Synthesis, crystal structure, cytotoxicity study, DNA/protein binding and molecular docking of dinuclear copper(II) complexes

Article abstract of DOI:10.1016/j.ica.2019.03.022

The Schiff base 2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) has been used for the synthesis of three dinuclear Cu(II) complexes, namely [Cu₂L₂(dca)₂] (1), [Cu₂L₂(2,5-pdc)₂]·6H_2<

Full text of DOI:10.1016/j.ica.2019.03.022

Accepted Manuscript
Research paper
Synthesis, crystal structure, cytotoxicity study, DNA / protein binding and mo-
lecular docking of dinuclear copper(II) complexes
Apurba Bhunia, Soumen Mistri, Rajesh Kumar Manne, Manas Kumar Santra,
Subal Chandra Manna
PII:
S0020-1693(18)31761-4
https://doi.org/10.1016/j.ica.2019.03.022
ICA 18827
DOI:
Reference:
Inorganica Chimica Acta
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Accepted Date:
21 November 2018
15 March 2019
15 March 2019
Please cite this article as: A. Bhunia, S. Mistri, R.K. Manne, M.K. Santra, S.C. Manna, Synthesis, crystal structure,
cytotoxicity study, DNA / protein binding and molecular docking of dinuclear copper(II) complexes, Inorganica
Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.03.022
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Synthesis, crystal structure, cytotoxicity study, DNA / protein binding and
molecular docking of dinuclear copper(II) complexes
Apurba Bhunia,a Soumen Mistri,a Rajesh Kumar Manne,b Manas Kumar Santra,b and Subal
Chandra Manna*,a
aDepartment of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India, E-mail: scmanna@mail.vidyasagar.ac.in, Fax: (91) (03222)
275329.
bNational Centre for Cell Science, NCCS Complex, Pune University Campus Ganeshkhind,
Pune-411 007, Maharashtra, India
Abstract
The Schiff base 2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) has been used for the
synthesis of three dinuclear Cu(II) complexes, namely [Cu2L2(dca)2] (1), [Cu2L2(2,5-
pdc)2]·6H2O (2), [Cu2L2(tp)] (3), where dca= dicyanamide, 2,5-pdc= pyridine-2,5-
dicarboxylate and tp= terephthalate. In 2, HL acts as bidentate (O, N) chelating ligand, while
tridentate (O, N, N) in 1 and 3. In all the complexes copper(II) is in its five coordination with
distorted trigonal bipyramidal geometry in 1, whereas distorted square pyramidal in 2 and 3.
Non covalent interactions give rise supramolecular architectures in all complexes.
Cytotoxicity of the complexes was examined using breast cell lines MCF7 and MDA-MB-
231. Complexes 1 and 2 show dose dependent suppression of MCF7 and MDA-MB- 231
cells and the related IC50 values are 26 ± 2.9 µM and 51 ± 4.7 µM for MCF7 cell, 16.4 ± 1.6
µM and 43.2 ± 3.1 µM for MDA-MB- 231 cell. Interaction of complexes 1-3 with bovine
serum albumin (BSA), human serum albumin (HSA) and calf thymus DNA have been
studied by electronic absorption and fluorescence spectroscopic technique. Molecular
docking study has been used to explain the interaction of complexes with serum albumins.
1

__________________________________________________________________________________
Keywords: Dinuclear Cu(II) complex; Cytotoxicity study; DNA binding; Serum albumin
binding; Molecular docking
__________________________________________________________________________________
Introduction
The most efficient and widely used approach for design and synthesis of coordination
compounds is the self-assembly of metal ions and ligands [1]. Schiff bases are most useful
ligands in the chemistry of 3d metal coordination compounds. Pseudo-halides and organic
carboxylates are good co-ligands to coordinate with metal sites. Transition metal Schiff base
compounds are important for their biological activity [2]. In addition to well known platinum
anticancer compounds, a good number of studies have been performed on transition metal
complexes for their ability to destroy cancer cells [3]. In comparison with other 3d metal
complexes, Cu(II) complexes are more suitable due to their less toxicity and important
cellular effects such as neurotransmission, cellular respiration, etc.[4]. Literature study
reveals that several synthetic Cu(II) complexes act as potential anticancer and cancer
inhibiting agents, and some of them have been found to be active both in vitro and in vivo
[4]. On the other hand bovine serum albumin and human serum albumin are most important
protein in blood plasma and they transport and distribute various endogenous and exogenous
substances, like nutriments, medicines, etc. Transportation and distribution of drugs depend
on the interaction of drugs with serum albumin (SA) molecules [5]. Moreover many Cu(II)
complexes have been observed to exhibit enhanced DNA binding and cleavage activities [6].
Therefore study of the kinetics of interaction of CT-DNA/serum albumin are important for
the development of Cu(II) Schiff base based metallo-pharmaceuticals.
2

2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) is a potential flexidentate chelating
ligand. Using this ligand and in combination with azide, three copper(II) complexes namely
[Cu2L2(N3)2], [Cu2L2(N3)2]·H2O and [CuL(N3)]n have been reported in the literature where
the ligand HL acts as N,N,O donor tridentate chelating ligand [7]. The Cu(II) centres in all
these three reported complexes possess five-coordinate square pyramidal geometries.
However till now there is no report on coppe(II) complexes of this ligand in combination
with dicarboxylate and dicyanamide. In the present contribution we have used HL in
combination with dicarboxylate / dicyanamide, and synthesized complexes [Cu2L2(dca)2]
(1), [Cu2L2(2,5-pdc)2]·6H2O (2) and [Cu2L2(tp)] (3) (where dca= dicyanamide, 2,5-pdc=
pyridine-2,5-dicarboxylate and tp= terephthalate). DNA/protein binding activities have been
studied. Anticancer activities in human breast cancer cell lines MCF7 and MDA-MB-231
have been investigated.
Experimental
Chemicals and instrumentation
N-methyl-1,3-diaminopropane and sodium dicyanamide were purchased from Sigma Aldrich
Chemical Company and all other chemical were of AR grade. Solvents used were purified by
standard methods [8].
1
Elemental analyses were performed using a Perkin-Elmer 240C elemental analyzer. HNMR
spectra of ligand recorded in CDCl3 on 400 MHz instrument. IR spectra were recorded as
KBr pellets on a Bruker Vector 22 FT IR spectrophotometer. ESI-MS spectra were obtained
from ESI mass spectrometry. UV-Vis spectra in room temperature were recorded on
Shimadzu UV-1601 UV-vis spectrophotometer. Emission spectra were recorded on a Hitachi
F-7000 spectrofluorimeter. Quantum yield was calculated according to the reported
method,[9] where phenol in water medium used as reference.
3

