Synthesis, spectroscopic characterization, X-ray structure, and in vitro antitumor activities of new triorganotin(IV) complexes with sulfur donor ligand

Article abstract of DOI:10.1007/s00044-013-0549-9

The discovery of the anticancer properties of cisplatin promoted a great deal of interest in the area of metal-based antitumor agents. With this aim, a new series of triorganotin(IV) derivatives: Ph₃SnL (1), BZ ₃SnL (2), where L = morpholine-1-carbodithioate (MCDT) have been synthesized by the reaction of triorganotin(IV) chlorides with the ligand-salt in the appropriate molar ratio. All the complexes have been characterized by FT-IR, ¹H, ¹³C, and ¹⁹Sn NMR spectroscopy. In addition, the molecular structure of complex 1 was determined by single crystal X-ray diffraction study. X-ray crystallographic analysis shows that in the compound [Ph₃Sn(MCDT)], the Sn ion is in a four-coordinate environment with a dithiocarbamate ligand coordinated to the tin(IV) in a monodentate fashion through the sulfur atom. Furthermore, the cytotoxic activity of the free ligand (MCDT) as well as its triorganotin(IV) complexes were tested against tumor cell lines human cervix carcinoma HeLa, human myelogenous leukemia K562 and normal immunocompetent cells, peripheral blood mononuclear cells PBMC. The cytotoxic results indicate that coupling of MCDT with R ₃Sn(IV) metal center results in a complex with important biological properties and remarkable cytotoxic activity, since we observe IC₅₀ values better to that of the antitumor drug cisplatin.

Full text of DOI:10.1007/s00044-013-0549-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0549-9
ORIGINAL RESEARCH
Synthesis, spectroscopic characterization, X-ray structure,
and in vitro antitumor activities of new triorganotin(IV)
complexes with sulfur donor ligand
Mojdeh Safari
Hilary A. Jenkins
Amir Amanzadeh
•
Mohammad Yousefi
Maryam Bikhof Torbati
•
•
•
Received: 29 November 2012 / Accepted: 16 February 2013
Springer Science+Business Media New York 2013
ꢀ
Abstract The discovery of the anticancer properties of
cisplatin promoted a great deal of interest in the area of
metal-based antitumor agents. With this aim, a new series
of triorganotin(IV) derivatives: Ph SnL (1), BZ SnL (2),
indicate that coupling of MCDT with R Sn(IV) metal
3
center results in a complex with important biological
properties and remarkable cytotoxic activity, since we
observe IC50 values better to that of the antitumor drug
cisplatin.
3
3
where L = morpholine-1-carbodithioate (MCDT) have
been synthesized by the reaction of triorganotin(IV) chlo-
rides with the ligand-salt in the appropriate molar ratio. All
Keywords Triorganotin (IV) complex ꢀ
Dithiocarboxylate ꢀ Crystal structure ꢀ NMR spectroscopy ꢀ
Antitumor activity ꢀ Cell lines
1
13
the complexes have been characterized by FT-IR, H, C,
1
9
and Sn NMR spectroscopy. In addition, the molecular
structure of complex 1 was determined by single crystal
X-ray diffraction study. X-ray crystallographic analysis
shows that in the compound [Ph Sn(MCDT)], the Sn ion is
3
Introduction
in a four-coordinate environment with a dithiocarbamate
ligand coordinated to the tin(IV) in a monodentate fashion
through the sulfur atom. Furthermore, the cytotoxic activity
of the free ligand (MCDT) as well as its triorganotin(IV)
complexes were tested against tumor cell lines human
cervix carcinoma HeLa, human myelogenous leukemia
K562 and normal immunocompetent cells, peripheral
blood mononuclear cells PBMC. The cytotoxic results
Research on the synthesis and applications of metal-based
antitumor drugs is currently considered one of the most
quickly-expanding areas in biomedical and inorganic
chemistry (Gomez-Ruiz et al., 2010; Gomez-Ruiz et al.,
2008). Cisplatin is a well-established metal-based antitu-
mor agent, however, its use is restricted due to acquired
cell resistance after continuous treatment and lethal side
effects such as nausea and organ failure typical of heavy
metal toxicity (Khan et al., 2011). More recently, different
studies with very interesting results on the in vitro antitu-
mor properties of organotin compounds against a wide
panel of tumor cell lines of human origin have been
reported and found to be as effective as, or even better than,
traditional heavy metal anticancer drugs (Kalu Cerovi
et al., 2010). Organotin(IV) compounds are among the
most widely used organometallic compounds owing to a
variety of industrial and agricultural applications, including
their use as pesticides, fungicides and anti-fouling agents
M. Safari ꢀ M. Yousefi (&)
Department of Chemistry, Shahr-e-Rey Branch, Islamic Azad
University, Tehran, Iran
e-mail: myousefi50@yahoo.com
H. A. Jenkins
Department of Chemistry & Chemical Biology, McMaster
University, Hamilton, ON, Canada
M. B. Torbati
Department of Biology, Shahr-e-Rey Branch, Islamic Azad
University, Tehran, Iran
(Rehman et al., 2011). Usually, triorganotin(IV) com-
