Structural aspects of a trimetallic CuII derivative: cytotoxicity and anti-proliferative activity on human cancer cell lines

Article abstract of DOI:10.1080/00958972.2019.1597973

A trimetallic Cu^II derivative, [Cu₃(L)₂(CF₃COO)₂] (1) (where H₂L = N,N′-bis(salicylidene)-1,3-propanediamine), was prepared and characterized. In 1, the two terminal Cu^II ions are linked to the central Cu^II by trifluoroacetato and doubly bridging phenoxido. Both the square-pyramidal and octahedral geometries are observed among two different Cu^II centers in the linear arrangement of the trimetallic unit. Compound 1 is characterized by IR and UV-Vis spectra. Compound 1 has high cytotoxic activity in breast adenocarcinoma (MCF-7), colorectal carcinoma (HCT116) and particularly, in ovarian carcinoma (A2780) cell line compared to a lung adenocarcinoma cell line. The IC₅₀ in A2780 cells is 25 times lower than the respective value for normal human primary fibroblasts demonstrating 1 has higher cytotoxicity towards cancer cells. Additionally, combination of DOX with 1 induces a higher loss of HCT116 cell viability compared with each drug alone.

Full text of DOI:10.1080/00958972.2019.1597973

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
II
Structural aspects of a trimetallic Cu derivative:
cytotoxicity and anti-proliferative activity on
human cancer cell lines
Kuheli Das, Amitabha Datta, Chiara Massera, Catarina Roma-Rodrigues,
Mariana Barroso, Pedro V. Baptista & Alexandra R. Fernandes
To cite this article: Kuheli Das, Amitabha Datta, Chiara Massera, Catarina Roma-Rodrigues,
Mariana Barroso, Pedro V. Baptista & Alexandra R. Fernandes (2019) Structural aspects of a
II
trimetallic Cu derivative: cytotoxicity and anti-proliferative activity on human cancer cell lines,
Journal of Coordination Chemistry, 72:5-7, 920-940, DOI: 10.1080/00958972.2019.1597973
To link to this article: https://doi.org/10.1080/00958972.2019.1597973
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JOURNAL OF COORDINATION CHEMISTRY
019, VOL. 72, NOS. 5-7, 920–940
https://doi.org/10.1080/00958972.2019.1597973
2
II
Structural aspects of a trimetallic Cu derivative:
cytotoxicity and anti-proliferative activity on human
cancer cell lines
a
a
b
c
Kuheli Das , Amitabha Datta , Chiara Massera , Catarina Roma-Rodrigues ,
c
c
c,d
Mariana Barroso , Pedro V. Baptista and Alexandra R. Fernandes
a
b
Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan; Dipartimento di Chimica,
c
Universita degli Studi di Parma, Parma, Italy; UCIBO, DCV, Faculdade de Ci ^e ncias e Tecnologia,
d
Universidade Nova de Lisboa, Caparica, Portugal; Centro de Qu ꢀı mica Estrutural, Complexo I,
Instituto Superior T ꢀe cnico, Universidade de Lisboa, Lisboa, Portugal
ABSTRACT
ARTICLE HISTORY
Received 13 November 2018
Accepted 8 March 2019
II
A trimetallic Cu derivative, [Cu (L) (CF COO) ] (1) (where H L ¼
3
2
3
2
2
0
N,N -bis(salicylidene)-1,3-propanediamine), was prepared and char-
II
acterized. In 1, the two terminal Cu ions are linked to the central
II
KEYWORDS
Cu by trifluoroacetato and doubly bridging phenoxido. Both the
Copper(II); trimetallic;
cytotoxicity; cancer cell
lines; DOX
square-pyramidal and octahedral geometries are observed among
two different Cu centers in the linear arrangement of the trime-
tallic unit. Compound 1 is characterized by IR and UV-Vis spectra.
II
Compound 1 has high cytotoxic activity in breast adenocarcinoma
(
MCF-7), colorectal carcinoma (HCT116) and particularly, in ovarian
carcinoma (A2780) cell line compared to a lung adenocarcinoma
cell line. The IC50 in A2780 cells is 25 times lower than the
respective value for normal human primary fibroblasts demon-
strating
1
has higher cytotoxicity towards cancer cells.
Additionally, combination of DOX with 1 induces a higher loss of
HCT116 cell viability compared with each drug alone.
GRAPHICAL ABSTRACT
CONTACT Amitabha Datta
15, Taiwan; Alexandra R. Fernandes
Supplemental data for this article can be accessed at https://doi.org/10.1080/00958972.2019.1597973
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
amitd_ju@yahoo.co.in
Institute of Chemistry, Academia Sinica, Nankang, Taipei
1
ma.fernandes@fct.unl.pt

JOURNAL OF COORDINATION CHEMISTRY
921
1. Introduction
Schiff base ligands, condensed products of aldehydes and diamines, have created
much attention in homogeneous and heterogeneous catalysis and bioinorganic chem-
istry [1–3]. Metal derivatives incorporating Schiff base precursors derived from aro-
matic carbonyl were widely used in connection with metalloprotein models because
of the versatile electronic and steric properties that could be tuned by selecting
II
appropriate amines and ring substituents [4]. Cu phenolato derivatives have promis-
ing involvement in different biological studies. These compounds have a vital role as
intermediates in the catalytic cycles of metalloenzymes e.g. tyrosinase and galactose
II
oxidase [5–8] and in the biogenesis of metalloenzyme cofactors. Generally Cu pheno-
II
lato derivatives display coordination numbers ranging from 4 to 6, typical for Cu
coordination chemistry. In most of these derivatives the phenolates are part of a multi-
dentate ligand system [9, 10]. Salen-based tetradentate ligands are prepared by reflux-
II
ing substituted salicylaldehydes and diamines. Several Cu derivatives are synthesized
incorporating N O -donor sets of salen-based ligands using the classical Werner-type
2
2
compartment [11, 12]. The spectral, magneto-structural and theoretical correlations of
II
II
homometallic Cu derivatives having diphenoxo-bridged Cu moieties [13, 14] and lin-
2
II
ear Cu units with a diphenoxo bridge connecting pairs of metal ions [15] are known
3
but not studied for bioactivities. Metal derivatives have influence on biological proc-
esses and have medical applications. Compounds derived from Schiff bases have been
described as antimicrobial agents and anticancer agents [16]. The lack of selectivity of
some anticancer agents towards cancer cells and the development of acquired resist-
ance to therapeutic drugs are major obstacles to cancer treatment [17]. After the suc-
cess of platinum(II) compounds in cancer therapy, several Cu(II) compounds were
studied as potential antitumor agents, toward the development of new anticancer
metallodrugs with improved specificity and decreased toxic side effects, to circumvent
the main disadvantages of the currently used chemotherapeutic agents [18].
II
Herein, we report the synthetic route to a new trimetallic Cu derivative,
0
[
Cu (L) (CF COO) ] (1) (where H L ¼ N,N -bis(salicylidene)-1,3-propanediamine) [19].
3
2
3
2
2
Both structural and spectral studies are carried out to characterize the compound.
Compound 1 induces high cytotoxic activity in ovarian carcinoma (A2780) cell line
compared to other human cancer cell lines. The relative IC50 in tumor cell lines are in
the order A2780 < HCT116 < MCF-7 < A549.
2. Results and discussion
2.1. Synthesis and characterization
The Schiff base precursor, H L, is the condensed product of salicylaldehyde and 1,3-
2
diaminopropane in methanol [19]. First we synthesize [CuL] by a one-step reaction
between anhydrous CuCl2 and H L in methanol. This N O -donor precursor could
2
2 2
II
encapsulate another Cu using its distinct coordination pocket. Thus, the stoichiomet-
ric reaction of metalloligand [CuL] with Cu(CF COO) ꢀ4H O in methanol renders the tri-
3
2
2
nuclear derivative 1 (see Scheme 1), which is subsequently crystallized.

