Design, spectral characterization, anti-tumor and anti-inflammatory activity of triorganotin(IV) hydroxycarboxylates, apoptosis inducers: In vitro assessment of induction of apoptosis by enzyme, DNA-fragmentation, acridine orange and comet assays Antitumor Active Organotin Compounds

Article abstract of DOI:10.1016/j.ica.2014.02.034

Interaction of triorganotin(IV) chlorides with sodium salt of hydroxycarboxylic acids results in the formation of triorganotin(IV) hydroxycarboxylates, R₃Sn(L) [R = Me, n-Bu and Ph; L = anion of glucuronic (HGlu), gallic (HGal) and mandelic (HMal) acid]. They exhibit trigonal-bipyramidal geometry which is well supported by elemental analysis, IR, ¹H, ¹³C, ¹¹⁹Sn NMR and ESI-MS spectral data. Triorganotin(IV) hydroxycarboxylates have been screened in vitro against five cancer cell lines of human origin viz. MCF-7, HEK-293, PC-3, HCT-15 and HepG-2. Title complexes are found to be cytotoxic to mildly cytotoxic, and exhibited IC₅₀ values in the range 4-30 μg/mL. The results of enzyme assays viz. glutathione reductase, glutathione peroxidase, total glutathione content and lipid peroxidase assay on MCF-7 cells indicate that the reactive oxygen species generated in the cancer cells by triorganotin(IV) hydroxycarboxylates is responsible for cell death. Marginal increase of lactate dehydrogenase suggests that necrosis is also occurring to a small extent. DNA (deoxyribonucleic acid) fragmentation assay, acridine orange assay and comet assay clearly support that the cell death is mainly due to apoptosis. The results obtained for the in vivo anti-inflammatory activity (% inhibition) and toxicity (LD₅₀ in mg/kg) suggested that the complexes have low toxicity and good anti-inflammatory activity.

Full text of DOI:10.1016/j.ica.2014.02.034

Inorganica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Design, spectral characterization, anti-tumor and anti-inflammatory
activity of triorganotin(IV) hydroxycarboxylates, apoptosis inducers:
In vitro assessment of induction of apoptosis by enzyme,
DNA-fragmentation, acridine orange and comet assays
a
b
Mala Nath a, , Monika Vats , Partha Roy
⇑
a Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
b Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Interaction of triorganotin(IV) chlorides with sodium salt of hydroxycarboxylic acids results in the
formation of triorganotin(IV) hydroxycarboxylates, R3Sn(L) [R = Me, n-Bu and Ph; L = anion of glucuronic
(HGlu), gallic (HGal) and mandelic (HMal) acid]. They exhibit trigonal–bipyramidal geometry which is
well supported by elemental analysis, IR, 1H, 13C, 119Sn NMR and ESI-MS spectral data. Triorganotin(IV)
hydroxycarboxylates have been screened in vitro against five cancer cell lines of human origin viz.
MCF-7, HEK-293, PC-3, HCT-15 and HepG-2. Title complexes are found to be cytotoxic to mildly cytotoxic,
Received 29 October 2013
Received in revised form 17 January 2014
Accepted 22 February 2014
Available online xxxx
Antitumor Active Organotin Compounds
and exhibited IC50 values in the range 4–30 lg/mL. The results of enzyme assays viz. glutathione reduc-
tase, glutathione peroxidase, total glutathione content and lipid peroxidase assay on MCF-7 cells indicate
that the reactive oxygen species generated in the cancer cells by triorganotin(IV) hydroxycarboxylates is
responsible for cell death. Marginal increase of lactate dehydrogenase suggests that necrosis is also
occurring to a small extent. DNA (deoxyribonucleic acid) fragmentation assay, acridine orange assay
and comet assay clearly support that the cell death is mainly due to apoptosis. The results obtained
for the in vivo anti-inflammatory activity (% inhibition) and toxicity (LD50 in mg/kg) suggested that the
complexes have low toxicity and good anti-inflammatory activity.
Keywords:
Anti-cancer activity
Apoptosis
DNA cleavage
Anti-inflammatory activity
Triorganotin(IV) hydroxycarboxylate
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
proliferation of cancer cells for which the replication of DNA is to
be arrested. A number of cancer chemotherapeutic drugs are
Globally cancer among the various deadly diseases is one of the
leading health concerns for humans, accounting for more than 15%
of human deaths. Recently, it has been reported by WHO that
about 27 million new cancer cases and 11.5 million deaths due to
cancer are expected in the next 2 decades [1]. Cancers, caused by
abnormal and uncontrolled cell division, are usually derived from
numerous tissues with multiple etiologies and endless combina-
tion of genetic and/or epigenetic alterations; therapies for cancers
are as diverse as the disease itself. The treatments for cancer
include surgery, radiotherapy, photodynamic therapy and chemo-
therapy, and chemotherapy is based on the inhibition of the rapid
commercially available for treatment of different types of cancers,
but each of which has one or other severe side effects [2]. That is
why; one of the most rapidly developing areas of pharmaceutical
research is the discovery, design and synthesis of robust, effective
and selective cancer chemotherapeutic drugs which should have
minimum side effects.
Further, cancer chemotherapy based on metallotherapeutic
drugs has gained momentum after the serendipitous discovery of
cis-platin (cis-diamminedichloroplatinum(II)) [3,4]. However,
despite its remarkable success, there are well-known drawbacks
associated with Pt drugs, viz. nephrotoxicity or neurotoxicity,
intrinsic acquired drug resistance and patient compliance [5,6].
Therefore, this discovery stimulated the search for other
metal-based chemotherapeutics with potential antitumor activity
having fewer side effects [7,8]. Among these, organotins have
emerged as potential biologically active metallopharmaceuticals,
Abbreviations: Bz-d6, deteuratedbenzene; HGlu, glucuronic acid; HGal, gallic
acid; HMal, mandelic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; DNA, deoxyribonucleic acid.
⇑
Corresponding author. Tel.: +91 9897135529.
E-mail addresses: malanfcy@iitr.ac.in, malanfcy@gmail.com (M. Nath).
http://dx.doi.org/10.1016/j.ica.2014.02.034
0020-1693/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Nath et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.02.034

2
M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
and several research groups are actively engaged in the field
[9–23]. Many organotin compounds have been established as
potential cancer chemotherapeutic agents because of their apopto-
tic inducing character [22–26], and they occupy an important place
in cancer chemotherapy reports [11,12,17b,24]. Furthermore, the
National Cancer Institute has tested about 2000 tin-based
compounds, the largest number ever tested among metal-based
drug candidates [27], and recently it has been reported that
four out of thirty interesting inorganic pharmaceuticals are tin
compounds [28]. Despite this, the exact mechanism of mode of
action of organotin compounds remains unestablished.
Organotin(IV) carboxylates are among the most widely studied
class of compounds owing to their rich structural chemistry rang-
ing from monomeric, dimeric, tetrameric, oligomeric to polymeric
motifs [13,22,23,29–31]. The structures can be easily controlled by
adjusting the carboxylato ligands, tin–R groups and metal-to-
ligand molar ratio [29,32,33]. In addition to their structural diver-
sity, the organotin(IV) carboxylates also present very promising
antitumor activities [11,12,22,23,27,34–42]. Among these, several
di-n-butyltin(IV) carboxylates have been found to be the most
active, and in some cases they exhibit much higher activity than
clinically used reference compounds such as cis-platin, doxorubi-
cine and methotrexate [11,12,27,37].
The systemic toxicity of chemotherapeutic drugs, their lack of
tumor localization and an even distribution throughout the body
including tumor tissues are the most significant challenges facing
effective cancer chemotherapy. Therefore, a rational drug design
is essentially required to optimize specific targeting features and
to control toxicity (side effects), by controlling thermodynamic
and kinetic processes of metal complexes viz., choice of metal ion
and its oxidation state [43] or tailoring of ligand scaffold. It is also
well known that the cytotoxicity is related to some extent to lipo-
philicity of the drugs i.e., the most lipophilic compounds are the
most cytotoxic [44]. Further, the ligands having biologically active
pharmacophore with biocompatible properties are tethered to
others having multifunctional N and O donors to obtain modulated
ligand scaffold which can mute the potential toxicity of metallo-
pharmaceuticals drugs [8].
Anti-tumor activity of several organotin compounds has been
reported by various workers, but their solubility has always
remained a problem. This can be solved by choosing ligands having
polar groups such as hydroxy groups, or other oxygen or nitrogen
containing groups or substituted fluorine which may enhance their
solubility. Glucuronic acid (HGlu), mandelic acid (HMal) and gallic
acid (HGal) are such hydroxycarboxylic acids which are highly sol-
uble in water, methanol, ethanol and other organic solvents and
possess various medicinal properties. Glucuronic acid (uronic acid)
( )-mandelic acid (HMal) and gallic acid (HGal) (Fig. 1), and inves-
tigate their biological activities, viz. in vitro anti-tumor activity
(against MCF-7, HEK-293, PC-3, HCT-15 and HepG-2), in vivo
anti-inflammatory activity and toxicity studies. Furthermore, to
view insight the mode of action of the studied complexes various
assays, such as lactate dehydrogenase, glutathione reductase, glu-
tathione peroxidase, total glutathione content, lipid peroxidase,
DNA fragmentation, acridine orange and comet assay have also
been conducted and discussed in this manuscript.
2. Material and methods
2.1. Materials
The specifications and makes of triorganotin(IV) compounds,
and methods to obtain various dried solvents are same as reported
previously [22]. Gallic acid, b-D-glucuronic acid and ( )-mandelic
acid were procured from Alfa Aesar and used as received. Further,
a large number of chemicals used for biological activities are also
same as reported in our previous manuscript [22]. Five cancer cell
lines of human origin, viz. MCF-7 mammary cancer, HEK-293 kid-
ney cancer, PC-3 prostate cancer, HCT-15 colon cancer and
HepG-2 liver cancer were purchased from National Center for Cell
Science (NCCS) Pune, India. For anti-inflammatory activity and
acute toxicity Swiss albino mice were procured from All India
Institute of Medical Science (AIIMS) Delhi, India.