Synthesis of the ligand
The ligands 2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) was prepared by
condensation of N-methyl-1,3-diaminopropane and 2-hydroxybenzaldehydein methanol at
room temperature.
A methanolic solution (10 ml) of N-methyl-1,3-diaminopropane (10 mmol, 0.882 g) was
added drop wise to a methanolic solution (10 ml) of 2-hydroxybenzaldehyde (10 mmol, 1.22
g) with constant stirring. Resulting yellow reaction mixture was stirred for 3 h. The yellow
color compound was separated using column chromatographic technique using 1:1 ethyl
1
acetate and hexane solvent and the compound was characterized adopting FT-IR, H NMR
and mass spectrometry techniques. Yield: 1.57g (82 %). C11H16N2O (192.25): C, 68.72; H,
8.38; N, 14.57%. Found: C, 68.71; H, 8.37; N, 14.58 (%). IR (KBr), cm−1: 3200-3600 (br,
vs), 2982 (s), 2943 (s), 1641 (vs), 1550 (vs), 1466 (s), 1413 (vs), 1372 (s), 1300 (s), 1281 (w),
1
1160 (w), 1112 (w), 1076 (s) 882 (w), 819 (w), 781 (w), 671 (s) 629 (s) 564 (w). H NMR
(CDCl3, 400 MHz, δ, ppm): 8.22 s (1H), 7.09-7.02 d (2H), 6.99-6.92 t (2H), 4.14 s (1H),
3.09-3.01 m (1H), 2.74-2.6 m (6H), 2.59-2.5 d (3H). MS (in MeOH, calcd m/z): 192.2511
(C11H16N2O), 177.1124 (C10H13N2O), 133.1394 (C8H7NO).
Synthesis of the Complexes
Synthesis of complexes 1-3 are schematically displayed in Scheme 1.
4

Scheme 1. Synthesis of 1, 2 and 3.
Synthesis of [Cu2L2(dca)2] (1).A methanolic solution (5 ml) of copper perchlorate
hexahydrate (1 mmol, 0.370 g) was added to a mixture of HL (1 mmol, 0.192 g) and
triethylamine (1 mmol, 0.10 g). To this resulting mixture an aqueous solution (5 ml) of
sodium dicyanamide (1 mmol, 0.089 g) was added. The resulting deep green reaction mixture
was stirred for 2 hours and filtered. Green single crystals suitable for X-ray diffraction quality
were obtained after a few days on keeping the solution in a refrigerator. Yield 0.233g (73 %).
C26H30Cu2N10O2 (641.70): C, 48.66; H, 4.71; N, 21.82 %. Found: C, 48.65; H, 4.72; N, 21.82
(%). IR (cm-1): 2975 (vs), 2944 (vs), 2885 (s), 2273 (s), 2219 (s), 2152 (s), 1643 (vs), 1554
(vs), 1466 (s), 1413 (vs), 1371 (s), 1298 (s), 1077 (s), 881 (s), 813 (s), 779 (s), 647 (s), 510
(s). MS (in MeOH, calcd m/z): 641.1170 (C26H30Cu2N10O2).
Synthesis of [Cu2L2(2,5-pdc)2]·6H2O (2) and [Cu2L2(tp)] (3).The complexes 2 and 3 were
synthesized by the same procedure as adopted for complex 1, using disodium pyridine-2,5-
5

dicarboxylate (1 mmol, 0.211 g) and disodium terephthalate (0.5 mmol, 0.105g), respectively,
instead of Sodium dicyanamide. For complex 2; Yield 0.385g (81 %). C36H50Cu2N6O16
(949.90): C, 45.52; H, 5.30; N, 8.80 %. Found: C, 45.51; H, 5.30; N, 8.81(%). IR (cm-1):
3600-3200 (br, vs), 2980 (vs), 2944 (vs), 2885 (s), 1643 (vs), 1555 (vs), 1466 (s), 1414 (vs),
1372 (s), 1299 (s), 1078 (s), 882 (s), 815 (s), 783 (s), 639 (s). MS (in MeOH, calcd m/z):
841.1275 (C36H38Cu2N6O10). For complex 3; Yield 0.263g (78 %). C30H34Cu2N4O6 (673.69):
C, 53.48; H, 5.08; N, 8.31%. Found: C, 53.48; H, 5.07; N, 8.30 %. IR (cm-1): 2977(vs),
2945(vs), 2893(s), 1643(vs), 1555(vs), 1466(s), 1413(vs), 1372(s), 1299(s), 1078(s), 882(s),
815(s), 783(s), 643(s). MS (in MeOH, calcd m/z): 674.1052 (C30H34Cu2N4O6).
Crystallographic data collection and refinement
Data collection of complexes 1, 2 and 3 were carried out by using a Nonius Kappa CCD
diffractometer with graphite monochromated Mo-K radiation, at room temperature. The
data sets were integrated with the Denzo-SMN package [10] and corrected for Lorentz,
polarization and absorption effects (SORTAV) [11]. The structures were solved by direct
methods using SIR97 [12] system of programs and refined using full-matrix least-squares
with all non-hydrogen atoms anisotropically and hydrogens included on calculated positions,
riding on their carrier atoms.All calculations were performed using SHELXL-97 [13] and
PARST [14] implemented in WINGX [15] system of programs. Graphical programs used are
those included in the WINGX System [15] and Diamond [16]. Crystal data and details of
refinements are given in Table 1.
Cytotoxicity assay
Cell culture
6

