmol) in THF
The X-ray diffraction experiments for the complexes were
made on a Bruker APEX area-detector diffractometer with
graphite monochromated Mo Ka radiation (k = 0.71073
(
25 ml) was treated dropwise under vigorous stirring with
1
23

Transition Met Chem (2013) 38:69–78
71
Table 1 Crystal data and structure refinement of the complexes
Complex 1 Complex 2
O
O
Cl
O
O
NH
Empirical formula C H ClCu N O ,
25 26
24 28 2 5 8
C H ClCu N O
OH
2
5
8
+
Formula weight
687.04
677.04
Crystal size (mm) 0.08 9 0.09 9 0.12
0.07 9 0.11 9 0.20
298
OEt
OEt
H
2
N
OH
Temperature (K)
298
˚
Wavelength (A)
0.71073
Triclinic
P-1
0.71073
Triclinic
P-1
+
H
2
N
N
Crystal system
Space group
˚
a (A)
10.468(5)
11.371(5)
11.619(5)
92.718(5)
97.366(6)
101.964(5)
1,337.9(10)
2
11.846(3)
12.302(4)
12.452(4)
63.410(3)
66.896(3)
67.701(3)
1,446.0(7)
2
˚
b (A)
+
+
Cu(ClO4)2
phen
˚
c (A)
Complex 1
Complex 2
O
O
NH
OH
a (°)
b (°)
c (°)
+
+
Cu(ClO4)2
bpy
NH
N
3
˚
Volume (A )
Z
3
Scheme 1 Synthesis routes for H pdmapo and the complexes
Density (calc)
-
1.705
1.555
3
(
g cm
)
˚
A) at a temperature of 298 K. The crystal structure was
solved by direct methods followed by Fourier syntheses.
Structure refinements were performed by full matrix least-
Mu (Mo Ka)
-
1.749
1.617
1
)
(
mm
F (000)
700
692
2
h range for data
collection
1.8–25.2
1.9–25.2
squares procedures using SHELXL-97 on F [26]. The
C-bound H atoms were placed in calculated positions with
˚
C–H distances of 0.93 (aryl), 0.96 (methyl), or 0.97 A
Limiting indices
-10 \ 12; -13 \ 13; -12 \ 14; -14 \ 14;
-12 \ 13
-14 \ 13
5,103, 3,926, 0.018
(
methylene) and refined in riding mode, with
Tol. Uniq. Data,
R(int)
4,737, 2,985, 0.028
U (H) = 1.2U (C) or 1.5U (methyl C). The O-bound H
iso
eq
eq
atoms were found in a difference Fourier map and were
2
treated as riding, with fixed U (H) = 0.08 A . In complex
Observed data
[I [ 2r(I)]
2,985
3,926
˚
iso
1
, O6, O7, and O8 of the perchlorate ion appeared to be
R, xR
2
0.0435, 0.1064
0.99
0.0851, 0.2575
1.13
disordered and were refined isotropically as two groups
sharing the same Cl1 (occupancy factors are 0.72 and 0.28,
respectively). The carbon atoms in dimethylpropylenedi-
amine fragment are disordered over two sets of positions
denoted by C10A to C12A and C10B to C12B, whose
occupancies were refined to 0.69 and 0.31. In complex 2,
the disordered perchlorate ion was treated as two sets of
positions denoted by Cl1, O5 to O8 and Cl2, O9 to O12
with constrained site occupation factors of 0.5. Further-
more, O6, O7, O8 and O10, O11, O12 were refined iso-
tropically as two groups with occupancy factors of 0.3 and
S
Max., av. Shift/
error
0.00, 0.00
0.00, 0.00
with 10 % (v/v) fetal bovine serum, 1 % (w/v) penicillin
(104 U/ml), and 10 mg/ml streptomycin. Cell lines were
maintained at 310 K in a 5 % (v/v) CO atmosphere with
2
95 % (v/v) humidity. Cultures were passaged weekly using
trypsin–EDTA to detach the cells from their culture flasks.