2.2. Physical measurements
The melting points, microanalyses (C, H and N) and tin content
were determined as described previously [22]. Molar conductance
measurements, infrared and far-infrared, nuclear magnetic reso-
nance (1H, 13C and 119Sn) spectra were recorded on the same
instruments as reported previously [22]. ESI-MS spectra were
recorded on Bruker MicroTOF-Q II mass spectrometer at a flow rate
of 180 lL/h on positive mode in water–acetonitrile mixture.
Optical density (OD) in MTT assay, gel electrophoresis, enzymes
assays, in vivo anti-inflammatory activity and toxicity were done
according to the reported procedures with slight modifications as
described in our previous manuscript [22].
2.3. Synthesis
2.3.1. Synthesis of sodium salt of hydroxycarboxylic acids
Hydroxycarboxylic acid (HGlu/HMal/HGal, 2.0 mmol) was
dissolved in 10 mL of specially dried ethanol under dry nitrogen
and added to sodium ethoxide, prepared by reacting sodium
(0.058 g, 2.5 mmol) with dry ethanol (25 mL). The resulting mix-
ture was refluxed giving a clear solution of sodium salt of the acid
within half an hour. Refluxing was continued for another 1–2 h
with constant stirring. The solution was concentrated and solid
was dried in vacuum.
exhibits anti-oxidant activity [45]. Mandelic acid (a-hydroxyl car-
boxylic acid) has been used for the treatment of skin problems, and
also possess anti-bacterial activity [46]. Gallic acid is a naturally
occurring plant phenol obtained by the hydrolysis of tannins and
is known to display some pharmacological activities such as anti-
cancer, fungicidal/fungi-static, antiviral, anti-inflammatory and
anti-oxidant properties [47].
Interaction of glucuronic acid with bis(tributyltin) oxide
(studied via NMR and mass spectroscopy) in solution [48], the
solution studies of tris(1-butyl)stannyl-D-glucuronate through
2.3.1.1. Na(Glu). IR (cmꢀ1):
187, as(COC) 1245m, s(COC) 1110m,
3400s, (OH-3) 3488s,
(OH-4) 3509s. 1H NMR (500.13 MHz,
mas(OCO) 1616s,
m
s(OCO) 1429m,
Dm
NMR spectral analysis [49], di-n-butyltin(IV) and diethyltin(IV)
complexes of methoxy analog of gallic acid [50], reaction of ethyl-
trichlorostannane with N,N-dimethylamide of mandelic acid [51]
and synthesis of trimethyltin derivative of mandelic acid [52] are
few reports available in literature. Moreover, the functional groups
attached to carboxylic acid may have pronounced influence
(increase/decrease) on the biological properties of organotin(IV)
carobxylates [22,34–37]. Therefore, it becomes indispensable to
m
m
m(OH-1) 3533s,
m(OH-2)
m
m
Bz-d6, ppm): d 4.63 (d, 1H, H-1, J1,2 = 8.0 Hz), 3.25 (dd, 1H, H-2,
J1,2 = 8.0 Hz, J2,3 = 9.1 Hz), 3.50 (dd, 1H, H-3, J2,3 = 9.1 Hz,
J3,4 = 9.2 Hz), 3.96 (dd, 1H, H-4, J3,4 = 9.2 Hz, J4,5 = 9.6 Hz), 3.93 (d,
1H, H-5, J4,5 = 9.6 Hz), 4.81 (s, 1H, OH-1), 3.55 (s, 1H, OH-2), 3.72
(s, 1H, OH-3), 3.86 (s, 1H, OH-4). 13C NMR (125.75 MHz, Bz-d6,
ppm): d 95.86 (C-1), 74.53 (C-2), 72.43 (C-3), 72.74 (C-4), 76.48
(C-5), 175.31 (C-6).
synthesize organotin(IV) derivatives of b-D-glucuronic acid (HGlu),
Please cite this article in press as: M. Nath et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.02.034

M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
3
Fig. 1. Molecular structures of hydroxycarboxylic acids.
2.3.1.2. HGlu. IR (cmꢀ1):
m
as(OCO) 1596s,
s(COC) 1115m, (OH-1) 3530s,
(OH-4) 3511s. 1H NMR (500.13 MHz, Bz-d6,
m
s(OCO) 1423s,
D
m
173,
(s, 2H, H-3, H-7), 3.71 (s, 1H, OH-4), 3.82 (s, 1H, OH-5), 3.76 (s,
1H, OH-6). 13C NMR (125.75 MHz, CD3OD, ppm): d 167.96 (C-1),
128.48 (C-2), 129.84 (C-3, C-7), 137.60 (C-4, C-6), 140.05 (C-5).
m
m
as(COC) 1249m,
(OH-3) 3486s,
m
m
m
m
(OH-2) 3405s,
ppm): d 12.27 (s, 1H, (COO)–H), 4.71 (d, 1H, H-1, J1,2 = 9.3 Hz),
3.35 (dd, 1H, H-2, J1,2 = 9.3 Hz, J2,3 = 9.0 Hz), 3.39 (dd, 1H, H-3,
J2,3 = 9.0 Hz, J3,4 = 9.1 Hz), 3.85 (dd, 1H, H-4, J3,4 = 9.1 Hz,
J4,5 = 9.5 Hz), 3.76 (d, 1H, H-5, J4,5 = 9.5 Hz), 4.77 (s, 1H, OH-1),
3.49 (s, 1H, OH-2), 3.62 (s, 1H, OH-3), 3.90 (s, 1H, OH-4). 13C
NMR (125.75 MHz, Bz-d6, ppm): d 95.61 (C-1), 74.49 (C-2), 72.32
(C-3), 72.68 (C-4), 76.39 (C-5), 173.22 (C-6).
2.3.2. Synthesis of triorganotin(IV) derivatives of hydroxycarboxylic
acids
A hot ethanolic solution (20 mL) of trimethyltin(IV) chloride
(0.398 g, 2.0 mmol) or tri-n-butyltin(IV) chloride (0.651 g,
2.0 mmol) or triphenyltin(IV) chloride (0.771 g, 2.0 mmol) was
added to the dry and hot ethanolic solution (10 mL) of the
preformed sodium salt of the acid as prepared in Section 2.3.1.
The resulting solution was refluxed with constant stirring for an-
other 5–6 h under dry nitrogen atmosphere. The resulting solution
was then centrifuged and filtered in order to remove the sodium
chloride formed during the reaction. The excess of solvent was re-
moved under reduced pressure and the products thus obtained
were washed with either ethanol–hexane or ethanol–petroleum
ether (b.p. 40–60 °C) mixture (1:3 v/v).
2.3.1.3. Na(Mal). IR (cmꢀ1):
177,
(OH-2) 3610s. 1H NMR (500.13 MHz, CD3OD, ppm): d 5.11
mas(OCO) 1597w, ms(OCO) 1420s, Dm
m
(s, 1H, H-2), 7.73 (m, 1H, H-4), 7.31 (m, 2H, H-5 + H-7), 7.21 (m,
1H, H-6), 7.52 (m, 1H, H-8), 3.30 (s, 1H, OH-2). 13C NMR
(125.75 MHz, CD3OD, ppm): d 175.33 (C-1), 75.22 (C-2), 136.19
(C-3), 127.21 (C-4), 127.41 (C-5), 126.21 (C-6), 127.33 (C-7),
126.06 (C-8).
2.3.1.4. HMal. IR (cmꢀ1):
(OH-2) 3621m. 1H NMR (500.13 MHz, CD3OD, ppm): d 11.81 (s,
1H, (OCO)–H), 5.01 (s, 1H, H-2), 7.70 (m, 1H, H-4), 7.23 (m, 2H,
H-5 + H-7), 7.19 (m, 1H, H-6), 7.49 (m, 1H, H-8), 3.50 (s, 1H, OH-
2). 13C NMR (125.75 MHz, CD3OD, ppm): d 173.33 (C-1), 75.12
(C-2), 136.12 (C-3), 127.13 (C-4), 127.36 (C-5), 126.16 (C-6),
127.29 (C-7), 125.96 (C-8).
mas(OCO) 1580m, ms(OCO) 1417m, Dm 163,
2.3.2.1. Ph3Sn(Glu). White solid. M.P.: 98 °C. Yield: 60%. Anal. Calc.
m
for C24H24O7Sn: Sn, 22.92; H, 4.42; C, 53.07. Found: Sn, 22.99; H,
4.40; C, 53.01%. Mw: 543. IR (cmꢀ1):
m
as(OCO) 1626s,
s(Sn–C) 240s, as(COC) 1150m, ms
(OH-1) 3480br, (OH-2) 3379w,
ms(OCO)
1422s,
(COC) 1005m,
(OH-3) 3360br,
D
m
204,
m
as(Sn–C) 277s,
m
m
m
m
(Sn–O) 546w,
m
m
m
(OH-4) 3410m. 1H NMR (500.13 MHz, CD3OD,
ppm): d 4.82 (d, 1H, H-1, J1,2 = 8.2 Hz), 3.37 (m, 1H, H-2), 3.40 (m,
1H, H-3); 3.97 (m, 1H, H-4), 3.86 (d, 1H, H-5, J4,5 = 9.3 Hz), 4.77
(s, 1H, OH-1), 3.94 (s, 1H, OH-2)a, 3.95 (s, 1H, OH-3), 3.99 (s, 1H,
2.3.1.5. Na(Gal). IR (cmꢀ1):
(OH-4) 3533br, (OH-5) 3440br,
m
as(OCO) 1629s,
ms(OCO) 1403s, Dm 226,
m
m
(OH-6) 3459br. 1H NMR
OH-4), 7.72 (d, 6H, H-b, J = 3.2 Hz), 7.51 (m, 6H, H-c), 7.32 (t, 3H,
H-d, J = 6.5 Hz). 13C NMR (125.75 MHz, CD3OD, ppm): d 97.24 (C-
(500.13 MHz, CD3OD, ppm): d 7.30, 7.89 (s, 2H, H-3, H-7), 3.81 (s,
1H, OH-4), 3.71 (s, 1H, OH-5), 3.90 (s, 1H, OH-6). 13C NMR
(125.75 MHz, CD3OD, ppm): d 169.68 (C-1), 127.98 (C-2), 130.01
(C-3, C-7), 137.89 (C-4, C-6), 140.13 (C-5).
