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Article history: A luminescent copper(II) complex [CuII(L)H2O]2SO4,1a was synthesized with the acyclic tridentate 2-
Received 22 August 2012 hydroxy naphthaldehyde-2-pyridylhydrazone ligand, HL, 1. The molecular structure of 1a clearly shows
Accepted 2 January 2013 that it is a monomeric copper(II) complex with CuN2OOW coordination with slightly distorted square pla-
Available online 11 January 2013
nar geometry. The asymmetric unit of 1a contains two independent complex molecules and a sulfate
anion. DNA binding interactions of 1a have been investigated by absorption, emission, viscosity and ther-
Keywords: mal denaturation studies. The cytotoxic activity of 1a was measured in vitro against the HeLa cells. The
Association constant
LD50 value was calculated as 4.97 lM. There was no nuclear fragmentation observed after treatment with
Structure
Luminescence
1a, which revealed that the copper(II) complex 1a was not capable of inducing apoptosis. The cell cycle
DNA binding analysis of 1a indicated a dose dependent decrease of G0/G1 and S-phase cell population and dose-depen-
Cytotoxicity dent increase of G2/M population compared with untreated control cell.
Ó 2013 Elsevier Ltd. All rights reserved.
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.01.002
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 229
2.2. Physical measurements and an average flow time was calculated. The rate of flow of the
Tris buffer (pH 7.2), DNA (100 lM) and DNA with the complex,
A Perkin Elmer 2400 C Elemental Analyzer was used to collect 1a at various concentrations were measured. The relative specific
microanalytical data (C, H, N). Sartorius CP64 balance was used viscosity was calculated using the equation g = (t t0)/t0, where
for weighing purpose. FTIR data were collected with the help of t0 is the flow time for the buffer and t is the observed flow time
FTIR Perkin Elmer L 120-000A. UV–Vis spectra were recorded on for DNA in the absence and presence of the complex 1a. Data are
Shimadzu UV-1700 spectrophotometer and corrected for back- presented as (g/g0)1/3 versus 1/R (where R = [complex]/[DNA]), g
ground due to solvent absorption. Emission spectra were carried is the viscosity of DNA in the presence of the complex and g0 is
out with the help of Perkin Elmer LS 50B Luminescence Spectrom- the viscosity of DNA alone [42,43].
eter. For binding constant measurements solutions were prepared
in methanol at a fixed concentration of HL, 1 (1.0 105 M) and at 2.5. Cell culture
a concentration of metal ions ranging from (1.0–10.0) 106 M at
room temperature. FACS Calibur™ (BD Bio-science, USA) fluores- Human cervical cancer cell line (HeLa) was obtained from Na-
cence-activated cell sorter was used for cell cycle analysis. Axio- tional Centre for Cell Sciences, Pune, India. HeLa cells were grown
skop 2 plus (Carl Zeiss) fluorescence microscope was used to study in MEM supplemented with 10% bovine serum (complete medium)
cell morphology and apoptosis. at 37 °C in humidified atmosphere containing 5% CO2.
Compound 1a
2.6. X-ray diffraction studies Empirical formula 2(C16H14CuN3O2)SO41.5(H2O)
Formula mass 810.77
A rod shaped single crystal of 1a (color green, 0.20 0.25 T (K) 293
k (Å) 0.71073
0.40 mm) was mounted on a glass fiber. Intensity data were
Crystal system triclinic
collected at 296 K on a Bruker CCD Diffraction System by the Space group (No. 2)
P 1Þ
x-scan method over the 2h range, 2.4–25.2° on a Bruker APEXII a (Å) 10.1966(18)
CCD diffractometer using Mo Ka radiation [47] equipped with a b (Å) 11.872(2)
four-circle goniometer and using Mo-Ka graphite monochromated c (Å) 13.858(3)
radiation (k = 0.71073 Å). Structure solution and refinement were a (°) 82.936(2)
b (°) 76.815(2)
done by direct methods using SHELXS-97 and SHELXL-97 programs c (°) 84.639(2)
[48]. The structure was successfully solved in space group P 1. Of V (Å3) 1617.2(5)
the 4844 independent reflections, 3742 satisfying I > 2r(I) were Z 2
used for structure solution. The water H-atoms were located from Dcalc (g/cm3) 1.655
l (mm1) 1.448
Fourier difference maps and refined with distance restraints. The
F(0 0 0) 822
remainders of the H-atoms were included in calculated positions Crystal dimension (mm) 0.20 0.23 0.50
and treated as riding atoms using SHELXL default parameters. All h Range (°) 1.5, 23.6
non-H atoms were refined anisotropically using weighted full- Limiting indices 11 < h < 11, 13 < k < 13, 15 < l < 15
Reflections measured 13 707
matrix least-squares on F2. A semi-empirical absorption correction
Reflections unique, Rint 4844, 0.0444
(multi-scan) was applied using SADABS. A region of disordered Reflections observed [I > 2r(I)] 3742
electron density was equated to 1.5H2O per molecule of the Refinement method Full-matrix least-squares on F2
complex 1a, using the SQUEEZE routine in PLATON [49]. Total potential Data/parameters 4844/454
solvent accessible void volume = 129.4 Å3; Electron count/cell = Goodness-of-fit (GOF) on F2 1.033
R1a, wR2b [I > 2r(I)] 0.0449, 0.1096
26; equated to 1.5H2O per molecule of 1a. Fig. 1 illustrates the
R1a, wR2b [all data] 0.0606, 0.1158
molecular structure and crystallographic numbering scheme, Residual electron density (e Å3) 0.47, 0.80
drawn using the program PLATON [49]. Details concerning crystal a
R1 = [R||Fo| |Fc||]/R|Fo| (based on F).
data and refinement are given in Table 1. b
wR2 = [[Rw (|Fo2 Fc2|)2]/[Rw(Fo2)2]]1/2 (based on F2).
