Poly organotin acetates against DNA with possible implementation on human breast cancer

Article abstract of DOI:10.3390/ijms19072055

Two known tin-based polymers of formula {[R₃ Sn(CH₃ COO)]_n} where R = n-Bu– (1) and R = Ph– (2),were evaluated for their in vitro biological properties. The compounds were characterized via their physical properties and FT

Full text of DOI:10.3390/ijms19072055

International Journal of
Molecular Sciences
Article
Poly Organotin Acetates against DNA with Possible
Implementation on Human Breast Cancer
1
1, ID
2, ID
George K. Latsis , Christina N. Banti * , Nikolaos Kourkoumelis *
,
3
3
3
Constantina Papatriantafyllopoulou , Nikos Panagiotou , Anastasios Tasiopoulos ,
4
5
4
1,
Alexios Douvalis , Angelos G. Kalampounias , Thomas Bakas and Sotiris K. Hadjikakou *
1
Section of Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina,
4
5110 Ioannina, Greece; glatsis@icloud.com
2
3
Medical Physics Laboratory, Medical School, University of Ioannina, 45110 Ioannina, Greece
Department of Chemistry, University of Cyprus, 1678 Nicosia, Cyprus;
constantina.papatriantafyllopo@nuigalway.ie (C.P.); panagiotou.nikos@ucy.ac.cy (N.P.);
atasio@ucy.ac.cy (A.T.)
Mössbauer Spectroscopy and Physics of Material Laboratory, Department of Physics, University of Ioannina,
4
4
5
5110 Ioannina, Greece; adouval@cc.uoi.gr (A.D.); tbakas@uoi.gr (T.B.)
Physical Chemistry Laboratory, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece;
akalamp@cc.uoi.gr
*
Correspondence: cbanti@cc.uoi.gr (C.N.B.); nkourkou@uoi.gr (N.K.); shadjika@uoi.gr (S.K.H.);
Tel.: +30-2651-008374 (S.K.H.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 16 June 2018; Accepted: 12 July 2018; Published: 14 July 2018
n
Abstract: Two known tin-based polymers of formula {[R Sn(CH COO)] } where R = n-Bu– (1)
3
3
and R = Ph– (2),were evaluated for their in vitro biological properties. The compounds were
1
19
1
characterized via their physical properties and FT-IR, Sn Mössbauer, and H NMR spectroscopic
data. The molecular structures were confirmed by single-crystal X-Ray diffraction crystallography.
0
The geometry around the tin(IV) ion is trigonal bi-pyramidal. Variations in O–Sn–O···Sn torsion
angles lead to zig-zag and helical supramolecular assemblies for 1 and 2, respectively. The in vitro cell
viability against human breast adenocarcinoma cancer cell lines: MCF-7 positive to estrogens receptors
ERs) and MDA-MB-231 negative to ERs upon their incubation with and was investigated. Their
toxicity has been studied against normal human fetal lung fibroblast cells (MRC-5). Compounds
and exhibit 134 and 223-fold respectively stronger antiproliferative activity against MDA-MB-231
than cisplatin. The type of the cell death caused by or was also determined using flow
cytometry assay. The binding affinity of and towards the CT-DNA was suspected from the
differentiation of the viscosity which occurred in the solution containing increasing amounts of
and . Changes in fluorescent emission light of Ethidium bromide (EB) in the presence of DNA
confirmed the intercalation mode of interactions into DNA of both complexes and which have
(
1
2
1
2
1
2
1
2
1
2
1
2
been ascertained from viscosity measurements. The corresponding apparent binding constants
4
(
7
K
app) of
1
and
2
towards CT-DNA calculated through fluorescence spectra are 4.9
×
10 (
and
1
2
) and
was
4
−1
.3 × 10 (2) M respectively. Finally, the type of DNA binding interactions with
1
confirmed by docking studies.
Keywords: biological inorganic chemistry; acetic acid; organotins; bio-polymer; anti-cancer activity;
cell cycle
1
. Introduction
Platinum-based compounds are at the focal point of research on potent anticancer drugs since the
discovery of the anticancer potential of cisplatin, back during 70’s [
1
–
4]. However, platinum-based
Int. J. Mol. Sci. 2018, 19, 2055; doi:10.3390/ijms19072055
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Int. J. Mol. Sci. 2018, 19, 2055
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cancer treatments are being dominated by serious side effects [
5]. Moreover, cancer cells resistance
against platinum based drugs is developed in a short while [6]. Nowadays, organometallic compounds,
with a different pharmacological profile than that of platinum one, are developed and tested against
various types of cancer cells [7].
During the last decades the anticancer activity of organotin compounds (OTCs) has been well
studied [
8
–
29]. Therefore, the investigation of new OTCs of low toxicity and improved anticancer
19]. Their activity can be coupled
activity are known to induce apoptosis in several cancer cell lines [17
–
to the lipophilicity of alkyl or aryl groups attached to the tin atoms [21]. In addition, due to their
lipophilicity, OTCs are able to permeate membranes and reach the cell nucleus, where the dissociable
ligands yield intermediate molecules capable of binding DNA [16,26].
The advantages of the use of polymeric drugs as anticancer agents have been described earlier [30].
This is because: (i) the polymers overcome cellular resistance mechanisms; (ii) the polymers could
be used as carriers in high-dose chemotherapy; (iii) polymers are filtered out by the kidneys more
slowly than small compounds increasing the body retention time; (iv) the size and structure of the
polymer provide more binding sites to cellular targets; (v) the polymers can be act as hybrid drugs
incorporating multiple anticancer agents against cells through different mechanisms; and (vi) the
polymers accumulate in solid tumors more than in normal tissues [30].
In the course of our studies on the design and synthesis of new metallodrugs [13
–29], the known
{
[R Sn(CH COO)] } where R = n-Bu– ( ) and R = Ph– ( ) compounds were isolated from the reaction
n
1
2
3
3
between acetic acid with tributyltin or triphenyltin oxides. The compounds were characterized via
their physical properties and their FT-IR, 1 Sn Mössbauer, and H NMR spectroscopic data, while their
19
1
structures were verified by single-crystal X-Ray diffraction crystallography. The enhancement on the
biological activity against tumor cells of the polymeric
1 and 2 is studied in relation to their polymeric
intermolecular architecture (helical and zig-zag). The presence of acetic acid is also, expected to adjust
the lipophilicity of the metallodrugs. The in vitro cell viability against MCF-7 (estrogen receptor (ER)
positive) and MDA-MB-231 (estrogen receptor (ER) negative) was evaluated. Their genotoxicity has
been studied against normal human fetal lung fibroblast cells (MRC-5). The type of the cell death
caused by
1 and 2 was studied by flow cytometry assay. Finally, conclusions on Structure Activity
Relationship are derived, in the light of the results obtained for OCT’s from our group up to now.
2
. Results and Discussion
2
.1. General Aspects
Complexes 1 and 2 were synthesized as pale white powders by refluxing a benzene solution of
tri-aryl-tin oxide and acetic acid (glacial) in a 1:1 molar ratio (Scheme 1), using a Dean–Stark water trap.
Scheme 1. Preparation route of 1 and 2.
The formulae of
the complexes and
acetone, and diethyl-ether.
1
and
2
were first deduced by melting point and spectroscopic data. Crystals of
and are soluble in DMSO, DMF, toluene, ethanol,
1
2
are stable in air. Complexes
1
2

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2
.2. Solid State
2
.2.1. Vibrational Spectroscopy
−
−1
The νas(COO ) vibrations are observed at 1584 (
1
) and 1574 (
2
) cm respectively, while the bands
−
1
−
−
−
at 1418 (
value is 166 (
1
) and 1416 (
2
) cm are assigned to νs(COO ) (Figure S1). The ∆ν
[
νas(COO ) − νs(COO )]
) cm 1, respectively. Monodentate coordination of the carboxylic group
v than those observed for the ionic compounds of the
v is considerably smaller than that observed for its
ionic compounds. For asymmetric bidentate coordination, the values are in the range of monodentate
one [20]. When the –COO group bridges metal ions, the v values are higher than that of the
chelating mode and nearly the same as that observed for ionic compounds [20]. In the case of sodium
−
1
) and 158 (2
results in significantly higher difference values
∆
ligand [20], while when the ligand chelates, the
∆
−
∆
−
1
acetate, the
and 158 ( ) cm ) is in the range of the corresponding one of sodium acetate (170 cm ) the bridging
coordination mode is concluded for the carboxylic group in and (Figure S1). Bands at 493 ( ) and
(Sn–O) bond vibrations [20], while the
∆
v[vas(COO
−
)
−
v
s
(COO
−
)] value is 170 cm
[31]. Since the ∆ν values in 1 and 2 (166 (1)
−
1
−1
2
1
ν
2
1
−
1
4
57 (
corresponding bands at 672, 614 (
symmetric vibrations of Sn–C bonds [29].
2
) cm in the spectra of
1
and
2
are assigned to the
−
1
1) and 730, 698 (
2
) cm are assigned to the antisymmetric and
2
.2.2. 119Sn Mössbauer Spectroscopy
119Sn Mössbauer spectra at 80 K are shown in Figure 1.
(
A)
(
B)
Figure 1. 119Sn Mössbauer spectra of 1 (A) and 2 (B) at 80 K.