1), 74.42 (C-2), 72.65 (C-3); 73.93b (C-4), 77.51 (C-5), 178.00 (C-
6), 129.93 (C-a
,1J(13C–119Sn) = 466.21 Hz, h = 117.65°), 138.12 (C-
b, 2J(13C–119Sn) = 44.33 Hz), 129.52 (C- 3J(13C–119Sn) = 68.00 Hz),
c,
130.24 (C-d). 119Sn NMR (186.50 MHz, CD3OD, ppm): d ꢀ141.44.
2.3.1.6. HGal. IR (cmꢀ1):
(OH-4) 3540m, (OH-5) 3509 w,
(500.13 MHz, CD3OD, ppm): d 12.23 (s, 1H, (OCO)–H), 7.28, 7.85
m
as(OCO) 1607m,
m
s(OCO) 1425 s,
D
m
182,
1H NMR (500.13 MHz, CDCl3, ppm):
d 4.82 (d, 1H, H-1,
m
m
(OH-6) 3499m. 1H NMR
J1,2 = 8.1 Hz), 3.32 (m, 1H, H-2), 3.35 (m, 1H, H-3), 3.86 (m, 1H,
H-4), 3.79 (d, 1H, H-5, J4,5 = 9.2 Hz), 3.99 (s, 1H, OH-1)a, 3.88
Please cite this article in press as: M. Nath et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.02.034

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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
(s, 1H, OH-2), 3.93 (s, 1H, OH-3), 3.97 (s, 1H, OH-4), 7.73 (d, 6H, H-
(s, 1H, H-2), 7.90 (m, 1H, H-4), 7.00 (m, 2H, H-5 + H-7), 6.98 (m,
b, J = 3.1 Hz), 7.50 (m, 6H, H-
c
), 7.33 (t, 3H, H-d, J = 6.5 Hz). 13C NMR
1H, H-6), 7.17 (m, 1H, H-8), 3.95 (s, 1H, OH-2), 7.60 (d, 6H, H-b,
(125.75 MHz, CDCl3, ppm): d 97.10 (C-1), 74.34 (C-2), 72.59 (C-3), 73.81b
J = 3.1 Hz), 7.42 (m, 9H, H-c
+ H-d). 13C NMR (125.75 MHz, CD3OD,
1
(C-4), 77.32 (C-5), 176.45 (C-6), 129.80 (C-
a
, J(13C–119Sn) = 350.33 Hz,
ppm): d 177.41 (C-1), 78.10 (C-2), 135.89 (C-3), 129.10 (C-4),
h = 107.46°), 138.00 (C-b, 2J(13C–119Sn) = 44.01 Hz), 129.13 (C-
c,
3J
127.62 (C-5), 126.25 (C-6), 127.46 (C-7), 127.12 (C-8), 139.60 (C-
(
13C–119Sn) = 67.71 Hz), 129.96 (C-d). 119SnNMR (186.50 MHz, CDCl3,
a), 137.33 (C-b), 130.14 (C-c
), 130.31 (C-d). 119Sn NMR
ppm): d –40.91. ESI-MS: 544 [M+H]+, 566 [M+Na]+ (100%), 350
(MꢀL)+, 274 [HSnPh2]+, 198 [H2SnPh]+.
(186.50 MHz, CD3OD, ppm): d ꢀ182.51. ESI-MS: 502 [M+H]+, 524
[M+Na]+ (100%), 350 (MꢀL)+, 851 [M+SnPh3]+, 274 [HSnPh2]+,
198 [H2SnPh]+.
2.3.2.2. n-Bu3Sn(Glu). Creamish semi-solid. Yield: 62%. Anal. Calc.
for C18H36O7Sn: Sn, 24.64; H, 7.46; C, 44.75. Found: Sn, 24.68; H,
2.3.2.5. n-Bu3Sn(Mal). White solid. M. P.: 170°C. Yield: 69%. Anal.
7.50; C: 44.79%. Mw: 483. IR (cmꢀ1):
m
as(OCO) 1625m,
s(Sn–C) 510s, as(COC) 1161m,
(OH-1) 3494br, (OH-2) 3373br,
(OH-4) 3419br. 1H NMR (500.13 MHz, CD3OD,
m
s(OCO)
Calc. for C20H34O3Sn: Sn, 26.98; H, 7.71; C, 54.42. Found: Sn,
1415s,
D
m
210,
s(COC) 1015m,
(OH-3) 3436 w,
m
m
as(Sn–C) 557sh,
m
m
m
26.93; H, 7.76; C, 54.49%; Mw: 441. IR (cmꢀ1):
m
as(OCO) 1609s,
s(Sn–C) 515 m, (Sn–O)
m
(OH-2) 3271br. 1H NMR (500.13 MHz, CD3OD, ppm): d
m
m
(Sn–O) 560w,
m
ms(OCO) 1396s,
D
m
213,
mas(Sn–C) 546s,
m
m
m
562w,
ppm): d 5.01 (d, 1H, H-1, J1,2 = 8.1 Hz), 3.40 (m, 1H, H-2); 3.43
(m, 1H, H-3), 3.89 (m, 1H, H-4), 3.79 (d, 1H, H-5, J4,5 = 9.0 Hz),
4.82 (s, 1H, OH-1), 3.83 (s, 1H, OH-2), 3.92 (s, 1H, OH-3), 3.98 (s,
5.90 (s, 1H, H-2), 7.71 (m, 1H, H-4), 7.30 (m, 2H, H-5 + H-7), 7.22
(mbr, 1H, H-6), 7.60 (m, 1H, H-8), 3.88 (s, 1H, OH-2), 1.35 (t, 6H,
H-a, J = 6.7 Hz), 1.61 (m, 6H, H-b), 1.11 (m, 6H, H-c), 0.83 (t, 9H,
1H, OH-4)a, 1.83 (t, 6H, H-
(m, 6H, H-
CD3OD, ppm): d 97.89 (C-1), 74.41 (C-2), 72.65 (C-3), 73.16b (C-
4), 77.44 (C-5), 176.61 (C-6), 20.26 (C-
, 1J(13C–119Sn) = 454.43 Hz,
a
, J = 6.8 Hz); 1.30 (m, 6H, H-b); 1.21
H-d, J = 6.7 Hz). 13C NMR (125.75 MHz, CD3OD, ppm): d 178.00
c
), 0.90 (t, 9H, H-d, J = 6.8 Hz). 13C NMR (125.75 MHz,
(C-1), 79.21 (C-2), 136.11 (C-3), 129.13 (C-4), 127.41
(C-5), 126.46 (C-6), 127.86 (C-7), 126.37 (C-8), 19.71 (C-
a
,
1J
),
a
(
13C–119Sn) = 471.04 Hz, h = 118.07°), 27.22 (C-b), 26.73 (C-
c
h = 116.62°), 27.62 (C-b), 27.02 (C-
c,
3J(13C–119Sn) = 82.00 Hz),
13.84 (C-d). 119Sn NMR (186.50 MHz, CD3OD, ppm): d ꢀ168.51.
ESI-MS: 442 [M+H]+, 464 [M+Na]+, 290 (MꢀL)+ (100%), 731
[M+SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+.
14.21 (C-d). 119Sn NMR (186.50 MHz, CD3OD, ppm): d ꢀ168.10.
1H NMR (500.13 MHz, CDCl3, ppm):
d 4.93 (d, 1H, H-1,
J1,2 = 8.0 Hz), 3.35 (m, 1H, H-2), 3.36 (m, 1H, H-3), 3.95 (m, 1H,
H-4), 3.85 (d, 1H, H-5, J4,5 = 9.3 Hz), 4.07 (s, 1H, OH-1), 3.78 (s,
1H, OH-2), 3.92 (s, 1H, OH-3), 3.97 (s, 1H, OH-4), 1.83 (t, 6H, H-a,
2.3.2.6. Me3Sn(Mal). White solid. M.P.: (123)d 118–120 °C; Yield:
65% Anal. Calc. for C11H16O3Sn: Sn, 37.77; H, 5.08; C, 41.90. Found:
J = 6.7 Hz), 1.31 (m, 6H, H-b), 1.22 (m, 6H, H-
c
), 0.90 (t, 9H, H-d,
Sn, 37.70; H, 5.09; C, 41.93%. Mw: 315. IR (cmꢀ1):
m
as(OCO) 1628s,
s(Sn–C) 566s, (Sn–O)
m
(OH-2) 3265br. 1H NMR (500.13 MHz, CD3OD, ppm): d
J = 6.8 Hz). 13C NMR (125.75 MHz, CDCl3, ppm): d 97.06 (C-1),
m
s(OCO) 1417m,
D
m
211,
m
as(Sn–C) 601s,
m
m
74.32 (C-2), 72.38 (C-3), 72.89b (C-4), 77.10 (C-5), 176.40 (C-6),
557w,
22.20 (C-
26.82 (C-
a
c
,
1J(13C–119Sn) = 334.65 Hz, h = 106.11°), 27.33 (C-b),
,
3J(13C–119Sn) = 81.11 Hz), 12.11 (C-d). 119Sn NMR
6.62 (s, 1H, H-2), 7.77 (m, 1H, H-4), 7.32 (m, 2H, H-5 + H-7), 7.25
(m, 1H, H-6), 7.64 (m, 1H, H-8), 3.94 (s, 1H, OH-2), 0.80 (s, 9H,
(186.50 MHz, CD3OD, ppm): d ꢀ45.12. ESI-MS: 484 [M+H]+, 506
[M+Na]+ (100%), 290 (MꢀL)+, 234 [HSnBu2]+, 178 [H2SnBu]+.
