Antiproliferative activity and apoptosis induction, of organo-antimony(III)–copper(I) conjugates, against human breast cancer cells

Article abstract of DOI:10.1007/s11030-019-10014-z

Abstract: Three known organo-antimony(III)–copper(I), mixed-metal small bioactive molecules (SBAMs) of formula [Cu(tpSb)₃Cl] (1), [Cu₂(tpSb)₄Br₂] (2) and [Cu₂(tpSb)₄I₂] (3) (tpSb = triphenylstibine) were used for the clarification of their antiproliferative activity against human breast cancer cells: MCF-7 (hormone-dependent cells) and MDA-MB-231 (hormone-independent cells). The in vitro toxicity of 1–3 was studied against normal human foetal lung fibroblast cells (MRC-5). The genotoxicity of 1–3 was determined by the presence of micronucleus. The type of the cell death caused by 1–3 was determined using cell cycle arrest. The molecular mechanism of action of 1–3 was defined by their binding affinity towards CT-DNA (calf thymus DNA) using UV spectroscopy and viscosity measurements. Docking studies depict the interactions between 1–3 and DNA. Computations were also employed in order to rationalize the activity of these compounds. This is based on the contribution of metal aromaticity in the case of compounds 2 and 3 where the short Cu···Cu distance (2.7724(6) (2) and 2.7251(11) (3) ?, respectively) suggests d¹⁰–d¹⁰ interaction between metal centres. Graphic abstract: The known small bioactive molecules of formula [Cu(tpSb)3Cl] (1), [Cu2(tpSb)4Br2] (2) and [Cu2(tpSb)4I2] (3) (tpSb = triphenylstibine) were used for the clarification of their antiproliferative activity against human breast cancer cells: MCF-7 (hormone-dependent (HD) cells) and MDA-MB-231 (hormone-independent (HI) cells).[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-10014-z

Molecular Diversity
https://doi.org/10.1007/s11030-019-10014-z
ORIGINAL ARTICLE
Antiproliferative activity and apoptosis induction,
of organo‑antimony(III)–copper(I) conjugates, against human breast
cancer cells
C. N. Banti1 · V. Tsiatouras1 · K. Karanicolas1 · N. Panagiotou2 · A. J. Tasiopoulos2 · N. Kourkoumelis3 ·
S. K. Hadjikakou1
Received: 25 September 2019 / Accepted: 2 November 2019
© Springer Nature Switzerland AG 2019
Abstract
Three known organo-antimony(III)–copper(I), mixed-metal small bioactive molecules (SBAMs) of formula [Cu(tpSb)3Cl]
(1), [Cu2(tpSb)4Br2] (2) and [Cu2(tpSb)4I2] (3) (tpSb=triphenylstibine) were used for the clarifcation of their antiproliferative
activity against human breast cancer cells: MCF-7 (hormone-dependent cells) and MDA-MB-231 (hormone-independent
cells). The in vitro toxicity of 1–3 was studied against normal human foetal lung fbroblast cells (MRC-5). The genotoxicity
of 1–3 was determined by the presence of micronucleus. The type of the cell death caused by 1–3 was determined using cell
cycle arrest. The molecular mechanism of action of 1–3 was defned by their binding afnity towards CT-DNA (calf thymus
DNA) using UV spectroscopy and viscosity measurements. Docking studies depict the interactions between 1–3 and DNA.
Computations were also employed in order to rationalize the activity of these compounds. This is based on the contribution
of metal aromaticity in the case of compounds 2 and 3 where the short Cu···Cu distance (2.7724(6) (2) and 2.7251(11) (3)
Ǻ, respectively) suggests d10–d10 interaction between metal centres.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-10014-z) contains
supplementary material, which is available to authorized users.
* C. N. Banti
cbanti@uoi.gr
* N. Kourkoumelis
nkourkou@uoi.gr
* S. K. Hadjikakou
shadjika@uoi.gr
1
Section of Inorganic and Analytical Chemistry, Department
of Chemistry, University of Ioannina, 45110 Ioannina,
Greece
2
Department of Chemistry, University of Cyprus,
1678 Nicosia, Cyprus
3
Medical Physics Laboratory, Medical School, University
of Ioannina, Ioannina, Greece
Vol.:(0123456789)
1 3

Molecular Diversity
Graphic abstract
The known small bioactive molecules of formula [Cu(tpSb)3Cl] (1), [Cu2(tpSb)4Br2] (2) and [Cu2(tpSb)4I2] (3) (tpSb=tri-
phenylstibine) were used for the clarifcation of their antiproliferative activity against human breast cancer cells: MCF-7
(hormone-dependent (HD) cells) and MDA-MB-231 (hormone-independent (HI) cells).
Keywords Metal biology · Metallotherapeutics · Copper(I)–antimony(III) complexes · Cytotoxic activity · Breast cancer
antiproliferative properties and have been tested in both nor-
mal and cancerous cells with promising results [8].