9
22
K. DAS ET AL.
Scheme 1. Formation of 1.
The infrared spectrum of 1 is consistent with the structural data presented in the
ꢁ
1
paper. The band at 1623 cm may be assigned to the imine stretch in the free ligand.
ꢁ
1
This band shifted 20–35 cm to lower wavenumbers upon complexation, which can
be attributed to the imine-N coordination [20–23]. In the free precursor, the phenolic
ꢁ
1
mAr–O is at 1201 cm ; after complexation, this band shifts to the lower vibration region
ꢁ
1
at 1177–1183 cm , evidence for metal-coordination via deprotonated phenolic-O [22].
ꢁ
ꢁ1
ꢁ
The typical trifluoroacetato stretch, m
(COO ) at 1590 cm
and msym(COO ) at
asym
ꢁ
1
1
1
445 cm , can be observed in 1. The difference Dm between these two vibrations is
45 cm , smaller than the 164 cm which is perceived in free trifluoroacetato anions.
ꢁ
1
ꢁ1
This is supporting evidence for the presence of a deprotonated trifluoroacetate which
ꢁ
1
bridges bidentate between two coppers [22, 23]. Sharp bands at 415 and 380 cm
correspond to the Cu–N and Cu–O stretching frequencies, respectively.
2.2. Crystal structure
The X-ray diffraction analysis of 1 reveals that it crystallizes on a monoclinic system
having space group P21/c. Relevant bond lengths and angles are summarized in Table
1
. The molecular structure of 1 (Figure 1) consists of a trinuclear unit having two sym-
II
0
metry-related moieties. Here, two terminal Cu ions, Cu1 and Cu1 , are connected to
the central Cu2 by means of a syn-anti trifluoroacetato bridging along with doubly
ꢂ
Table 1. Selected geometric parameters (Å, ) for 1.
Cu1–N1
Cu1–N2
Cu1–O1
Cu1–O2
Cu1–O3
Cu2–O1
1.977(4)
1.987(4)
1.997(3)
1.922(3)
2.261(3)
1.996(3)
Cu2–O2
Cu2–O4
Cu1ꢀꢀꢀCu2
O1–Cu1–N2
O2–Cu1–N1
O1–Cu2–O2
2.290(4)
2.010(3)
3.094(6)
171.9(1)
167.56(1)
73.4(1)

JOURNAL OF COORDINATION CHEMISTRY
923
Figure 1. Molecular structure of 1 with partial atom labeling scheme. Hydrogens are omitted
for clarity.
Figure 2. CHꢀꢀꢀp interactions (green) stabilizing the crystal structure of 1.
0
phenoxido bridging. Cu2 is on an inversion center, connecting O4 and O4 from the tri-
0
0
fluoroacetato unit and four phenoxido oxygens (O1, O2, O1 , and O2 ). Thus, it pos-
sesses an octahedral environment. The corresponding bond angles O4–Cu2–O2,
0
ꢂ
ꢂ
ꢂ
0
O2–Cu2–O1, and O1–Cu2–O4 are 92.8 , 73.4 , and 91.0 . The Cu1 and Cu1 ions are
five-coordinate with square-pyramidal geometry. The apical positions are occupied by
0
trifluoroacetato O3 and O3 , respectively, whereas the equitorial plane is formed by two
0
0
Nimine atoms (N1, N2 or N1’ and N2’) and two Ophenoxido atoms (O1, O2 or O1 and O2 ).
The ligand deviates from planarity, with an overall concave geometry. The crystal
structure is further stabilized by weak CHꢀꢀꢀp interactions (Figure 2) giving sets of par-
allel chains running along a axis of the unit cell. The shortest inter-chain Cu–Cu separ-
ation is 10.573(4) Å.
2.3. UV-Vis spectra
The UV-vis spectrum for H L in HPLC grade methanol has sharp bands at 215 (e: 37.1
2
3
ꢁ1
ꢁ1
3
ꢁ1
3
ꢁ1
ꢁ1
3
ꢁ1
ꢁ1
E mol .L.cm ), 255 (e: 19.5 E mol .L.cm ) and 316 (e: 6.5 E mol .L.cm ) nm
ꢁ1
ꢃ
and a weak band at 400 nm (e: 1.8 E mol .L.cm ) which correspond to the p!p
ꢃ
and n!p transitions (Figure 3) [24]. Compound 1 exhibits a strong band at 218 nm

9
24
K. DAS ET AL.
Figure 3. UV-vis spectra for H L and 1.
2
Figure 4. Solid state emission spectra of 1 measured at room temperature upon excitation at
250 nm. The inset shows a weak hump appearing at 555 nm.
3
ꢁ1
ꢁ1
(
2
e: 146 E mol .L.cm ) with a hump at 234 nm as well as a very weak vibration at
3
ꢁ1
ꢁ1
3
ꢁ1
ꢁ1
73 (e: 7.3 E mol .L.cm ) and 360 nm (e: 3.1 E mol .L.cm ) [25]. A broad band is
observed at 595–610 nm that could be ascribed to the d-d transition, typical for Schiff
base Cu(II) compounds [26].
2
.4. Emission spectra
At room temperature, 1 has a strong blue luminescence with emission maxima at
90 nm and 395 nm, upon excitation at 250 nm in the solid state. Emissions coming
from H L are observed around the same wavelengths, thus the luminescent behavior
2
2

JOURNAL OF COORDINATION CHEMISTRY
925
(A) A2780
(B) HCT116
1
1
1
40.0
20.0
00.0
0.0
0.0
0.0
0.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
*
8
6
4
2
*
*
*
*
* * * * *
* * * * *
0.0
0.050.0750.1 0.5
1
5
10 25 50 75 100
0.1 0.5
1
2
4
5
10 25 50 75 100
Concentraꢀon (μM)
Concentraꢀon (μM)
(C) MCF-7
(D) A549
1
1
1
40.0
20.0
00.0
0.0
0.0
0.0
0.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
*
8
*
*
6
4
2
*
*
* * * *
*
*
* * *
0.0
0.1 0.5
1
2
4
5
10 25 50 75 100
0.1 0.5
1
2
4
5
10 25 50 75 100
Concentraꢀon (μM)
Concentraꢀon (μM)
Figure 5. Cytotoxicity of 1 in (A) A2780, (B) HCT116, (C) MCF-7, and (D) A549 cell lines (at 48 h).
The data were normalized against the control treated with 0.1% (v/v) DMSO and expressed as
ꢃ
mean ± SEM of three independent assays. The symbol in the figure means that the results are
statistically significant with a p < 0.05 (as compared to control for each cell line).
Table 2. Relative IC50 values for 1 in the human cancer cell lines (A2780,
HCT116, MCF-7, and A549) and in normal primary human fibroblasts.
Cell line
IC50 (mM)
A2780
HCT116
MCF-7
A549
Fibroblasts
0.4 ± 0.05
3.3 ± 0.1
3.6 ± 0.1
6.2 ± 0.2
10.2 ± 0.3
of the compound can be rationalized as intraligand n–pꢃ and p–pꢃ charge transfer
transitions. An additional weak absorption is seen for 1 at 555 nm (inset in Figure 4),
corresponding to the metal-to-ligand charge transfer where the electrons are trans-
ferred from the Cu-3d to O-2p and N-2p states [27].
2.5. Cytotoxicity study
The cytotoxic activity of 1 was assessed by MTS assay on representative human cancer
cell lines: ovarian cisplatin sensitive (A2780), breast adenocarcinoma (MCF7), colorectal
carcinoma (HCT116), lung adenocarcinoma (A549), and in normal human primary fibro-
blasts. A decrease of the cell viability in a dose-dependent manner was observed for
all tumor cell lines after 48 h exposure to 1 (Figure 5). Compound 1 displayed higher