Int. J. Mol. Sci. 2018, 19, 2055
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The spectrum of
1
consists of one asymmetric Lorentzian doublet. The absorption line intensity
asymmetry could be attributed to the recoilless fraction (f) asymmetry, which could be a consequence
of vibrational-bond anisotropic involving the Sn ions [32]. Preferred orientation of the crystallites in
the powdered sample cannot also be excluded in order to justify this asymmetry. The occurrence of
one Lorentzian double, indicates either the existence of one type of Sn atom in
isomer [25 29]. The corresponding spectrum of consists by two symmetric Lorentzian doublets.
The occurrence of two symmetric Lorentzians, however, indicates two kinds of tin centers under
different environment in with 85–15% molar ratio [25 29]. The values of the Isomer Shifts
I.S.) of +1.44 ( ), 1.28 (2A) and 1.15 (2B) mm corresponds to the (4+) oxidation state [25 29].
The quadrupole splitting parameter ( Eq) values are 3.57 ( . Therefore,
trigonal-bibyramidal (tbp) geometry should be concluded for tin(IV) ions in and 2A as in the case of
tbp R Sn(IV) (eg-R = alkyl) geometry where the Eq values lie between 2.50–4.00 mm
the contrary, tetrahedral (tet) geometry R Sn(IV) results in to Eq values of 1.30–3.00 mm
tet conformation should be attributed to the tin(IV) atoms in 2B.
1 or one structural
,
2
2
,
−
1
(
1
·
s
,
−
), 3.35 (2A) and 1.33 (2B) mm s
1
∆
1
·
1
−
1
∆
·
s
[
s
25,29]. On
3
3
−
1
∆
·
[25,29],
3
2
.2.3. Crystal and Molecular Structures of [Bu SnCH COO]n (1) and [Ph SnCH COO]n (2)
3 3 3 3
Crystals suitable for X-ray analysis were obtained by slow evaporation of diethyl-ether solutions
and . Their formula was confirmed here by single crystal X-ray diffraction analysis at ambient
of
1
2
conditions. The structure of
1
is identical to that already reported by M. Adeel Saeed et. al. [33]. Thus
◦
1
crystallizes in P2 /c space group, a = 10.1845(3), b = 20.2542(7), c = 16.2466(6) Å,
β
= 94.739(3) ,
1
3
V = 3339.87 Å ; while the reported one crystallizes in P2 /c space group, a = 10.386(4), b = 20.924(3),
1
◦
3
c = 16.584(6) Å,
β
= 92.87(2) , V = 3599(2) Å [33]. Although,
2
crystallizes in Pn space group with
◦
a = 16.7427(6), b = 10.0426(2), c = 25.5119(8) Å,
V = 4211.68 Å3, the already reported crystallizes in P2 /c, space group, a = 8.969(4), b = 10.146(5),
α
= 89.999(2),
β
= 100.936(3),
γ
= 89.998(2) ,
1
◦
3
c = 19.540(7) Å,
β
= 93.70(4) , V = 1774.5 Å [34] suggesting a polymorphism between
2 and the
published one. However, the extended disorder on the density observed in
2
prevent its accurate
refinement allowing only qualitative conclusions to be drawn. Although the obtained X-ray data do
not allow discussion about bond lengths and angles, they are of sufficient quality to determine the
connectivity and the packing of 2. The molecular diagrams of 1 and 2 are shown in Figure 2.
(
A)
(B)
Figure 2. Molecular diagrams of 1 (A) and 2 (B).
Three C atoms from the alkyl groups and two O atoms from two de-protonated CH COOH
3
molecules form the trigonal bipyramidal arrangement around the Sn ions in
1. The average C–O bond
lengths found in
1
and
2
(1.260
±
0.020 Å), indicates a bond order of 1.5 e. This delocalization of the
−
electron density in the –COO group suggests a charge distribution shown in Scheme 2.

Int. J. Mol. Sci. 2018, 19, 2055
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Scheme 2. Charge distribution.
2
.3. Solution Studies
1H-NMR Spectroscopy
1
The H-NMR spectrum of free acetic acid in DMSO-d is dominated by a single resonance signal
6
at 1.91 (s, H) ppm for the methyl protons, which is shifted upon its coordination to the tin(IV) ion at
1
.776 (s, H) ppm in
1 and at 1.758 (s, H) ppm in 2 (Figure S2). In the case of 1 four additional signals
a
a
b
b
are observed at 1.52 ppm (t, H (– CH –Sn)), 1.27 ppm (m, H (– CH –CH –Sn)), at 1.00 ppm (m,
H ((– CH –CH –CH –Sn)), and 0.84 ppm (t, H (( CH –CH –CH –CH –Sn)) of the butyl substituent
2
2
2
c
c
d
d
2
2
2
3
2
2
2
bind on tin(IV) ions (Figure S2). These four signals (1.52–0.84 ppm) have been replaced by the signals
at 7.79–7.45 ppm in the spectrum of (Figure S2), which were attributed into the aromatic protons
2
of phenyl substituent of organotin moieties. Since the cells were incubated for 48 h the stability of
1
1
and 2 is checked for this period with H-NMR spectroscopy. No changes were observed between the
initial spectra of freshly prepared solutions and the corresponding spectra when measured after 48 h
confirming the retention of the structures in solution (Figure S2).
2
.4. Biological Tests
2
.4.1. Anti-Proliferative Activity
Organotin compounds
breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231, by the mean of sulforhodamine B (SRB)
assay [16 17]. The cells were incubated for 48 h with and . Since ERs are expressed in 65% of human
breast cancer, (a hormone dependent malignancy) the MCF-7 and MDA-MB-231 cells were used in
order to ascertain the influence of the ER’s in the mechanism of action of and 16 17]. MCF-7
cells serve as a valuable model system to elucidate pathways of hormone response and resistance.
Especially, the MCF-7 cells were used for studying estrogen response both in vitro and in vivo 35].
1 and 2 were tested for their in vitro cytotoxic activity against human
,
1
2
1
2 [ ,
[
MDA-MB-231 human breast cancer cells, on the other hand, are used as a model of ER-negative breast
cancers [36].
The IC50 values of
and 0.21 0.01 M respectively, while their corresponding IC50 values against MDA-MB-231 cells
are 0.20 0.01 and 0.12 0.01 M. The activity of follows reverse order to the corresponding one
of against these cell lines suggesting no interference of the estrogen receptors to their mechanism.
By taking into account the IC50 value of cisplatin against MCF-7 and MDA-MB-231 cells (5.5 0.4
and 26.7 1.1 M respectively), both and exhibit extremely cytotoxic activity against these cell
lines. These values indicate 22 and 26-fold higher activity of and against MCF-7 cells than cisplatin
and 134 and 223-fold against MDA-MB-231 cells. Despite their strong activity against tumor cells,
1
and
2
against MCF-7 cells lie in the range of nM and they are 0.25
±
0.02
±
µ
±
±
µ
1
2
±
±
µ
1
2
1
2
1
0
and
2
also exhibit high toxic activity against MRC-5 cells with IC50 values of 0.22
±
0.01 (1) and
.11 ± 0.01 (2) µM, respectively. The IC50 values against MCF-7, MDA-MB-231, and MRC-5 cells of
organotin compounds studied from our group, are summarized in Table 1. The following conclusions
are made: (i) tri-organotin derivatives are more active than the corresponding di-organtin ones;
(ii) organotin derivatives of carboxylic acids are generally more active than those of other types of