The compounds and cisplatin were dissolved in DMSO and
diluted to the required concentrations with culture medium
before use. The content of DMSO in the final concentrations
didnotexceed0.1 %andwasfoundtobenon-toxictothecells
tested. Rapidly growing cells were harvested, counted, and
incubated at the appropriate concentration in 96-well micro-
plates for 24 h. The test compounds dissolved in culture
medium were then applied to the culture wells to achieve final
0
.2, respectively. Crystal data and refinement conditions
are summarized in Table 1.
In vitro cytotoxicity evaluation
In vitro cytotoxicities of the free ligand (H pdmapo), the
3
binuclear Cu(II) complexes, and of cisplatin were evaluated
against two cancer cell lines including human hepatocellular
carcinoma cell line (SMMC-7721) and human lung adeno-
carcinomacellline(A549)usingtheSulforhodamine B(SRB)
assay. All cells were cultured in RPMI 1640 supplemented
-
3
2
concentrations ranging from 10 to 10 lg/ml. Control wells
were prepared by the addition of culture medium without
cells. The plates were incubated at 310 K in a 5 % CO2
atmosphere for 48 h. Upon completion of the incubation, the
123

7
2
Transition Met Chem (2013) 38:69–78
cells were fixed with ice-cold 10 % trichloroacetic acid
100 ml) for 1 h at 277 K, washed five times with distilled
binucleating ligand. The second is to use a ‘‘complex
ligand’’ that contains a potential donor group capable of
coordinating to another metal [30]. In this study, our pur-
pose was to obtain Cu(II)–Cu(II) binuclear complexes and
the former synthetic method was adopted. Both of these
(
water and dried in the air, then stained with 0.4 % SRB in 1 %
acetic acid (100 ml) for 15 min. The cells were washed four
times with 1 % acetic acid and air-dried. The stain was solu-
bilizedin10 mMunbufferedTrisbase(100 ml)andtheODof
each well was measured at 540 nm on a microplate spectro-
photometer. The IC50 values were calculated from the curves
constructed by plotting cell survival (%) versus the concen-
trations of the test compounds.
binuclear complexes are soluble in H O, DMF, and DMSO
2
to give stable solutions at room temperature and are
moderately soluble in methanol, ethanol, and acetone. The
molar conductance values of the two complexes (72 and 79
-
1
-2
-1
-3
X
cm mol
in 1 9 10 M DMF solutions for
complexes 1 and 2, respectively) fall in the expected range
for 1:1 electrolytes [31], which is consistent with the results
of the X-ray diffraction analysis.
DNA-binding experiments
All the experiments involving the interaction of the com-
pounds with DNA were carried out in Tris buffer (pH
In order to clarify the mode of bonding, the IR spectra of
the free ligand (H pdmapo) and its binuclear Cu(II) com-
3
7
.24). The calf thymus DNA (CT-DNA) concentration was
plexes were assigned on the basis of a careful comparison
determined by absorption spectroscopy using the molar
of the complexes with the free ligand. The sharp strong
-1
-
1
-1
absorption coefficient of 6,600 M cm at 260 nm [27].
The absorption titration experiments were performed by
keeping the concentration of the compound constant while
varying the CT-DNA concentration. While measuring the
absorption spectra, equal amounts of DNA were added to
both the compound solution and the reference solution to
eliminate the absorbance of DNA itself. Fluorescence
quenching experiments were carried out with DNA pre-
treated with ethidium bromide (EB) for 30 min. To this
pretreated DNA solution, various amounts of compound
were added and their effect on the emission intensity was
measured. Samples were excited at 522 nm and the emis-
sion was observed between 540 and 650 nm. Viscosity
measurements were taken using an Ubbelodhe viscometer
immersed in a thermostatic water bath maintained at
band (3,319 cm ) observed for the free ligand H pdmapo
3
is the result of overlapping between m(O–H) of and
m(N–H). It is missing in the binuclear complexes because
of the loss of both protons, indicating the phenolic oxygen
and amido nitrogen atoms both take part in coordination.
