Synthesis, structure, DNA-binding, and cytotoxic activities of N-(5-chloro-2-hydroxyphenyl)-N″-[3-(2-hydroxyethylamino) propyl]oxamide and its dicopper(II) complex

Article abstract of DOI:10.1080/00958972.2011.638063

A dissymmetrical N,N″-bis(substituted)oxamide, N-(5-chloro-2- hydroxyphenyl)-N″[3-(2- hydroxyethylamino)propyl]oxamide (H ₃oxpep), and its dicopper(II) complex, [Cu2(oxpep)(phen)]ClO ₄ (1) (phen=1,10-phenanthroline), were synthesized

Full text of DOI:10.1080/00958972.2011.638063

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Synthesis, structure, DNA-binding,
and cytotoxic activities of N-(5-
chloro-2-hydroxyphenyl)-N′-[3-(2-
hydroxyethylamino)propyl]oxamide
and its dicopper(II) complex
Shi-Hao Cui a , Man Jiang b , Yan-Tuan Li a , Zhi-Yong Wu c & Xiao-
Wen Li c
a Marine Drug & Food Institute, Ocean University of China , 5
Yushan Road, Qingdao, 266003 , PR China
b Qindao Municipal Medical Group , Qindao , Shandong, 266011 ,
PR China
c Key Laboratory of Marine Drug, Chinese Ministry of Education,
Ocean University of China , Qingdao , PR China
Published online: 22 Nov 2011.
To cite this article: Shi-Hao Cui , Man Jiang , Yan-Tuan Li , Zhi-Yong Wu & Xiao-Wen Li (2011)
Synthesis, structure, DNA-binding, and cytotoxic activities of N-(5-chloro-2-hydroxyphenyl)-N′-
[3-(2-hydroxyethylamino)propyl]oxamide and its dicopper(II) complex, Journal of Coordination
Chemistry, 64:23, 4209-4224, DOI: 10.1080/00958972.2011.638063
To link to this article: http://dx.doi.org/10.1080/00958972.2011.638063
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Journal of Coordination Chemistry
Vol. 64, No. 23, 10 December 2011, 4209–4224
Synthesis, structure, DNA-binding, and cytotoxic activities of
N-(5-chloro-2-hydroxyphenyl)-N9-[3-(2-hydroxyethylamino)
propyl]oxamide and its dicopper(II) complex
SHI-HAO CUIy, MAN JIANGz, YAN-TUAN LI*y, ZHI-YONG WUx
and XIAO-WEN LIx
yMarine Drug & Food Institute, Ocean University of China, 5 Yushan Road,
Qingdao, 266003, PR China
zQindao Municipal Medical Group, Qindao, Shandong, 266011, PR China
xKey Laboratory of Marine Drug, Chinese Ministry of Education,
Ocean University of China, Qingdao, PR China
(Received 13 August 2011; in final form 20 October 2011)
A
dissymmetrical N,N0-bis(substituted)oxamide, N-(5-chloro-2-hydroxyphenyl)-N0[3-(2-
hydroxyethylamino)propyl]oxamide (H3oxpep), and its dicopper(II) complex,
[Cu2(oxpep)(phen)]ClO4 (1) (phen ¼ 1,10-phenanthroline), were synthesized. The crystal
structure of 1 was determined by single-crystal X-ray diffraction. In 1, Cu1 and Cu2 are
bridged by cis-oxpep3ꢀ with Cu ꢁ ꢁ ꢁ Cu separation of 5.2007(6) A. Cu1 is in a distorted square-
˚
pyramidal environment, while Cu2 has a square-planar coordination geometry. The 3-D
supramolecular structure of 1 is formed through ꢀ–ꢀ stackings and hydrogen bonds. The
DNA-binding properties and cytotoxic activities of the two compounds were investigated. The
results suggest that the two compounds can interact with HS-DNA by intercalation with
binding affinities following the order 1 4 H3oxpep, which is consistent with their anticancer
activities.
Keywords: Dissymmetrical N,N0-bis(substituted)oxamide; Dicopper(II) complex; Crystal
structure; DNA-binding property; Cytotoxic activity
1. Introduction
Deoxyribonucleic acid (DNA) plays an important role in cells since it contains all the
genetic information for cellular function. Although there are numerous biological targets
in cancer cells, including DNA, RNA, enzyme, and other protein receptors, it is generally
accepted that DNA is the primary target for many anticancer drugs [1, 2]. Cisplatin is a
widely used anticancer drug [3, 4] and the detailed molecular mechanism of its action has
been elucidated, which involves covalent binding to DNA [5]. Although it is curative in
some tumor types, its usage is limited by both side-effects and acquired cellular resistance
[6]. Hence, more-efficacious, target-specific, less-toxic, and non-covalently DNA-binding
*Corresponding author. Email: yantuanli@ouc.edu.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.638063