Int. J. Mol. Sci. 2018, 19, 2055
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ligands; (iii) organotin compounds are also highly toxic, even more toxic than cispaltin; (iv) however
the selectivity index, which is defined as the IC50 value against MRC-5 towards the corresponding
value against MCF-7, and is an indicator of the therapeutic potency of a compound (the higher the
value is, the better potency), shows that
1
and
)) than cisplatin (TPI = 0.20). (v) The tri-n-butyl tin compound of thiobarbituric acid
n-Bu) Sn(o-HTBA)(H O) exhibits the higher TPI value of 1.6 (Table 1).
2 are more potent therapeutics (TPI values of 0.88
(1) and 0.52 (2
(
3
2
Table 1. Bioactivity data recorded for
1
and
2
in comparison with those of other reported
organotin compounds.
IC50 (µM)
Compound
MCF-7
MDA-MB-231
MRC-5
TPI *
Ref.
1
2
0.25 ± 0.02
0.21 ± 0.01
0.030
0.142 ± 0.043
0.108 ± 0.0026
0.724 ± 0.0054
0.121 ± 0.0037
0.325 ± 0.0023
0.103
0.20 ± 0.01
0.12 ± 0.01
0.22 ± 0.01
0.11 ± 0.01
0.88
0.52
[present]
[present]
[18]
[19]
[19]
[19]
[19]
[19]
[17]
[17]
[14]
[14]
[14]
{
[Ph3Sn]2(mna)·[(CH3)2CO]}
>0.200
[
Me2Sn(Sal)2]
0.0975 ± 0.00015
0.1041 ± 0.0002
0.0981 ± 0.0001
0.0945 ± 0.000.2
0.0784 ± 0.0002
0.130
0.69
0.96
0.14
0.78
0.24
1.26
1.59
[
[
(n-Bu)2Sn(Sal)2]
(n-Bu)3Sn(Sal)]
Ph3Sn(Sal)]
(n-Bu)3Sn(pHbza)]
[Ph3Sn(o-HTBA)]}n
n-Bu)3Sn(o-HTBA)(H2O)
(tert-Bu–)2(HO–Ph)]2SnCl2
(tert-Bu–)2(HO–Ph)]2Sn(PMT)2
[
[
{
0.203
0.106
(
0.068
0.108
[
3.12 ± 0.38
7.86 ± 0.87
0.58 ± 0.1
>30
[
[
{
(tert-Bu–)2(HO–Ph)]2Sn(MPMT)2
[(tert-Bu–)2(HO–Ph)]2SnCl(PYT)}
[14]
[
(tert-Bu–)2(HO–Ph)]2SnCl(MBZT)}
Ph3SnCl
>30
0.130
0.070
>10
[14]
[16]
[16]
[16]
0.166
0.165
>10
0.141
0.090
>10
1.08
1.29
[
Ph3SnOH]n
[
(Ph2Sn)4Cl2O2(OH)2]
Me2Sn((tert-Bu–)2(HO–Ph–S))2
Et2Sn(((tert-Bu–)2(HO–Ph–S))2
n-Bu)2Sn–(((tert-Bu–)2(HO–Ph–S))2
19.20 ± 1.70
6.20 ± 0.80
0.40 ± 0.06
6.20 ± 0.80
>30
19.50 ± 1.40
7.30 ± 0.60
0.61 ± 0.07
12.40 ± 1.40
>30
1.02
1.18
1.53
2.00
[15]
[15]
[15]
[15]
(
Ph2Sn(((tert-Bu–)2(HO–Ph–S))2
[
(tert-Bu–)
2
(HO–Ph)]
2
Sn(((tert-Bu–)
2
(HO–Ph–S))
2
>30
[15]
Me3Sn((tert-Bu–)2(HO–Ph–S))
Ph3Sn(((tert-Bu–)2(HO–Ph–S))
Cisplatin
4.90 ± 0.50
0.25 ± 0.03
5.5 ± 0.4
3.36 ± 0.13
0.22 ± 0.01
1.1 ± 0.2
0.69
0.88
0.20
[15]
[15]
[37]
26.7 ± 1.1
*
TPI = IC50(MRC-5)/IC50(MCF-7), mna = 2-mercapto-nicotinic acid, salH = salicylic acid, pHbzaH =
p-Hydroxyl-benzoic acid, H TBA = 2-thiobarbituric acid, PMTH = 2-mercapto-pyrimidine, MPMTH =
-mercapto-4-methyl-pyrimidine, PYTH = 2-mercapto-pyridine, MBZTH = 2-mercapto-benzothiazole.
2
2
2
.4.2. Evaluation of Genotoxicity by Micronucleus Assay In Vitro
Micronucleus assay is a reliable and an accessible technique to evaluate the appearance of
genetic damage on a cell. The detection of micronucleus (MN) indicates mutagenic, genotoxic, or
teratogenic effects [37]. In the presence of exogenous genotoxic factors, the MN is formed due to
the metaphase–anaphase transition of the mitotic cycle. The possible induction of micronucleus
frequencies was evaluated when MRC-5 cells were treated by
IC50 values. The micronucleus frequency in the MRC-5 cell culture without treatment is 0.91
while it is 1.0 0.1% upon treatment with DMSO. However, the micronucleus frequencies are slightly
increased when the cells are incubated with and to 2.10 0.04% ( ) and 2.19 0.03% ( ) (Figure S3).
The compounds and show a slightly increase in micronucleus frequency in contrast to the control or
to cisplatin (1.6%) at its IC50 value of 26 M [37]. Cisplatin is used as a reference control. Doxorubicin
has also been used as reference control from other groups. MRC-5 cells show slight increasing MN’s
when they are treated with and than the case of cisplatin indicating higher genotoxocity indeed.
However, the treatment of MRC-5 cells with 0.18 M or 0.014 M of doxorubicin increases the percent
of micronucleus at (94.7 20.0)% and (17.00 1.73)%, respectively, in contrast to the control ones
15.0 2.64)% [38]. Thus, despite its higher genotoxicity of and towards MRC-5 cells than cisplatin
1
and
2
at the concentrations of their
±
0.02%,
±
1
2
±
1
±
2
1
2
µ
1
2
µ
µ
±
±
(
±
1
2
both exhibit significant lower MN percentage than doxorubicin a medication against tumors in humans
in used. It is therefore considered that 1 and 2 are nongenotoxic substances.

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2
.4.3. Cell Cycle Studies
Internucleosomal DNA fragmentation has been described as one of the main characteristics of the
apoptotic process and can be identified by a sub-G peak on DNA frequency histograms [37]. Therefore,
1
the apoptotic type of the cell deaths caused by the regulation of cell cycle progression because of
1 and
2
can be evaluated by flow cytometric analysis since these cells give a sub-G peak [37]. The percentage
1
of cells in the phases of the cell cycle was analyzed after 48 h exposure of MCF-7 cells with 1 and 2 at
their IC50 values.
The effect on the cell cycle which is illustrated in Figure 3, as the number of cells towards DNA
content in sub-G , G /G , S, and G /M phases is caused by
1
and
2. The untreated cells are spread
1
0
1
2
in 6.1% sub-G1 phase, 46.5% in G /G , 18.3% in S, and 28.9% in G /M phases. After incubation of
0
1
2
MCF-7 cells with
1 and 2, a significant increase in the number of apoptotic cells in sub-G phase (14.4%
1
(1
), 24.1% ( ), respectively) was observed towards the control group (6.1%). In the case of 1, the cells
2
in G /G phase are reduced to 37.9%, on the contrary, with 46.5% for the untreated cells while in the
0
1
case of
2
, the corresponding value decrease to 35.8%. However, the percentage of MCF-7 cells in S
), 22.9% ( ). Finally, the percentage of MCF-7 cells in G /M phase was
) and 17.1% ( ), respectively. In the DMSO-treated cells, the distribution in phases
sub-G (6.5%), G /G (42.7%), S (20.6%), and G /M (29.5%) were similar to the corresponding ones of
phase, was increased to 25.1% (
reduced to 22.1% (
1
2
2
1
2
1
0
1
2
control cells’. All the data obtained in cell cycle studies of 1 and 2 are summarized in Table 2.
Table 2. Cell cycle studies data of 1 and 2.
Phases of cell cycle
Description
Sub-G1
G /G
S
G /M
2
0
1
Untreated cells
Treated cells with
6.1%
46.5%
42.7
18.3%
20.6
28.9%
29.5
6
.5
DMSO
1
2
14.4
24.1
37.9%
35.8%.
25.1
22.9
22.1
17.1
In conclusion,
inhibiting DNA synthesis, in accordance to other anticancer agents (resveratrol, mitomycin C, and
hydroxyurea) [37]. Likewise, cisplatin causes cell cycle arrest at S and G /M phases and the percentage
1 and 2 stimulate S-phase cell cycle arrest, thus suppressing cell proliferation by
2
of MCF-7 cells in sub-G phase is increased, exhibiting an increasing number of apoptotic cells [37].
1
To summarize, metal drugs of this type induce cell cycle arrest either in G /G or in S phase (like
0
1
cisplatin), resulting in MCF-7 cell growth inhibition. Therefore, the reduced cell growth caused by
1
and is attributed to the apoptotic type of cell death, in accordance to the way of action of cisplatin [37].
2

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Figure 3. Number of MCF-7 cells in sub-G , G /G , S, and G /M phases, upon their treatment with
1
1
0
1
2
and 2. The meaning of color labeling is white= Sub-G1, blue= G0/G1, green= S, pink= G2/M.
2
.4.4. Detection of the Loss of the Mitochondrial Membrane Permeabilization (MMP Assay)
The releasing of cytochrome c in the cytosol through the loss of mitochondrial membrane
permeability activates the mitochondrion cell apoptosis pathway [37]. The induction of loss in
mitochondrial membrane permeability in tumor cells is one of the main accomplishments of targeted
chemotherapy. The MMP assay is based on the cationic hydrophobic mitochondrial potential dye which
accumulates in normal mitochondria. When cells are treated with a metallo-agent, the mitochondrial
membrane permeability collapses, and the fluorescence emission of the dye decreases simultaneously.
The MCF-7 cells were treated with
the MMP assay dye decreased by 6.71% (
treated with cisplatin at its IC50 values (5.5
4.9% [37]. Therefore, the MMP assay should not support mitochondrial membrane permeability loss.
Thus, and cause cell death by a different mechanism. Since apoptosis has been observed from cell
cycle studies, it should be activated by a different mechanism than mitochondrion pathways.
1
and
) and 6.52% (
M) the fluorescence of the MMP assay dye decreased by
2
at their IC50 values, for 48 h, and the fluorescence of
1
µ
2), respectively. When the MCF-7 cells are
5
1
2
2
.4.5. DNA Binding Studies
DNA is the main target of the successful chemotherapeutics, the interaction of 1 and 2 towards
CT-DNA was investigated by viscosity measurements and fluorescence spectroscopic studies.

