Tri- and diorganotin(IV) complexes of biologically important orotic acid: Synthesis, spectroscopic studies, in vitro anti-cancer, DNA fragmentation, enzyme assays and in vivo anti-inflammatory activities

Article abstract of DOI:10.1016/j.ejmech.2012.11.023

Tri-and diorganotin(IV) orotates of general formula, R_nSn(H ₂Or)_m [n = 3/2, m = 1/2, R = Me, n-Bu, n-Oct and Ph; H₂Or^- = monoanion of orotic acid (H₃Or)] (n-Bu₂Sn(HOr) as an exception) have been synthesized. On the basis of various spectroscopic studies it is revealed that R₃Sn(H ₂Or) and R₂Sn(H₂Or)₂ exhibit distorted trigonal-bipyramidal and distorted octahedral geometry, respectively, and n-Bu₂Sn(HOr) shows both five and six coordination geometry around tin. In vitro anti-cancer screening against MCF-7 (mammary), HEK-293 (kidney), PC-3 (prostate), HCT-15 (colon) and HepG-2 (liver) cancer cell lines suggest that the n-Oct₂Sn(H₂Or)₂ is the most active complex among all of the studied complexes. DNA fragmentation and antioxidant enzyme assays suggest that cytotoxic effect of the complexes is selectively mediated through the induction of apoptosis. They also exhibit low toxicity and good anti-inflammatory activity (in vivo).

Full text of DOI:10.1016/j.ejmech.2012.11.023

Accepted Manuscript
Tri- and diorganotin(IV) complexes of biologically important orotic acid: synthesis,
spectroscopic studies, in vitro anti-cancer, DNA fragmentation, enzyme assays and in
vivo anti-inflammatory activities
Mala Nath, Monika Vats, Partha Roy
PII:
S0223-5234(12)00692-7
10.1016/j.ejmech.2012.11.023
EJMECH 5849
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2012
Revised Date: 17 September 2012
Accepted Date: 15 November 2012
Please cite this article as: M. Nath, M. Vats, P. Roy, Tri- and diorganotin(IV) complexes of biologically
important orotic acid: synthesis, spectroscopic studies, in vitro anti-cancer, DNA fragmentation, enzyme
assays and in vivo anti-inflammatory activities, European Journal of Medicinal Chemistry (2012), doi:
10.1016/j.ejmech.2012.11.023.
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ACCEPTED MANUSCRIPT
Tri- and diorganotin(IV) complexes of biologically important orotic acid:
synthesis, spectroscopic studies, in vitro anti-cancer, DNA fragmentation,
enzyme assays and in vivo anti-inflammatory activities
a*
a
b
Mala Nath , Monika Vats and 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
*
Author for correspondence e-mail; malanfcy@iitr.ernet.in
Graphical abstract
Organotin(IV) orotates have been synthesized and characterized. Di-n-octyltin(IV) orotate is
the most active among the studied complexes and its cytotoxicity is selectively mediated
through apoptosis. Complexes exhibit good anti-inflammatory activity .