-
1
The m(C=O) vibration of H pdmapo (1,668 cm ) is red-
3
-
shifted to 1,639 and 1,638 cm for complexes 1 and 2,
1
respectively. This indicates coordination of the oxygen
-
atom (oxamidate group). The bands at 1,471–1,428 cm
1
in the spectra of the complexes are attributed to the ter-
minal ligands (phen and bpy, respectively). Bands at
-
1
approximately 1,100 and 625 cm , typical for a non-
coordinated perchlorate group [32], are also observed in the
spectra of both complexes.
The electronic spectra of the ligand and its complexes
were recorded in the UV–visible region using methanol as
solvent. The electronic spectrum of the ligand has a band at
297 nm. For the complexes, intense bands at 225 and
234 nm are assigned to the intra-ligand (p–p*) transitions
associated with phen and bpy for 1 and 2, respectively. The
bands found at 272–313 nm are typical of charge transfer
transitions between the ligand (phen or bpy) and metal
(LMCT) [33, 34]. Much weaker, less defined absorption
bands at lower energy (610–670 nm) in the spectra of the
complexes are associated with the d–d transitions of Cu(II)
[35]. The spectroscopic data of the two Cu(II) complexes
are consistent with their crystal structures, as now
described.
2
0.0(± 0.l) °C. DNA samples of approximately 200 base
pairs in length were prepared by sonication in order to
minimize complexities arising from DNA flexibility [28].
Flow times were measured with a digital stopwatch; each
sample was measured three times and an average flow time
was calculated. Relative viscosities for DNA in the pres-
ence and absence of the compounds were calculated from
the relation n = (t-t )/t , where t is the observed flow time
0
0
of DNA-containing solution and t is that of Tris–HCl
0
1
/3
buffer alone. Data are presented as (n/n0) versus binding
ratio [29], where n is the viscosity of DNA in the presence
of compound and n is the viscosity of DNA alone.
0
Crystal structure of complex 1
Results and discussion
The molecular structure of complex 1 consists of a cis
asymmetric binuclear Cu(II) cation and one non-coordi-
nated perchlorate anion (Fig. 1). Selected bond lengths
and angles are listed in Table 2. The asymmetric pdm-
Synthesis and general properties
Two synthetic strategies are generally available for the
preparation of binuclear complexes. The first is to use a
3
-
apo
ligand bridges two Cu(II) atoms in the cis
1
23

Transition Met Chem (2013) 38:69–78
73
˚
Table 2 Selected bond distances (A) and angles (°) in complexes 1
and 2
Complex 1
Cu1–N1
Cu1–N3
1.940(4)
2.009(4)
1.940(3)
2.330(3)
1.993(4)
1.277(5)
1.284(5)
83.05(15)
83.18(14)
165.93(13)
83.67(17)
93.43(15)
95.83(15)
91.35(13)
94.97(13)
114.4(3)
115.5(3)
112.8(3)
Cu1–N2
1.981(3)
1.953(3)
Cu1–O1
Cu2–O2