4210
S.-H. Cui et al.
anticancer drugs are required to be developed. Inspired by cisplatin, number of
researchers now focus on the development of new metal-based anticancer drugs targeting
DNA with non-covalent binding modes. Transition-metal complexes can bind to DNA
non-covalently by intercalation, electrostatic interaction, and groove binding. These
binding modes are important in the development of drugs, new biochemical tools, etc.
Many complexes possessing these binding modes have been synthesized and studied for
DNA-binding properties and cytotoxic activities [7–9].
Copper is a biologically relevant element and many enzymes depend on copper for
their activities. Copper(II) complexes also possess numerous biological activities, such
as antitumor [10], antimicrobial [11], DNA cleavage [12], DNA, and protein binding
[13] activities. Among the above biological activities, attention has been paid to the
research of DNA-binding properties and cytotoxic activities since copper(II) complexes
are regarded as promising alternatives to cisplatin [13]. Compared with the number of
studies dealing with mononuclear copper complexes [7, 12, 13], relatively few studies on
binuclear copper complexes have been reported [14, 15]. N,N0-bis(substituted)oxamides
are good candidates as bridging ligands in forming di- or polynuclear complexes
because they have potential coordination sites that permit the formation of metal
complexes. Coordination abilities toward metal ions can be modified and tuned by
changing the nature of the amide substituents [16, 17]. A series of complexes
with symmetrical N,N0-bis(substituted)oxamides as bridging ligands have been reported
[18–20]. However, studies on complexes of dissymmetrical N,N0-bis(substituted)
oxamides are limited [21], due to synthetic difficulty. Exploration of interactions
between complexes and DNA stimulates us to synthesize new dicopper(II) complexes
with bridging dissymmetrical N,N0-bis(substituted)oxamides to evaluate and understand
the factors in DNA-binding properties and cytotoxic activities.
Herein, we report the synthesis, structure, DNA-binding properties, and cytotoxic
activities of a dissymmetrical N,N0-bis(substituted)oxamide, N-(5-chloro-2-hydroxy-
phenyl)-N0-[3(2-hydroxyethylamino)propyl]oxamide (H3oxpep), and its dinuclear
copper(II) complex, [Cu2(oxpep)(phen)]ClO4 (1), which was structurally characterized
by X-ray crystallography.
2. Experimental
2.1. Materials
All chemicals used in the synthesis were of reagent grade and obtained commercially
without purification. Doubly-distilled water was used to prepare buffers. Ethidium
bromide (EB) and herring sperm DNA (HS-DNA) were purchased from Sigma Corp.
and used as received.
2.2. Synthesis of H3oxpep
A total of 2.22 mL (20 mmol) of ethyl oxalyl chloride in 10 mL THF (tetrahydrofuran)
was added dropwise into a 40 mL THF solution containing 2.86 g (20 mmol) of 2-amino-
4-chlorophenol at 0ꢂC. The mixture was stirred for 2 h at room temperature. Then, the
mixture was added dropwise into solution which contained 30 mL of absolute ethanol

Dicopper(II) complex
4211
and 4.8 mL (40 mmol) of 2-(3-amino-propylamino)ethanol at 0ꢂC with stirring.
The resulting solution was stirred for 5 h at room temperature. H3oxpep, precipitated
as yellow powder, was recrystallized from ethanol and dried under vacuum. Yield: 88%.
Anal. Calcd for C13H18N3O4Cl (%): C, 49.45; H, 5.75; N, 13.31. Found (%): C, 49.55;
1
H, 5.61; N, 13.32. MS(ES-MS), m/z (%): 316.1 (100) [MH]þ. H NMR (DMSO-d6,
600 MHz): ꢁ 9.82 (br, 1 H), ꢁ 9.26 (t, J ¼ 5.52 Hz, 1H), ꢁ 8.15 (s, 1H), ꢁ 6.96 (d, J ¼ 8.82 Hz,
1H), ꢁ 6.81 (d, J ¼ 8.82 Hz, 1H), ꢁ 5.58 (br, 2H), ꢁ 3.48 (t, J ¼ 6.06, Hz, 2H), ꢁ 3.25
(dt, J ¼ 5.52, 6.06 Hz, 2H), ꢁ 2.65-2.61 (m, 4H), ꢁ 2.50 (quintet, J ¼ 1.80 Hz, 1H), ꢁ 1.67
(quintet, J ¼ 6.60 Hz, 2H).
2.3. Synthesis of 1
A total of 0.0315 g (0.1 mmol) of H3oxpep was dissolved in 5 mL of methanol and
deprotonated by 0.0255 g (0.3 mmol) of piperidine in 2 mL of methanol. To this solution,
a 5 mL methanol solution of 1,10-phenanthroline (0.1 mmol, 0.0180 g) was added. After
stirring for 10 min, a solution of Cu(ClO4)2 ꢁ 6H2O (0.2 mmol, 0.0742 g) in 5 mL methanol
was further added dropwise. The mixture was stirred vigorously at 60ꢂC for 6 h, and then
filtered. The filtrate was allowed to stand at room temperature for several days to give
dark brown crystals. Yield: 60%. Anal. Calcd for Cu2C25H23Cl2N5O8 (%): C, 41.73;
H, 3.22; N, 9.73. Found (%): C, 41.65; H, 3.25; N, 9.84.
2.4. Physical measurements
Carbon, hydrogen, and nitrogen analyses were performed with a Perkin-Elmer
elemental analyzer Model 240. The mass spectrum (ES–MS) was measured with a
1
Waters Q-TOF GLOBLE mass spectrometer. H NMR spectrum was measured on a
JEOL JNM-ECP600 spectrometer with TMS as an internal standard when DMSO-d6
was used as solvent. Molar conductance was measured with a Shanghai DDS-11 A
conductometer. The infrared spectrum was recorded with samples as KBr pellets in a
Nicolet model Impact 470 FTIR spectrophotometer. The UV-Vis spectrum was
recorded in a 1 cm path length quartz cell on a Cary 300 spectrophotometer.
Fluorescence was tested on an Fp-750w Fuorometer. Viscosity measurements were
carried out using an Ubbelodhe viscometer immersed in a thermostated water bath
maintained at 16.0(ꢃ0.1)ꢂC.
2.5. Crystal structure determination of 1
The X-ray diffraction experiment for 1 was conducted on a Bruker APEX area-detector
˚
diffractometer with graphite monochromated Mo-Kꢂ radiation (ꢃ ¼ 0.71073 A) at
296 K. The crystal structure was solved by direct methods followed by Fourier
synthesis. Structure refinements were performed by full matrix least-squares using
SHELXL-97 on F2 [22]. The oxygens in perchlorate and the carbons in hydro-
xyethylaminopropyl (heap) group of oxpep3ꢀ are both disordered into two parts.
The occupancies of the two perchlorates were equal to 0.5, while those of heap were
refined freely at first and then fixed at 0.75 (suffix A) and 0.25 (suffix B), respectively.
The hydrogen of hydroxyl was located in a difference Fourier map and refined freely.