MCF7 cells were grown in DMEM and MDA MB-231 cells were RPMI medium as
monolayer supplemented with 10 % FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at
37°C in a humid and 5% CO2 atmosphere.
In vitro cytotoxicity assay
Cells were seeded at 5 × 103 cells per well in 96-well plates. Next day, cells were incubated
with complexes 1 and 2, with different concentrations (0 - 100 µM) for 48 hours and DMSO
used as vehicle control. All the treatments were given in triplicate. MTT solution (20 µl of 5
mg/ml MTT) was added to each well following incubation with compounds for 48 hours and
further incubated for 3.5 h at 37°C in a humid, 5% CO2 atmosphere. Then, solution
containing media was replaced by MTT solvent (iso-propanol, HCl and Triton X-100) and
incubated for 15 min at room temperature with gentle rocking. The absorbance was read in
Thermo Pierce Elisa plate reader at 570 nm. The results were expressed as percentage
inhibition relative to the cells treated with DMSO (considered as 0 %). All the experiments
were repeated for three times.
Immunoblotting assay
MDA-MB-231 cells were treated with the complex 1 (25 μM) and collected different time
points (24 and 48 h) to check the apoptotic markers by immunoblotting. Cells were lysed and
whole cell protein extracts were separated by SDS-PAGE and transferred onto PVDF
membranes. After blocking with 3% nonfat dry milk in TBST for 1 h, the membranes were
incubated with primary antibody against the indicated proteins for overnight at 4 °C. After
washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated
secondary antibody for 1 h. Blots were developed using chemiluminescence methods in GE
4000 quantum chemiluminescence.
7

Cell cycle analysis
MDA-MB-231 cells were incubated with either with 25 µM complexes 1 or with 25 µM 2 for
48 h. Cells incubated with 0.1% DMSO were considered as control. At the end of the
treatment, the cells were washed with ice cold PBS, fixed in 95% chilled ethanol and kept at
4°C for 24 h. On the day of FACS analysis, the fixed cells were again washed with ice cold
PBS to remove the trace amount of ethanol and stained with the staining solution comprising
50 μg/ ml propidium iodide and 50 μg/ml RNAse in PBS. Cells were acquired on BD FACS
Calibur instrument and the data was analysed using Cell Quest Pro software.
Albumin Binding studies
Electronic absorption spectral study
To maintain the physiological pH, solutions of bovine serum albumin (BSA)[17,18] (2.3
µM), human serum albumin (HSA)[18] (2.3 µM) and complexes (4 µM) solution were
prepared in HEPES buffer. The interaction of complexes with serum albumins were studied
by recording the change of electronic absorption spectra of BSA/HSA (2 ml 2.3 µM) with
gradual addition of 10 µL 4 µM complexes at room temperature. The apparent association
constant (Kapp) [19] were calculated adopting the following equation
where, Aobs is the observed absorbance at 280 nm (characteristics band of BSA/HSA) at
different concentration of complex. A0 is the absorbance of serum albumin only.
Emission spectral study
8

BSA and HSA show characteristic emission bands at 341 nm (λex, 280 nm) and 329 nm (λex,
280 nm), respectively. The interaction of complexes with BSA (2.3 µM) and HSA (6.2 µM)
were studied by gradual addition of 10 µL, 4 µM aqueous Cu(II) solution to 2 ml serum
albumin solutions. The Stern-Volmer constants (Ksv) [20] and quenching rate constants (kq)
were calculated using the equations F0/F = 1 + Ksv[complex] and Ksv = kqτ0, where F0 and F
are the fluorescence intensity in the absence and in the presence of the complex, and τ0 is the
lifetime [21] of serum albumin (5 × 10-9 s). To calculate the binding constant of the complex
with serum albumin, the following equation have been used [(Fo–F)/F] = log Kb + n
log[complex], where Kb is the binding constant of the complex with serum albumin and n is
the number of binding sites per albumin[22].
DNA binding experiments
Electronic absorption spectral study
Interaction of complexes 1-3 with CT-DNA were investigated using UV-vis absorption
spectroscopic studies. For all the complexes, interaction have been studied by gradual
addition of 10 µL, 15.6 µM solution of CT-DNA to 2 mL, 4 µM aqueous solution of
complexes. Intrinsic binding constants (Kib) [23] were determined using the following
equation
where, [DNA] is the concentration of CT-DNA, is the extinction co-efficient value of the
complex at a given CT-DNA concentration, and are the extinction co-efficient of the
complex, in free solution and when fully bound to CT-DNA, respectively.
9

Competitive Ethidium bromide-DNA binding fluorescence measurement
When excited at 500 nm, ethidium bromide (EB) bounded CT-DNA shows emission band at
600 nm. Interaction of CT-DNA with complexes were studied flurometrically upon gradual
addition of 10 µL 4 µM solution of complexes to the aqueous solution of EB bound CT-DNA
(2 mL 8.5 µM) in HEPES buffer (pH 7.2). The Stern-Volmer constants (Ksv) were calculated
using Stern-Volmer equation [20].
Molecular docking
Serum albumin - complex docking were performed by using AutoDock 4.2 software [24].
Crystal structure of BSA (PDB ID: 4f5s) and HSA (PDB ID: 1ao6), were downloaded from
protein
data
bank
(http://www.rcsb.org/pdb),
and
the
Chimera
program
(http://www.cgl.ucsf.edu/chimera/) was used for receptor preparation. During docking
calculation, the SAs were kept rigid and complexes being docked were kept flexible. The
docking results were visualized by Chimera (http://www.cgl.ucsf.edu/chimera/) and PyMol
software.
Results and discussion
Crystal structure description
Solid state structural analysis of 1-3 exhibit that all the compounds are dinuclear with five
coordinated Cu(II) centre. Molecular structures of the complexes are shown in Figs.1-3. Bond
length and bond angle are summarized in Table 2. In complex 1, two [CuL(dca)] asymmetric
units are connected through phenolic oxygens (O1a) and Cu(II) centres and form a base plane
with phenyl ring (Fig. 1). Two dicyanamide (dca) units are placed in opposite direction and
the dca units are perpendicular to the base plane. Largest bond length and bond angle of 1 are
[Cu(1)-N(3), 2.087(4)Å] and [O(1a)-Cu(1)-N(1), 169.44(12)°], respectively and smallest
10