H-a,
2J(1H–119Sn) = 64.10 Hz, h = 114.93ꢀ). 13C NMR (125.75 MHz,
CD3OD, ppm): d 178.50 (C-1), 76.11 (C-2), 135.61 (C-3), 130.74
(C-4), 128.10 (C-5), 126.12 (C-6), 127.42 (C-7), 129.00 (C-8),
2.3.2.3. Me3Sn(Glu). White solid. M.P.: 73 °C. Yield: 58%. Anal. Calc.
16.85 (C-a,
1J(13C–119Sn) = 476.12 Hz, h = 118.52°). 119Sn NMR
for C9H18O7Sn: Sn, 33.35; H, 5.04; C, 30.25. Found: Sn, 33.38; H,
(186.50 MHz, CD3OD, ppm): d ꢀ149.15. ESI-MS: 316 [M+H]+, 338
[M+Na]+, 164 (MꢀL)+ (100%), 479 [M+SnMe3]+, 150 [HSnMe2]+,
136 [H2SnMe]+.
5.08; C, 30.28%. Mw: 357. IR (cmꢀ1):
m
as(OCO) 1626s, s(OCO)
s(Sn–C) 575s, as(COC) 1154m,
(OH-1) 3484br, (OH-2) 3370br,
(OH-4) 3421br. 1H NMR (500.13 MHz, CD3OD,
1413m,
D
m
213,
s(COC) 1019m,
(OH-3) 3429 w,
m
as(Sn–C) 590s,
m
m
m
m
m
m
(Sn–O) 550w,
m
m
2.3.2.7. Ph3Sn(Gal). White solid. M.P.: 221–226 °C. Yield: 61%. Anal.
ppm): d 5.08 (d, 1H, H-1, J1,2 = 8.3 Hz), 3.37 (m, 1H, H-2), 3.39 (m,
1H, H-3), 3.92 (m, 1H, H-4), 3.75 (d, 1H, H-5, J4,5 = 9.2 Hz,), 4.81
(s, 1H, OH-1), 3.89 (s, 1H, OH-2), 3.90 (s, 1H, OH-3), 3.91 (s, 1H,
Calc. for C25H20O5Sn: Sn, 22.93; H, 3.85; C, 57.80. Found: Sn, 22.87;
H, 3.89; C, 57.79%. Mw: 519. IR (cmꢀ1):
m
as(OCO) 1642 m,
ms(OCO)
1413s,
D
m
229,
m
as(Sn–C) 288s,
m
s(Sn–C) 225m, (Sn–O) 556w,
m
OH-4)a, ꢀ0.40, 0.11 (s, 9H, H-
a
,
2J(1H–119Sn) = 66.12 Hz,
m(OH-4) 3444br, m(OH-5) 3440br, m
(OH-6) 3409br. 1H NMR
h = 116.51°). 13C NMR (125.75 MHz, CD3OD, ppm): d 97.83 (C-1),
(500.13 MHz, CD3OD, ppm): d 7.75, 7.88 (s, 2H, H-3, H-7)c, 4.07
74.45 (C-2) 72.26 (C-3), 75.16b (C-4), 77.41 (C-5), 178.92 (C-6),
(s, 1H, OH-4), 3.79 (s, 1H, OH-5), 4.66 (s, 1H, OH-6), 8.20 (d, 6H,
ꢀ4.3 (C-
a
,
1J(13C–119Sn) = 447.10 Hz, h = 115.97° 119Sn NMR
H-b, J = 3.3 Hz), 7.74 (m, 6H, H-c
), 7.46 (t, 3H, H-d, J = 7.1 Hz). 13C
(186.50 MHz, CD3OD, ppm): d ꢀ183.01. 1H NMR (500.13 MHz,
CDCl3, ppm): d 4.94 (d, 1H, H-1, J1,2 = 8.1 Hz), 3.37 (m, 1H, H-2),
3.39 (m, 1H, H-3), 3.90 (m, 1H, H-4), 3.70 (d, 1H, H-5,
J4,5 = 9.3 Hz), 4.98 (s, 1H, OH-1), 3.82 (s, 1H, OH-2), 3.92 (s, 1H,
NMR (125.75 MHz, CD3OD, ppm): d 172.43 (C-1), 123.72 (C-2),
110.02 (C-3), 144.04 (C-4), 141.41 (C-5), 137.87 (C-6), 109.00 (C-
7), 130.15 (C-
b), 129.66 (C-
a,
1J(13C–119Sn) = 484.57 Hz, h = 119.21°), 137.54 (C-
c
), 130.34 (C-d). 119Sn NMR (186.50 MHz, CD3OD,
OH-3), 3.98 (s, 1H, OH-4), ꢀ0.42, 0.09 (s, 9H, H-
a
,
2J(1H–119-
ppm): d ꢀ132.19. ESI-MS: 520 [M+H]+, 542 [M+Na]+, 350 (MꢀL)+
Sn) = 43.82 Hz, h = 106.47°). 13C NMR (125.75 MHz, CDCl3, ppm): d
(100%), 869 [M+SnPh3]+, 274 [HSnPh2]+, 198 [H2SnPh]+.
97.78 (C-1), 74.05 (C-2), 72.00 (C-3), 75.00b (C-4), 77.24 (C-5),
177.86 (C-6), ꢀ2.2 (C-
a,
1J(13C–119Sn) = 329.07 Hz, h = 105.62°].
2.3.2.8. n-Bu3Sn(Gal). Yellowish solid. M.P.: 95 °C. Yield: 51%. Anal.
119Sn NMR (186.50 MHz, CDCl3, ppm): d ꢀ49.21. ESI-MS: 358
[M+H]+, 380 [M+Na]+, 164 (MꢀL)+ (100%), 150 [HSnMe2]+, 136
[H2SnMe]+.
Calc. for C19H32O5Sn: Sn, 25.93; H, 6.97; C, 49.67. Found: Sn, 25.90;
H, 6.92; C, 49.63%. Mw: 459. IR (cmꢀ1):
mas(OCO) 1637s,
ms(OCO)
1410s,
D
m
227,
m
as(Sn–C) 550s,
m
s(Sn–C) 521s, (Sn–O) 565w,
m
m
(OH-4) 3446br, m(OH-5) 3437br, m
(OH-6) 3412br. 1H NMR
2.3.2.4. Ph3Sn(Mal). White solid. M.P.: 190 °C. Yield: 61%; Anal. Calc.
(500.13 MHz, CD3OD, ppm): d 7.70, 7.86 (s, 2H, H-3, H-7)c, 4.18
for C26H22O3Sn: Sn, 23.75; H, 4.39; C, 62.28. Found: Sn, 23.70; H,
(s, 1H, OH-4), 3.77 (s, 1H, OH-5), 4.52 (s, 1H, OH-6), 1.05 (t, 6H,
4.33; C, 62.20%; Mw: 501. IR (cmꢀ1):
m
as(OCO) 1650w,
s(Sn–C) 230s, (Sn–O) 555w,
(OH-2) 3268br. 1H NMR (500.13 MHz, CD3OD, ppm): d 4.81
m
s(OCO)
H-
(m, 6H, H-
CD3OD, ppm): d 173.51 (C-1), 124.02 (C-2), 109.72 (C-3), 143.30
a,
2J(1H–119Sn) = 56.17 Hz, h = 110.05°), 1.56 (m, 6H, H-b), 1.21
1427s,
D
m
223,
m
as(Sn–C) 291s,
m
m
c
), 0.87 (t, 9H, H-d, J = 6.7 Hz). 13C NMR (125.75 MHz,
m
Please cite this article in press as: M. Nath et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.02.034

M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
5
(C-4), 141.63 (C-5), 137.47 (C-6), 109.06 (C-7), 21.05 (C-
a
), 29.16
10 mM Tris, 1 mM of EDTA (pH 8.5), and 50
1 h at 37 °C.
For agarose gel electrophoresis, 0.4 g of agarose was added to
50 mL of 1x TAE buffer (containing tris base, acetic acid and EDTA)
and the mixture was heated in a microwave oven till the agarose
was completely dissolved. When the solution becomes lukewarm,
l
g/mL of RNAase for
(C-b), 27.35 (C-
c
:), 13.17 (C-d). 119Sn NMR (186.50 MHz, CD3OD,
ppm): d ꢀ161.43. ESI-MS: 460 [M+H]+, 482 [M+Na]+, 290 (M-L)+
(100%), 749 [M+SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+.
2.3.2.9. Me3Sn(Gal). White solid. M.P.: 230–235 °C. Yield: 68%. Anal.
2 lL of ethidium bromide (10 mg/mL stock) was added. This solu-
Calc. for C10H14O5Sn: Sn, 35.74; H, 4.20; C, 36.04. Found: Sn, 35.77;
tion was then poured into the gel casting tray fitted with appropri-
ate comb and allowed to solidify. This gel was used for the
submerged electrophoresis for separating the DNA samples using
suitably 1ꢁ or 0.5ꢁ TAE as the running buffer. DNA samples were
mixed with gel loading dye and loaded into the wells along with
the controls. Gel was run at 80 V for 1–2 h till the dye front reached
the last one-third of the gel and was observed in a UV illuminator.
H, 4.26; C, 36.09%. Mw: 333. IR (cmꢀ1):
mas(OCO) 1643s,
ms(OCO)
1421m,
D
m
222,
m
as(Sn–C) 587m,
m
s(Sn–C) 568 s, (Sn–O) 551w,
m
m
(OH-4) 3441br, m(OH-5) 3432br, m
(OH-6) 3415br. 1H NMR
(500.13 MHz, CD3OD, ppm): d 7.85, 7.92 (s, 2H, H-3, H-7)c, 4.17
(s, 1H, OH-4), 3.75 (s, 1H, OH-5), 4.73 (s, 1H, OH-6), 0.04, 0.08 (s,
9H, H-
123.91 (C-2), 110.18 (C-3), 144.52 (C-4), 142.31 (C-5), 137.28
a
). 13C NMR (125.75 MHz, CD3OD, ppm): d 174.10 (C-1),
(C-6), 109.36 (C-7), ꢀ4.12 (C-
a
,
1J(13C–119Sn) = 447.50 Hz,
h = 116.01°]. 119Sn NMR (186.50 MHz, CD3OD, ppm): d ꢀ202.77.
ESI-MS: 334 [M+H]+, 356 [M+Na]+, 164 (MꢀL)+ (100%), 497
[M+SnMe3]+, 150 [HSnMe2]+, 136 [H2SnMe]+.