4212
S.-H. Cui et al.
The Cl–O lengths of ClOꢀ4 were limited to 1.4 A with DFIX and the O ꢁ ꢁ ꢁ O distances
˚
were restrained by SADI instruction for the reasonable geometry. The C–O, C–N, and
˚
C–C in heap were similarly restrained by DFIX instruction to 1.43, 1.47, and 1.54 A,
respectively. The other hydrogen atoms were placed in calculated positions with
˚
˚
C–H ¼ 0.97 (methylene), 0.93 A (aromatic) and N–H ¼ 0.91 A, and included in the final
cycles of refinement in the riding mode, with Uiso(H) ¼ 1.2 Ueq of the carrier atoms.
Crystal data and structural refinement parameters for 1 are summarized in table 1 and
selected bond distances and angles are listed in tables 2 and 3.
2.6. DNA-binding experiments
All experiments involving herring sperm DNA (HS-DNA) were performed in tris-
(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer solution (pH ¼ 7.16). Tris-HCl
buffer was prepared using deionized and sonicated triply-distilled water. Solution
of HS-DNA in Tris-HCl buffer gave a ratio of UV absorbance at 260 and 280 nm,
A260/A280, of ca 1.9, indicating that the DNA was sufEciently free of protein [23].
The concentration of HS-DNA was determined according to its absorbance at 260 nm
adopting ꢄ260 ¼ 6600 (mol L
)
ꢀ1 ꢀ1 cmꢀ1 [24]. All stock solutions of HS-DNA were stored
at 4ꢂC and used after no more than 4 days. Concentrated stock solutions of H3oxpep
and 1 were prepared by dissolving in DMSO and suitably diluted with Tris-HCl buffer
to required concentrations for all the experiments. Electronic absorption spectral
titration experiments were performed keeping concentrations of the two compounds
constant while varying the HS-DNA concentration. Equal solution of HS-DNA was
added to every solution of H3oxpep, 1, and the reference to eliminate the absorbance of
Table 1. Crystal data and details of the structure determination for 1.
Empirical formula
Formula weight
Crystal system
Space group
Cu2C25H23N5O8Cl2
719.46
Monoclinic
P21/c
ꢂ
˚
Unit cell dimensions (A, )
a
b
c
12.9484(7)
23.8073(14)
8.8829(5)
ꢂ
ꢅ
ꢆ
90.00
105.5880(10)
90.00
2637.6(3), 4
1.812
1.877
3
˚
Volume (A ), Z
Calculated density (g cm3)
Absorption coefficient (mmꢀ1
F(0 0 0)
)
1456
0.07 ꢄ 0.09 ꢄ 0.31
Crystal size (mm3)
Temperature (K)
˚
Wavelength (A)
Limiting indices
296
0.71073
ꢀ16 ꢅ h ꢅ 14;
ꢀ31 ꢅ k ꢅ 19;
ꢀ11 ꢅ l ꢅ 11
15,525
6030[R(int) ¼ 0.038]
1.84–27.57
3983
Reflections collected
Independent reflection
ꢇ range for data collection (ꢂ)
Observed data [I 4 2ꢈ(I)]
R, wR2, S
0.0400, 0.0984, 1.02
0.000, 0.000
Max., avg. shift/error

Dicopper(II) complex
4213
HS-DNA itself. In the ethidium bromide (EB) Fuorescence displacement experiment,
5 mL of the EB Tris-HCl solution (1 mmol Lꢀ1) was added to 1 mL of HS-DNA solution
(at saturated binding levels) [25] and stored in the dark for 2 h before use. Then solution
of each compound was titrated into the DNA/EB mixture and diluted in Tris-HCl
buffer to 5 mL, producing solutions with varied mole ratio of each compound to
HS-DNA. Before measurement, the mixture was shaken up and incubated at room
temperature for 30 min. Fuorescence spectra bound to HS-DNA were obtained at an
emission wavelength of 581 nm in the Fluorometer. Viscosity experiments were
performed using an Ubbelodhe viscometer immersed in a thermostated water bath
maintained at 16.0(ꢃ0.1)ꢂC. HS-DNA samples of approximately 200 base pairs in
length were prepared by sonication in order to minimize complexities arising from
DNA Fexibility [26]. Flow times were measured with a digital stopwatch, each
compound was measured thrice, and an average Fow time was calculated. Relative
viscosities for HS-DNA in the presence and absence of each compound were calculated
from the relation ꢉ ¼ (t ꢀ t0)/t0, where t is the observed Fow time of DNA-containing
solution and t0 is that of Tris-HCl buffer alone. Data were presented as (ꢉ/ꢉ0)1/3 versus
binding ratio [27], where ꢉ is the viscosity of DNA in the presence of H3oxpep or 1 and
ꢉ0 is the viscosity of DNA alone.
2.7. In vitro antitumor activity evaluation by SRB assays
In vitro antitumor activities of H3oxpep, 1, and cisplatin were evaluated against two
cancer cell lines, including SMMC-7721 and A549, by using the Sulforhodamine B
ꢂ
˚
Table 2. Selected bond distances (A) and angles ( ) for 1.
Cu1–O1
Cu1–N1
Cu1–N3
Cu2–O3
Cu2–N5
1.981(2)
1.955(3)
1.998(3)
1.904(2)
1.983(3)
Cu1–O4
Cu1–N2
Cu2–O2
Cu2–N4
CL1–C4
2.402(3)
1.980(3)
1.964(2)
1.975(3)
1.740(3)
O1–Cu1–O4
O1–Cu1-N2
O4–Cu1–N1
O4–Cu1–N3
N1–Cu1–N3
O2–Cu2–O3
O2–Cu2–N5
O3–Cu2–N5
93.52(9)
159.54(10)
105.56(10)
77.02(10)
177.35(11)
86.55(9)
O1–Cu1–N1
O1–Cu1–N3
O4–Cu1–N2
N1–Cu1–N2
N2–Cu1–N3
O2–Cu2–N4
O3–Cu2–N4
N4–Cu2–N5
82.55(9)
97.98(9)
104.20(10)
82.88(11)
95.96(11)
98.36(10)
173.77(11)
83.12(11)
173.82(10)
91.60(11)
ꢂ
˚
Table 3. Hydrogen-bonding geometries for 1 (A, ).
D–HA
D–H
H ꢁ ꢁ ꢁ A
D ꢁ ꢁ ꢁ A
D–H ꢁ ꢁ ꢁ A
O4–H4 ꢁ ꢁ ꢁ O1i
0.91
0.93
0.93
0.93
0.93
0.93
0.93
1.82(4)
2.46
2.50
2.57
2.45
2.44
2.46
2.705(3)
165(2)
130.3
133.6
140.7
153.2
148.1
162.9
C14–H14 ꢁ ꢁ ꢁ O6A
C14–H14 ꢁ ꢁ ꢁ O6B
C15–H15 ꢁ ꢁ ꢁ O7A
C15–H15 ꢁ ꢁ ꢁ O8B
C16–H16 ꢁ ꢁ ꢁ O7Aii
C16–H16 ꢁ ꢁ ꢁ O8Bii
3.142(11)
3.211(10)
3.339(11)
3.305(9)
3.271(11)
3.362(12)
i
ii
Symmetry codes: x, ½ ꢀ y, ꢀ1/2 þ z; ꢀx, ꢀy, ꢀz.