Introduction
In the course of our studies in the development
of novel metallodrugs [2, 9–17], the known organo-
antimony(III)–copper(I) mixed-metal small bioac-
tive molecules (SBAMs) of formula [Cu(tpSb)3Cl] (1),
[Cu2(tpSb)4Br2] (2) and [Cu2(tpSb)4I2] (3) (tpSb=triphenyl-
stibine, Scheme 1) were tested against MCF-7 (hormone-
dependent (HD)) and MDA-MB231 (hormone-independ-
ent (HI)) cells. Their toxicity of 1–3 was evaluated against
MRC-5. Further, their genotoxicity was determined by the
presence of micronucleus. The type of the cell death caused
by metallodrugs was determined using cell cycle arrest
assay. The molecular mechanism of their action was evalu-
ated towards CT-DNA (calf thymus DNA) using UV spec-
troscopy and viscosity measurements.
The most frequent malignancy in women worldwide is
breast cancer [1]. The molecular features of breast can-
cer include activation of human epidermal growth factor
receptor 2, activation of hormone receptors (oestrogen
receptor and progesterone receptor) and/or mutations
in the tumour suppressor genes [1, 2]. Most breast can-
cers are oestrogen receptors (ER) positive, which have a
relatively good prognosis [3]. However, limited targeted
therapies are available for the most aggressive ER-negative
breast cancers, “triple negative” breast cancers (TNBC)
[3]. Human breast cancer MCF-7 and MDA-MB-231
cells are both breast carcinoma cells, which have, how-
ever, many phenotypic/genotypic diferences: MCF7 are
hormone-dependent (both positive to oestrogen (ER) and
progesterone (PR) receptors) [4], while MDA-MB-231
are triple negative that lack oestrogen and progesterone
receptors and human epidermal growth factor receptor 2
(HER2) [5].
Results and discussion
General aspects Although 1–3 are known, they were
obtained here by an alternative method which is described
briefy. CuX (Cl(1), Br(2) and I(3)) react with Ph3Sb in 1:3
(1) and 1:2 (2 and 3) molar ratios in methanol/acetonitrile
solution under refux (Reaction 1). Pure 1–3 in crystalline
form were obtained by slow evaporation of the methanol/
acetonitrile solution which remains after the fltration of the
Copper(II) compounds, on the other hand, have been
intensively studied; however, the corresponding ones of
Cu(I) have been scarcely tested as antimicrobial, antiviral,
antifungal and anticancer drugs due to their signifcant lower
stability in water media [6]. Moreover, copper(I) complexes
are expected to be less toxic to normal cells in contrast to
tumour ones, due to the physiological properties of copper
as endogenous metal [7]. Antimony(III) compounds exhibit
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Molecular Diversity
b = 15.5253(5), c = 21.8157(8) Å, α = 77.178(3),
β = 84.177(3), γ = 81.118(3)°, on the other hand, varied
from the corresponding one of COWBOF; space group P-1,
a=13.827(3), b=14.279(3), c=14.399(3) Å, α=84.43(2),
β=87.39(2), γ=75.18(1)°, R=0.00636 [21] due to the sol-
vent molecule which is co-crystallized in this case. Thus,
although the molecular formula of 1 has been reported by
Reichle, previously [22], its crystal structure is a polymor-
phic one to COWBOF reported by Rheingold et.al [21].
Therefore, the preparation and crystallization media used
in case of COWBOF (hexane/dichloromethane), towards
methanol/acetonitrile used in 1, sets the crystallization sys-
tem. This is why the crystal structure determination of 1 was
Scheme 1 Molecular formula of tpSb
reaction solution. Air-stable crystals of 1–3 were analysed
by X-ray difraction crystallography.
Reaction 1. Preparation routes of 1– 3
refned and reported here again. Moreover, since no biologi-
cal data are available for these compounds (1–3) up to now,
their antiproliferative activities were evaluated.
Solid‑state studies
Crystal structure of [Cu(tpSb)3Cl] (1), [Cu2(tpSb)4Br2] (2)
and [Cu2(tpSb)4I2] (3) The molecular and crystal struc-
tures of 2–3 are known. The unit cell of 2 is: space group
P 21/c, a = 24.1564(8), b = 14.0156(4), c = 19.8556(7) Å,
β = 110.116(4)°, while the corresponding of the known
NUHQOW is: P21/c, a = 24.336(7), b = 14.214(4),
c = 20.097(7) Å, β = 109.91(2)° [18]. The unit cell of
3: space group P 21/c, a = 24.4375(4), b = 13.9106(2),
c = 20.2194(3) Å, β = 111.169(2)°; NUHQUC: P21/c,
a=24.620(9), b=14.090(4), c=20.382(9) Å, β=110.65(3)°
[18], NUHQUC01: space group P21/a, a = 20.436(5),
b = 14.125(3), c = 24.683(3) Å, β = 110.67(1)° [19],
NUHQUC02: space group P21/c, a = 24.4463(6),
b = 13.9088(3), c = 20.2168(5) Å, β = 111.241(3)° [20].
