Structure-activity relationships of new Organotin(IV) anticancer agents and their cytotoxicity profile on HL-60, MCF-7 and HeLa human cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2019.07.047

There is a growing interest in the cancer cell growth inhibitory effects of organotin (IV) compounds and, accordingly, a new series of dimethyl-, di-(n-butyl)-, diphenyl- and chloro-phenyl tin(IV) complexes with a Schiff base core were prepared. Their bin

Full text of DOI:10.1016/j.ejmech.2019.07.047

Accepted Manuscript
Structure-activity relationships of new Organotin(IV) anticancer agents and their
cytotoxicity profile on HL-60, MCF-7 and HeLa human cancer cell lines
Hussain Ullah, Viola Previtali, Helene B. Mihigo, Brendan Twamley, Muhammad
Khawar Rauf, Fatima Javed, Amir Waseem, Robert J. Baker, Isabel Rozas
PII:
S0223-5234(19)30668-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.047
EJMECH 11544
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2019
Revised Date: 27 June 2019
Accepted Date: 15 July 2019
Please cite this article as: H. Ullah, V. Previtali, H.B. Mihigo, B. Twamley, M.K. Rauf, F. Javed, A.
Waseem, R.J. Baker, I. Rozas, Structure-activity relationships of new Organotin(IV) anticancer agents
and their cytotoxicity profile on HL-60, MCF-7 and HeLa human cancer cell lines, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.047.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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Structure-Activity Relationships of New Organotin(IV)
Anticancer Agents and their Cytotoxicity Profile on
HL-60, MCF-7 and HeLa Human Cancer Cell Lines
a,b
c
c
b
Hussain Ullah, Viola Previtali, Helene B. Mihigo, Brendan Twamley, Muhammad
a
a,§
a,
b,
c,
Khawar Rauf, Fatima Javed, Amir Waseem, * Robert J. Baker * and Isabel Rozas *
a
Department of Chemistry, Quaid-i-Azam University Islamabad-45320, Pakistan.
b
School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
c
School of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160
Pearse Street, Dublin 2, Ireland
§
Present address: Department of Chemistry, Shaheed Benazir Bhutto Women University,
Peshawar 25000, Khyber Pakhtunkhwa, Pakistan.
Co-corresponding Authors: Amir Waseem, email: amir@qau.edu.pk;Robert J. Baker, email:
bakerrj@tcd.ie; Isabel Rozas, email: rozasi@tcd.ie.
*

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Graphical Abstract
Highlights
•
•
•
•
Organotin(IV) complexes have been prepared with diverse lipophilic substituents
Five of these complexes’ X-ray structures were solved confirming their coordination
Bis-(2-hydroxybenzylidene)malonohydrazone Sn(IV) complex do not bind to DNA
Di-(n-butyl) complexes show good cytotoxicity in HL-60, MCF-7 and HeLa cancer
cells
•
Bis-(2-hydroxybenzylidene)malonohydrazone Sn(IV) complex strongly induces
apoptosis

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Abstract
There is a growing interest in the cancer cell growth inhibitory effects of organotin (IV)
compounds and, accordingly, a new series of dimethyl-, di-(n-butyl)-, diphenyl- and chloro-
phenyl tin(IV) complexes with a Schiff base core were prepared. Their binding to DNA was
assessed by UV thermal denaturation showing no interaction and by UV-vis titration
exhibiting moderate interaction by intercalation. Complexes having n-butyl substituents were
more potent and cytotoxic against human leukemia, breast and cervical cancer cell lines than
other organotin(IV) complexes tested. Unfortunately, some of these compounds showed
similar cytotoxicity in a non-cancerous cell line. We may conclude that cytotoxic activity was
dependent on the nature (lipophilicity and size, according to the structure-activity relationship
studies) and substitution pattern on the different structures. These results may aid in the
rational design of metallodrugs, expanding the scope of organotin complexes in formulating
new metal based drugs with dibutyl moieties.
Keywords
Organotin(IV)complexes; anticancer agents; cell growth inhibition; HL-60; MCF-7; HeLa;
apoptosis; structure-activity relationships.
Abbreviations used
DNA: deoxyribonucleic acid; IC : concentration required for 50% inhibition of cell growth;
5
0
HL-60: human Caucasian promyelocytic leukemia cell line; MCF-7: human breast carcinoma
cell line; HeLa: human cervical cancer cell line; HEK293: human embryonic kidney cells;
∆Tm: change in DNA thermal melting temperature; st-DNA: salmon testes DNA; ct-DNA:
calf thymus DNA.

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1
. INTRODUCTION
The enormous success of cisplatin as antitumor agent triggered the search for new metal-
based complexes with application as anticancer therapies [1,2]. Metallo-organic complexes
are very versatile because of the inherent characteristics of both the metal core and the organic
ligand [3]. Moreover, because of their cationic nature, metal centres can nucleophilically
interact with some amino acids and nucleotides [4], thus targeting proteins and nucleic acids
such as DNA. Furthermore, if the organic moiety has aromatic character, DNA groove
binding or intercalation will be facilitated [5]. Additionally, organometallic complexes as a
whole can produce DNA cleavage by induction of reactive oxygen species (ROS) [6,7].
Among the non-platinum metal based drugs, organotin(IV) complexes are very appealing
because of their antimicrobial, antifungal and anticancer activities [8] and, compared to other
main-group metal complexes, they seem to show the most potent cytotoxic activities [9].
It has been reported that organotin(IV) complexes showed toxicity from moderate to high
levels based on the number, nature (polar or lipophilic) and bulkiness of the ligands attached
to the Sn(IV) atom [10]. In terms of the number of substituents and according to the literature,
the bioactivity potency of organotin compounds decrease in the following order:
3
+
2
2+
3+
R Sn >R Sn >RSn [11]. Different ligands have been used to form these bioactive Sn
complexes such as hydroxamic acids [12,13], carboxylates [14,15], cyclohexyl or thiolate
[16]. In particular, hydrazones, which by themselves can present a wide range of biological
applications as antimicrobial [17] and cytotoxic [18] agents, when coordinated to Sn(IV) by
the azomethine (R C=NNR ) moiety have shown to inhibit cancer cell growth [19]. Similarly,
2
2
Sn(IV) complexes of N(4)-phenyl-2-benzoylpyridine thiosemicarbazone have shown very
interesting cytotoxic activity against the MCF-7 (human breast carcinoma), TK-10 (human

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renal carcinoma) and UACC-62 (human melanoma) tumour cell lines [20]. Moreover, the
choice of ligands is crucial because they play a key role in transporting and directing
organotin complexes to the target site and determine their solubility and bioavailability.
All this previously reported evidence on the structural and biological diversity of organotin
derivatives inspired us to prepare a new series of Sn(IV) complexes. Thus, Schiff’s base
derivatives with the salicylaldehyde acyldihydrazone core and variable linker lengths were
chosen as ligands and different coordination were explored to investigate their potential as
DNA binders and cancer cell growth inhibitors. Thus, their binding to salmon testes DNA (st-
DNA) was studied using UV thermal melting experiments. Additionally, their activity against
human Caucasian promyelocytic leukemia (HL-60), human breast carcinoma (MCF-7) and
human cervical cancer (HeLa) cell lines was studied as well as their potential as anti-apoptotic
agents. These complexes conjugate both the presence of Sn, which is known to exert
cytotoxicity, along with aromatic systems that could facilitate DNA binding as well as
aliphatic ligands that could modulate the lipophilicity of the systems.
2
. RESULTS AND DISCUSSION
2
.1. Synthesis and characterization of the Sn complexes 1-10
The tin complexes 1-10 were readily prepared in high yields (Scheme 1) following a known
procedure [21] by deprotonation of the corresponding parent Schiff’s base ligands (11-14)
followed by the addition of either R SnCl or RSnCl (R = methyl, n-butyl, or phenyl) in
2
2
3
toluene (Scheme 1).