4214
S.-H. Cui et al.
(SRB) assays. All cells were cultured in RPMI 1640 supplemented with 10% (v/v) fetal
bovine serum, 1% (w/v) penicillin (104 U mLꢀ1), and 10 mg mLꢀ1 streptomycin.
Cell lines were maintained at 37ꢂC in a 5% (v/v) CO2 atmosphere with 95% (v/v)
humidity. Cultures were passaged weekly using trypsin-EDTA to detach the cells from
their culture Fasks. The three compounds were dissolved in DMSO and diluted to the
required concentration with culture medium when used. The content of DMSO in the
Enal concentrations did not exceed 0.1%. At this concentration, DMSO was found to
be non-toxic to the cells tested. Rapidly growing cells were harvested, counted, and
incubated at the appropriate concentration in 96-well microplates for 24 h.
The compounds dissolved in culture medium were then applied to the culture wells
to achieve Enal concentrations ranging from 10ꢀ3 to 102 mg mLꢀ1. Control wells were
prepared by the addition of culture medium without cells. The plates were incubated at
37ꢂC in a 5% CO2 atmosphere for 48 h. Upon completion of the incubation, the cells
were Exed with ice-cold 10% trichloroacetic acid (100 mL) for 1 h at 4ꢂC, washed Eve
times in distilled water and allowed to dry in air, and stained with 0.4% SRB in 1%
acetic acid (100 mL) for 15 min. The cells were washed four times in 1% acetic acid and
air-dried. The stain was solubilized in 10 mmol Lꢀ1 unbuffered Tris base (100 mL) and
the OD of each well was measured at 540 nm on a microplate spectrophotometer. The
IC50 values were calculated from the curves constructed by plotting cell survival (%)
versus the complex concentration (mg mLꢀ1).
3. Results and discussion
3.1. Synthetic routes and general properties of H3oxpep and 1
In general, two synthetic strategies are available for the preparation of binuclear
complexes. The first is to use a designed polynucleating ligand, which offers
coordination geometry or ligand Eeld strength suitable for metal ions. The second is
to use a complex as ‘‘ligand’’ that contains a potential donor group capable of
coordinating to another metal ion. In this study, our aim was to obtain a ꢊ-oxamido-
bridged dicopper(II) complex, therefore, the first synthetic strategy was adopted. For
that, a new dissymmetrical N,N0-bis(substituted)oxamide, N-(5-chloro-2hydroxy-
phenyl)-N0-[3-(2-hydroxyethylamino)propyl]oxamide (H3oxpep), was chosen as bridg-
ing ligand. Simultaneously, 1,10-phenanthroline (phen) was used as terminal ligand. In
the course of preparing the dicopper(II) complex, the use of piperidine as base makes
the bridging ligand coordinate to copper(II) through deprotonated oxamido nitrogen.
Elemental analyses indicate that the reaction of H3oxpep with Cu(ClO4)2 ꢁ 6H2O and
phen in 1 : 2 : 1 ratio yields the dicopper(II) complex [Cu2(oxpep)(phen)]ClO4 (1), as
expected. The synthetic pathway for H3oxpep and 1 may be represented by scheme 1.
Both H3oxpep and 1 are insoluble in non-polar solvents, moderately soluble in water
and methanol, and very soluble in DMF and DMSO to give stable solutions at room
temperature. In the solid state, the two compounds are fairly stable in air to allow
physical measurements. The molar conductance value (81 ꢀꢀ1 cmꢀ2 molꢀ1) of 1 in
DMF solution falls in the expected range for 1 : 1 electrolyte [28]. These observations
coincide with the following spectral characterization.

Dicopper(II) complex
4215
Cl
NH2
NH
Cl
HO
OH
OH
Ethyl oxalyl chloride
OH
O
O
NH OH
O
O
NH
Cl
NH2
NH NH
H3oxpep
OEt
Cl
O
Cu
Cu(ClO ) •
6H2O
4 2
N
N
O
O
N
N
–
ClO
4
1,10-phenanthroline
Cu
HO
NH
[Cu2(oxpep)phen]ClO4
Scheme 1. Synthesis of H3oxpep and 1.
3.2. IR and electronic spectra
The IR spectra were taken from 4000 to 400 cmꢀ1. The carbonyl stretching vibration at
1657 cmꢀ1 for H3oxpep is considerably shifted to lower frequency 1629 cmꢀ1 in 1,
implying that the carbonyl oxygens of H3oxpep take part in coordination. This shift has
often been used as proof of an oxamido-bridge [29]. The ꢋ(C¼N) at 1523 for phen is
found in 1, indicating the coordination of phen to copper(II) [30]. A broad and intense
band centered at 1108 cmꢀ1, and a strong sharp band at 628 cmꢀ1 for the antisymmetric
stretching vibration and the antisymmetric bending vibration of non-coordinated
perchlorate [31] are also observed for 1.
The UV-Vis absorption spectra of H3oxpep and 1 were measured in the UV-Vis
region (200–800 nm) using DMSO as solvent. For H3oxpep, there is an intense band at
229 nm which can be assigned to the ꢀ–ꢀ* transition of the substituted benzene. In the
spectrum of 1, four absorption bands with varied intensities are observed. The three
bands at 229, 269, and 288 nm can be assigned to ꢀ–ꢀ* transitions of coordinated
oxpep3ꢀ and phenanthroline [32, 33]. A broad band is observed at 671 nm in the
spectrum of 1, corresponding to the d–d transition of copper(II) [34].
3.3. Crystal structure of 1
As shown in figure 1, two copper(II) ions are bridged by cis-oxpep3ꢀ and the Cu ꢁ ꢁ ꢁ Cu
˚
separation through the oxamido-bridge is 5.2007(6) A. The cis-oxamido group coordi-
nates to Cu1 and Cu2 in the usual chelating mode, with bite angles of 82.89(10) and
86.55(9)ꢂ, respectively. Cu1 is in a distorted square-pyramidal coordination geometry
with a ꢌ value of 0.30 [35], while Cu2 has a square-planar coordination geometry. Cu1
resides in an inner site of the cis-oxpep3ꢀ and the basal plane is formed by O1, N1, N2,
and N3 from oxpep3ꢀ. The maximum displacement from the coordination plane is