9
26
K. DAS ET AL.
Fibroblasts
1
1
1
40.0
20.0
00.0
0.0
0.0
0.0
0.0
*
8
6
4
2
*
*
* * *
0.0
0.1 0.5
1
2
4
5
10 25 50 75 100
Concentraꢀon (μM)
Figure 6. Cytotoxicity of 1 in normal human primary fibroblasts (at 48 h). The data were normal-
ized against the control treated with 0.1% (v/v) DMSO and are expressed as mean ± SEM of three
ꢃ
independent assays. The symbol in the figure means that the results are statistically significant
with a p < 0.05 (as compared to control).
Figure 7. Hoechst staining of A2780 cell line for visualization of apoptotic nuclei. Cells were grown
in the presence of: (A) DMSO control, (B) 1 (at IC ). Three random microscopic fields per sample
50
with circa 50 nuclei were counted. (C) % of apoptotic cells after exposition of A2780 cells to con-
ꢃ
trol vehicle (DMSO) or 1. p-value < 0.05 relative to apoptosis in cells incubated with DMSO.
cytotoxic activity in A2780 cancer cell line after 48 h of treatment in comparison to
the other cancer cell lines studied (HCT116, MCF-7, and A549) (Figure 5 and Table 2).
Cytotoxicity of 1 is the same order of magnitude as the cytotoxicity presented by
doxorubicin (DOX), a common chemotherapeutic agent, in A2780 cell line. In fact,
using the same experimental conditions at 48 h incubation, the relative IC50 values

JOURNAL OF COORDINATION CHEMISTRY
927
found for DOX in A2780 and HCT116 were 0.1 ± 0.02 mM and 0.3 ± 0.03 mM, respect-
ively (Figure S1A and B). Interestingly, 1 displays lower cytotoxic effect in normal
human primary fibroblasts, which shows its selectivity towards cancer cells, in particu-
lar to ovarian cancer cells (relative IC50 25ꢄ lower) (Figures 5 and 6 and Table 2). The
relative IC50 of DOX in human primary fibroblasts is much lower than that of 1 at 48 h
(1.3 ± 0.2 mM compared to 10.2 ± 0.3 mM, respectively) (Supplementary Figure S1 and
Table 2). These results highlight 1’s cytotoxicity towards ovarian carcinoma cell line
with lower side effects in normal healthy fibroblasts (compared to DOX). To get insight
into the mechanism of cytotoxic action induced after 48 h of exposure of A2780 cell
line to 1, apoptosis and autophagy were assessed. Hoechst nuclei staining of A2780
cell line in absence (DMSO as control) or presence of 1 (at its IC ) was assessed
50
0
0
(
Figure 7). Hoechst 33258 (2 -[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]2,5 -bi-1H-ben-
zimidazole trihydrochloride trihydrate) is a cell-permeable DNA stain that binds prefer-
entially to adenine–thymine (A–T) regions of DNA and can detect gradations of
nuclear damage and apoptosis. Normal nuclei show non-condensed chromatin uni-
formly distributed over the entire nucleus, whereas apoptotic nuclei show condensate
or fragmented chromatin. Some cells form apoptotic bodies [28]. Figure 7 shows a
clear increase of apoptotic markers, such as chromatin condensation, nuclear structure
abnormalities and apoptotic bodies (arrows in Figure 7A and B) in A2780 cells incu-
bated with 1. The level of cell death due to apoptosis observed in Figure 7C for 1 is
almost 60%. To understand if this compound may also induce other types of pro-
gramed cell death (PCD), Type II – autophagic cell death was evaluated.
Autophagy is a catabolic process that digests cellular contents within lysosomes
and may be accelerated by a variety of cellular stressors such as nutrient starvation,
Figure 8. (A) Autophagic cell death evaluation by fluorescence microscopy. Nuclei were stained
with DAPI (in blue) and autofagolysosomes were stained in green. Merge images are superim-
posed. White arrows point to accumulation of autofagolysosomes and orange arrows point to cells
in apoptosis. (B) % of Autophagic A2780 cells in the presence of 1. DMSO was used as vehicle con-
trol and rapamycin as an autophagic positive marker.

9
28
K. DAS ET AL.
A1.4
1
.2
1
0
µM CT-DNA + Compound 1
15 µM CT-DNA + Compound 1
5 µM CT-DNA + Compound 1
50 µM CT-DNA + Compound 1
0
0
0
0
.8
.6
.4
.2
0
2
2
40
290
340
390
440
Wavelenght (nm)
B 10
0
-10
-20
-30
-40
-50
5
5
0 µM CT-DNA + Compound 1
0 µM CT-DNA
2
30
250
270
290
310
330
350
Absorbance
Figure 9. (A) Absorption spectra of 1 (25 mM) in the presence of increasing amounts of CT-DNA
15–50 mM). Arrow indicates the absorbance changing upon increase of CT-DNA concentrations. (B)
(
CD spectra of CT-DNA (50 mM) and 1 (25 mM) bound to CT-DNA (50 mM).
DNA damage, and organelle damage. Figure 8 shows that all compounds can induce
autophagolysosomes accumulation, a characteristic of autophagy. As observed in
Figure 8 incubation of A2780 cells with 1 (at its IC ) does not induce autophagy com-
50
pared to DMSO control cells (Figure 8A and B). Rapamycin was used as a positive
marker for autophagy (Figure 8). These results indicate that the loss of viability
observed in the presence of 1 (at its IC ) (Figure 5) may be attributed to the induc-
50
tion of apoptosis (Figure 7), which has been observed for other Schiff base-derived
copper complexes [29–31]. Several compounds induce their mechanism of action with
cancer cells via the interaction with DNA (e.g. DOX, cisplatin) [32]. To get insight into
the mechanism of action of 1 in A2780 cancer cells, its interaction with CT-DNA was
analyzed by UV-Vis spectroscopy, an efficient method of examining the mode and
extent of binding of a metal complex with DNA [33] (Figure 9A). The UV spectra of 1
ꢃ
exhibited an intense absorption with maximum at 271 nm due to intraligand (IL) p–p
transitions, and a second band, at 351 nm, associated to mixed metal-to-ligand charge
transfer (MLCT) and IL transitions (Figure 9A – solid gray line; 0 mM CT-DNA þ 1). The
absorption intensity of the compound decreases (hypochromism) with increasing CT-
DNA concentrations (Figure 9A). Also, a red shift (271 nm to 276 nm) is observed with
increasing CT-DNA concentrations (Figure 9A). Hypochromism and the red shift in the

JOURNAL OF COORDINATION CHEMISTRY
929
A
B
Compound 1
1
1
20
00
8
6
4
2
0
0
0
0
0
relaxed circular
Supercoiled
pDNA
DMSO
5 µM
25 µM
50 µM
75 µM
100 µM
M
2
3 Kbp
.4 Kbp
.4 Kbp
9
4
Relaxed circular
Supercoiled
2.3 Kbp
.0 Kbp
2
Figure 10. Agarose gel electrophoresis of pUC18 DNA incubated without or with 1 for 24 h at
ꢂ
3
7 C in 5 mM Tris-HCl and 50 mM NaCl buffer (pH 7.2). Lane 1: pUC18 (pDNA); Lane 2: pUC18
incubated with 1% (v/v) DMSO (DMSO); Lanes 3–7: pUC18 incubated with increasing concentra-
tions of 1 (5, 25, 50, 75, and 100 lM); Lane 8: k/HindIII molecular marker (M).
absorption spectra indicated a strong electronic interaction between 1 and the DNA
bases. These spectral characteristics were consistent with intercalative binding of this
compound involving a p-stacking interaction between the compound and DNA base
pairs [34].
4
ꢁ1
An intrinsic binding constant of Kb ¼ 2.3 ꢄ 10 M
was calculated by plotting
[
DNA/(e ꢁe )] vs. [DNA]) for 1 [35], indicating that 1 has good binding affinity to CT-
a
f
4
ꢁ1
DNA [36], of the same order of magnitude of cisplatin (5.73 ± 0.45 ꢄ 10 M ) [37] but
0 times lower compared to other Cu(II) metal complexes that bind DNA by intercala-
tive modes and 100–1000 times lower than that reported for classical intercalators,
ethidium bromide and [Ru(phen)DPPZ] whose binding constants are 10 –10 M [38].
To further confirm this interaction CD spectral technique was used (Figure 9B). CD
spectral technique gives information on how the bound compound influences the
conformation of the CT-DNA chain. The CD spectrum of CT-DNA has a positive band
at 274 nm due to base stacking and a negative band at 243 nm due to helicity and it
is also characteristic of DNA in a right-handed B form [39].
1
6
7
ꢁ1
The changes in CD spectra indicated that 1 induces perturbation on the base stack-
ing and helicity of B-DNA (Figure 9B), which agrees with previous UV-Visible observa-
tions (Figure 9A). Both the positive and negative bands of the CD spectra are affected
by the presence of 1, with flattening of the positive band and a more intense negative
band. This indicates that 1 probably induces an over-winding of the helix with a nar-
row and deep minor groove and a wide and shallow major groove, facilitating external
access to the nucleobases. This effect has also been observed in the presence of cer-
tain organic amines and polyamines, high salt and other compounds [40–42].

