Structural and in vitro biological studies of organotin(iv) precursors; Selective inhibitory activity against human breast cancer cells, positive to estrogen receptors

Article abstract of DOI:10.1071/CH12448

Crystals of Ph₃SnCl (1) were grown from a methanol/acetonitrile solution. Compounds [Ph₃SnOH]_n (2) and [(Ph ₂Sn)₄Cl₂O₂(OH)₂] (3) were crystallized from diethyl ether/methanol/acetonitrile and hot acetone/water solutions respectively, of the white precipitation, formed by adding KOH to solutions of 1 and [Ph₂SnCl₂] in 1:1 and 1:2 molar ratios respectively. Complex 1 was characterized by X-ray crystallography. X-ray structure determination of compounds 2 and 3 confirmed the previously reported identities. The molecular structure of 1, reported here, is a new polymorphic form of the known one for Ph₃SnCl. Four independent [Ph ₃SnCl] molecules constitute the crystal structure of 1. The moieties are packed in two pairs in a tail-to-tail arrangement. Complexes 1-3 were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (human cervical), MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (kidney carcinoma), 786-O (renal adenocarcinoma), K1 (thyroid carcinoma), and the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) versus, the normal immortalized human mammary gland epithelial cell line MTSV17 with a sulforhodamine B (SRB) assay. The results show potent cytotoxic activity of the complexes against all cell lines used, which was superior to that of cisplatin (CDDP). Compounds 1-3 showed higher activity against breast cancer cells MCF-7 (ER positive) than against of MDA-MB-231 (ER negative). These findings prompted us to search for possible interaction of these complexes with other cellular elements of fundamental importance in cell proliferation. The influence of these complexes 1-3 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX), as well as their binding affinity towards calf thymus-DNA, were kinetically and theoretically studied.

Full text of DOI:10.1071/CH12448

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH12448
Structural and In Vitro Biological Studies of Organotin(IV)
Precursors; Selective Inhibitory Activity Against Human
Breast Cancer Cells, Positive to Estrogen Receptors
A
A B
,
C
Vasilis I. Balas, Christina N. Banti,
Nikolaos Kourkoumelis,
A H
,
D
Sotiris K. Hadjikakou,
Despina Sahpazidou, Louise Male, Mike B. Hursthouse,
Barbara Bednarz, Maciej Kubicki, Konstantinos Charalabopoulos,
and Nick Hadjiliadis
George D. Geromichalos,
D
E
E
A F
,
F
B G
,
A H
,
A
Section of Inorganic and Analytical Chemistry, Department of Chemistry,
University of Ioannina, 45110 Ioannina, Greece.
B
Department of Experimental Physiology, Medical School, University of Ioannina,
45110 Ioannina, Greece.
C
Medical Physics Laboratory, Medical School, University of Ioannina, 45110 Ioannina,
Greece.
D
Cell Culture, Molecular Modeling and Drug Design Laboratory, Symeonidion Research
Center, Theagenion Cancer Hospital, 54007 Thessaloniki, Greece.
E
Department of Chemistry, University of Southampton, Highfield, Southampton,
SO17 1BJ, UK.
F
Department of Chemistry, Adam Mickiewicz University, ul. Grunwaldzka 6,
60-780 Poznan, Poland.
G
H
Department of Physiology, Democritus University Medical School,
68100 Alexandroupolis, Greece.
Corresponding authors. Email: shadjika@uoi.gr, nhadjis@uoi.gr
Crystals of Ph3SnCl (1) were grown from a methanol/acetonitrile solution. Compounds [Ph3SnOH]n (2) and
[(Ph2Sn)4Cl2O2(OH)2] (3) were crystallized from diethyl ether/methanol/acetonitrile and hot acetone/water solutions
respectively, of the white precipitation, formed by adding KOH to solutions of 1 and [Ph2SnCl2] in 1 : 1 and 1 : 2 molar
ratios respectively. Complex 1 was characterized by X-ray crystallography. X-ray structure determination of compounds 2
and 3 confirmed the previously reported identities. The molecular structure of 1, reported here, is a new polymorphic form
of the known one for Ph3SnCl. Four independent [Ph3SnCl] molecules constitute the crystal structure of 1. The moieties are
packed in two pairs in a tail-to-tail arrangement.
Complexes 1–3 were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa
(human cervical), MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, ER negative), A549 (lung),
Caki-1 (kidney carcinoma), 786-O (renal adenocarcinoma), K1 (thyroid carcinoma), and the normal human lung cell line
MRC-5 (normal human fetal lung fibroblast cells) versus, the normal immortalized human mammary gland epithelial cell
line MTSV17 with a sulforhodamine B (SRB) assay. The results show potent cytotoxic activity of the complexes against
all cell lines used, which was superior to that of cisplatin (CDDP). Compounds 1–3 showed higher activity against breast
cancer cells MCF-7 (ER positive) than against of MDA-MB-231 (ER negative). These findings prompted us to search for
possible interaction of these complexes with other cellular elements of fundamental importance in cell proliferation. The
influence of these complexes 1–3 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the
enzyme lipoxygenase (LOX), as well as their binding affinity towards calf thymus-DNA, were kinetically and
theoretically studied.
Manuscript received: 28 September 2012.
Manuscript accepted: 24 October 2012.
Published online: 26 November 2012.
Introduction
In particular, triphenyltin hydroxide is used as a fungicide for
potatoes, sugar beets, and pecans, while the main application for
triphenyltin chloride is as a fungicide and antifoulant.[2a,2b] The
proven toxicity of these compounds, however, have led to
Stannanes are used in a wide range of applications such
as biocides, insecticides, pesticides, fungicides, chemical
intermediates, anti-fouling agents, and as catalysts.[1a,1b]
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