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Scheme 1
Conditions: (i) Et N, Toluene, 30 min; (ii) R SnCl or RSnCl , reflux, 6 hr
3
2
2
3
Complexes 2, 4-7 were further characterized by single crystal X-ray diffraction; single
crystals were grown by slow evaporation of a chloroform/hexane solution. All show the same
structural features and the structure of 2 is shown in Figure 1 as an example; the remainder are
collated in the supporting information (Figures S1-S7, ESI). The structure confirms our
proposed formulation of the products: the geometry around the tin in 2 and 4-7 is distorted
trigonal bipyramidal with the oxygen atoms in the axial position and the distortion from a
pure trigonal bypyramid (τ = 1) quantified [22] and similar to other examples in the literature
[23]; the remaining metric parameters are unexceptional compared to a survey of the
Cambridge Structural Database.

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…
Figure 1. (A) Solid state structure of 2, (B) spacefilling structure of 2 and (C) Sn O tetrel
interactions highlighted by red lines. Colour code: red = O; blue = N; grey = C; Sn = yellow;
hydrogen atoms omitted for clarity.
The structures of related compounds have been reported by us previously and we noted
…
intramolecular Sn O contacts that were shorter than the van der Waal radii and we
…
characterized as a tetrel Sn O bond [24,25]. This bonding is also noted in 2 (Figure 1C) and 4
(Figure S2, ESI) and the distances are given in Table 1. In 5 this interaction is not present and
we can ascribe this to the fact that short linkers between the oxidobenzyliden Schiff bases
gives pseudo-cis geometry whilst the longer linker in 5 gives a pseudo-trans displacement of
the tin atoms. Space filling models show that the tin is not completely sterically saturated and
the buried volume [26] can be calculated from the structures (Table 1); there are only small
variations in this metric.

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Table 1. Selected metric and structural parameters from the solid state structures of 2, 4-7
…
Complex
2
Sn-O (Å)
Sn-N (Å)
Sn-C (Å)
Sn O (Å)
τ
%Vbur
2.093(4)
2.196(5)
2.204(5)
2.127(7)
2.120(7)
2.113(6)
2.136(7)
3.461(4)
0.118
79.1
2.177(4)
2.190(4)
2.114(4)
4
2.070(4)
2.181(5)
2.191(5)
2.138(6)
2.127(6)
2.140(6)
2.131(6)
3.318(5)
0.007
80.1
2.130(4)
2.156(4)
2.104(4)
5
6
2.1196(17)
2.178(2)
2.150(3),
2.125(2)
2.122(2)
none
none
0.020
0.098
80.0
81.9
2
.1468(16)
2.067(3)
2.113(4)
2.117(3)
2.116(4)
2.119(4)
2.144(3)
2.140(2)
2.079(3)
7
2.0692(14) 2.1665(18)
2.122(2)
2.114(2)
2.115(2)
2.120(2)
none
0.120
82.7
2
2
2
.1359(14) 2.1649(17)
.0656(14)
.1210(14)
IR spectroscopy confirms the solid state structure by the disappearance of the OH, NH, and
CO bands from the precursors and small shifts of the ν(C=N) to lower wavenumbers and ν(N-
N) to higher wavenumbers consistent with a redistribution of electron density within the
-1
ligand. Additionally, new bands ascribed to ν(Sn-O) and ν(Sn-N) appear in the 400-200 cm
region [16,27,28].
Given that the biological testing (vide infra) will be in solution, multinuclear NMR
spectroscopy can be used to shed light on the solution structures of these species [29]; this is
1
particularly important given the σ-hole that is present at the tin centers in 2 and 4. The H
NMR spectra show the expected downfield shifts in the CH=N proton and the appearance of
1
19
2
Sn satellites to the Sn-CH R fragment; the size of these coupling constants ( J
≈ 58-
119Sn-1H
2
o
7
9 Hz for 1-5) suggest a C-Sn-C angle of 130 , using the work of Lockhart and Manders on

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1
Me Sn compounds [30,31], and indicative of a 5-coordinate geometry. The J
2
119Sn-13C
satellites (590-654 Hz) are also in agreement with this geometry [24]. More diagnostic has
1
19
been the Sn NMR spectra in both coordinating (d -DMSO) and non-coordinating (CDCl )
6
3
1
19
solvents. For reference, the Sn chemical shift of δ = -440 to -540 ppm indicates a seven
Sn
coordination number; δ = -210 to -400 ppm indicates a six-fold coordination geometry; δ
Sn
Sn
=
-90 to -190 ppm, penta-coordinate and δSn = 200 to -60 ppm is related to a coordination
number of 4 [25,32]. In all complexes a single resonance was observed shifted significantly
from the starting materials {δSn = +31 ppm (Me SnCl ); -27 ppm (Ph SnCl ); -61 ppm
2
2
2
2
(PhSnCl )} [10(c),26,29,33]. In compounds 1-5 this was observed at δ = -150 to -191 ppm
3 Sn
[34] and indicates that in solution the coordination number is 5 as expected from the structure
in the solid state and the tetrel interaction is not present in solution. For compounds 6-10 δ =
Sn
-330 ppm, although the change in the R groups can influence this. Thus, compound 11 had
limited solubility in CDCl , but in DMSO δ = -500 ppm, indicative of the coordination of
3
Sn
this solvent [35] to the coordinatively unsaturated tin center.
2
.2. DNA binding studies
Thermal denaturation experiments with unspecific st-DNA were performed, as previously
described by Rozas’ group [36], to explore the mechanism of action of the prepared
complexes. In general, the melting temperature (T ) of DNA increases upon ligand binding
m
because the stabilization provided to the double strand [37] and the shift in T (ꢀT ) is a
m
m
measure of the DNA-ligand interaction’s strength. Compound 2 was selected for this study as
a representative of the complexes prepared and it was found that at a 15 µM concentration this
complex did not induce an increment in T and thus no further thermal melting studies were
m
performed.

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Considering that UV-visible spectroscopy can provide clear indications of the interaction of
small molecules with DNA [36,38], the UV-vis titration of compound 2 was carried out with
ct-DNA. Thus, the spectra obtained from this experiment (Figure 2) shows that the addition of
DNA causes a rapid change of the sample’s spectrum due to the interaction established. The
characteristic UV-vis spectrum of 2 contains two bands; a strong absorption band in the near
UV region (300–330 nm) with λmax at 308 nm (triplet excited state of phenyl ring based π–π*
electronic transitions) and another band in the visible region (350-460 nm) with a lower
energy shoulder and λmax at 386 nm (π–π* and n–π* transition of the tridentate ligand).
Figure 2. Absorption spectra of 0.1 mM compound 2 in the absence and presence of ct-DNA.
[
(
DNA] = 0, 10, 20, 30, 40, 50, 60 µM. The downward arrows show the changes in absorbance
hypochromic effect) with respect to an increase in the ct-DNA concentration.