Int. J. Mol. Sci. 2018, 19, 2055
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(a) Viscosity measurements: DNA length changes upon its incubation with anticancer agents
affect strongly affecting the viscosity of its solution. Thus (i) if the agent intercalates in the DNA strands,
this results in its lengthening and a viscosity increase; (ii) if the agent interacts electrostatically with the
DNA, no effect on DNA length is caused and therefore no significant change in viscosity is observed;
(
iii) however, in case the DNA strands are cleaved by an agent, the length of the DNA decreases, and
also, the viscosity decreases significantly (iv) bending of the DNA helix caused by the agent reduces
the viscosity. Therefore, viscosity exhibits high sensitivity to changes in the DNA and it is used for
the study of the binding modes of an agent towards DNA [39]. The solution of CT-DNA (10 mM) is
incubated with increasing amounts of
1 and 2 so that the [compound]/[DNA] molar ratio reaches
r = 0.27. The relative viscosity of the solution which contains the agent/DNA/buffer, towards the
corresponding one which contains DNA/buffer, increased for both compounds (Figure 4), suggesting
an intercalation mode of interaction between 1 and 2 and CT-DNA.
◦
Figure 4. Effect of increasing concentrations of
[DNA] = 10 mM, r = [compound]/[DNA], n is the viscosity of DNA in the presence of
the viscosity of DNA alone).
1
and
2
on the relative viscosity of CT-DNA at 25 C.
(
o
1 or 2 and n is
(
b) Fluorescence Spectroscopic Studies: In order to verify the intercalation mode of 1 and 2 towards
CT-DNA, fluorescence spectroscopic studies were carried out. In the fluorescence spectroscopic studies
the dye ethidium bromide (EB) is used. In the presence of DNA, EB emits, due to its strong intercalation
between the adjacent DNA base pairs [37]. The displacement of EB during titration with the agent
suggests an intercalative binding mode. The emission data of the solutions of EB with CT-DNA at
6
10 nm, with increasing concentrations of
recorded (Figure 5). The decreasing percent in fluorescence upon increasing of concentration of
and at 610 nm was 31.5% ( ) and 30.1% ( ), respectively, confirming that both compounds can
1 and 2 (0–250 µM) upon their excitation at 532 nm, were
1
2
1
2
interact with DNA by the intercalation mode. The corresponding apparent binding constants (Kapp
)
4
of
7.3
tri-n-butyltin acetates.
1
and
2
towards CT-DNA calculated through fluorescence spectra are (4.9
±
0.5)
×
10 (
1
) and
4
−1
(
±
1.3)
×
10 (2) M , respectively, indicating stronger binding affinity of the triphenyltin than the

Int. J. Mol. Sci. 2018, 19, 2055
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1
1
1
1
.6
.5
.4
.3
140000000
120000000
100000000
1.2
1.1
1
0
0
.9
.8
0
50
100
150
200
250
300
(B)
Concentration (μΜ)
80000000
60000000
40000000
20000000
0
550
600
650
700
750
800
Wavelength (nm)
(Α)
Compound 1
CT-DNA
25 μΜ
100 μΜ
200 μΜ
250 μΜ
(
A)
1
1
1
1
1
1
1
.7
.6
.5
.4
.3
.2
.1
1
140000000
120000000
100000000
0.9
.8
0
0
50
100
150
200
250
8
0000000
0000000
0000000
0000000
(Β)
Concentration (μΜ)
6
4
2
0
550
600
650
700
750
800
Wavelength (nm)
(Α)
Compound 2
CT-DNA
50 μΜ
100 μΜ
150 μΜ
200 μΜ
(
B)
Figure 5. Emission spectrum of EB bound to DNA (peak around 610 nm) decreases in order of the
concentration of the complex ( ) and )). The arrows show the intensity changing upon increasing
complex concentration. Inset shows the plots of emission intensity Io/I vs. [complex].
1
(A
2 (B
(
c) Computational studies—molecular docking: In order to verify the type of interaction between
DNA with or molecular docking calculations were performed. Small aromatic molecules can
1
2
typically bind to DNA by intercalation. Generally, either a planar molecule or a fragment is inserted
between two adjacent DNA base pairs to form a hydrophobic pocket in the DNA structure. This
non-covalent interaction is usually stabilized by π-interactions; at the same time, additional interactions
with the groves are possible. Statistically, the CG intercalation site is preferable for intercalation [40].
Upon ligand binding, the DNA structure is slightly elongated and accommodates a small gap between
two consecutive base pairs for the intercalators. Consequently, the latter can act as potent antitumor

Int. J. Mol. Sci. 2018, 19, 2055
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drugs and mutagens, inhibiting DNA replication and transcription. Targeting DNA sequences is a
challenging task for docking software, especially when intercalation sites are unavailable, because gap
openings cannot be simulated with rigid receptors. Moreover, it has been shown that popular docking
software fails to predict the intercalation into canonical B-DNA when analogous solved structures are
lacking [41]. For these reasons, the DNA target chosen for our study was 1DSC (PDB: www.rcsb.org)
which is an octamer d(GAAGCTTC) complexed with actinomycin D [42]. Actinomycin D is a
2
potent antibiotic with high antibacterial and antitumor activity. It intercalates to DNA by localizing
a phenoxazone ring at a GpC sequence while the two cyclic polypeptides of the drug bind to the
DNA minor groove. 1DSC has been already used successfully for docking of metal complexes [43
]
and organic compounds [44] acting as DNA intercalators. The geometry of compounds and
1
2
is trigonal bipyramidal (TBP) in the solid state. However, upon solvation in aqueous media their
structure are converted to tetrahedral by cleavage of the Sn-O(acetate) dative bond. Tetrahedral metal
complexes, unlike those with TBP geometry, can effectively bind to DNA by intercalation, although
they normally cannot penetrate as deep as square planar complexes [45]. The optimized structures of
the two organotin compounds [R Sn(CH COO)], (R = n-Bu (1), Ph– (2)) were used as ligands for DNA
3
3
docking using AutodockVina. Molecular docking evaluates affinity potentials through a precalculated
grid finding favorable binding positions for a flexible ligand towards a rigid macromolecular target.
Complexes
1 and 2 were successfully docked into the B-DNA duplex and the lowest energy poses are
depicted in Figure 6. The tri-phenyl derivative can adopt two different conformations intercalating in
the GC region through either one pnenyl-ring (Figure 6(2a)) or the carboxylic moiety (Figure 6(2b))
with computed binding free energies
−
5.5 and
−
5.1 kcal/mol, respectively. The planarity of the
phenyl ring favors stacking interactions with the DNA base pairs, while additional hydrogen
π-π
bonding interactions with the carboxylate O (G4:N2-Lig O, 2.97 Å and Lig O-C5O4, 3.1 Å) and with the
pi-orbitals of the phenyl ring (G12:N2-Lig pi, 2.96 Å and G4:N2-Lig pi, 3.6 Å). On the other hand, the
minimum energy conformation of the tri-n-butyl-derivative shows a semi-intercalation into the same
GC region (Figure 6(
1
)) with lower binding affinity ( 3.7 kcal/mol). The structure is also stabilized by
−
hydrogen bonding through the carboxylic moiety (G4:N2-Lig O, 3.13 Å). Possibly, the bulk and agile
butyl groups significantly increase the steric hindrance and prevent the intercalation. These results
support the experimental results showing that the phenyl derivative exhibits higher antitumor activity
possibly through its intercalative mode of action (Table 1).
Figure 6. DNA docking and H-bonding interactions between B-DNA and compounds 1 and (2a, 2b).