The crystal structure of 1; space group P-1, a=14.1646(5),
Molecular diagrams of 1–3 are shown in Fig. 1.
Vibrational spectroscopy The vibration bands at
3066 cm−1 in FT-IR spectra of 1–3 (Figures S1-S2) are
attributed to the C-H bond of the aromatic ring of tpSb.
The corresponding one for free ligand tpSb is observed at
3062 cm−1. The vibration band at 448 cm−1 in the FT-IR
spectrum of tpSb which is attributed to the νsym(C–Sb) is
shifted at 453 (1), 461 (2) and 461 (3) cm−1, suggesting
coordination of the ligand in the metal centre [23].
Solution studies
Stability studies The retention of the structures of 1–3 in
solution was checked by 1H-NMR spectroscopy, in dmso-
d6 solution (Figures S3–S5). No changes were observed
1 3

Molecular Diversity
between the initial spectrum and the corresponding after
48 h.
the hormone-dependent MCF-7 ones is observed. Moreo-
ver, the IC50 of cisplatin, a commercial drug of clinical
used, is 5.5 0.4 μΜ, and against MDA-MB 231 cells, it is
26.7 1.1 μΜ. Therefore, 1–3 show two- to fourfold higher
activity against MBA-MB 231 cells than cisplatin.
1H NMR studies The multiple resonance signals at
7.39–7.30 ppm are due to the Haromatic of free tpSb, which
are shifted at 7.43–7.36 (1), 7.43–7.36 (2) and 7.42–7.36 (3)
ppm, respectively, in the case of the spectra of 1–3 (Figure
S6).
The homoleptic compounds 1–3, on the other hand,
exhibit stronger activity than the corresponding heteroleptic
ones of formulae [CuX(μ2-S)-tzdtH)(tpSb)]2 (X=Cl, Br and
I; tzdtH=2-mercapto-thiazolidine) (Table 1). This activity is
four times higher against MDA-MB 231 and two times when
MCF-7 cells are used [23]. Therefore, the substitution of one
ligand in the coordination sphere of Cu(I) by a tzdtH ligand
weakens their biological activity [23]. Thus, compounds 1–3
not only exhibit selectivity against the breast cancer cells
without hormone receptors (MDA-MB 231), which is higher
even than that of cisplatin as well, but also exhibit stronger
activity than the corresponding one of the heteroleptic Cu(I)
complexes of tpSb [23].
Nucleus-Independent Chemical Shifts (NICS) studies The
cuprophilicity which is observed in 2 and 3 by unexpected
short Cu···Cu distances (2.7724(6) (2) and 2.7251(11) (3) Ǻ,
respectively) prompts us to apply NICS [24] for the inves-
tigating these Cu–Cu bonding interactions by magnetic cri-
teria. The electron density delocalization of the molecular
orbitals involved in the Cu2Br2 and Cu2I2 cores can possibly
provide increased stability to the overall molecular struc-
tures. Clusters with transition‐metal can exhibit d‐orbital
aromaticity or δ‐aromaticity due to δ bonding interactions.
Yet, for d‐orbital aromaticity to occur signifcant d–d bond-
ing interactions are essential. The tendency of d orbitals to
participate in chemical bonding depends strongly on the
coordination environment due to their spatial arrangement
[25]. In this work, the four-membered aromatic clusters
are stabilized by two bridging Br or I atoms adjacent to Cu
atoms exhibiting Cs symmetry but with the I analog organ-
ized in a wider area with increased Cu–I bond lengths. The
distance d(Cu–I) is approximately 0.2 Å larger than the cor-
responding d(Cu–Br). NICS indices are the negative iso-
tropic values of the absolute NMR shielding at distances
ranging from 2.0 to + 2.0 Å along the vertical C2 axis
(Fig. 2, white spheres) of the clusters. NICS(0) values near
− 8 ppm, indicate aromaticity since, at the same level of
theory, the corresponding value for benzene is −8.5 ppm,
while NICS(0) values close to zero represent non-aromatic-
ity. NICS(0) (i.e., at 0 Å) of the Br analog was calculated to
−2.4 ppm, while for the I analog it was −6.6 ppm. NICS(1)
values are lower by 4.2 ppm for complex 3 and practically
zero at NICS(2). The results suggest the presence of induced
diatropic ring current at the molecular plane of Cu2I2 and
not at Cu2Br2, confrming that metal aromaticity is strongly
afected by the metal coordination and halogen type.
