Synthesis, structure, DNA/protein binding, molecular docking and in vitro anticancer activity of two Schiff base coordinated copper(II) complexes

Article abstract of DOI:10.1016/j.poly.2019.06.049

Two Schiff bases HL and HL′, having potential tridentate O,N,N′ donor sets, have been used for the synthesis of two copper(II) complexes, namely [Cu(HL)(pdc)]₂ (1) and [Cu(L′)₂]₂ (2′), where HL = 2-([2-(piperazin-yl)ethyli

Full text of DOI:10.1016/j.poly.2019.06.049

Accepted Manuscript
Synthesis, structure, DNA/protein binding, molecular docking and in vitro an-
ticancer activity of two Schiff base coordinated copper(II) complexes
Subal Chandra Manna, Soumen Mistri, Apu Patra, Manas Kumar Mahish, Dama
Saren, Rajesh Kumar Manne, Manas Kumar Santra, Ennio Zangrando, Horst
Puschmann
PII:
S0277-5387(19)30460-7
https://doi.org/10.1016/j.poly.2019.06.049
POLY 14040
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2018
17 June 2019
18 June 2019
Please cite this article as: S. Chandra Manna, S. Mistri, A. Patra, M. Kumar Mahish, D. Saren, R. Kumar Manne,
M. Kumar Santra, E. Zangrando, H. Puschmann, Synthesis, structure, DNA/protein binding, molecular docking and
in vitro anticancer activity of two Schiff base coordinated copper(II) complexes, Polyhedron (2019), doi: https://
doi.org/10.1016/j.poly.2019.06.049
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Synthesis, structure, DNA/protein binding, molecular docking and in vitro
anticancer activity of two Schiff base coordinated copper(II) complexes
Subal Chandra Manna,*,[a] Soumen Mistri,[a] Apu Patra,[a] Manas Kumar Mahish,[a] Dama
Saren,[a] Rajesh Kumar Manne,[b] Manas Kumar Santra,[b] Ennio Zangrando,[c] Horst
Puschmann[d]
[a]
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India, E-mail: scmanna@mail.vidyasagar.ac.in, Fax: (91) (03222)
275329.
[b]
National Centre for Cell Science, NCCS Complex, Pune University Campus Ganeshkhind,
Pune-411 007, Maharashtra, India.
[c] Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste,
Italy
[d] Department of Chemistry, University of Durham, South Road, Durham DH1 3LE, UK.
Abstract
Two Schiff bases HL and HL', having potential tridentate O,N,N’ donor sets, have been
used for the synthesis of two copper(II) complexes, namely [Cu(HL)(pdc)]2 (1) and [Cu(L')2]2
(2'), where HL = 2-([2-(piperazin-yl)ethylimino]methyl)phenol, pdc = py-2,5-dicarboxylate and
HL' = 2-(((2-(di-isopropylamino)ethyl)imino)methyl)phenol. X-ray single crystal analysis of
complex 1 shows it is a centro-symmetric dimer which crystallizes with a number of lattice water
molecules that form a network of H-bonds, also involving the protonated piperazinium fragment,
giving rise to a 3D supramolecular architecture. Complex 2' also crystallizes as dinuclear,
formed through mutual bridging phenol oxygen atoms as [Cu(L')2]2, whilst an ESI mass
spectrometry study evidences that in solution the complex exists as mononuclear [Cu(L')2] (2).
1

The interaction of complexes 1 and 2 with calf thymus DNA (CT-DNA) and with bovine serum
albumin (BSA) was investigated using electronic absorption and fluorescence spectroscopic
techniques. In both studies the results show a higher binding affinity of complex 1 in comparison
to 2. The anticancer activity of the complexes against human breast (MCF7) cancer cell lines
reveals that complex 1 has moderate growth suppression activity against these cells with an IC50
value of 24 ± 6.24 M.
Keywords: Copper(II) complexes; Crystal structure; DNA / protein binding; Molecular docking;
Cytotoxicity studies
1. Introduction
Among the 3d metal ions, copper plays an important role in many biological and essential
processes, such as electron transfer and oxygen transport, etc. [1]. Recently copper complexes
have been widely investigating due to their potential application as metallo-pharmaceuticals [2],
and the efficiency of these metal complexes as drugs depends not only on the oxidation states of
the metal ions, but also on the ligand framework. The binding affinity of metal complexes with
multidentate Schiff base ligands towards DNA and serum albumin is noteworthy in order to
access the type of interaction [3]. Moreover, the presence of other co-ligands along with the
Schiff base may affect the copper atom’s coordination geometry in the complexes, thus
controlling the binding affinity with DNA / proteins [4]. A literature survey reveals that plenty of
copper complexes have been reported with the aim of investigating their interactions with bio
2

