Synthesis, crystal structure, cytotoxicities and DNA-binding properties of a tetracopper(II) complex with N-benzoato-N0-[2-(2-hydroxyethylamino)ethyl- amino]oxamide ligand

Article abstract of DOI:10.1007/s11243-012-9623-2

A new dissymmetrical N,N'-bis(substituted)- oxamide ligand, N-benzoato-N'-[2-(2- hydroxyethylamino)- ethyl]oxamide (H₃bhyox), and its tetranuclear copper(II) complex, [Cu₄(bhyox)₂(dabt) ₂](ClO₄)₂ (1) have been synthesized and characterized (dabt = 2,2'-diamino- 4,4'-bithiazole). The crystal structures of H₃bhyox and complex 1 have been determined by X-ray single-crystal diffraction. Complex 1 has a cyclic tetranuclear cation with an embedded inversion center, in which the Cu-Cu separations through the oxamido and carboxyl bridges are 5.2246(6) and 5.3141(6) A , respectively. Both Cu(II) centers at the inner and outer sites of the cis-bhyox^3- ligand have square pyramidal geometries. There is a 3D hydrogen-bonding network in the crystal. The cytotoxicities and DNA-binding properties of H3bhyox and complex 1 were studied. Both compounds can interact with HS- DNA by intercalation, and the DNA-binding affinities are consistent with their in vitro cytotoxicities. Springer Science+Business Media B.V. 2012.

Full text of DOI:10.1007/s11243-012-9623-2

Transition Met Chem (2012) 37:569–577
DOI 10.1007/s11243-012-9623-2
Synthesis, crystal structure, cytotoxicities and DNA-binding
properties of a tetracopper(II) complex with N-benzoato-N0-
[2-(2-hydroxyethylamino)ethyl-amino]oxamide ligand
•
Wen-Cai Chen Ling-Dong Wang
•
•
Yan-Tuan Li Zhi-Yong Wu Cui-Wei Yan
•
Received: 20 April 2012 / Accepted: 8 June 2012 / Published online: 28 June 2012
Ó Springer Science+Business Media B.V. 2012
Abstract A new dissymmetrical N,N’-bis(substituted)-
oxamide ligand, N-benzoato-N0-[2-(2- hydroxyethylamino)-
ethyl]oxamide (H3bhyox), and its tetranuclear copper(II)
complex, [Cu4(bhyox)2(dabt)2](ClO4)2 (1) have been syn-
thesized and characterized (dabt = 2,20-diamino- 4,40-bi-
thiazole). The crystal structures of H3bhyox and complex 1
have been determined by X-ray single-crystal diffraction.
Complex 1 has a cyclic tetranuclear cation with an
embedded inversion center, in which the Cu–Cu separa-
tions through the oxamido and carboxyl bridges are
Introduction
DNA is the primary target for many anticancer drugs
since it contains all the genetic information required for
cellular function [1]. Cisplatin is one of the most suc-
cessful drugs used in the clinic for the treatment of tes-
ticular, bladder, lung, and ovarian cancers and has been
shown to act on DNA by numerous studies [2]. Despite
its remarkable pharmacological activity, cisplatin has
severe disadvantages such as a relatively limited spec-
trum of activity and high toxicity [3]. With the hope of
finding highly effective, target-specific and less toxic
drugs, much effort has been devoted to the development
of metal-based anticancer agents in recent years. Transi-
tion metal complexes can interact with DNA by non-
covalent modes such as intercalation, groove binding, and
electrostatic effects. Intercalation has attracted special
interest due to its various applications in cancer therapy
and molecular biology [4, 5]. In general, the intercalating
ability not only correlates with the planarity of a ligand
but is also related to the coordination geometry of the
metal center and donor atom types of the ligand [6]. In
addition, both the type of metal and its valency play
important roles in deciding the binding extent of com-
plexes to DNA [7]. Many copper(II) complexes have
been studied in this context, since copper, as a biologi-
cally active metal, has many correlations with endoge-
nous oxidative DNA damage associated with aging and
cancer [8]. Compared to mono- and binuclear copper(II)
complexes [9, 10], relatively few studies on tetranuclear
copper(II) complexes have been reported to date [11].
However, the reported enhancement of DNA-binding
activity and cytotoxicities for tetranuclear complexes
prompted us to design and synthesize new tetracopper(II)
complexes.
˚
5.2246(6) and 5.3141(6) A, respectively. Both Cu(II)
centers at the inner and outer sites of the cis-bhyox3-
ligand have square pyramidal geometries. There is a 3D
hydrogen-bonding network in the crystal. The cytotoxici-
ties and DNA-binding properties of H3bhyox and complex
1 were studied. Both compounds can interact with HS–
DNA by intercalation, and the DNA-binding affinities are
consistent with their in vitro cytotoxicities.
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-012-9623-2) contains supplementary
material, which is available to authorized users.