9
30
K. DAS ET AL.
4
3
2
1
0
.5
4
.5
3
*
.5
2
.5
1
.5
0
DMSO
H2O2
Compound 1
Figure 11. ROS production normalized to control cells treated with 0.1% DMSO in A2780 cells
ꢃ
treated for 48 h with 1 (at its IC ). H O was used as positive control of ROS induction. p-value
50
2 2
<
0.05 relative to apoptosis in cells incubated with DMSO.
B
A
DOX Resistant HCT116 Cells
DOX Resistant HCT116 Cells
1
20
00
120
100
80
60
40
20
0
1
*
8
6
4
2
0
0
0
0
0
*
*
*
*
0.05
0.1
0.5
1
2.5
5
10
25
50
0.05
0.1
0.5
1
2.5
5
10
25
50
DOX [μM]
Compound 1 [μM]
C
a
b
Figure 12. Cytotoxicity of DOX (A) and 1 (B) in DOX resistant HCT116 cells. The data was normal-
ized against the control treated with 0.1% (v/v) DMSO (control) and expressed as mean ± SEM of
ꢃ
three independent assays. The symbol in the figure means that the results are statistically signifi-
cant with a p < 0.05 (as compared to control). Hoechst staining of DOX resistant HCT116 cells (C)
for visualization of apoptotic nuclei. Resistant cells were grown in the presence of: (a) DMSO con-
trol and (b) 1 (at IC ). Green arrows indicate markers of apoptosis namely chromatin condensation
50
and/or apoptotic bodies.

JOURNAL OF COORDINATION CHEMISTRY
931
A
HCT116 sensiꢀve cells
B
a
b
1
20
00
1
80
60
40
20
0
*
*
c
d
*
Control
Compound 1
DOX
Compound 1 + DOX
Figure 13. (A) Cytotoxicity of DOX (at IC , 0.3 mM), 1 (at IC , 1.4 mM), and DOX þ 1 (both at
50
50
IC ) in HCT116 sensitive cells (at 48 h). The data were normalized against the control treated with
5
0
0
.1% (v/v) DMSO (control) and expressed as mean ± SEM of three independent assays. The symbol
in the figure means that the results are statistically significant with a p < 0.05 (as compared to
ꢃ
control). (B) Hoechst staining of HCT116 sensitive cells for visualization of apoptotic nuclei. Cells
were grown in the presence of: (a) DMSO control, (b) DOX (at IC ), (c) 1 (at IC ) and d) DOX þ 1
5
0
50
(
both at IC ). Green arrows indicate markers of apoptosis namely chromatin condensation and/or
50
apoptotic bodies.
Remarkably, a shift from 249 nm to 240 nm and from 284 nm to 291 nm is observed
for CT-DNA incubated with 50 mM of 1 (compared to CT-DNA alone) (Figure 9B).
Besides interacting with DNA, several Cu(II) compounds promote their cellular
effects by being capable to induce DNA cleavage [43], therefore, we evaluated the in
vitro interaction of plasmid DNA (pDNA) with 1 to assess its capability to cleave pDNA
(Figure 10). The incubation of pDNA with 1 resulted in an increase of relaxed circular
form (nicked) and a decrease of supercoiled form of pDNA, nevertheless, this single
strand cleavage was not concentration dependent (Figure 10). Indeed, 5 mM of 1
induce the same level of cleavage as the maximum tested concentration (100 mM). For
all the tested concentrations no induction of double strand cleavage was observed
(Figure 10; no linear band between supercoiled and relaxed circulated band
is observed).
To gain more insight into the mechanism by which 1 induces pDNA cleavage, its
0
0
ability to generate reactive oxygen species (ROS) was investigated using the 2 ,7 -
dichlorodihydrofluorescein diacetate (DCFH-DA) assay [44]. As shown in Figure 11
treatment of the A2780 cells with 1 for 48 h resulted in a marked increase of the intra-
cellular ROS levels. This increase correlates with p-DNA single strand induced cleavage
(shown in Figure 10).
One of the major concerns in cancer treatment is the development of multidrug
resistance by cancer patients [45]. We have developed HCT116 cells resistant to DOX
by continuous exposure of HCT116 sensitive cells to increasing concentrations of DOX
during several passages. The development of resistance was accomplished (Figure
12A), where 3.6 mM of DOX do not induce any effect in HCT116 cell viability in com-
parison to the relative IC50 of DOX in HCT116 sensitive cells (0.3 ± 0.03 mM) – see
Supplementary Figure S1B. Interestingly, DOX resistant HCT116 cells are very sensitive

9
32
K. DAS ET AL.
to 1 (with a relative IC50 of 4.3 mM), demonstrating the possibility of its application to
counteract colorectal carcinoma drug resistance (Figure 12B). The observed cytotox-
icity of 1 is due to an increase of apoptosis in DOX resistant HCT116 cells (Figure 12C).
We have also observed simultaneous addition of 1 and DOX (at their IC50 alone) to
HCT116 sensitive cells induce increased loss of cell viability compared to both com-
pounds alone (Figure 13A). Once again, the observed cytotoxicity is due to an increase
of apoptosis in HCT116 sensitive cells (Figure 13B).
3. Conclusion
Combination of a “metalloligand” [CuL] with copper(II) salt affords the trifluoroacetato-
and phenoxido-bridged trinuclear Cu(II) derivative. The spectral data are consistent with
the compound formation. From the biological point of view, 1 can induce a high cyto-
toxic activity in ovarian carcinoma (A2780) cell line compared to other human cancer cell
lines. The relative IC50 in tumor cell lines were in the order A2780 < HCT116 < MCF-
7
< A549; the IC50 of 1 in A2780 cells is of the same order of magnitude of the respective
value for DOX making it an interesting compound for further investigation. Additionally, 1
relative IC50 in A2780 cells is 25 times lower than the respective value for normal human
primary fibroblasts demonstrating its higher cytotoxicity towards A2780 cells, with low
side effects in non-tumor cells (at its IC ). Also, when comparing with the effect of DOX
50
in normal human primary fibroblasts, it is clear that 1 induces much lower side effects in
this type of normal cells. Preliminary investigation on the mode of action of 1 indicated
that the loss of cell viability is mainly due to the induction of A2780 cell apoptosis.
Compound 1 can bind to CT-DNA by intercalation involving a p-stacking interaction
between the compound and DNA base pairs that was also confirmed using CD. This bind-
ing to DNA and its ability to induce ROS may indicate induction of single strand breaks,
observed after the incubation of 1 with pDNA. The high toxic side effects and acquired
resistance of cancer cells to common antitumor drugs such as DOX, limits, in certain
cases, applications in clinical settings. Our preliminary data suggest that 1 can induce the
loss of viability of DOX resistant HCT116 cells (IC50 in the low micromolar range, 4.3 mM)
via the induction of apoptosis. Also, combination of DOX with 1 induces higher loss of
HCT116 cell viability compared with each drug alone. This work provides a good candi-
date compound for further anti-tumor research via improving tumor cell targeted delivery
using nano-formulations and ultimately in vivo efficacy studies.
4. Experimental
4.1. Materials
All chemicals and solvents used for synthesis were of reagent grade, obtained com-
mercially and used as received. Salicylaldehyde, 1,3-diaminopropane and anhydrous
CuCl were purchased from Aldrich Chemicals. Hydrated copper(II) trifluoroacetate was
2
prepared by the treatment of copper(II) carbonate basic hydrate, CuCO ꢀ2Cu(OH) (AR
3
2
grade, E. Merck), with trifluoroacetic acid (AR grade, E. Merck), followed by slow evap-
oration on a steam bath. It was then filtered through a fine glass frit and stored in
CaCl desiccators.
2