molecules (nucleic acids and serum albumins), but relatively few studies involve Schiff base
compounds [4].
DNA and proteins are the main molecular targets for drugs and metal complexes, where these
compounds exhibit therapeutic effects. Therefore, the study of the interaction of metal complexes
with biomolecules is a vital step in the design and synthesis of metal based drugs as anticancer
therapeutics. It is well established that transition metal complexes can interact non-covalently
with DNA through van der Waals, electrostatic interactions, groove binding and intercalation
modes [5]. Among these interactions, the intercalation mode attracts significant attention due to
its strong binding ability and wide applications in molecular biology and cancer therapy [6]. On
the other hand, drug-proteins interactions also influence the absorption, metabolism and
excretion properties of drugs [7]. Serum albumin acts as drug carrier and plays a very important
role in drug delivery processes due to its outstanding binding properties. Out of human serum
albumin (HSA) and bovine serum albumin (BSA), the second is widely used as a model protein
for studying its interactions with molecular species due to its lower cost.
Over the last few years, our group has been involved in the design and synthesis of Schiff
base copper(II) coordination compounds. Here we choose a Schiff base bearing a piperazinyl
moiety, 2-([2-(piperazin-yl)ethylimino]methyl)phenol (HL), and an ethylene diamine based one,
2-(((2-(di-isopropylamino)ethyl)imino)methyl)phenol (HL'), for the synthesis of [Cu(HL)(2,5-
pdc)]2 (1) and [Cu(L')2]2 (2'), respectively. The interaction of these complexes with CT-DNA and
BSA was studied using spectroscopic techniques. We have also tested the cytotoxicity of the
complexes against MCF7 cancer cell lines. The molecular docking technique has been used to
access the mode of interaction of these complexes with DNA/protein.
2. Experimental section
3

2.1. Materials
High purity 2-ethylaminopiperazine and N,N-diisopropyl ethylene diamine were purchased from
the Aldrich Chemical Co. Inc. and used as received. All other chemicals used were of analytical
grade. Solvents used for spectroscopic studies were purified and dried by standard procedures
before use [8].
2.2. Physical measurements
Elemental analyses (carbon, hydrogen and nitrogen) were performed using a Perkin-Elmer 240C
elemental analyzer. IR spectra were recorded as KBr pellets on a Bruker Vector 22FT IR
spectrophotometer operating from 400 to 4000 cm–1. NMR spectra of the ligand were recorded
on a Bruker 400 MHz instrument. The ESI-MS spectrum of compound 1 (in dichloromethane)
was recorded on a LC-MS/MS system, Q-Tof Premier mass spectrometer. The ESI-MS spectrum
of compound 2' (in acetonitrile) was recorded with a Waters QtoF Model YA 263 mass
spectrometer. Electronic absorption spectra were obtained with a Shimadzu UV-1601 UV-vis
spectrophotometer at room temperature. Quartz cuvettes with a 1 cm pathlength and a 3 cm3
volume were used for all measurements. Emission spectra were recorded on a Hitachi F-7000
spectrofluorimeter. Room temperature (300 K) spectra were obtained in dichloromethane
solution using a quartz cell of 1 cm path length. The slit width was 2.5 nm for both excitation
and emission.
2.3. Synthesis of the ligands
4

The
ligands
2-([2-(pyperazin-1-yl)ethylimino]methyl)phenol
(HL)
and
2-(((2-(di-
isopropylamino)ethyl)imino)methyl)phenol (HL') were prepared by the condensation reaction of
salicyldehyde and the related amines in a 1:1 ratio in methanol for 3h. Details of the synthesis
and characterization of the ligands are given in a supplementary file. 1H and 13C NMR spectra of
HL and HL' in CDCl3 are shown in Figs. 1S-4S.
2.4. Synthesis of the complexes
Caution! The perchlorate salts were used in small quantities and handled with care since
explosions may occur.
2.4.1. Synthesis of [Cu(HL)(2,5-pdc)]2 (1)
The ligand HL (1 mmol, 0.23 g) in methanol was deprotonated with triethylamine and added into
a methanolic solution of Cu(ClO4)2·6H2O (1 mmol, 0.37 g), stirring the mixture for 2h. A
methanol-water solution of disodium pyridine-2,5-dicarboxylate (Na2pdc) (1 mmol, 0.21 g) was
slowly added to the resulting deep green solution and stirred for an additional 1h. The resulting
filtrate was kept in air. Deep green needle shaped single crystals suitable for X-ray diffraction
were obtained by slow evaporation of methanol solution after two days. Yield: 0.720 g (75%).
Anal. Calc. For C40H44Cu2N8O10.12H2O (1140.10): C, 42.14; H, 6.01; N, 9.83 % Found: C,
42.01; H, 6.03; N, 9.75 %. IR (cm-1): 3800-3000 (br,s), 2975(s), 2943(w), 1652(w), 1561(s),
1467(s), 1419(s), 1370(s), 1251(vw), 1124(vw), 1079(s), 1008(w), 885(s), 817(s), 652(w),
509(w).
2.4.2. Synthesis of [Cu(L')2]2 (2')
The ligand HL' (2 mmol, 0.49 g) in methanol (10 mL) was deprotonated with triethylamine and
added dropwise into a methanolic solution (10 mL) of Cu(ClO4)2·6H2O (1 mmol, 0.37g) under
5