W.-C. Chen ꢀ L.-D. Wang ꢀ Y.-T. Li (&)
Marine Drug and Food Institute, Ocean University of China,
5 Yushan Road, Qingdao 266003, People’s Republic of China
e-mail: yantuanli@ouc.edu.cn
Z.-Y. Wu
Key Lab. Marine Drug, Chinese Minist. Educ., Ocean University
of China, Qingdao, People’s Republic of China
C.-W. Yan (&)
College of Marine Life Science, Ocean University of China,
Qingdao 266003, People’s Republic of China
e-mail: cuiweiyan@ouc.edu.cn
123

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Transition Met Chem (2012) 37:569–577
Synthesis of H3bhyox
A promising strategy to design and synthesize poly-
nuclear complexes is the use of bridging ligands. In this
regard, N,N’-bis(substituent)oxamides have played a key
role as bridging ligands because their coordinating ability
toward transition metals can be modified by varying the
amide substituents. Following this synthetic approach, a
series of polynuclear complexes with interesting struc-
tures based the bridging symmetrical N,N0-bis(substi-
tuted)oxamides have been reported [12, 13]. However,
the studies on dissymmetrical N,N’-bis(substituted)oxam-
ides are limited [14], due to the difficulties of their
synthesis. We therefore decided to synthesize new tetra-
copper(II) complexes with bridging dissymmetrical
N,N’-bis(substituted)oxamides in order to evaluate the
factors effecting their DNA-binding properties and
cytotoxicities.
A solution of ethyl oxalyl chloride (1.11 mL, 10 mmol) in
THF (10 mL) was added dropwise to a solution of
anthranilic acid (1.37 g, 10 mmol) in THF (mL) at 273 K.
The mixture was stirred rapidly for 1 h at room tempera-
ture, and then, absolute ethanol (20 mL) was added. After
0.5 h, the mixture was added dropwise to a solution of
2-(2-aminoethylamino)ethanol (1.1 mL, 10 mmol) in
absolute ethanol (20 mL) at 273 K. The resulting solution
was stirred for 8 h, whereupon H3bhyox precipitated as a
white powder which was filtered off, washed with absolute
ethanol and dried under vacuum. Yield: 2.33 g (79 %).
Colorless crystals of H3bhyox suitable for X-ray analysis
were obtained from an ethanol/water (3:2) mixture by slow
evaporation for 2 weeks at room temperature. Anal. Calc.
for C13H20.4N3O5.7(%): C, 47.8; H, 6.3; N, 12.8. Found(%):
C, 47.7; H, 6.2; N, 12.9. IR (KBr pellet, cm-1): 3,311
[m(O–H)]; 1,675 [m(C=O)]. ES-MS, m/z (%): 296.1(100)
[M ? H]?. 1H NMR (600 MHz, DMSO-d6, d, ppm): 14.65
(s, 1H), 8.99 (t, J = 5.82 Hz, 1H), 8.53 (d, J = 2.28 Hz,
1H), 8.00 (dd, 1H), 7.37 (d, J = 3.36 Hz, 1H), 7.08 (s, 1H),
5.92 (m, 1H), 3.69 (m, 2H), 3.32 (m, 4H), 2.50 (m, 4H),
1.92 (m, 2H).
With these thoughts in mind, in this paper, a new dis-
symmetrical N,N’-bis(substituted)oxamide ligand, namely
N-benzoato-N0-[2-(2-hydroxylethylamino)ethyl]oxamide
(H3bhyox), and its tetracopper(II) complex, [Cu4(bhyox)2-
(dabt)2](ClO4)2 (1) (dabt = 2,20-diamino-4,40-bithiazole)
have been synthesized and structurally characterized by
single-crystal X-ray diffraction. In vitro cytotoxicities and
the reactivities toward DNA of the two compounds are
reported.
Synthesis of complex 1
To a stirred solution of Cu(ClO4)2ꢀ6H2O (0.0371 g,
0.1 mmol) in ethanol (5 mL), a solution of H3bhyox
(0.0148 g, 0.05 mmol) and piperidine (0.0128 g,
0.15 mmol) in ethanol (10 mL) was added dropwise at
room temperature. After stirring for 0.5 h, a solution of
dabt (0.0099 g, 0.05 mmol) in ethanol (5 mL) was added
dropwise. The mixture was stirred vigorously at 333 K for
5 h, the resulting green solution was filtered and green cube
crystals of the complex suitable for X-ray analysis were
obtained by slow evaporation at room temperature. Yield:
0.0330 g (64 %). Anal. Calc. for Cu4C38H40N14O18S4Cl2
(%): C, 31.8; H, 2.7; N, 13.7. Found(%): C, 31.8; H, 2.8; N
13.6. IR (KBr pellet, cm-1): 1,632 [m(C=O) ? mas(COO)];
1,365 [m(C=N)]; 1,107, 623 [m(ClO4)]. Molar conductance:
KM (DMF solution): 146 X-1 cm-2 mol-1. ES-MS, m/z
(%): 1,236.2 (100) [M-2ClO4]?.