Antiproliferative studies The metallodrugs 1–3 were
tested for their in vitro antiproliferative activity against
human breast adenocarcinoma cell lines: MCF-7 (hormone-
dependent cells), MDA-MB-231 (hormone-independent
cells) and the non-cancerous cell line, MRC-5 (normal
human foetal lung fibroblast cells), by the sulforhoda-
mine B (SRB) assay [23] (Table 1). The cells were incu-
bated for 48 h with the metallodrugs. The IC50 values of
1–3 towards MCF-7 cells are 11.1 0.4 (1), 9.2 0.5 (2)
and 18.4 1.4 (3) μΜ, respectively, while against MDA-
MB 231 cells are 7.3 0.3 (1), 6.4 0.2 (2) and 11.2 1.1
(3) μΜ, respectively. Therefore, a selectivity of 1–3 towards
the hormone-independent MBA-MB 231 cancer cells than
The in vitro toxicity of 1–3 was tested against the non-
cancerous cell line, MRC-5 (normal human foetal lung
fbroblast cells) (Table 1). The IC50 values against MRC-5
cells are 9.9 0.5 (1), 5.1 0.1 (2) and 8.1 0.2 (3) μΜ,
respectively. The therapeutic potency index (TPI) which is
defned as the IC50 value against normal cells towards the
IC50 against cancerous ones is 1.4 (1), 0.8 (2) and 0.7 (3),
respectively, in case of MDA-MB 231 and 0.9 (1), 0.6 (2)
and 0.4 (3), respectively, against MCF-7 cells (Table 1) [9].
The corresponding TPI values of cisplatin against MDA-
MB-231 and MCF-7 cells are 0.8 and 0.9, respectively.
Therefore, the efectiveness of 1–3 is better than correspond-
ing one of cisplatin.
The micronucleus (MN) assay was employed for the
evaluation of the in vitro genotoxicity of 1–3. The MRC-5
cells were incubated for 48 h, with 1–3 at their IC50 val-
ues, and the micronucleus frequencies were checked (Fig-
ure S7). The micronucleus frequency (%) in the case of the
non-treated cells is 1.0 0.2. This remains unchanged when
MRC-5 cells were treated with 1–3, 1.0 0.1 (1), 1.3 0.4
(2) and 1.2 0.2 (3) %, respectively. When MRC-5 cells
are incubated with cisplatin, the MN frequency is 1.6% [9].
Therefore, 1–3 are causing less genetic damage against the
normal MRC-5 than cisplatin.
Cell cycle arrest One of the main characteristics of apop-
tosis is the internucleosomal DNA fragmentation, which can
be identifed by the presence of sub-G1 peak on DNA con-
tent histograms [26]. In order to evaluate the efect of the
cell cycle arrest of MCF-7 cells, upon their treatment with
1–3 at IC50 values, fow cytometric analysis was performed.
The percentage of cells in various phases of the cell cycle
was analysed and it is presented as the number of cells ver-
sus DNA content in diferent phases of cell cycle (sub-G1,
G0/G1, S and G2/M) (Fig. 3).
1 3

Molecular Diversity
Fig. 1 Molecular diagrams
together with the number-
ing scheme of 1 (A), 2
(B) and 3 (C) compounds.
Selected bond distances
(Å) are: 1_MOLECULE-
A: Cu1-Cl1=2.2367(15),
1_MOLECULE-B:
Cu2-Cl2=2.2699(15);
2: Cu1-Br1=2.5079(7),
Cu2-Br1=2.4703(6),
Cu1-Br2=2.4507(6),
Cu2-Br2=2.4989(7),
Cu1-Cu2=2.7724(6);
3: Cu1-I1=2.6502(9),
Cu2-I1=2.6188(9),
Cu1-I2=2.6338(7), Cu2-
I2=2.6424(9)
1 3

Molecular Diversity
phase, presumably allowing cells to repair DNA lesions
prior to mitosis [28]. The percentage of cells is also increas-
ing in the S phase (21.2%) towards to the untreated cells
(15.3%) in the case of 3. Thus, 3 accumulates the cells in
G2/M and S phases simultaneously; as a result, it suppresses
the cell proliferation by inhibiting DNA synthesis.
The cisplatin causes cell cycle arrest at S and G2/M
phases and the percentage of MCF-7 cells in sub-G1 phase is
increased, exhibiting an increasing number of apoptotic cells
[9, 29]. Overall, these metallodrugs reduce the cell growth
by induction of apoptotic type of cell death, in a similar
manner to cisplatin.
DNA binding studies Metallodrugs bind either covalently
or non-covalently to DNA. Non-covalent modes by which
metal complexes bind to DNA include among others inter-
calation, groove binding, intercalation between the stacked
base pairs of native DNA, etc [9]. However, the type of the
particular specifc molecular interactions of copper com-
pounds with DNA is still not clearly established [30, 31]. In
order to elucidate how copper compounds 1–3 interact with
DNA, their binding studies were performed by UV absorp-
tion spectroscopy and viscosity.
Fig. 2 Superimposed structures with the Cu2Br2 and Cu2I2 cores and
the vertical axis along which NICS values have been calculated by
placing “ghost” atoms
The untreated cells are spread by 7.39% in sub-G1 phase,
69.7% in G0/G1, 15.3% in S and 6.11% in G2/M phases
(Fig. 3). However, upon incubation of MCF-7 cells with
the 1–3, the percentage of the cells in the sub-G1 phase is
increased at 25.4% (1), 14.4% (2) and 15.3% (3), respec-
tively, in respect of the untreated cells which are in sub-G1
phase (7.39%), indicating the apoptotic type of the death of
MCF-7 cells.