pounds display a higher biological activity than their
di- and monoorganotin(IV) counterparts, which has been
related to their ability to bind to proteins (Gomez-Ruiz
M. Safari ꢀ A. Amanzadeh (&)
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
e-mail: amir_amanzadeh@yahoo.com
123

Med Chem Res
et al., 2010). In recent years, considerable efforts have been
made to synthesize and characterize organotin compounds
with O, N, S donor ligands, and many studies have been
focused on their structure–activity correlations (Sedaghat
and Jalilian, 2009). Among the various organosulfur sys-
tems, dithiocarboxylates and their metal complexes are of
special significance due to their use as catalysts in the
rubber industry and in pesticides (Shaheen et al., 2012;
Rehman et al., 2009a). Moreover, dithiocarboxylates have
generated interest because of their antibacterial, antiviral,
and antitumor activities (Rehman et al., 2011). Dithio-
carboxylates as ligands are well known to bind strongly
and selectively to many metal ions (Husain et al., 2010;
Fuentes-Martinez et al., 2009). Crystallographic studies
have revealed that the coordination geometry at the tin
atom depends not only on the factors such as R-radical
stereochemistry, but also whether the 1,1-dithiolates
behave as monodentate or bidentate ligands and whether
the complexes are monomeric or oligomeric (Yin et al.,
compounds at room temperature by the reaction of the
ligand (MCDT) with the selected triorganotin(IV) chloride
(R=C H , CH –C H ) in 1:1 mol ratio. Spectroscopic
6
5
2
6 5
studies agree with the proposed formulation for the com-
plexes. The mechanisms for the formation of the ligands
and complexes are as follows (Scheme 1):
Infrared spectroscopy
FT-IR band values related to different functional groups
have been assigned by comparison of the FT-IR spectra of
the complexes (1–2) with the free ligand (MCDT). The two
bands indicative of C–S and C–N stretching vibrations give
valuable information about the coordination behavior of
O
NH
+
CS
2
a) Dry MeOH
b) 4 h stirring
c) 273 K
2
004; Rehman et al., 2009b). Nowadays, the dithiocarb-
amato moiety has been exploited as a useful structural
motif for metal directed self- assembled systems with a
range of structures such as nano-sized resorcarene-based
assemblies, catenanes, assorted macrocycles and cryptands
(
Husain et al., 2010). Organotin(IV) dithiocarboxylates
continue to attract significant attention because of their
broad spectrum of biological applications as fungicides,
bactericides, insecticides and antitumor agents (Shaheen
et al., 2012; Hadjikakou and Hadjiliadis, 2009). Further-
more, the significance of the presence of tin was illustrated
by the fact that all the organotin compounds investigated
had greater biological activities than those exhibited by the
uncoordinated dithiocarboxylate ligand (Menezes et al.,
S
O
N
SH
O
NH
..
2
008). Although many efforts have been made to explore
S
S
the mechanism of the biological action of organotin com-
plexes, none of them have been entirely successful
O
N
(
Metsios et al., 2012). Keeping all these factors in mind,
H N
2
O
we aimed to synthesize and characterize new organotin(IV)
derivatives of a ligand morpholinium morpholine-1-car-
bodithioate (MCDT) and to screen them for their antitumor
activity.
Dry MeOH
-
Salt 6h reflux
R SnCl
3
Results and discussion
R
S
Syntheses of ligand-salt (MCDT) and complexes 1–2
+
N
O
Salt = ClH N
O
R
Sn
2
The reaction of CS with a secondary amine (morpholine)
2
S
in dry methanol at about 0 ꢁC leads to the formation of
morpholinium morpholine-1-carbodithioate. The synthesis
of the free ligand (MCDT) and the full spectroscopic
characterization have been previously reported (Yousefi
et al., 2012). The complexes were obtained as air stable
R
R= C H (1), CH -C H (2)
6
5
2
6 5
Scheme 1 Synthetic mechanism of ligand-salt and complexes
1
23

Med Chem Res
1
Scheme 1. In the H-NMR spectrum of compound 1, two
the dithiocarboxylate ligand to the Sn atom, and thus give a
clue about the structure of the complexes. The presence of
sets of signals at 7.78–7.76 and 7.46–7.42 ppm corre-
sponding to the protons of the phenyl groups of the SnPh3
moiety, were observed. The protons of the benzyl part
resonate in two regions; a singlet at 2.69 ppm due to CH2
protons and two sets (7.23–7.2, 7.09–6.85 ppm) of the
phenyl moiety. The coordination around Sn atom was
-
1
single band in the region of 900–1,000 cm is charac-
teristic of a bidentate nature for the dithiocarbamate moi-
ety, while the splitting of the same band within a difference
-
1
of[20 cm in the same region is due to the monodentate
binding of dithiocarboxylate ligand (Khan et al., 2008). In
our present study, the appearance of two bands for C–S, in
complexes 1 and 2, is in agreement with monodentate
bonding of 1,1-dithiolate moiety with Sn. The presence of
delocalized double bond in NCS2 moiety was further
confirmed by X-ray single crystal analysis for complex 1.