4216
S.-H. Cui et al.
Figure 1. A view of 1 with the atom numbering scheme. Displacement ellipsoids are drawn at 30%
probability and hydrogen atoms are shown as small spheres of arbitrary radii. Dashed lines indicate non-
classical hydrogen bonds.
˚
0.1175(14) A (N1). Cu1 displaces 0.1469(13) A out of the plane. The apical position is
˚
˚
occupied by O4 with Cu1–O4 distance of 2.402(2) A, which is longer than the equatorial
˚
one (Cu1–O1: 1.981(2) A). Cu2 coordinates to the exo-oxygens (O3 and O4) of the cis-
oxpep3ꢀ and nitrogens (N4 and N5) from phenanthroline. Maximum deviation of O3,
˚
˚
O4, N4, and N5 from basal plane is 0.022(16) A (N5) and Cu2 is 0.08(6) A out of the
plane. The cis-oxpep3ꢀ coordinates pentadentate to Cu1 and results in the formation of
three five-membered rings and one six-membered ring.
In the crystal structure, two kinds of ꢀ–ꢀ stacking interactions connect the complex
molecules, forming 1-D chains parallel to the c axis (figure 2). One is observed between
a phenanthroline and another one at 1 ꢀ x, ꢀy, ꢀziii. The nearest separation is
iii
˚
3.338(4) A for C14 to the N5-pyridine ring of phenanthroline. The other is observed
between the quinoline ring of phenanthroline and the substituted benzene ring of
oxpep3ꢀ with the symmetry operation of 1 ꢀ x, ꢀy, 1ꢀziv. The nearest separation is
iv
˚
3.318(4) A for C6 to the middle benzene ring of phenanthroline. These 1-D chains are
linked through classical hydrogen bonds O4–H4 ꢁ ꢁ ꢁ O1i [symmetry code: ix, ½ ꢀ y, ꢀ1/
2 þ z] into a 2-D network parallel to the plane b0c. Adjacent layers are further
assembled by relatively weak non-classical hydrogen bonds C16–H16 ꢁ ꢁ ꢁ O7ii [symme-
ii
try code: ꢀx, ꢀy, ꢀz] to form a 3-D supramolecular structure which is revealed
in figure 3.

Dicopper(II) complex
4217
Figure 2. A 2-D network of 1 parallel to the b0c plane, formed by classical O4–H4 ꢁ ꢁ ꢁ O1i hydrogen bonds
(dashed lines) and two kinds of ꢀ–ꢀ stacking interactions (double dashed lines). The ClOꢀ4 1 and hydrogen
i
atoms not involved in hydrogen bonds have been omitted for clarity. [Symmetry codes: x, ½ ꢀ y, ꢀ1/2 þ z;
iii1ꢀx, ꢀy, ꢀz; iv1ꢀx, ꢀy, 1 ꢀ z].
3.4. DNA-binding studies
3.4.1. Electronic absorption titration. The application of electronic absorption
spectrometry in DNA-binding studies is one of the most useful techniques to examine
the binding mode and strength of a compound to DNA. Usually, the binding of an
intercalative molecule to DNA is accompanied by hypochromism and red-shift in the

4218
S.-H. Cui et al.
Figure 3. A view of the non-classical hydrogen bonds in 1 (dashed lines). Hydrogen atoms not involved in
ii
hydrogen bonds have been omitted for clarity. [Symmetry codes: ꢀx, ꢀy, ꢀz].
absorption spectrum due to stacking interactions between aromatic chromophores of
the molecule and DNA base pairs. The extent of hypochromism and red-shift are
commonly proportional to the intercalative binding strength [36, 37]. The absorption
spectra of H3oxpep and 1 in the absence and presence of HS-DNA are given in figure 4.
As shown in figure 4, absorption spectra of H3oxpep and 1 at 229 nm show
hypochromic (7.9% for H3oxpep and 10.9% for 1) and red-shift of 3 nm on the
addition of increasing amounts of HS-DNA. According to electronic absorption
spectra, we can deduce that both H3oxpep and 1 bind to DNA by intercalation.
In order to further evaluate the binding strength of H3oxpep and 1 with HS-DNA,
the intrinsic binding constants Kb were determined by monitoring changes in
absorbance at 229 nm adopting the following equation [38]:
½DNAꢆ=ð"a ꢀ "f Þ ¼ ½DNAꢆ=ð"b ꢀ "f Þ þ 1=Kbð"b ꢀ "f Þ
ð1Þ
where [DNA] is the concentration of DNA, "a, "f, and "b correspond to the extinction
coefEcients, respectively, for each addition of DNA to the compounds, for the free
compounds, and for the compounds in fully bound form. From the [DNA]/("a ꢀ "f)
versus [DNA] plots (insets in figure 4), the binding constants for H3oxpep and 1 are
estimated to be 6.67 ꢄ 104 (mol Lꢀ1 ꢀ1
)
(R ¼ 0.9951 for six points) and 4.50 ꢄ 105
(mol Lꢀ1 ꢀ1
than H3oxpep may be due to the phenanthroline ring in 1 which is expected to be
)
(R ¼ 0.9934 for six points), respectively. The better binding constant of 1

