Organotin(IV) complexes derived from Schiff base N′-[(1E)-(2-hydroxy-3-methoxyphenyl)methylidene]pyridine-4-carbohydrazone: Synthesis, in vitro cytotoxicities and DNA/BSA interaction

Article abstract of DOI:10.1016/j.ejmech.2014.08.070

Five organotin(IV) compounds were synthesized from Ng′-[(1E)-(2-hydroxy-3-methoxyphenyl)methylidene]pyridine-4-carbohydrazone and the corresponding dialkyltin(IV) or trialkyltin(IV) precursor. Solid state structures were determined by IR, elemental analys

Full text of DOI:10.1016/j.ejmech.2014.08.070

European Journal of Medicinal Chemistry 86 (2014) 550e561
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Organotin(IV) complexes derived from Schiff base N0-[(1E)-(2-
hydroxy-3-methoxyphenyl)methylidene]pyridine-4-carbohydrazone:
Synthesis, in vitro cytotoxicities and DNA/BSA interaction
,
,
Min Hong a *, Honglin Geng a, Meiju Niu a, Fei Wang a, Dacheng Li a b, Jifeng Liu a,
Handong Yin a
,
*
a Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng
University, Liaocheng 252059, China
b Collaborative Innovation Center of Antibody Drugs, Liaocheng University, Liaocheng 252059, China
a r t i c l e i n f o
a b s t r a c t
Five organotin(IV) compounds were synthesized from N0-[(1E)-(2-hydroxy-3-methoxyphenyl)methyl-
idene]pyridine-4-carbohydrazone and the corresponding dialkyltin(IV) or trialkyltin(IV) precursor. Solid
state structures were determined by IR, elemental analysis, NMR spectroscopy, and for 1, 2, 4 and 5 single
crystal X-ray diffraction analysis. Compounds 1, 2 and 4 are monomers with the tin atoms five-
coordinated in distorted trigonal bipyramid, of which the deprotonated Schiff base ligand chelate to
tin center in the enolic tridentate mode. Differently, in compound 5, the enolization does not occur for
the Schiff base ligand, and only the pyridinyl N atom and the deprotonated phenol hydroxyl oxygen atom
participate in the coordination. Fascinatingly, six trimethyltin(IV) coordination units are linked by the
Sn/N weak interaction atoms and form a 72-membered crown-like macrocycle. Preliminary in vitro
cytotoxicity studies on five human tumor cells lines (HL-60, A549, HT-29, HCT-116 and Caco-2) by MTT
assay reveal that di-n-butyltin(IV) complex 2 and diphenyltin(IV) complex 4 triggered significant anti-
proliferative effects in cultured tumor cells, and their cytotoxic activity correlates with intracellular
organotin(IV) concentration. The interaction of the complexes with calf thymus DNA (CT-DNA) has been
explored by absorption and emission titration methods, which revealed that complexes 2 and 4 interact
with CT-DNA through groove-binding and partial intercalation of the extended planar ligand with the
DNA base stack. Further, the albumin interactions of complexes 2 and 4 were investigated using fluo-
rescence quenching spectra and synchronous fluorescence spectra. Studies reveal that di-n-butyltin(IV)
complex 2 with higher cytotoxicity show stronger DNA/BSA interaction than diphenyltin(IV) complex 4.
© 2014 Elsevier Masson SAS. All rights reserved.
Article history:
Received 1 October 2013
Received in revised form
27 August 2014
Accepted 27 August 2014
Available online 28 August 2014
Keywords:
Organotin(IV)
Isonicotinoyl hydrazone
MTT assay
DNA/BSA-binding
Spectroscopy study
1. Introduction
activities, in some cases being more effective than cisplatin in vitro
tests [2e4]. A huge number of tin derivatives have been prepared
It is well known that cancer is currently the second leading
cause of death in industrialized countries, accounting for roughly a
quarter of all deaths. Hence, the development of new anticancer
therapies is one of the fundamental goals in medicinal chemistry
[1]. Organotin(IV) compounds have attracted more and more
attention for their characteristics, such as pharmic value, antitumor
activity and biological activity. Among the main-group metal
compounds, they appear to exhibit the most potent antitumor
and tested in the past as possible antitumor agents [5e9] and many
of them exhibited interesting activity in specific cancer models. In
general, the biochemical activity of organotin compounds is influ-
enced greatly by the structure of the molecule and the coordination
number as well as the bound alkyl groups of the tin atom. Usually,
n-butyltin(IV) complexes display a larger array of biological activity
than their methyl-, phenyl- or benzyltin(IV) analogs [10,11]. But the
exact antitumor mechanism of organotin(IV) compounds remains
unknown.
DNA is the most important target for antitumorals because of
its central role on replication and transcription. Different types of
interactions are possible, such as intercalation between base-pairs
(such as doxorubicin or daunorubicin), minor (such as netropsin,
* Corresponding authors.
E-mail addresses: hongminlcu@163.com (M. Hong), handongyin@163.com
(H. Yin).
http://dx.doi.org/10.1016/j.ejmech.2014.08.070
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
551
Scheme 1. The reaction procedures.
berenil and pyrrole-imidazole polyamides) or major groove
2. Results and discussion
binding (such as triplex-forming oligonucleotides and cisplatin),
or catalysis of DNA cleavage through hydrolytic or oxidative re-
actions [12]. According to the previous reports by Tabassum and
co-workers, most of the organotins tested are DNA-targeted and
mitotic, the action mode occurring via a gene-mediated pathway
[13,14]. Generally, the interaction between organotin(IV) com-
pounds and DNA vary with their configuration and planarity of the
ligand. Barbieri et al. reported that the 1:1 complex/DNA con-
densates are derived from an electrostatic interaction between
the cations Me2SnCl(SPy)þ and Me2SnCl(SPym)þ (HSPy ¼ 2-
mercaptopyridine; HSPym ¼ 2-mercaptopyrimidine) and the
phosphate {O} of phosphodiester groups [15]. Differently, our
studies reveal that organotin(IV) compounds coordinated with
naphthalene-contained Schiff base ligand can bind to CT-DNA via
an intercalative mode [16]. To enrich this aspect of studies and
explore the anticancer mechanism of organotin(IV) compounds,
further works are needed.
It is well known that serum albumins are the class of proteins
involved in the transport of metal ions and metal complexes
along with drugs through the blood stream [17]. The binding
ability of drugealbumin in blood stream has significant impact
on the distribution, free concentration, metabolism and toxicity
of drugs. Generally, weak binding leads to a shorter lifetime or
poor distribution, while strong binding decreases the concen-
tration of free drug in plasma. Studies on this aspect can provide
information on the structural feature that determines the ther-
apeutic effectiveness of drugs and standardized screens for pro-
tein binding in new drug design and for fixing dose limits
[18e20]. Therefore, the binding of drugs to serum albumin
in vitro, considered as a model in protein chemistry to study the
binding behavior of proteins, has been an interesting research
field in chemistry, life sciences, and clinical medicine [21].