stirring. A methanol-water solution of disodium pyridine-2,5-dicarboxylate (Na2pdc) (1mmol,
0.21 g) was slowly added to the resulting deep green solution and stirred for additional 2h, then
filtered. The green color filtrate was kept in air for slow evaporation and single crystals suitable
for X-ray diffraction were obtained after a few days. Yield: 0.468 g (84 %). Anal. Calc. For
C60H92Cu2N8O4 (1116.49): C, 64.48; H, 8.23; N, 10.03 %. Found: C, 64.45; H, 8.26; N, 10.06 %.
IR (cm-1): 2960(w), 2907(w), 1628(s), 1540(s), 1443(w), 1243(vw), 1396(vw), 1208(w),
1064(w), 754(s), 568(w).
2.5. Crystallographic data collection and refinement
Data collection for complexes 1 and 2' was carried out with Mo-Kα radiation (λ = 0.71073 Å) on
an Xcalibur, Sapphire3 and on a Bruker APEX-II CCD diffractometer, respectively. Experiments
were performed at 120(2) K and at ambient temperature, for 1 and 2', respectively. Cell
refinement, indexing and scaling of the data sets were done by using the CrysAlis PRO package
[9] and BrukerAPEX2 and SAINT programs [10]. Both the structures were solved by direct
methods and subsequent Fourier analyses [11,12] and refined by the full-matrix least-squares
method based on F2 with all observed reflections [12]. In compound 1, one of water lattice
molecules was found to be disordered over two positions and occupancies were refined to
0.54(6)/0.46(6). Hydrogen atoms were placed at calculated positions, except those of the water
molecules, detected on the difference Fourier map and refined with restrained O-H distances of
0.85 Å. Publication material was prepared with the Olex2 program [13] and WinGX package
[14]. Crystal data and details of the refinements are given in Table 1.
6

2.6. CT-DNA binding experiments
Electronic absorption spectral titrations were carried out at a fixed concentration of the
complexes (3 mL, 0.068 mM) in dichloromethane and with varying concentrations of CT-DNA.
The intrinsic binding constant (Kib) of the complexes with CT-DNA was determined using the
equation [15]:
[DNA]
[DNA]
1
=
+
(εf ‒ εa) (εf ‒ εb) Kib(εf ‒ εb)
where [DNA] is the concentration of CT-DNA, εa is the extinction coefficient of the complex at
a given CT-DNA concentration, εf and εb are the extinction coefficients of the complex in
solution and when fully bound to CT-DNA, respectively. A plot of [DNA]/(εf-εa) vs [DNA] gives
a straight line with 1/(εf-εb) and 1/[Kib(εf-εb)] as the slope and intercept, respectively. The value
of Kib was calculated from the ratio of the slope to the intercept.
2.7. BSA binding studies
A stock solution of bovine serum albumin (BSA) was prepared in 55 mM HEPES buffer (pH
7.2). The solutions of the copper complexes were prepared by dissolving the compounds in
water:HEPES buffer (1:99) mixture. The interaction of the compounds with serum albumin was
studied by recording the tryptophan fluorescence of BSA. Complexes 1 and 2 were added at
room temperature to the BSA solution (3.263 µM) and the quenching of the emission intensities
at 340 nm (λex, 280 nm) was recorded with gradual addition of a dichloromethane solution (0.068
Mm) of the complexes.
7

2.8. Cell culture
The breast cancer cell line MCF7 was cultured in DMEM medium as monolayers supplemented
with 10 % FBS, 100 U/mlpenicillin and 100 µg/ml streptomycin at 37 °C in a humid atmosphere
of 5% CO2.
2.9. In vitro cytotoxicity assay
The cytotoxicity of complexes 1 and 2 was determined by an MTT (3-(4,5-dimethylthiazol-2-yl)-
2-5 diphenyltetrazolium bromide) assay. MCF7 cells were seeded in 96-well plates at 5 x 103
cells per well. Cells were treated with different concentrations (0.01 – 100 µM) in triplicate for
48 hours. The MTT solution was then added to each well and further incubated for 3.5 h at 37 °C
in a humid atmosphere, 5% CO2. Finally, the MTT containing media was discarded and solvent
(iso-propanol, HCl and Triton X-100) was added to each well and incubated for 15 minutes at
room temperature with gentle rocking. The absorbance was read in a Thermo Pierce Elisa plate
reader at 570 nm, and the results were expressed as the percentage inhibition relative to the cells
treated with DMSO (considered as 0 %). Each experiment was repeated three times.
2.10. Molecular docking
2.10.1. DNA-complex docking
We used the HEX 6.3 program to study the DNA-complex interaction and Spherical Polar
Fourier Correlations to perform docking. The B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB
ID: 1bna) (http://www.rcsb.org./pdb) was used as a model compound for CT-DNA [16a]. The
molecular structures of 1 and 2 were optimized using Gaussian 09 [16b]. The geometries of the
8