bond length and bond angle are [Cu(1)-N(1), 1.945(4)Å] and [O(1)-Cu(1)-O(1a), 79.53(12)°],
respectively. In 2, asymmetric unit of [CuL(2,5-pdc)] possesses planer structure and
dimerises through phenolic oxygen (O1a) and Cu(II) centre with relatively larger copper
oxygen bond length, 2.680Å (Fig. 2). Cu(1)-O(1a)[2.680(3)Å] and Cu(1)-O(1)[1.906(2)Å]
are largest and smallest bond length, respectively. On the other hand O(1)-Cu(1)-
O(2)[171.56(11)°] and O(1)-Cu(1)-O(1a) [79.70(9)] are largest and smallest bond angles,
respectively. Crystal structure of 3 exhibits that Cu(L) units are connected through
terephthalate ion(tp) (Fig. 3) and in this compound tp is perpendicular to copper(II)-Schiff
base plane. Largest and smallest bond length in 3 are Cu(1)-O(3)[2.680(2)Å] and Cu(1)-
O(1)[1.905(2)Å], respectively. O(2)-Cu(1)-N(1) [170.34(10)°] and O(2)-Cu(1)-
O(3)[54.38(8)°] are largest and smallest bond angle, respectively.
Five coordinated complexes may possess either regular trigonal bipyramidal (TBP) or square
pyramidal (SP) geometries. τ parameter [25] calculation allows to estimate deviations from
TBP or SP ideal geometries. Regular TBP structure with D3h symmetry has τ = 1 and for a
regular SP structure with C4v geometry has τ = 0. The τ values are calculated from the
structural data and the values are 0.681, 0.060 and 0.163 for 1, 2 and 3, respectively (Table
2). The τ value indicate that 1 possesses TBP geometry, whereas 2 and 3 exhibit SP
geometries, and in between 2 and 3, complex 2 is more close to ideal SP geometry. It is
interesting to note that most of the five coordinated copper(II) complexes possess SP
geometry, however copper(II) centre in complex 1 possesses rare TBP coordination
environment.
Packing structure of 1 exhibits that the dimeric units of 1 are connected through H-bonding
[26] interactions (Table 1S) between the terminal dca nitrogen (N5) and imine hydrogen of
Schiff base (N5∙∙∙H26=2.212Å), forming a 1D supramolecular chain structure. These
supramolecular 1D chains are connected through C-H...π interaction (H∙∙∙Cg= 3.232Å) (Table
11

2S), and form 2D supramolecular sheet like structure (Fig.1S). 2D supramolecular sheets are
again interact with another type of C-H..π interaction [27] (H∙∙∙Cg= 3.626Å),(Fig. 2S) which
results 3D structure (Figs. 4 and 3S). Packing structure of 2 exhibits that the dimeric units are
connected through H-bonding (Table 1S) between the free carboxylate of 2,5-pdc and imine
hydrogen of Schiff base (O4∙∙∙H19=2.180Å, O5∙∙∙H19=2.115Å), forming 1D supramolecular
chain (Fig. 4S). This 1D chains are again interacts through H-bonding with the lattice water
molecules and form 2D supramolecular sheet like structure (Figs. 5 and 5S). It is interesting
to note that the six lattice water molecules interact through H-bonding, and produced a twelve
member cyclic supramolecular water cluster (Fig. 6S), where the oxygen atoms of six water
molecules are arranged in a chair like conformation. Packing structure of 3 exhibits that the
molecular units are connected through H-bonding interactions (O1∙∙∙H2A = 2.018) (Table 1S)
between phenolic oxygen and imine hydrogen of Schiff base, and form1D supramolecular
chain structure. These 1D chains are again connected through C-H∙∙∙π interaction (H∙∙∙Cg=
2.755Å) (Table 2S), forming 2D supramolecular sheet like structure (Fig. 6). The 2D
supramolecular sheets are again connected through another type of C-H..π interaction
(H∙∙∙Cg= 3.126Å) (Fig.7S) and build 3D supramolecular structure (Fig. 8S).
ESI mass spectrometry
ESI mass spectra of complexes were recorded using their methanolic solutions. Methanolic
solutions of complexes were prepared 24 h before the mass spectral study. ESI-mass spectra
of complexes show peaks at m/z = 641.1170 (for 1), 841.1275 (for 2) and 674.1052 (for 3)
corresponds to [Cu2L2(dca)2] ˙+ (calc. = 641.70, for 1), [Cu2L2(2,5-pdc)2] ˙+ (calc. = 841.81,
for 2) and [Cu2L2(tp)] ˙+(calc. = 673.69, for 3) (Figs. 9S-11S). Mass spectral results indicate
that complexes are stable in methanol and retain their dinuclear structure in solution.
Cytotoxic activity
12