Experimental
Materials and measurements
Ethidium bromide (EB) and herring sperm DNA (HS–
DNA) were purchased from Sigma Corp. and used as
received. All other chemicals were of reagent grade and
obtained commercially.
The C, H, and N microanalyses were obtained on a
Perkin-Elmer 240 elemental analyzer. Molar conductances
were measured with a Shanghai DDS-11A conductmeter.
Mass spectra (ES-MS) were measured with a Waters
Q-TOF GLOBLE mass spectrometer. NMR spectra were
recorded with a JEOL-ECP 600 spectrometer with TMS as
internal standard and DMSO-d6 as solvent, and chemical
shifts are reported as d values. IR spectra were recorded
on
a Nicolet-470 spectrophotometer in the range
4,000–400 cm-1 as KBr pellets. Electronic spectra (in
DMF) were recorded on a Cary 300 spectrophotometer.
Fluorescence spectra were measured with an Fp-750w
Fluorometer. Viscosity experiments were carried out using
an Ubbelodhe viscometer maintained at a constant tem-
perature of 298.0 ( 0.1) K in a thermostatic water bath.
Caution: Although we have not encountered any prob-
lems, perchlorate compounds are potentially explosive and
should be handled with care.
X-ray crystallography
The X-ray diffraction experiments were made on a Bruker
APEX area-detector diffractometer with graphite mono-
˚
chromatic Mo-Ka radiation (k = 0.71073 A) at 296 K.
The crystal structures were solved by direct methods fol-
lowed by Fourier syntheses. Structure refinement was
performed by full matrix least-squares procedures using
SHELXL-97 on F2 [15]. For the free ligand, the H atoms on
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Transition Met Chem (2012) 37:569–577
571
passaged weekly using trypsin–EDTA to detach the cells
from their culture flasks. The two compounds and cisplatin
were dissolved in DMSO and diluted to the required con-
centration with culture medium before use. The content of
DMSO in the final 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, coun-
ted, and incubated at the appropriate concentration in
96-well microplates for 24 h. The test two compounds
dissolved in culture medium were then applied to the cul-
ture wells to achieve final concentrations ranging from
10-3 to 102 lg/mL. Control wells were prepared by addi-
tion 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 cells were fixed
with ice-cold 10 % trichloroacetic acid (100 mL) for 1 h at
277 K, washed five times with distilled water and allowed
to dry 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
solubilized in 10 mM unbuffered Tris base (100 mL) and
the OD of each well was measured at 540 nm on a
microplate spectrophotometer. The IC50 values were cal-
culated from the curves constructed by plotting cell sur-
vival (%) versus the concentrations of the test compound.
Table 1 Crystal data and details of the structure determination for
H3bhyox (L) and complex 1
Empirical formula
C13H17N3O5ꢀ
1.7(H2O) (L)
C38H40Cu4N14O10S4ꢀ
2(ClO4)(1)
Formula weight
Crystal system
Space group
325.92
1,434.22
Monoclinic
C2/c
Monoclinic
P2(1)/n
˚
Unit cell dimensions (A °)
a
b
c
a
b
c
12.7747(2)
14.0035(13)
24.654(2)
15.5419(14)
90
19.1528(3)
14.0693(2)
90.00
110.287(1)
90.00
105.501(2)
90
3
˚
Volume (A ), Z
D(calc) (g cm3)
l (MoKa) (mm-1
F(0 0 0)
3,228.82(9), 8
1.341
5,170.6(8), 4
1.842
)
0.109
1.975
1,384
2,896
Crystal size (mm3) 0.30 0.25 0.15
0.10 9 0.25 9 0.30
0.71073
˚
Radiation (A)
0.71073
1.9–27.7
(Mo-Ka)
h range
1.65–27.59
Tot., uniq. data
16,369, 7,546, 0.030 15,316, 5,926, 0.0351
Observed data
[I [ 2d(I)]
4,361
4,260
R indices
S
0.0606, 0.2091
1.02
0.0403, 0.1099
1.022
DNA-binding studies
Max., av.