2
119
1
deduced from the [ J( Sn, H)] coupling constant of
70 Hz for compound 2, thus confirming five coordinated
Sn atom in solution (Rehman et al., 2008). Owing to the
complex peak patterns observed for the organic groups
bonded to Sn, (just give the compound number here, either
1 or 2) no tin-proton satellites were observed and hence
-
1
The strong bands observed in the 1,465–1,489 cm
1
3
region for both complexes (1–2) are attributed to the m(C–
N) stretching vibration. This band is observed at a lower
assignment of the geometry is not possible. The C NMR
data exhibited explicitly the resonances of all the distinct
carbon atoms present in the compounds. The important
chemical shift of the carbon in dithiocarbamate compound
is thion carbon (CS ). The signal due to the CS carbon
-
1
frequency in the free ligand (1,450 cm ) and indicates an
increase of the carbon–nitrogen double bond character,
caused by electron delocalization toward the metal center
upon coordination to the metal atoms (Cea-Olivares et al.,
2
2
atom in the free ligand appears at 213.6 ppm. However, in
the spectra of the corresponding tin complexes, these
appear at 196 and 201 ppm, respectively. Small chemical
shifts in the position of C(1) were noted, resulting from
deshielding of this particular carbon due to disconnection
of cationic part of the ligand and coordination of the CS2
moiety through the sulfur atom to the tin moiety.
2
000). In addition, the absorption bands appeared in the
-
1
-1
region 444–446 cm
and 542–543 cm
and can be
assigned to the Sn–C and Sn–S stretching mode of vibra-
tions, respectively (Scheme 2).
NMR spectroscopy
A characteristic feature of the triphenyltin derivative is
13
the observation of the C chemical shift of the ipso-carbon
1
In the H-NMR spectra of the complexes (1–2), the ratio of
the integrals of the signals from protons of the ligand to
those from protons of the organic groups on the Sn atom
provided a reliable measure of the metal-to-ligand ratio in
at about 142 ppm, which is attributed to a five-coordinate
1
Sn atom (Shahzadi et al., 2006). C NMR spectral data for
3
the studied compounds are listed along with their syntheses
119
in the experimental section. Sn NMR spectroscopy has
1
the synthesized complexes. The H-NMR spectrum of
MCDT displayed the expected resonances with appropriate
multiplicities and intensities. The morpholine protons show
up as four triplets (4.34–4.36, 3.76–3.78, 3.60–3.62, and
also been very supportive for the revelation of environment
around Sn atom in the organotin(IV) dithiocarboxylates.
1
19
The presence of single
Sn peak in the spectra of all
3
.06–3.08 ppm) in the aliphatic region, as expected for the
compounds is consistent with the formation of a single
1
19
structure. The structure of the MCDT salt is shown in
species. In general,
Sn chemical shifts move to lower
frequency with increasing coordination number. Although
the shift ranges are somewhat dependent on the nature of
the substituents at the tin atom, the following ranges have
been proposed for some tin(IV) dithiocarbamato com-
plexes: ?120 to -145 ppm for four-coordinate com-
pounds, -150 to -250 ppm for five-coordinate compounds
and -300 to -500 ppm for six-coordinate compounds
a
3
2
S
S
2
a
3a
O
N
1
H N
2
O
3
'
2'
(
Tarassoli et al., 2001). The organotin(IV) complexes 1 and
exhibit a singlet at -174 and -237 ppm, respectively,
2
'a 3'a
2
thus indicating only existence of five-coordinate tin atoms
in solution, which are not accordance with the structures in
the solid state.