JOURNAL OF COORDINATION CHEMISTRY
933
Table 3. Crystal data and structure refinement information for 1.
Empirical formula
38 32 4 8 6 3
C H N O F Cu
M
977.29
Crystal system
Space group
a/Å
b/Å
c/Å
Monoclinic
P21/c
9.553(1)
10.105(1)
19.128(2)
94.607(2)
1840.5(3)
2
b/deg
3
V/Å
Z
T/K
q/g cm
m/mm
293(2)
1.763
1.809
ꢁ
1
3
–
F(000)
986
Total reflections
Unique reflections (Rint
24398
4364 (0.0720)
2957
1.001
0.0515, 0.1428
)
o o
Observed reflections [F >4r(F )]
2
a
GOF on F
b
R
indices [F
o
>4r(F
o
)] R
1
, wR
2
–
3
Largest diff. peak and hole (eÅ )
1.088, ꢁ0.941
a
2
2 2
1/2
Goodness-of-fit S ¼ [Rw(F
o
ꢁ F
c
) /(nꢁp)] , where n is the number of reflections and p the number
of parameters.
b
2
2 2
2 2 1/2
R
1
¼ RjjF
o
j ꢁ jF
c
jj/RjF
o
j, wR
2
¼ [R[w(F
o
ꢁ F
c o
) ]/R[w(F ) ]] .
4.2. Physical measurements
Microanalytical data (C, H, and N) were collected on a Perkin–Elmer 2400 CHNS/O
elemental analyzer. FTIR spectra were recorded on a Perkin-Elmer RX-1 spectropho-
ꢁ
1
tometer from 4000–400 cm as KBr pellets. Electronic spectra were measured on a
Lambda 25 (U.V.–Vis.–N.I.R.) spectrophotometer. Solid state emission measurements
were performed on a F-4500 FL spectrophotometer using 5.0 nm slit width, scan speed
of 1200 nm/min, and PMT voltage of 700 V.
4.3. Synthesis of the ligand and 1
4
.3.1. Synthesis of the Schiff-base ligand (H L)
2
A 10 mL methanol solution of salicylaldehyde (1.05 mL, 10 mmol) was first mixed with
0 mL methanolic solution of 1,3-propanediamine (0.42 mL, 5 mmol). Then, the result-
1
ing mixture was refluxed for ca. 2 h and cooled. The desired yellow ligand was filtered
off and dried in vacuum. Anal. calc. for C H N O : C, 72.30; H, 6.43; N, 9.92. Found: C,
1
7 18 2 2
1
7
2.51; H, 6.78; N, 10.17%. H NMR (DMSO-d , d ppm): 13.51 (bs, 2H; Ar-OH), 8.59 (s, 2H;
6
N ¼ CH), 7.45–7.31 (m, 4H; Ar-H), 6.92–6.88 (m, 4H; Ar-H), 3.69 (t, 4H, J ¼ 6.74 Hz; N-
CH ), 2.03 (t, 2H, J ¼ 6.74 Hz; CH -CH -CH ). IR spectra: 1623, 1448, 1201, 976, 832,
2
2
2
2
771, 643.
4.3.2. Preparation of the “metalloligand” [CuL]
A mixture of H L (1.432 g, 5 mmol) in 10 mL methanol and ammonia solution (10 mL,
2
2
5
0%) was added dropwise to a 20 mL methanolic solution of anhydrous CuCl (0.670 g,
mmol) to yield [CuL]. After mixing, the resulting solution turns pale green.
2

9
34
K. DAS ET AL.
4
.3.3. Synthesis of [Cu (L) (CF COO) ] (1)
3 2 3 2
1
5 mL of a methanolic solution of Cu(CF COO) ꢀ4H O (0.610 g, 2 mmol) was mixed to a
3
2
2
methanolic solution (10 mL) of [CuL] (1 mmol), under constant stirring. The resulting
green solution was kept boiling for 20 mins and then left undisturbed at room tem-
perature. Dark-green, square-shaped single crystals of 1 were formed after a few days.
Shiny crystals were separated by filtration and air-dried before collection of X-ray data.
Yield: 0.62 g. Anal. calc. for C H N O F Cu : C, 46.66; H, 3.30; N, 5.73. Found: C, 46.89;
38
32
4
8 6
3
H, 3.57; N, 5.59%. IR spectra: 1585, 1445, 1303, 1220, 1177, 1183, 980, 678, 544,
ꢁ
1
4
15, 380 cm .
4.4. X-ray crystallography
The crystal structure of 1 was determined by X-ray diffraction analysis. Intensity data
and cell parameters were recorded at 293(2) K on a Bruker APEX II equipped with a
CCD area detector and a graphite monochromator (MoKa radiation k ¼ 0.71073 Å).
The raw frame data were processed using SAINT and SADABS to yield the reflection
data file [46]. The structures were solved by Direct Methods using the SIR97 program
2
[
[
47] and refined on Fo by full-matrix least-squares procedures using SHELXL-2014/7
48] in the WinGX suite v.2014.1 [49, 50]. All non-hydrogen atoms were refined with
anisotropic atomic displacements. The weighting schemes used in the last cycle of
2
2
2
2
2
refinement were w ¼ 1/[r F þ (0.0940P) þ 0.9360P] where P ¼ (Fo þ 2F )/3.
o
c
Geometric calculations were performed with the PARST97 program [51]. Crystal data
and experimental details for data collection and structure refinement are reported in
Table 3. Crystallographic data for the structures reported have been deposited with
the Cambridge Crystallographic Data Center as supplementary publication no. CCDC-
1493436 and can be obtained free of charge on application to the CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (Fax: þ44-1223-336-033; E-mail deposit@ccdc.cam.ac.uk
or http://www.ccdc.cam.ac.uk).
4.5. Biological assays
4.5.1. Cell culture
Human ovarian carcinoma (A2780), colorectal carcinoma (HCT116), and lung adenocar-
cinoma (A549) cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM)
(
Invitrogen Corp., Grand Island, NY, USA) supplemented with 10% fetal bovine serum
ꢂ
and 1% antibiotic/antimycotic solution (Invitrogen Corp.) and maintained at 37 C in a
humidified atmosphere of 5% (v/v) CO [52]. MCF-7 cell line derived from pleural effu-
2
sion of breast adenocarcinoma from a female patient was grown in similar conditions,
supplemented with 1% MEM non-essential amino acids (Invitrogen Corp.) [52b].
Normal human primary fibroblasts were grown in the same conditions as MCF7 cell
line [35b]. All cell lines were purchased from ATCC (www.atcc.org).
4.5.2. Compounds exposure for dose-response curves
Cells were plated at 5000 cells/well in 96-well plates. Medium was removed 24 h after
platting and replaced with fresh media containing: 0.1–50 mM of 1, 0.01–50 mM of