Int. J. Mol. Sci. 2018, 19, 2055
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3
. Experimental
3
.1. Materials and Instruments
All solvents used were of reagent grade, (Aldrich, Merck, Darmstadt, Germany) and they were
−
1
used with no further purification. Infrared spectra in the region of 4000–370 cm were obtained in
1
KBr pellets with a Jasco FT-IR-6200 spectrometer. The H-NMR spectra were recorded on a Bruker
AC 250, 400 MHFT-NMR instrument in DMSO-d . Chemical shifts are given in ppm using H-TMS as
1
6
internal reference. Elemental analysis for C, H, N, and S were carried out with a Carlo Erba EA MODEL
19
1
8
108 (Waltham, MA, USA). The 1 Sn M o˝ ssbauer spectra were collected at sample temperature of
0 K using a constant acceleration spectrometer equipped with a Ca119mSnO source kept at room
3
temperature. The isomer shift values of the components used to fit the spectra are given relative to
119
SnO at room temperature. The Sn M o˝ ssbauer spectra were recorded with Constant acceleration
2
WissEl-Wissenschaftliche Elektronik GmbH spectrometer (Starnberg, Germany).
3
.2. Synthesis and Crystallization of {[(n-Bu) Sn(CH COO)]n} (1) and {[Ph Sn(CH COO)]n} (2)
3
3
3
3
Although the synthesis of these compounds is already known [33
,
34] we briefly described the
procedure follows here. 0.5 mmol of tri-n-butyltin oxide (C H OSn , 0.298 g) for
1, or triphenyltin(IV)
24
54
2
hydrooxide (C H OSn, 0.183 g) for
2, were diluted with 0.5 mmol acetic acid, in 20 mL benzene
24
16
in a 100-mL spherical flask. The flask was fitted with a Dean–Stark moisture trap and the reaction
mixture was refluxed for 3 h. The solution was filtered and the clear filtrate was concentrated to
dryness. Crystals of
ether solution.
1
and
2
, suitable for X-ray analysis, were formed by slow evaporation of a diethyl
◦
1: Yield: 40%; m.p: 75–76 C; (C H O Sn)n·(MW = 349.04); elemental analysis: found C = 48.23,
14
30
2
−
1
H = 6.65%; calcd: C = 48.18, H = 8.66%. MID-IR (cm ) (KBr): 3045 w, 2330 w, 2295 s, 1959 w, 1821
w, 1659 s, 1643 w, 1573 w, 1555 s, 1428 s, 1334 vs, 1261 vs, 1077 vs, 1025 w, 997 w, 729 w, 696 w, 665 w,
1
6
09 w, 499 w, 455 w. H NMR (ppm) in DMSO-d : 1.776 (s), 1.578–1.501 (q), 1.327–1.236 (q), 1.043–1.001
6
(
t), 0.880–0.843 (t).
◦
2: Yield: 40%; m.p: 110–115 C; (C H O Sn) (MW = 409.02); elemental analysis: found
n
20
18
2
−
1
C = 58.91, H = 4.40%; calcd: C = 58.73, H = 4.43%. MID-IR (cm ) (KBr): 2400 w, 1574 w, 1556 s, 1415 w,
1
1
384 s, 1015 s, 867 w, 671 w, 612 s. H NMR (ppm) in DMSO-d : 7.850–7.707 (q), 7.452–7.390 (q), 1.758
6
(single), 1.114–1.079 (t).
3
.3. X-Ray Structure Determination
Single crystal X-ray diffraction data for
diffractometer, equipped with a CCD area detector utilizing Cu Kα (λ
1
and
2
were collected on an Oxford-Diffraction Supernova
= (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 corrections (multiscan based on
symmetry-related measurements) were applied using CrysAlis RED software [46]. The structures were
2
solved by direct methods using SIR2004 [47] and refined on F using full-matrix least-squares with
SHELXL-2014/7 [48]. Software packages used were as follows: CrysAlis CCD for data collection [46],
CrysAlis RED for cell refinement and data reduction [46], WINGX for geometric calculations [49]. The
non-H atoms were treated anisotropically, whereas the aromatic H atoms were placed in calculated,
ideal positions and refined as riding on their respective carbon atoms. The single crystals of compound
2
exhibited a fairly poor diffraction pattern. As a result moderate quality X-ray data were collected
which did not lead to a publishable crystal structure. For this reason the X-ray data of
here and have not been deposited in Cambridge Structural Database.
2 are not quoted
Supplementary data (1_bu3sn2o_asp_checkcif_new.pdf and 1_bu3sn2o_asp_FINAL.cif) are
available from CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (e-mail:deposit@ccdc.cam.ac.uk), on
request, quoting the deposition numbers CCDC-1846946 (1).

Int. J. Mol. Sci. 2018, 19, 2055
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1
: (C H O Sn)
n
: MW = 349.04, Monoclinic, space group P21/c, a = 10.1845(3), b = 20.2542(7),
14
30
2
◦
3
−3
c = 16.2466(6) Å,
= 1.522 mm
β
= 94.739(3) , V = 3339.9(2) Å , Z = 4, T = 100 K,
ρ(calc) = 1.388 g
·
cm ,
−
1, F
µ
(000) = 1440. 22782 reflections measured, 5864 unique (Rint = 0.041), 5131 with
2
I > 2
σ
(I). The final R = 0.0265 (for 5131 reflections with I > 2
σ
(I)) and wR (F ) = 0.0678 (all data),
1
2
S = 1.09.
3
.4. Biological Tests
Biological experiments were carried in dimethyl sulfoxide Dulbecco’s Modified Eagle’s Medium
solutions (DMEM) DMSO/DMEM (0.02–0.2% v/v) for the complexes . Stock solutions of the
complexes , (0.01 M) in DMSO were freshly prepared and diluted in with cell culture medium to
the desired concentrations (0.05–0.4 M). Results are expressed in terms of IC50 values, which is the
1–2
1–2
µ
concentration of drug required to inhibit cell growth by 50% compared to control, after 48 h incubation
of the complexes towards cell lines. Since there is no “universal” correct time point to find the IC50
value of a given compound. Generally the inhibitory concentration of the 50% of the cells is determined
at once at the time of the cells (of interest) doubling time, and twice the time of the doubling time. In
cell culture, the vast majority of adherent cell lines display a doubling time between 18 and 24 h. In our
research unit, we prefer to use 48 h since 24 h is too short to determine a reliable IC50 concentration.
The cell viability was determined by SRB assay as previously described [37] and it is briefly described
here: Cells were plated (100 µL per well) in 96-well flat-bottom microplates at various cell inoculation
densities (MCF-7, MDA-MB-231 and MRC-5: 6000, 6000 and 2000 cells/well respectively). Cells were
◦
incubated for 24 h at 37 C and they were exposed to tested agents for 48 h afterwards, followed by
the addition of an equal volume (100 µL) of complete culture medium only in the well containing the
controls, or twice the final drug concentrations diluted in complete culture medium in the wells where
the compounds are tested. Drug activity was measured by means of a SRB colorimetric assay giving
the percent of the survival cells towards the control (untreated cells) absorbance. The culture medium
was aspirated before fixation and 50
to the wells. Microplates were left for 30 min at 4 C, washed five times with deionized water and
µ
L of 10% cold trichloroacetic acid (TCA) were gently added
◦
left to dry at room temperature for at least 24 h. Subsequently, 70 µL of 0.4% (w/v) sulforhodamine
B (Sigma, Darmstadt, Germany) in 1% acetic acid solution was added to each well and left at room
temperature for 20 min. SRB was removed and the plates were washed five times with 1% acetic acid
before air-drying. Bound SRB was solubilized with 200
µL of 10 mM un-buffered Tris-base solution.
Absorbance was read in a 96-well plate reader at 540 nm.
Evaluation of genotoxicity by micronucleus assay, cell cycle studies, detection of the loss of the
Mitochondrial Membrane Permeabilization (MMP assay), and fluorescence spectral studies were
performed as previously reported [37]. However, they are all quoted here in brief: (a) Micronucleus:
4
MRC-5 cells were seeded (at a density of 2
×
10 cells/well) in glass cover slips which were afterwards
placed in six-well plates, with 3 mL of cell culture medium and incubate for 24 h. MRC-5 cells
exposed with and in IC50 values for a period of 48 h. After the exposure of and , the cover slips
1
2
1
2
were washed three times with PBS and with a hypotonic solution (75 Mm KCl) for 10 min at room
temperature. The hypotonized cells were fixed by at least three changes of 1/3 acetic acid/methanol.
The cover slips were also washed with cold methanol containing 1% acetic acid. The cover slips were
◦
stained with acridine orange (5
µ
gr/mL) for 15 min at 37 C. After, the cover slips were rinsed three
times with PBS to remove any excess acridine orange stain. The number of micronucleated cells per
5
1
000 cells was determined. (b) Cell cycle: MCF-7 cells were seeded at a density of 10 cells/well in
◦
six-well plates at 37 C for 24 h. Cells were treated with
1
and
2
at the indicated IC50 values for 48 h.
The cells were then trypsinized and washed twice with phosphate-buffered saline (PBS) and separated
by centrifugation. With the addition of 1 mL of cold 70% ethanol, the cells were incubated overnight
◦
at
(
−
20 C. For analysis, the cells were centrifuged and transferred into PBS, incubated with RNase
0.2 mg/mL) and propidium iodide (0.05 mg/mL) for 40 min at 310 K and then analyzed by flow
cytometry using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). For each