However, it is a pity that limited data on BSA interaction of
organotin compounds are as yet available.
2.1. Syntheses of complexes 1e5
The synthesis procedures of complexes 1e5 are given in Scheme
1. All the complexes are prepared under atmospheric conditions.
Complexes 1e5 are yellow crystalline solids and are stable in air,
soluble in organic solvents (chloroform, methanol, acetone, DMSO,
mixture of water/DMSO).
2.2. Spectroscopic data
2.2.1. IR spectra
For complexes 1e4, the IR spectra show absorption bands
resulting from the vibration of the C]NeN]C group at
1590e1617 cmꢀ1 [25]. The disappearance of yC ¼ O and yNeH confirm
that the ligand coordinates with tin atom in the enolic form [26]
and the disappearance of yOeH indicates the deprotonation of the
eOH and its subsequent coordination to the center tin atom. The
appearance of 578e564 and 471e480 cmꢀ1 are assigned to SneO
and SneN absorption, respectively [27]. Compared with the ligand,
there is little change in complex 5 except for the disappearance of
OeH absorption and appearance of SneO absorption.
2.2.2. NMR spectra
All compounds gave satisfactory NMR spectra. The signals
observed at
d
¼ 12.27 ppm and 11.17 ppm are assigned to the
protons of eOH and eNH in the ligand, respectively. They are all
absent in the 1H NMR in complexes 1e4, indicating deprotonation
of the eNH and eOH groups and conforming the coordination to
the Sn atom. In addition, the signals of azomethine protons in 1e4
shift to downfield about 0.2 ppm due to the coordination of eNH
with tin atom and the formation of C]NeN]C conjugated system.
For complex 5, there is little change in its NMR except for the
disappearance of the eOH signal. The 13C NMR spectra of all com-
pounds show a significant downfield shift of all carbon resonances
compared with the free ligand because of an electron-density
transfer from the ligand to the metal atom, which is consistent
with that reported in the literature [28,29].
Hydrazone Schiff base has long been paid considerable attention
for its fascinating chemical behavior and biological essentiality
[22e24]. Motivated by the great potential of hydrazone complexes
in drug design and by our recent results with organotin(IV) species
[25e27], we designed and synthesized a series of organotin(IV)
complexes with N0-[(1E)-(2-hydroxy- 3-methoxyphenyl)methyl-
idene]pyridine-4-carbohydrazone ligand involving different alkyl
groups. The target compounds are based on the hydrazone core and
the R2Sn2+ or R3Sn2+ active site for inhibiting the growth of cancer
cells. The synthesis, structural characterization, in vitro cytotoxic-
ities and DNA/albumin interactions are reported here and
demonstrate a huge potential of this type of bioorganometallics in
medicinal chemistry.
The 119Sn NMR data show only one signal under the range
of ꢀ147 to ꢀ326 ppm for diorganotin(IV) compounds 1e4, and
their tinecarbon coupling constant, 1
J
, are equal to 654.8,
119Sne13CÞ
ð
368, 332 and 344 Hz, respectively, on the basis of Lockarts's
equation [30]. Thus, in solutions probably complex 1 is five-
coordinate and complexes 2, 3 and 4 are four-coordinate suggest-
ing cleavage of, e.g. SneN bond in solutions. Generally, typical
values of 119Sn chemical shifts for four-coordinate SnR3OR0
(R
¼
alkyl) compounds are 80e150 ppm and 1J
ð
119Sne13CÞ

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M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
330e390 Hz and for five-coordinate SnR3 complexes chemical
shifts are lower (in the range þ50 to ꢀ30 ppm) and 1J
nitrogen atom occupying the equatorial positions. The Schiff base
coordinates to the Sn atom as a tridentate ligand via the azome-
thine N atom, the deprotonated hydroxyl O atom and the depro-
tonated enolic hydroxyl O atom with Sn(1)eO(1) [2.192(3) Å for 1,
2.288(16) Å for 2, 2.132(13) Å for 4] and Sn(1)eO(2) [2.159(3) Å for
1, 2.301(15) Å for 2, 2.058(11) Å for 4]. The Sn(1)eN(3) distance
[2.199(4) Å for 1, 2.222(19) Å for 2, 2.138(15) Å for 4] is close to the
sum of the non-polar covalent radii (2.15 Å), indicating strong
Sn/N interaction. The sum of the angles subtended at the tin atom
in the equatorial plane is 359.43ꢁ for 1 and 359.60ꢁ for 4. So for
Sn(1), two carbon atoms and one nitrogen atom, which occupy the
equatorial positions, are almost in the same plane. However, the
equatorial plane for 2 is only 354.9ꢁ. The angle of the axial O(1)e
Sn(1)eO(2) [154.49(13)ꢁ for 1, 156.5(6)ꢁ for 2, 156.1(5)ꢁ for 4] de-
viates from the linear angle of 180ꢁ.
The bond length of C(6)eO(1) lies between double and single-
bond lengths. The CeNeNeC chain shows conjugation, as evi-
denced by the intermediate values for the bond lengths (Table S1).
These data indicate that the ligand functions as a tridentate
chelate with ONO atoms in the deprotonated enolic form and
forms a C]NeN]C conjugated system, which is introduced into
the inner coordination sphere. Besides, the tridentate ligand forms
a five- and six-membered chelate ring with the central Sn atom.
These interactions contribute to the crystal stability and
compactness.
ð
119Sne13CÞ
440e500 Hz. For complex 5, tin chemical shift is observed around
5.78 ppm with the tinecarbon coupling constant 462.2 Hz, indi-
cating the five-coordination environment for the tin atom being
still retained in solution.
2.3. Description of crystal structures
2.3.1. Structural description of complexes 1, 2 and 4
Compounds 1, 2 and 4 possess similar structure features. The
molecular structures of compounds 1, 2 and 4 are illustrated in
Fig. 1. Crystal data is listed in Table 1. Selected bond distances and
angles are listed in Table S1.
Structural analysis of compounds 1, 2 and 4 show that each
complex is a monomer, in which the Sn atom adopts five-
coordinated geometry. For compounds 1, 2 and 4, the coordina-
tion environment is a distorted trigonal bipyramid with two oxygen
atoms occupying the axial positions and two carbon atoms, one
2.3.2. Structural description of complex 5
The repeating unit and network structure of complex 5 are
illustrated in Figs. 2 and 3, respectively. Selected bond distances
and angles are listed in Table S2. In complex 5, the trimethyltin(IV)
groups are linked by a hydroxyl group from one ligand and a N atom
from another ligand. Six ligands are linked by six metal centers into
a 72-membered crown-like macrocycle with three ligands higher
and the other three ligands lower.