2.4.2. Acridine orange assay
Acridine orange staining is one of the acceptable assays for
evaluating the apoptosis by analysing the plasma-membrane
permeability, nuclear morphology as well as the chromatin con-
densation [55,56]. In the assay, the cancer cells were stained with
Mw: molecular weight; IR: s, strong; m, medium; w, weak; sh,
shoulder; br, broad;
D
m
=
m
as(OCO) ꢀ
ms(OCO);
NMR: hydrogen and carbon number according to Fig. 1; s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; angle h
calculated from Eq.: \C–Sn–C = 0.0161|2J|2 ꢀ 1.32|2J| + 133.4 [32]
and |(1J(13C119Sn)| = 11.4h ꢀ 875; avery weak intensity signal; bbroad
acridine orange (AO)/ethidium bromide (EB) dye mixture (100
lg/
mL of AO and 100
lg/mL of EB) and assay was performed according
to the method described by Takahashi et al. [56]. Briefly, 0.5 ꢁ 106
cancer (MCF-7) cells were seeded in a 6-well plate and incubated
with the respective IC50 value of n-Bu3Sn(Gal) and cis-platin
(positive control) for 24 h and then washed with PBS. After
c
signal; the signals of the two hydrogens may interexchange;
dAccording to Ref. [52];
washing 500 lL of AO/EB dye mixture dissolved in PBS was added
to the plate and the cells were observed under fluorescent
microscope (Zeiss, Axiovert 25, Germany).
2.4. Biological studies
2.4.3. Comet assay
Comet assay is a very simple technique yet highly sensitive to
detect DNA damage in individual cells [57]. Cytotoxicity effect of
n-Bu3Sn(Gal) (test compound) which leads to DNA damage was
also validated by comet assay (single cell gel electrophoresis).
The assay was performed according to the method reported earlier
[58] with slight modifications. In brief, 5 ꢁ 105 cancer (MCF-7)
cells were treated with test compound and incubated for 24 h at
37 °C and then washed thoroughly with PBS. Fresh media was
added after proper washing and then the cells were harvested after
24 h. Slides were prepared with 1% normal melting agarose, where,
The synthesized triorganotin(IV) hydroxycarboxylates have
been screened for their in vitro anti-cancer (MTT assay) and
in vivo anti-inflammatory activity, and have also been investigated
for acute toxicity according to the reported procedures which are
described in our previous manuscript [22]. Further, various en-
zyme assays, viz. lipid peroxidase, glutathione peroxidase, glutathi-
one reductase, total glutathione and lactate dehyrogenase assays
against MCF-7 cell lines by n-Bu3Sn(Gal) were carried out to deter-
mine the effect of the synthesized complexes on cancerous cell
lines, compared to control group of cells (non-treated). The details
of methodologies employed are the same as reported previously
[22]. DNA cleavage, acridine orange and comet assays against
MCF-7 by n-Bu3Sn(Gal) have also been carried out according to
the procedures as described below.
0.3 ꢁ 106 cells were mixed with 50
lL of 1% low melting agarose at
37 °C in PBS and sandwiched between the previously prepared
slide with 1% normal melting agarose. The prepared slides were
stored at 4 °C for 10 min before incubating them in lysis buffer con-
sisting 2.5 M of NaCl, 100 mM of EDTA (pH 10), 10 mM of Tris-base,
1% sodium lauryl sarcosinate and 1% triton X-100 for 2 h at 4 °C.
Electrophoresis was carried out for 5 min at 25 V in 300 mM of
NaOH and 1 mM of EDTA. After that the slides were neutralized
with tris base at pH 7.5. The nuclei stained with ethidium bromide
were visualized under fluorescent microscope.
2.4.1. DNA fragmentation assay
Natural DNA fragmentation generally occurs during pro-
grammed cell death and is a landmark for apoptosis. The DNA
cleavage pattern occurring due to cytotoxic effect of n-Bu3Sn(Gal)
(test compound) was analyzed through agarose gel electrophoresis
[53,54]. 3 ꢁ 106 cancer (MCF-7) cells were exposed to the IC50 va-
lue of the test compound. Cells were centrifuged and then washed
with phosphate buffer saline (PBS), and the pellet was lysed with
2.4.4. Statistical analysis
The results (for biological data) have been expressed as
mean standard error and unpaired two tailed t-test has been
performed to measure the significance in the results obtained.
Student’s t-test is a statistical method of testing hypotheses about
the mean of a small sample when the population standard devia-
tion is not known.
The unpaired two tailed t-test performed has been used to
analyse the significance level at p < 0.5 for anti-inflammatory activ-
ity and enzyme activity data. The statistically significant p value
has been calculated through GraphPad Prism 5 version.
400
7.5), 1 mM of EDTA and 0.2% triton X-100) for 15 min at room tem-
perature, and then centrifuged at 13000 rpm for 15 min. 350 L of
the supernatant was again lysed in 106 L of second lysis buffer
lL hypotonic buffer solution (containing 10 mM of tris (pH
l
l
(150 mM NaCl, 10 mM Tris–HCl (pH 8.0), 40 mM EDTA, 1% SDS
and 0.2 mg/mL of proteinase K, at final concentration) for 4 h at
37 °C. The DNA was extracted with phenol/chloroform/isoamyl
alcohol (25:25:1 v/v/v), and the pellet thus obtained was washed
with ethanol and resuspended for RNAase digestion in 15 lL of
Please cite this article in press as: M. Nath et al., Inorg. Chim. Acta (2014), http://dx.doi.org/10.1016/j.ica.2014.02.034

6
M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
found to be comparable with that of sodium salt of the gallic acid,
3. Results and discussion
which indicates that the carboxylate group is bidentate and
bridging [22,59,60]. The disappearance of a broad band (2833–
2957 cmꢀ1) in the spectra of the triorganotin(IV) hydroxycarboxy-
lates (which was present in the free acids as a weak intensity
band), suggests the deprotonation of the free (COOH) group upon
3.1. Synthetic aspects
The reaction of triorganotin chloride with sodium salt of
hydroxycarboxylic acid (formed according to Eq. (1)) in a 1:1 M
ratio led to the formation of complexes according to Eq. (2).
complexation. Furthermore, appearance of
556 10 cmꢀ1 supports the bonding of carboxylate oxygen with
the tin atom. The s(Sn–C) and as(Sn–C) have been observed at
their corresponding frequencies and are in accordance with the lit-
erature values [21–23]. The (O–H) of complexes broadens and
a
m(Sn–O) at
HL þ NaOEt ꢀ! NaL þ EtOH
HL ¼ HGlu; HMal and HGal
1:1
ð1Þ
m
m
m
shifts towards lower frequency, indicating the involvement of hy-
droxy (OH) group in intra-/intermolecular hydrogen bonding.
It has been observed from the IR spectra of R3Sn(Glu) that the
R3SnCl þ NaLꢀ!R3SnðLÞ þ NaCl
R ¼ Me; n-Bu and Ph
ð2Þ
mas(COC) and ms(COC) shift towards lower frequencies in compari-
son to the free acids. On this basis, it can be inferred that the ring
oxygen may also be participating in weak interaction with organo-
tin(IV) moiety. The carboxylate groups in organotin(IV) carboxyl-
ates generally adopt a bridge structure in the solid state, unless
the organic substituent at the tin atom is bulky or carboxylate
The reaction in Eq. (1) required 1–2 h and Eq. (2) required 5–6 h
of refluxing. The resulting products were obtained in good yield
(51–68%). From the analytical data it can be inferred that the
resulting triorganotin(IV) hydroxycarboxylates crystallized with
1:1 stoichiometry. R3Sn(Glu) are soluble in water/DMSO mixture,
methanol, ethanol, ether, DMSO, benzene and chloroform, whereas
R3Sn(Mal) and R3Sn(Gal) are soluble in DMSO and water/DMSO
mixture, show low solubility in ethanol, methanol, and insoluble
in non-polar solvents. The synthesized complexes are found
to be stable towards air and moisture. Diorganotin(IV) derivatives
of these hydroxycarboxylic acids have not been obtained
in pure form under the conditions used for triorganotin(IV)
hydroxycarboxylates.
group is branched at the
a-carbon [29]. Mandelic acid (
a-hydroxy-
carboxylic acid) is branched at
a-carbon, therefore, due to its
expected steric property; the bridging through carboxylate group
can be excluded [29]. But there is a possibility of hydroxy (OH)
group participation in bridging or interaction with tin, which has
been supported by the relative lowering of
m
(OH) of the complexes
(3265–3271 cmꢀ1
(3621 cmꢀ1) [52].
) in comparison with free mandelic acid
3.4. Multinuclear (1H, 13C and 119Sn) NMR spectral studies
3.4.1. 1H NMR spectra
3.2. Molar conductance measurements
The characteristic resonance peaks in the 1H NMR spectra of the
mandelic and gallic acid and their triorganotin(IV) derivatives have
Very low molar conductance values of 10ꢀ3 M solutions (in
methanol) of the synthesized triorganotin(IV) hydroxycarboxylates
are in the range 8–12 ohmꢀ1 cm2 molꢀ1, which suggest their
non-electrolytic nature.
been recorded in CD3OD. The 1H NMR spectra of b-
D-glucuronic
acid has been recorded in benzene-d6 (because of its poor solubility
in methanol) and the triorganotin(IV) derivatives of glucuronic
acid have been recorded in both CDCl3 and CD3OD. The absence
of 1H NMR signal corresponding to acidic hydrogen (CO(OH)) in
the spectra of triorganotin(IV) hydroxycarboxylates, suggests the
coordination of triorganotin(IV) moiety with the carboxylate
group.
3.3. Infrared and far-infrared spectral studies
The structures of hydroxycarboxylic acids (HGlu, HMal and
HGal) raise several questions such as whether there is a coordina-
tion to tin through the carboxylic oxygen or the hydroxy oxygen,
what is the coordination number of tin atom due to these interac-
tions, and the detailed geometry around the tin atom where the
possibility of isomerism exists [29,30]. Furthermore, due to poly-
dentate nature of these acids, it is essential to differentiate the
monomeric structures (formed through chelation/intra-molecular
bonding) and polymeric structures (formed through bridging/
intermolecular bonding), where the later may further adopt one/
two dimensional structure or cyclic oligomeric structure [29,30].