B
V. I. Balas et al.
c
b
a
Ph
Ph
MOH/MeCN
KOH
Ph3SnCl
Cl
Sn
O
O
Sn
Ph
(2)
MOH/MeCN
n
(1)
Ph
Ph
Ph
Ph
Sn
HO
O
Sn
O
OH
Sn
Ph
Acetone/water
4Ph2SnCl2 ꢀ 4KOH
Cl
ꢀ 4KCl ꢀ 2HCl
Cl
Ph
Sn
Ph
Ph
(3)
Scheme 1.
environmental concern because they are persistent organic
pollutants.[3] Given the important role of organotin compounds
in pollution and toxicology, there is limited literature dealing
with their binding to biological macromolecules but it is clear
that organotin compounds and especially triorganotins show
significant biological action.[1a] Biological studies also
include organotin interactions with haemoglobin,[4a–d] liver
mitochondria,[5a–e] and ATPase,[6] and the effects of di- and tri-
alkyltin compounds on membrane stability[7a,7b] and their
interactions with nuclear bases[8a,8b] and DNA fragments.[8c–g]
The mechanisms of organotin biological activity postulate
interactions with sulfur-containing proteins.[9a–c]
During the past few years organotin(IV) compounds have an
important place in cancer chemotherapy reports.[10–13] Recently,
a group of organotin(IV) complexes with 2,6-dimethoxynicotinic
acid (DMNIH), 1,4-benzodioxane-6-carboxylic acid (BZDOH),
and 2,5-dimethyl-3-furoic acid (DMFUH) of formulae
[SnCy3(DMNI)], [SnCy3(BZDO)], [SnCy3(DMFU)], and
[SnPh2(BZDO)2] (where Cy ¼ cyclohexyl) were tested for their
cytotoxicity against pancreatic carcinoma (PANC-1), erythro-
leukemia (K562), and two glioblastoma multiform (U87 and
LN-229) human cell lines.[14a] The compounds showed
very high antiproliferative activity, with IC50 values in the
150–700 nm range.[14a] Distribution of cellular DNA upon
treatment with these compounds revealed that whereas
arachidonic acid through the activation of phospholipase A2.[15]
Lipoxygenase (LOX) is an enzyme which catalyzes the oxida-
tion of arachidonic acid to leukotrienes, an essential mechanism
for the cell life involving inflammation.[16a,b] LOX inhibition is
found to induce apoptosis.[16c] Thus, LOX inhibition provides a
potential novel target for the treatment and chemoprevention for
several different cancers.
In this paper, we report our studies on the structural properties
of [Ph3SnCl] (1), [Ph3SnOH]n (2), and [(Ph2Sn)4Cl2O2(OH)2]
(3). Although, the molecular structures of 1–3 are known,[17–19]
a new polymorphic form of 1 is reported here, while 2–3 were
shown to be identical to the ones already reported.[18,19] Com-
plexes 1–3 were evaluated for their in vitro cytotoxicity (cell
viability) against human cervical, breast, lung, kidney, renal,
and thyroid cancer cell lines (along with two non-tumour human
breast and lung cell lines) with a sulforhodamine B (SRB) assay.
The following cell lines were used: HeLa (human cervical),
MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER nega-
tive), A549 (lung), Caki-1 (kidney carcinoma), 786-O (renal
adenocarcinoma), and K1 (thyroid carcinoma). The non-tumour
cell lines were MRC-5 (normal human fetal lung fibroblast
cells) and MTSV17 (normal immortalized human mammary
gland epithelial cell line). The LOX inhibitory activity originated
by complexes 1–3 and their interaction with calf thymus
(CT)-DNA were studied by means of kinetic and theoretical
calculations.
compounds
[SnCy3(DMNI)],
[SnCy3(BZDO)],
and
[SnCy3(DMFU)] induce apoptosis in most of the cell lines,
compound [SnPh2(BZDO)2] does not affect cell viability in any
cell line tested, indicating a possible difference in cytotoxic
mechanism.[14a] Moreover, the organotin(IV) complexes of thio-
amides [(n-Bu)3Sn(O-HTBA)H2O] and {[Ph3Sn(O-HTBA)ꢀ0.7
H2O]}n, (H2TBA ¼ 2-thiobarbituric acid) showed high activity
against MCF-7 (estrogen receptor, ER, positive) but not against
breast cancer cells (MDA-MB-231) (breast, ER negative),
indicating that estrogen receptors may be involved in their
antitumour mechanism.[13a]
Results and Discussion
General Aspects
Crystals of complex 1 were grown from methanol/acetonitrile
solution (Scheme 1). The reaction of Ph3SnCl with an equimolar
amount of KOH resulted in the formation of compound
[Ph3SnOH]n (2) which was crystallized from diethylether/
methanol/acetonitrile 6 : 1 : 1 solution (Scheme 1). Finally,
crystals of the complex [(Ph2Sn)4Cl2O2(OH)2] (3) were grown
from hot acetone/water solution, itself formed by the action of
KOH on [Ph2SnCl2] (Scheme 1).
Organotin compounds have been shown to affect cell signal-
ing by activating protein kinase C,[14c,d] while they increase free