In complex 5, as a result of the bidentate mode of coordination
of the ligand, the central tin atom is essentially five-coordinated,
bound to three alkyl groups, one deprotonated hydroxyl oxygen
atom, and one nitrogen atom, so the geometry of the tin atom can
be described as a distorted trigonal bipyramidal geometry in which
the apical position is occupied by the oxygen atom and the nitrogen
atom. The axial O(2)eSn(1)eN(1)#1 [#1: ꢀy þ 1/3, x ꢀ y þ 2/3,
z ꢀ 1/3] is not 180ꢁ but 167.5(3)ꢁ, which proves the distortion of the
geometry (Table S2). The sum of the angles subtended at the Sn
atom in the equatorial plane is 354.7ꢁ. The bond length of Sn(1)e
O(2) distance [2.076(7) Å] is slightly shorter than the sum of the
covalent radii of Sn and O (2.13 Å) and should be considered as
strong bonding interaction. The Sn(1)eN(1)#1 bond distance
[2.768(10) Å] approaches similar intermolecular Sn / N (2.787 Å)
bond length in trimeric [Me3SnSCN4Ph]3 unit [31] and is midway
between the sums of the van der Waals and covalent radii of Sn and
N, 3.74 and 2.15 Å [32]. The SneC distances [2.096(12)e2.136(11) Å]
are equal within experimental error and are close to the single-
bond value for a trigonal bipyramidal tin atom [33].
2.4. In vitro cytotoxicities
The in vitro cytotoxicities of complexes 1e5 were determined by
MTT-based assays. This study has been carried out to understand
the possible relationship between the different tin(IV) moieties
(bearing dimethyl, di-n-butyl, di-n-octyl, diphenyl and trimethyl
groups) and the cytotoxicities. Complexes were tested for their
in vitro cytotoxicities on five tumor cell lines: human lung cancer
cell line (A549), human leukemia cell line (HL-60) and human colon
cell line (HT-29, HCT-116, Caco-2). The corresponding IC50 values
are listed in Table 2. The inhibition effects of compound 4 on the
Fig. 1. The molecular structure of complexes 1, 2 and 4 (the thermal ellipsoids are
drawn at 30% probability). All H atoms are omitted for clarity.

M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
553
Table 1
Crystal data and structure refinement parameters for compounds 1, 2, 4 and 5.
Complex
1
2
4
5
Empirical formula
Formula weight
Wavelength (Å)
Crystal system
Space group
a (Å)
C32H34N6O6Sn2
836.03
0.71073
Monoclinic
C2/c
26.035(3)
9.5250(9)
13.5262(15)
90
102.5850(10)
90
3273.6(6)
4
1.696
1.579
C88H116N12O12Sn4
2008.69
0.71073
Monoclinic
P2(1)/c
23.463(2)
17.3302(15)
11.4409(11)
90
94.5640(10)
90
4637.4(8)
2
1.439
1.128
C26H21N3O3Sn
542.15
0.71073
Triclinic
P-1
8.9241(7)
13.8249(11)
18.6528(15)
81.5950(10)
87.397(2)
85.7830(10)
2269.0(3)
4
C17H21N3O3Sn
434.06
0.71073
Trigonal
R-3
31.643(8)
31.643(8)
12.0832(19)
90
90
120
10,478(4)
18
1.238
b (Å)
c (Å)
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
V (Å3)
Z
Dcalc (Mg/m3)
1.587
1.160
m
(mmꢀ1
)
1.112
F(000)
1664
2048
1088
3924
Crystal size (mm)
Reflections collected
Unique reflections [Rint
0.28 ꢂ 0.17 ꢂ 0.06
0.31 ꢂ 0.24 ꢂ 0.18
0.31 ꢂ 0.19 ꢂ 0.08
0.30 ꢂ 0.28 ꢂ 0.18
8120
22,681
11,698
14,300
]
2889 [R(int) ¼ 0.0653]
2889/0/211
0.828
8173 [R(int) ¼ 0.1592]
8173/0/529
1.009
7904 [R(int) ¼ 0.1255]
7904/3/597
0.980
4114 [R(int) ¼ 0.2027]
4114/6/221
0.880
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2
R indices (all data)
s
(I)]
R1 ¼ 0.0401
wR2 ¼ 0.0865
R1 ¼ 0.0634
wR2 ¼ 0.0962
R1 ¼ 0.0983
wR2 ¼ 0.2342
R1 ¼ 0.2153
wR2 ¼ 0.3330
R1 ¼ 0.1151
wR2 ¼ 0.2718
R1 ¼ 0.2039
wR2 ¼ 0.3291
R1 ¼ 0.0792
wR2 ¼ 0.1381
R1 ¼ 0.2178
wR2 ¼ 0.1806
12-fold improvement of the antitumor potency in HL-60 cells
than Caco-2 cells, 7-fold improvement than A549 cells, 2.5-fold
improvement than HT-29 cells and 3-fold improvement than
HCT-116 cells.
2) Every tumor cell line reported here exhibits different resistance
to different organotin compounds. All compounds exhibit
prominent in vitro antitumor activity toward HL-60 cells, while
A549 and Caco-2 cells exhibit somewhat low sensitivity to these
compounds. The sensitivity of the five cells to compounds 2 and
3 can be listed as: HL-60 > HT-29 > HCT-116 > A549 > Caco-2
and HL-60 > HCT-116 > HT-29 > A549 > Caco-2 for compounds 4
and 5.
3) As for the alkyl chain bound with tin atom, the length of the
organic groups has a great influence on the growth suppression
activity of diorganotin complexes on tumor cells. In our exper-
iments, the IC50 values of complexes 1, 2 and 3 follow the trends
2 > 3 > 1 for all cell lines. Diorganotin derivatives with a too
short (methyl) or a too long (n-octyl) carbon chain length tends
to exhibit lower activities. This result emphasizes the impor-
tance of the n-butyl moiety that is present in complex 2. Indeed,
di(n-butyl)tin compound is found to be the most active one in
diorganotin complexes, which is based upon the optimal bal-
ance between the cytotoxicity, solubility and lypophilicity
[14,35,36]. As for the identical coordination geometry for the
four diorganotin complexes in the present study, the activity
follows the consistent order: n-Bu > Ph > n-Oct > Me, for most of
the tumor cells.
4) With regard to the data published on all the Sn(IV) derivatives, it
can be concluded that R2Sn(IV)2þ compounds generally exhibit
higher antitumor activity than those of corresponding mono-,
tri- and tetraorganotin(IV) or the inorganic Sn(IV) derivatives,
and within the diorganotin(IV) class, the highest activity is
exerted by the Bu2Sn(IV)2þ and Ph2Sn(IV)2þ complexes [7].
Conversely, trimethyltin(IV) compound shows a slightly higher
activity than the dimethyltin(IV) derivative herein.