complexes under study were optimized using standard 6-31G basis sets for N, C, H and O atoms
and LANL2DZ for the Cu atom. The binding energy of the complexes with DNA was minimized
using the semi-empirical AM1 method of the Chimera program. The following parameters were
used for docking: correlation type - shape only, FFT mode - 3D, grid dimension - 0.6, receptor
range - 180, ligand range - 180, twist range - 360, distance range - 40. PyMol software was used
for visualization of the docked pose.
2.10.2. BSA-complex docking
The AutoDock4.2 [17] software was used to perform BSA-complex docking. The structure of
BSA (PDB ID: 4f5s), downloaded from the Protein Data Bank (http://www.rcsb.org/pdb), was
used for docking and the Chimera program (http://www.cgl.ucsf.edu/chimera/) was used for
receptor preparation. The molecular structures of 1 and 2 were optimized by using Gaussian 09
[16b]. The geometries of the complexes under study were optimized using standard 6-31G basis
sets for the N, C, H and O atoms and LANL2DZ for the Cu atom. In order to recognize the
binding sites in BSA, the grid size was set to 100, 100 and 100 along the X-, Y- and Z-axes, with
a
0.375
Å
grid spacing. The docking results were visualized by Chimera
(http://www.cgl.ucsf.edu/chimera/) and PyMol software.
3. Results and discussion
3.1. Synthetic aspects
The multisite coordinating ligands HL and HL' were prepared by the condensation reaction of
the corresponding amine and aldehyde in methanol under reflux conditions, and characterized by
9

NMR (Figs. 1S-4S) and FT-IR (Figs. 5S and 6S). Using HL and HL', complexes 1 and 2' were
synthesized by adopting the procedures as schematically displayed in Scheme 1.
Scheme 1. Synthesis of complexes 1 and 2'.
3.2. Crystal structure description
3.2.1. [Cu(HL)(2,5-pdc)]2 (1)
10

The X-ray structural analysis of complex 1 evidences a μ-phenoxo bridged dimer, affording a
planar Cu2O2 moiety arranged about a crystallographic inversion center, crystallizing with twelve
lattice water molecules. The molecular structure of the complex is shown in Fig. 1 and selected
bond lengths and angles are listed in Table 2. Each symmetry related copper center exhibits a
coordination environment best described by an approximately square-pyramidal geometry, with
the basal plane sites occupied by the chelating Schiff base ligand through the phenol oxygen and
the imino nitrogen atoms, and by the pyridine dicarboxylate anion. The coordination is
completed by the phenoxo oxygen atom of the symmetry related complex unit at the apical
position. Here the propensity of the pyridine dicarboxylate anion to act as a chelating ligand
induces the Schiff base to behave as bidentate and the piperazine nitrogen atoms do not
participate in coordination, the ethyl-piperazinium fragment being oriented far away from the
metal center, although in some structurally characterized examples this ligand acts as an O,N,N-
tridentate chelating species, also involving the piperazine nitrogen atom [18]. The two Cu-O as
well as Cu-N bond distances are slightly different, being 1.897(5), 1.968(5) and 1.959(7),
2.016(6) Å, respectively The apical Cu-O(1) bond in the square-pyramidal environment is the
longest, as expected, with a length of 2.503(6) Å. The bond angles do not deviate significantly
from those of a regular geometry and the O(2)-Cu-N(4) angle, of 81.6(2)°, has the largest
deviation from the ideal value. The Cu2O2 core of the complex dimer is close to a square, with a
Cu-O(1)-Cu' bond angle of 96.7(2)° that giv a metal-metal separation of 3.312 Å. It is worth
noting that the nitrogen atom at position 4 of the piperazine ring is protonated, so that the ligand
is neutral in its zwitter ionic form. This protonated nitrogen atom N(3) is involved in rather
strong H-bonds with a water molecule and a carboxylate oxygen atom (NH…Ow of 2.757(9) and
2.749(10) Å), and N(2) acts as an acceptor with respect to the water molecule O(1w)
11