Structural and In Vitro Biological Studies of Organotin(IV) Precursors
C
Table 1. The unit cell parameters of the reported structures for 1–3
Complex
Unit cell
a, b, g [8]
19.0027(2), 18.5503(3), 18.3714(2) 90.00, 98.6150(10), 90.00
Space
group
Z, Z0 Temperature Solvent
Ref
[K]
˚
a, b, c [A]
A
1
P21/c
P21/a
P21/a
P21/a
R-3
16, 4 120
8, 2 rt
CH3CN/MeOH
TPSNCLB
18.65(3), 9.59(2), 19.02(3)
90, 105.5(5), 90
Benzene
Benzene
Benzene
Ethanol
[17a]
[17b]
[17b]
[17c]
A
TPSNCL01B 18.664(4), 9.721(4), 18.983(5)
TPSNCL02B 18.410(4), 9.5593(19), 18.704(5)
90, 105.601(20), 90
90, 105.201(19), 90
8, 2 rt
8, 2 110
24, 2 298
TPSNCL03B 24.4935(4), 24.4935(4), 19.1219(6) 90, 90, 120
2
8.2705(2), 10.2202(2), 17.6775(3) 90.00, 90.00, 90.00
18.053(8), 10.382(5), 8.303(1) 90.00, 90.00, 90.00
P212121 4, 0 100
P212121 4, 0 283–303
P212121 4, 0 120
Et2O/MeOH/MeCN 6 : 1 : 1
MeCN
HXTPSN
[18a]
[18b]
A
HXTPSN01 8.2573(2), 10.2229(2), 17.6996(5) 90.00, 90.00, 90.00
Ethanol ꢁ water 97 : 3
Aceton/water
Ethanol ꢁ water 97 : 3
DMF
3
10.1080(3), 10.7141(3), 11.6629(3) 78.183(2), 67.573(2), 75.802(2) P-1
1,0
2
120
295
293
293
173
CIJJEK1
JIGJAK
LAZVUD
11.758(2), 19.327(3), 11.649(1)
17.231(4), 17.552(2), 18.345(3)
10.295(3), 10.791(3), 11.827(4)
90.00, 92.87(1), 90.00
90.00, 90.00, 90.00
P21/c
Pbca
P-1
[19a]
[19b]
[19c]
[19d]
4
77.74(3), 66.49(2), 75.63(2)
Water
LAZVUD01 10.643(2), 11.570(4), 10.106(2)
112.82(2), 104.56(2), 77.95(2) P-1
AThis work.
BTPSNCL, TPSNCL01, and TPSNCL02 are the same polymorph, while 1 and TPSNCL03 are different polymorphs.
In order to assure the retention of the geometry adopted by
the complexes in DMSO solution, 1H NMR spectra of the
complexes in [D6]DMSO solutions were recorded. The stability
of the complexes was also tested after 48 h. This period was
chosen since biological experiments require 48 h of incubation
with complexes. The resonance signals of phenyl protons in the
1H NMR spectra of complexes 1–2 in [D6]DMSO solutions
(Figs. S1, S2) are observed at d 7.85–7.82 ppm, 6H, d (for H-a)
and 7.49–7.41 ppm, 9H, m (for H-b and H-c) for 1 (Scheme 1),
and at d 7.71–7.70 ppm, 6H, d and 7.47–7.27 ppm, 9H, m for 2
(Scheme 1). This data confirms the retention of the Sn-Ph bond
in solution. The low solubility of 3 in [D6]DMSO prohibits the
NMR spectrum recording. Also, no changes were observed
between the initial spectrum and the corresponding one mea-
sured after 48 h (Figs. S3, S4).
Compounds 2, HXTPSN, and HXTPSN01 correspond to the
same polymorphic forms of (Ph3SnOH)n, which is crystallized
in the P212121 space group.[18] Three polymorphic forms have
been detected up to now for [(Ph2Sn)4Cl2O2(OH)2] which are
crystallized in P-1 (for 3, LAZVUD, and LAZVUD01), in P21/c
(for CIJJEK1), and in Pbca (for JIGJAK).[19]
Biological Tests
Cytotoxicity
The 50 % of cell viability (IC50) values for complexes 1–3
against HeLa (human cervical), MCF-7 (breast, ER positive),
MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1
(kidney carcinoma), 786-O (renal adenocarcinoma), K1 (thy-
roid carcinoma), MRC-5 (normal human fetal lung fibroblast
cells), and MTSV17 (normal immortalized human mammary
gland epithelial cell line) are shown in Table 3. Cell survival was
estimated by means of SRB assay, after 72 h of their exposure to
increasing concentrations of the tested compounds. The dose–
response curves for all tested compounds are shown in Fig. 3.
The results revealed that complexes 1–2 caused a dose-
dependent inhibition of cell proliferation. MCF-7, MTSV-17,
and MRC-5 cells were found to be the most sensitive over the
action of Ph3SnCl (1) and (PhSnOH)n (2) with IC50 values
ranging between 0.070 and 0.135 mM, while A-549 and HeLa
cells were the most resistant. Complex 2 exhibits slightly better
cytotoxic activity (lower IC50 values) compared with 3. On the
contrary, 3 showed almost no inhibitory effect up to maximum
concentration used (10 mM). It is also noteworthy that for all
tested cell lines, 1 and 2 showed a far better cytotoxic activity
than the clinically established chemotherapeutic cisplatin (76.2
and 141.4-fold lower IC50 for 1 and 2 respectively against MCF-
7 cells; almost 49.5-fold against MDA-MB-231; 24.7 and
33.3-fold respectively against MTSV17 cells; 44.8 and 45.4-fold
respectively times against A-549; 25.5 and 40.0-fold respectively
for MRC-5; 48.9 and 55.7-fold respectively against K1; 11.0 and
11.6-fold respectively against HeLa; 14.3 and 15.3-fold respec-
tively against 786-O; and 15.1 and 18.5-fold respectively
against Caki-1; Table 3).
Crystal Structures of [Ph3SnCl] (1), [Ph3SnOH]n (2),
and [(Ph2Sn)4Cl2O2(OH)2] (3)
The structures of complexes 1–3 were determined by X-ray
diffraction at 120 K. Although the molecular structures of 1–3
were known,[17–19] compound 1 crystallized in a new poly-
morphic form, while the full refinement of the structures of 2–3
proved their coincidence with the previous determinations.[18,19]
Table 1 summarizes the unit cell parameters of complexes 1–3
solved up to now and Figs. 1 and 2 show the molecular structures
of the complexes. In the case of 1, three different polymorphs
have been found up to now (Table 1).[17] Compounds TPSNCL,
TPSNCL01, and TPSNCL02 belong to the same polymorphic
form crystallizing in the P21/a space group, while 1 and
TPSNCL03 constitute two new polymorphic forms of Ph3SnCl
which are crystallized in P21/c and R-3 respectively.[17]
The asymmetric unit of 1 contains four independent mole-
cules, which is the only structure among its polymorphs where
the moieties are packed in a tail-to-tail arrangement (Fig. 1b).
The rest of the polymorphs contain two independent molecules
in their asymmetric units (Table 1).[17] Table 2 summarizes
significant bond distances and angles of 1.
˚
The Sn-Cl bond distance varies between 2.32 and 2.38 A,
while the geometry around the tin atom is disordered tetrahedral
in all cases since the Cl–Sn–C bond angles are close to ideal
(108.08–102.48).
Both complexes 1 and 2 appeared to be equipotent against
MDA-MB-231, A-549, HeLa, 786-O, and K1 cells, while for
Caki-1 cells, 1 exhibited better cytotoxic activity than 2 (Table 3).

D
V. I. Balas et al.
C(39)
C(58)
(a)
C(57)
C(56)
C(40)
C(41)
C(59)
C(60)
Cl(1)
C(9)
C(38)
C(8)
C(7)
C(10)
C(44)
C(37)
Sn(3)
C(45)
C(46)
Sn(1)
C(13)
C(11)
C(18)
Cl(3)
C(55)
C(1)
C(62)
C(6)
C(17)
C(16)
C(28)
C(5)
Sn(4)
Cl(4)
C(61)
C(66)
C(20)
C(24)
C(4)
C(26)
C(49)
C(30)
C(67)
C(68)
C(15)
C(25)
C(65)
C(19)
C(33)
C(50)
C(71)
Sn(2)
C(34)
C(31)
C(53)
C(51)
C(70)
C(36)
C(52)
C(35)
C(69)
Cl(2)
(b)
Fig. 1. (a) ORTEP view of 1 with ellipsoids drawn at the 50 % probability level. Hydrogen atoms have been omitted for clarity. (b) Space
filling plot of two of the four crystallographically unique molecules showing the tail-to-tail arrangement.
The observed cell line chemosensitivity order was revealed
cellular elements of fundamental importance in cell prolifer-
ation, such as mitochondria and nuclei. Thus, the influence of
complexes 1–3 on the oxidation of linoleic acid by the enzyme
LOX was studied over a wide concentration range. The degree
of LOX activity (A, %) in the presence of these complexes was
calculated according to the method described previously.[12a]
The IC50 values found for the complexes were: 50.8 (for 1),
47.4 (for 2), and 34 mM (for 3). The corresponding IC50 value
for cisplatin is 65.9 mM. Thus, four or five substituted tri-
phenyl or di-pheny tin(IV) precursor compounds exhibit
inhibitory activity towards LOX which, however, is lower to
the one found for other organotin compounds.[12,13] LOX
inhibition activity of organotin(IV) complexes is related with
their high cytotoxicity.[10a,12–13] This is due to their interfer-
ence with the programmed cell death mechanism through the
cellular organelle mitochondrion, where LOX is mainly dis-
tributed.[20] However, the significant cytostatic activity shown
by 1 and 2 (see Cytotoxicity above), along with their moderate
inhibitory activity towards LOX, indicates involvement of a
different cytostatic mechanism which may not only invoke
mitochondrion.
to be the following: MCF-7 . MRC-5 . Caki-1 $ MDA-MB-
231 . MTSV-17 .786-O $ K1 . A-549 . HeLa for 1 and
MCF-7 . MRC-5 . MTSV-17 . MDA-MB-231 . K1 .786-
O . Caki-1 . HeLa . A-549 for 2. For both complexes,
MCF-7 and MRC-5 cell lines were found to be the most
sensitive.
It has been shown that diorganotin(IV) complexes exhibit
lower activity than those of triorganotin(IV).[10] Moreover, 1 and
2 showed selective cytotoxic activity against carcinoma cells,
especially against MCF-7 (human breast adenocarcinoma,
Table 3). This strong selective activity of complexes against
MCF-7 (ER positive) is not observed when they were tested
against breast cancer cells (MDA-MB-231) (breast, ER nega-
tive), indicating that estrogen receptors may also be involved in
their antitumour mechanism.
Study of the Peroxidation of Linoleic Acid by the Enzyme
Lipoxygenase in the Presence of Complexes 1–3
The high cytostatic activity caused by 1 and 2 prompted us to
search for possible interactions of these complexes with other