JOURNAL OF COORDINATION CHEMISTRY
935
DOX or 0.1% (v/v) DMSO (vehicle control). All previous solutions were prepared from
concentrated stock solutions (in DMSO) of the compounds.
4.6. Viability assays
After 48 h of cell incubation in the presence or absence of 1, cell viability was eval-
V
R
uated with CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega,
Madison, WI, USA) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)2H-tetrazolium, inner salt (MTS) as previously described [53]. In brief, this
is a homogeneous, colorimetric method for determining the number of viable cells in
V
R
proliferation, cytotoxicity or chemosensitivity assays. The Cell Titer 96 Aqueous Assay
is composed of solutions of MTS and an electron coupling reagent (phenazinemetho-
sulfate, PMS). MTS is bioreduced by cells into a formazan product that is soluble in tis-
sue culture medium. The absorbance of the formazan product at 490 nm can be
measured directly from 96-well assay plates without additional processing. The conver-
sion of MTS into the aqueous soluble formazan product is accomplished by dehydro-
genase enzymes found in metabolically active cells. The quantity of formazan product
was measured in a Bio-Rad microplate reader Model 680 (Bio-Rad, Hercules, CA, USA)
at 490 nm, as absorbance is directly proportional to the number of viable cells
in culture.
4.7. Assessment of apoptosis through Hoechst 33258 staining
HCT116 cells grown as described above were plated at 7500 cells/mL and incubated
for 48 h in culture medium containing 1 or 0.1% (v/v) DMSO (vehicle control). Hoechst
staining (excitation and fluorescence emission at 352 and 461 nm, respectively) was
used to detect apoptotic nuclei as previously described [52b]. Briefly, medium was
removed, cells were washed with phosphate-buffered saline 1X (PBS) (Invitrogen), fixed
with 4% (v/v) paraformaldehyde in PBS 1X (10 min in the dark) and incubated with
Hoechst dye 33258 (Sigma, Missouri, USA; 5 mg/mL in PBS 1X) for another 10 min.
After washing with PBS 1X, cells were mounted using 20 mL of PBS:glicerol (3:1; v/v)
solution. Fluorescent nuclei were sorted per the chromatin condensation degree and
characteristics. Normal nuclei showed non-condensed chromatin uniformly distributed
over the entire nucleus. Apoptotic nuclei showed condensate or fragmented chroma-
tin. Some cells formed apoptotic bodies. Plates were photographed in an AXIO Scope
(Carl Zeiss, Oberkochen, Germany), and three random microscopic fields per sample
with ca. 50 nuclei were counted. Mean values were expressed as the percentage of
apoptotic nuclei [52b].
4.8. Autophagy
5
For autophagy analysis, 0.5 ꢄ 10 of HCT116 cells were seeded on top of a sterilized
cover slide and let to adhere for 24 h. The supernatant was substituted with fresh
medium containing 1 (at its IC ). For control purposes, one cover slide with cell
50
monolayer was treated with 0.1% (v/v) DMSO in fresh medium, and another cover

9
36
K. DAS ET AL.
slide was treated with fresh medium for 24 h, when rapamycin was added to a final
concentration of 50 mg/mL. After 48 h of incubation of cells with 1, DMSO (control for
1
) or Rapamycin (positive control), medium was removed and cells were stained
according to the instructions of CYTO-ID Autophagy detection kit (ENZO, NY, USA).
Stained cells were imaged using a fluorescence microscope (Carl Zeiss) and autophagy
V
R
measured using the CYTO-ID Green dye (excitation and fluorescence emission at 463
and 534 nm, respectively; DAPI was used to counterstain the nucleus (excitation and
fluorescence emission at 358 and 461 nm, respectively) and respective software (ZEN
Blue edition, 2011). The total number of cells and the number of cells with autophago-
lysosomes were counted in at least 5 different images of each sample to calculate the
%
of cells in autophagocytosis.
4
.9. DNA UV-titrations
The interaction mechanism and binding affinity of 1 with calf thymus DNA (CT-DNA)
Invitrogen Corp.) was studied by UV spectroscopy as previously described [54].
(
Compound 1 (25 mM) was incubated in the absence or presence of CT-DNA (15–50
ꢂ
mM) in Tris-HCl 5 mM, NaCl 50 mM buffer (pH 7.02) for 24 h at 37 C and UV spectra
were recorded from 230 to 600 nm. The dilution effect as the result of the addition of
the DNA solution was corrected and the affinity constants were calculated according
to Equation (1):
½
DNAꢅ
½DNAꢅ
eb ꢁ e
1
¼
þ
f
(1)
ea ꢁ e
Kbðeb ꢁ ef Þ
f
where [DNA] is the concentration of CT-DNA (per nucleotide phosphate), e ¼ Abs/
a
[
complex], e is the extinction coefficient for the free complex and e is the extinction
f
b
coefficient for the Co(II) complex when fully bound to DNA. CT-DNA concentration
expressed as molarity of phosphate groups) was determined in a NanoDrop2000
(
ꢁ
1
ꢁ1
spectrophotometer assuming e260 ¼ 6600 M cm [55].
4.10. Circular dichroism
Interaction of 1 with DNA was performed using calf thymus DNA (CT-DNA, Sigma-
Aldrich). Samples were prepared in 5 mM Tris-HCl pH 8.2 with 50 mM NaCl using a
range of concentrations of CT-DNA between 0 and 50 mM and a fixed concentration
of 1, 25 mM, or 1% DMSO as control. Circular dichroism (CD) measurements were per-
formed in triplicate in a Chirascan qCD from AppliedPhotophysics (Surrey, UK) using a
1
cm (1mL) quartz cuvette from 230 and 500 nm. Spectra were obtained using 5 mM
Tris-HCl pH 8.2 with 50 mM NaCl buffer as baseline. Each spectrum was collected after
averaging over at least 3 accumulations using a scan speed of 100 nm per min and a
1
s response time. The values of absorbance are measured in ellipticity, CD (mdeg).
4.11. Electrophoretic analysis of DNA-compounds interactions
Plasmids were obtained from E. coli transformed cells, grown overnight in an LB liquid
ꢁ
1
medium (Applichem, Darmstadt, Germany) with 100 lg mL
Ampicillin (Bioline,