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DNA intercalation usually results in hypochromism and strong bathochromic shift (red shift)
of the ligand’s UV-vis spectrum due to the stacking interaction between the ligand’s planar
aromatic chromophore and the DNA base pairs [39,40]. In our case, by adding DNA the
absorption spectra of 2 show significant hypochromism at 386 nm resulting from the lowering
of π–π* and n–π* transition energy of the ligand in 2 in agreement with a stacking between
the DNA base pairs. However, instead of a red shift, a small blue shift from 386 to 381 nm
was observed. Thus, we cannot conclude that the mode of binding between 2 and DNA is
intercalative and other binding modes should not be discarded. The value of the binding
3
constant (K) was calculated to be 3.5 10 indicating a moderate interaction between complex 2
-1
and DNA [41]. Further, ∆G was assessed to be -18.5 kJ mol indicating a spontaneous
binding to DNA that could be due to an ionic interaction with the polyanionic phosphate
backbone [38].
2
.3. Cell growth inhibition and apoptosis studies
Considering the anticancer activity previously reported for Sn(IV) hydrazones [19,20], cell
viability studies were performed with some of the complexes prepared. Thus, AlamarBlue®
assays were carried out with three cancer cell lines (human Caucasian promyelocytic
leukaemia, HL-60; human breast carcinoma, MCF-7; and human cervical cancer, HeLa) using
carboplatin as control. The results are presented in Table 2. Excellent cell growth inhibition
was obtained for Sn(IV) complexes carrying n-butyl ligands (2-5) with IC50 values mostly
under the µM range and more effective than the control, carboplatin. The length of the linker
separating the Sn(IV) cores seems not to play any important role in the activity of these
complexes with very similar IC values. Diphenyl Sn(IV) complex 7 showed poor activity in
5
0
HL-60 and MCF-7 cell lines and, for that reason, was not tested in HeLa cells. Even though
Sn(IV) complexes carrying Cl ligands (9 and 10) were not highly cytotoxic, they showed an

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interesting selectivity, inhibiting cell growth more actively in MCF-7 and HL-60 than in HeLa
(
IC >10 µM). Compounds 2-5 were also more active in MCF-7 and HL-60 than in HeLa and
50
a number of factors can explain the different results obtained with the solid tumour cell lines.
For example, HeLa cells are folate receptor positive whereas MCF-7 cells are folate receptor
negative [42]; additionally, even though both have a wild type p53 gene, HeLa cells have
absent or at least diminished p53 function because of the HPV18-E6 presence which leads to
p53 degradation [43].
Table 2. Cell viability results (IC ± SEM, µM) obtained for complexes 2-5, 7 and 9-10
5
0
tested in HL-60 (leukemia), MCF-7 (breast) and HeLa (cervical) human cancer cell lines
1
2
N
1
2
3
4
2
2
3
R
R
HL-60
MCF-7
HeLa
0.53 ± 0.03
0.35 ± 0.02
0.41 ± 0.04
0.83 ± 0.07
NT
2
3
4
5
7
9
-(CH
2
2
2
2
)
3
CH
CH
CH
CH
3
3
3
3
-(CH
2
2
2
2
)
3
CH
CH
CH
CH
3
3
3
3
0.17 ± 0.06
0.13 ± 0.02
0.17 ± 0.02
0.20 ± 0.07
31.46 ± 3.84
5.59 ± 0.17
4.90 ± 0.71
26.45 ± 2.66
0.21 ± 0.01
0.17 ± 0.04
0.19 ± 0.04
0.14 ± 0.02
44.19 ± 3.21
4.48 ± 0.24
3.56 ± 0.53
115.5 ± 6.39
-(CH
-(CH
-(CH
)
3
)
3
)
3
-(CH
-(CH
-(CH
)
3
)
3
)
3
Ph
Ph
Ph
Ph
Cl
Cl
17.42 ± 1.31
18.06 ± 1.02
NT
1
0
Carboplatin
NT: Not tested
Next, flow cytometric analysis on HL-60 cells was performed to assess potential induction of
apoptosis (Figure 3 and Table 3). Thus, considering the IC values obtained and the slightly
5
0
better selectivity exhibited by 2 towards the HL-60 cell line, this compound was chosen for
the study.

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Vehicle – H O
Carboplatin (25 µM)
Carboplatin (50 µM)
2
Vehicle - EtOH
Compound 2 (0.1 µM)
Compound 2 (1 µM)
Figure 3. Plots representing the percentage of cells in each step of the cell cycle in HL-60
cells for carboplatin and 2. Cells with sub-diploid DNA contents were deemed apoptotic.
Results represent the mean ± S.E.M for at least 3 experiments.

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Thus, we observed that after 72 hours, both at 25 or 50 µM, carboplatin had little effect on
cell viability as indicated by the proportion of cells in the apoptotic (M1) peak (Table 3).
However, at much lower concentrations (0.1 and 1 µM), compound 2 showed a potent
apoptosis induction with 13.4 ±3.4% and 82.1 ±4.4% of cells in M1, respectively; as a result,
there was a reduction in the number of cells in the M3 S phase and M2 gap phase (Table 3).
No significant change was detected in the polyploidy DNA content (G peak) during the dose-
n
response experiment with carboplatin and 2.
Table 3. Effects of the control (carboplatin) and Sn(IV) complex 2 on the cycle of the HL-60
cells. Cells were treated with different concentrations of each compound and after 72 hours
analysed by flow cytometry as described in the text and experimental section. Mean values ±
S.E.M. of the proportion of cells of each phase of the cell cycle from at least three
independent experiments are shown with M1 = pre-G (<2N DNA), M2 = G /G (2N DNA),
1
0
1
M3 = S (2N - 4N DNA), M4 = G /M (4N DNA) and M5 = G (>4N DNA). Percentage
2
n
apoptosis is determined from the M1 (pre-G1) peak.
Conc. (µM)
Carboplatin
Vehicle
M1 (pre-G ) M2 (G -G )
M3 (S)
M4 (G -M)
M5 (Gn)
1
0
1
2
2.7 ± 1.3
61.5 ± 1.3
62.6 ± 1.1
76.5 ± 1.5
15.6 ± 1.7
5.4 ± 0.6
4.3 ± 0.9
18.3 ± 2.6
5.4 ± 1.7
3.2 ± 1.1
4.6 ± 0.8
2.1 ± 0.7
1.4 ± 0.5
2
5
5
0
28.1 ± 1.3
20.1 ± 3.0
2
Vehicle
.1
8.4 ± 1.0
13.4 ± 3.4
82.1 ± 4.4
58.3 ± 1.2
56.9 ± 2.2
10.8 ± 1.4
17.9 ± 0.4
15.1 ± 0.9
4.8 ± 1.8
14.8 ± 0.3
14.0 ± 0.6
2.3 ± 1.4
1.4 ± 0.2
1.9 ± 0.1
0.0 ± 0.0
0
1
Therefore, compound 2 induces apoptosis in an 82% at a concentration of 1 µM indicating
that the very good cell growth inhibition observed is exerted by programmed cell-death.
Considering the high structural similarity among complexes 2 to 5 it seems reasonable to
consider that all these compounds induce the strong cytotoxicity observed through apoptosis.