Structural and In Vitro Biological Studies of Organotin(IV) Precursors
E
(a)
C24
C15a
C23
C15b
C22
C33a
C32a
C25
C26
C14a
C16a
C33b
C34b
C14b
C13b
C16b
C34a
C13a
C32b
O1
C21
C35b
C31a
C35a
C12a
C31b
Sn1
C31
C12b
Sn1a
C36
Sn1b
O1a
C12
O1b
C35
C11
C32
C21a
C13
C26a
C16
C34
C21b
C33
C26b
C22a
C25a
C14
C15
C22b
C25b
C23a
C24b
C24a
C23b
(b)
C16a
C10
C15a
C17a
C21
C5a
C4a
C3a
C9
C22
C18a
C11
C14a
C8
C12
C7
C6a
C23
C24
C13a
C20
C2a
C1a
C19
Cl 1
O1a
Sn2a
O2
Sn1
Sn1a
O2a
C19a
O1
Sn2
Cl 1a
C7a
C9a
C2
C24a
C23a
C20a
C13
C1
C6
C12a
C11a
C8a
C3
C4
C18
C14
C15
C22a
C21a
C5
C10a
C17
C16
Fig. 2. (a) ORTEP view of 2 and (b) 3 with ellipsoids drawn at the 50 % probability level.
Computational Studies-LOX Docking
Asn534, Asn769, Asp768, Cys127, Glu244, His515, His531,
Lys526, Pro530, Thr529, Trp772, Tyr525, Tyr532, Val126, and
Val762 (Fig. 4a). Complex 3 however, binds away from this site
to a pocket defined by Ala426, Ala710, Arg712, Asn424,
Asn550, Asp275, Gln267, Glu271, Glu554, Ile281, Ile738,
Ile746, Leu423, Leu558, Leu742, Phe274, Phe557, Pro559,
Pro743, Ser425, Ser560, Thr555, Thr556, and Thr739, showing
a hydrogen bonding interaction with the O atom of Thr555.
The moderate inhibitory activity towards LOX, shown by
compounds 1–3 prompted us to investigate their behavior the-
oretically. Thus, in order to determine the interaction degree
between the complexes under study and the LOX enzyme (E) we
have performed theoretical docking calculations. Complexes 1
and 2 preferred the largest hydrophobic pocket of the enzyme
which is constructed by the residues Arg533, Arg767, Asn128,

F
V. I. Balas et al.
Table 2. Selected bond lengths and angles for complex 1
Molecule B Molecule C
Molecule A
Molecule D
˚
(a) Bond lengths [A]
˚
(a) Bond lengths [A]
˚
(a) Bond lengths [A]
˚
(a) Bond lengths [A]
Cl1-Sn1
C1-Sn1
C7-Sn1
C13-Sn1
2.3664(9)
2.127(4)
2.125(3)
2.119(4)
Cl2-Sn2
2.3643(9)
2.121(4)
2.125(3)
2.127(3)
Cl3-Sn3
2.3754(9)
2.127(3)
2.125(3)
2.121(3)
Cl4-Sn4
2.3723(10)
2.120(3)
2.123(3)
2.125(3)
C19-Sn2
C25-Sn2
C31-Sn2
C37-Sn3
C43-Sn3
C49-Sn3
C55-Sn4
C61-Sn4
C67-Sn4
(b) Bond angles [8]
(b) Bond angles [8]
(b) Bond angles [8]
(b) Bond angles [8]
C13-Sn1-C7
C13-Sn1-C1
C7-Sn1-C1
C13-Sn1-Cl1
C7-Sn1-Cl1
C1-Sn1-Cl1
114.42(13)
113.09(14)
110.03(13)
106.26(9)
108.02(9)
104.31(9)
C19-Sn2-C25
C19-Sn2-C31
C25-Sn2-C31
C19-Sn2-Cl2
C25-Sn2-Cl2
C31-Sn2-Cl2
113.02(13)
113.25(13)
114.79(13)
103.66(9)
106.22(9)
104.58(9)
C49-Sn3-C43
C49-Sn3-C37
C43-Sn3-C37
C49-Sn3-Cl3
C43-Sn3-Cl3
C37-Sn3-Cl3
110.13(13)
114.91(13)
117.55(13)
104.72(9)
104.37(9)
103.46(9)
C55-Sn4-C61
C55-Sn4-C67
C61-Sn4-C67
C55-Sn4-Cl4
C61-Sn4-Cl4
C67-Sn4-Cl4
112.72(13)
115.05(13)
114.14(13)
105.47(9)
103.45(10)
104.49(9)
Table 3. IC50 values for cell viability found for complexes 1–3 against HeLa (human cervical), MCF-7 (breast, ER positive), MDA-MB-231 (breast,
ER negative), A549 (lung), Caki-1 (kidney carcinoma), 786-O (renal adenocarcinoma), K1 (thyroid carcinoma) and additionally, the normal human
lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line (MTSV17)
cell lines
Compounds
IC50 values [mM]A
Cell lines
MCF-7
MDA-MB-231
MTSV-17
A-549
MRC-5
K1
HeLa
786-O
Caki-1
Ph3SnCl (1)
0.130
0.070
0.166
0.165
0.182
0.135
0.218
0.221
0.141
0.090
0.205
0.180
0.220
0.210
.10
2.430
0.203
0.190
0.163
0.200
(PhSnOH)n (2)
(Ph2Sn)4Cl2O2(OH)2 (3)
Cisplatin
.10
.10
.10
.10
9.900
.10
.10
10.03
.10
2.900
.10
9.900
8.200
4.500
3.600
3.010
AIC50 values were derived from the corresponding dose-effect curves drawn from sextuplicate determinations with CV lower than 5 %.
1
2
MRC-5
MRC-5
A-549
1.0
0.5
0
1.0
0.5
0
A-549
K1
K1
MDA-MB-231
Caki-1
MTSV-17
786-O
MDA-MB-231
Caki-1
MTSV-17
786-O
HeLa
HeLa
MCF-7
MCF-7
0
0.001
0.01
0.1
1
10
0
0.001
0.01
0.1
1
10
Concentration [µM]
Concentration [µM]
3
CDDP
MRC-5
A-549
K1
1.0
0.5
0
1.0
0.5
MDA-MB-231
MRC-5
A-549
Caki-1
MTSV-17
786-O
HeLa
K1
MDA-MB-231
Caki-1
MTSV-17
786-O
HeLa
MCF-7
MCF-7
0
0
0.001
0.01
0.1
1 10
0
0.01
0.1
1
10
100
Concentration [µM]
Concentration [µM]
Fig. 3. Dose-effect survival plots for complexes 1, 2, 3 and cisplatin (CDDP) against a panel of human cancer and normal cell lines 72 h after the
administration of the agents. Cytotoxicity was estimated via SRB assay (each point represents mean of six replicate wells).