b
γ
β
γ
δ
α
H
C
α
2
β
δ
ε
X-ray crystallography
,
Crystals suitable for X-ray diffraction studies of the com-
Scheme 2 Numbering scheme of ligand-salt and organic groups
a, b)
(
plex (1) were grown by slow evaporation of a methanol
123

Med Chem Res
solution of the complex at room temperature. The molec-
ular structure and packing of the molecule in a unit cell of
the complex are depicted in Figs. 1 and 2, while selected
bond lengths and bond angles and crystal data are given in
Tables 1 and 2, respectively. X-ray crystallographic study
of compound 1, has clearly shown that the Sn(IV) atom is
located at the center of a distorted tetrahedron, where it is
surrounded by three phenyl groups and one dithiocarba-
mate ligand (Fig. 2). The tin center in the complex 1 is
four-coordinate, with the dithiocarbamate group in a
monodentate chelating coordination mode. The complex
crystallizes in the monoclinic space group P2(1)/c with
four molecules in the unit cell. In the synthesized complex,
two different lengths of nitrogen-carbon bonds are
˚
observed, the single N–C bond length of 1.4701 (18) A in
the heterocycle of the dithiocarbamate ligand is signifi-
˚
cantly longer than the exocyclic in 1.3326 (16) A for N(1)–
C(1) suggesting a partial double bond character. The N(1)–
˚
C(1) distance of 1.3326 (16) A is similar to those of
3
Fig. 2 Packing of molecules in a unit cell of Ph Sn(MCDT)
complex [(C H ) Sn(S CNRC H )] (R=CH3, C H )
6
5 3
2
6
11
2 5
˚
1.324 A) (Awang et al., 2011), but longer than that of
(
˚
complex Me Sn [(S CN(CH )(i–C H )] (1.309 A) (Aw-
Pharmacology: antiproliferative activity in vitro
2
2
3
3 7 2
ang et al., 2010). The two C–S bond lengths [C1–
˚
S1 = 1.7576 (12) A and C1–S2 = 1.6932 (13) A] are
˚
The in vitro cytotoxicity of the free ligand (MCDT) and
triphenyltin(IV) complexes 1–2 against tumor cell lines
human cervix carcinoma HeLa, human myelogenous leu-
kemia K562 and on normal immunocompetent cells,
peripheral blood mononuclear cells (PBMC), were deter-
mined using the MTT microculture colorimetric assay. On
the basis of this study, the present investigation was
designed to investigate organotin(IV) complexes with
improved in vitro antitumor activity. Indeed, an improved
activity was observed for the triphenyltin(IV) complex
of the present investigation. The results of the in vitro
cytotoxicity tests performed with morpholinium morpho-
line-1-carbodithioate (MCDT) and its triorganotin(IV)
complexes, are summarized in Table 3 and the screening
results are compared with the standard drug (cisplatin) that
is in current clinical use as antitumor agent. From the
derived IC50 values, it is concluded that the cytotoxic effect
of the triorganotin complexes and the free ligand on tested
cells was strictly concentration dependent (Fig. 3). The
free ligand (MCDT) is less toxic compared to triorganotin
complexes and more toxic than cisplatin against the studied
cell lines. Complexes 1 and 2 present lower IC50 values
than those of cisplatin, which indicates their higher activity
against the tumoral cell lines evaluated. The IC50 values for
compound 1 against HeLa and K562 cell lines are 0.11 and
unequal, attesting the coordination of S1 of ligand to Sn
˚
atom. The Sn(1)–S(1) bond length is 2.4673 (4) A, almost
equal to the bond distance of complex (C H ) Sn
6
5 3
˚
C H N S O ) (2.4683(8) A) (Rehman et al., 2010). It is
8 13 2 2 2
(
worth noting that the values of the final R indices in the
studied triorganotin(IV) complex are R1 = 0.0207,
wR2 = 0.0471. Furthermore, the absence of intermolecu-
lar Sn….S interactions in complex 1, can be explained on
the basis of steric reasons of the bulky phenyl groups.
Attempts to prepare suitable crystals of complex 2 were
unsuccessful.