Fig. 2. The repeating unit of complex 5 (the thermal ellipsoids are drawn at 30%
probability). All H atoms are omitted for clarity.
five cell lines at different concentrations are listed in Fig. 4 and
inhibition effects of compounds 1, 2, 3, and 5 on the five cell lines at
a concentration of 1
mg/mL and 10 mg/mL are listed in Fig. 5,
respectively. From the derived data, we can see that there is a good
doseeeffect relationship for all complexes, the percentage inhibi-
tion grows with the increasing of the concentration of organo-
tin(IV) complexes. As observed in previous studies [11,25,34], the
organo-ligand R plays an important role. Indeed, di-n-butyl (IV)
compound 2 and diphenyl (IV) compound 4 were more potent than
dimethyltin compound 1, di-n-octyltin compound 3 and trime-
thyltin compound 5 in inhibiting the growth of all cell lines tested
here. Possible structureeactivity relationships could be recognized
as follows:
1) Analyzed organotin compounds exhibit different inhibitions for
different tumor cells. Complex 2 seems to be the most efficient
antitumor agent for A549, HT-29, HCT-116 and HL-60 cells,
while complex 4 exhibit the highest activities against Caco-2
cells. This makes them promising anticancer agents. The most
outstanding result is obtained from the activity of compound 2
5) As the five human cancer cell lines we chose are cisplatin-
resistant, cisplatin exhibits almost non-inhibition for cellular
proliferation here.
in HL-60 tumor cells (0.176 mg/mL). For compound 2, it is about

554
M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
Fig. 3. The supramolecular structure of complex 5 formed by intermolecular Sn/N bonding interactions.
The cytotoxicities shown by these compounds against five
pairs of DNA [38,39]. The changes in the spectra of complexes 2 and
4 caused by the addition of DNA are quite different from this latter
description, suggesting that the binding mode of complexes 2 and 4
may be other than complete intercalative, i.e., by the DNA groove
binding or electrostatic interactions [39]. And this should be
demonstrated by other experimental methods.
To compare the binding strength of the complexes with CT-DNA,
the intrinsic binding constant Kb were calculated according to the
equation [37]:
cancer cell lines indicate the synergistic effect of hydrazone Schiff
base ligand with R2Sn(IV) metal center results in important bio-
logical properties and remarkable antitumor activity. Compounds 2
and 4 can be considered as agents with potential antitumor activity
and therefore be candidates for further stages of screening in vivo.
As trimethyltin compound 5 show moderate activities, the effect of
coordination geometry to tin center in the present compounds
toward their antitumor activities may not be rules out.
.ꢀ
ꢁ
.ꢀ
ꢁ
.
ꢀ
ꢁ
DNA εa ꢀ εf ¼ DNA ε0 ꢀ εf þ 1 Kb ε0 ꢀ εf
2.5. CT-DNA binding effects on UVevisible, fluorescence and
circular dichroism spectra
in which [DNA] is the concentration of DNA in base pairs, εa is the
ratio of the absorbance/[Sn(IV)], εf is the extinction coefficient of
the free Sn(IV) complex, and ε0 is the extinction coefficient of the
complex in the fully bound form [12]. The ratio of slope to intercept
in the plot of [DNA]/(εa ꢀ εf) versus [DNA] gives the value of K (inset
Fig. 5). Complex 2 has a slightly higher affinity to DNA than complex
4 with Kb values of 2.83 ꢂ 104 and 1.88 ꢂ 104, respectively. These
values are comparable to those reported to other metal complexes
For evaluating the anticancer property of any newly synthesized
complex, DNA binding is the predominant property looked for in
pharmacology and hence, the interaction between DNA and metal
complexes is of paramount importance in understanding the
mechanism. Thus, the mode and propensity for binding of the
higher cytotoxicities compounds 2 and 4 to CT-DNA were studied
with the help of electronic absorption, fluorescence and circular
dichroism (CD) techniques.
(ranging from 102 to 105
M
ꢀ1) [12,38,39].
2.5.1. UVeVis absorption studies
The absorption spectra of complexes 2 and 4 displayed two well
resolved bands ~350 nm and ~430 nm, which are assigned to
intraligand charge transfer (ILCT) transitions and ligand-to-metal
charge transfer (LMCT) transitions, respectively. We have regis-
tered the spectra of solutions of the compounds 2 and 4 in the
absence and presence of increasing concentrations of CT-DNA
which are shown in Fig. 6, respectively. From the electronic ab-
sorption spectral data, it was clear that increasing the concentra-
2.5.2. Fluorescence spectral studies
Investigation of the effect of adding DNA on the luminescence of
metal complexes have been widely used to provide information
about binding constants as well as the type of DNA binding modes.
The fluorescence spectra of complex 2 and 4, exhibiting a broad
emission band in the range 440e640 nm, were monitored at a fixed
concentration of 100
mM. Enhanced fluorescence without wave-
length shift were observed (Fig. 7A), when CT-DNA was added into
the complex 2 solution, but for complex 4 the fluorescence
enhancing is negligible (Fig. 7B). The result suggests that the
stronger enhancement of fluorescence intensity for the complex 2
may be largely due to the interaction between adjacent base pairs
of CT-DNA and the complex. As we know that, the protection of the
intercalative moieties by the DNA from forming intermolecular
hydrogen bonds increased hydrophobility, and the rigidity of the
tion of CT-DNA (0e40 mM) added to the organotin(IV) complexes 2
and 4, both of the above mentioned absorption bands showed
hypochromism accompanied with a minor hypsochromic shifts
(2e5 nm). Interaction of a complex to DNA through intercalation
usually results in hypochromism and strong bathochromic shift
due to the intercalative binding involving a strong stacking inter-
action between the planar aromatic chromophore and the base

M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
555
Table 2
significant change in the base stacking but can not unwind the DNA
helix conformation.
Half maximal inhibitory concentration (
lines.
mg/mL) of the compounds against tumor cell
Based on the above analysis (UVevisible, fluorescence and cir-
cular dichroism spectra), we can draw the conclusion that dio-
rganotin(IV) compounds 2 and 4 could bind to CT-DNA in the
groove-binding and partial intercalation mode due to the
planarity of the chelating ligand N0-[(1E)-(2-hydroxy-3-
Compound IC50 (mg/mL)
Caco-2
A549
HT-29
HCT-116
HL-60
1
2
3
4
>10
>10
4.73 0.68 >10
2.58 0.41
2.12 0.33 1.23 0.09 0.43 0.36 0.57 0.25 0.18 0.75
>10 7.44 0.87 3.79 0.58 3.97 0.60 0.69 0.16
1.75 0.24 1.61 0.21 1.18 0.07 1.09 0.03 0.18 0.74
methoxyphenyl)methylidene]pyridine-4-carbohydrazone.
Both
the binding constant obtained by UVevis and fluorescence phe-
nomenon reveals the stronger DNA-binding for complex 2 than 4,
which is accordance with their in vitro cytotoxicities.
5
>10
>60
10.33 1.01 5.37 0.73 4.20 0.62 1.79 0.25
>60 >60 53 >60
Cisplatin
2.6. Protein binding studies
2.6.1. Fluorescence quenching studies of BSA
Serum albumins are important blood proteins which have the
ability to transport a multitude of ligands. The binding ability of
drugealbumin in the blood stream has significant impact on the
distribution, free concentration, metabolism and toxicity of drugs
[46,47]. Thus, it is important to understand the mechanism of
interaction of a bioactive compound with serum albumins. Bovine
serum albumin (BSA) is the most extensively studied serum albu-
min owing to its structural homology with human serum albumin.