Structural and In Vitro Biological Studies of Organotin(IV) Precursors
G
Leu246, Leu249, Lys110, Met15, Phe108, Ser129, and Trp130
while for 2 the binding site is constructed by Ala76, Arg533,
Arg767, Asn128, Asn769, Asp760, Asp768, Gln766, Glu78,
Glu761, Gly75, Gly247, His248, Leu249, Lys110, Met15,
Phe108 Pro770, Ser759, Tyr763, and Val762.
Complex 3 binding behavior probably reveals a non-
competitive inhibition which does not interfere with the binding
of the substrate. In this case the extent of the inhibition depends
only on the concentration of the inhibitor.
DNA Binding Studies
DNA interaction with metal complexes may cause DNA frag-
mentation leading to cell death through a mechanism known as
apoptosis. In order to ascertain whether the high cytostatic
activity showed by 1 and 2 may be attributed to their interaction
with DNA bases, their binding properties to CT-DNA were
studied using electronic absorption spectroscopy.[22a] Hyper-
chromism or hypochromism observed between the UV spectra
of the CT-DNA-compound and the corresponding one of free
CT-DNA is related with the configuration of the double helix
structure of DNA.[22a]
Fig. 5 shows the UV spectra of CT-DNA (at constant
concentration) in buffer solution in the absence and presence
of 1–3 at various r values (r ¼ [complex]/[DNA], [DNA] ¼
5 ꢃ 10ꢂ5 M) (a) and their plots of A/Ao versus [complex] at
lmax ¼ 261 nm (b). Table 4 summarizes the DNA binding
constants (Kb) for the complexes 1–3. In case of complexes 1
and 2, a total gradual hyperchromism in the CT-DNA absorption
at 261 nm was observed by increasing of the [Complex]/
[CT-DNA] molar ratio (Fig. 5a). The observed hyperchromic
effect of 10.3 % (1) and 22.7 % (2) respectively suggests binding
of the complexes to CT-DNA,[22] which is attributed to either
external contact or to the uncoiling of the helix structure of DNA
by these complexes (Fig. 5 and Table 4).[22] However, in case of
3, the hyperchromic effect could not be detected due to the
overlay absorption of both 3 and CT-DNA at 260 nm.
Fig. 4. (a) Binding site of the inhibitors 1 and 2 studied in the EI complex.
(b) Displacement of complex 1 in the presence of the substrate.
The large volume of this structure probably prohibits its fitting
into the binding pocket which has been recognized as the active
site of the enzyme.[12c,13a] Linoleic acid (S) acts as the substrate
entering the enzymes active site and forming the ES complex.
The binding affinity of S, expressed by energetic factors,
The binding constants (Kb) of complexes 1–3 towards
CT-DNA were evaluated according to the method described in
ref. [22a], by monitoring the changes in absorbance of the UV
spectra of the complexes 1–3, ([1] ¼ 10 ꢃ 10ꢂ5 M, [2] ¼ 15 ꢃ
10ꢂ5 M, [3] ¼ 2 ꢃ 10ꢂ5 M), at 300–310 nm, with increasing
concentration of CT-DNA (Fig. 6). The calculated Kb values
for 1–3 are: (5.9 ꢁ 0.8) ꢃ 104 (1), (10 ꢁ 0) ꢃ 104 (2), and
(11.2 ꢁ 2) ꢃ 104 (3) Mꢂ1 respectively, suggesting interaction
with CT-DNA in accordance to the low hyperchromic effect of 1
and 2, observed. The corresponding Kb value of cisplatin is
was calculated to ꢂ100.2 kJ molꢂ1 [21]
.
The corresponding
values for 1, 2, and 3 were ꢂ75.4, ꢂ74.3, and ꢂ87.0 kJ molꢂ1
respectively. These values indicate a stronger interaction
between E and S than between E and the reversible inhibitors (I).
The inhibitory action of a compound is an approximation of the
interaction strength between the pose and the macromolecule.
Therefore these theoretical calculations confirm the experi-
mental results showing minor cytotoxic activity of 3 towards
cancer cells. Results also show that complexes 1 and 2 partici-
pate in a competitive inhibition mechanism where the substrate
and inhibitor compete for access to the enzyme’s active site.
Although they bind to the same pocket, described in the case of
the EI formation, ESI binding energy is significantly decreased
probably due to higher affinity of the substrate which displaces
(5.73 ꢁ 0.45) ꢃ 104 Mꢂ1 [22b]
.
Thus, the binding affinity the
complexes 1–3 towards CT-DNA is higher than that of cisplatin.
The higher Kb value found for 2 in contrast to that of 1 might
be attributed to the intermolecular hydrogen bonds between the
hydroxyl group of 2 with the DNA bases (see Computational
Studies). The stronger DNA-complex interaction observed for 2
towards 1 leads to lower IC50 values for 2 against the cell lines
tested (see Cytotoxicity above). Also, 3 binds to CT-DNA
strongly due to the presence of the hydrogen bonds formed
between the hydroxyl group and DNA.
˚
the binding site by almost 10 A (shifting distance of the metal
centres between EI and ESI complexes) (Fig. 4b). Binding
orientation is also significantly altered although no specific
interaction is found regarding this parameter for each of the
complexes.
The vicinity of compound 1 participating in the ESI complex
is characterized by the residues Arg242, Arg533, Asn128,
Asn245, Asp243, Asp760, Asp768, Glu244, Gly247, His248,
Computational Studies-DNA Docking
The type and the possible differentiation of interaction between
the synthesized complexes and CT-DNA were studied by DNA
docking studies since it has been suggested that the binding of
small molecules to DNA plays a crucial role regarding the action
of relevant drugs.[23]