JOURNAL OF COORDINATION CHEMISTRY
937
ꢂ
London, UK), at 37 C with stirring. Plasmid extractions were performed using the
InvisorbVR Spin Plasmid Mini Two Kit (Invitek, Berlin, Germany) and DNA quantified by
spectrophotometry with NanoDrop 2000 (Thermo Scientific, Massachusetts, USA). The
interactions between Co(II) complex and pUC18 (2686 bp) (Fermentas, USA) were
determined as previously described [37].
For the concentration-dependent studies, pUC18 (100 ng in a final volume of 20 mL)
was incubated in the presence (5–100 mM) or absence [vehicle DMSO; 1% (v/v)] of 1
ꢂ
for 24 h at 37 C in reaction buffer (5 mM Tris-HCl, 50 mM NaCl pH 7.02). Untreated
plasmid DNA was incubated in the same conditions in the absence of 1 or DMSO
(
referred to as pDNA in figure). After the incubation period, reactions were quenched
ꢂ
by keeping the samples at ꢁ20 C followed by addition of 4 lL of loading buffer
(
25 mM Tris-HCl, 25 mM EDTA (pH ¼ 8.0), 50% glycerol, 0.1% of bromophenol blue).
Samples were then loaded on 0.8% agarose gel (p/v) (Agarose SeaKemVR LE, Maine,
USA) dissolved in TAE buffer 1 ꢄ (4.84 g Tris-Base (Merck), EDTA (Riedel-de Ha e€ n) 0.5
M, 1.142 mL acetic acid (Panreac), pH 8.0). Electrophoresis was performed at 80 V con-
stant voltage for 2 h in 1 ꢄ TAE buffer. DNA was stained by immersing the agarose gel
ꢁ
1
in an ethidium bromide solution (0.5 mg L in distilled water) for 20 min; afterwards
the gel was washed in distilled water for 10 min and the results were analyzed and
photographed using a UVI TEC transilluminator (Cambridge, UK) coupled to a Kodak
Alpha-DigiDoc camera (Alpha Innotech, California, USA).
4.12. Reactive oxygen species (ROS)
For ROS quantification, A2780 cells were seeded in a cell culture slide with a density
4
of 7.5 ꢄ 10 cells/mL. After 24 h, the medium was replaced with fresh culture medium
supplemented with: i) the IC50 concentration of 1, ii) with 0.1% (v/v) DMSO, iii) with 50
mM H O , or with non-supplemented medium. After 48 h, the medium was removed,
2
2
cells were washed 3 times with phosphate buffer saline (PBS, 10 mM phosphate buffer
pH 7.4, 2.7 mM KCl and 137 mM NaCl), covered with pre-warmed solution of 10 mM
H DCF-DA in PBS (Invitrogen, ThermoFisher Scientific, Waltham, MA USA) and incu-
2
ꢂ
bated 20 min at 37 C, 5% (v/v) CO2 and 99% (v/v) relative humidity. Cells were
washed 3 times with PBS and fixed for 20 min with 4% (w/v) formaldehyde (Sigma-
Aldrich) at RT. After washing 3 times with PBS, cell images were obtained using a
fluorescence microscope (Carl Zeiss) and respective software (Zen Blue edition, 2011).
The relative fluorescence intensity was measured using FIJI software for each individ-
ual cell. The correct total cell fluorescence (CTCF) was calculated using the formula:
CTCF ¼ integrated density ꢁ (area of selected cell ꢄ mean fluorescence of back-
ground). Final fluorescence intensity values for treated cells were normalized with the
corresponding CTCF values of untreated cells.
4.13. Statistical analysis
All data were expressed as mean ± SEM from at least three independent experiments.
Statistical significance was evaluated using the Student’s t-test; p < 0.05 was consid-
ered statistically significant.

9
38
K. DAS ET AL.
Acknowledgments
AD would like to express their appreciation to the Ministry of Science and Technology, Taiwan
for financial assistance. The authors thank P. Martins for preliminary induction of DOX resistant
HCT116 cells. Our sincere thanks to Prof. Chen-Hsiung Hung for his valuable suggestion on
manuscript preparation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was also supported by the Unidade de Ci ^e ncias Biomoleculares Aplicadas-UCIBIO
which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by
the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).
References
[
1] I. Correa, A. Dornyi, T. Jakusch, F. Avecilla, T. Kiss, J.C. Pessoa. Eur. J. Inorg. Chem., 2819
2006).
(
[2] K. Das, C. Patra, C. Sen, A. Datta, C. Massera, E. Garribba, M.S. El Fallah, B.B. Beyene, C.-H.
Hung, C. Sinha, T. Askun, P. Celikboyun, D. Escudero, A. Frontera. J. Biol. Inorg. Chem., 22,
481 (2017).
[3] K. Das, A. Datta, S. Nandi, S.B. Mane, S. Mondal, C. Massera, C. Sinha, C.-H. Hung, T.
Askun, P. Celikboyun, Z. Cant u€ rk, E. Garribba, T. Akitsu. Inorg. Chem. Frontier, 2, 749
(2015).
[
[
4] N. Mondal, S. Mitra, V. Gramlich, S.O. Ghodsi, K.M.A. Malik. Polyhedron, 20, 135 (2001).
5] J.W. Whittaker, Metalloenzymes Involving Amino Acid Residue Related Radicals, H. Sigel, A.
Sigel (Eds.), vol. 30, p. 315, Marcel Dekker, New York (1994).
[6] P.F. Knowles, N. Ito, Perspectives in Bio-inorganic Chemistry, vol. 2, p. 207, Jai Press,
London (1994).
[7] J.W. Whittaker, M.M. Whittaker. Pure Appl. Chem., 70, 903 (1998).
[8] E.I. Solomon, U.M. Sundaram, T.E. Machonkin. Chem. Rev., 96, 2563 (1996).
[9] H. Higashimura, M. Kubota, A. Shiga, K. Fujisawa, Y. Morooka, H. Uyama, S. Kobayashi.
Macromolecules, 33, 1986 (2000).
[10] B.A. Jazdzewski, W.B. Tolman. Coord. Chem. Rev., 200–202, 633 (2000).
[11] O. Rotthaus, O. Jarjayes, C. Philouze, C. Perez Del Valle, F. Thomas. Dalton Trans., 1792
(2009).
[
[
12] Y. Shimazaki, T.D.P. Stack, T. Storr. Inorg. Chem., 48, 8383 (2009).
13] L.K. Thompson, S.K. Mandal, S.S. Tandon, J.N. Bridson, M.K. Park. Inorg. Chem., 35, 3117
(1996).
[
14] O. Wichmann, H. Sopo, E. Colacio, A.J. Mota, R. Sillanpa €a . Eur. J. Inorg. Chem., 4877 (2009).
[
15] L. Botana, J. Ruiz, J.M. Seco, A.J. Mota, A. Rodrıguez-Dieguez, R. Sillanpa €a , E. Colacio.
Dalton Trans., 40, 12462 (2011).
[16] (a) R. Kurtaran, L.T. Yildirim, A.D. Azaz, H. Namli, O. Atakol. J. Inorg. Biochem., 99, 1937
(
2005). (b) G.B. Bagihalli, S.A. Patil. J. Enzyme Inhib. Med. Chem., 25, 430 (2010). (c) S.
Chandra, S. Bargujar, R. Nirwal, K. Qanungo, S.K. Sharma. Spectrochim. Acta, Part A, 113,
64 (2013). (d) Z. Li, H. Yan, G. Chang, M. Hong, J. Dou, M. Niu. J. Photochem. Photobiol.
B, 163, 403 (2016).
1