Int. J. Mol. Sci. 2018, 19, 2055
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sample, 10,000 events were recorded. The resulting DNA histograms were drawn and quantified
using the FlowJo software (version FlowJo X 10.0.7r2, Tree Star, Ashland OR, USA). (c) Detection of
the loss of the Mitochondrial Membrane Permeabilization: MCF-7 cells were treated with
IC50 values. After 48 h of incubation period of and , the cell medium was removed and added the
Dye Loading Solution. The cells were incubated in 5% CO at 37 C for 30 min. Afterwards, 50
1 and 2 at
1
2
◦
µL
2
of Assay Buffer B was added of each well and are incubated for 30 min. The fluorescence intensity is
measured at λex = 540 and λem = 590 nm. The MMP assay kit used was purchased from sigma Aldrich
“Mitochondria Membrane Potential Kit for Microplate Readers, MAK147”. (d) Fluorescent studies:
The fluorescence spectroscopy method using ethidium bromide (EB) was employed to determine the
relative DNA binding properties of complexes
of the spectra of EB (2.3 M) solutions which contain CT-DNA (26
various concentrations of complexes and (0–250 M) were recorded upon their excitation at 532 nm
Figure 5). The apparent binding constant (Kapp) was calculated using the equation:
1
and
2
into CT-DNA. The emission data at 610 nm
µ
µM) in the absence or presence of
1
2
µ
(
K [EB] = Kapp[drug]
EB
()
7
−1
,
where [drug] is the concentration of the complex at a 50% reduction of the fluorescence, KEB = 10 M
and [EB] = 2.3 M. The concentration of the drug at a 50% reduction of the fluorescence is calculated
from the diagram I /I vs. the concentration of the complex [Q] (Figure 5), where, I and I are the
fluorescence intensities of the CT-DNA in the absence and presence of complexes and , respectively.
The fitting of the experimental I /I ratio as a function of concentration (C) was performed utilizing
µ
o
o
1
2
o
x
the linear least-squares fitting algorithm. However, the fitting procedure depends strongly on the
weighting of the data points, i.e., on the knowledge of the standard deviation (SD) of every point in
the experimental spectrum (<1%) since all spectra represent the average of 5000 individually recorded
2
spectra. As a measure for the goodness of the fit, we used the R statistics.
The slope of the I
o
/I
x
ratio as a function of C was estimated equal to a = 2100.9
±
204.8 and
1
a = 3142.4 ± 561.4 for 1 and 2 case, respectively.
2
The SD of the Kapp is calculated using the standard error propagation variance formula [50]:
s
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
2
2
2
∂
∂
f
x
∂ f
∂y
ꢂ ꢃ2
∂ f
∂z
2
2
sf =
(sx) +
sy
+
(sz) + · · ·
In general, s represents the standard deviation of the function f, s
x
represents the standard
represents the standard deviation of y, and so forth. This formula is based on
the linear characteristics of the gradient of f and therefore it is a good estimation for the standard
f
deviation of x, s
y
deviation of f. Using the above methodology, the parameter Kapp is calculated as 48,950.97
±
4771.84
−
1
2
and 73,217.92
±
13,080.62 M for
1
and
2, respectively. More details concerning the calculation of R ,
the error propagation issue and other statistical parameters, see e.g., reference [50]
3
.5. Viscosity Measurements
The kinematic viscosity of
1 and 2 was measured with the use of an Ubbelohde-type glass
capillary viscosity-meter. The value of kinematic viscosity was measured as an average from triplicate
measurements of viscosity with an accuracy of 2% at constant temperature. Temperature was set at
2
98 K by means of an ultra-thermostat.
3
.6. Computational Studies—Molecular Docking
Molecular docking studies were carried out with AutodockVina [51] along with the graphical
interface AutodockTools (ADT 1.5.6) [52] using the Lamarckian genetic algorithm (LGA). The DNA
structure (PDB file 1DSC) was considered as a rigid target while no torsional restraints were applied
to the complexes. Docking was performed to the optimized organotin derivatives [R Sn(CH COO)],
3
3

Int. J. Mol. Sci. 2018, 19, 2055
15 of 18
(
R = n-Bu (
package [53]. Prior to docking, water molecules and the co-crystallized ligand were removed. The
search space was set as a grid box of 40 30 40 Å and spacing of 1 Å centered at the CG rich area.
1), Ph– (2)) at the B3LYP/3-21G*/LANL2DZ(Sn) level using the Gaussian03Wsoftware
×
×
Default parameters were used as described in the Vina manual except from “exhaustiveness” which
was set to 24.
4
. Conclusions
The discovery of the antitumor activity of organotin compounds goes back to the 1980s when
Gielen first reports on the subject [ 11 12]. Later on, the antitumor properties of orgnaotin
8,9, ,
compounds were reviewed by Saxena and Hubert [10] and Hadjiliadis et al. [13]. Continuing
our studies on the antitumor activity of organotin compounds, two known compounds of formula
3 3
n
{[R Sn(CH COO)] } where R = n-Bu– (1) and R = Ph (2) with trigonal bipyramidal geometry around
each metal center, were used to investigate their antitumor activity against MCF-7 (positive to ERs)
and MDA-MB-231 (negative to ERs). The mechanism of their action against human breast tumor cells
was also investigated. Compounds 1 and 2 inhibit strongly both cell lines, while their IC50 values are
lei in nanomolar range (Table 1). The estrogen receptors play no significant role in their action. It is
also concluded that (i) the tri-organotin derivatives exhibit stronger activity than the corresponding
di-organotin ones (Table 1); (ii) the organotin derivatives of carboxylic acids are generally more active
than those of other type of ligands (Table 1); (iii) organotin compounds are also highly toxic against
normal MRC-5 cells (Table 1); (iv) the selectivity index, however, shows that
therapeutics (TPI values of 0.88( ) and 0.52( )) than cisplatin (TPI = 0.20) (Table 1) and (v) among all
organotins tested from our group the strongest activity against MCF-7 cells has been obtained from
the triphenyltin derivative of 2-mercapto-nicotinic acid {[Ph Sn] (mna) [(CH ) CO]} (IC = 30 nM)
1 and 2 are more potent
1
2
·
3
2
3 2
50
and the tri-n-butyl tin compound of thiobarbituric acid (n-Bu) Sn(o-HTBA)(H O) (IC = 68 nM and
3
2
50
TPI = 1.6) (Table 1). The mitochondrial membrane permeability loss is not supported by MMP assay.
Since and are acting through apoptosis on tumor cells (cell cycle studies) this should therefore be
1
2
activated through a different pathway than that of the mitochondrion, such as the extrinsic apoptotic
pathway or direct interaction with DNA. The relative viscosity measurements and fluorescence
spectroscopic studies of DNA solutions suggest an intercalation mode of interaction between
1 and 2
and CT-DNA. Docking studies verify the intercalative mode, while the bulk and agile n-butyl groups
significantly increase the steric hindrance prohibiting the intercalation, resulting in higher antitumor
activity of the phenyl derivative instead of the corresponding n-Bu– (Table 1).
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/
2
055/s1.
Author Contributions: Conceptualization, S.K.H.; Methodology, C.N.B., N.K., and S.K.H.; Validation, S.K.H.,
A.T., and T.B.; Investigation, G.K.L., C.N.B., N.K., C.P., N.P. A.D., and A.G.K.; Writing-Original Draft Preparation,
C.N.B., N.K., and S.K.H.; Writing-Review & Editing, S.K.H.; Supervision, S.K.H.
Funding: This research received no external funding.
Acknowledgments: (i) This work was carried out in partial fulfilment of the requirements for the graduate studies
of G.L. under the supervision of S.K.H. The International Graduate Program in “Biological Inorganic Chemistry”,
which operates at the University of Ioannina within the collaboration of the Departments of Chemistry of the
Universities of Ioannina, Athens, Thessaloniki, Patras, Crete, and the Department of Chemistry of the University
of Cyprus, is acknowledged for the stimulating discussions forum. (ii) C.N.B. and S.K.H. would like to thank
the Unit of Bioactivity Testing of Xenobiotics, of the University of Ioannina, for providing access to the facilities.
(
iii) C.N.B. and S.K.H. would like to thank the Atherothrombosis Research Centre of the University of Ioannina for
providing access to the fluorescence microscope and flow cytometry. (iv) A.G.K acknowledges the Laser Center of
Ioannina University for the access to its infrastructure. S. Kaziannis (Lecturer) is also acknowledged by A.G.K. for
his useful assistance during the luminescence experiments.
Conflicts of Interest: The authors declare no conflicts of interest.