(a) UV absorption spectroscopy In order to confrm the
apoptotic mechanism of cells death which has been sug-
gested by cell cycle arrest, the interaction of 1–3 interac-
tion with CT-DNA was examined by UV–visible absorp-
tion spectroscopy. The confguration of the double-helix
structure of DNA due to interaction with metallodrugs can
be assigned either by hypochromism or by hyperchromism
[9, 14]. Thus, hypochromism is attributed to intercalated
or electrostatic binding mode, while hyperchromism is
assigned to the breakage of hydrogen bonds which stabi-
lized the secondary structure of DNA [9]. Upon the increas-
ing of r values (r=[complex]/[DNA], [DNA]=10−4 M) of
1–3, a hyperchromism is observed at λmax =258 nm for all
complexes (Figure S8). The hyperchromism determined for
The percentage of cells in G2/M phase is increasing in the
metallodrugs 1–3 (19.0% (1), 15.7% (2) and 10.7% (3)), in
contrast to 6.11% for the untreated cells. Metallodrugs 1–3
accumulate cancer cells at the G2/M phase of their cycle by
delaying or inhibiting cell cycle progression at the G2/M
phase [27]. The damaged cells stop DNA replication at G2
Table 1 Geometric parameters and IC50 values of the complexes against two adenocarcinoma breast cell lines MCF-7 (hormone-dependent),
MDA-MB 231 (hormone-independent) and one normal human foetal lung fbroblast cells (MRC-5 cells)
Metallodrugs
Volumes IC50 values (μΜ)
TPI
Kb (×104) (M−1
)
References
MCF-7
MDA-MB-231 MRC-5
MCF-7 MDA-MB-231
1
2
3
1131.6
1564.8
1588.9
11.1 0.4
9.2 0.5
18.4 1.4 11.2 1.1
22.6 0.6 29.8 0.9
24.2 0.5 24.9 1.0
21.5 0.7 26.1 1.8
5.5 0.4 26.7 1.1
7.3 0.3
6.4 0.2
9.9 0.5 0.9
1.4
0.8
0.7
0.6
0.8
0.2
0.04
14.4 1.1
14.2 1.7
12.0 3.9
*
*
[23]
[23]
[23]
[23]
[23]
5.1 0.1 0.6
8.1 0.2 0.4
17.4 0.7 0.8
19.3 0.6 0.8
5.9 0.3 0.3
1.1 0.2 0.2
[CuCl(μ2-S)-tzdtH)(tpSb)]2 1074.4
[CuBr(μ2-S)-tzdtH)(tpSb)]2 1093.7
[CuI(μ2-S)-tzdtH)(tpSb)]2
1107.3
–
Cisplatin
*This work
tzdtH, 2-mercapto-thiazolidine
1 3

Molecular Diversity
Fig. 3 Efects on cell cycle arrest against MCF-7 cells; Untreated cells (a), 1 (b), 2 (c) and 3 (d). The relative number of cells within each cell
cycle was determined by fow cytometry. Number of cells in sub-G1, G0/G1, S and G2/M phase are indicated
1–3 is (17.1 8.8) % (1), (13.7 1.6) % (2) and (16.6 1.9)
% (3), respectively. Therefore, a breakage of the hydrogen
bonds which stabilized the secondary structure of DNA is
suggested [9].
1 > 2 > 3. The corresponding Kb values for silver(I) com-
plexes with tri-aryl-pnictogens (Ar3E, E = P, As, Sb and
Ar = Ph-, p-tolyl-, m-tolyl, o-tolyl) of the general formula
[Ag(D)(Ar3E)n] (D=salicylic acid, aspirin, diclofenac, nap-
roxen, nimesulide, etc) lie from 5.3×104 to 68.2×104 M−1
for tri-aryl phosphines from 4.0×104 to 25.0×104 M−1 for
triphenylarsine and 8.9 2.0 × 104 M−1 for triphenylanti-
mony complex [32]. Therefore, the binding afnity of 1–3
towards DNA is within the range of those already found for
silver(I) compounds.