JOURNAL OF COORDINATION CHEMISTRY
939
[
17] (a) C. Marzano, M. Pellei, F. Tisato, C. Santini. Anticancer Agents Med. Chem., 9, 185 (2009).
b) M.L. Teyssot, A.S. Jarrousse, M. Manin, A. Chevry, S. Roche, F. Norre, C. Beaudoin, L.
(
Morel, D. Boyer, R. Mahiou, A. Gautier. Dalton Trans., 35, 6894 (2009). (c) A. Blasina, J.
Hallin, E. Chen, M.E. Arango, E. Kraynov, J. Register, S. Grant, S. Ninkovic, P. Chen, T.
Nichols, P. O’Connor, K. Anderes. Mol. Cancer Ther., 7, 2394 (2008). (d) D.L. Wheeler, S.
Huang, T.J. Kruser, M.M. Nechrebecki, E.A. Armstrong, S. Benavente, V. Gondi, K.T. Hsu,
P.M. Harari. Oncogene, 27, 3944 (2008).
[18] (a) F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini. Med. Res. Rev., 30, 708 (2010).
(b) C. Marzano, M. Pellei, F. Tisato, C. Santini. Anticancer Agents Med. Chem., 9, 185 (2009).
[19] J. Reglinski, S. Morris, D.E. Stevenson. Polyhedron, 21, 2167 (2002).
[20] S.H. Rahaman, R. Ghosh, T.-H. Lu, B.K. Ghosh. Polyhedron, 24, 1525 (2005).
[
21] M. Dolaz, M. T u€ mer, M. Digrak. Transition Met. Chem., 29, 528 (2004).
[22] Z.-L. You, H.-L. Zhu. Z. Anorg. Allg. Chem., 630, 2754 (2004).
[23] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds,
Theory and Applications in Inorganic Chemistry, 5th edn, John Wiley and Sons, Inc., New
York (1997).
[
24] A. Golcu, M. T u€ mer, H. Demirelli, R.A. Wheatley. Inorg. Chim. Acta, 358, 1785 (2005).
[
25] M. T u€ mer. Synth. React. Inorg. Met. Org. Chem., 30, 1139 (2000).
[26] M. Dieng, I. Thiam, M. Gaye, A.S. Sall, A.H. Barry. Acta Chim. Slov., 53, 417 (2006).
[27] B. Chen, G. Qian, Metal-Organic Frameworks for Photonic Applications, Springer,
Heidelberg (2014).
[
[
28] X. Zhang, J. Chen, B. Davis, F. Kiechle. Arch. Pathol. Lab. Med., 123, 921 (1999).
29] M. Hajrezaie, M. Paydar, S.Z. Moghadamtousi, P. Hassandarvish, N.S. Gwaram, M.
Zahedifard, E. Rouhollahi, H. Karimian, C.Y. Looi, H.M. Ali, N.A. Majid, M.A. Abdulla. Sci.
World J., 463 (2014).
[
[
30] H.-Q. Chang, L. Jia, J. Xu, Z.-Q. Xu, R.-H. Chen, W.-N. Wua, H.-Y. Bie, T.-F. Zhu, T.-L. Mac, Y.
Wang, Inorg. Chem. Commun., 57, 8–10 (2015).
ꢁ
31] K. Ko nꢁ arikov ꢀa , G.A. Perdikaris, H. Gbelcova, L. Andrez ꢀa lov ꢀa , M. Sv ꢀe da, T. Ruml, L.
ꢁ
Laubertov ꢀa , L. Zit nꢁ anov ꢀa . Mol. Med. Rep., 14, 4436–4444 (2016).
[
[
[
[
32] M. Sui, W. Liu, Y. Shen. J. Control Release, 155, 227 (2011).
33] P.P. Utthra, N. Pravin, N. Raman. J. Photochem. Photobiol. B: Biol., 158, 136 (2016).
34] Y.-B. Wei, Y.-X. Li, H. Song, X.-J. Feng. J. Enzyme Inhib. Med. Chem., 5, 722 (2014).
35] (a) D.V. Lu ꢀı s, J. Silva, A.I. Tomaz, R.F.M. De Almeida, M. Larguinho, P.V. Baptista, L.M.D.R.S.
Martins, T.F.S. Silva, P.M. Borralho, C.M.P. Rodrigues, A.S. Rodrigues, A.J.L. Pombeiro, A.R.
Fernandes. J. Biol. Inorg. Chem., 19, 787 (2014). (b) J. Silva, A.S. Rodrigues, P.A. Videira, J.
Lasri, A.J. Charmier, A.J.L. Pombeiro, A.R. Fernandes. Inorg. Chim. Acta, 423, 156 (2014).
36] K. Thimmaiah, A.G. Ugarkar, E.F. Martis, M.S. Shaikh, E.C. Coutinho, M.C. Yergeri.
Nucleosides Nucleotides Nucleic Acids, 34, 309 (2015).
[
[
[
0
37] C.N. N soupkpoe Kossi, C. Descoteaux, E. Asselin, H.A.T. Riahi, G. Berube. Cell Biol., 27, 101
(
2008).
38] (a) M. Cory, D.D. McKee, J. Kagan, D.W. Henry, J.A. Miller. J. Am. Chem. Soc., 107, 2528
1985). (b) M.J. Waring. J. Mol. Biol., 13, 269 (1965).
(
[
[
[
[
39] V.I. Ivanov, L.E. Minchenkova, A.K. Schyolkina, A.I. Poletayer. Biopolymers, 12, 89 (1973).
40] W.A. Baase, W.C. Johnson. Nucleic Acids Res., 6, 797 (1979).
41] J. Portugal, J.A. Subirana. EMBO J., 4, 2403 (1985).
42] A.O. Lenis-Rojas, C. Roma-Rodrigues, A.R. Fernandes, F. Marques, D. P ꢀe rez-Fern ꢀa ndez, J.
Guerra-Varela, L. S ꢀa nchez, D. V ꢀa zquez-Garc ꢀı a, M. L oꢀ pez-Torres, A. Fern ꢀa ndez, J.J.
Fern ꢀa ndez. Inorg. Chem., 56, 7127 (2017).
[43] Z. Ma, B. Zhang, M.F. Guedes da Silva, J. Silva, A S. Mendo, P.V. Baptista, A.R. Fernandes,
A.J. Pombeiro. Dalton Trans., 45, 5339 (2016).
[
[
44] X. Feng, Q. Xia, L. Yuan, X. Yang, K. Wang. Neurotoxicology, 31, 391 (2010).
45] G. Khamisipour, F. Jadidi-Niaragh, A.S. Jahromi, K. Zandi, M. Hojjat-Farsangi. Tumour Biol.,
3
7, 10021 (2016).

9
40
K. DAS ET AL.
[
46] A. Datta, K. Das, S.B. Mane, S. Mendiratta, M.S. El Fallah, E. Garribba, A. Bauz ꢀa , A. Frontera,
C.-H. Hung, C. Sinha. RSC Adv., 6, 54856 (2016).
[
47] SADABS Bruker AXS; Madison, WI (2004); SAINT, Software Users Guide, Version 6.0; Bruker
Analytical X-ray Systems, Madison, WI (1999). G.M. Sheldrick, SADABS v2.03: Area-Detector
Absorption Correction, University of G o€ ttingen, G o€ ttingen, Germany (1999).
[
[
[
[
[
48] A. Altomare. J. Appl. Cryst., 32, 115 (1999).
49] G.M. Sheldrick. Acta Crystallogr., A, Found. Crystallogr., 64, 112 (2008).
50] L.J. Farrugia. J. Appl. Crystallogr., 45, 849 (2012).
51] P. van der Sluis, A.L. Spek. Acta Cryst. Sect A, 46, 194 (1990).
52] (a) A. Silva, D. Luis, S. Santos, J. Silva, A.S. Mendo, L. Coito, T.F. Silva, M.F. da Silva, L.M.
Martins, A.J. Pombeiro, P.M. Borralho, C.M. Rodrigues, M.G. Cabral, P.A. Videira, C.
Monteiro, A.F. Fernandes. Drug Metabol. Drug Interact., 28, 167 (2013). (b) O.A. Lenis-
Rojas, A.R. Fernandes, C. Roma-Rodrigues, P.V. Baptista, F. Marques, D. P eꢀ rez-Fern ꢀa ndez, J.
Guerra-Varela, L.E. Sanchez, D. Vazquez-Garcia, M. Lopez-Torres, A. Fern ꢀa ndez, J.J.
Fern ꢀa ndez S ꢀa nchez. Dalton Trans., 45, 19127 (2016).
[
53] (a) K.T. Mahmudov, M.F.C. Guedes da Silva, A. Mizar, M.N. Kopylovich, A.R. Fernandes, A.
Silva, A.J.L. Pombeiro. J. Organomet. Chem., 760, 67 (2014). (b) T.F.S. Silva, L.M.D.R.S.
Martins, M.F.C. Guedes da Silva, M.L. Kuznetsov, A.R. Fernandes, A. Silva, C-J. Pan, J.-F.
Lee, B.-J. Hwang, A.J.L. Pombeiro. Chem. Asian J., 9, 1132 (2014).
[
[
54] T.F.S. Silva, L.M.D.R.S. Martins, M.F.C. Guedes da Silva, A.R. Fernandes, A. Silva, P.M.
Borralho, S. Santos, C.M.P. Rodrigues, A.J.L. Pombeiro. Dalton Trans., 41, 12888 (2012).
55] C.D. Kanakis, P.A. Tarantilis, M.G. Polissiou, S. Diamantoglou, H.A. Tajmir-Riahi. Cell.
Biochem. Biophys., 49, 29 (2007).