The ligand HL, 1, is tridentate in nature with NNO as potential elemental analysis data are consistent with the proposed empirical
donor sites. The 1:1 reaction of HL, 1 with copper(II) sulfate penta- formulae. The room temperature (298 K) magnetic moment of the
hydrate in methanol at room temperature afforded green colored complex 1a is found to be 1.84 lb (S = 1/2, t26e3) which is expected
complex [CuII(L)H2O]2SO4, 1a in excellent yield. The ligand and for d9 system. The complex 1a is non-conducting in acetonitrile.
the complex are soluble in methanol and were characterized by There is no change in absorption and emission pattern of 1a in so-
elemental analyses, FTIR, absorption, emission spectroscopy. The lid and solution phases, indicating the stability of the structure in a
Fig. 1. Perspective view and atom labeling scheme for [CuII(L)H2O]2SO4, 1a. The thermal displacement ellipsoids are drawn at the 50% probability level.
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 231
solution phase. The single crystal of the complex 1a has been iso- Table 3
lated and its crystal structure was solved successfully. Hydrogen bonding (Å) and angles (°) for [CuII(L)H2O]2SO4, 1a.
Complex, 1a
D–H A D–H H A D A D–H A
O1W–H1WA O12 0.89(3) 1.71(3) 2.587(4) 168(5) 1_455
O1W–H1WB O2 0.86(4) 1.92(4) 2.767(5) 171(5) 2_656
N2–H2 N O14 0.8600 1.9000 2.729(5) 161.00 2_665
O2W–H2WA. O1 0.86(3) 1.87(3) 2.714(4) 170(6) 2_656
O2W–H2WB O11 0.90(2) 1.69(3) 2.571(4) 167(4) 2_656
N5–H5 N O13 0.8600 1.8800 2.716(5) 164.00
C8–H8 O11 0.9300 2.3600 3.292(6) 177.00 2_665
C11–H11 O11 0.9300 2.3700 3.271(5) 163.00 2_665
C16–H16 O12 0.9300 2.5800 3.421(6) 150.00 1_455
C24–H24 O12 0.9300 2.4200 3.308(5) 160.00
C27–H27 O12 0.9300 2.3200 3.238(5) 171.00
3.4.1. Absorption spectral studies of CT-DNA with 1a 3.4.2. Ethidium bromide induced emission spectral studies
UV–Vis spectroscopy is an effective tool to determine the bind- Competitive ethidium bromide (EB) binding study was carried
ing modes of metal complexes with DNA. The spectral changes out to understand the mode of DNA interaction of 1a. The molecu-
(hypochromism or hyperchromism) reflect the corresponding lar fluorophore EB emits intense fluorescence at 600 nm in the
alteration of DNA in its conformation and structure after the metal presence of CT-DNA due to its strong intercalation between the
complex bound to DNA [53]. We have investigated the DNA bind- adjacent DNA base pairs. Addition of a second molecule, which
ing activity of the ligand HL, 1 and the complex 1a. In the case of 1, binds to DNA more strongly than EB, would quench the DNA-in-
no significant interaction was observed. In copper(II) complex, 1a, duced EB emission [57,58]. The extent of quenching of the fluores-
the decrease in absorption intensity (hypochromism) with increas- cence of EB bound to DNA would reflect the extent of DNA binding
ing concentration of CT-DNA (0.0–4.0 105 M) was observed of the second molecule. Here, upon addition of the complex, 1a, in
(Fig. 3) [54]. Tris HCl–NaCl buffer (pH 7.2) to CT-DNA (4.0 105 M) pretreated
In order to further investigate the intensity of interactions be- with EB the emission intensity decreases almost up to 50% (Fig. 4).
tween 1a and CT-DNA, we have calculated the intrinsic binding The quenching of the EB bound to DNA by the complex 1a is in
constant, Kb, from the plot of [DNA]/(ea ef) versus [DNA] using agreement with the linear Stern–Volmer equation, Eq. (3) [58],
the following Eq. (2) [55,56] where, I0 and I represent the fluorescence.
Fig. 4. Variations of emission spectra of EB with CT- DNA (4.0 105 M) on addition of the complex, 1a. Inset: plot of I0/I vs r.
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 233
Fig. 7. Nuclear fragmentation detection under fluorescence microscope – (A) untreated control HeLa cells and (B) represents HeLa cells treated with 10 lM of 1a for 17 h.
Table 4
Cell cycle distribution in HeLa cells by FACS. Cells were treated with different concentrations of 1a (0–10 lM). The percentages of cell cycle distribution were evaluated by ModFit
LT software using PI staining. Values represent the means ± SD of three independent experiments. p-Values were calculated with respect to untreated control for each phase and
shown within the brackets.
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