The antiproliferative activity of 1 and 2 against human breast cancer cell line MCF7 and
MDA-MB-231 has studied by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Results demonstrated that 1 has better growth suppressor activity as
compared to the 2 in both the cell lines. For example, the IC50 value of complexes 1 and 2 in
MDA-MB0231 is 16.4 ± 1.6 and 43.2 ± 3.1 µM, respectively (Fig. 7). In addition, IC50
values of these compounds demonstrated that both compounds have more potent cytotoxic
effect against triple negative breast cancer cell line MDA-MB-231 (more aggressive
metastatic cell line) as compared to luminal A cell line MCF7 (less aggressive metastatic cell
line). To understand whether the potent cytotoxic effect of these compounds is due to the
induction of apoptosis, we performed cell cycle analysis. MDA-MB-231 cells were grown in
the absence and presence of complex 1 for the indicated time periods and then analyzed the
cell population by florescence activated cell sorting (FACS) analysis (Fig. 8a). Results
revealed that population of cells at sub-G1 significantly increased with increasing periods of
incubation complex 1 indicating that the complex might be promoting apoptosis through
induction of DNA damage (Fig. 8a). To examine whether cytotoxic effect complex 1 is due
to induction of apoptosis, immunoblotting was performed to examine the levels of pro-
apoptotic and anti-apoptotic proteins following exposure of complex 1 for indicated time
periods (Fig. 8b). Immunoblot results showed that expression levels of pro-apoptotic proteins
are increased and in contrast expression levels of anti-apoptotic Bcl2 declined following
exposure of complex 1 (Fig. 8b). Increased levels of p53, Bax and Apaf1 indicating that p53
might be playing important role in induction pro-apoptotic protein Bax at the transcriptional
level. Increased Bax levels facilitate the polarization of mitochondrial membrane potential-
mediated release cytochrome C. Released cytochrome C is then form complex with Apaf1 to
promote the apoptosis induction. Results taken together suggest that complex 1 inhibits the
13

growth of cancer cells through induction of apoptosis-mediated cell death. A comparison of
IC50 values of reported Cu(II) complexes are shown in Table 3.
Protein binding studies
Electronic absorption spectra of bovine serum albumin (BSA) and Human serum albumin
(HSA) show the increase of absorbance with bathochromic shift (~20 nm) upon the gradual
addition of complexes to serum albumin solutions (Figs. 9, 12S-16S). This bathochromic
shift in electronic absorption spectra confirms ground state association of complexes with
serum albumins. The calculated values of apparent association constants (Kapp, L mol-1) are
8.36 ± 1.62 × 105 (1-BSA), 16.90 ± 1.25 × 105 (1-HSA), 7.31 ± 1.05 × 105 (2-BSA), 11.00 ±
1.96 × 105 (2-HSA), 9.38 ± 1.34 × 105 (3-BSA), 17.19 ± 2.14 × 105 (3-HSA) (Table 4). The
observed order of apparent association constant can be explain on the bases of the size of the
molecules. Due to the small size, complex 1 is more interactive than 2. On the other hand
relatively high interaction affinity of 3 is due to its linear structure which favors the
incorporation inside the protein moiety.
Fluorescence intensity of BSA and HSA considerably decreases (with slight blue shift) upon
gradual increasing concentration of complexes (Figs. 9, 12S-16S). The Stern-Volmer
constants (Ksv) and quenching rate constants (kq) (Fig. 17S) indicate strong interaction
between complex and serum albumin (Table 4). As electronic spectral change of serum
albumins in presence of complexes indicate ground state association, and hence this
fluorescence quenching is static in nature. Calculated values of binding constants (Kb, L mol-
1) are 2.42 ± 0.01 × 106 (1-BSA), 46.91 ± 0.02 × 106 (1-HSA), 2.33 ± 0.02 × 106 (2-BSA),
37.33 ± 0.02 × 106 (2-HSA), 7.22 ± 0.02 × 106 (3-BSA), 76.12 ± 0.03× 106 (3-HSA) (Table
3).
DNA binding studies of complexes
14

Aqueous solution of 1-3 show electronic absorption bands at 217 nm, 234 nm, 266 nm and
354 nm for 1; 216 nm, 264 nm, 270 nm and 343 nm for 2; 218 nm, 235 nm, 264 nm and 354
nm for 3. On gradual addition of CT-DNA, for 1, electronic absorbance of 266 nm band
gradually increases with blue shift and finally the band appears at 259 nm, whereas
absorbance of 354 nm band gradually decreases. An isobestic point in the titration curve (Fig.
10) at 336 nm indicates the presence of more than two species in the medium [30]. Upon
gradual addition of CT-DNA to the solutions of 2 and 3 the absorbance at 264 nm band
gradually increases with blue shift and finally the band appear at 258 nm and 254 nm,
respectively (Figs. 18S and 19S). Calculated values of intrinsic binding constants (Kib) are
1.52 ± 0.08× 105, 2.50 ± 0.12× 105 and 10.07 ± 0.56× 105 L mol-1 for complexes 1, 2 and 3,
respectively (Table 5). Kib of the complexes are determined with respect to the wavelength
259 nm, 258 nm and 254 nm for 1, 2 and 3, respectively.
Fluorescence intensity of ethidium bromide (EtBr) increases strongly in presence of CT-DNA
due to its strong intercalation with CT-DNA [32]. On excitation at 500 nm, CT-DNA
bounded EtBr shows emission at 608 nm. A quenching of fluorescence intensity is observed
when complex solutions were added to the aqueous solutions of EtBr-CT-DNA (Fig. 20S-
22S). This is due to the interaction of complexes with DNA, where complex molecule
displaces the ethidium bromide molecules from the interactive side of DNA. From the Stern-
Volmer equation linear relationships (Fig. 23S) were obtained from the titration of CT-DNA
bounded EtBr, using complexes as quencher. The calculated values of binding constants (Ksv)
are 13.63 ± 0.01 × 106, 13.80 ± 0.02× 106 and 16.69 ± 0.02 × 106 L mol-1 for 1, 2 and 3,
respectively (Table 5).Protein-complex docking
Molecular docking studies have been carried out to understand the nature of binding nature in
between complexes and serum albumins. Chimera scores of the individual complexes with
different poses have been tabulated in Table 3S. Since BSA and HSA have two different
15