Dicopper(II) complex
4219
(a) 1.6
1.2
6
5
4
3
0.8
2
4
6
8
10
5
–1
[DNA] ×10 molL
0.4
250
275
300
325
350
Wavelength (nm)
(b) 0.8
0.6
8
7
6
5
4
3
2
0.4
2
4
6
8
–1
10
5
[DNA] ×10 molL
0.2
0.0
250
275
300
325
350
Wavelength (nm)
Figure 4. Absorption spectra of H3oxpep (a) and 1 (b) upon titration of HS-DNA. Arrows indicate changes
upon increasing HS-DNA concentration. Insets: Plots of [DNA]/("a ꢀ "f) vs. [DNA] for absorption titration
of HS-DNA to H3oxpep (a) and 1 (b).
stacked between base pairs upon interaction with DNA. Electrostatic interaction may
be another factor for 1 to enhance the binding strength to DNA, which is lacking in
H3oxpep [39]. Comparing the intrinsic binding constant of 1 with those of some
mononuclear copper(II) complexes containing 1,10-phenanthroline (table 4), we
conclude that 1 can strongly bind to DNA and its binding constant is remarkable.
Furthermore, the Kb value of 1 is also higher than those of some dicopper(II) complexes
containing 1,10-phenanthroline, such as [Cu2(pdmp)(phen)](ClO4)2 [31], Cu2(phen)2Cl4
[33], and [Cu2(bipp)(phen)2](ClO4)4 [46], as listed in table 4. The main differences
between these dicopper(II) complexes are in their bridging ligands and this indicates
that diverse bridging ligands have a profound effect on DNA-binding ability, as
revealed by the different binding constants.
3.4.2. Fluorescence titration. In order to further investigate the interactions between
the two compounds and HS-DNA, the ethidium bromide (EB) Fuorescence displace-
ment experiments were employed. Ethidium bromide (EB) is a well-known Fuorescence

4220
S.-H. Cui et al.
Table 4. The intrinsic binding constants (Kb) with DNA.
Compound
Kb ((mol Lꢀ1 ꢀ1
)
)
Reference
H3oxpep
Complex 1
Cu(phen)2Cl2
6.67 ꢄ 104
4.50 ꢄ 105
2.75 ꢄ 103
5.10 ꢄ 103
2.24 ꢄ 103
3.86 ꢄ 103
4.08 ꢄ 103
6.35 ꢄ 103
4.20 ꢄ 103
4.90 ꢄ 103
3.47 ꢄ 104
0.90 ꢄ 105
4.40 ꢄ 104
4.75 ꢄ 104
1.60 ꢄ 104
This work
This work
[40]
[Cu(l-leucine)(phen)(H2O)]NO3
[Cu(l-isoleucine)(phen)(H2O)]NO3
[Cu(l-proline)(phen)(H2O)]ClO4
[Cu(l-tyrosine)(phen)]ClO4
[Cu(l-threonine)(phen)(H2O)]ClO4
[Cu(HisLeu a)(phen)]Cl
[Cu(HisSer b)(phen)]Cl
[41]
[41]
[42]
[43]
[44]
[45]
[45]
[30]
[7]
[31]
[33]
[46]
c
Cu(saltyr )(phen)
[Cu(tdp d)(phen)]ClO4ꢁCH3OH
e
[Cu2(pdmp )(phen)](ClO4)2
Cu2(phen)2Cl4
[Cu2(bipp )(phen)2](ClO4)4
f
aHisLeu ¼ histidylleucine.
bHisSer ¼ histidylserine.
cSaltyr ¼ salicylidene tyrosine.
dHtdp ¼ 2-[(2-(2-hydroxyethylamino)ethylimino)methyl]phenol.
epdmp ¼ 6,60-piprazine-1,4-diyl-dimethylene bis(4-methyl phenol).
fbipp ¼ 2,9-bis(2-imidazo[4,5-f][1,10]phenanthroline)-1,10-phenanthroline.
probe that can bind to DNA [47]. In general, the Fuorescence intensity of DNA is very
low, and that of EB in Tris-HCl buffer is also not high because of being quenched by
the solvent molecules. But when EB is intercalated into DNA base pairs, the
Fuorescence intensity is greatly increased. The Fuorescence can be quenched by the
addition of another molecule [39, 48] owing to the replacement of EB Fuorophores.
Thus, EB can be used to probe the interactions of compounds with DNA.
In our experiment, as illustrated in figure 5, the Fuorescence intensity of EB bound to
HS-DNA at 581 nm shows obvious decreasing trend upon increasing the concentration
of 1, indicating that 1 can bind to HS-DNA, and some EB molecules are released into
solution. Similar trend was also observed for H3oxpep (figure 5). The Fuorescence
quenching of EB bound to HS-DNA by the two compounds is in agreement with the
linear Stern–Volmer equation [49]:
I0=I ¼ 1 þ Ksv½Qꢆ
ð2Þ
where I0 and I represent the Fuorescence intensities in the absence and presence of
quencher, respectively. Ksv is a linear Stern-Volmer quenching constant, Q is the
concentration of quencher. In the Stern–Volmer plots (insets in figure 5), Ksv values are
given by the slopes. The Ksv values for H3oxpep and 1 are 3.42 ꢄ 104 (R ¼ 0.9942 for 11
points) and 9.75 ꢄ 104 (R ¼ 0.9972 for 11 points), respectively. The quenching ability of
the two compounds to the EB-DNA system follows the order: 1 4 H3oxpep. The data
show that the DNA-binding interaction of 1 is stronger than that of H3oxpep,
consistent with the above electronic absorption spectral results.
3.4.3. Viscosity measurement. Viscosity measurement is sensitive to length changes
and regarded as the least ambiguous and the most critical test of DNA-binding modes
in solution, providing reliable evidence for intercalative binding [50, 51]. To further

Dicopper(II) complex
4221
(a)
80
60
40
20
0
1.6
1.4
1.2
1.0
0
5
10
15
20
6
–1
[H oxpep] ×10 molL
3
560
580
600
620
640
660
Wavelength (nm)
(b)
100
80
60
40
20
0
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0
2
4
6
8
10
6
–1
[Complex 1] ×10 molL
560
580
600
620
640
660
Wavelength (nm)
Figure 5. Emission spectra of HS-DNA-EB system upon titration of H3oxpep (a) and 1 (b). Arrows show
the changes upon increasing concentrations of the two compounds. Insets: Plots of I0/I vs. [compound] for the
titration of H3oxpep (a) and 1 (b) to the HS-DNA-EB system.
clarify the binding modes of the two compounds with HS-DNA, viscosity studies were
carried out. For either compound, the relative viscosity of HS-DNA increased with the
increasing concentration of H3oxpep or 1 (figure 6). The increase of viscosity can be
ascribed to the extension of HS-DNA resulting from intercalation of H3oxpep or 1
between adjacent DNA base pairs. The increased degree of viscosity follows the order:
1 4 H3oxpep. The obviously increased viscosity by 1 may arise from the phenanthroline
ring which makes it intercalate more deeply into HS-DNA base pairs than H3oxpep.
Thus, the results of viscosity studies validate those obtained from electronic absorption
titration and fluorescence titration.
If we compare the DNA-binding of 1 with some structurally-similar polynuclear
copper(II) complexes reported earlier by us, using N,N0-bis(substituted)oxamides as