0.00/0.00
0.00/0.00
All experiments involving Herring Sperm DNA (HS–DNA)
were performed in tris-(hydroxymethyl)aminomethane-HCl
(Tris–HCl) buffer solution (pH = 7.35). Tris–HCl buffer
was prepared using deionized and sonicated triple distilled
water. The solution of HS–DNA in Tris–HCl buffer gave a
nitrogen and oxygen atoms were located in a difference
Fourier map (DFP) and refined freely for N atoms or
treated as riding for O atoms, with Uiso(H) = 1.5 Ueq
(waters). H atoms on C atoms were placed in calculated
ratio of UV–visible absorbance at 260 and 280 nm, A260
A
/
˚
positions, with C–H = 0.93 (aromatic), 0.97 A (methy-
280, of ca. 1.9, indicating that the DNA was sufficiently free
lene), and refined in riding modes, with Uiso(H) = 1.2 Ueq
(carrier atoms). For complex 1, the H atoms on nitrogen
and oxygen atoms were found in a DFP and then refined
with a riding model (atoms O5, N6 and N7) or freely (atom
N3). Other H atoms on carbon atoms were treated as for the
ligand. Crystal data and structure refinement parameters are
summarized in Table 1.
of protein. The concentration of HS–DNA was determined
by UV–visible absorbance at 260 nm. The molar absorption
coefficient, e260, was taken as 6,600 M-1 cm-1. Stock
solution of HS–DNA was stored at 277 K and used after no
more than 3 days. Concentrated stock solutions of the
compounds were prepared by dissolving samples in DMSO
and diluted suitably with Tris–HCl buffer to the required
concentrations. Absorption spectral titrations were per-
formed by keeping the concentration of the test compound
constant while varying the HS–DNA concentration. Equal
solutions of HS–DNA were added to the test compound and
reference solutions to eliminate the absorbance of HS–DNA
itself. In the ethidium bromide (EB) fluorescence displace-
ment experiments, 5 lL of EB Tris–HCl solution (1 mM)
was added to 1 mL of HS–DNA solution (at saturated
binding levels), and the mixture stored in the dark for 2 h,
then titrated into the DNA/EB mixture and diluted in Tris–
HCl buffer to 5 mL, producing a set of solutions with varying
In vitro cytotoxicity evaluation
In vitro cytotoxicities of the free ligand (H3bhyox), com-
plex 1 and cisplatin were evaluated against two cancer cell
lines including SMMC-7721 and A549 using the Sulfo-
rhodamine B (SRB) assay. All cells were cultured in RPMI
1640 supplemented with 10 % (v/v) fetal bovine serum,
1 % (w/v) penicillin (104 U/mL) and 10 mg/mL strepto-
mycin. Cell lines were maintained at 310 K in a 5 % (v/v)
CO2 atmosphere with 95 % (v/v) humidity. Cultures were
123

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Transition Met Chem (2012) 37:569–577
mole ratio of the test compound to HS–DNA. Before mea-
surement, the mixture was shaken and incubated at room
temperature for 30 min. The fluorescence spectra were
obtained at an emission wavelength of 584 nm in the fluo-
rometer. For the viscosity measurements, HS–DNA samples
approximately 200 base pairs in length were prepared by
sonication in order to minimize complexities arising from
DNA flexibility. Flow times were measured with a digital
stopwatch. Each sample was measured three times, and an
average flow time was calculated. Relative viscosities for
HS–DNA in the presence and absence of the test compounds
were calculated from the relation g = (t - t0)/t0, where
t is the observed flow time of DNA-containing solution and
t0 is that of Tris–HCl buffer alone. Data were analyzed as
(g/g0)1/3 versus binding ratio, where g is the viscosity of
DNA in the presence of the compound and g0 is the viscosity
of DNA alone.
allows the bridging ligand (H3bhyox) to coordinate to cop-
per(II) through the deprotonated oxamido nitrogen atoms.
Elemental analyses indicate that the reaction of H3bhyox
with Cu(ClO4)2ꢀ6H2O plus dabt yielded the tetracopper(II)
complex [Cu4(bhyox)2(dabt)2](ClO4)2 (1). The synthetic
pathway for H3bhyox and complex 1 are shown in Scheme 1.
H3bhyox is soluble in water, DMF and DMSO, and
slightly soluble in methanol and ethanol at room tempera-
ture. Complex 1 is insoluble in common polar solvents, but
very soluble in DMF and DMSO to give stable solutions at
room temperature, consistent with its polymeric nature [16].
The molar conductance value (146 X-1 cm-2 mol-1) of
complex 1 in DMF solution falls in the expected range for a
1:2 electrolyte [17], suggesting that the tetracopper(II)
complex consists of a tetracopper(II) complex cation
[Cu4(bhyox)2(dabt)2]2? and two uncoordinated perchlorate
anions in solutions. The stability of complex 1 as a whole
tetranuclear entity in solution was further supported by the
ES-MS results (see ‘‘Experimental’’).