active sites (Tyr 149 and Tyr 410 for BSA; Tyr 150 and Tyr 411 for HSA) for binding, we
performed docking of each complex with two binding sites individually in order to
investigate effective binding site of BSA and HSA for our complexes. Based on the chimera
score it is clear that complexes 1-3 selectively bind with BSA at the site of Tyr 149, and that
of HSA at the site of Tyr 150. Moreover, chimera score also indicate that complex 3 binds
more effectively with serum albumins in comparison to 1 and 2. Binding interaction of the
complexes with the RCSB protein are shown in Figs. 11, 24S-34S.The dock results indicate
that complexes 1-3 form hydrogen bonding with HSA when interact with its Tyr150 site. But
only 2 forms hydrogen bond with HSA when interacts at Tyr411 site of HSA (Table 6).
In addition to hydrogen bonding interaction, we also analyzed the hydrophobic and van der
Waals interaction of complexes with serum albumins and the results are tabulated in Table
4S. The analysis indicate that hydrophobic amino acids of SA that are interacting with
complexes are: for complex 1, Leu237 (active site Tyr149) and Leu386 (active site Tyr410)
of BSA; for complex 2, Phe148, Ala290 (active site Tyr149) and Leu386, Leu406, Leu429,
Leu452 (active site Tyr410) of BSA and Val241, Ala291 (active site Tyr150) and Ala194,
Val455 (active site Tyr411) of HSA; for complex 3,Phe148, Val186, Leu237, Ala290 (active
site Tyr149) and Leu386, Leu406, Leu429, Leu452 (active site Tyr410) of BSA and Ala191,
Leu238 (active site Tyr150) and Ala194, Val455 (active site Tyr411) of HSA. The polar
amino acids that are interacting with complexes are: for 1,Tyr149, Glu152, Tyr156, Trp213,
His241 (active site Tyr149) and Asn390, Tyr410 (active site Tyr410) of BSA and Cys448,
Tyr452 (active site Tyr411) of HSA; for 2,Tyr149, Glu152, Tyr156, Trp213, His241 (active
site Tyr149) and Asn390, Tyr410, Ser488 (active site Tyr410) of BSA and Gln196 (active site
Tyr150) and Trp214, Tyr452 (active site Tyr411) of HSA; for 3,Tyr149, Glu152, Tyr156,
Ser191, Ser192, Gln195, His241, His287 (active site Tyr149) and Asn390, Tyr410, Thr448,
16

Ser488 (active site Tyr410) of BSA and Tyr150, Ser192, Trp214, His242 (active site Tyr150)
and Gln459, Tyr452 (active site Tyr411) of HSA.
Conclusion
We have presented here synthesis, crystal structure and biological activities of three dinuclear
2-[(3-Methylamino-propylimino)-methyl]-phenol (HL) coordinated copper(II) complexes
using N/O donor anionic ligands. Copper(II)-L in combination with anionic ligands dca (1),
2,5-pdc (2) and tp (3) generates discrete dinuclear complexes. In 1 and 3, HL shows a
tridentate (N,N,O) chelating coordination mode, whereas in 2, it functions as bidentate (N,O)
chelating ligand. Copper(II) centres in complexes 2 and 3 possess square pyramidal (SP)
geometry whereas in complex 1 copper(II) centres possess rare trigonal bipyramidal (TBP)
geometry. 3D supramolecular architecture in 1 and 3 are formed with H-bonding and C-H…π
interactions, whereas only H-bonding interactions results 2D supramolecular structure in 2.
Anticancer activities of 1 and 2 in human breast cancer cell lines MCF7 and MDA-MB-231
show that 1 has better growth suppressor activity in comparison to 2 through induction of
apoptosis. Study of the kinetics of interactions of 1-3 with CT-DNA and serum albumins
show that the complexes effectively binds with CT-DNA and serum albumins and the order
of interaction is 3>1>2. This order of interaction has been explained on the basis of size and
shapes of the compounds. Molecular docking study indicates that complexes 1 and 3 interact
with Tyr150 site of human serum albumin, whereas complex 2 interacts with Tyr150 and
Tyr411 sites of human serum albumin through hydrogen bonding interaction. In addition to
H-bonding, hydrophobic and van der Waals interactions are also responsible for albumin and
complex interactions.
Acknowledgements
17

This work was supported by the UGC Innovative Research Program (VU / Innovative / Sc /
08 / 2015) of Vidyasagar University, India. Dr. S. C. Manna thanks UGC-SAP and DST-
FIST New Delhi and Vidyasagar University for infrastructural facilities. Authors are also
thankful to Prof. V. Bertolasi (Universita di Ferrara) for structural data collection of complex
3.
Supplementary information
CCDC 1813748-1813750contain the supplementary crystallographic data for this article.
These
data
can
be
obtained
free
of
charge
viahttp://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-
mail: deposit@ccdc.cam.ac.uk. Results of spectroscopic studies (electronic absorption
spectra, emission spectra, IR spectra, NMR, ESI mass), Figures of crystal structures and
biological studies are provided as supplementary material.
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Table 1. Crystal data and structure refinement for complexes 1-3.
Complex
1
2
3
Empirical formula
C26H30Cu2N10O2 C36H50Cu2N6O16 C30H34Cu2N4O6
Formula mass, g mol–1 641.70
949.90
Triclinic
P-1
673.69
Monoclinic
P21/c
Crystal system
Space group
a, Å
Triclinic
P-1
8.883(5)
9.080(5)
10.432(5)
97.961(5)
112.838(5)
111.247(5)
1
7.9884(16)
11.211(2)
12.760(3)
69.66(3)
84.98(3)
80.76(3)
1
9.5456(9)
10.7836(13)
14.0349(16)
90
b, Å
c, Å
α, deg
β, deg
99.524(8)
90
γ, deg
Z
2
V, Å3
683.5(6)
1.559
1057.0(4)
1.492
1424.8(3)
1.570
D(calcd), g cm–3
(Mo-Ka), mm–1
F(000)
1.600
1.083
1.544
330
494
696
θmin-θmax, deg
No. of collected data
No. of unique data
Rint
4.3-25
4606
4.1-25
14835
3711
3.4-27.5
8749
2394
3052
0.029
0.160
0.032
Observed reflections 2193
2520
2479
[I> 2σ(I)]
Goodness of fit (F2)
Parameters refined
R1, wR2 (all data) [a]
Residuals, e Å–3
1.087
0.852
1.041
186
281
191
0.0418, 0.1135
-0.54, 1.60
0.0563, 0.1492
-0.65, 0.71
0.0431, 0.1150
-0.41, 0.55
½
[a]R1(Fo) = Fo–Fc / Fo, wR2(Fo2) = [w (Fo 2 – Fc 2) 2/ w (Fo 2) 2 ]
22