The binding constants (Kb) of 1–3 towards CT-DNA
were determined by monitoring the absorbance changes
at 300–310 nm of spectra of 1–3 (25 μΜ) with increas-
ing concentration of CT-DNA ([CT-DNA = 10–100 μΜ)
(Figure S9). The Kb values have been obtained from
Wolfe–Shimer equation [9, 10, 13–15] and they are cal-
culated (14.4 1.1) × 104 (1), (14.2 1.7) × 104 (2) and
(12.0 3.9)×104 M−1 (3), respectively. Thus, the strength
order of the binding affinity of 1–3 towards DNA is:
(b) Viscosity measurements DNA length changes upon its
incubation with an anticancer agent, and the viscosity of its
solution is strongly afected. Thus, (i) if an agent intercalates
1 3

Molecular Diversity
Fig. 4 Efect of increasing
concentrations of 1–3 on the
relative viscosity of CT-DNA
([DNA]=10 mM, r=[com-
pound]/[DNA], n is the viscos-
ity of DNA in the presence of
1–3 and nO is the viscosity of
DNA alone)
1.05
1
3
1
0.95
0.9
0.85
0.8
2
0.75
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
[Complex]/[DNA]
with the DNA strands, this results in DNA lengthening and
the viscosity increase; (ii) if an agent interacts electrostati-
cally with the DNA, no efect on DNA length is caused,
and therefore, no signifcant change in viscosity is observed;
(iii) in case that DNA strands are cleavage by an agent, the
length of the DNA decreases and the viscosity decreases
also signifcantly; and (iv) bending of the DNA helix caused
by the agent reduces the viscosity [11]. Therefore, viscosity
exhibits high sensitivity to the changes on DNA and it is
used for the study of the binding modes of an agent towards
DNA [11]. The relative DNA solutions viscosity (η/η0) is
correlated with the DNA length (L/L0) by Eq. 1 [11, 33, 34]:
L∕L0 = (ꢀ∕ꢀ0)1∕3
(1)
The solution of CT-DNA (10 mM) is incubated with
an increasing amounts of 1–3 that the [compound]/[DNA]
molar ratio reaches the r= 0.32. Figure 4 shows the rela-
tive specifc viscosity (η/η0)1/3 versus binding ratio. The
trend of lowering in the viscosity of DNA, upon increasing
Fig. 5 Docking poses of 1 (a) and 2 (b) at major groove of B-DNA. The corresponding CG region is marked as charged surface
1 3

Molecular Diversity
concentrations of 1–3, suggests either cleavage of the DNA
strands or bending of the DNA helix [11]. This is in accord-
ance with the breakage of hydrogen bonds that stabilize the
secondary structure of DNA, which is concluded by UV
measurements (see above).
women with TNBC are only managed with standard chemo-
therapy, such as platinum-based compounds [37]. Therefore,
1–3 might be candidates for the development new targeted
chemotherapeutics against TNBC. However, upon substitu-
tion of a tpSb ligand from the coordination sphere of the
homoleptic Cu(I)-Sb(II), 1–3 complexes by a tzdtH one
weaken the biological activity of the compounds. The in
vitro genotoxicity assay shows that the MN frequency of
1–3 is less than cisplatin, suggesting that the compounds
can cause less genetic damage. Moreover, the metallodrugs
reduce growth of MCF-7 cells through apoptosis. This is due
to their DNA binding afnity. UV, viscosity measurement
and computational data suggest breakage of DNA hydrogen
bonds. Although electronegative AT bases sequence are nar-
rower and ofer better van der Waals interaction space com-
pared to GC bases, the bulk dimensions of the complexes
favour GC binding (Table 1) [14].
Docking studies Docking studies attempted to depict the
interactions between the synthesized metal complexes and
DNA as the type of binding and interaction plays a major
role in drug design and action [35]. Figure 5 shows the
lowest energy binding site of complexes 1 and 2 with the
DNA sequence d(ACCGACGTCGGT)2. Complexes 2 and
3 adopt similar confguration due to their similar structure.
All complexes were loosely stabilized at the major groove
so that the abundant phenyl rings make favourable π–π and
T-shaped electrostatic interactions with C9, C14, C15, G7
and G16. The lowest binding energy was calculated to -6,
-5.8 and -5.6 kcal/mol for complexes 1, 2 and 3, respectively.
Therefore, the afnity of the binding interaction between
1–3 and DNA is: 1 > 2 > 3 which follows the correspond-
ing one which is determined from Kb. (The binding afn-
ity of 1–3 towards DNA order is: 1>2>3; see above.) An
important aspect of binding is van der Waals interactions
and hydrophobic contacts. Although electronegative AT
bases sequence are narrower and ofer better van der Waals
interaction space compared to GC bases, here we found that
the bulk dimensions of the complexes favour GC binding.
Santini et.al [36] have shown that copper–DNA binding is
dependent on copper complex size, electron afnity and
geometry of the formed adduct, inducing an irreversible
modifcation of the DNA conformational structure. This
rationalize the diferentiation observed of 1–3 binding site
towards DNA with respect to the corresponding derived by
energy criterion.
Experimental
Materials and instruments All solvents used were of rea-
gent grade; triphenyl antimony (Sigma-Aldrich, Merck)
was used without further purifcation. Dulbecco’s modifed
Eagle’s medium (DMEM), foetal bovine serum, glutamine
and trypsin were purchased from Gibco, Glasgow, UK.
Phosphate bufer saline (PBS) and CT-DNA and propidium
iodide were purchased from Sigma-Aldrich. Dimethyl sul-
foxide and boric acid were purchased from Riedel-de Haen.