Results and discussion
Spectroscopic characterization
Synthesis and properties
Comparison of the IR spectra of free H3bhyox and its
tetracopper(II) complex provide some information regard-
ing the mode of coordination in the tetra- copper(II)
complex. It is noteworthy that the antisymmetric stretching
vibration of the carboxylate group [mas(COO)], together with
the carbonyl stretching vibration of the oxamidate group
(mC=O) of the free H3bhyox at 1,675 cm-1 are considerably
shifted toward lower frequency (1,632 cm-1) in the
The new ligand, N-benzoato-N0-[2-(2-hydroxyethyl-
amino)ethyl]oxamide (H3bhyox), is expected to function as a
bridging ligand because it may coordinate to metals through
not only its oxamido oxygens and nitrogens but also the
oxygens of the carboxyl group. In this work, 2,20-diamine-
4,40-bithiazole (dabt) was used as terminal co-ligand. In
order to prepare complex 1, the use of piperidine as base
Scheme 1 The synthetic
pathway for H3bhyox and
complex 1
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Transition Met Chem (2012) 37:569–577
573
complex, implying that not only the oxygens of the
oxamidate group but also the oxygens of the carboxylate
group are coordinated with copper(II) to form a tetranu-
clear complex [18]. In addition, the skeletal vibration of the
bithiazole is reasonably red-shifted, being observed at
1,365 cm-1 in complex 1, indicating that the dabt ligand is
involved in terminal coordination to the tetracopper(II)
complex [19]. Furthermore, a broad and intense band
centered at 1,088 cm-1, plus a strong sharp band at
624 cm-1 assigned to the antisymmetric and antisymmetric
bending vibrations of the non-coordinated perchlorate
group [20] are also observed for complex 1.
The electronic spectrum of the tetracopper(II) complex was
recorded in the UV–Vis region (200–800 nm). Spectra
obtained for complex
1 at different concentrations
(1.6 9 10-5 to 6 9 10-6 M in DMSO) obeyed the Beer-
Lambert law, indicating that the tetracopper(II) complex stays
intact at these concentrations. The complex shows two
absorption bands, the band at 230 nm (e = 8.02 9
104 M-1 cm-1) can be assigned to the p-p* transition of the
coordinated oxamide group. The second, broad band at
671 nm(e = 697 M-1 cm-1) isassignedtothed–dtransition
of copper(II) [21]. For the free ligand H3bhyox, there is an
intense band at 231 nm and a less intense band at 276 nm
which can be assigned to the intramolecular and intermolec-
ular p-p* transitions of the substituted benzene ring,
respectively.
Crystal structure of H3bhyox
As shown in Fig. 1a, the asymmetric unit of H3bhyox con-
tains two molecules [LA (C1–C13) and LB (C14–C26)] plus
3.4 solvent water molecules. Both H3bhyox molecules are in
the trans- conformation; the torsion angles C9–N2–C10–
C11 and C22–N5–C23–C24 are 68.4(3)° and 74.9(3)°,
respectively. The torsion angles of N2–C10–C11–N3
[61.4(3)°] and N5–C23–C24–N6 [64.6(3)°] indicate gauche
conformations for the bonds C10–C11 and C23–C24. LA and
LB form a dimer as shown in Fig. 1b. In this array, each
molecule has four sites interacting with its neighbor, by two
classical hydrogen bonds, one non-classical hydrogen bond
(Table 2) and one p–p stacking interaction. The benzene
rings of LA and LB subtend a dihedral angle of 7.78(18)° and
Fig. 1 a An ORTEP view of ligand L with the thermal ellipsoids at
30 % probability level. H atoms are shown as small spheres of
arbitrary radii. Hydrogen bonds are shown as dotted lines. b A view of
the couple formed by LA and LB. In such an array, the ligand
molecules, respectively have five sites interacting with each other
[two classical hydrogen bonds, two non-classical hydrogen bonds,
one p–p stacking interaction]
(Fig. 2a). The Cu–Cu separations through the oxamido and
˚
the carboxyl bridges are 5.2246(6) and 5.3141(6) A,
respectively. The octadentate bhyox3- ligand adopts a cis-
conformation. Both the inner and the outer copper(II) centers
(atoms Cu1 and Cu2, respectively) are in square pyramidal
coordination geometries with s values [22] of 0.03 and 0.06,
respectively. Atom Cu1 in an {N3O2} environment is dis-
placed from the basal plane (O1, N1, N2 and N3) 0.1965(13)
˚
the distance between ring centroids is 4.1182(16) A.
The molecule pairs link each other to form a chain parallel
to the [1 0 -1] direction via the hydrogen bonds involving
hydroxyl groups [N3–H3BꢀꢀꢀO10i and O5–H5CꢀꢀꢀO2i, sym-
metry code (i) = x ? 1/2, 1/2 - y, z - 1/2. Fig. S1].
˚
A forward the apical hydroxyl group (O5). The Cu1–N3
bond is longer than Cu1–N1 and Cu1–N2 (Table 3), which is
consistent with the stronger donor abilities of the deproto-
nated amide N atoms compared to the primary amine N
atoms. Atom Cu2 has an {N2O3} coordination and deviates
Crystal structure of complex 1
The complex consists of a centrosymmetric tetranu-
clear cation and two uncoordinated perchlorate anions
˚
0.1629(12) A from the basal plane.