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Finally, the cytotoxicity of compound 2 (as an example of the organotin compounds prepared)
towards a non-cancerous cell line (HEK293, human embryonic kidney cells) was studied.
Unfortunately, compound 2 showed to be completely toxic in this cell line.
2
.4. Structure Activity Relationships
In order to established some structure activity relationships (SAR) of the compounds studied,
different physicochemical and ADME properties have been calculated at theoretical level with
the help of tools such as Marvin (ChemAxon [44]) and SwissADME [45]. The parameters
calculated for compounds 2-5, 7 and 9-10 are presented in Table S4 (ESI). According to these
parameters, all compounds were insoluble to poorly soluble in water (as found
experimentally), showed a promising gastro-intestinal absorption potential, no BBB crossing
ability and were not substrate for different members of the CYP family. In addition, they did
not show a great potential in terms of drug or lead likeness. However, for all compounds
tested we found that lipophilicity parameters as well as van der Waals volume and molar
refractivity (steric parameters) are linearly related to the cytotoxicity [log(IC )] for the three
5
0
cancer cell lines. An exception was compound 7 which was an outlier and, considering its low
cytotoxicity, was excluded.
Thus, regarding lipophilicity, the best correlations were found with the Marvin “cLogP”
parameter [(IC :HL-60) = -5.75 ln(“cLogP”) + 8.54, R² = 0.97; (IC :MCF-7) = -4.41
5
0
50
ln(“cLogP”) + 6.58, R² = 0.98; and (IC :HeLa) = -19.15 ln(“cLogP”) + 28.51, R² = 0.94].
5
0
These indicate that “cLogP” values between 3.9 and 4.7 are ideal for cell-growth inhibition of
the cancer cell lines studied. The “Consensus-LogP” parameter (SwissADME) also correlated
well with the cytotoxicity values [ (IC :HL-60) = -13.26 ln(“Consensus-LogP”) + 25.26, R² =
5
0
0
.95; (IC :MCF-7) = -10.36 ln(“Consensus-LogP”) + 19.33, R² = 0.96; and (IC :HeLa) = -
50 50

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4
4.41 ln(“Consensus-LogP”) + 84.65, R² = 0.94] also indicating that large “Consensus-logP”
values favour cancer cell cytotoxicity. Regarding steric factors a trend was also found; thus,
3
molecules with van der Waals volumes (Marvin) between 622-674 Å gave lower IC values
5
0
3
in the three cell lines than those with values between 520-555 Å . Additionally, compounds
3
-1
with large molar refractivity (MR, SwissADME) values, i.e. 197-216 m mol , show better
3
-1
anticancer activity than those with MR values between 180-189 m mol .
Even though a clear target cannot be identified to explain the cell-growth inhibitory activity of
the compounds tested, considering the σ-hole intramolecular interactions that were observed
in compounds 2 and 4, such a non-covalent interaction cannot be discarded. This effect has
been previously observed in a number of biological interactions, in general, and tin-protein
complexes, in particular. Thus, non-covalent carbon bonding interactions through σ-holes
have been reported by Biswal and collaborators [46]; the σ-hole of thiophene has been
suggested as the driving force for pre-organised specific recognition of G-C in the DNA
minor groove [47]; Frontera et al. have reported theoretical and experimental evidence of the
presence of S···Sn tetrel bonds that may play an important role in the interaction between
PPARs (peroxisome proliferator-activated receptors) and organotin compounds [48].
3
. CONCLUSSIONS
Tin(IV) complexes of bis-Schiff base cores (separated by linkers of different lengths) with
dimethyl-, di-(n-butyl)-, diphenyl- and chloro-phenyl ligands have been prepared and fully
1
13
1
119
characterised by spectroscopic methods ( H, C{ H},
Sn NMR, HRMS, and IR
spectroscopies) and x-ray crystal structure analysis.

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Their potential binding to DNA as a therapeutic target was assessed by means of UV DNA
thermal denaturation finding that these compounds do not interact with DNA. Further, the
interaction of compound 2 to DNA was assessed with UV-vis absorption titrations, which
3
showed a moderate binding (with a constant in the order of 10 ).
Cytotoxicity studies against three cell lines (HL-60, MCF-7 and HeLa) by means of cell
viability assays showed that compounds with n-butyl ligands (2-5) gave better cytotoxicity
responses (100 nM level) than those carrying a phenyl and a chloride (10-11, IC values at
5
0
µM level). Additionally, the distance between the Schiff bases cores did not affect the
biological response. Compound 7 gave very poor results both in HL-60 and MCF-7 cancer
cell lines. Compound 2 induces 82% apoptosis in HL-60 cells at a concentration of 1 µM after
7
2 h. Unfortunately, high toxicity was observed with the non-cancerous cell line HEK293.
SAR analysis of the relation between different physicochemical and ADME properties with
the biological response indicates that both lipophilicity and size play a positive role in the
cell-growth inhibition. Even though no clear target can be identified to explain the good
biological results obtained, a mechanism of action such as σ-hole non-covalent interactions
cannot be discarded. These results will enable us to rationalise the design parameters for new
organotin(IV) derivatives with improved anticancer activity.
4
. EXPERIMENTAL SECTION
4
.1. Materials and methods
All chemicals including solvents and reactant materials were purchased from sigma Aldrich
1
13
1
119
and were used without further purification. H, C{ H} and Sn NMR spectra were obtained

ACCEPTED MANUSCRIPT
on a Bruker Avance 400 and Bruker DPX 400, resonance operating at 400.23, 155.54 and
1
13
1
49.2 MHz respectively, and were referenced to the residual H and C resonances of the
solvent used and internal standard SiMe . The δ values were expressed in ppm units
4
andassignments were verified using two dimensional (2D) experiments. Proton-carbon
positions of the aromatic systems are indicated by Arabic numbers while those for the
methylene bridges are indicated by Greek letters. Infrared (IR) spectra were recorded on
Bruker Tensor II spectrometer with attenuated total reflectance (ATR) accessory.High
resolution mass spectrometry analysis were carried out using a Premier Waters Maldi-
quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with Z-spray electrospray
ionization (ESI) and Mareiz Assisted Laser Desorption Ionization (MALDI) sources.
For all crystal data reported, the X-ray intensity data Bruker D8 Quest Eco using Mo Kα
radiation (λ = 0.71073 Å). Each sample was mounted on a MiTeGen cryoloop and data
collected at 290(2) K for 2 and 100(2) K for 4-7 using an Oxford Cryostream cryosystem.
Bruker APEX [49] software was used to collect and reduce data and determine the space
group. The structures were solved using intrinsic phasing [50] and refined with least squares
minimization [51] in Olex2 [52]. Absorption corrections were applied using SADABS [53].
Details of data collections as well as crystal data, and refinement are given in Supporting
Information (Table S3). CCDC 1907744-1907748 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif
The ligands were prepared via literature procedures [54]. All chemicals and solvents were
obtained from commercial sources and used as received.