Int. J. Mol. Sci. 2018, 19, 2055
16 of 18
References
1.
2.
3.
4.
5.
Rosenberg, B. Platinum coordination complexes in cancer chemotherapy. Die Naturwissenschaften 1973, 60,
99–406. [CrossRef] [PubMed]
Lippert, B. Impact of Cisplatin on the recent development of Pt coordination chemistry: A case study.
Coord. Chem. Rev. 1999, 182, 263–295. [CrossRef]
Arnesano, F.; Natile, G. Mechanistic insight into the cellular uptake and processing of cisplatin 30 years after
its approval by FDA. Coord. Chem. Rev. 2009, 253, 2070–2081. [CrossRef]
Arnesano, F.; Pannunzio, A.; Natile, G. Effect of chirality in platinum drugs. Coord. Chem. Rev. 2015, 284,
3
2
86–297. [CrossRef]
Astolfi, L.; Ghiselli, S.; Guaran, V.; Chicca, M.; Simoni, E.; Olivetto, E.; Lelli, G.; Martini, A. Correlation of
adverse effects of cisplatin administration in patients affected by solid tumours: A retrospective evaluation.
Oncol. Rep. 2013, 29, 1285–1292. [CrossRef] [PubMed]
6
.
.
Galluzzi, L.; Senovilla, L.; Vitale, I.; Michels, J.; Martins, I.; Kepp, O.; Castedo, M.; Kroemer, G. Molecular
mechanisms of cisplatin resistance. Oncogene 2012, 31, 1869–1883. [CrossRef] [PubMed]
Huang, K.-B.; Wang, F.-Y.; Tang, X.-M.; Feng, H.-W.; Chen, Z.-F.; Liu, Y.-C.; Liu, Y.-N.; Liang, H.
Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline Induce ER-Stress-Mediated
Apoptosis and Pro-Death Autophagy in A549 Cancer Cells. J. Med. Chem. 2018, 61, 3478–3490. [CrossRef]
7
[PubMed]
8
.
.
Haiduc, I.; Silvestru, C.; Gielen, M. Organotin compounds: New organometallic derivatives exhibiting
anti-tuequr activity. Bull. Soc. Chim. Belg. 1983, 92, 187–189. [CrossRef]
Gielen, M.; Melotte, M.; Atassi, G.; Wrllem, R. Synthesis, characterization and antitumour activity of
9
7
,7-di-n-butyi-5,9-dioxo-6,6-dioxa-7-stanna-spiro[3,5]nonane, a di-n-butyltin(lV) analog of “paraplatin”, and
of a series of di-n-butyitin(lV) derivatives of mono- and disubstituted malonic acids. Tetrahedron 1989, 45,
219–1229. [CrossRef]
0. Saxena, A.K.; Huber, F. Organotin compounds and cancer chemotherapy. Coord. Chem. Rev. 1989, 95, 109–112.
CrossRef]
1
1
[
1
1
1. Gielen, M. Tin-based antitumour drugs. Coord. Chem. Rev. 1996, 151, 41–51. [CrossRef]
2. Gielen, M. Organotin compounds and their therapeutic potential: A report from the Organometallic
Chemistry Department of the Free University of Brussels. Appl. Organomet. Chem. 2002, 16, 481–494.
[CrossRef]
1
1
3. Hadjikakou, S.K.; Hadjiliadis, N. Antiproliferative and anti-tumor activity of organotin compounds.
Coord. Chem. Rev. 2009, 253, 235–249. [CrossRef]
4. Shpakovsky, D.B.; Banti, C.N.; Beaulieu-Houle, G.; Kourkoumelis, N.; Hadjikakou, S.K.; Manoli, M.;
Manos, M.J.; Tasiopoulos, A.J.; Milaeva, E.R.; Charalabopoulos, K.; et al. Synthesis, structural
characterization and in vitro inhibitory studies against humanagainst human breast cancer of the
bis-(2,6-di-tert-butylphenol)tin(IV) dichloride andits complexes. Dalton Trans. 2012, 41, 14568–14582.
[CrossRef] [PubMed]
1
1
1
5. Shpakovsky, D.B.; Banti, C.N.; Mukhatova, E.M.; Gracheva, Y.A.; Osipova, V.P.; Berberova, N.T.; Albov, D.V.;
Antonenko, T.A.; Aslanov, L.A.; Milaeva, E.R.; et al. Synthesis, antiradical activity and in vitro cytotoxicity of
novel organotin complexes based on 2,6-di-tert-butyl-4-mercaptophenol. Dalton Trans. 2014, 43, 6880–6890.
[CrossRef] [PubMed]
6. Balas, V.I.; Banti, C.N.; Kourkoumelis, N.; Hadjikakou, S.K.; Geromichalos, G.D.; Sahpazidou, D.; Male, L.;
Hursthouse, M.B.; Bednarz, B.; Kubicki, M.; et al. Structural and in vitro biological studies of organotin(IV)
precursors; selective inhibitory activity against human breast cancer cells, positive to estrogen receptors.
Austr. J. Chem. 2012, 65, 1625–1637. [CrossRef]
7. Balas, V.I.; Verginadis, I.I.; Geromichalos, G.D.; Kourkoumelis, N.; Male, L.; Hursthouse, M.B.; Repana, K.H.;
Yiannaki, E.; Charalabopoulos, K.; Bakas, T.; et al. Synthesis, structural characterization and biological
studies of the triphenyltin(IV)complex with 2-thiobarbituric acid. Eur. J. Med. Chem. 2011, 46, 2835–2844.
[CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 2055
17 of 18
1
8. Verginadis, I.I.; Karkabounas, S.; Simos, Y.; Kontargiris, E.; Hadjikakou, S.K.; Batistatou, A.; Evangelou, A.;
Charalabopoulos, K. Anticancer and cytotoxic effects of a triorganotin compound with 2-mercapto-nicotinic
acid in malignant cell lines and tumor bearing Wistar rats. Eur. J. Pharm. Sci. 2011, 42, 253–261. [CrossRef]
[PubMed]
1
2
2
2
2
2
9. Agiorgiti, M.S.; Evangelou, A.; Vezyraki, P.; Hadjikakou, S.K.; Kalfakakou, V.; Tsanaktsidis, I.; Batistatou, A.;
Zelovitis, J.; Simos, Y.V.; Ragos, V.; et al. Cytotoxic effect, antitumour activity and toxicity of organotin
derivatives with ortho- or para-hydroxy-benzoic acids. Med. Chem. Res. 2018, 27, 1122–1130. [CrossRef]
0. Abdellah, M.A.; Hadjikakou, S.K.; Hadjiliadis, N.; Kubicki, M.; Bakas, T.; Kourkoumelis, N.; Simos, Y.V.;
Karkabounas, S.; Barsan, M.M.; Butler, I.S. Synthesis, characterization, and biological studies of organotin(IV)
derivatives with o- or p-hydroxybenzoic acids. Bioinorg. Chem. Appl. 2009, 12. [CrossRef] [PubMed]
1. Xanthopoulou, M.N.; Hadjikakou, S.K.; Hadjiliadis, N.; Milaeva, E.R.; Gracheva, J.A.; Tyurin, V.-Y.;
Kourkoumelis, N.; Christoforidis, K.C.; Metsios, A.K.; Karkabounas, S.; et al. Biological studies of new
organotin(IV) complexes ofthioamide ligands. Eur. J. Med. Chem. 2008, 43, 327–335. [CrossRef] [PubMed]
2. Balas, V.I.; Hadjikakou, S.K.; Hadjiliadis, N.; Kourkoumelis, N.; Light, M.E.; Hursthouse, M.; Metsios, A.K.;
Karkabounas, S. Crystal structure and antitumor activity of the novel zwitterionic complex of
tri-n-butyltin(IV) with 2-thiobarbituric acid. Bioinorg. Chem. Appl. 2008, 654137. [CrossRef] [PubMed]
3. Xanthopoulou, M.N.; Hadjikakou, M.N.; Hadjiliadis, N.; Kourkoumelis, N.; Milaeva, E.R.; Gracheva, J.A.;
Tyurin, V.-Y.; Verginadis, I.I.; Karkabounas, S.; Baril, M.; et al. Biological studies of organotin(IV) complexes
with 2-mercaptopyrimidine. Russ. Chem. B 2007, 56, 767–773. [CrossRef]
4. Xanthopoulou, M.N.; Hadjikakou, S.K.; Hadjiliadis, N.; Kubicki, M.; Karkabounas, S.; Charalabopoulos, K.;
Kourkoumelis, N.; Bakas, T. Synthesis and characterization of a new chloro-di-phenyltin(IV) complex
with 2-mercapto-nicotinic acid: Study of its influence upon the catalytic oxidation of linoleic acid to