Complexes 2 and 4 were chosen to study the interaction between
organotin and BSA for their remarkable antitumor activities and
potential applications in further study. The interaction of com-
plexes 2 and 4 with BSA was monitored by studying the quenching
the fluorescence of BSA with increasing concentrations of organotin
complexes at room temperature.
Fig. 4. The inhibition effects of compound 4 on the five cells lines at different con-
centration (0.01, 0.1, 0.5, 1, 10 g/mL).
m
environment by DNA groove-binding all may contribute to the
DNA-induced emission enhancement observed [40,41].
In general, the planarity of the main ligand plays a dominant
role in determining the DNA-binding mode of a drug molecule.
However, for different compounds with the same coordination
mode and the main ligand, the steric hindrance caused by two
phenyl groups bound with tin center in 4 decreased the DNA-
binding interaction obviously, and thus made little fluorescence
enhancing.
Usually, DNA-EB system was used to understand the interaction
pattern of the complex with DNA. A significant maximum absorp-
tion peak at 590 nm in the DNA-EB system could be observed. As
compounds 2 and 4 tested here show appreciable interfering
fluorescence in the spectral region, so DNA-EB system can not be
used here.
A solution of BSA (1.0 mM) was titrated with increasing con-
centrations of the compounds. The effects of the compounds on the
fluorescence emission spectrum of BSA are shown in Fig. 9. As seen,
in both cases, the fluorescence intensities of the proteins are
decreased regularly with increasing concentrations of the probe
compounds, which confirm the interaction between complexes and
BSA have occurred. Addition of the compounds to BSA resulted in a
significant decrease in the fluorescence intensity of BSA at 346 nm,
up to 30.22% and 50.17% of the initial fluorescence intensity of BSA
for complexes 2 and 4, respectively, with slight hypsochromic shift
due to the formation of a complex between the test compounds and
BSA. Commonly, fluorescence quenching can be described by the
following SternꢀVolmer equation:
ꢂ
ꢃ
ꢂ
ꢃ
F0=F ¼ 1 þ Ksv
Q
¼ 1 þ kqt0 Q :
where F0 and F are the fluorescence intensities in the absence and
presence of quencher, respectively, Ksv is the SterneVolmer
quenching constant, [Q] is the total concentration of quencher, kq is
the biomolecular quenching constant and t0 is the average lifetime
of protein in the absence of quencher, and its value is 10ꢀ8 s [48].
Fig. 10A displays the SterneVolmer plots of the quenching of BSA
fluorescence by different complexes and Ksv can be obtained by a
slope from the plot of F0/F vs [Q]. The calculated values of Ksv and kq
for the interaction of the complexes with BSA are given in Table 3.
All plots represent good linear relationship. As is known, linear
SternꢀVolmer plots represent a single quenching mechanism,
either static (the formation of a complex between quencher and
fluorophore) or dynamic (a collisional process) [49]. The kq values
2.5.3. Circular dichroism (CD) spectroscopy
This technique has been widely used to examine the non-
covalent DNA binding interactions of metal complexes, owing to
its ability to provide binding constants as well as information about
nucleic acid conformation. The CD spectrum of DNA exhibits a
positive peak at 273 nm because of base stacking and a negative
peak at 246 nm due to right handed helicity, this is a characteristic
of the B-form of CT-DNA [42,43]. The changes in the CD spectra can
be attributed to the corresponding changes in the CT-DNA struc-
ture. The CD spectra show almost no change in the case of groove
binding and electrostatic binding, whereas the intercalative bind-
ing affects both the positive and negative bands, as observed for
classical intercalators, such as ethidium bromide and methylene
blue [44]. The observed CD spectra of CT-DNA in the presence of
complex are shown in Fig. 8. In the presence of complexes 2 and 4,
both the positive (ca. 278 nm) and negative (ca. 246 nm) peaks
increased in intensity, this behavior is characteristic of intercalation
between complexes 2 and 4 and CT-DNA [45]. Further, we can see
that the binding of the complexes 2 and 4 to CT-DNA lead to a
(~1012
M
ꢀ1 sꢀ1) of the complexes 2 and 4 are much higher than the
maximum scatter collision-quenching constant of diverse kinds of
quenchers for biopolymers fluorescence (2 ꢂ 1010 Mꢀ1
s
ꢀ1), indi-
cating the existence of static quenching mechanism [50].
For the static quenching interaction, the binding constant (Kbin
)
and the number of binding sites (n) can be determined according to
the Scatchard equation: log[(F0 ꢀ F)/F] ¼ logKbin þ nlog[Q]. The n

556
M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
Fig. 5. The inhibition effects of compounds 1, 2, 3, and 5 on the five cells lines at a concentration of 1 mg/mL (left) and 10 mg/mL (right), respectively.
Fig. 6. UVevis absorption spectra of complexes 2 (A) and 4 (B) (40
in absorbance with respect to an increase in the DNA concentration.
m
M) in the absence and presence of CT-DNA. [DNA] ¼ 0, 5, 10, 15, 20, 25, 30, 35, 40
mM. Arrows show the changes
and Kbin can be calculated by the slope and the intercept of the
double logarithm regression curve of log(F0 ꢀ F)/F versus log[Q]
(Fig. 10B and Table 3). The values of binding constant (Kbin) suggest
BSA has moderate affinity to organotin 2 and 4, because the
documented Kbin values of noncovalent association of BSA with
drugs mostly range from 104e106 Mꢀ1 [51]. The binding constant
between the organotin complexes and BSA suggest that these
compounds can easily be stored in protein and can be released in
desired target areas [52]. Complex 2 exhibits stronger binding
constant than complex 4. The low Kbin of complex 4 may be
attributed to its poor planarity and large steric effects, which might
decrease the accessibility of the organotin center to the active site
in BSA [53]. The values of n are basically associated with binding
constants, which implies a direct relation between the binding
constant and number of binding sites. To some extent, the binding
ability of organotin complexes with BSA is in accord with the cy-
totoxicities tested above, we can speculate that the antitumor ac-
tivities of organotin compounds may be partly due to the
interaction between organotin compounds and some proteins in
cancer cells.
decrease in the fluorescence intensity of BSA at 286 nm, up to
63.84% (complex 2) and 80.85% (complex 4) of the initial fluores-
cence intensity of BSA with no shift in their emission wavelength
maxima. At the same time, in the case of Dl ¼ 60 nm, additions of
the compounds result in a significant decrease in the fluorescence
intensity of BSA at 282 nm, up to 41.04% (complex 2) and 57.44%
(complex 4) of the initial fluorescence intensity of BSA with slight
blue shift in their emission wavelength maxima. As can be seen, the
binding strength of complex 2 with tyrosine or tryptophan is higher
than complex 4.