In the 1H NMR spectra of R3Sn(Glu) and R3Sn(Gal), the protons
of hydroxyl groups (OH) have been observed as a weak, broad sig-
nal which either remained at nearly same resonance or deshielded,
indicating intra-/intermolecular hydrogen bonding. In 1H NMR
spectra of R3Sn(Mal) a significant change in OH resonance has been
observed in comparison to that of free mandelic acid, which sug-
gests that the bonding of tin with oxygen atom of OH group may
also be possible along with carboxylate group. The resonances
due to all the magnetically non-equivalent protons in the studied
complexes have been successfully identified, and the total
numbers of protons calculated from the integration curve have
been found to be in good agreement with those calculated from
the proposed molecular formula.
The stretching frequencies of carboxylic group (m(OCO)) has
been utilized to confirm the mode and nature of coordination
through carboxylate oxygen (monodentate, bidentate or bridging)
in the studied triorganotin(IV) hydroxycarboxylates. The IR
absorption spectra of the studied complexes indicate that the mas
(OCO) shift towards higher frequencies, whereas the corresponding
Further, the 2J(1H119Sn) coupling constants obtained from the
resolved satellites for Me3Sn(Mal) and n-Bu3Sn(Gal) are 64.10 Hz
and 56.17 Hz, respectively. These values suggest penta-coordi-
nated geometry for the complexes. The \CSnC calculated using
Lockhart and Manders equation [61] are 115.39° and 110.05° for
Me3Sn(Mal) and n-Bu3Sn(Gal), respectively. These values further
suggest that the complexes exhibit trigonal–bipyramidal geome-
try, with more electronegative (oxygen) groups in axial position
and organic groups in equatorial plane. The 2J(1H119Sn) values for
Me3Sn(Glu) have been observed at 43.82 Hz in CDCl3, while at
66.12 Hz in CD3OD. These values indicate the four-coordinated
m
s(OCO) absorption frequencies either remain at the same value or
move to lower frequencies in comparison to free acids. The magni-
tude of (mas s)OCO ( ) has been found to be useful in the identi-
–m
Dm
fication of structural features [22,23,59,60]. For R3Sn(Glu) and
R3Sn(Mal),
D
m
(ꢂ200 cmꢀ1) has been observed to be greater than
that of the sodium salt of the corresponding acid, which indicates
that the carboxylate group is monodentate [21,59,60] but bridging
or chelation through other groups of the ligands cannot be ex-
cluded. The magnitude of
Dm
for R3Sn(Gal) (>200 cmꢀ1) has been
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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
7
geometry for Me3Sn(Glu) in non-coordinating solvent and
five-coordinated geometry in coordinating solvent. The \CSnC
calculated for Me3Sn(Glu) in CDCl3 (106.47°) and CD3OD
(116.51°) further support pseudo-tetrahedral geometry in CDCl3
and distorted trigonal–bipyramidal geometry in CD3OD.
midal geometry about tin. The proposed structures for the studied
triorganotin(IV) hydroxycarboxylates have been presented in
Fig. 2.
3.5. ESI-MS spectrometric studies
3.4.2. 13C NMR spectra
The ESI-MS spectral data for triorganotin(IV) hydroxycarboxy-
lates have been recorded in the range m/z 50–1000, using metha-
nol as solvent and further diluting with acetonitrile and water
mixture. In the ESI-MS spectra of triorganotin(IV) hydroxycarboxy-
lates, the weak molecular ion peak ([M+H]+) appears, which has
been used for determining the molecular weight (Mw) of the com-
plexes. Mw for Ph3Sn(Glu), n-Bu3Sn(Glu) and Me3Sn(Glu) have
been observed at 543, 483 and 357, respectively; for Ph3Sn(Mal),
n-Bu3Sn(Mal) and Me3Sn(Mal) at 501, 441 and 315, respectively;
and for Ph3Sn(Gal), n-Bu3Sn(Gal) and Me3Sn(Gal) at 519, 459,
and 333, respectively. The observed molecular weights (Mw) of tri-
organotin(IV) hydroxycarboxylates are in excellent agreement
with the calculated values, which verify the proposed molecular
formula for the complexes. In the ESI-MS spectra of triorgano-
tin(IV) hydroxycarboxylates, [SnR3]+ ion peaks have been observed,
which are due to loss of one mole of ligand from one mole of com-
plex, involving the cleavage of weak Sn–O bond yielding two com-
plementary ions, where the cationic part of the complex is
measured in positive mode [63]. Triorganotin(IV) hydroxycarboxy-
lates often adopt polymeric structure with five-coordinated tin and
the peak corresponding to [M+SnR3]+ provides additional proof for
penta-coordinated linear polymeric motif [64]. The [M+SnR3]+
peaks have also been observed for R3Sn(Mal) and R3Sn(Gal), which
suggest penta-coordinated polymeric nature of the complexes. The
subsequent fragment ions [HSnR2]+ and [H2SnR]+ resulted from
[SnR3]+ have been reported earlier in the literature for other
triorganotin(IV) carboxylates [65], similar peaks have also been ob-
served in ESI-MS spectra of the synthesised triorganotin(IV)
hydroxycarboxylates. The ESI-MS spectrometric data do not pro-
vide any crucial information about the structure of the complexes,
except that R3Sn(Mal) and R3Sn(Gal) may be polymeric in nature,
however the data helped in confirming the Mw and empirical for-
mula of the complexes.
From 13C NMR spectral data of hydroxycarboxylic acids and
their triorganotin(IV) derivatives it has been inferred that the
signals attributable to –(OCO) show significant downfield shift
upon complex formation, which suggests coordination of triorga-
notin(IV) moiety with carboxylate group.
R3Sn(Glu) shows an interesting coordination behavior. The
carbon atom C4 exhibits downfield shift, while C2 remaines unaf-
fected, and C1 and C3 show only slight change in the chemical
shifts. A small downfield shift for the C5 has also been observed,
indicating that weak interaction of ring oxygen with triorgano-
tin(IV) moiety may be possible [49,50]. However, the shifts are
very small and hence the interactions between ring oxygen and
tin atom can be excluded.
In R3Sn(Mal), the C2 shows a significant downfield shift, which
supports the weak interaction between oxygen of hydroxyl group
(OH group) and tin atom. All the other carbon atoms of the acid
either remained unaffected or very small change in chemical shifts
have been observed for them. A significant change in the chemical
shift of C4 and C6 in 13C NMR spectra of R3Sn(Gal) has been ob-
served, which suggests hydrogen bonding through OH groups at
meta positions of the acid.
The 13C NMR chemical shifts of methyl, n-butyl and phenyl
carbons attached to tin are observed at comparable positions as re-
ported previously. The heteronuclear coupling constant
1J(13C–119Sn) provides useful information about the coordinating
environment and geometry of organotin(IV) complexes. The satel-
lites have been resolved for most of the complexes and the
observed coupling constants 1J(13C–119Sn) are found to be in the
range 465.60 20 Hz. These values suggest penta-coordinated
organotin moiety for R3Sn(Mal) and R3Sn(Gal). Further, the calcu-
lated values of \CSnC (116.01–119.21°) with the help of Lockhart
and Manders equation [62] {Eq. 1J(13C–119Sn) = 11.4h ꢀ 875}, sug-
gest distorted trigonal–bipyramidal geometries for the complexes.
The 1J(13C–119Sn) values for R3Sn(Glu) have been observed in
the range 329.07–350.33 Hz in CDCl3, while 454.43–466.21 Hz in
CD3OD, indicating four-coordinated geometry in non-coordinating
solvent and five-coordinated geometry in coordinating solvent for
the complexes. The \CSnC calculated for R3Sn(Glu) in CDCl3
(107.46–105.61°) and CD3OD (117.68–115.97°) further support
the pseudo-tetrahedral geometry in CDCl3 and distorted trigonal–
bipyramidal geometry in CD3OD.
3.6. Biological studies
All the three hydroxycarboxylic acids viz. glucuronic acid,
mandelic acid and gallic acid are biologically important molecules
and individually known for their therapeutic applications. Thus, it
becomes indispensable to evaluate the biological behavior of syn-
thesized triorganotin(IV) hydroxycarboxylates especially for their
anti-cancer and anti-inflammatory activities.
3.4.3. 119Sn NMR spectra
3.6.1. Cytotoxicity assay (MTT assay)
The 119Sn NMR shifts are very sensitive to coordination number
of tin and greatly shift upfield on bonding to Lewis base. The 119Sn
chemical shifts increase markedly with the increase in coordina-
tion number, ca. d ꢀ50 to +200 ppm for four-coordinated tin; d
ꢀ100 to ꢀ200 ppm for five-coordinated tin; d ꢀ200 to ꢀ330 ppm
for six-coordinated tin compounds [22].
To study the cytotoxicity effect of synthesized complexes, all of
the triorganotin(IV) hydroxycarboxylates along with standard
drugs, viz. cis-platin and 5-fluorouracil have been screened
in vitro against five cell lines of human origin, viz. MCF-7 (mam-
mary cancer), HEK-293 (kidney cancer), PC-3 (prostate cancer),
HCT-15 (colon cancer) and HepG-2 (liver cancer) and their
observed IC50 values are presented in Table 1. n-Bu3Sn(Gal) has
been observed to be the most active among the studied triorgano-
tin(IV) hydroxycarboxylates against all cancer cell lines, and it
exhibits even higher activity in comparison to cis-platin, whereas
it shows lower potential in comparison to 5-fluorouracil against
all cell lines except HCT-15. Ph3Sn(Glu) exhibits lowest activity
amongst the studied complexes and lower potential in comparison
to cis-platin as well as 5-fluorouracil.
The 119Sn chemical shifts for R3Sn(Glu) have been observed in
the region ꢀ40.91 to ꢀ49.21 ppm in CDCl3, suggesting four-coordi-
nated tin. However, these complexes exhibit five-coordinated tin
in CD3OD (ꢀ141.44 to ꢀ183.01 ppm), suggesting that these
complexes exhibit additional coordination through inter- or
intramolecular association/interaction or from complexation with
coordinating solvent [50].
