3856
X.-W. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 3851e3857
3.06 (m, 1H), 2.98: (m, 2H), 0.97 (m, 3H). UVevis (in DMF):
lmax(nm) [emax (L molꢀ1 cmꢀ1)]: 297(12700).
Rapidly growing cells were harvested, counted, and incubated at
the appropriate concentration in 96-well micro plates for 24 h. The
four compounds and cis-platin dissolved in culture medium were
then applied to the culture wells to achieve final concentrations
4.2.2. Synthesis of [Cu2(apopoxd)(bpy)](ClO4)$H2O (1)
To
a
stirred methanol solution (5 mL) containing Cu-
ranging from 10ꢀ4e102
mg/mL. Control wells were prepared by
(ClO4)2$6H2O (0.0742 g, 0.2 mmol) was added dropwise a methanol
solution (10 mL) of H3apopoxd (0.0237 g, 0.1 mmol) and piperidine
(0.0256 g, 0.3 mmol) at room temperature. After stirring for 30 min,
a methanol solution (5 mL) of bpy (0.0156 g, 0.1 mmol) was added
dropwise. The mixture was stirred quickly at 333 K for 6 h, the
resulting brown solution was filtered and brown cube crystals of
the complex suitable for X-ray analysis were obtained by slow
evaporation at room temperature. Yield: 0.0375 g (59%). Anal. Calc.
for Cu2C21H22N5O8Cl: C, 39.72; H, 3.49; N, 11.03%. Found: C, 39.70;
addition of culture medium without cells. The plates were incu-
bated at 310 K in a 5% CO2 atmosphere for 48 h. Upon completion of
the incubation, the cells were fixed with ice-cold 10% trichloro-
acetic acid (100 mL) for 1 h at 277 K, washed five times in distilled
water and allowed to dry in the air and stained with 0.4% SRB in 1%
acetic acid (100 mL) for 15 min. The cells were washed four times in
1% acetic acid and air-dried. The stain was solubilized in 10 mM
unbuffered Tris base (100 mL) and the OD of each well was
measured at 540 nm on a microplate spectrophotometer. The IC50
values were calculated from the curves constructed by plotting cell
H, 3.47; N, 11.08%. IR (KBr pellet, cmꢀ1): 1654 [
(C]N)]; 1107, 623 [ (ClO4)]. Molar conductance: LM (DMF
solution): 72
ꢀ1$cmꢀ2$molꢀ1. UVeVisible (in DMF): lmax(nm)
emax(L$molꢀ1$cmꢀ1)]: 640(701), 299(40125), 244(42300).
n(C]O)]; 1548
[
n
n
survival (%) versus the compounds concentration (mg/mL).
U
[
4.5. DNA-binding studies
4.2.3. Synthesis of [Cu2(apopoxd)(dabt)](ClO4)$2H2O (2)
This complex as dark brown crystals suitable for X-ray single-
crystal analysis was obtained by the same procedure and same
amount of reagents as above but using dabt (0.0199 g, 0.1 mmol)
instead of bpy (0.0156 g, 0.1 mmol). Yield: 0.0500 g (72%). Anal. Calc.
for Cu2C17H22N7S2O9Cl: C, 39.38; H, 3.19; N, 14.11%. Found: C, 39.33;
All experiments involving HS-DNA were performed in TriseHCl
buffer solution (pH ¼ 7.18). Solutions of HS-DNA in TriseHCl buffer
gave a ratio of UV absorbance at 260 and 280 nm, A260/A280, of z1.9,
indicating that the DNA was sufficiently free of protein [44]. The
concentration of DNA was determined by UV absorbance at
H, 3.14; N, 14.07%. IR (KBr pellet, cmꢀ1): 1660 [
(C]N)]; 1108, 623 [
solution): 78
ꢀ1$cmꢀ2$molꢀ1. UVevisible (in DMF): lmax(nm)
emax(L$molꢀ1$cmꢀ1)]: 672(648), 280(36250), 218 (64250).