(N(2)…O(1w) of 2.832(9) Å). The crystal packing shows a 3D structure for a network of H-
bonds to the numerous lattice water molecules (Fig. 2), the geometries of which are reported in
Table 1S.
3.2.2. [Cu(L')2]2 (2')
The structural analysis of [Cu(L')2]2 (2') reveals in solid state discrete centro-symmetric dimer
species where the copper atom is bis-chelated by the deprotonated Shiff base ligands through the
phenol oxygen and the imino nitrogen donors, but the phenol oxygen atoms of one chelating
ligand and of its symmetry counterpart act as bridging donors, with the formation of a Cu2O2
central rhomboid moiety. The metal environment can be best described as distorted square
pyramidal. A selection of bond lengths and angles of this complex is reported in Table 2 and an
Ortep drawing is shown in Fig.3. The Cu-O bond lengths in basal plane are 1.8861(19) and
1.9214(18) Å, the latter being to the oxygen atom that acts as bridging atom, and Cu-N ones are
of 2.019(2) and 2.000(2) Å, in agreement with those measured in 1. The oxygen atom at the
apical position is at 2.3120(19) Å. The Cu2O2 rhomboid unit is slightly different from that of 1
with a Cu-O(1)-Cu' angle of 104.73(8)° and with the metal atoms separated by 3.361 Å. Due to
the steric hindrance of the di-isopropyl amino groups, but also to accomplish the short apical
bond distance for Cu-O1' (2.3120(19) Å), the coordination square of the copper atom is
significantly distorted towards a tetrahedral configuration, having O-Cu-O and N-Cu-N
coordination bond angles of 169.14(8) and 139.12(9)°, the latter considerably deviating from the
ideal value of 180°.
The reported observations indicate the tendency of these complexes to form μ-phenoxo bridged
dimers and, in addition to complex 1, different examples have been reported elsewhere [19, 20].
12

3.3. IR and electronic spectra
IR spectra of HL, HL' and of complexes 1 and 2' are shown in Figs. 5S, 6S, 7S and 8S,
respectively, and the most important IR bands with their tentative assignments are given in Table
2S. The IR spectrum of complex 1 exhibits a broad band in the region 3800-3000 cm-1, assigned
to the ν(O-H) stretching vibrations of water molecules [21a]. Bands at 2982, 2992, 2975 and
2
2985 cm-1 for HL, HL', 1 and 2', respectively, correspond to ν(Csp -H) stretching frequencies and
3
those at 2918, 2920, 2943 and 2960 cm-1 agree with ν(Csp -H). The band at 1652 cm-1 for 1 is due
to ν(COO). Aromatic ν(C=C) stretching vibrations appear in the region 1561-1443 cm-1, and the
bands in the range 1254-1243 and 1082-1064 cm-1 correspond to νs(C–O)phenolic and ν(C–N)aliphatic
stretching, respectively.
The UV-vis spectra of 1, 2, HL and HL' were recorded in dichloromethane (Figs. 4, 11S, 9S and
10S). The spectra show significant transitions at 263 nm (ε ~ 4.647 × 103 L mol-1) for 1, at 258
nm (ε ~4.557×103 L mol-1) for 2, at 265 nm (ε ~ 8.213 × 103 L mol-1) and 360 nm (ε ~ 3.517 ×
103 L mol-1) for HL, and finally at 265 nm (ε ~ 8.581 × 103 L mol-1) and 352 nm (ε ~ 3.720 × 103
L mol-1) for HL'. The bands in the region 258-265 nm are due to * of benzene ring / intra
ligand charge transfer (ILCT) transitions and the bands in the region 352-360 nm may due to
n* transitions. [21b].
3.4. ESI mass spectrometry
The ESI mass spectrometric data (Fig. 12S) of 1 show a peak (m/z) at 922.45, corresponding to
·+
the radical cation [Cu(HL)(pdc)]2 (for 1, calc. m/z = 925.93). On the other hand, the ESI mass
spectrometric data (Fig. 13S) of 2' shows peaks (m/z) at 681.00, 558.11, 312.00 and 249.09,
corresponding to the radical cations {[Cu(L')2]·+ + 3CH3CN}, [Cu(L')2]·+ , [Cu(L')]·+ and [HL']·+,
13