Table 2. Experimental coordination bond lengths (Å) and angles (°) for complexes 1-3.
1
2
3
Cu(1)-O(1)
Cu(1)-N(1)
Cu(1)-N(2)
Cu(1)-N(3)
Cu(1)-O(1a)
2.041(3)
1.945(4)
2.030(4)
2.087(4)
1.965(3)
Cu(1)-O(1)
Cu(1)-O(2)
Cu(1)-N(1)
Cu(1)-N(2)
Cu(1)-O(1a)
1.906(2)
1.963(2)
1.958(4)
2.017(4)
2.680(3)
Cu(1)-O(1)
Cu(1)-O(2)
Cu(1)-O(3)
Cu(1)-N(1)
Cu(1)-N(2)
1.905(2)
1.990(2)
2.680(2)
1.955(3)
2.009(3)
O(1)-Cu(1)-N(1)
O(1)-Cu(1)-N(2)
O(1)-Cu(1)-N(3)
90.56(12)
O(1)-Cu(1)-O(2)
171.56(11) O(1)-Cu(1)-O(2) 85.36(9)
128.54(13) O(1)-Cu(1)-N(1)
105.38(13) O(1)-Cu(1)-N(2)
94.73(13)
91.27(13)
79.70(9)
93.48(13)
81.03(13)
96.74(9)
O(1)-Cu(1)-O(3) 87.17(9)
O(1)-Cu(1)-N(1) 93.54(10)
O(1)-Cu(1)-N(2) 160.52(10)
O(2)-Cu(1)-O(3) 54.38(8)
O(2)-Cu(1)-N(1) 170.34(10)
O(2)-Cu(1)-N(2) 88.28(10)
O(1)-Cu(1)-O(1a) 79.53(12)
O(1)-Cu(1)-O(1a)
O(2)-Cu(1)-N(1)
O(2)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
N(1)-Cu(1)-N(3)
89.74(14)
93.48(14)
O(1a)-Cu(1)-N(1) 169.44(12) O(1a)-Cu(1)-O(2)
N(2)-Cu(1)-N(3) 125.97(15) N(1)-Cu(1)-N(2)
167.94(15) O(3)-Cu(1)-N(1) 116.01(10)
101.33(12) O(3)-Cu(1)-N(2) 103.98(9)
O(1a)-Cu(1)-N(2) 93.57(14)
O(1a)-Cu(1)-N(3) 92.70(14)
O(1a)-Cu(1)-N(1)
O(1a)-Cu(1)-N(2)
90.03(11)
N(1)-Cu(1)-N(2) 95.71(11)
Cu(1)-O(1)-Cu(1a) 100.47(13) Cu(1)-O(1)-Cu(1a) 100.30(10)
τ5 parameter
0.681
0.060
0.163
23

Table 3. IC50 values of Cu(II) complexes at μM level.
Complex
IC50
Ref
MCF7
MDA-MB-231
26±2.9
51±4.7
16.4±1.6
This work
This work
[28a]
1
43.2±3.1
2
[Cu(Tf-PIP)2(H2O)]2+
[Cu(PYIP)2(H2O)]2+
[Cu(CN-PIP)2(H2O)]2+
[Cu(PIP)2(H2O)]2+
[Cu(L1)(diimine)]+
[Cu(L2)(H2O)]∙ClO4
[Cu(L3)(H2O)]∙ClO4
[Cu(L4)(H2O)] ∙ClO4
{[Cu(HL5)(pdc)(H2O)]∙
[Cu(HL5)(pdc)]∙10.5(H2O)}
0.6 ± 0.03
26.5 ± 1.5
40.0 ± 4.6
0.1 ± 0.005
[28a]
[28a]
[28a]
0.59 ± 0.01 0.96 ± 0.02
31.07±6.67
42.63±7.13
>100
[28b]
[28c]
[28c]
[28c]
47 ± 5.2
[28d]
IP= Imidazophenanthroline ligands, HL1 = 2-[(2-dimethylaminoethylimino)methyl]phenol,
diimine
=
dipyrido[3,2-a:20,30-c]phenazine
(dppz),
=
HL2
2-{[2-(2-aminoethylamino)
=
o-{[2-(2-
aminoethylamino)ethylimino]methyl}phenol;
ethylimino]methyl}-6-methoxyphenol;
aminoethylamino)ethylimino]ethyl}phenol,
HL3
HL4
=
o-{1-[2-(2-
HL5=
6-methoxy-2-{[2-(1-
piperazinyl)ethylimino]methyl}phenol, pdc = pyridine 2,5-dicarboxylate.
24