Cu2–O3
1.973(3)
Cu2–O4
Cu2–N4
2.005(4)
Cu2–N5
C7–N1
1.299(5)
C7–O2
C8–N2
1.299(5)
C8–O3
C7–C8
1.530(6)
N1–Cu1–N2
N1–Cu1–O1
O1–Cu1–N2
N5–Cu2–N4
O2–Cu2–N4
O3–Cu2–N5
O3–Cu2–O4
N4–Cu2–O4
C6–N1–Cu1
C7–N1–Cu1
C8–N2–Cu1
Complex 2
Cu1–N1
N1–Cu1–N3
N2–Cu1–N3
O1–Cu1–N3
N5–Cu2–O2
O3–Cu2–N4
O3–Cu2–O2
O2–Cu2–O4
N5–Cu2–O4
C6–N1–C7
C8–N2–C9
C9–N2–Cu1
172.45(16)
97.97(15)
96.06(14)
172.40 (15)
173.68(15)
86.27(12)
95.31(15)
91.95(14)
130.1(4)
Fig. 1 ORTEP drawing of complex 1, with 30 % probability
displacement ellipsoids. The two parts of the disordered perchlorate
3-
anion and pdmapo ligand are represented with different styles for
clarity
118.3(4)
128.8(3)
conformation and the CuꢀꢀꢀCu separation through the
˚
oxamide bridge is 5.2046(18) A. The coordination envi-
1.929(7)
2.009(7)
1.960(6)
2.249(7)
1.976(8)
1.270(10)
1.279(10)
83.2(3)
Cu1–N2
1.980(7)
1.972(6)
1.946(6)
1.977(8)
1.277(11)
1.318(11)
1.500(13)
174.3(3)
98.2(3)
Cu1–N3
Cu1–O1
ronments of the two Cu(II) atoms are different. Cu1
occupies an inner site of the trideprotonated ligand, with a
Cu2–O2
Cu2–O3
CuN O distorted square-planar environment. The devia-
3
Cu2–O4
Cu2–N4
tions of the ligating atoms from the mean plane are
Cu2–N5
C7–N1
0
.092(2) (N1), 0.079(2) (N2), 0.069(2) (N3), and 0.082(2)
C7–O2
C8–N2
˚
O1) A, while Cu1 is 0.048(2) A out of the plane. The
˚
(
C8–O3
C7–C8
˚
Cu1–N3 [2.009(4) A] bond distance is longer than that of
˚
Cu1–N1 and Cu1–N2 [1.940(4) and 1.981(3) A, respec-
N1–Cu1–N2
N1–Cu1–O1
O1–Cu1–N2
N5–Cu2–N4
O2–Cu2–N4
O3–Cu2–N5
O3–Cu2–O4
N4–Cu2–O4
C2–N1–Cu1
C7–N1–Cu1
C8–N2–Cu1
N1–Cu1–N3
N2–Cu1–N3
O1–Cu1–N3
N5–Cu2–O2
O3–Cu2–N4
O3–Cu2–O2
O2–Cu2–O4
N5–Cu2–O4
C2–N1–C7
C8–N2–C9
C9–N2–Cu1
83.1(3)
tively], which is consistent with the stronger donor abil-
ities of the deprotonated amide N atoms compared to the
primary amine N atoms [36, 37]. The coordination envi-
ronment of the Cu2 atom is a square pyramid with a s
parameter of 0.02 [38]. The basal plane is built from two
164.5(3)
81.8(3)
96.2(3)
94.2(3)
167.9(3)
167.0(3)
99.0(3)
95.8(3)
85.5(2)
91.0(3)
3
-
carbonyl oxygen atoms (O2 and O3) of pdmapo and
two nitrogen atoms (N4 and N5) of phen, while the apical
position is occupied by water (O4). The four basal plane
atoms are practically coplanar with deviations from the
100.6(3)
114.3(5)
115.7(6)
111.2(6)
94.0(3)
129.9(8)
119.3(7)
129.3(6)
˚
least-squares plane lower than 0.007(2) A. Atom Cu2 is
displaced out of this basal plane toward the apical site by
˚
0
.116(2) A. The oxamide bridge is planar within experi-
non-classical hydrogen bonds stabilize the complex into a
one-dimensional chain (Fig. 2).
mental uncertainties. The phen ligand is nearly planar and
coordinates to Cu2 in the usual bidentate chelate fashion
with a bite angle of 83.67(17)°.