ACCEPTED MANUSCRIPT
All tissue culture medium and constituents were purchased from Gibco Europe Ltd. (Paisley,
Scotland) and tissue culture plastic was obtained from Cruinn Diagnostic Ltd. (Dublin,
Ireland). Carboplatin was purchased from Sigma Aldrich (St Louis, MO, USA).
AlamarBlue® Cell Viability Reagent was purchased from Thermo Fisher. The salmon sperm
DNA [(ss)DNA] was purchased from Sigma Aldrich (St Louis, MO, USA) and has an
-1
-1
extinction coefficient ε = 6600 M cm .
2
60
4
4
.2.
Synthesis
.2.1. Synthesis of Complexes 1 to10
Organotin(VI) complexes 1-10 were prepared in good to excellent yields following the
procedure previously reported [24]. The ligand (0.30 g, 1 equiv.) was reacted with 4 equiv. of
Et N in dry toluene and stirred for 30 min at room temperature. Two equivalents of R SnCl
2
3
2
or RSnCl were added and the mixture was refluxed for 6 h to yield a yellow solution and a
3
white precipitate. Filtration and removal of the solvent afforded the crude product, which was
recrystallized from a 3:2 vol/vol mixture of chloroform and hexane. Single crystals suitable
for X-ray diffraction were obtained by slow evaporation of the solvent mixture.
4
.2.1.1. Bis-[dimethyltin(IV)]-N',N'-4-bis(-2-oxidobenzylidene)malonohydrazide (1)
o
-1
Yield: 0.25 g, 83%. M.p. 238-240 C. IR ν (cm ): 1607 (s, C=N), 1544 (m, C-O), 1503 (m),
1
473 (m), 1442 (m), 1364 (m), 1361 (m), 1295 (s, Ar-O),1162 (m), 997 (w, N-N), 970 (w),
04 (m), 758 (m), 603 (m, Sn-C), 564 (m), 556 (m, Sn-O), 524 (m, Sn-C), 465 (m, Sn-N), 327
9
1
2
119/117
1
(
m), 320 (m). H NMR (400 MHz,CDCl ) δ(ppm): 0.81 (s, 12H, J (
Sn- H) = 79/76 Hz,
3
3
3
SnCH ), 3.35 (s, 2H, α), 6.74 (t, 2H, J
= 7.9 Hz, H4), 6.77 (d, 2H, J
= 7.9 Hz, H2),
3
H-H
H-H
3
3
119/117
1
7
.15 (d, 2H, J = 7.9 Hz, H5), 7.35 (t, 2H, 7.9 Hz, H3), 8.64 (s, 2H, J (
Sn- H) = 45
H-H
13
1
1
119/117
13
Hz, H7). C{ H} NMR (155 MHz, CDCl ) δ(ppm): 1.6 ( J (
Sn- C) = 644/ 620 Hz,
3

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SnCH ) 40.6 (Cα), 116.3 (C6), 117.3 (C4), 121.7 (C2), 134.3 (C5), 135.5 (C3), 162.2 (C7),
3
1
19
1
66.4 (C1), 174.8 (C8). Sn NMR (149 MHz, CDCl ) δ(ppm): -152.74. HRMS (MALDI):
3
+
calculated for C H N O Sn [M ]: 635.9842, found: 635.9936.
2
1
24
4
4
2
4
.2.1.2. Bis-[di-n-butyltin(IV)-N',N'-4-bis(-2-oxidobenzylidene)malonohydrazide (2)
o
-1
Yield 0.27 g, 90%.M.p. 100-101 C. IR ν (cm ): 1605 (s, C=N), 1542 (m, C-O), 1514 (m),
471 (m), 1443 (m), 1360 (m), 1348 (m),1300 (s, Ar-O),1200 (m), 1148 (m), 1128 (m) 1000
1
(w, N-N), 962 (w), 900 (m), 752 (m), 673 (m), 601 (m, Sn-C), 564 (m, Sn-O), 515 (m, Sn-C),
1
4
86 (m, Sn-N), 453 (m), 340 (m), 302 (m). H NMR (400 MHz,CDCl ) δ(ppm): 0.88 (t, 12H,
3
3
3
4
119/117
1
JH-H = 7.4 Hz, δ'), 1.35 ( sextet , 8H, J
= 6.73 Hz, J(
Sn- H) = 64/52 Hz, γ'), 1.50
H–H
3
3
119
1
2
119/117
1
(
m, 8H, J
= 8.4 Hz, J( Sn- H) = 28 Hz, β'), 1.66 (m, 8H, J(
Sn- H) = 74/70 Hz,
H-H
3
3
α'), 3.36 (s, 2H, α), 6.72 (t, 2H, J = 8.0 Hz, H4), 6.77 (d, 2H, J = 8.0 Hz, H2), 7.12 (d,
H-H
H-H
3
3
119
1
2
H, JH-H = 8.0 Hz, H5), 7.34 (t, 2H, J
= 8.0 Hz, H3), 8.63 (s, 2H, J( Sn- H) = 43 Hz,
H-H
1
3
1
1
119/117
13
13
H7). C{ H} NMR (155 MHz, CDCl ) δ(ppm): 13.6 (Cδ'), 22.2 ( J(
Sn- C) = 598/578
3
3
119/117
13
2
119/117
Hz , Cα'), 26.5 ( J(
Sn- C) = 96/86 Hz, Cγ'), 26.8 ( J(
Sn- C) = 34 Hz, Cβ'), 40.5
(Cα), 116.4 (C6), 116.9 (C4), 121.7 (C2), 134.3 (C5), 135.3 (C3), 161.7 (C7), 167.8 (C1),
1
19
1
71.2 (C8). Sn NMR (149 MHz,CDCl ) δ(ppm): -190.3. HRMS (MALDI): calculated for
3
+
C H N O Sn [M ]: 804.1720, found: 804.1855.
33
48
4
4
2
4
.2.1.3. Bis-[di-n-butyltin(IV)]-N',N'-4-bis(-2-oxidobenzylidene)succinohydrazide (3)
-1
Yield: 0.23 g, 76%. Oil. IR ν (cm ): 1606 (s, C=N), 1544 (m, C-O), 1515 (m), 1472 (m),
1
9
444 (m), 1362 (m), 1349 (m),1302 (s, Ar-O),1202 (m), 1150 (m), 1129 (m), 1001 (w, N-N),
64 (w), 899 (m), 754 (m), 674 (m), 603 (m, Sn-C), 566 (m, Sn-O), 514 (m, Sn-C), 487 (m,
1
3
Sn-N), 454 (m), 341 (m), 303 (m). H NMR (400 MHz, CDCl ) δ(ppm): 0.84 (t, 12H, JH-H
=
3
3
4
119
1
7
.5 Hz, δ'), 1.31 (sextet, 8H, J
= 6.73 Hz, J( Sn- H) = 62/50 Hz, γ’), 1.44 (t, 8H,
H–H