0.01 lg/mL, which are 12 and 171 times more active than
cisplatin, respectively. According to these results, 1 is the
most active complex of this study. Although the complex 1
in the present investigation possess quite high in vitro
cytotoxicity, it should be noted that the triphenyltin(IV)
3
Fig. 1 Molecular structure of Ph Sn(MCDT)
1
23

Med Chem Res
˚
Table 1 Selected bond lenghts (A) and bond angles (ꢁ) for
Ph Sn(MCDT)
Table 2 Crystallographic data for Ph
3
Sn(MCDT)
3
Empirical formula
C
23
H23NOS Sn
2
Sn(1)–C(11)
2.1342(12)
Formula weight
512.23
Sn(1)–C(21)
2.1427(12)
2.1587(12)
2.4673(4)
1.7576(12)
1.6932(13)
1.3326(16)
117.18(5)
105.38(5)
104.77(5)
113.67(3)
118.50(4)
92.55(3)
Temperature
296(2) K
Sn(1)–C(31)
˚
Wavelength
0.71073 A
Monoclinic
P2(1)/c
Sn(1)–S(1)
Crystal system
S(1)–C(1)
Space group
S(2)–C(1)
Unit cell dimensions
N(1)–C(1)
˚
7.2077(7) A
a
C(11)–Sn(1)–C(21)
C(11)–Sn(1)–C(31)
C(21)–Sn(1)–C(31)
C(11)–Sn(1)–S(1)
C(21)–Sn(1)–S(1)
C(31)–Sn(1)–S(1)
C(1)–S(1)–Sn(1)
˚
10.7209(7) A
b
˚
11.0037(7) A
c
a
83.2310(10)ꢁ
79.7290(10)ꢁ
71.5540(10)ꢁ
4
b
c
Z
95.82(4)
3
Density (calculated)
Crystal size
1.584 Mg/m
3
0.27 9 0.24 9 0.08 mm
Reflections collected
Independent reflections
Final R indices
R indices (all data)
Data/restraints/parameters
34,587
(
morpholine-1-carbodithioate) is even more cytotoxic than
7,517 [R(int) = 0.0259]
diphenyltin(IV)bis(morpholine-1-carbodithioate) which is
well consistent with the results reported in the former lit-
erature (Yousefi et al., 2012). IC50 values of complex 2
against K562 and HeLa cell line are similar, which are 10
times better than that of the antitumor drug cisplatin. In
order to determine the selectivity in the in vitro cytotox-
icity of MCDT and complexes (1–2) some additional
experiments were conducted on stimulated PBMCs. The
IC50 values for MCDT, complex 1 and 2 against stimulated
PBMCs are 0.99, 0.004, and 0.33 lg/mL, respectively. It is
worth noticing that the triorganotin(IV) complexes 1 and 2
bearing very similar moieties showed notable differences
in the cytotoxic activity. From those results, one can
envisage that small structural changes, (replacement of
phenyl with benzyl group) led to a significant decrease of
the final cytotoxic activity. As these results are preliminary,
further study on the antitumor effects of these complexes is
highly recommended.
R
R
1
1
= 0.0207, wR
= 0.0245, wR
2
2
= 0.0471
= 0.0489
7,517/0/345
1
13 119
H, C, Sn NMR were obtained by a Bruker AVANCE
500 spectrometer. The splitting of proton resonances in the
1
reported H-NMR spectra are defined as s = singlet,
d = doublet, t = triplet, q = quadruplet, m = multiplet.
X-ray: Crystals suitable for analysis were selected and
mounted on a MiTeGen head and placed in the cold stream
of the diffractometer (at 100 K). Data were collected on a
Bruker Apex2 diffractometer, using MoKa radiation
˚
source (k = 0.7107 A) at the MAX Diffraction Facility at
McMaster University. Unit cell parameters were deter-
mined using data from three different x scans; the full
dataset was collected using x and u scans. The data were
corrected for absorption (Apex2 programing system, 2012,
Bruker, Madison WI). Data were solved using the Apex2
software suite. All non-hydrogen atoms were refined aniso-
tropically, and hydrogen atoms were located and then held
as riding on their constituent atoms in subsequent refine-
ments. The analysis of crystal packing and structure visu-
alization were done using the Mercury and Ortep programs.