Crystal structure of complex 2
There are two sets of hydrogen bonds in the crystal
structure of the complex (Table 3). One classical O–HꢀꢀꢀO
hydrogen bond between the coordinated water ligand (O4)
The structure of complex 2 is very similar to that of
complex 1 (Fig. 3). It also consists of a cis asymmetric
binuclear Cu(II) cation and one non-coordinated perchlo-
rate anion. Selected bond lengths and angles are listed in
Table 2. The asymmetric cis-oxamide bridging ligand
3
-
and the phenol oxygen atom of pdmapo is observed.
In addition, there is also a non-classical hydrogen bond
ii
[
C18–H18ꢀꢀꢀO4 (ii = -x, -y, 1-z)]. These classical and
123

7
4
Transition Met Chem (2013) 38:69–78
3
-
pdmapo chelates two Cu(II) atoms with different coor-
dination numbers. Cu1 is coordinated to three nitrogen
atoms (N1, N2, and N3) and one oxygen atom (O1) of
As shown in Fig. 4, the supramolecular structure of
complex 2 can be described in terms of both intermolecular
hydrogen bonds and p–p stacking interactions. One
O–HꢀꢀꢀO hydrogen bond between the coordinated methanol
i
3
-
pdmapo in a slightly distorted square-planar arrange-
ment. The deviations of the coordinating atoms from their
3
-
ligand (O4) and the phenol oxygen atom of pdmapo [O1
mean plane are 0.118(4) (N1), 0.102(4) (N2), 0.088(3)
˚
(i = 2-x, -y, 1-z.)] links the molecules to form a dimer.
The second structural motif that characterizes the crystal
packing is two types of strong p–p interactions, involving
the benzene ring of pdmapo–bpy (ii = x, -1 ? y, z.) and
iii
bpy–bpy (iii = 1-x, 1-y, 1-z). The shortest interplanar
(
N3), and 0.104(4) A (O1). Atom Cu1 is displaced out of
˚
this plane by 0.007(4) A. Cu2 is five-coordinated in a
ii
square pyramidal environment with a s parameter of 0.01.
Two oxygen atoms (O2 and O3) of pdmapo and two
3
-
˚
distances are 3.446(11) and 3.382(11) A, respectively.
nitrogen atoms (N4 and N5) of bpy form the coordination
˚
basal plane, with the maximum deviation being 0.001(4) A
With these hydrogen bonds and p–p stacking interactions,
a two-dimensional network structure is constructed.
for N5, while atom O4 of the methanol ligand occupies the
˚
apical position. Atom Cu2 is displaced by 0.212(4) A out
of the basal plane toward the apical site. The CuꢀꢀꢀCu
In vitro cytotoxicities
˚
separation through the oxamide bridge is 5.207(2) A. The
bpy ligand is nearly planar, with a dihedral angle between
the two pyridyl rings of 2.4(6)°. It coordinates to the Cu2
atom in the usual bidentate chelate fashion with a bite
angle of 81.8(3)°.
The in vitro cytotoxicities of the free ligand (H pdmapo),
3
the binuclear Cu(II) complexes, and cisplatin against two
cancer cell lines (SMMC-7721 and A549) were assayed.
The free ligand H pdmapo has no cytotoxicities against
3
either of these cancer cell lines, whereas both complexes
displayed significant cytotoxicities. The IC50 values of
complex 1 are 29.2 ± 1.6 and 49.0 ± 2.0 lg/ml for
SMMC-7721 and A549, respectively, while for complex 2,
the IC50 values are 75.4 ± 2.4 and 70.2 ± 3.0 lg/ml,
respectively. Hence, the in vitro cytotoxicities of complex
˚
Table 3 Hydrogen bonds geometries (A, °) of complexes 1 and 2
D–H
A
D–H
HꢀꢀꢀA
DꢀꢀꢀA
D–HꢀꢀꢀA
1
are higher than those of complex 2, although the cyto-
Complex 1
O4–H4A
O4–H4B
C18–H18
Complex 2
O4–H4A
C21–H21
toxicities of both complexes are less than those of cisplatin
(IC50 values of 5.4 and 7.6 lg/ml, respectively), Never-
theless, the inhibition of cell proliferation by these binu-
clear Cu(II) complexes is still notable.