respectively. The observed ESI mass spectral peaks indicate that in solution the metal
coordination environment of complex 1 is the same as in its crystal structures, but for complex
2', in solution it exists as mononuclear [Cu(L')2] (2).
3.5. DNA binding studies
Interactions of complexes 1 and 2 with CT-DNA were investigated using the electronic
absorption spectroscopic technique. Figs. 4 and 12S show the UV-vis spectral changes of 1 and 2
upon gradual addition of 10 µL of an aqueous solution of 22.72 µMCT-DNA. It is to be noted
that the absorbances of 1 and 2 gradually decrease upon addition of CT-DNA, keeping the
absorption wavelength fixed. The observed hypochromism indicates intercalative binding [22].
The hypochromicity is mainly attributed to electrostatic interactions between the compounds and
the DNA bases [23].
The intrinsic binding constants (K ) of 1 and 2 [calculated from the [DNA]/(
푎) vs [DNA]
휀푓 ‒ 휀
ib
plot (inset of Figs. 4 and 11S)] are 5.29(±0.13) × 105, 4.18(±0.21) × 105 and 4.35(±0.14) × 105,
2.70(±0.16) × 105 L mol-1, respectively.
3.6. BSA binding studies
The UV-vis spectral changes (at pH 7.2) of BSA upon gradual addition of 1 and 2 are shown in
Figs. 5(a) and 16S(a). It is to be noted that the UV-vis spectral band at 280 nm of BSA
undergoes a hypsochromic shift (9 nm for 1 and 11 nm for 2) with an increase of the absorbance
in the presence of 1 and 2. The apparent association constants (Kapp) for the interaction of the
compounds with BSA were calculated by using the following equation:
14

1
1
1
=
+
(
)
(퐴표푏푠 ‒ 퐴0) (퐴푐 ‒ 퐴0)
퐾푎푝푝 퐴푐 ‒ 퐴0 [푐표푚푝푙푒푥]
where Aobs is the observed absorbance at 280 nm of the solution containing different
concentrations of the complex, A0 and Ac are the absorbance of BSA only and of serum albumin
with the complex. The calculated apparent association constants for 1 and 2 are 41.24 (±0.13) ×
108 and 2.88 (±0.15) × 108L mol-1, respectively (Table 3).
We also studied BSA-compound interaction by fluorimetric analysis. Complexes 1 and 2 are
non-fluorescent in dichloromethane solution. Upon excitation at 280 nm, an aqueous solution of
BSA exhibits fluorescence at 340 nm. Figs.5(b) and 16S(b) show quenching of the fluorescence
intensity of BSA upon gradual addition of 20 µL of a dichloromethane solution of 0.068 mM
complex 1. The quenching of fluorescence intensity of BSA mainly occurs due to complex
formation with metal species, denaturation or conformational change of the serum albumins [24].
The Stern-Volmer constant values (Ksv) for complexes 1 and 2 interacting with BSA [25] have
been calculated and the Stern-Volmer plots I/It vs [complex] are shown in Figs. 20S-21S. The Ksv
values (Table 3) indicate that compounds show significant serum albumin quenching ability. The
UV-vis spectroscopic behavior of BSA upon gradual addition of the complexes reveals that the
electronic absorption spectra of BSA are influenced by the addition of the complexes, thus
indicating a static interaction between BSA and the compounds.
When the complex independently binds in a static mode to serum albumin in a set of equivalent
sites, the equilibrium between free and bound compounds is expressed by the Scatchard equation
[26]:
(F0 ‒ F)
log
= log kb + 푛log [compound]
F
15

where kb is the binding constant of compound with serum albumin, n is the number of binding
sites per serum albumin molecule. Plots of
(F ‒ F) vs log[compound] are shown in the inset of
o
log
F
Figs. 5(b) and 16S(b) for 1 and 2, respectively, and the calculated values of kb and n are given in
Table 3. It is interesting to note that complex 1 presents one binding site whereas two are
observed for 2. However, 1 donates a relatively higher binding strength for BSA in comparison
to 2. The values of the binding constants are high enough (~107 M-1) to indicate a strong affinity
of the complexes with the BSA [27] and the results are supported by the dock prediction.
3.7. Cytotoxicity assay
The MTT assay data reveal that complex 1 has a moderate growth suppression activity against
MCF7 cells. It inhibits the growth of MCF7 cells in a dose dependent manner with an IC50 value
of 24 ± 6.24 M (Fig.6). On the other hand, complex 2 exhibits a very mild effect on the growth
of the breast cancer cell line MCF7. For example, compared to vehicle treated MCF cells, only
15% growth was inhibited in the presence of a 1 μM solution of complex 2.
3.8. Molecular docking
3.8.1. DNA-complex docking
Molecular docking is a key tool to determine the mode of DNA drug interaction [28]. Figs. 7 and
21S show the minimum energy conformation for 1 and 2, respectively. The binding of 1 with
DNA occurs through N-H…O hydrogen bonding interactions with A5 and A17, and
intermolecular interactions with A5, A6, G16, A17, and A18, whereas 2 binds with DNA
through an intermolecular interaction with C15 and G16. In the docked structure, both
complexes interact with DNA in an intercalation mode, which is in good agreement with the UV-
16