ACCEPTED MANUSCRIPT
3
119
1
3
J( Sn- H) = 28 Hz, β'), 1.61 (m, 8H, α'), 2.65 (s, 4H, α), 6.66 (t, 2H, J
= 8.0 Hz, H4),
H-H
3
3
3
6
.73 (d, 2H, J = 8.0 Hz, H2), 7.0 (d, 2H, J = 8.0 Hz, H5), 7.30 (t, 2H, J = 8.0 Hz,
H-H
H-H
H-H
3
119
1
13
1
H3), 8.56 (s, 2H, J( Sn- H = 43 Hz, H7). C{ H} NMR (155 MHz, CDCl ) δ(ppm): 13.5
3
1
119/117
13
3
119/117
13
(
Cδ'), 22.0 ( J (
Sn- C) = 604/576 Hz, Cα'), 26.5 ( J (
Sn- C) = 98/86 Hz, Cγ),
2
119/117
13
2
6.8 ( J (
Sn- C) = 36 Hz, Cβ'), 30.7 (Cα), 116.4 (C6), 116.8 (C4), 121.9 (C2), 134.0
1
19
(
C5), 134.9 (C3), 160.9 (C7), 166.9 (C1), 174.8 (C8). Sn NMR (149 MHz,CDCl ) δ(ppm): -
3
+
1
91.34. HRMS (MALDI): calculated for C H N O Sn [MH ]: 819.1954, found: 819.1951.
3
4
51
4
4
2
4
.2.1.4. Bis-[di-n-butyltin(IV)]-N',N'-4-bis(-2-oxidobenzylidene)glutarohydrazide (4)
o
-1
Yield: 0.23 g, 76%. M.p. 70-71 C. IR ν (cm ): 1607 (s, C=N), 1550 (m, C-O), 1516 (m),
1
9
469 (m), 1444 (m), 1363 (m), 1339 (m),1300 (s, Ar-O),1205 (m), 1160 (m),) 1014 (w, N-N),
93 (w), 901 (m), 760 (m), 732 (m), 674 (m), 693 (m, Sn-C), 573 (m, Sn-O), 513 (m, Sn-C),
1
4
64 (m, Sn-N), 440 (m), 348 (m), 284 (m). H NMR (400 MHz,CDCl ) δ(ppm): 0.88 (t, 12H,
3
3
3
4
119
1
JH-H = 7.2 Hz, δ'), 1.34 (sextet, 8H, J
= 7.5 Hz, J( Sn- H) = 64, 48 Hz, γ’), 1.47 (m, 8H,
H–H
3
119
1
2
119
1
3
J( Sn- H) = 27 Hz, β'), 1.63 (m, 8H, J( Sn- H) = 70, 64 Hz, α'), 2.00 (quint, 2H, J
=
H-H
3
3
3
8
.1 Hz, β), 2.39 (t, 4H, J = 8.1 Hz, α), 6.70 (t, 2H, J = 7.9 Hz, H4), 6.76 (d, 2H, J
H-H
H-H
H-H
3
3
3
=
7.9 Hz, H2), 7.12 (d, 2H, JH-H = 7.9 Hz, H5), 7.3 (t, 2H, J = 7.9 Hz, H3), 8.58 (s, 2H, J
H-H
1
19
1
13
1
1
(
(
(
(
Sn- H) = 42 Hz, H7). C{ H} NMR (155 MHz, CDCl ) δ(ppm): 13.6 (Cδ'), 22.0 ( J
3
1
1
19/117
13
3
119/117
13
2
Sn- C) = 602/575 Hz, Cα'), 23.0 (Cβ), 26.5 ( J(
Sn- C) = 88 Hz, Cγ), 26.9 ( J
19/117
13
Sn- C) = 35 Hz, Cβ'), 30.1 (Cα), 116.5 (C6), 116.8 (C4), 121.6 (C2), 134.2 (C5), 135.0
1
19
C3), 160.3 (C7), 166.8 (C1), 175.5 (C8). Sn NMR (149 MHz,CDCl ) δ(ppm): -191.85.
3
+
HRMS (MALDI): calculated for C H N O Sn [MH ]: 833.2111, found: 833.2148.
3
5
53
4
4
2

ACCEPTED MANUSCRIPT
4
.2.1.5. Bis-[di-n-butyltin(IV)]-N',N'-4-bis(-2-oxidobenzylidene)adipohydrazide (5)
o
-1
Yield: 0.25 g, 83%. M.p. 85-86 C. IR ν (cm ): 1604 (s, C=N), 1547 (m, C-O), 1516 (m),
1
9
473 (m), 1449 (m), 1364 (m), 1339 (m), 1297 (s, Ar-O), 1202 (m), 1156 (m), 999 (w, N-N),
97 (w), 899 (m), 757 (m), 729 (m), 694 (m, Sn-C), 574 (m, Sn-O), 514 (m, Sn-C), 465 (m,
1
3
Sn-N), 440 (m), 352 (m), 281 (m). H NMR (400 MHz,CDCl ) δ(ppm): 0.88 (t, 12H, JH-H
=
3
3
4
119/117
1
7
.5 Hz,δ'), 1.35 (sextet, 8H, J
= 7.29 Hz, J(
Sn- H) = 60, 50 Hz, γ'), 1.47(m,
H–H
3
119
1
2
119/117
1
J( Sn- H) = 34 Hz, β'), 1.63 (m, 8H, J(
Sn- H) = 70/60 Hz, α'), 1.75 (m, 4H, β), 2.35
3
3
(
m, 4H,α), 6.71 (t, 2H, JH-H = 8.1 Hz, H4), 6.76 (d, 2H, J
= 8.1 Hz, H2), 7.13 (d, 2H,
H-H
3
3
3
119/117
1
JH-H = 8.1 Hz, H5), 7.32 (t, 2H, J = 8.1 Hz, H3), 8.58 (s, 2H, J (
Sn- H) = 42 Hz,
H-H
1
3
1
1
119/117
13
H7). C{ H} NMR (155 MHz, CDCl ) δ(ppm): 13.6 (Cδ'), 22.0 ( J (
Sn- C) = 606/578
3
3
119/117
13
2
119/117
13
Hz, Cα')), 26.2 (Cβ), 26.5 ( J (
Sn- C) = 86 Hz, Cγ'), 26.9( J (
Sn- C) = 35 Hz,
Cβ'), 34.3 (Cα), 116.5 (C6), 116.8 (C4), 121.6 (C2), 134.2 (C5), 135.0 (C3), 160.6 (C7),
119
1
66.8 (C1), 175.9 (C8). Sn NMR (149 MHz,CDCl ) δ(ppm): -191.4. HRMS (MALDI):
3
+
calculated for C H N O Sn [MH ]: 847.2267, found: 847.2309.
3
6
55
4
4
2
4
.2.1.6. Bis-[diphenyltin(IV)][N',N'-4-bis(-2-oxidobenzylidene)malonohydrazide] (6)
o
-1
Yield: 0.24 g, 82%. M.p.184-186 C. IR ν (cm ): 1604 (s, C=N), 1541 (m, C-O), 1512 (m),
471 (m), 1442 (m), 1410, 1356 (m), 1338 (m), 1298 (s, Ar-O), 1200 (m), 1148 (m), 1013 (w,
1
N-N), 999 (w), 898 (m), 760 (s), 731 (m), 661 (m), 574 (m), 565 (m, Sn-O), 507 (m), 456 (m,
1
Sn-N), 421 (m), 346 (s), 309 (m), 271 (m, Sn-C), 232 (m, Sn-C). H NMR (CDCl ) δ(ppm):
3
3
3
3
2
.69 (s, 2H, α), 6.80 (t, 2H, J
= 7.7 Hz, H4), 7.1 (d, 2H, J
= 7.7 Hz, H2), 7.14 (d,
H−H
H−H
3
3
H, J
= 7.7 Hz, H5), 7.36 (m, 4H, para-Ar), 7.4 (m, 8H, meta-Ar), 7.46 (t, 2H, J
= 7.7,
H−H
H−H
3
2
119/117
1
3
119
1
H3), 7.87 (d, 8H, J =7.5, J (
Sn- H) = 87/73 Hz, ortho-Ar), 8.56 (s, 2H, J ( Sn- H)
H-H
1
3
1
=
50 Hz, H7). C{ H} NMR (CDCl ) δ(ppm): 40.8 (Cα), 116.4 (C6), 117.5 (C4), 122.0 (C2),
3
3
119/117
13
4
119/117
13
1
28.8 ( J (
Sn- C) = 87 Hz, meta-Ar), 130.5 ( J (
Sn- C) = 17 Hz, para-Ar), 134.4