Melting points were measured in open tubes with a Stuart
Scientifc apparatus and are uncorrected. IR spectra in the
region of 4000–370 cm−1 were obtained from KBr discs,
with a PerkinElmer Spectrum GX FT-IR spectrophotom-
eter. The 1H NMR spectra were recorded on a Bruker AC
400 MHz FT-NMR instrument in DMSO-d6 solution. A
UV-1600 PC series spectrophotometer of VWR was used
to obtain electronic absorption spectra. FACSCalibur fow
cytometer (Becton Dickinson, San Jose, CA, USA) was
obtained for the cell cycle. MDA-MB-231, MCF-7 and
MRC-5 cells were purchased from the American Type Cul-
ture Collection (ATCC, Rockville, MD, USA) and the Impe-
rial Cancer Research Fund (ICRF), London.
Thus, compounds 1–3 initially bind in DNA with conse-
quent cleavage of the hydrogen bonds, which are stabilizing
its secondary structure [14]. This leads to the decrease of
DNA length, resulting in lowering of its viscosity value [14].
Conclusions
The known small bioactive molecules of formula
[Cu(tpSb)3Cl] (1), [Cu2(tpSb)4Br2] (2) and [Cu2(tpSb)4I2]
(3) were tested against MCF-7 (hormone-dependent (HD))
cells and MDA-MB-231 (hormone-independent (HI)) cells.
A selectivity of 1–3 towards the hormone-independent
MBA-MB 231 cancer cells than the hormone-dependent
MCF-7 ones is observed. Compounds 1–3 are more active
than cisplatin against MDA-MB 231 cells. It is noteworthy
to mention here that MBA-MB 231 is triple negative breast
cancer cells (TNBC). TNBC are very aggressive cancers that
lack ER, PR and HER2 receptors and they do not respond
to targeted treatment agents. Chemotherapy options for
Synthesis and crystallization of 1–3 complexes 1–3 were
synthesized by reacting 1.5 mmol Ph3Sb (0.352 g) with
0.5 mmol of CuCl (0.049 g) (1) or 0.75 mmol of CuBr
(0.071 g) (2) and CuI (0.095 g) (3), in 20 mL of methanol/
acetonitrile solution under refux for 3 h. A clear solution
was fnally formed. The solution was fltered of, and the
clear solution was kept in the darkness at room temperature.
Pale yellow crystals of 1–3 suitable for X-ray analysis were
grown from the fltrate after few days.
1: Melting point: 145–150°C; Elemental analysis found:
C: 56.30; H: 4.01%; calculated for C54H45ClCuSb3: C:
1 3

Molecular Diversity
56.00; H: 3.91%. IR (cm−1) (KBr): 3043 m, 1576 m, 1477 s,
1428 vs, 1304 m, 1182 m, 1156 m, 1065 vs, 1019 s, 998 s,
916 m, 852 m, 725 vs, 691 vs, 446 vs; 1H-NMR (ppm) in
DMSO-d6: 7.42–7.37 ppm (m, tpSb).
Z=4, T=105 K, ρ(calc)=1.858 g cm−3, μ=21.771 mm−1,
F(000)=3440. 23921 refections measured, 11426 unique
(Rint = 0.038), 9600 with I > 2σ(I). The fnal R1 = 0.0335
(for 9600 refections with I> 2σ(I)) and wR2(F2) = 0.0819
(all data), S=0.99.
2: Melting point: 181–183 °C; Elemental analysis found:
C: 51.05; H: 3.41%; calculated for C72H60Br2Cu2Sb4: C:
50.90; H: 3.56%. IR (cm−1) (KBr): 3043 m, 1576 m, 1478 s,
1429 vs, 1303 m, 1180 m, 1156 m, 1067 vs, 1019 s, 995 s,
911 m, 852 m, 726 vs, 690 vs, 450 vs; 1H-NMR (ppm) in
DMSO-d6: 7.41–7.38 ppm (m, tpSb).
Supplementary data are available from CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (e-mail: mailto:deposit@
ccdc.cam.ac.uk), on request, quoting the deposition num-
bers CCDC-1917278 (1), CCDC-1917279 (2) and CCDC-
1917280 (3).
3: Melting point: 171–174 °C; Elemental analysis found:
C: 48.50; H: 3.22%; calculated for C72H60Cu2I2Sb4: C:
48.23; H: 3.37%. IR (cm−1) (KBr): 3045 m, 1570 m, 1477 s,
1429 vs, 1301 m, 1182 m, 1156 m, 1065 vs, 1021 s, 995 s,
911 m, 850 m, 726 vs, 691 vs, 446 vs; 1H-NMR (ppm) in
DMSO-d6: 7.40–7.36 ppm (m, tpSb).
Biological tests
Solvents used: Stock solutions of 1–3 (0.01 M) in DMSO
were freshly prepared and diluted with cell culture medium
to the desired concentration. The biological experiments
including the SRB assay, cell cycle and micronucleus assay
were carried out in DMSO/DMEM solutions 0.005–0.3%
v/v DMSO in DMEM for 1–3. For DNA binding studies,
the experiments were carried out in DMSO/bufer solutions
(0.00025–0.005% v/v DMSO).