n(C]O)]; 1547
260 nm. The molar absorption coefficient, e260, was taken as
[
n
n
(ClO4)]. Molar conductance: LM (DMF
6600 L,molꢀ1,cmꢀ1 [45]. Stock solution of DNA was stored at 277 K
and used after no more than 4 days. Concentrated stock solution of
the three bicopper(II) complexes and the free ligand were prepared
by dissolving them in the TriseHCl buffer solution to required
concentrations for all the experiments, respectively. Absorption
spectral titration experiment was performed by keeping the
concentration of the compounds constant while varying the HS-
DNA concentration. Equal solution of HS-DNA was added to the
compounds solution and reference solution to eliminate the
absorbance of DNA itself. In the ethidium bromide (EB) fluores-
U
[
4.2.4. Synthesis of [Cu2(apopoxd)(phen)2](ClO4) (3)
Dark green cube crystals of the complex suitable for X-ray
analysis were obtained by the same procedure and same amount of
reagents as complex 1 except using phen (0.0198 g, 0.1 mmol)
instead of bpy (0.0156 g, 0.1 mmol). Yield: 0.0542 g (66%). Anal. Calc.
for Cu2C35H28N7O9Cl: C, 51.19; H, 3.44; N, 11.94%. Found: C, 51.13;
H, 3.41; N, 11.97%. IR (KBr pellet, cmꢀ1): 1644 [
(C]N)]; 1104, 623 [
solution): 76
ꢀ1$cmꢀ2$molꢀ1. UVevisible (in DMF): lmax(nm)
emax(L$molꢀ1$cmꢀ1)]: 622(701), 270(48750), 229(50300).
n
(C]O)]; 1516
cence displacement experiment, 5 mL of the EB TriseHCl solution
[
n
n
(ClO4)]. Molar conductance: LM (DMF
(1 mmol,Lꢀ1) was added to 1 mL of DNA solution (at saturated
binding levels) [38], stored in the dark for 2 h. Then the solution of
the compounds was titrated into the DNA/EB mixture and then
diluted in TriseHCl buffer to 5 mL, producing the solutions with the
varied mole ratio of the compound to HS-DNA. Before measure-
ments, the mixture was shaken up and incubated at room
temperature for 30 min. The fluorescence spectra were obtained at
an excitation wavelength of 522 nm and an emission wavelength of
584 nm in the Fluorometer. In viscosity measurement, DNA
samples approximately 200 base pairs in length were prepared by
sonication in order to minimize complexities arising from DNA
flexibility [46]. Flow time was measured with a digital stopwatch,
and each sample was measured three times, and an average flow
time was calculated. Relative viscosities for DNA in the presence
and absence of the compound were calculated from the relation
s ¼ (t-t0)/t0, where t is the observed flow time of DNA-containing
solution and t0 is that of TriseHCl buffer alone. Data was pre-
U
[
4.3. X-ray crystallography
The X-ray diffraction experiments for complexes 1 to 3 were
made on a Bruker APEX area-detector diffractometer with graphite
monochromatic Mo K
a
radiation (
l
¼ 0.71073 Å). The crystal
structures were solved by the directed method followed by Fourier
syntheses. Structure refinements were performed by full matrix
least-squares procedures using SHELXL-97 on F2 [43].
4.4. In vitro anticancer activity evaluation by SRB assays
In vitro anticancer activities of the free ligand and the three
bicopper(II) complexes together with cis-platin were evaluated
against two cancer cell lines including SMMC-7721 and A549 by
using the Sulforhodamine 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 streptomycin. Cell lines
are maintained at 310 K in a 5% (v/v) CO2 atmosphere with 95% (v/v)
humidity. Cultures were passaged weekly using trypsineEDTA to
detach the cells from their culture flasks. The four compounds and
cis-platin were dissolved in DMSO and diluted to the required
concentration with culture medium when used. 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.
sented as (s/s )1/3 versus binding ratio [47], where s is the viscosity
0
of DNA in the presence of complex and s is the viscosity of DNA
0
alone.
Acknowledgments
This project was supported by the 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 Teamin University (IRT0944).
Li, Xiao-Wen