O5
O1
O4
0.87
0.94
0.93
2.16
1.81
2.47
2.976(5)
2.749(5)
3.335(7)
155
175
155
i
ii
i
O1
O5
0.90
0.93
1.76
2.57
2.620(10)
3.276(19)
160
133
Electronic absorption titrations
ii
The application of electronic absorption spectroscopy in
DNA-binding studies is one of the most useful techniques.
The absorption spectra of the free ligand, complex 1 and
Symmetry codes for complex 1: i = 1 - x, -y, -z; ii = -x, -y,
- z. Symmetry codes for complex 2: i = 2 - x, -y, 1 - z;
ii = 1 - x, 1 - y, -z
1
Fig. 2 The one-dimensional
chain structure of complex 1.
Dotted lines indicate hydrogen
bonds [symmetry codes:
i 1 - x, -y, -z, ii -x, -y,
1
- z]
1
23

Transition Met Chem (2013) 38:69–78
75
complex 2 in the absence and presence of CT-DNA are
given in Fig. 5a–c, respectively. For the free ligand,
increasing DNA concentrations resulted in only small
hypochromism for the band at 297 nm. Complexes 1 and 2
showed greater hypochromism, as evidenced from the
decreasing intensity of the peaks at 272 nm (complex 1)
and 300 nm (complex 2), accompanied by slight redshifts
of 3 and 2 nm, respectively, in the absorbance maxima. It
is well known that redshift and hypochromism in the
absorption bands are indicative of intercalative binding of
molecules to DNA. Also the spectra of intercalators are
more perturbed than those of groove binders, and the extent
of hypochromism generally parallels the strength of inter-
calative binding. Hence, these spectral characteristics
suggest that the ligand binds to DNA via a groove binding
mode [39], while the binuclear complexes interact with
DNA most likely through a stacking interaction between
the aromatic chromophore and the base pairs of DNA [40].
These observations can be rationalized by the following
considerations. When the binuclear complexes intercalate
the base pairs of CT-DNA, the p* orbital of the intercalated
ligands (phen or bpy) in the two binuclear complexes can
couple with the p orbitals of the base pairs, thus decreasing
*
the p–p transition energy and resulting in bathochromism.
Furthermore, the coupling p - orbital is partially filled by
electrons, thus decreasing the transition probabilities and
giving rise to hypochromism.
In order to compare quantitatively the binding strength
of the compounds, the intrinsic binding constants K of the
b
compounds with CT-DNA were obtained by monitoring
the changes in absorbance at 297 nm for the ligand,
2
72 nm for complex 1, and 300 nm for complex 2 with
increasing concentrations of DNA, using the following
equation [41]:
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ
where [DNA] is the concentration of DNA, and the
apparent absorption coefficients e , e and e correspond to
a
f
b
Aobsd/[Compound], the extinction coefficient for the free
compound, and the extinction coefficient for the compound
in the fully bound form, respectively. The binding con-
stants, K for complexes 1 and 2 were determined from the
b
Fig. 3 ORTEP drawing of complex 2, with 30 % probability
displacement ellipsoids. The perchlorate anion is disordered at four
positions (both the occupancies of perchlorate anions containing Cl1
and Cl2 are 0.5)
plots of [DNA]/(e - e ) versus [DNA] as 7.16 ± 0.13
a
f
4
4
-1
910 ) and 2.14 ± 0.10 (910 ) M , respectively, while
(
Fig. 4 Crystal packing of
complex 2. Dashed lines
indicate hydrogen bonds and
p–p stacking interactions
[
symmetry codes: i 2 - x, -y,
1
1
- z, ii x, -1 ? y, z, iii 1 - x,
- y, 1 - z.]