hydroperoxylinoleic acid by the enzyme lipoxygenase. J. Organomet. Chem. 2006, 691, 1780–1789. [CrossRef]
5. Xanthopoulou, M.N.; Hadjikakou, S.K.; Hadjiliadis, N.; Schurmann, M.; Jurkschat, K.; Michaelides, A.;
Skoulika, S.; Bakas, T.; Binolis, J.; Karkabounas, S.; et al. Synthesis, structural characterization and
in vitro cytotoxicity of organotin(IV) derivatives of heterocyclic thioamides, 2-mercaptobenzothiazole,
2
5
-chloro-2-mercaptobenzothiazole, 3-methyl-2-mercaptobenzothiazole and 2-mercaptonicotinic acid. J. Inorg.
Biochem. 2003, 96, 425–434. [CrossRef]
2
2
2
6. Banti, C.N.; Gkaniatsou, E.I.; Kourkoumelis, N.; Manos, M.J.; Tasiopoulos, A.J.; Bakas, T.; Hadjikakou, S.K.
Assessment of organotins against the linoleic acid, glutathione and CT-DNA. Inorg. Chim. Acta 2014, 423,
9
8–106. [CrossRef]
7. Xanthopoulou, M.N.; Kourkoumelis, N.; Hadjikakou, S.K.; Hadjiliadis, N.; Kubicki, M.; Karkabounas, S.;
Bakas, T. Structural and biological studies of organotin(IV) derivatives with 2-mercapto-benzoic acid and
-mercapto-4-methyl-pyrimidine. Polyhedron 2008, 27, 3318–3324. [CrossRef]
8. Hadjikakou, S.K.; Jurkschat, K.; Schurmann, M. Novel organotin(IV) compounds derived from
bis(organostannyl)methanes: Synthesis and crystal structures of bis[diphenyl(pyridin-2-onato)stannyl]methane
and bis[bromophenyl(pyrimidine-2-thionato) stannyl]methane C H . J. Organomet. Chem. 2006, 691,
7
8
1
637–1642. [CrossRef]
2
9. Xanthopoulou, M.N.; Hadjikakou, S.K.; Hadjiliadis, N.; Kubicki, M.; Skoulika, S.; Bakas, T.; Baril, M.;
Butler, I.S. Synthesis, structural characterization, and biological studies of six and five-coordinate
organotin(IV) Complexes with the Thioamides 2-Mercaptobenzothiazole, 5-Chloro-2-mercaptobenzothiazole,
and 2-Mercaptobenzoxazole. Inorg. Chem. 2007, 46, 1187–1195. [CrossRef] [PubMed]
3
0. Carraher, C.E., Jr.; Roner, M.R. Organotin polymers as anticancer and antiviral agents. J. Org. Chem. 2014
51, 67–82. [CrossRef]
,
7
3
1. Jones, L.H.; McLaren, E. Infrared Spectra of CH COONa and CD COONa and Assignments of Vibrational
3
3
Frequencies. J. Chem. Phys. 1954, 22, 1796–1800. [CrossRef]
3
3
2. Greenwood, N.N.; Gibb, T.C. Mossbauer Spectroscopy; Chapman and Hall: London, UK, 1971.
3. Saeed, M.A.; Badshah, A.; Rauf, M.K.; Craig, D.C.; Ali, S. catena-Poly[tributyltin(IV)-l-acetato]. Acta Cryst.
2
006, E62, m469–m471. [CrossRef]
3
3
4. Molloy, K.C.; Purcell, T.G.; Quill, K. Organotin biocides 1. The structure of triphenyltin acetate. J. Organomet.
Chem. 1984, 267, 237–241. [CrossRef]
5. Lee, A.V.; Oesterreich, S.; Davidson, N.E. MCF-7 Cells—Changing the Course of Breast Cancer Research and
Care for 45 Years. JNCI J. Nat. Cancer Inst. 2015, 107, Djv073. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 2055
18 of 18
3
3
6. Rochefort, H.; Glondu, M.; Sahla, M.E.; Platet, N.; Garcia, M. How to target estrogen receptor-negative breast
cancer? Endocr. Relat. Cancer 2003, 10, 261–266. [CrossRef] [PubMed]
7. Banti, C.N.; Papatriantafyllopoulou, C.; Manoli, M.; Tasiopoulos, A.J.; Hadjikakou, S.K. Nimesulide silver
metallodrugs, containing the mitochondriotropic, triaryl derivatives of pnictogen; Anticancer activity against
human breast cancer cells. Inorg. Chem. 2016, 55, 8681–8696. [CrossRef] [PubMed]
3
8. HCilião, L.; Ribeiro, D.L.; Camargo-Godoy, R.B.O.; Specian, A.F.L.; Serpeloni, J.M.; Cólus, I.M.S. Cytotoxic
and genotoxic effects of high concentrations of the immunosuppressive drugs cyclosporine and tacrolimus
in MRC-5 cells. Exp. Toxicol. Pathol. 2015, 67, 179–187.
3
9. Gao, E.; Lin, L.; Liu, L.; Zhu, M.; Wang, B.; Gao, X. Two new palladium(II) complexes: Synthesis,
characterization and their interaction with HeLa cells. Dalton Trans. 2012, 41, 11187–11194. [CrossRef]
[PubMed]
4
4
4
0. Boer, D.R.; Canals, A.; Coll, M. DNA-binding drugs caught in action: The latest 3D pictures of drug-DNA
complexes. Dalton Trans. 2009, 399–414. [CrossRef] [PubMed]
1. Gilad, Y.; Hanoch, S. Docking studies on DNA intercalators. J. Chem. Inf. Model. 2013, 54, 96–107. [CrossRef]
[
PubMed]
2. Lian, C.; Robinson, H.; Wang, A.-H. Structure of actinomycin D bound with (GAAGCTTC)2 and
GATGCTTC) and its binding to the (CAG) :(CTG) triplet sequence as determined by NMR analysis. J. Am.
(
n
n
2
Chem. Soc. 1996, 118, 8791–8801. [CrossRef]
4
3. Icsel, C.; Yilmaz, V.T.; Kaya, Y.; Samli, H.; Harrison, W.T.A.; Buyukgungor, O. New palladium (II)
0
and platinum (II) 5, 5-diethylbarbiturate complexes with 2-phenylpyridine, 2, 2 -bipyridine and 2,
0
2
-dipyridylamine: Synthesis, structures, DNA binding, molecular docking, cellular uptake, antioxidant
activity and cytotoxicity. Dalton Trans. 2015, 44, 6880–6895. [CrossRef] [PubMed]
4
4
4. Lauria, A.; Patella, C.; Dattolo, G.; Almerico, A.M. Design and Synthesis of 4-Substituted Indolo [3, 2-e][1,
2
, 3] triazolo [1, 5-a] pyrimidine Derivatives with Antitumor Activity. J. Med. Chem. 2008, 51, 2037–2046.
CrossRef] [PubMed]
5. Pages, B.J.; Ang, D.L.; Wright, E.P.; Aldrich-Wright, J.R. Metal complex interactions with DNA. Dalton Trans.
015, 44, 3505–3526. [CrossRef] [PubMed]
[
2
4
4
6. Oxford Diffraction. CrysAlis CCD and CrysAlis RED; Oxford Diffraction Ltd.: Abingdon, UK, 2008.
7. Burla, M.C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G.L.; de Caro, L.; Giacovazzo, C.;
Polidori, G.; Spagna, R. SIR2004: An improved tool for crystal structure determination and refinement. J.
Appl. Cryst. 2005, 38, 381–388. [CrossRef]
4
4
5
5
5
8. Sheldrick, G.M. SHELXL-2014/7, Program for Refinement of Crystal Structures; University of Göttingen:
Göttingen, Germany, 2014.
9. Farrugia, L.J. WinGX suite for small-molecule single-crystal crystallography. J. Appl. Cryst. 1999, 32, 837–838.
[CrossRef]
0. Bevington, P.R.; Robinson, D.K. Data Reduction and Error Analysis for the Physical Sciences; McGraw Hill: New
York, NY, USA, 1992; pp. 194–197.
1. OTrott, O.; Olson, A.J. AutoDockVina: Improving the speed and accuracy of docking with a new scoring
function, efficient optimization and multithreading. J. Comput. Chem. 2010, 31, 455–461.
2. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. Autodock4
and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 16,
2
785–2791. [CrossRef] [PubMed]
5
3. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Montgomery, J.A., Jr.;
Vreven, T.; Kudin, K.N.; Burant, J.C.; et al. GAUSSIAN 03 (Revision C.02); Gaussian, Inc.: Wallingford, CT,
USA, 2004.
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