4222
S.-H. Cui et al.
2.2
2.0
1.8
1.6
1.4
1.2
1.0
a
b
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16
[Compound] / DNA
Figure 6. Effects of the increasing amounts of 1 (a) and H3oxpep (b) on the relative viscosity of HS-DNA at
16.0(ꢃ0.1)ꢂC, [HS-DNA] ¼ 0.2 mmolLꢀ1
.
Table 5. In vitro antitumor activities against SMMC-7721 and A549.
IC50 (mg mLꢀ1
)
Compound
SMMC-7721
A549
H3oxpep
Complex 1
Cisplatin
4100
4100
32.1 ꢃ 0.1
28.0 ꢃ 0.2
5.4 ꢃ 0.2 ng mLꢀ1
7.6 ꢃ 0.4 ng mLꢀ1
bridging ligands and polypyridines as terminal ligands [52, 53], we find that not only the
interaction modes but also the DNA-binding ability of these ꢊ-oxamido-bridged
polynuclear complexes are different. These results confirm that interactions of this class
of complexes toward HS-DNA may be tuned by changing the bridging or terminal
ligands. Such strategy should be important in designing new ꢊ-oxamido-bridged
polynuclear complexes and understanding the binding properties between oxamido
complexes and DNA. Indeed, further investigations using various bridging and terminal
ligands are still required in order to confirm the effect and are in progress in our
laboratory.
3.5. In vitro antitumor activity studies
In vitro antitumor activities of H3oxpep and 1 against two cancer cell lines, human
hepatocellular carcinoma cell line SMMC-7721 and human lung adenocarcinoma cell
line A549, were investigated in comparison with the widely used drug cisplatin by using
SRB assays. As revealed in table 5, though 1 has higher IC50 values than those of
cisplatin, the inhibition of cell proliferation produced by 1 is still rather active with its
IC50 values falling in the 28–32 mg mLꢀ1 range, and better than those of H3oxpep.
The cytotoxicities of compounds vary with the planarity of different ligands and the
extent of their intercalation into DNA base pairs [7, 54, 55] and the significant
cytotoxicity of 1 may be partially attributed to its strong intercalation.

Dicopper(II) complex
4223
4. Conclusions
In order to investigate the interactions of binuclear complexes with DNA, we synthesized
and structurally characterized a new ꢊ-oxamido-bridged dicopper(II) complex
[Cu2(oxpep)(phen)]ClO4 (1) by employing N-(5-chloro-2-hydro-xyphenyl)-N0
[3-(2-hydroxyethylamino)propyl]oxamide (H3oxpep) as bridging ligand and
1,10-phenanthroline (phen) as terminal ligand. DNA-binding properties of the two
compounds were studied through absorption and emission spectrometry and viscometry.
Both compounds interact with HS-DNA by intercalation. The DNA-binding ability of 1
is better than free H3oxpep, which can be attributed to the electrostatic and
stronger stacking interactions between 1 and DNA. Complex 1 has higher
DNA-binding constant than some known copper(II) complexes containing
1,10-phenanthroline. The cytotoxicities of H3oxpep and 1 against SMMC-7721 and
A549 cell lines were tested in vitro by SRB assays. Complex 1 has good cytotoxicity,
which may be partially explained by strong intercalation. These results should be
valuable in designing new ꢊ-oxamido-bridged polynuclear complexes and understanding
the DNA-binding properties and anticancer activities of this class of complexes.
Supplementary material
Crystallographic data for the structural analysis of the copper(II) complex has been
deposited with the Cambridge Crystallographic Data Center, CCDC No. 823655.
Copies of this information may be obtained free of charge from The Director, CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK (Fax: C44 1233 336 033; Email:
deposit@ccdc.cam.ac.uk).
Acknowledgments
This project was supported by the National Natural Science Foundation of China (No.
21071133), the Natural Science Foundation of Qingdao City (No. 09-1-3-73-jch), and
the Program for Changjiang Scholars and Innovative Research Team in University
(IRT0944).
References
[1] C.X. Zhang, S.J. Lippard. Curr. Opin. Chem. Biol., 7, 481 (2003).
[2] C.H. Wu, D.H. Wu, X. Liu, G. Guoyiqibayi, D.D. Guo, G. Lv, X.M. Wang, H. Yan, H. Jiang, Z.H. Lu.
Inorg. Chem., 48, 2352 (2009).
[3] T. Boulikas, M. Vougiouka. Oncol. Rep., 10, 1663 (2003).
[4] E. Wong, C.M. Giandomenico. Chem. Rev., 99, 2451 (1999).
[5] E.R. Jamieson, S.J. Lippard. Chem. Rev., 99, 2467 (1999).
[6] D. Wang, S.J. Lippard. Nat. Rev. Drug Discov., 4, 307 (2005).
[7] V. Rajendiran, R. Karthik, M. Palaniandavar, V.S. Periasamy, M.A. Akbarsha, B.S. Srinag,
H. Krishnamurthy. Inorg. Chem., 46, 8208 (2007).