ACCEPTED MANUSCRIPT
2
119/117
13
(
C5), 135.6 (C3), 136.3 ( J(
Sn- C) = 52 Hz, ortho-Ar), 138.8 (ipso-Ar), 161.8 (C7),
1
19
1
67.0 (C1), 171.1(C8). Sn NMR (CDCl ) δ(ppm): -330.2. HRMS (MALDI): calculated for
3
+
C H N O Sn [M ]: 884.0468, found: 884.0485.
41
32
4
4
2
4
.2.1.7. Bis-[diphenyltin(IV)][N',N'-4-bis(-2-oxidobenzylidene)succinohydrazide] (7)
o
-1
Yield: 0.24 g, 82%. M.p. 244-246 C. IR ν (cm ): 1606 (s, C=N), 1524 (m, C-O), 1470 (m),
1
9
442 (m), 1431 (m), 1358 (m), 1298 (s, Ar-O), 1262 (m), 1200 (m), 1150 (m), 1013 (w, N-N),
97 (w), 899 (m), 795 (s), 757 (m), 730 (m), 694 (m), 661(m), 568 (m, Sn − O), 514 (m), 445
1
(
m, Sn-N), 385 (m), 336 (m), 282 (m, Sn − C), 230 (m, Sn − C). H NMR (CDCl ) δ(ppm):
3
3
3
2
2
8
.98 (s, 4H, α), 6.80 (t, 2H, J
= 8.2 Hz, H4), 7.1 (d, 2H, J
= 8.2 Hz, H2), 7.15 (d,
H−H
H−H
3
3
H, J
= 8.2 Hz, H5), 7.38 (m, 4H, para-Ar), 7.40 (m, 8H, meta-Ar), 7.45 (t, 2H, J
=
H−H
H−H
3
119/117
3
3
.2 Hz, H3), 7.84 (d, 8H, J
Sn−H= 88/73 Hz, J
= 7.5 Hz, ortho-Ar), 8.56 (s, 2H, J
H−H
1
19
1
13
1
(
Sn- H) = 52 Hz, H7). C{ H} NMR (CDCl ) δ(ppm): 30.8 (Cα), 116.6 (C6), 117.5 (C4),
3
3
119/117
13
4
119/117
13
1
22.0 (C2), 128.9 ( J (
Sn- C)= 86 Hz, meta-Ar), 130.5 ( J (
Sn- C) = 18 Hz, para-
2
119/117
13
Ar), 134.4 (C5), 135.4 (C3), 136.2 ( J (
Sn- C) = 54 Hz, ortho-Ar), 139.0 (ipso-Ar),
1
19
1
61.0 (C7), 167.0 (C1), 174.6 (C8). Sn NMR (CDCl ) δ(ppm):-332.2. HRMS (MALDI):
3
+
calculated for C H N O Sn [M ]: 898.0624, found: 898.0667.
4
2
34
4
4
2
4
.2.1.9. Bis-[phenylchlorotin(IV)][N',N'-4-bis(-2-oxidobenzylidene)malonohydrazide] (8)
-1
Yield: 0.22 g, 80%. Oil. IR ν (cm ): 1606 (s, C=N), 1543 (m, C-O), 1518 (m), 1472 (m),
1
9
444 (m), 1431 (m), 1359 (m), 1294 (s, Ar-O), 1260 (m), 1200 (m), 1157 (m), 1034 (w, N-N),
99 (w), 908 (m), 804 (s), 759 (m), 738 (m), 696 (m), 615(m), 585 (m, Sn − O), 454 (m, Sn-
1
N), 325 (m, Sn-Cl), 276 (m, Sn − C), 258 (m), 236 (m, Sn − C). H NMR (CDCl ) δ(ppm):
3
3
3
3
.65 (s, 2H, α), 6.77 (t, 2H, J
= 7.5 Hz, H4), 6.86 (d, 2H, J
= 7.5 Hz, H2), 7.11 (d, 2H,
H−H
H−H
3
3
JH-H = 7.5 Hz, H5), 7.37 (m, 6H, para-Ar, meta-Ar), 7.54 (t, 2H, JH−H = 7.5 Hz, H3), 7.87

ACCEPTED MANUSCRIPT
3
3
119/117
1
3
119/117
1
(
d, 4H, J
= 6.5, J (
Sn- H)= 75 Hz, ortho-Ar), 8.51 (s, 2H, J (
Sn- H) = 53 Hz,
H−H
1
3
1
H7). C{ H} NMR (CDCl ) δ(ppm): 45.6 (Cα), 117.35 (C6), 118.2 (C4), 122.0 (C2), 128.8
3
3
119/117
13
2
(
(
J (
Sn- C) = 80 Hz, meta-Ar), 130.5 (para-Ar), 134.5 (C5), 135.6 (C3), 136.2 ( J
1
19/117
13
119
Sn- C, ortho-Ar) = 50 Hz), 138.0 (ipso-Ar), 161.0 (C7), 167.0 (C1), 171.1 (C8). Sn
NMR (CDCl ) δ(ppm): -330.0.
3
4
.2.1.10. Bis-[phenylclorotin(IV)][N',N'-4-bis(-2-oxidobenzylidene)succinohydrazide] (9)
o
-1
Yield: 0.25 g, 85%. M.p. 166-168 C. IR ν (cm ): 1604 (s, C=N), 1544 (m, C-O), 1516 (m),
470 (m), 1445 (m), 1430 (m), 1358 (m), 1290 (s, Ar-O), 1262 (m), 1199 (m), 1156 (m), 1036
1
(w, N-N), 997 (w), 905 (m), 801 (s), 757 (m), 735 (m), 694 (m), 612(m), 582 (m, Sn-O), 450
1
(
m, Sn-N), 326 (m, Sn-Cl), 274 (m, Sn-C), 254 (m), 234 (m, Sn-C). H NMR (DMSO-d )
6
3
3
δ(ppm): 2.72 (s, 4H, α), 6.79 (t, 2H, J
= 7.7 Hz, ArH4), 7.2 (d, 2H, J
= 8.2 Hz,
H–H
H–H
3
ArH2), 7.4 (d, 2H, J
= 7.7 Hz, ArH5), 7.50-7.56 (m, 4H, para-Ar , H3), 7.71 (m, 4H,
H–H
3
3
119
1
3
meta-Ar), 7.83 (d, 4H, J
= 8.3 Hz, J( Sn- H) = 122/110 Hz, ortho-Ar), 8.80 (s, 2H, J
H–H
1
19/117
1
13
1
(
Sn- H) = 84 Hz, H7). C{ H} NMR (DMSO-d ) δ(ppm): 30.66 (Cα), 117.7 (C6),
6
3
119/117
13
4
119/117
13
1
18.2 (C4), 122.2 (C2), 128.9 ( J (
Sn- C) = 123 Hz, meta-Ar), 130.1 ( J (
Sn- C)
2
119/117
13
=
16 Hz, para-Ar), 134.7 (C5), 135.1 (C3), 135.2 ( J (
Sn- C) = 67 Hz, ortho-Ar),144.0
1
19
(ipso-Ar), 158.0 (C7), 165.0 (C1), 172.1 (C8).
Sn NMR (DMSO-d ) δ(ppm): -513.4.
6
+
HRMS (MALDI): calculated for C H Cl N O Sn Na [M-Na ]: 836.9116, found: 836.9098.
3
0
24
2
4
4
2
4
.2.1.11.
hydrazide] (10)
Yield: 0.22 g, 80%. Oil.IR ν (cm ): 1604 (s, C=N), 1552 (m, C-O), 1517 (m), 1468 (m), 1444
Bis-[phenylchlorotin(IV)]
[N',N'-4-bis(-2-oxidobenzylidene)glutataro
-1
(
m), 1363 (m), 1339 (m),1301 (s, Ar-O),1205 (m), 1160 (m),) 1034 (w, N-N), 993 (w), 901
m), 760 (m), 732 (m), 674 (m), 693 (m, Sn-C), 573 (m, Sn-O), 512 (m, Sn-C), 463 (m, Sn-
(