As for complex 4, synchronous fluorescence spectral clearly
suggest the tyrosine residues in BSA are not affected obviously, but
the significant decrease along with blue shift of the fluorescence
intensity of tryptophan has been observed. For complex 2, syn-
chronous fluorescence reveal that both tyrosine and tryptophan
residues in BSA are affected. These results suggest that the inter-
action of compound 4 with BSA mainly affects the conformation of
tryptophan while both tyrosine and tryptophan residues are
significantly affected by complex 2. All in all, compound 2 may be is
more suitable for further research in vivo for its excellent antitumor
property and fine binding ability with serum albumins to be
transported through the blood stream.
2.6.2. Synchronous fluorescence spectroscopic studies of BSA
The synchronous fluorescence spectrum has been reported to be
an effective approach to distinguish the tryptophan (Trp)
3. Conclusions
(
Dl ¼ 60 nm) and tyrosine (Tyr) (Dl ¼ 15 nm) residues in protein
[54,55] for this technique can give information about the molecular
environment in the vicinity of the fluorophore molecules at low
concentrations under physiological conditions [56]. The effects of
organotin complexes on BSA synchronous fluorescence spectros-
copy with Dl ¼ 15 nm and Dl ¼ 60 nm are shown in Figs. 11 and 12,
respectively.
In this article, five organotin compounds derived from N0-[(1E)-
(2-hydroxy-3-methoxyphenyl)methylidene]pyridine-4-
carbohydrazone are synthesized. Structural analyses show that
three compounds (1, 2 and 4) adopt distorted trigonal bipyramid
geometries while compound 5 show 72-membered macrocycle
ring structure. In vitro cytotoxicities of the five complexes against a
panel of human cancer cell lines show that the cytotoxicities of
organotin compounds are greatly affected by the organo-ligand R
In the synchronous fluorescence spectra of BSA at Dl ¼ 15 nm,
additions of the compounds to the BSA solution result in small

M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
557
Fig. 7. Fluorescence spectra of complexes 2 (A) and 4 (B) (100
with respect to an increase in the DNA concentration.
m
M) in the absence and presence of CT-DNA. [DNA] ¼ 0, 20, 40, 60, 80, 100
mM. Arrows show the changes in emission
bound with tin atom. Di-n-butyltin(IV) complex 2 and diphenyl (IV)
complex 4 display potent efficiency, making them promising anti-
cancer agents in cisplatin resistant cell lines. Absorption and
emission spectral studies indicated that diorganotin(IV) com-
pounds 2 and 4 could bind to CT-DNA in the groove-binding and
partial intercalation mode due to the planarity of the chelating
ligand. BSA-binding investigations reveal that complex 2 exhibits
higher BSA-binding ability and synchronous fluorescence spectra
show that organotin complexes can be transported by binding with
tryptophan in protein. These results provide a valuable tool for
understanding the interactions between protein/DNA and organo-
tin complexes. More detailed mechanism of antitumor activities on
organotin(IV) compounds will be further studied from several re-
spects, including the expressions for anti-apoptosis proteins Bcl-2
and Bcl-xL, different interactions viz. p53 mitochondrial gene, the
inhibition on the tyrosine kinase and the effects on cellular meta-
bolism activity etc.
atoms and refined with fixed thermal factors. The refinement fac-
tor, R-values for compounds 2, 4 and 5 are quite high, which should
be caused by rapid crystallization under the high room
temperature.
4.3. Preparation of the ligand
20 mL ethanol solution of 2-hydroxy-3-methoxybenzaldehyde
(1.52 g, 10 mmol) was added slowly to an ethanol solution
(50 mL) containing pyridine-4-carbohydrazide (1.37 g, 10 mmol)
under stirring. The mixture was heated under reflux with stirring
for 3 h and the crude product was precipitate. The precipitate was
filtered, washed with ethanol and an amount of yellow powder can
be obtained (see: Scheme 1). Yield: 85%. M.p.: 243e244 ꢁC. Anal.
Calc. for C14H13N3O3: C 61.99, H 4.83, N 15.49%. Found: C 62.12, H
4.74, N 15.65%. 1H NMR (DMSO-d, ppm): 12.27 (1H, s, OH), 11.17(1H,
s, NH), 8.77 (2H, d, J ¼ 4.0 Hz, H2, H2’), 8.64 (1H, s, N]CHe), 7.85
(2H, d, J ¼ 4.4 Hz, H3, H3’), 7.06 (1H, d, J ¼ 8 Hz), 6.97 (1H, d,
J ¼ 8 Hz), 6.87 (1H, t, J ¼ 8 Hz), 3.88 (3H, s, eOCH3). 13C NMR
(DMSO-d, ppm): 161.86 (C]O), 150.90, 150.61, 148.45, 148.33,
140.45, 122.34, 121.94, 119.38, 118.69, 114.41 (aromatic-C), 56.41
4. Experimental details
4.1. Materials and measurements
(eOCH3). IR (KBr, cmꢀ1
C]O), 1630(m, C]N).
) y: 3433(s, NeH), 3260 (m, OeH), 1689(s,
Dimethyltin dichloride, di-n-butyltin, dichloride, diphenyltin
dichloride, di-n-butyltin oxide, di-n-octyltin oxide and trimethyltin
chloride were commercially available, and they were used without
further purification. N0-[(1E)-(2-hydroxy-3-methoxyphenyl)meth-
ylidene] pyridine-4-carbohydrazone was prepared according to
reported methods [57]. Elemental analyses were performed on a
PE-2400-II elemental analyzer. IR spectra were recorded on a
Nicolet-5700 spectrophotometer using KBr discs. 1H, 13C and 119Sn
NMR spectra were recorded on a Varian Mercury Plus-400 NMR
spectrometer. Chemical shifts were given in ppm relative to Me4Si
(1H, 13C) and Me4Sn (119Sn) in CDCl3 or DMSO solvent. UVevis was
performed on a UV-2550 ultraviolet spectrophotometer. Fluores-
cence spectra were recorded on a PE-LS-55 spectrophotometer.
Circular dichroism spectral measurements were taken on a Jasco J-
810 spectropolarimeter.
4.2. X-ray crystallography
Diffraction data for the title compounds were obtained on a
Bruker Smart 1000 CCD diffractometer (graphite monochromized
Mo K
a
radiation,
l
¼ 0.71073 Å). All data were corrected using
SADABS method and the final refinement was performed by full-
matrix least-square methods with anisotropic thermal parame-
ters for non-hydrogen atoms on F2 using SHELX-97 program. The
hydrogen atoms were added theoretically, riding on the concerned
Fig. 8. Circular dichroism spectra of CT-DNA (100
complexes 2 (—) and 4 (-∙-∙-) (50 M).
mM) in the absence and presence of
m

558
M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
Fig. 9. Effects of complexes 2 (A) and 4 (B) on the fluorescence spectra of BSA system. [BSA] ¼ 1.0
mM, [VOL] ¼ 0, 5, 10, 15, 20, 25, 30
mM, respectively.