123

7
6
Transition Met Chem (2013) 38:69–78
a
4
4
4
4
.5
.4
.3
.2
a
a
f
1.12
0
.3
2
2
1
50
00
50
1
.08
1.04
0
.2
4.1
1
.00
4
3
.0
.9
0.00
0.04
0.08
0.12
0.16
0.20
[Compound]/[DNA]
2
4
6
8
10
12
100
[
DNA]× 10
0.1
5
0
0
0.0
560
580
600
620
640
660
300
350
400
450
Wavelength(nm)
Wavelength(nm)
a
2.0
1.8
1.6
1.4
b
3
00
b
2.0
f
1.8
250
0
.8
1
.6
1.2
.0
2
1
00
50
1
1.4
0.00
0.04
0.08
0.12
0.16
0.20
0
0
0
.6
.4
.2
[Complex]/[DNA]
1.2
2
4
6
8
5
10
12
100
[
DNA]× 10
5
0
0
5
60
580
600
620
640
660
Wavelength(nm)
2
60
280
300
Wavelength(nm)
1.6
a
c
3.4
3.2
3.0
2.8
1
1
1
.4
.2
.0
c
250
f
0.6
0.5
0.4
0.3
2
1
1
00
50
00
0
.00
0.04
0.08
0.12
0.16
0.20
[Complex]/[DNA]
2
4
[
6
8
10
12
DNA]× 10
5
0
0
5
60
580
600
620
640
660
Wavelength(nm)
2
60
280
300
320
Wavelength(nm)
Fig. 6 Fluorescence changes that occur when the CT-DNA-EB
system is titrated with the compounds: ligand (a), complex 1 (b) and
-6
-1
Fig. 5 Absorption spectra of the ligand (a), complex 1 (b) and
complex 2 (c) in Tris–HCl buffer upon addition of calf thymus DNA.
Arrow shows the absorbance changing upon increasing DNA
complex 2 (c). k = 522 nm; [EB] = 2 9 10 mol l ; [DNA] =
ex
-5 -1 -6 -6
5 9 10 mol l ; [Compound]: a 0, b 2 9 10 , c 4 9 10
-6
,
d 6 9 10 , e 8 9 10 , f 1 9 10 mol l . Arrow shows the
-6
-5
-1
concentrations. Inset plots of [DNA]/(e
a
f
- e ) versus [DNA] for the
intensity change upon the increasing complex concentrations
titration of DNA with the compound
Phen possesses a greater planar area and more extended p
system compared to bpy, which will lead to phen penetrating
more deeply into, and stacking more strongly with the base
pairs of DNA.
4 -1
K for the free ligand [1.03 ± 0.07 (910 ) M ] is very
b
small. Complex 1 intercalate more strongly than complex 2,
which can be explained by their different terminal ligands.
1
23

Transition Met Chem (2013) 38:69–78
77
Fluorescence quenching experiments
1.12
Ligand
Complex 2
Complex 1
In order to further investigate the interactions between the
compounds and CT-DNA, fluorescence quenching experi-
ments were carried out. The intrinsic fluorescence intensity
of DNA is very low, as is that of EB in Tris buffer due to
quenching by the solvent molecules. However, on addition
of DNA, the fluorescence intensity of EB is enhanced
because of its intercalation into the DNA. Thus, EB can be
used to probe the interactions of complexes with DNA,
since its fluorescence intensity can be quenched by the
addition of another molecule due to the displacement of EB
from DNA [42]. The emission spectra of EB bound to
DNA in the absence and presence of the free ligand,
complex 1 and complex 2 are given in Fig. 6a–c, respec-
1
1
.10
.08
1.06
1
1
.04
.02
0
.00
0.02
0.04
0.06
0.08
0.10
0.12
[
Compound]/[DNA]
tively. For increasing H pdmapo concentrations, only a
3
Fig. 7 Effect of increasing amounts of the ligand and its binuclear
complexes on the relative viscosity of CT-DNA at 20.0 ± 0.1 °C
slight decrease was observed in the emission intensity of
DNA pretreated with EB (Fig. 6a). For complexes 1 and 2,
the addition of each complex causes an obvious reduction
in emission intensity (Fig. 6b, c), indicating displacement
of some EB molecules into solution. The observed changes
are characteristic of intercalation [43]. According to the
classical Stern–Volmer equation [44]:
while the viscosity of the DNA increases steadily with
increasing amounts of each complex. Such behavior is
consistent with that observed for other intercalators [49].