Experimental
Material and physical measurement
The reagents, triphenyltin(IV) chloride, morpholine, and
CS were purchased from commercial sources and were
2
Synthesis of morpholinium morpholine-1-
carbodithioate (MCDT)
used without further purification. Tribenzyltin(IV) chloride
was prepared by a standard method reported in the litera-
ture (Sisido and Takeda, 1961). Melting points were
determined in open capillaries and were uncorrected. FT-
IR spectra were recorded on a Bomem MB-100 FT-IR
Dropwise addition of CS (in excess, 1.7 mL,23 mmol) in
2
methanol (30 mL) to morpholine (1 mL, 11.5 mmol) in
methanol (30 mL) followed by stirring for 4 h at 0 ꢁC gave
the white product. The white precipitates obtained were
-
1
spectrometer, using KBr pellets (400–4,000 cm ). The
123

Med Chem Res
Table 3 IC50 (lg/mL) for the 72 h of action of the studied com-
pounds and cisplatin on HeLa, K562 and PBMC stimulated with PHA
determined by MTT test
MCDT
100
8
6
4
2
0
0
0
0
-
Compound
HeLa
K562
PBMC ? PHA
HeLa
MCDT
0.38
0.11
0.21
1.32
0.031
0.01
0.21
1.71
0.99
0.004
0.33
7.8
K562
Complex (1)
Complex (2)
PBMC+PHA
a
Cisplatin
a
Standard drug, cis-[Pt(NH
3
)
2
(Cl)
2
]
0
0.001 0.01 0.1
1
10
Concentration µg/ml
filtered and washed with diethyl ether. M.P.: 195 ꢁC; FT-
IR (KBr): m = 1019 (C–S), 1450 (C–N) cm ; H-NMR
-
1 1
Complex 1
1
00
80
60
3
4H, JH-
(
500 MHz, DMSO): d = 4.35 (t, H3,3
0
,
3
, 4H, JH-H = 10 Hz), 3.60 (t,
H = 10 Hz), 3.77 (t, H3
0
a,3 a
HeLa
3
H2,2
H = 10 Hz) ppm;
d = 213.6 (C-1), 66.94 (C-3,3 ), 64.23 (C-3a,3 a), 50.68
0
, 4H, 3JH-H = 10 Hz), 3.07 (t, H
0
, 4H, JH-
2a,2 a
K562
1
3
C
NMR (500 MHz, CDCl3):
4
2
0
0
-
PBMC+PHA
0
0
0 0
C-2,2 ), 43.71 (C-2,2 a) ppm.
(
0
0.001 0.01 0.1
1
10
Synthesis of triphenyltin(IV) (morpholine-1-
carbodithioate) (1)
Concentration µg/ml
Complex 2
100
A solution of Ph SnCl in methanol (0.23 g, 0.6 mmol) were
3
added dropwise to the methanolic solution of ligand-salt
8
0
0
0
(
0.15 g, 0.6 mmol). The reaction mixture was refluxed for 6 h
6
4
HeLa
with constant stirring and the solvent was evaporated, then the
colorless crystals were separated out 5 days later. M.P.:
K562
1
1
60 ꢁC; FT-IR (KBr): m = 1068, 1112 (C–S), 1465 (C–N),
PBMC+PHA
-1 1
427–1572 m(C=C), 446 (Sn–S), 543 (Sn–C) cm ; H-NMR
20
-
0
3
(
500 MHz, DMSO): d = 4.14 (t, H , 4H, J
= 10 Hz),
3
,3
H–H
0
3
3
.79 (t, H2,2 , 4H, J
= 10 Hz), 7.76–7.78 (H , SnC H ,
H–H O 6 5
0
0.001 0.01
0.1
1
10
1
H), 7.46–7.42 (m,Hm,p, SnC H , 9H) ppm; C NMR
3
6
6
5
Concentration µg/ml
0
(
500 MHz, CDCl ): d = 196.8 (C-1), 66.1 (C-3,3 ), 52.3 (C-
3
0
,2 ), 142 (C ), 136 (C ), 129 (C ), 128 (C ) ppm; Sn NMR
a b d c
119
Fig. 3 Cytotoxicity graphs from typical MTT assay showing the
effect of MCDT and triorganotin(IV) complexes on the viability of
K562, HeLa and PBMC ? PHA (PBMC stimulated with PHA) cells
2
(
500 MHz, DMSO): d = -174.5 ppm.
Synthesis of tribenzyltin(IV)(morpholine-1-
carbodithioate) (2)
In vitro antitumor screening
The synthesis of compound 2 was carried out in an iden-
tical manner as described for 1 using tribenzyltin (IV)
chloride (0.25 g, 0.6 mmol) and ligand-salt (0.15 g,
Preparation of the studied agents
Stock solutions of the studied tin complexes were prepared
in dimethyl sulfoxide (DMSO, Sigma Aldrich) at a con-
centration of 1,000 lg/mL, sterilized by filtration through
Millipore filter, 0.22 lm, before use, and diluted by cell
culture medium to various working concentration. DMSO
was used due to solubility problems. Nutrient medium was
RPMI-1640 (Gibco BRL, Scotland) supplemented with
10 % fetal bovine serum (FBS-Gibco BRL/Scotland).
MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide was dissolved (5 mg/mL) in phosphate buf-
fer saline pH 7.2, and filtered through Millipore filter,
0
.6 mmol) and L-salt (0.1 g 0.64 mmol). M.P.:140 ꢁC; FT-
IR (KBr): m = 1111,1088 (C–S), 1489 (C–N), 1427–1595
m(C=C), 444 (Sn–S), 542 (Sn–C) cm
-
1
1
;
H-NMR
3
0
(
500 MHz, DMSO): d = 4.14 (t, H , 4H, JH–H
=
3
,3
0
3
0 Hz), 3.79 (t, H2,2 , 4H, J
1
= 10 Hz), 2.69 (s, H , 6H,
H–H a
2
JSn–H = 70 Hz), 7.23–7.20 (H , SnCH C H , 6H),
2 6 5
O
13
7
.09–6.85 (m, Hm,p, Sn-CH C H , 9H) ppm; CNMR
2 6 5
0
(
500 MHz, DMSO): d = 201 (C-1), 65.6 (C-3,3 ), 43 (C-
0
,2 ), 29 (C ), 140 (C ), 128 (C ), 123 (C ), 127 (C ) ppm;
a b c e d
2
1
19
Sn NMR (500 MHz, DMSO): d = -237 ppm.
1
23

Med Chem Res
0
.22 lm, before use. HeLa cells were split using 0.25 %
TRED (Trypsin/EDTA-Gibco BRL, Scotland) medium
in 96 well microtiter plates and 4 h later, the compounds
were added to the wells, in quadruplicate, to 5 final con-
centrations, except to the control wells where enriched cell
culture medium only was added to the cells. The incubation
time was 72 h, during the period the control cells showed
exponential growth.
prior to reaching 80 % confluence.
Human cell culture
The HeLa (human cervix carcinoma) cells (NCBI C115)
and K562 (human myelogenous leukemia) cells (NCBI
C122), were obtained from National Cell Bank of Iran,
Pasteur Institute of Iran. Cells were cultured in RPMI-1640
medium supplemented with 10 % heat inactivated fetal calf
serum (FCS- Gibco BRL, Scotland) 2 mM L-Glutamine
Cytotoxicity studies: assay in the presence of MTT
Cell survival was determined by MTT test according to the
method of (Mosmann (1983) and modified by Ohno and
Abe (1991), which measures the reduction of the yellow
tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,
5-diphenyltetrazolium bromide; SIGMA) into a purple
formazan crystal, mainly by the activity of the mitochon-
drial enzymes, cytochrome oxidase, and succinate dehy-
drogenase. Briefly, cells were incubated for 72 h and then,
20 ll of MTT solution (5 mg/mL) in phosphate buffer
saline (1/10 of total volume in a well) was added to wells.
Samples were incubated for further 4 h at 37 ꢁC in
humidified atmosphere with 5 % CO2 (Fig. 3). Superna-
tants were removed and 100 lL of DMSO was added to the
plate as a solvent to each well. The plate was shaken for
15 min by a shaker incubator to dissolve the formazan
crystals.
(
Gibco BRL, Scotland) and antibiotics, including strepto-
mycin (100 lg/mL) and penicillin (100 IU/mL) (Sigma,
USA) and incubated at 37 ꢁC in a humidified 5 % CO2
atmosphere.
HeLa cells were cultured as monolayers in the com-
pleted RPMI 1640 medium, while K562 cells were main-
tained as suspension culture. The cells were grown at 37 ꢁC
in 5 % CO2 and humidified air atmosphere. Peripheral
blood mononuclear cells (PBMC) were separated from
whole heparinized blood from a healthy volunteer by den-
sity gradient centrifugation using lymphoprep (Nycomed,
Oslo, Norway). Isolated cells, washed three times with PBS,
then were counted and resuspended in nutrient medium.
Cells were counted by Neubauer slide and Trypan blue dye
exclusion method.
The 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 control cells).
Trypan blue exclusion
Finally, the absorbance at 570 nm (test wavelength) and
with a reference filter of 630 nm was measured using an
ELISA microplate reader (Stat Fax-2100, USA). All
experiments were performed three times and the percent-
age of cytotoxicity was calculated according to following
formula:
The loss of membrane integrity, as a morphological char-
acteristic for cell death, was assayed by trypan blue dye
exclusion (Moldeus et al., 1978). The alive cells were
estimated through a hematocytometer and phase-contrast
microscopy.
mean absorbance of toxicant treated cells
%
Cytotoxicity ¼ 1 ꢁ
ꢂ 100
mean absorbance of negative control
Viability ¼ 100 ꢁ % Cytotoxicity
Cell sensitivity analysis
%
HeLa cells were seeded (5,000 cells per well) into 96-wells
flat-bottom microtiter plates and incubated for 4 h prior to
the addition of filtered 5 different concentrations of the
studied compounds. Final concentrations achieved in
treated wells were 0.001, 0.01, 0.1, 1, and 10 lg/mL. Each
concentration was tested in quadruplicate on each cell line.