H
V. I. Balas et al.
1.12
1.10
1.08
1.06
1.04
1.02
1.00
(a)
(b)
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
Concentation of 1 [ꢁ106M]
Comp. 1
0
230
250
270
290
310
330
350
370
390
410
430
450
Wavelength [nm]
r ꢂ 0
r ꢂ 0.05
r ꢂ 0.07
r ꢂ 0.09
r ꢂ 0.11
r ꢂ 0.13
(a)
(b) 1.25
1.20
0.5
1.15
1.10
0.4
0.3
0.2
0.1
0
1.05
0
1
2
3
4
5
6
7
Concentation of 2 [ꢁ106M]
Comp. 2
240
270
300
330
360
390
420
450
Wavelength [nm]
r ꢂ 0
r ꢂ 0.05
r ꢂ 0.07
r ꢂ 0.09
r ꢂ 0.11
r ꢂ 0.13
1.2
(a)
1.0
0.8
0.6
0.4
0.2
0
Comp. 3
230
250
270
290
310
330
350
370
390
410
430
450
Wavelength [nm]
r ꢂ 0
r ꢂ 0.05
r ꢂ 0.07
r ꢂ 0.09
r ꢂ 0.11
r ꢂ 0.13
Fig. 5. (a) UV spectra of calf thymus (CT)-DNA in buffer solution in the absence and presence of 1–3 at r values of 0,
0.05, 0.07, 0.08, 0.11, and 0.13 (r ¼ [complex]/[DNA], [DNA] ¼ 5 ꢃ 10ꢂ5 M). (b) Plot of A/Ao vs [complex] at
lmax ¼ 261 nm.
anchoring the minor groove in the vicinity of the area defined by
cytosine C21 and guanine G7 (Fig. 7). Complex 3 binds to the
major groove, which in B-DNA is rather wide and therefore
accessible to quite bulky agents like 3, at the vicinity of guanine
G19 and thymine T20 (Fig. 7). The conformation energies were
Table 4. The DNA binding constants (Kb) for the complexes 1–3
H [%] ¼ percentage of hyperchromism [%]
Complexes
H [%]
Dl [nm]
Kb [Mꢂ1
]
1
2
3
10.3
22.7
–
–
–
–
(5.9 ꢁ 0.8) ꢃ 104
(10 ꢁ 0) ꢃ 104
calculated to be ꢂ49.3 and ꢂ84.8 kcal molꢂ1 (1 kcal molꢂ1
¼
4.186 kJ molꢂ1) for 1 and 3, respectively. Significant H-bonding
interactions appear to stabilize complex 2 (dimer structure)
inside the major groove near adenine A17 and guanine G17
with conformation energy of ꢂ87.3 kcal molꢂ1 (Fig. 7).
H-bonding is evident between the O of the structure with the
N atom of the imidazole ring and the amine N of A17 at 2.02
(11.2 ꢁ 2) ꢃ 104
The complexes under study have diverse structures and
volumes and therefore we expected different modes of action
towards DNA. We concluded that, minimal electrostatic inter-
actions are present for complexes 1 and 3, with complex 1
˚
and 2.03 A respectively. Calculated binding energies are very