X-ray structure determination Single-crystal X-ray
difraction data for 1–3 were collected on an Oxford Dif-
fraction Supernova difractometer, equipped with a CCD
area detector utilizing Cu Kα (λ = (1.5418 Å) radiation.
A suitable crystal was mounted on a Hampton cryoloop
with Paratone-N oil and transferred to a goniostat where it
was cooled for data collection. Empirical absorption cor-
rections (multiscan based on symmetry-related measure-
ments) were applied using CrysAlis RED software [38].
The structures were solved by direct methods using SIR2004
[39] and refned on F2 using full-matrix least squares with
SHELXL-2014/7 [40]. Software packages used were as fol-
lows: CrysAlis CCD for data collection [38], CrysAlis RED
for cell refnement and data reduction [38] and WINGX for
geometric calculations [41]. The non-H atoms were treated
anisotropically, whereas the aromatic H atoms were placed
in calculated, ideal positions and refned as riding on their
respective carbon atoms.
SRB assay, Micronucleus, Cell cycle and DNA binding
studies were performed in accordance with the previous
reported methods [9, 11, 14, 42].
Computational methods Calculations were carried out
with the Gaussian03 W program package [43] at RHF/3-
21G* level of theory, based on the molecular geometry
acquired via X-ray difraction methods. All structures were
fully optimized without constraints but to the bond lengths
of the central 4-membered rings of complexes 2 and 3. Simi-
larly, magnetic shielding tensors for ghost atoms placed at
diferent positions along the C2 axis of the Cu2X2 (X: halo-
gen) cluster were computed using the gauge-independent
atomic orbital (GIAO) method.
1: C54H45ClCuSb3, MW = 1158.14, monoclinic, space
group P-1, a = 14.1646(5), b = 15.5253(5), c = 21.8157(8)
Å, α = 77.178(3), β = 84.177(3), γ = 81.118(3)°,
V=4610.5(3) Å3, Z=4, T=101 K, ρ(calc)=1.669 g cm−3,
μ=15.104 mm−1, F(000)=2272. 30933 refections meas-
ured, 16414 unique (Rint=0.038), 13045 with I>2σ(I). The
fnal R1=0.0368 (for 13045 refections with I>2σ(I)) and
wR2(F2)=0.0961 (all data), S=0.97.
For the DNA docking studies, we used the sequence
5′-D(*AP*CP*CP*GP*AP*CP*GP*TP*CP*GP*GP*T)-3′
based on the 423D structure of non-complexed B-DNA
found in the PDB database [44]. Molecular docking simula-
tions were performed with Autodock Vina [45] using the
default parameters. The search space covered the whole
structure of the receptor and exhaustiveness was set to 12.
Acknowledgements This work was carried out for the fulflment of
the requirements for the B.Sc. thesis of Mr. K.K. according to the
curriculum of the Department of Biological Applications and Tech-
nology of the University of Ioannina under the supervision of SKH.
CNB and SKH would like to thank the Unit of Bioactivity Testing of
Xenobiotics of the University of Ioannina for providing access to the
facilities. CNB and SKH would like to thank the Atherothrombosis
Research Centre of the University of Ioannina for providing access to
the fow cytometer and to the fuorescence microscopy. This research
has been co-fnanced by the European Union and Greek national funds
through the Operational Program Competitiveness, Entrepreneurship
and Innovation, under the call RESEARCH – CREATE – INNOVATE
(Project Code: T1EDK-02990).
2: C72H60Br2Cu2Sb4, MW=1699.10, monoclinic, space
group P21/c, a=24.1564(8), b=14.0156(4), c=19.8556(7)
Å, α = 90, β = 110.116(4), γ = 90°, V = 6312.4(4) Å3,
Z=4, T=106 K, ρ(calc)=1.788 g cm−3, μ=3.657 mm−1,
F(000)=3296. 32598 refections measured, 14044 unique
(Rint=0.036), 12250 with I>2σ(I). The fnal R1=0.0360
(for 12250 refections with I>2σ(I)) and wR2(F2)=0.0801
(all data), S=1.08.
3: C72H60Cu2I2Sb4, MW = 1793.08, monoclinic, space
group P21/c, a=24.4375(4), b=13.9106(2), c=20.2194(3)
Å, α = 90, β = 111.169(2), γ = 90°, V = 6409.57(19) Å3,
1 3

Molecular Diversity
Bakas T, Hadjikakou SK (2018) Poly organotin acetates against
DNA with possible implementation on human breast cancer. Int
J Mol Sci 19:2055–2072. https://doi.org/10.3390/ijms19072055
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M, Tasiopoulos AJ, Hadjikakou SK (2015) Novel mixed metal
Ag(I)-Sb(III)-metallotherapeutics of the NSAIDs, aspirin and
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Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
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