Fig. 10. SterneVolmer (A) and Scatchard plots (B) of the fluorescence titration of complexes 2 and 4 with BSA.
4.4. Syntheses of organotin(IV) complexes
dichloromethane/petroleum ether (5:2) at room temperature.
Yield: 82%. M.p.: 109e111 ꢁC. Anal. Calc. for C88H116N12O12Sn4: C
52.62, H 5.82, N 8.37%. Found: C 52.54, H 6.10, N 8.38%. 1H NMR
(CDCl3, ppm): 8.76 (1H, s, N]CH), 8.72 (2H, d, 2,6-pyridine-H,
J ¼ 4 Hz), 7.94 (2H, d, 3,5-pyridine-H, J ¼ 4 Hz), 6.95 (1H, d, J ¼ 8 Hz),
6.82(1H, d, J ¼ 8 Hz), 6.69 (1H, t, aromatic-H, J ¼ 8 Hz), 3.87(3H, s,
eOCH3), 1.82e1.34 (m, 12H, SneCH2CH2CH2), 0.94 (t, 6H, eCH3,
4.4.1. [(CH3)2Sn(C14H11N3O3)] (1)
An ethanol solution (2 mL) of EtONa (0.136 g, 2.0 mmol) was
added to a solution of Schiff base ligand (0.270 g, 1.0 mmol) in
methanol (60 mL) with constant stirring. After 0.5 h, dimethyltin
dichloride (0.219 g, 1.0 mmol) was then added to the reaction
mixture. The resulting solution was heated under reflux conditions
for 8 h under atmospheric conditions. After removing the formed
sodium chloride by filtration, the solvent of the resulting suspen-
sion was removed under reduced pressure to give the crude
product. Light yellow block crystals of compound 1 were obtained
by recrystallizing from dichloromethane/petroleum ether (5:2) at
room temperature. Yield: 76%. M.p.: 268e269 ꢁC. Anal. Calc. for
J ¼ 8 Hz). 13C NMR (CDCl3, ppm):
d 167.39(CeO), 163.36 (C]NeN),
158.34, 151.64, 149.65, 142.03, 126.25, 121.96, 116.59 (aromatic-C),
56.54(eOCH3), 27.02, 26.60, 23.22, 13.72 (SneCH2CH2CH2CH3,
1J
¼ 368 Hz). 119Sn NMR (CDCl3, ppm): e185.30. IR (KBr,
119Snꢀ13CÞ
ð
cmꢀ1
) y: 1616,1596 (m, C]NeN]C), 571 (m, SneO), 474 (w, SneN).
C
32H34N6O6Sn2: C 45.97, H 4.10, N 10.05%. Found: C 46.01, H 4.04, N
4.4.3. [(C8H17)2Sn(C14H11N3O3)] (3)
10.21%. 1H NMR (CDCl3, ppm): 8.77 (1H, s, N]CH), 8.70 (2H, d, 2,6-
pyridine-H, J ¼ 4 Hz), 7.88 (2H, d, 3,5-pyridine-H, J ¼ 4 Hz), 6.95
(1H, d, J ¼ 8 Hz), 6.84(1H, d, J ¼ 8 Hz), 6.71 (1H, t, J ¼ 8 Hz), 3.88 (3H,
Compound 3 was prepared by the similar method as compound
2 by the reaction of the ligand with di-n-octyltin oxide in 1:1
stoichiometry in methanol. Yellow precipitate was obtained by
recrystallizing from dichloromethane/petroleum ether (5:2) at
room temperature. Yield: 85%. M.p.: 50e51 ꢁC. Anal. Calc. for
s, eOCH3), 0.92 (s, 6H, eCH3Sn,2J
¼ 80 Hz). 13C NMR
1
ð
119Snꢀ HÞ
(DMSO, ppm): 167.50(CeO), 163.42 (C]NeN), 157.48, 151.53,
150.34, 141.10, 127.17, 121.64, 116.91 (aromatic-C), 56.41 (eOCH3),
C30H45N3O3Sn: C 58.64, H 7.38, N 6.84%. Found: C 58.67, H 7.30, N
2.73 (SneCH3, 1J
¼ 654:8 Hz). 119Sn NMR (DMSO, ppm):
) y: 1617, 1590 (m, C]NeN]C), 574 (m,
119Snꢀ13CÞ
6.88%. 1H NMR (CDCl3, ppm): 8.80 (1H, s, N]CH), 8.70 (2H, d, 2,6-
pyridine-H, J ¼ 4 Hz), 7.89 (2H, d, 3,5-pyridine-H, J ¼ 4 Hz), 6.94
(1H, d, J ¼ 8 Hz), 6.82(1H, d, J ¼ 8 Hz), 6.68 (1H, t, J ¼ 8 Hz), 3.86 (3H,
s, eOCH3), 1.61e1.17 (m, 28H, Sne(CH2)7), 0.83 (t, 6H, J ¼ 8 Hz,
eCH3). 13C NMR (CDCl3, ppm): 167.71(CeO), 163.13 (C]NeN),
158.20, 151.64, 150.29, 141.31, 126.66, 116.45(aromatic-C), 56.49(-
OCH3), 33.52, 32.00, 29.32, 29.24, 24.89, 23.60, 22.83 (Sne(CH2)7e,
ð
e147.45. IR (KBr, cmꢀ1
SneO), 478 (w, SneN).
4.4.2. [(C4H9)2Sn(C14H11N3O3)] (2)
A mixture of Schiff base ligand (0.270 g, 1 mmol) and di-n-
butyltin oxide (0.249 g, 1 mmol) was refluxed in methanol for 8 h.
The solvent of the resulting reaction was removed under reduced
pressure to give the crude product. Light yellow block crystals of
1J
¼ 332 Hz), 14.29 (eCH3). 119Sn NMR (CDCl3,
119Snꢀ13CÞ
ð
ppm): ꢀ184.89. IR (KBr, cmꢀ1
(m, SneO), 471 (w, SneN).
) y: 1615, 1596 (m, C]NeN]C), 572
compound
2
were obtained by recrystallizing from

M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
559
Found: C 47.11, H 4.82, N 9.59%. 1H NMR (CDCl3, ppm): 11.16 (1H, s,
NH), 8.77(2H, d, 2,6-pyridine-H, J ¼ 4 Hz), 8.65 (1H, s, N]CH), 7.85
(2H, d, 3,5-pyridine-H, J ¼ 4 Hz), 7.07 (1H, d, J ¼ 8 Hz), 6.98 (1H, d,
J ¼ 8 Hz), 6.86 (1H, t, J ¼ 8 Hz), 3.88 (3H, s, eOCH3), 0.44 (s,
SneCH3). 13C NMR (DMSO, ppm): 161.82 (CeO), 150.67, 150.57,
148.48, 148.28, 140.46, 122.16, 121.96, 119.41, 118.87, 114.47 (aro-
Table 3
The quenching constant, binding constant and number of binding sites for the in-
teractions of complexes 2 and 4 with BSA.