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1
3
N), 440 (m), 348 (m), 284 (m). H NMR (DMSO-d ) δ(ppm): 1.27 (t, 8H, J = 7.2 Hz),
6
H-H
3
2
119/117
1
3
.14 (sextet, 8H, J =7.5 Hz, γ), 1.31(t, 8H, = 7.8 ( J (
Sn- H)= 58/43 Hz, α'), 6.90 (m,
H-H
3
3
3
ArH4), 7.06 (d, 2H, J = 8.6 Hz, ArH2), 7.02 (d, 2H, J = 7.9 Hz, H5), 7.40 (t, 2H, J
H-H
H-H
H-H
1
3
1
=
7.6 Hz, H3), 7.51 (m, 4H, Ph) 7.93(m, 4H Ph), 8.26 (m, 2H, Ph). C{ H} NMR (DMSO-
1
119/117
13
3
d ) δ (ppm):20.1 (Cδ), 22.3 (Cα, J (
Sn- C)= 606/578 Hz), 22.9 (β), 27.3 (Cγ, J
6
1
19/117
13
(
Sn- C)= 87 Hz), 33.5 (Ca), 116.2 (C6), 117.2 (C4), 117.9 (i-Ph, JSn-C= 300 Hz), 121.9
1
19
(C2), 129.6 (Ph), 134.2 (C5), 134.6 (C3), 165.3 (C7), 170.2 (C1), 173.6 (C8).
Sn NMR
+
(
DMSO-d δ(ppm): -500.77. HRMS (MALDI): calculated for C H Cl N O Sn [M ]:
6
31 27
2
4
4
2
8
28.9453, found: 828.9510.
4
4
.3. Biochemistry
.3.1. Cell viability (AlamarBlue®) assays
After 72 h incubation, 20 µL of AlamarBlue® was added to each well. The plates were
incubated in darkness at 37 °C for 4-5 h. Using a Molecular Devices microplate reader, the
fluorescence (F) was then read at an excitation wavelength of 544 nm and an emission
wavelength of 590 nm. Cell viability was then determined by subtracting the mean blank
fluorescence (Fb) from the treated sample fluorescence (Fs) and expressing this as a
percentage of the fluorescence of the blanked vehicle control (Fc). This is demonstrated in the
equation below. The results were then plotted as a nonlinear regression, sigmoidal dose-
response curves on Prism, from which the IC value for each drug was determined.
5
0
Fs − Fb
Fc − Fb
ꢀxꢀ100 = %ꢀCellꢀViability
4
.3.2. Flow Cytometry (determination of DNA content)
HL-60 cells were seeded at 200,000 cells/mL in 6-well plates and treated with appropriate
drug concentrations and ddH2O as vehicle control (20 µL of drug or ddH O in 2mL of cells)
2

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for 72 h. Cells were harvested at each time-point by centrifugation at 1600 rpm for 10 min
(brake 3). Media was drained and the pellets resuspended in 50 µL non-sterile PBS. The cells
were then fixed by a drop-wise addition of 1 mL of ice-cold 70% EtOH/PBS while vortexing.
Following overnight fixation at -20 °C, 1 mL of non-sterile FBS was added to the cells and
the solutions centrifuged at 2400 rpm for 10 min (brake 1). The ethanol was then drained and
the cell pellet resuspended in 200 µL PBS, 12.5 µL, 10 mg/mL RNase A (which destroys
RNA so that only DNA can be observed) was then added. Next, 37.5 µL, 1 mg/mL propidium
iodide (PI) (a DNA binding dye which fluoresces when activated and thus allows the amount
of DNA present to be determined) was also added to each sample. Cells were vortexed and
incubated in the dark at 37 °C for 30 min. The PI fluorescence was measured on a linear scale
using a BD Accuri™ C6 Plus. Data collections (10,000 events per sample) were gated to
exclude cell debris and cell aggregates. The amount of fluorescence is proportional to the
amount of DNA present and hence the population of cells in each phase of the cell cycle can
be determined. Cells are gated as follows: pre-G (<2N DNA), G /G (2N DNA), S (2N – 4N
1
0
1
DNA), G /M (4N DNA). Apoptotic cells are hypoploid (< 2N DNA). Therefore, apoptosis
2
was determined from the peak in pre-G1. All data was recorded and analysed using the BD
Accuri C6 software.
4
4
.4. DNA Binding
.4.1. UV Thermal Denaturation
Thermal melting experiments were conducted with UV-VIS Lambda 35, Perkin Elmer
TempLAB software). Temperatures were monitored with a thermistor inserted into a 1 mL
(
quartz cuvette containing the same volume of water as in the sample cells. Absorbance
changes at 260 nm were monitored from a range of 30 to 90 °C with a heating rate of 1 °C
-1
min and a data collection rate of five points per °C. A quartz cell with a 1 cm path length

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was filled with a 1 mL solution of DNA or DNA-ligand complex. The st-DNA oligomer (150
µM base) and ligand (15 µM) solutions were prepared in a phosphate buffer [0.01 M
K HPO /KH PO ], adjusted to pH 7 so that a ligand to DNA base ratio of 0.1 was obtained.
2
4
2
4
The difference in thermal melting temperature (∆T , °C) of the DNA and DNA-ligand
m
complex obtained from the first derivative of the melting curves are reported.
4
.4.2. UV absorption titration DNA binding studies
UV-visible spectra were recorded on UV-visible spectrophotometer Shimadzu 1800. An
amount of 0.01 g of sodium salt of ct-DNA was dissolved in 100 mL of distilled water stirred
at room temperature for 24 hours and filtered. The prepared stock solution of DNA was stored
below 4 °C and used within 4 days. From the stock solution 1 mL solution was taken and
diluted up to 50 mL in phosphate buffer of 7.4 pH (0.051 g of NaH PO and 0.169 g of
2
4
Na HPO dissolved in 100 mL distilled water) and its concentration was measured by
2
4
−
1
−1
spectrophotometry using 6600 M cm molar absorption coefficient. The purity of DNA was
measured by the appearance of absorption peaks at 260 and 280 nm (ratio = 1.86). The
interaction of ligand to DNA was assessed by using aqueous ethanol (containing 20% water
and 80% ethanol) and phosphate buffer of pH 7. The spectrum of the compound was recorded
in the absence and presence of varying concentration of DNA [55].
Based upon the decrease in absorbance, the binding constant was calculated according to the
equation [37]
A0
εG
εG
εG εH −G
1
=
+
A− A0 εH −G
−
−
εG
K
[
DNA
]
were A and A are the absorbance of test sample, at λmax, in the absence and presence of DNA
0
respectively. Similarly εG and εH−G are the molar absorptivity of test sample, at λmax, in the

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absence and presence of DNA, respectively. The value of binding constant (K) was calculated
from the intercept to slope ratio of the linear plot between A /(A − A ) and 1/[DNA]
0
0
5
. ACKNOWLEDGMENTS
H.U. and A.W. thank the Higher Education Commission of Pakistan. V.P. thanks the School
of Chemistry at Trinity College Dublin (Ireland) for a postgraduate award. H.B.M. thanks the
Irish Research Council (Ireland) for a postgraduate scholarship. The authors also express their
sincere thanks to Drs. John O'Brien, Manuel Ruether, Gary Hessman and Peter Brien (School
of Chemistry, TCD) for their invaluable cooperation.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data to this article can be found online at
CONFLICTS OF INTEREST
There are no conflicts of interest to report.
REFERENCES
[
1] M.J. Hannon, Metal-based anticancer drugs: From a past anchored in platinum chemistry
to a post-genomic future of diverse chemistry and biology, Pure Appl. Chem. 79 (2007)
2
243-2261.