4224
S.-H. Cui et al.
[8] L. Messori, F. Abbate, G. Marcon, P. Orioli, M. Fontani, E. Mini, T. Mazzei, S. Carotti, T. Connell,
P. Zanello. J. Med. Chem., 43, 3541 (2000).
[9] J. Liu, W.J. Zheng, S. Shi, C. Tan, J. Chen, K. Zheng, L. Ji. J. Inorg. Biochem., 102, 193 (2008).
[10] M. Chauhan, K. Banerjee, F. Arjmand. Inorg. Chem., 46, 3072 (2007).
[11] J.D. Saha, U. Sandbhor, K. Shirisha, S. Padhye, D. Deobagkar, C.E. Ansond, A.K. Powelld. Bioorg.
Med. Chem. Lett., 14, 3027 (2004).
[12] R. Ren, P. Yang, W. Zheng, Z. Hua. Inorg. Chem., 39, 5454 (2000).
[13] S.S. Bhat, A.A. Kumbhar, H. Heptullah, A.A. Khan, V.V. Gobre, S.P. Gejji, V.G. Puranik. Inorg.
Chem., 50, 545 (2011).
[14] C.Y. Gao, X.F. Ma, J. Lu, Z.G. Wang, J.L. Tian, S.P. Yan. J. Coord. Chem., 64, 2157 (2011).
[15] N. Raman, A. Sakthivel, R. Jeyamurugan. J. Coord. Chem., 63, 1080 (2010).
[16] R. Ruiz, J. Faus, F. Lloret, M. Julve, Y. Journaux. Coord. Chem. Rev., 193, 1069 (1999).
[17] M.C. Dul, E. Pardo, R. Lescouezec, Y. Journaux, J. Ferrando-Soria, R. Ruiz-Garcia, J. Cano, M. Julve,
F. Lloret, D. Cangussu, C.L.M. Pereira, H.O. Stumpf, J. Pasan, C. Ruiz-Perez. Coord. Chem. Rev., 254,
2281 (2010).
[18] Z.L. Liu, L.C. Li, D.Z. Liao, Z.H. Jiang, S.P. Yan. Cryst. Growth Des., 5, 783 (2005).
[19] E.Q. Gao, H.Y. Sun, D.Z. Liao, Z.H. Jiang, S.P. Yan. Polyhedron, 21, 359 (2002).
[20] Y.H. Zhang, M. Yu, Q.L. Wang, G.F. Xu, M. Hu, G.M. Yang, D.Z. Liao. Polyhedron, 27, 3371 (2008).
[21] X.W. Zhang, Y.J. Zheng, Y.T. Li, Z.Y. Wu, C.W. Yan. J. Coord. Chem., 63, 2985 (2010).
[22] G.M. Sheldrick. Acta Cryst., A64, 112 (2008).
[23] J. Marmur. J. Mol. Biol., 3, 208 (1961).
[24] M.E. Reichmann, S.A. Rice, C.A. Thomas, P.J. Doty. J. Am. Chem. Soc., 76, 3047 (1954).
[25] J.B. Chaires, N. Dattagupta, D.M. Crothers. Biochemistry, 21, 3933 (1982).
[26] G. Cohen, H. Eisenberg. Biopolymers, 8, 45 (1969).
[27] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro. J. Am. Chem. Soc., 108, 2081 (1986).
[28] W.J. Geary. Coord. Chem. Rev., 7, 81 (1971).
[29] H. Ojima, K. Nonoyama. Coord. Chem. Rev., 85, 92 (1988).
[30] P.R. Reddy, A. Shilpa. Polyhedron, 30, 565 (2011).
[31] S. Anbu, M. Kandaswamy. Polyhedron, 30, 123 (2011).
[32] L.F. Chin, S.M. Kong, H.L. Seng, K.S. Khoo, R. Vikneswaran, S.G. Teoh, M. Ahmad, S.B.A. Khoo,
M.J. Maah, C.H. Ng. J. Inorg. Biochem., 105, 221 (2011).
[33] Q.Q. Zhang, F. Zhang, W.G. Wang, X.L. Wang. J. Inorg. Biochem., 100, 1344 (2006).
[34] P. Camurlu, A. Yilmaz, L. Tatar, D. Kisakurek, D. Ulku. Cryst. Res. Technol., 40, 271 (2005).
[35] A.W. Addison, T.N. Rao, J. Reedijk, J.V. Rijin, G.C. Verschoor. J. Chem. Soc., Dalton Trans., 1349
(1984).
[36] D. Cremer, J.A. Pople. J. Am. Chem. Soc., 97, 1354 (1975).
[37] T.M. Kelly, A.B. Tossi, D.J. McConnell, T.C. Strekas. Nucleic Acids Res., 13, 6017 (1985).
[38] A. Wolf, G.H. Shimer Jr, T. Meehan. Biochemistry, 26, 6392 (1987).
[39] Y.T. Li, C.Y. Zhu, Z.Y. Wu, M. Jiang, C.W. Yan. J. Coord. Chem., 62, 3795 (2009).
[40] T. Gupta, S. Dhar, M. Nethaji, A.R. Chakravarty. J. Chem. Soc., Dalton Trans., 1896 (2004).
[41] R. Rao, A.K. Patra, P.R. Chetana. Polyhedron, 27, 1343 (2008).
[42] P.R. Reddy, N. Raju, P. Manjula, K.V.G. Reddy. Chem. Biodiv., 4, 1565 (2007).
[43] S. Ramakrishnan, V. Rajendiran, M. Palaniandavar, V.S. Periasamy, B.S. Srinag, H. Krishnamurthy,
M.A. Akbarsha. Inorg. Chem., 48, 1309 (2009).
[44] S.C. Zhang, Y.G. Zhu, C. Tu, H.Y. Wei, Z. Yang, L.P. Lin, J. Ding, J.F. Zhang, Z.J. Guo. J. Inorg.
Biochem., 98, 2099 (2004).
[45] P.R. Reddy, P. Manjula. Chem. Biodiv., 4, 468 (2007).
[46] J.Z. Wu, L. Yuan, J.F. Wu. J. Inorg. Biochem., 99, 2211 (2005).
[47] J. Olmsted, D.R. Kearns. Biochemistry, 16, 3647 (1977).
[48] E.B. Veale, T. Gunnlaugsson. J. Org. Chem., 75, 5513 (2010).
[49] O. Stern, M. Volmer. Z. Phys., 20, 183 (1919).
[50] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires. Biochemistry, 31, 9319 (1992).
[51] L. Jin, P. Yang. J. Inorg. Biochem., 68, 79 (1997).
[52] H.H. Lu, Y.J. Zheng, Y.T. Li, Z.Y. Wu, K. Zheng, C.W. Yan. J. Coord. Chem., 64, 1360 (2011).
[53] X.W. Li, M. Jiang, Y.T. Li, Z.Y. Wu, C.W. Yan. J. Coord. Chem., 63, 1582 (2010).
[54] A. Barve, A. Kumbhar, M. Bhat, B. Joshi, R. Butcher, U. Sonawane, R. Joshi. Inorg. Chem., 48, 9120
(2009).
[55] J.H. Tan, Y.J. Lu, Z.S. Huang, L.Q. Gu, J.Y. Wu. Eur. J. Med. Chem., 42, 1169 (2007).