Complex 1 shows a greater effect than complex 2, which is
again consistent with the result of our spectroscopic studies.
I0=I ¼ 1 þ Ksvr
where I and I represent the fluorescence intensities in the
0
Conclusion
absence and presence of the complex, respectively. Ksv is a
linear Stern–Volmer quenching constant, and r is the ratio of
the total concentration of compound to that of DNA. In the
linear fit plot of I /I versus [Compound]/[DNA], K is given
The design and synthesis of asymmetric ligands has
become more and more interesting. Herein, a new asym-
0
0
sv
metric N,N -bis(substituent)oxamide ligand, H pdmapo,
3
by the ratio of the slope to intercept. The Stern–Volmer
and two of its binuclear Cu(II) complexes have been syn-
thesized and characterized. Our intention was to study the
influence of the terminal ligands on the biological proper-
ties of these complexes. Electronic absorption, fluores-
cence, and viscosity measurements indicate that the ligand
binds to DNA via a groove binding mode, while the two
complexes are DNA intercalators. Complex 1, which
contains phen as co-ligand has stronger binding affinity for
DNA than its bpy counterpart (complex 2), since phen
possesses a greater planar area and more extended p system
than bpy. The DNA-binding affinities are consistent with
their in vitro cytotoxicities. These results are valuable in
understanding the biological properties of such compounds
as well as laying a foundation for the rational design of
novel, powerful agents for targeting nucleic acids. Further
studies on the physicochemical and pharmacokinetic
investigations of these complexes are being pursued.
quenching constants for the free ligand, complexes 1 and 2 are
.51 ± 0.02, 5.33 ± 0.10 and 2.77 ± 0.08, respectively.
0
Hence,theinteractionofcomplex1withDNAisthestrongest,
followed by complex 2 and then by the free ligand, which is
consistent with the above absorption spectral results.
Viscosity measurements
Measurement of viscosity, which is sensitive to changes in
the length of the DNA molecules, is regarded as the least
ambiguous and most critical test of a binding model in
solution in the absence of crystallographic structural data
[
45]. Intercalating agents are expected to elongate the double
helix by the accommodation of the ligands between the base
pairs, leading to an increase in the viscosity of DNA. In
contrast, compounds that bind exclusively in the DNA
grooves typically cause less pronounced (positive or nega-
tive) or no changes in DNA solution viscosity [46]. Figure 7
-
4
shows the relative viscosity of DNA (10 M) in the pres-
ence of varying amounts of the free ligand and the com-
plexes. The ligand shows relatively small changes in DNA
viscosity consistent with DNA groove binding [47, 48],
Supplementary materials
CCDC No. 689821 and 689903 contain the supplementary
crystallographic data for this paper. These data can be
123

7
8
Transition Met Chem (2013) 38:69–78
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
23. Srinivasan S, Annaraj J, Athappan PR (2005) J Inorg Biochem
9
9:876–882
2
2
4. Tan LF, Chao H (2007) Inorg Chim Acta 360:2016–2022
5. Mart ´ı nez-Mart ´ı nez FJ, Padilla-Mart ´ı nez II, Brito MA, Geniz ED,
Rojas RC, Saavedra JBR, H o¨ pfl H, Tlahuextl M, Contreras R
Acknowledgments This project was supposed by the National
Natural Science Foundation of China (No. 21071133), the Program
for Science and Technology of Shandong Province (2011GHY
11521), and the Youth Science Foundation of Huanggang Normal
University (No. 2011CQ132).
(
1998) J Chem Soc Perkin Trans 2:401–406
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