Table 4. Kinetic parameters of BSA / HSA interaction of Cu(II) complexes.
Complex-albumin
Kapp
Ksv
Kb
kq
n
Ref
(L mol-1)
(L mol-1)
(L mol-1)
(L mol-1 s-1)
1-BSA
8.36 ± 1.62 11.60 ± 0.03 2.42 ± 0.01 × 23.20 × 1014 0.96
This
× 105
× 106
106
work
1-HSA
16.90
1.25 × 105
±
16.40 ± 0.08 46.91 ± 0.02 32.80 × 1014 1.09
× 106 × 106
This
work
2-BSA
7.31 ± 1.05 9.87 ± 0.01 2.33 ± 0.02 × 19.75 × 1014 0.94
This
× 105
× 106
106
work
2-HSA
11.00
1.96 × 105
±
16.00 ± 0.06 37.33 ± 0.02 32.00 × 1014 1.09
× 106 × 106
This
work
3-BSA
9.38 ± 1.34 12.07 ± 0.07 7.22 ± 0.02 × 24.15 × 1014 1.01
This
× 105
× 106
106
work
3-HSA
17.19
±
17.14 ± 0.10 76.12 ± 0.03 34.28 × 1014 1.13
This
2.14 × 105
× 106
× 106
work
{[Cu2(L1)2(fum)]∙
1.34 × 104
2.09 × 105
7.83 × 105
4.1866
1013
×
1.34
[29a]
(H2O)∙(MeOH)}n-
BSA
{[Cu2(L1)2(fum)]∙
1.81 × 104
1.39 × 105
7.55 × 105
2.794 × 1013 1.14
[29a]
(H2O)∙(MeOH)}n-
HSA
25

[Cu2(L2)2(N3)2]-BSA
[Cu2(L2)2(N3)2]-HSA
8.24 × 104
7.57 × 104
4.4 × 105
2.92 × 105
1.28 × 105
4.7 × 105
5.15 × 1012
4.73 × 1012
4.4 × 1013
1.06
1.39
0.70
[29b]
[29b]
[29c]
[Cu2(L3)2(mb)]·ClO4-
BSA
1.7 × 105
1.6 × 105
5.7 × 105
6.9 × 105
[Cu2(L3)2(mb)]·ClO4-
HSA
4.8 × 105
5.2 × 105
4.9 × 105
4.9 × 105
5.8 × 105
5.7 × 105
4.8 × 1013
5.2 × 1013
4.9 × 1013
0.70
0.76
1.02
[29c]
[29c]
[29c]
[Cu2(L4)2(mb)]·ClO4-
BSA
[Cu2(L4)2(mb)]·ClO4-
HSA
HL1 derived from the condensation reaction of 2-amino-1-butanol and salicylaldehyde;
fum2−= fumarate; HL2 derived from the condensation reaction of 2-acetylpyridine and
thiosemicarbazide; HL3 = 2-[(2-diethylaminoethylimino)methyl]phenol; HL4 = 2-[1-(2-
diethylaminoethylimino)propyl]phenol; mb = 4-methylbenzoate
Table 5. Kinetic parameters of CT-DNA interaction of Cu(II) complexes.
Complex
Kb
Ksv (L mol-1)
Ref
1.52 ± 0.08 × 105
2.50 ± 0.12 × 105
10.07 ± 0.56 × 105
1.68 × 105
13.63 ± 0.01 × 106 This work
13.80 ± 0.02 × 106 This work
16.69 ± 0.02 × 106 This work
1
2
3
[Cu(L1)(pa)]
[Cu(L1)(mb)]
14.05 × 104
19.90 × 104
1.71 × 105
4.73 × 104
6.12 ×104
3.80 ×104
[31a]
[31a]
[29a]
[31b]
[31c]
[31c]
2.27 × 105
{[Cu2(L2)2(fum)](H2O)(MeOH)}n
2.96 × 105
[Cu4(μ-L3)2(μ1,1,3,3-O2CH)](OH)6H2O 3.37 ×104
[Cu(L4)(phen)(NO3)]
[Cu(L4)(bpy)(NO3)]
5.81 ×104
2.50 ×104
26

[Cu(L4)(DACH)(NO3)]
[Cu(bba)(bpy))]2+
[Cu(bba)(phen)]2+
[Cu(bba)(5,6-dmp)]2+
[Cu(bba)(dpq)]2+
3.05 ×104
4.68 ×104
[31c]
[6]
(0.06 ± 0.07) × 105 1.7 × 105
(0.28 ± 0.04) × 105 3.6 × 105
(1.09 ± 0.39) × 105 1.08 × 106
(2.20 ± 0.34) × 105 5.1 × 105
[6]
[6]
[6]
HL1= o-{(3-morpholinopropylimino)methyl}phenol, pa= 3-phenylacrylate, mb= p-
methylbenzoate, fum= fumarate, Schiff base (HL2) derived from the condensation reaction of
2-amino-1-butanol and salicylaldehyde. H3L3 = 1,3-bis [3-aza-3-(1-methyl-3-oxobut-1-
enyl)prop-3-en-1-yl]-2-(2-hydroxyphenyl)-1,3-imidazolidine, L4= 3-formylchromone, phen=
1,10-phenanthroline, bpy= 2,2′-bipyridine, DACH= 1R,2R-DACH, bba= N,N-
bis(benzimidazol-2-ylmethyl)-amine, 5,6-dmp= 5,6-dimethyl-1,10-phenanthroline, dpq=
dipyrido[3,2-d:2,3-f ] quinoxaline.
Table 6.Hydrogen bonding interactions for complexes 1-3 with HSA.
Complex
H bonds
D···A (Å) D-H···A (Å)
1 (Active site: Tyr150) N-H (His816)···N
2 (Active site: Tyr150) N-H (Lys773)···O
2 (Active site: Tyr411) N-H (Lys436)···O
3 (Active site: Tyr150) N-H (Gln196)···O
2.436
3.307
2.739
3.016
1.641
2.547
1.956
2.100
27

Fig. 1. Molecular structure of complex 1.
Fig. 2. Molecular structure of complex 2 (water molecules are omitted for structural clarity).
28

Fig. 3. Molecular structure of complex 3.
Fig. 4. 3D supramolecular structure of 1.
29