123

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Transition Met Chem (2012) 37:569–577
˚
Table 2 Hydrogen-bonding geometries (A °) for the ligand
D–HꢀꢀꢀA
d(D–H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA)
\(DHA)
N1–H1AꢀꢀꢀO1
0.96(3)
0.98(3)
0.88(3)
0.94(3)
0.88(3)
0.93(3)
0.97(3)
0.96
1.84(3)
1.80(3)
2.04(3)
1.78(3)
2.33(3)
1.85(3)
1.97(3)
1.66
2.625(3)
2.774(3)
2.789(3)
2.576(3)
3.033(3)
2.747(3)
2.867(3)
2.614(3)
2.726(3)
2.814(3)
2.989(6)
2.756(3)
2.832(3)
2.889(5)
3.170(7)
3.033(6)
2.636(10)
3.291(3)
3.375(4)
137(2)
172(3)
142(3)
141(2)
136(2)
161(2)
153(2)
173.4
164.7
171.2
140.8
178.4
168.8
160
N3–H3AꢀꢀꢀO6
N3–H3BꢀꢀꢀO10i
N4–H4AꢀꢀꢀO6
N5–H5AꢀꢀꢀO12ii
N6–H6AꢀꢀꢀO1
N6–H6AꢀꢀꢀO11ii
O5–H5BꢀꢀꢀO2i
O10–H10CꢀꢀꢀO12iii
O11–H11CꢀꢀꢀO7
O11–H11DꢀꢀꢀO14
O12–H12CꢀꢀꢀO5
O12–H12DꢀꢀꢀO7
O13–H13CꢀꢀꢀO3
O13–H13DꢀꢀꢀO7
O14–H14CꢀꢀꢀO2iv
O14–H14DꢀꢀꢀO13
C24–H24BꢀꢀꢀO4
C25–H25AꢀꢀꢀO8v
0.96
1.77
0.93
1.89
0.99
2.16
0.94
1.81
0.88
1.97
0.86
2.06
0.92
2.31
155.4
146.9
155.9
150.9
149.3
0.88
2.25
0.87
1.82
0.97
2.41
0.97
2.50
Symmetry codes: (i) x ? 1/2, 1/2 - y, z - 1/2; (ii) x - 1/2, 1/2 - y,
z ? 1/2; (iii) x, y, z ? 1; (iv) x ? 1, y, z; (v) x ? 1/2,1/2 - y, z ? 1/2
Around atom Cu1, the five-membered chelate rings (Cu1–
N2–C10–C11–N3 and Cu1–N3–C12–C13–O5) have enve-
lope conformations with the Pucker parameters [23] of
Fig. 2 a An ORTEP view of complex 1 with the thermal ellipsoids at
30 % probability level. H atoms are shown as small spheres of arbitrary
radii. Hydrogen bonds are shown as dotted lines. Symmetry codes:
(i) = 1/2 - x, 1/2 - y, 1 - z. b A view of the one-dimensional
hydrogen-bonding structure parallel to [1 0 1] direction. Symmetry
codes: (i) = 1/2 - x, 1/2 - y, 1 - z; (iii) = 1 - x, y, 3/2 - z
˚
˚
Q = 0.418(3) A, u = 115.8(4)° and Q = 0.491(3) A,
u = 65.7(3)°, respectively. The six-membered chelate ring
formed by the carboxylato group folds 16.32(12)° along
˚
C1ꢀꢀꢀN1 (Q = 0.232(3) A, h = 82.2(7)° and u =
292.0(6)°). The carboxylate group bridges the copper(II)
centers in a skew–skew fashion, with torsion angles of Cu1–
O1–C1–O2 = 163.4(2)°and Cu2i–O2–C1–O1 = -94.7(3)°
[symmetry code: (i) = 1/2 - x, 1/2 - y, 1 - z], which are
similar to those found in related complexes [24].
complex 1 displayed significant cytotoxicities with IC50
values of 22.0 0.4 and 36.0 0.6 lg/mL for SMMC-
7721 and A549, respectively. Though the in vitro cyto-
toxicities for complex 1 are less than those of cisplatin
(IC50 values of 5.4 and 7.6 lg/mL, respectively), the
inhibition of cell proliferation produced by complex 1 is
still rather active. According to these results, we conclude
that complexation may be the major cause of the enhanced
effect on the viability of cancer cells [25]. These findings
encouraged us to explore the DNA-binding properties of
the two compounds.
In the crystal, the tetranuclear cations and the perchlo-
rate anions make up a hydrogen-bonding network parallel
to the bc plane (Fig. S3, Table 4). Moreover, the cations
form a chain extending along the [1 0 1] direction through
the hydrogen bonds between the hydroxyl and carboxylate
groups (Fig. 2b). Finally, the networks and chains together
complete a three-dimensional supramolecular structure.