vis spectrometric DNA-complex interaction study. The binding energies of docking are -342.8
and -296.5 Kcal/mol for complexes 1 and 2, respectively, indicating a higher binding affinity for
the former with respect to complex 2.
3.8.2. BSA-complex docking
In order to corroborate our experimental results, the molecular docking technique has been used
to recognize the complex protein interactions. The Chimera scores of the individual complexes
with different poses have been tabulated in Table 3S. Since BSA has two different active sites
for binding (Tyr 149 and Tyr 410), we performed docking of each complex with each of these
binding sites in order to investigate the effective interaction of BSA with our complexes. Based
on the Chimera score (Table 3S), both complexes selectively bind with BSA at site Tyr 149. The
interactions between the complexes and RCSB protein are shown in Figs.8-9 and 22S, 23S.
The results of the docking study indicate the formation of N-H···O and N-H···N bonds between
the complexes and the protein, as presented in Table 4. Thus, both complexes form two hydrogen
bonds with BSA when they bind at the Tyr149 site. On the other hand, at the Tyr410 site, only
complex 2 exhibits one hydrogen bond with BSA. In addition to H-bonding interactions, we also
analyzed hydrophobic and van der Waal’s interactions (Table 4S). The analysis indicates that 1
undergoes hydrophobic interactions with Leu237, Val240 (active site Tyr149) and Leu386,
Asn390, Phe402, Leu406, Leu429, Leu452, Leu456, Phe487 (active site Tyr410), and 2
undergoes hydrophobic interactions with Phe148, Leu237, Val240, Ala256, Ala290 (active site
Tyr149) and Leu386, Leu429, Leu452, Leu490, Pro492 (active site Tyr410). The polar amino
acids interacting with complexes are: Tyr149, Tyr156, Ser191, Trp213, His241, Cys244, His287
17

(active site Tyr149) and Tyr410, Ser488 (active site Tyr410) for 2 and Tyr149, Tyr156, Ser191,
Gln195, His287 (active site Tyr149) and Asn390, Tyr410, Ser488 (active site Tyr410) for 1.
4. Conclusion
In summary, we have presented here the synthesis, crystal structure, DNA / protein binding,
cytotoxicity studies and molecular docking of two copper(II) complexes. Crystal structure
analysis reveals that in the solid state both [Cu(HL)(pdc)]2 (1) and [Cu(L')2]2 (2') are dinuclear,
and in both the complexes the Schiff base acts as a bidentate ligand. These complexes manifest a
tendency to form dimers so as to modify the coordination geometry from square planar to square
pyramidal. The ESI mass spectrometric analysis reveals that in solution complex 1 retains its
dinuclear composition, whereas, complex 2', exists as mononuclear [Cu(L')2] (2). Spectroscopic
and molecular docking studies indicate that both complexes 1 and 2 interact with serum albumin,
binding BSA at the Tyr149 site. In addition, both 1 and 2 interact with CT-DNA via the
intercalation binding mode. The MTT assay results indicate that complex 1 shows higher
anticancer activity on the human breast cancer cell line MCF7.
Acknowledgements
The authors gratefully acknowledge the financial assistance given by the CSIR, Government of
India, to Dr. Subal Chandra Manna (Project No. 01 (2743)/13/EMR-II). Dr. S. C. Manna thanks
UGC-SAP and DST-FIST New Delhi and Vidyasagar University for infrastructural facilities.
Supplementary information
18

CCDC 1839877 and 1019784 contain the supplementary crystallographic data for 1 and 2'.
These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/ or on
application to CCDC, Union Road, Cambridge, CB2 1EZ, UK [fax: (+44)1223-336033, e-mail:
deposit@ccdc.cam.ac.uk]
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Table 1 Crystal data and details of structure refinement for complexes 1 and 2'.
Complex
1.12H2O
2'
Empirical formula
C40H68Cu2N8O22
C60H92Cu2N8O4
Formula mass, g mol–1 1140.10
1116.49
Monoclinic
P21/n
16.054(6)
9.283(3)
20.130(8)
90.0
Crystal system
Space group
a, Å
Triclinic
P
1
7.7383(6)
11.5130(12)
14.7643(16)
76.790(9)
75.401(8)
79.109(8)
1227.1(2)
1
b, Å
c, Å
α, deg
β, deg
94.52(3)
90.0
γ, deg
V, Å3
Z
2991(2)
2
D(calcd), g cm–3
1.543
1.240
(Mo-Kα), mm–1
F(000)
0.956
598
0.762
1196
Theta range, deg
No. of collected data
No. of unique data
Rint
2.11 - 25.35
12821
2.4172- 23.56
62658
4265
0.1226
4439
0.0597
3769
Obs reflects [I> 2σ(I)] 2891
Goodness of fit (F2)
1.045
371
1.043
Parameters refined
R1, wR2 (I >2σ(I)) [a] 0.0928, 0.2129
R1, wR2 (all data)
342
0.0387, 0.0983
0.0481, 0.1056
0.1388, 0.2407
Residuals, e Å–3
1.094, –0.823
0.789, –0.371
½
[a]R1(Fo) = Fo–Fc / Fo, wR2(Fo2) = [w (Fo2 – Fc 2) 2/ w (Fo2) 2 ]
22