The final concentrations (\0.1 %) of DMSO were non-
toxic to the cells. Only complete medium was added to the
cells in the control wells. The studied compounds were
added to a suspension of leukemia K562 cells (10,000 cells
per well) 4 h after cell seeding, in the same final concen-
trations applied to HeLa cells. Each assay included a blank
containing complete medium without cells. PBMC were
seeded (200,000 cells per well) in complete medium enri-
ched with (2.5 lg/mL) phytohemaglutinin (Sigma, USA)
Statistical analysis
After subtracting the solvent toxicity, the concentration
giving 50 % inhibition (IC ) was determined for the test
5
0
samples by nonlinear regression analysis of curves. Graph
Pad Prism version 4.00 was used to calculate IC . Mean
5
0
difference among groups was calculated by paired t test,
one-way and repeated measures ANOVA (p \ 0.05).
Conclusions and outlook
A new series of triorganotin(IV) complexes containing the
dithiocarboxylate ligand have been synthesized and
123

Med Chem Res
structurally characterized by different analytical tech-
niques. Detailed studies of the structures and spectra of
these complexes indicate different structural chemistry in
solution and solid state. Both complexes have five-coor-
dinate tin as evidenced by the multinuclear solution NMR
Hadjikakou SK, Hadjiliadis N (2009) Antiproliferative and anti-tumor
activity of organotin compounds. Coord Chem Rev 253:235–249
Husain A, Nami SAA, Siddiqi KS (2010) Synthesis, characterization
and biocidal activities of heterobimetallic complexes having
tin(IV) as a padlock. J Mol Struct 970:117–127
Kalu Cerovi GN, Paschke R, Prashar S, Go
Synthesis, characterization and biological studies of 1-D poly-
meric triphenyltin(IV) carboxylates. Organomet Chem
95:1883–1890
Khan S, Nami SAA, Siddiqi KS (2008) Mononuclear indolyldithioc-
arbamates of SnCl and R SnCl : spectroscopic, thermal char-
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93:1049–1057
´
mez-Ruiz S (2010)
1
119
(
H and
Sn) while FT-IR spectra clearly demonstrated
J
that the ligand (MCDT) behaves as a monodentate che-
lating agent for coordination to tin atom, so the effective
coordination number in solid state is four. Furthermore,
based on the X-ray single crystal analysis, a tetrahedral
structure can be attributed to compound 1.
6
4
2
2
6
Khan H, Badshah A, Murtaz G, Said M, Rehman Z, Neuhausen C,
Todorova M, Jean-Claude BJ, Butler IS (2011) Synthesis,
characterization and anticancer studies of mixed ligand vdithioc-
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The in vitro cytotoxic activity has been evaluated against
human cancer cell lines: HeLa, K562 and on normal cells
PBMC. The screening results have shown that the triog-
anotin(IV) complexes have better anticancer activity than
the free ligand (MCDT). From the studied cell lines, com-
plexes 1 and 2 exhibited much higher cytotoxicity than
cisplatin. Compound 1 (Which contains phenyl group) is the
most active complex against the evaluated tumor cell lines.
Therefore, complex 1 seems to be a very promising
candidate for further in vivo tests which will be carried out
in the near future.
4
071–4077
Menezes DC, Vieira FT, de Lima GM, Wardell JL, Cort e´ s ME,
Ferreira MP, Soares MA, Vilas Boas A (2008) The in vitro
antifungal activity of some dithiocarbamate organotin(IV) com-
pounds on Candida albicans—amodelforbiological interaction of
organotin complexes. Appl Organomet Chem 22:221–226
Metsios A, Verginadis I, Simos Y, Batistatou A, Peschos D, Ragos V,
Vezyraki P, Evangelou A, Karkabounas S (2012) Cytotoxic and
anticancer
effects
of
the
triorganotin
compound
(C Sn(cmbzt)]: an in vitro, ex vivo and in vivo study.
[
Eur J Pharm Sci 47:490–496
6 5 3
H )
Moldeus T, Hogberg J, Orrhenius S, Fleischer S, Packer L (1978)
Isolation and use of liver cells. Methods Enzymol 52:60–71
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survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
Ohno M, Abe T (1991) Rapid colorimetric assay for the quantification
of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6).
J Immunol Methods 145:199–203
Acknowledgments The authors like to express their profound
gratitude to the Azad University, Shahr-e-Rey Branch, Iran and
National Cell Bank of Pasteur Institute of Iran for providing necessary
research facilities and financial assistance. The authors also gratefully
acknowledge the valuable help of Dr. Mohammad Ali Shokrgozar,
head of National Cell Bank of Iran, Pasteur Institute of Iran.
Rehman W, Kaleem Baloch M, Badshah A (2008) Synthesis, spectral
characterization and bio-analysis of some organotin(IV) com-
plexes. Eur J Med Chem 43:2380–2385
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