In vitro cytotoxicities
UV–visible absorption titration
In vitro cytotoxicities of the two compounds together with
cisplatin against two cancer cell lines, namely human
hepatocellular carcinoma cell line SMMC-7721 and human
lung adenocarcinoma cell line A549 were conducted in our
study. The results indicate that the free ligand H3bhyox has
no cytotoxicities against the two cancer cell lines, whereas
Electronic absorption spectroscopy is widely used to study
the binding modes of metal complexes with DNA. Gen-
erally, the observations of hypochromism and red-shift
caused by adding DNA to solutions of test compounds, are
attributed to intercalation of the aromatic chromophore of
the test compound between the base pairs of DNA [26].
123

Transition Met Chem (2012) 37:569–577
575
˚
0.40
0.35
0.30
0.25
0.20
0.15
Table 3 Selected bonds (A) and angles (°) for the complex 1
3
2.5
2
Cu1–N1
1.972(2)
2.047(3)
Cu1–N2
1.920(2)
1.909(2)
1.965(3)
2.345(2)
1.959(2)
93.78(9)
97.80(11)
99.48(10)
84.14(10)
82.34(11)
94.87(9)
92.97(10)
168.79(10)
96.75(10)
82.64(11)
-94.7(3)
Cu1–N3
Cu1–O1
Cu1–O5
2.371(2)
Cu2–N4
Cu2–O2i
Cu2–N5
1.981(3)
1.5
Cu2–O3
1.968(2)
Cu2–O4
1
2
3
4
5
[DNA]×105 mol/L
O1–Cu1–O5
O1–Cu1–N2
O5–Cu1–N1
O5–Cu1–N3
N1–Cu1–N3
O2i–Cu2–O3
O2i–Cu2–N4
O3–Cu2–O4
O3–Cu2–N5
O4–Cu2–N5
Cu1–O1–C1–O2
96.63(10)
163.55(11)
108.86(9)
78.33(10)
165.61(11)
90.69(8)
O1–Cu1–N1
O1–Cu1–N3
O5–Cu1–N2
N1–Cu1–N2
N2–Cu1–N3
O2i–Cu2–O4
O2i–Cu2–N5
O3–Cu2–N4
O4–Cu2–N4
N4–Cu2–N5
Cu2i–O2–C1–O1
100.38(9)
84.00(8)
240
260
280
300
Wavelength(nm)
95.07(10)
172.11(11)
163.4(2)
Fig. 3 Absorption spectra of ligand L upon the titration of HS–DNA.
Arrow indicates the change upon increasing the DNA concentration.
Inset Plot of [DNA]/(ea – ef) versus [DNA] for the absorption titration
of HS–DNA with ligand
Symmetry code: (i) = 1/2 - x, 1/2 - y, 1 - z
complex were calculated as 4.00 9 104 M-1 (r = 0.9962
for five points) and 6.67 9 104 M-1 (r = 0.9984 for six
points), respectively. These Kb values are lower than those
˚
Table 4 Hydrogen-bonding geometries (A °) for the complex 1
D–HꢀꢀꢀA
d(D–H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA)
\(DHA)
observed for classical intercalators (e.g. EB, *106 M-1
)
N6–H6AꢀꢀꢀO4
N6–H6BꢀꢀꢀO7ii
N7–H7AꢀꢀꢀO3
N7–H7BꢀꢀꢀO9
O5–H5ꢀꢀꢀO2iii
0.85
0.87
0.89
0.89
0.87
2.14
2.39
2.05
2.02
1.85
2.890(4)
3.094(4)
2.858(4)
2.890(4)
2.718(3)
146.2
137.8
149.1
167.3
173.9
[28], but are similar to those of some well-established
intercalation agents (*104 M-1) [29, 30]. The binding
ability of complex 1 is better than that of the free ligand
L. This may be mainly due to the contribution of the thi-
azole rings of the terminal ligands in the complex, which
are expected to be stacked between base pairs upon inter-
action with DNA. Electrostatic interactions may be another
factor for the complex to enhance the binding strength to
DNA compared to H3bhyox alone [8].
Symmetry codes: (ii) = 1/2 - x, y - 1/2, 1/2 - z; (iii) = 1 - x, y,
3/2 - z
The absorption plots of H3bhyox and its complex in the
absence and presence of HS–DNA are given in Figs. 3 and
4, respectively. As shown in the two figures, when titrated
with HS–DNA, both compounds presented significant
hypochromism accompanied by slight red-shifts of 1 nm in
the absorbance maxima. These characteristics reveal that
both compounds interact with HS–DNA most likely
through the intercalation mode [27].
0.8
1.25
1.05
0.85
0.6
For the purpose of quantitatively evaluating the binding
strength of the two compounds with HS–DNA, the intrinsic
binding constants Kb were determined by monitoring the
changes in absorbance at 276 nm for H3bhyox and 230 nm
for the complex, using the following equation [27]:
0.65
0.45
1
2
3
4
5
6
[DNA]×105 mol/L
0.4
0.2
ꢀÀ
Á
ꢀÀ
Á
ꢀ
ÀÀ
ÁÁ
½DNAꢁ ea ꢂ ef ¼ ½DNAꢁ eb ꢂ ef þ 1 Kb eb ꢂ ef
ð1Þ
where [DNA] is the concentration of DNA, ef, ea and eb
correspond to the extinction coefficients, respectively, for
the free compounds, for each addition of HS–DNA to their
solutions and for the compounds in the fully bound form.