The 119Sn NMR shifts for R3Sn(Mal) and R3Sn(Gal) are observed
in the region ꢀ202.77 to ꢀ132.19 ppm. These chemical shifts
clearly suggest that they exhibit five-coordinated trigonal–bipyra-
It has been reported [66] that the compounds exhibiting IC50 in
the range 1.0–10.0 lg/mL are cytotoxic and 10.0–30.0 lg/mL are
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8
M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Fig. 2. Probable structures of triorganotin(IV) hydroxycarboxylates: (a) triorganotin(IV) glucuronates in coordinating solvent (R3Sn(Glu)ꢃsolvent), (b) triorganotin(IV)
mandelates (R3Sn(Mal)) and (c) triorganotin(IV) gallates (R3Sn(Gal)); where R = Ph, n-Bu and Me.
Table 1
In vitro cytotoxicity data (IC50 expressed as lg/mL SEM) of triorganotin(IV) hydroxycarboxylates.
Complex
MCF-7
HEK-293
PC-3
HCT-15
HepG-2
Ph3Sn(Glu)
n-Bu3Sn(Glu)
Me3Sn(Glu)
Ph3Sn(Mal)
n-Bu3Sn(Mal)
Me3Sn(Mal)
Ph3Sn(Gal)
n-Bu3Sn(Gal)
Me3Sn(Gal)
Cis-platin
29.95 0.6
22.77 2.2
23.26 2.5
13.36 1.1
10.18 1.3
12.62 0.9
9.03 0.9
2.04 0.1
6.81 0.9
8.97 0.10
<0.65
29.93 0.8
21.22 1.7
25.55 1.5
16.10 0.8
10.15 1.4
11.36 0.8
5.99 0.7
5.16 0.4
6.28 0.5
6.72 0.13
1.61 0.05
26.66 1.1
20.78 0.8
21.50 3.1
17.60 1.1
11.38 0.8
16.0 0.1
6.45 0.7
5.30 0.2
5.85 0.8
5.99 0.05
1.41 0.05
27.96 0.6
23.99 0.6
25.21 1.1
17.02 1.1
12.70 1.1
15.76 0.4
12.99 0.3
3.16 0.5
28.46 0.8
20.74 0.4
25.53 1.4
23.83 0.3
20.99 0.3
21.19 1.2
18.24 1.7
3.98 0.2
15.84 0.3
5.97 0.10
<0.65
11.70 1.1
3.71 0.05
7.17 0.13
5-Fluorouracil
moderately cytotoxic in nature. Triorganotin(IV) hydroxycarboxy-
lates exhibit activity in the range 4–30 g/mL. Therefore, it can
xybenzo-18-crown-6 and -15-crown-5 are less active than the
corresponding triphenyltin derivatives but they are more active
than di-n-butyltin analogues [12b]. Triphenyltin derivatives of
substituted-phenylacetic acid/-furoic acid/1,4-benzodioxane-6-
carboxylic acid are more active than diphenyltin analogues [39].
However, in the present study tri-n-butyltin carboxylates are more
active than the corresponding trimethyltin derivatives which are in
turn more active than the corresponding triphenyltin derivatives.
The highest activity of the tri-n-butyltin carboxylates may also
be related to the most lipophilic character of the n-butyl groups
as the lipophilicity of the more stable C-bound groups on tin is
important in controlling their toxicity [27]. Further, the studied tri-
organotin derivatives of gallic acid are more active than those of
mandelic acid which are in turn more active than those of glucu-
ronic acid.
l
be concluded that the complexes are cytotoxic to moderately cyto-
toxic in nature. R3Sn(Gal) (gallic acid derivatives) are cytotoxic
against MCF-7, HEK-293 and PC-3 and moderately cytotoxic
against HCT-15 and HepG-2. R3Sn(Mal) and R3Sn(Glu) (mandelic
and glucuronic acid derivatives) exhibit moderate activity against
all cell lines. It has been observed that the activity increases as
the alkyl carbon chain length increases, and further, the alkyl
derivatives are more active than phenyl derivatives, similar results
have also been reported earlier [27].
The first systematic structure–activity relationships on several
di- and tri-organotin(IV) carboxylates made by Gielen and his
co-workers [12,27] reveals that di-n-butyltin(IV) carboxylates are
the most active compounds, and some of them exhibit much high-
er potency [11,12,27,37]. Further, tri-n-butyltin difluorobenzoates
are less active than the corresponding triphenyltin derivatives
and also less active than the corresponding di-n-butyltin
derivatives [12a]. Similarly, tri-n-butyltin 3,6-dioxaheptanoates/
3,6,9-trioxadecanoates and tri-n-butyltin derivatives of 4-carbo-
The structure–activity correlation for the studied triorgano-
tin(IV) hydroxy-carboxylates reveals that the substituent attached
to OAC@O group has pronounced effect on the activity. Moreover,
the activity of organotin compounds also depends on availability of
coordination positions around the tin atom, stability of the Sn–O
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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
9
bond towards hydrolytic cleavage (as well as Sn–C stability, phenyl
derivatives are prone to hydrolytic cleavage than alkyl ones), and
lipophilic and hydrophilic properties of the complexes [24,27,29].
Further, the conformation assumed by the ligand in the cytoplasm
may play an important role in activity of the complexes. Hence, the
transportation of R3Sn+ moiety or triorganotin(IV) hydroxycarb-
oxylate as a single entity across the cellular membrane may be
responsible for their activity.
3.6.2. Enzymatic assays
The observed data for enzymatic assays of n-Bu3Sn(Gal) against
MCF-7 are presented in Table 2. The graphical representation of the
data is expressed in Fig. 3–5. Necrosis and apoptosis are two ways
through which cell death can occur. Anti-tumor compounds gener-
ally arrest the cell growth through apoptosis. Apoptosis is a mode
of cell death that differs from necrosis. Apoptosis is PCD
(programmed cell death) while necrosis is PMCD (premature cell
death).
Fig. 4. Total glutathione content in MCF-7 after the treatment with n-Bu3Sn(Gal)
(mean SEM).
Non-specific necrosis leads to cell death with in short interval of
time, mediated through membrane toxicity, which results in
sudden and marginal increase in lactate dehydrogenase (LDH)
Table 2
Enzymatic assays of n-Bu3Sn(Gal) against MCF-7.
Enzymes
Activity
Untreated
Treated
Induction fold
Lipid peroxidationa
4.58 0.65
8.31 0.03
6.31 0.57
6.31 0.027
7.82 0.03
9.89 0.11
9.81 0.06
6.52 1.25
4.95 0.12
8.05 0.18
2.16
1.18
1.03
0.78
1.03
Glutathione peroxidaseb
Total Glutathionec
Glutathione reductaseb
Lactate dehydrogenaseb
a
b
c
nmol/mg protein.
lmol/mg protein/min.
ng/mg protein; Induction fold: (mean of treated/mean of untreated); the
experiment was performed in triplicate and mean SEM of three independent
experiments are shown here.
Fig. 5. Lipid peroxidation in MCF-7 after the treatment with n-Bu3Sn(Gal)
(mean SEM). ⁄Represents the significant increase in lipid peroxidation with
respect to the control (p < 0.05), for MCF-7.
concentration. The result shows a very small increment in LDH
concentration, therefore, a minor role of necrosis in causing cell
death cannot be excluded. Substantial folds increase in enzymatic
activity suggests the generation of reactive oxygen species (ROS) in
the cells, leading to oxidative stress, which may be responsible for
cytotoxicty.
Lipid peroxidation is a common assay to measure oxidative
damage. n-Bu3Sn(Gal) treatment to MCF-7 cells results in high lipid
peroxidation which initiates the oxidative reactions, ensuring the
generation of reactive oxygen species (ROS) in the cells. The exces-
sive ROS generation may be responsible for cytotoxicity of the
complex. Various drugs are known in literature [67,68], which
disturb the intracellular mitochondrial membrane potential via
generation of reactive oxygen species (ROS).
Redox system of cells is regulated by certain enzymes such as
glutathione peroxidase and glutathione reductase [69]. Any change
in enzyme concentration of these enzymes may affect the
oxidative balance of cells and consequently subjecting the cells
to oxidative stress [70] and it is reported that the oxidative stress
plays an important role in apoptosis [71]. Induction of apoptosis
due to increase in glutathione peroxidase activity has also been
reported in literature [72]. Glutathione peroxidase (GPx), glutathi-
one reductase (GR) activity and total glutathione content show
alteration in their activity for MCF-7 cells treated with n-Bu3-
Fig. 3. Enzymatic activity: lactate dehydrogenase (LDH); glutathione peroxidase
(GPx); glutathione reductase (GR) in MCF-7 after the treatment with n-Bu3Sn(Gal).
⁄⁄
##
The results are expressed as mean SEM.
and
represent the significant
difference in glutathione reductase and glutathione peroxidase activity with respect
to control (p < 0.05), respectively.
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10
M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Sn(Gal) with respect to control. Thus, n-Bu3Sn(Gal) may lead cell
death through induction of apoptosis.
Further, there are various mechanisms through which cell death
may be induced. One of them is cell membrane damage, resulting
in necrosis. Oxidative stress is the other possible way which may
affects cell survival and hence inhibiting cell growth. Alternatively,
n-Bu3Sn(Gal) might has induced peroxides generation, which are
metabolized by glutathione peroxidase (GPx), consequently form-
ing oxidized glutathione (GSSG) from reduced form of glutathione
(GSH). Decrease in GSH concentration and NADPH oxidation are
related to alteration of Ca2+ homeostasis, which is a crucial event
in loss of cell viability induced by peroxides [73]. Organotins also
influence [Ca2+] and mitochondrial activity, resulting in oxidative
stress in tumour cells leading to apoptosis [74]. Alteration in mito-
chondrial membrane potential may also be responsible for causing
apoptosis [75].