Table 2 Coordination bond distances (Å) and angles (°) for complexes 1 and 2'.
1
2'
Cu-O(1)
Cu-O(2)
Cu-N(1)
Cu-N(4)
Cu-O(1')
1.897(5)
1.968(5)
1.959(7)
2.017(6)
2.503(6)
1.9214(18)
1.8861(19)
2.019(2)
2.000(2)
2.3120(19)
O(1)-Cu-O(2)
O(1)-Cu-N(1)
O(1)-Cu-N(4)
O(2)-Cu-N(4)
N(1)-Cu-O(2)
N(1)-Cu-N(4)
O(1)-Cu-O(1')
O(2)-Cu-O(1')
N(1)-Cu-O(1')
N(4)-Cu-O(1')
Cu-O(1)-Cu'
172.0(2)
93.7(2)
90.7(2)
81.5(2)
94.2(2)
172.0(2)
83.3(2)
95.2(2)
94.3(2)
92.9(2)
96.7(2)
83.3(2)
169.14(8)
89.80(8)
93.80(9)
92.44(9)
91.29(9)
139.12(9)
75.27(8)
94.53(8)
115.21(8)
105.04(8)
104.73(8)
75.28(7)
O(1)-Cu-O(1')
Primed atoms at 1-x, 1-y, -z for 1, and at 1-x, 1-y, 1-z for 2'.
Table 3 Kinetic parameters for the interaction of BSA with complexes 1 and 2.
Kapp (L mol-1)
Ksv (L mol-1)
kb (L mol-1)
n
HL
HL'
1
42.29 (±0.17) × 108 0.21 (±0.11) × 108
13.01 (±0.14) × 108 0.19 (±0.18) × 108
15.12 (±0.16) × 108
12.98 (±0.19) × 108
12.41 (±0.11) × 108
11.70 (±0.17) × 108
1.34
1.03
1.06
2.59
41.24 (±0.13) × 108
2.88 (±0.15) × 108
0.14 (±0.21) × 108
0.12 (±0.23) × 108
2
23

Table 4 Hydrogen bonding interactions of complexes 1 and 2 with B-DNA and BSA.
Complex
B-DNA
1
Bonds formed
D···A (Å)
D-H···A (Å)
N-H (A5)···O
N-H (A17)···O
2.927
3.226
2.010
2.396
BSA
1 (Active site: Tyr149)
N-H (Arg198)···O
N-H (Arg198)···O
N-H (Arg198)···N
N-H (Arg409)···N
3.202
2.612
2.812
3.599
2.307
1.755
1.977
2.700
2 (Active site: Tyr149)
2 (Active site: Tyr410)
Fig.1. ORTEP drawing (thermal ellipsoids at 40% probability) of dimeric species of 1 (primed
atoms at 1-x, 1-y, -z).
24

Fig.2. 3D structure of 1 through hydrogen bonding interactions.
Fig.3. ORTEP drawing of 2' (thermal ellipsoids at 20% probability, H atoms omitted for clarity)
with the atomic labels for the crystallographic independent unit. Primed atoms at 1-x, 1-y, 1-z.
25

Fig.4. Electronic absorption spectra of complex 1 (3 ml, 0.068 mM) in HEPES buffer upon
gradual addition of 10 µL of an aqueous solution of 22.72 µM CT-DNA.
Fig.5. (a) UV-vis absorption spectra of BSA (3 ml 3.26 µM aqueous solution) upon gradual
addition of 20 µL of a dichloromethane solution of 0.068 mM complex 1 at 300K and (b)
fluorescence spectra of BSA (2 ml 3.26 µM) upon gradual addition of 20 µL of a
dichloromethane solution of 0.068 mM complex 1 at room temperature (λex = 280 nm, λem = 330
nm).
26

Fig.6. Dose dependent suppression of cell viability of human breast cancer cell line MCF7 by
complexes 1 and 2.
Fig.7. Molecular docking of 1 with B-DNA, showing the binding sites, (a) hydrogen bonding
and (b) intermolecular interactions.
27

Fig.8. Molecular docking of 1 with BSA (binding site: Tyr149) showing the binding sites and
hydrogen bonding interactions.
Fig.9. Molecular docking of 2 with BSA (binding site: Tyr149) showing the binding sites and
hydrogen bonding interactions.
28

29