From the plots of [DNA]/(ea - ef) versus [DNA] (insets in
Fig. 4), the binding constants for free H3bhyox and the
240
260
280
300
320
Wavelength(nm)
Fig. 4 Absorption spectra of complex 1 upon the titration of HS–
DNA. Arrow indicates the change upon increasing the DNA
concentration. Inset Plot of [DNA]/(ea – ef) versus [DNA] for the
absorption titration of HS–DNA with the complex
123

576
Transition Met Chem (2012) 37:569–577
Fluorescence titration
80
60
40
20
0
1.65
1.45
1.25
1.05
In order to further investigate the interaction modes of the
two compounds with HS–DNA, ethidium bromide fluo-
rescence displacement experiments were carried out. For
test compounds that intercalate into DNA, the binding sites
of DNA available for EB will be reduced, and hence the
fluorescence intensity of EB will be quenched [31]. In our
experiments, as illustrated in Figs. 5 and 6, the fluores-
cence intensities of EB bound to DNA at 584 nm show a
notable decrease with increasing concentrations of the two
compounds, indicating that some EB molecules were
delivered into solution after an exchange with the test
compounds, resulting in fluorescence quenching of EB.
This observation is often the characteristic of intercalation.
In order to understand quantitatively the magnitude of
the binding strength of the two compounds with HS–DNA,
the linear Stern–Volmer equation was employed [31]:
1
3
5
7
[complex]×106 moL/L
560
580
600
620
640
Wavelength(nm)
Fig. 6 Emission spectra of the HS–DNA–EB system upon the
titration of complex 1. Arrow shows the change upon the increasing
complex 1 concentration. Inset Plot of I0/I versus [complex] for the
titration of the complex to HS–DNA–EB system
I0=I ¼ 1 þ Ksv½Qꢁ
ð2Þ
Viscosity measurements
where I0 and I represent the fluorescence intensities in
the absence and presence of quencher, respectively. Ksv is
the linear Stern–Volmer quenching constant, and Q is the
concentration of quencher. In the quenching plots (inset in
Figs. 5, 6, respectively) of I0/I versus [compound], the Ksv
values are given by the ratio of the slope to intercept. The
Ksv values for H3bhyox and the complex are 1.97 9 104
(r = 0.9985 for five points) and 1.02 9 105 (r = 0.9940
for seven points), respectively. The order of these values is
consistent with the results derived from the absorption
spectra.
Viscosity measurement that is sensitive to length changes,
is regarded as the least ambiguous and most critical test of
DNA-binding modes in solution, and provides reliable
evidence for the intercalative binding mode [32, 33]. As
illustrated in Fig. 7, on increasing the amount of both test
compounds, the relative viscosity of HS–DNA increased
steadily, providing further evidence that both compounds
bind to HS–DNA by intercalation [32, 33]. Once again, the
complex shows a bigger effect than the free ligand.
1.45
1.4
1.3
100
1.25
1.35
1.3
1.2
1.15
80
1.1
1.05
1.25
1.2
1
3
5
7
[complex]×106 moL/L
60
40
20
0
1.15
1.1
1.05
1
560
580
600
620
640
0
0.02
0.04
0.06
0.08
0.1
Wavelength(nm)
[compound]/[DNA]
Fig. 5 Emission spectra of the HS–DNA–EB system upon the
titration of ligand L. Arrow shows the change upon the increasing
ligand concentration. Inset: Plot of I0/I versus [ligand] for the titration
of the ligand to HS–DNA–EB system
Fig. 7 Effect of the increasing amount of the two compounds
(triangle for L, diamond for 1) on the relative viscosity of herring
sperm DNA at 298.0 ( 0.1) K, [DNA] = 0.2 mM
123

Transition Met Chem (2012) 37:569–577
577
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A new dissymmetrical N,N’-bis(substituted)oxamide ligand
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their structures were determined by single-crystal X-ray
analysis. Both of the free ligand and the complex can
interact with HS–DNA by intercalation, and the complex
has better DNA-binding ability than the free ligand. In
addition, the tetracopper(II) complex shows significant
levels of cytotoxicity. These results should be valuable in
understanding the binding properties of such compounds
with DNA as well as laying a foundation for the rational
design of novel, powerful agents for targeting nucleic
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Crystallographic data have been deposited at the Cam-
bridge Crystallographic Data Centre with the deposition
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complex, respectively. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif.
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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 Natural Science Foundation of Qingdao City [No.
11-2-4-1-(9)gch) and 12-1-3-52-(1)-nsh].
´
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