Structural and In Vitro Biological Studies of Organotin(IV) Precursors
I
low, even for 2, which is further stabilized by H-bonds show-
ing minimal DNA interaction. Electrostatic interaction of the
metal with the pyrophosphate O atoms may also take place,
contributing to the binding modes proposed, in accordance with
the results of nucleobase-Sn(IV) interactions, as previsously
reported.[8a,b]
A diversity of cytostasis mechanism is often employed
by organotin compounds.[12–14] Organotin(IV) complexes of
2-thiobarbituric acid [(n-Bu)3Sn(O-HTBA)H2O] and {[Ph3Sn(O-
HTBA)ꢀ0.7H2O]}n, on the other hand, possess high activity
against MCF-7 (ER positive), with simultaneous low activity
against breast cancer cells (MDA-MB-231) (breast, ER nega-
tive), indicating that estrogen receptors may be involved in their
antitumour mechanism.[13a] These compounds mediate strong
cytotoxic response to cell lines tested through apoptosis and
induce cell cycle arrest in the S phase of the cell cycle,
suggesting DNA intercalation (direct or indirect) with the
complexes.[13a] Similarly, in case of organotin(IV) complexes
with 2,6-dimethoxynicotinic acid (DMNIH), 1,4-benzodioxane-
6-carboxylic acid (BZDOH), and 2,5-dimethyl-3-furoic acid
(DMFUH) of formulae [SnCy3(DMNI)], [SnCy3(BZDO)],
[SnCy3(DMFU)], and [SnPh2(BZDO)2] the high activity against
cancer cell lines[14a] was attributed to the apoptosis caused by
the first three only.
Conclusions
Three organotin compounds 1–3, which are used as common
processors in organotin synthetic chemistry, were structurally
characterized by X-ray diffraction patterns analysis and
evaluated for their in vitro cytotoxicity against HeLa (human
cervical), MCF-7 (breast, ER positive), MDA-MB-231 (breast,
ER negative), A549 (lung), Caki-1 (kidney carcinoma), 786-O
(renal adenocarcinoma), K1 (thyroid carcinoma) and also
against the normal cell lines MRC-5 (normal human fetal lung
fibroblast cells) and MTSV17 (normal immortalized human
mammary gland epithelial cell line).
Triorganotin(IV) complexes 1 and 2 exhibit significantly
higher activity than that of diorganotin complex 3. This is
attributed to the availability of additional coordination positions
around the four coordinated Sn(IV) ions in 1 and 2, while the
negligible antitumour activity shown by compound 3, on the
other hand, to their absence.[10,12,13] Moreover among 1 and 2,
the hydroxyl group enhances the cytotoxic activity in contrast to
the coordinated chloride anion due to the strong hydrogen
bonding interaction of hydroxyl with cellular biomolecules such
as DNA or LOX.
Moreover, 1 and 2 showed selective cytotoxic activity
against carcinoma cells, especially against MCF-7 (human
breast adenocarcinoma, Table 3). The strong selective activity
of these complexes against MCF-7 (ER positive) is not observed
when they were tested against breast cancer cells (MDA-MB-
231) (breast, ER negative), indicating that estrogen receptors
may also be involved in their antitumour mechanism. Addition-
ally, adenocarcinoma cells (HeLa, MCF-7, MDA-MB-231, and
786-O) are relatively more sensitive to 1 and 2 than carcinoma
cells (Cak-1, A-549, and K1) (Table 3). Also 1 and 2 showed a
far better cytotoxic activity than cisplatin which against MCF-7
is 76.2 and 141.4-fold higher respectively.
Experimental
Materials and Instruments
All solvents used were of reagent grade, organotin chlorides
(Aldrich, Merck) were used with no further purification.
Crystallization of Ph3SnCl (1), [Ph3SnOH)]n (2),
and [(Ph2Sn)4Cl2O2(OH)2] (3)
Crystals of complex 1 were grown from a methanol/acetonitrile
solution of Ph3SnCl. The reaction between Ph3SnCl (1 mmol,
0.347 g) with an equimolar amount of potassium hydroxide
solution (1 cm3, 1 N) results in the formation of a white precipite,
which was filtered off. The precipitate was first crystallized in
toluene/dichloromethane (1 : 1) and then in methanol/aceto-
nitrile (1 : 1) solution. After slow evaporation of the solvents, an
oily product was formed. Crystals of complex 2 suitable for
X-ray analysis was formed by the slow evaporation of diethyl
ether/methanol/acetonitrile (6 : 1 : 1) solution. Finally, crystals
of complex [(Ph2Sn)4Cl2O2(OH)2] (3) were grown from hot
acetone/water solution, of the white precipitate, filtered off,
from the partial neutralization of [Ph2SnCl2] (1 mmol, 0.344 g)
by KOH (0.5 cm3, 1 N).
Compounds 1–3 showed inhibitory activity towards LOX.
Since LOX inhibition of organotin(IV) complexes was found to
be related with their high cytotoxicity by interfering to mito-
chondrion,[10a,12–13] the significant high cytostatic activity
shown by 1 and 2 (see Cytotoxicity above), along with the
moderate inhibitory activity towards LOX, implies an additional
inhibition mechanism. The binding constants Kb towards
CT-DNA indicates interference of 1–3 with DNA. It is well
known that cytotoxic drugs such as cisplatin kill tumour cells[24]
by interacting with DNA bases. Interaction with mitochondrion
may also cause apoptosis.[24] Compounds 1 and 2 on the other
hand, involve interaction with both DNA and mitochondrion.
The superior cytostatic activity of 1 and 2 shown towards
MCF-7 and the high activity found for the rest of cancerous
cell lines (Table 3) is reinforced by their selectivity to estrogen
receptors. However, compound 3 does not affect cell viability in
any cell line tested, although it interferes with DNA (higher Kb
value) and shows strongest LOX inhibition (lower IC50 inhibi-
tory value of LOX activity) among 1–3, and showed no
selectivity to positive estrogen receptor cell lines. Therefore,
a difference in cytotoxic mechanism is expected for 3. This also
occurs in the case of the [SnPh2(BZDO)2] studied previously.[14a]
X-Ray Structure Determination
Intensity data for the pale brown crystals of 1 and 3 were col-
lected on a Nonius Kappa CCD area detector and the crystals of
2 on an Agilent Technologies SuperNova diffractometer
with Atlas detector, (f and v scans to fill the asymmetric unit)
in the range 3.08ꢂ27.488 (for 1), 3.00ꢂ29.008 (for 2), and
3.23ꢂ27.488 (for 3), using graphite-monochromated MoKa
˚
radiation (l ¼ 0.71073 A) at 120(2) K. The structures were
solved using direct methods with SHELXS-97[25a] or
Sir2004[25b] and refined by full-matrix least-squares procedures
on F2 with SHELXL-97.[25a] Hydrogen atoms were fixed as
riding models. In case of 3, H1, the hydrogen atom bonded to
O1, was located from the electron density and refined. All other
hydrogen atoms were fixed as riding models.
Compound 1: C18H15ClSn, M ¼ 385.44, monoclinic, space
˚
group P21/c, a ¼ 19.0027(2), b ¼ 18.5503(3), c ¼ 18.3714(2) A,
3
˚
b ¼ 98.615 (1)8, V ¼ 6402.96 (14) A , Z ¼ 16, T ¼ 120 (2),
r(calc) ¼ 1.599 g cmꢂ3
,
m ¼ 1.750 mmꢂ1
,
F(000) ¼ 3040,
84110 reflections measured, 14653 unique (Rint ¼ 0.0514)
which were used in all calculations. The final R1 ¼ 0.0365

J
V. I. Balas et al.
40
35
30
25
20
15
10
5
(b)
1
(a)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0
20
40
60
80
100
120
[DNA] ꢁ 106 [M]
Comp. 1
230
250
270
290
310
330
350
370
390
410
430
450
Wavelength [nm]
r ꢂ 1
r ꢂ 0.5
r ꢂ 0.25
r ꢂ 0.17
r ꢂ 0.125
r ꢂ 0.1
1
(a)
2
(b)
30
25
20
15
10
5
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
0
0
20
40
60
80
100
120
[DNA] ꢁ 106 [M]
Comp. 2
240
270
300
330
360
390
420
450
Wavelength [nm]
r ꢂ 1
r ꢂ 0.5
r ꢂ 0.25
r ꢂ 0.17
r ꢂ 0.125
r ꢂ 0.1
2
3
(a)
14
12
10
8
6
4
(b)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
2
0
0
20
40
60
80
100
120
[DNA] ꢁ 106 [M]
Comp. 3
295
310
325
340
355
370
385
400
Wavelength [nm]
r ꢂ 1
r ꢂ 0.5
r ꢂ 0.25
r ꢂ 0.17
r ꢂ 0.125
r ꢂ 0.1
3
Fig. 6. (a) UV spectra of complexes 1–3 in the absence and presence of calf thymus (CT)-DNA at r values of 1, 0.5, 0.25,
0.17, 0.125, and 0.1 (r ¼ [complex]/[DNA], [1] ¼ 10 ꢃ 10ꢂ5 M, [2] ¼ 15 ꢃ 10ꢂ5 M, [3] ¼ 2 ꢃ 10ꢂ5 M, [CT-DNA] ¼
10–100 mM. (b) Graphical plot of [DNA]/(ea – ef) versus [DNA].
(for 11819 reflections with I . 2s(I)) and wR(F2) ¼ 0.0992
(all data).
unique (Rint ¼ 0.015) which were used in all calculations.
The final R1 ¼ 0.0193 (for 3397 reflections with I . 2s(I))
and wR(F2) ¼ 0.0391 (all data).
Compound 2: C18H16OSn, M ¼ 366.99, orthorhombic, space
˚
group P212121, a ¼ 8.2705(2), b ¼ 10.2202(2), c ¼ 17.6775(3)ꢂA3,
Compound 3: C48H42Cl2O4Sn4, M ¼ 1228.48, triclinic,
space group P-1, a ¼ 10.1080(3), b ¼ 10.7141(3), c ¼
3
˚
V ¼ 1494.21(5) A , Z ¼ 4, T ¼ 100 K, r(calc) ¼ 1.560 g cm
,
m ¼ 1.7 mmꢂ1, F(000) ¼ 728, 7489 reflections measured, 3397
11.6629(3) A, a ¼ 78183(2)8, b ¼ 103.197(4)8, g ¼ 75802(2)8,
˚