Compound
Ksv (Mꢀ1
)
kq (Mꢀ1 Sꢀ1
)
n
Kbin
2
4
7.842 ꢂ 104
7.842 ꢂ 1012
1.07538
1.04908
1.748 ꢂ 105
6.503 ꢂ 104
3.969 ꢂ 104
3.969 ꢂ 1012
Fig. 11. Synchronous spectra of BSA (1.0
mM) in the presence of increasing amounts of complexes 2 (A) and 4 (B) at a wavelength difference of Dl ¼ 15 nm.
Fig. 12. Synchronous spectra of BSA (1.0
mM) in the presence of increasing amounts of complexes 2 (A) and 4 (B) at a wavelength difference of Dl ¼ 60 nm.
4.4.4. [(C6H5)2Sn(C14H11N3O3)] (4)
matic-C), 56.42 (-OCH3), 3.11 (SneCH3, 1J
¼ 462:2 Hz).
119Snꢀ13CÞ
ð
Compound 4 was also prepared by the similar method as
compound 2 by the reaction of the ligand with di-phenyltin oxide
in 1:1 stoichiometry in methanol. Yellow block crystals of 4 were
recrystallized from dichloromethane/petroleum ether (5:2) at
room temperature. Yield: 78%. M.p.: 226e227 ꢁC. Anal. Calc. for
119Sn NMR (DMSO, ppm): 5.78. IR (KBr, cmꢀ1
) y: 3425(s, NeH), 1682
(s, C]O), 1625 (m, C]N). 577 (m, SneO).
4.5. In vitro cytotoxicities
C
26H21N3O3Sn: C 57.60, H 3.90, N 7.75%. Found: C 57.48, H 3.87, N
4.5.1. Cell lines and culture conditions
7.80%. 1H NMR (CDCl3, ppm): 8.77 (1H, s, N]CH), 8.05 (2H, d, 2,6-
pyridine-H, J ¼ 4 Hz), 7.88 (2H, d, 3,5-pyridine-H, J ¼ 4 Hz),
7.43e6.75 (13H, m, aromatic-H), 3.99 (3H, s, eOCH3). 13C NMR
(CDCl3, ppm): 167.27(CeO), 163.47 (C]N), 158.42, 152.12, 150.31,
141.23, 138.81, 136.37, 130.90, 129.20, 126.30, 121.80, 117.35, 116.69
The cell lines, human lung cancer cell line (A549), human leu-
kemia cell line (HL-60) and human colon cell line (HT-29, HCT-116,
Caco-2) were used for screening. Cell lines were maintained in the
logarithmic phase at 37 ꢁC in a 5% carbon dioxide atmosphere using
the following culture media containing 10% fetal bovine serum and
¼ 344 Hz), 56.83 (eOCH3). 119Sn NMR
119Snꢀ13CÞ
1% antibiotics (50 units$mLꢀ1 penicillin and 50 g mLꢀ1 strepto-
m
(aromatic-C, 1J
ð
(CDCl3, ppm): ꢀ326.42. IR (KBr, cmꢀ1): 1609, 1594 (m, C]NeN]C),
575 (m, SneO), 476 (w, SneN).
mycin): (a) RPMI-1640 medium for HL-60 cells, (b) D-MEM (Dul-
becco's Modified Eagle Medium) medium for A549 cells. (c)
McCoy's 5A medium for HT-29 and HCT-116 cells. (d) I-MDM me-
dium for Caco-2 cells.
4.4.5. [(CH3)3Sn(C14H12N3O3)]6 (5)
Compound 5 was obtained by the reaction of the ligand/
Me3SnCl/EtONa in a 1:1:1 M ratio in methanol. The preparing
procedure was similar to that of compound 1. Light yellow block
crystals of compound 5 were slowly formed from dichloromethane/
petroleum ether (5:2) at room temperature. Yield: 70%. M.p.:
255e256 ꢁC. Anal. Calc. for C17H21N3O3Sn: C 47.04, H 4.88, N 9.68%.
4.5.2. Preparation of drug solutions
Stock solutions of the studied organotin compounds were pre-
pared in dimethyl sulfoxide (DMSO, Sigma Aldrich) at concentra-
tions of 10 mg/mL and diluted by cell culture medium to various
working concentration. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-

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M. Hong et al. / European Journal of Medicinal Chemistry 86 (2014) 550e561
diphenyl tetrazolium bromide was dissolved (5 mg/mL) in phos-
phate buffer saline pH 7.2, and filtered through millipore filter
(0.22 mm) before use.
manually by using a trace syringe and the fluorescence spectra
were recorded with excitation wavelength of BSA at 280 nm and
the emission at 290e450 nm by keeping the concentration of BSA
constant (1.0
0 to 30 M at room temperature. For synchronous fluorescence
spectra, a solution of BSA (1.0 M) was titrated with increasing
concentrations of the compounds (0, 5, 10, 15, 20, 25, 30 M for
mM) while varying the complex concentration from
4.5.3. MTT assays
m
All cells were seeded into a 96-well plate at cell densities of
m
1000e1500 cells/well, respectively, in 100
and were incubated for 24 h. The medium was then removed, and
200 L of new growth medium containing various concentrations
of the organotin complexes was added. After 48 h, the medium was
removed, 100 L of a 0.5 mg/mL solution of MTT in medium was
added, and the plate was incubated for an additional 4 h. The
medium/MTT mixture was aspirated, and 100 L of DMSO were
m
L of growth medium
m
Dl ¼ 60 nm and 0, 5, 10, 15, 20 M for Dl ¼ 15 nm) with excitation
m
m
wavelength at 280 nm and the emission at 200e350 nm.
m
Acknowledgments
m
We acknowledge the National Natural Foundation of China
(21105042) and the Natural Science Foundation of Shandong
added to dissolve the purple formazan crystals. The plate was
shaken for 10 min on a plate shaker to ensure complete dissolution.
The absorbance of the plates was read at 490 nm using ELx808
Absorbance Microplate Reader (Bio-Tek Co., USA). IC50 values were
extrapolated from the resulting curves. The reported IC50 values are
the averages from at least three independent experiments, each of
which consisted of three replicates per concentration level.
Province
(ZR2013BM017,
ZR2011BM007,
ZR2011BQ015,
ZR2010BQ021) for financial support. And this work was supported
by Shandong “TaieShan Scholar Research Fund”.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.070.
4.6. DNA binding studies
All the experiments involving the interaction of compound with
CT DNA were conducted in Tris buffer (0.01 M TriseHCl/50 mM
NaCl, pH 7.2). The purity of the DNA was determined by monitoring
the value A260/A280 about 1.8e1.9:1, indicating that the DNA was
sufficiently free of protein. The DNA concentration was determined
by absorption spectroscopy using the molar absorption coefficient
(6600 Mꢀ1 cmꢀ1) at 260 nm.
Spectroscopic titrations were carried out at room temperature
to determine the binding affinity between DNA and organotin(IV)
complexes. Initially, the solutions (3 mL) of the blank buffer and
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m
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