From the observed results of the enzyme assays it may be in-
ferred that both necrosis and apoptosis (apnosis) are responsible
for cell toxicity. Many anti-cancer drugs like cladribine, 5-fluoro-
uracil, doxorubicin, phyllanthus and cis-platin, have been reported
which lead to cell death through both apoptosis and necrosis [76].
3.6.3. DNA fragmentation assay
Natural DNA fragmentation generally occurs during pro-
grammed cell death and is a landmark for apoptosis [22]. DNA
fragmentation assay is among the most reliable method for distin-
guishing apoptosis from necrosis. DNA ladder/marker of 100 base
pair (lane 1), DNA extracted from untreated MCF-7 cells (lane 2),
DNA extracted from MCF-7 cells treated with n-Bu3Sn(Gal) (lane
3) and DNA extracted from MCF-7 cells treated with cis-platin as
reference drug (lane 4) were taken for DNA fragmentation assay
(as in Fig. 6). Gel electrophoresis experiment was conducted and
recorded on Biorad-UV-transilluminator after the addition of
ethidium bromide. The image was taken with the help of Gel
Doc-software. DNA–ladder formation (smearing) has been
Fig. 6. DNA ladder formation through gel electrophoresis in MCF-7 cells; lane 1:
DNA marker; lane 2: control; lane 3: n-Bu3Sn(Gal); lane 4: cis-platin.
Fig. 7. Acridine orange assay for (a) MCF-7 cells (control), (b) cis-platin treated MCF-7 cells, (c) n-Bu3Sn(Gal) treated MCF-7 cells. Unfilled arrows and filled arrows indicate
live cells and apoptotic cells, respectively. The experiment was performed in triplicate and a representative experiment is presented.
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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
11
observed for n-Bu3Sn(Gal) treated MCF-7 cells (Fig. 6), which sug-
gests that apoptotic pathway is mainly responsible for the cell
death.
under fluorescent microscope. The Fig. 8 represents the single cell
gel (alkaline) electrophoresis showing DNA fragmentation in MCF-
7 cells treated with n-Bu3Sn(Gal) in comparison to cis-platin and
control. The DNA breaks or fragmentation from individual cell
has been observed as comet tails (marked by arrows). However,
3.6.4. Acridine orange assay
Morphological changes have been observed by acridine orange
fluorescent staining assay [55]. The Fig. 7 represents the induction
of apoptosis followed by membrane blebbing and chromatin
condensation by n-Bu3Sn(Gal) on MCF-7 cells. The cells were
incubated for 24 h with IC50 value of test compound and cis-platin.
Nuclear staining was visualized under fluorescent microscope
(Zeiss, Axiovert 25, Germany). n-Bu3Sn(Gal) induced cell death
has been considered to be apoptotic by simply observing the typi-
cal morphological change in the cells due to AO/EB staining. Live
cells and apoptotic cells can be easily distinguished by the percent-
age uptake of AO/EB but necrosis cannot be excluded completely.
In view of the fact that AO permeates all live cells, and hence ap-
peared green. EB is taken up only when the cells have lost their
cytoplasmic membrane integrity and hence appear red. It has been
observed (Fig. 7) that live cells have green nucleus (shown by un-
filled arrows), while apoptotic cells have orange nucleus (shown by
filled arrows).
3.6.5. Comet assay
Cytotoxicity leading to DNA damage has also been validated by
single cell gel electrophoresis. Comet assay or single cell gel elec-
trophoresis is simple yet sensitive technique which detects DNA
damage to the level of single cell [57]. The nuclei of MCF-7 cells
(treated as well as control) were stained with EB and visualized
Fig. 9. Percentage of DNA in comet tail in n-Bu3Sn(Gal) treated MCF-7 cells (A) in
comparison to cis-platin (B) and control (C).
Fig. 8. Comet assay for n-Bu3Sn(Gal) treated MCF-7 cells in comparison to cis-platin and control. The DNA breaks from individual cells were visualized as comet tails (marked
by arrows). The experiment was performed in triplicate and a representative experiment is presented.
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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Table 3
Anti-inflammatory activity and toxicity data of triorganotin(IV) hydroxycarboxylates.
Complex/standard drug
Anti-inflammatory activity dose (50 mg/kg)
Toxicity LD50
(mg/kg)
Paw vol. before (A)
(mean SEM)
Paw vol. after (B)
(mean SEM)
Paw vol. difference
(mean SEM)
p Valueb
% Inhibition
Ph3Sn(Glu)
n-BuSn(Glu)
Me3Sn(Glu)
Ph3Sn(Mal)
n-Bu3Sn(Mal)
Me3Sn(Mal)
Ph3Sn(Gal)
n-Bu3Sn(Gal)
Me3Sn(Gal)
Ibuprofen
8.52 0.28
8.30 0.24
8.06 0.10
8.48 0.25
8.56 0.24
8.26 0.29
8.24 0.16
8.40 0.21
8.20 0.26
9.30 0.23
9.00 0.35
9.76 0.11
9.46 0.16
9.70 0.23
9.92 0.20
10.06 0.16
9.86 0.17
9.98 0.34
10.30 0.38
10.14 0.28
9.58 0.14
10.06 0.61
10.04 0.32
10.64 0.26
9.82 0.38
11.68 0.19
11.46 0.31
11.60 0.18
1.40 0.11
1.76 0.15
1.80 0.17
1.50 0.16
1.74 0.18
1.88 0.16
1.34 0.09
1.66 0.18
1.84 0.09
1.34 0.13
0.82 0.09
1.92 0.08
2.00 0.07
1.90 0.05
0.0024
0.0005
0.0007
0.0038
0.0013
0.0003
0.0015
<0.0001
0.0004
–
28.57
10.20
8.16
23.47
11.22
4.08
31.6 3
15.31
6.12
36.19
60.95
8.57
>200
>400
>200
>600
>600
>600
>600
>800
>600
>2000
>1000
>400
>400
>400
Indomethacina
Ph3SnCl
–
–
–
–
n-Bu3SnCl
Me3SnCl
4.76
9.52
a
Dose (5 mg/kg); paw volume before (A) and after (B) carrageenan injection.
calculated through unpaired t-test at significant level p < 0.05 with respect to Indomethacin.
b
vehicle treated cells (control) do not show any fragmentation.
Induction of DNA fragmentation (as shown in Fig. 8) has been
further validated by the comet assay.
Fig. 9 represents the % DNA in comet tail (58%) in MCF-7 cells
treated with n-Bu3Sn(Gal). The results suggest that n-Bu3Sn(Gal)
treated MCF-7 cells show a high level of DNA tailing in comparison
to cis-platin (51%) treated cells and control (1.32%) cells. The
results further support that apoptosis is the main cause for cell
death.
4. Conclusions
Triorganotin(IV) hydroxycarboxylates have been synthesized
through the sodium salt method. IR, 1H, 13C and 119Sn NMR spectral
and ESI-MS studies of all the synthesized triorganotin(IV) hydroxy-
carboxylates suggest a distorted trigonal–bipyramidal geometry.
Anti-cancer screening of these triorganotin(IV) hydroxylcarboxy-
lates suggests that n-Bu3Sn(Gal) is the most active complex among
the studied complexes in this manuscript, which exhibits potent
anticancer activity against all the cell lines. Significant increase in
glutathione peroxidase activity and lipid peroxidation assay sup-
port that cells undergo oxidative stress. Marginal increase in LDH
suggests that necrosis may also occur to a small extent. Further-
more, DNA ladder formation, AO and comet assays indicate that
these complexes inhibit the cancer cell growth through apoptosis.
Taken together, the results indicate that the complexes possess
dual capability of cell death: necrosis (minor) and apoptosis
(major). However, further experiments are needed to establish
the fact more strongly. An anti-cancer agent is expected to possess
an anti-inflammatory activity. It has been observed that title com-
plexes show good anti-inflammatory activity. Among the studied
complexes, Ph3Sn(Gal) exhibits the highest anti-inflammatory
activity followed by Ph3Sn(Glu). In vitro cytotoxicity assays,
in vivo anti-inflammatory activity and toxicity results of the stud-
ied complexes predict their potency as metal based anti-cancer
drugs.
%
DNA = TA ꢁ TAI ꢁ 100/[TA ꢁ TAI) + (HA ꢁ HAI)];
where
TAI = tail area intensity, HAI = head area intensity, TA = tail area
and HA = head area [77].
3.6.6. Anti-inflammatory activity
Cancer is related to inflammation to the some extent and there-
fore, it was intriguing to see if the studied complexes also exhibit
anti-inflammatory activity. The anti-inflammatory activity data
(% inhibition) of synthesized triorganotin(IV) hydroxycarboxylates
are presented in Table 3. The results obtained indicate the order of
the anti-inflammatory activity of the studied triorganotin(IV)
hydroxycarboxylates as: Ph3Sn(Gal) > Ph3Sn(Glu) > Ph3Sn(Mal) >
n-Bu3Sn(Gal) > n-Bu3Sn(Mal) > n-Bu3Sn(Glu) > Me3Sn(Glu) > Me3
Sn(Gal) > Me3Sn(Mal).
Ph3Sn(Gal) and Ph3Sn(Glu) have been found to exhibit potential
anti-inflammatory activity (31.63% and 28.57% inhibition, respec-
tively). Ph3Sn(IV)+ moiety, is membrane-active and highly lipo-
philic, can interact with lipid fraction and cytoplasmic
membrane. The swift and frequent formation of Ph3Sn(IV)+ and
its transportation across the cellular membrane as a part of the
mechanism may be responsible for the higher efficacy [21–23].
Acknowledgements
We are thankful to technical staff of the Chemistry Department,
I.I.T. Roorkee, for spectroscopic studies and elemental analysis.
Thanks are also due to The Head I.I.C. for providing NMR facility.
Ms. Monika Vats is thankful to MHRD, New Delhi, for financial
Assistance.
3.6.7. Toxicity
The LD50 values of the studied triorganotin(IV) hydroxycarboxy-
lates are found to be in the range >200 to >800 mg/kg in albino
mice (Table 3), which suggest that these complexes have moderate
toxicity with minimum adverse effects and wider safety margin.
This property is desirable from pharmaco-economic point of view.
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