Structural and In Vitro Biological Studies of Organotin(IV) Precursors
K
1
2
3
Fig. 7. Binding sites of 1–3 towards calf thymus (CT)-DNA.
3
ꢂ3
,
˚
V¼ 1123.12 (6) A , Z¼ 1, T¼ 120 (2) K, r(calc) ¼ 1.816gcm
trypsin (Gibco) – 0.02 % EDTA (Sigma) mixture and incubation
for 2–5 min at 378C. The loss of membrane integrity, as a
morphological characteristic for cell death, was assayed by
Trypan Blue exclusion.[26a] The number of cells that were alive
was estimated through a haematocytometer and phase-contrast
microscopy. Each result represented the mean of four indepen-
dent measurements and used for the inoculation of cells in the
microplates. All chemicals and solvents used were of high purity
and purchased from Sigma or Merck.
m ¼ 2.360 mmꢂ1, F(000) ¼ 596, 13189 reflections measured,
5088 unique (Rint ¼ 0.0490) which were used in all calculations.
The final R1 ¼ 0.0359 (for 5088 reflections with I . 2s(I)) and
wR(F2) ¼ 0.0860 (all data).
Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre and have been allo-
cated the deposition numbers: CCDC 778236 (1), 902582 (2),
and 902865 (3). Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (fax: (þ44) 1223–336–033; email: deposit@ccdc.cam.ac.uk.
Experimental Agents
Tin complexes and the chemotherapeutic drug cisplatin used as
a reference Pt complex (CDDP, PtCl2(NH3)2) were tested in
nine graduated sextuplicate dilutions in complete growth
medium, starting with a peak concentration of 10 and 50 mM,
respectively. The cytotoxic activity of all agents was tested in
concentrations covering the range of 0.001 to 10 mM for Sn
complexes and 0.005 to 50 mM for CDDP. Tin complexes were
dissolved in DMSO to a concentration of 10 mM (for 1 and 2)
and 2 mM (for 3). The final concentration of DMSO was less
than 0.1 %, a concentration that exhibits no effect on cell growth
and proliferation, as was experimentally confirmed. Stock
solutions were sterilized via filtration (0.22 mm) and kept at
2–48C. CDDP (cisplatin) was a ready solution of 1.7235 mM in
concentration, stored at room temperature. A concentrated
solution of each agent was prepared in complete growth medium
and used to make serial dilutions immediately after the agent
was dissolved.
Biological Studies
In Vitro Cytotoxic Activity-Cell Lines and Culture
Maintenance
Human cancer cell lines used as targets were HeLa (cervical),
MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER nega-
tive), A549 (lung), Caki-1 (kidney carcinoma), 786-O (renal
adenocarcinoma), K1 (thyroid carcinoma) and additionally, the
normal human lung cell line MRC-5 and normal immortalized
human mammary gland epithelial cell line MTSV-17. All cells
were obtained from the American Type Culture Collection
(ATCC, Rockville, MD, USA) and the Imperial Cancer
Research Fund (ICRF), London. Cells were routinely grown
as monolayer cell cultures in T-75 flasks (Costar) in an atmo-
sphere containing 5 % CO2 in air, and 100 % relative humidity at
378C and subcultured twice a week, restricting the total number
of cell passages below 20. The culture medium used was
Dulbecco’s modified Eagle’s medium, DMEM (GIBCO BRL
Glasgow, UK), supplemented with 10 % fetal bovine
serum (GIBCO BRL Glasgow, UK), 2 mM glutamine (Sigma),
100 mg mLꢂ1 streptomycin, and 100 IU mLꢂ1 penicillin. Cell
passages were carried out by detaching adherent cells at a
logarithmic growth phase by addition of 2–3 mL of a 0.05 %
Cell Inoculation – Drug Exposure – SRB
Cytotoxicity Assay
For the experiments, cells were plated (100 mL per well) in
96-well flat-bottom microplates (Costar-Corning, Cambridge)
at various cell inoculation densities (Hela and MRC-5: 5000
cells/well, A-549, K1, MDA-MB-231, Caki-1, MTSV-17,
786-O, and MCF-7: 10000 cells/well) so that untreated cells

L
V. I. Balas et al.
were in exponential growth phase at the time of cytotoxicity
evaluation. Cells were left for 24 h at 378C to resume expo-
nential growth and stabilization and afterwards exposed to
tested agents for 72 h by the addition of an equal volume (100 mL)
of either complete culture medium (control wells), or twice the
final drug concentrations diluted in complete culture medium
(test wells). Drug cytotoxicity was measured by means of a SRB
colorimetric assay estimating the survival fractions (SF) as the
percent of control (untreated cells) absorbance. The SRB assay
was carried out as previously described[26b] and modified by our
group.[26c] In brief, culture medium was aspirated before fixa-
tion using a microplate-multiwash device (Tri-Continent Sci-
entific, Inc., Grass Valley, CA) and 50 mL of 10 % cold (48C)
trichloroacetic acid (TCA) were gently added to the wells.
Microplates were left for 30 min at 48C, washed five times with
deionized water and left to dry at room temperature for at least
24 h. Subsequently, 70 mL of 0.4 % (w/v) sulforhodamine B
(Sigma) in 1 % acetic acid solution were 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 mL of 10 mM
unbuffered Tris-base solution and plates were left on a plate
shaker for at least 10 min. Absorbance was read in a 96-well
plate reader at 492 nm subtracting the background measurement
at 620 nm. The test optical density (OD) value was defined as the
absorbance of each individual well, minus the blank value
(‘blank’ is the mean optical density of the background control
wells, n ¼ 8). Mean values and the coefficient of variation (CV)
from six replicate wells were calculated automatically. Results
were expressed as the SF, as shown below.
Parameters like flexible torsions, bond orders, and charges were
assigned automatically to the complexes while no geometric
constraints were imposed to the initial structures obtained by
X-ray diffraction.
DNA docking interactions were performed with ICM-Pro 3.5
software[27b,c] which is based on a biased-probability Monte
Carlo algorithm performing a global optimization using a
modified version of the ECEPP/3 force field.[26d] The three-
dimensional coordinates of DNA were constructed according to
the sequence 50-D(*AP*CP*CP*GP*AP*CP*GP*TP*CP*GP*
GP*T)-30 based on the structure 423D from Protein Data Bank.
The Monte Carlo thoroughness was set to 1 and the conforma-
tion selected was the one with the lowest energy. A grid potential
˚
map for the receptor was created at a grid size of 0.5 A and was
allocated along the whole DNA molecule. Potential binding
sites were specified automatically by the software.
DNA Binding Studies
This study was performed as we previously reported.[22a]
Supplementary Material
1H-NMR spectra of the complexes 1–3 are available on the
Journal’s website.
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