ACCEPTED MANUSCRIPT
Tri- and diorganotin(IV) complexes of biologically important orotic acid:
synthesis, spectroscopic studies, in vitro anti-cancer, DNA fragmentation,
enzyme assays and in vivo anti-inflammatory activities
a*
a
b
Mala Nath , Monika Vats and 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
Author for correspondence e-mail; malanfcy@iitr.ernet.in
*
Abstract:
n 2 m
Tri- and diorganotin(IV) orotates of general formula, R Sn(H Or) [n = 3/2, m = 1/2, R =
−
Me, n-Bu, n-Oct and Ph; H Or = monoanion of orotic acid (H Or)] (n-Bu Sn(HOr) as an
2
3
2
exception) have been synthesized. On the basis of various spectroscopic studies it is revealed
that R Sn(H Or) and R Sn(H Or) exhibit distorted trigonal-bipyramidal and distorted
octahedral geometry, respectively, and n-Bu Sn(HOr) shows both five and six coordination
geometry around tin. In vitro anti-cancer screening against MCF-7 (mammary), HEK-293
kidney), PC-3 (prostate), HCT-15 (colon) and HepG-2 (liver) cancer cell lines suggest that
the n-Oct Sn(H Or) is the most active complex among all of the studied complexes. DNA
3
2
2
2
2
2
(
2
2
2
fragmentation and anti-oxidant enzyme assays suggest that cytotoxic effect of the complexes
is selectively mediated through the induction of apoptosis. They also exhibit low toxicity and
good anti-inflammatory activity (in vivo).
Key Words: Organotin Carboxylates, Anti-cancer, Enzyme Assay, DNA Ladder, Anti-
inflammatory
1
. Introduction
Orotic acid (6-uracilic acid or 1,2,3,6-tetrahydro-2,6-di-oxo-4-pyrimidine carboxylic
acid, abbreviated as H Or; Fig. 1) is very much important in the `de nuovo' biosynthesis of
3
pyrimidine bases of nucleic acids in living organisms [1]. The synthesis of uridylate (UMP,
1

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uridine monophosphate, the basic pyrimidine nucleotides) is a two step process: OMP
(orotidine 5′-monophosphate) synthesis via phosphorylation of orotic acid, for which divalent
magnesium cations are necessary [2] followed by decarboxylation [3]. Besides its importance
1−
2−
in biology, the mono- and dianion of orotic acid (H Or and HOr ) are potential
2
multidentate ligands, since co-ordination may occur through two nitrogen atoms of the
pyrimidine ring, the carbonyl oxygens or through carboxyl group. It is not only a multidentate
ligand [4], but also behaves differently at different pH. The orotic acid has three pKa values
1
3
in aqueous solution, viz. 2.8, 9.28 and 13 assigned to carboxlic group, (N H) and N H,
1−
respectively [5]. At pH 5–9, orotic acid is present as the H Or anion (monoprotic) and at pH
2
2
−
>
9.3 as HOr anion (diprotic). Only at strong alkaline pH or with very strong Lewis acids,
3
deprotonation of N H (pKa > 13) may occur, where orotic acid behaves as a triprotic ligand,
3
−
Or anion [5, 6].
A detailed study of the coordination chemistry of orotic acid, H
3
Or has shown a wide
range of its application from bioinorganic to pharmaceutical and materials chemistry. Due to
the interesting behaviour of orotic acid and its complexes, it has gained much attention in the
area of medicine and drugs. Some metal complexes of orotic acid display therapeutic
properties and are widely used in medicine [7, 8] as well as for other purposes [9]. Metal
orotates, such as platinum, palladium, and zinc orotates have been widely used in medicine
and screened as potent anti-cancer agents [10−12]. It has been proposed that orotates might
also be used as a carrier in living organisms to cure deficiency syndromes of some metal ions,
such as calcium, magnesium, iron and zinc [12−15]. Different metal-binding modes of
orotate ligand have been reported, such as the monoanionic form of orotic acid behaving as a
monodentate ligand in tris(aqua)bis(orotato)dioxouranium(VI) {U(VI) binds to the ligand via
OCO} [16], bidentate bridging ligand in copper(I) bis(triphenylphosphine) orotate {Cu(I)
2
binds to the ligand via OCO and O } [17], and µ ligand in aqua(orotato)lithium(I) {Li(I)
3
2

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2
4
binds to ligand via OCO, O and O } [15]. The complexes of uranyl(II), vanadyl(II) and
zirconyl(II) moieties with orotic acid have been reported in which orotic acid is in its
monoanion form bonded to oxometal ions through the carboxylic group [18]. Several studies
revealed that at neutral or slightly acidic pH, in the complexes of Cu(II), Mn(II), VO(II),
Zn(II), Hg(II), Cd(II), Fe(III), Cr(III) and Ag(I), orotic acid coordinates through the
carboxylate group [19] while crystal structures of Ni(II) and Cu(II) complexes confirm that
1
3
orotic acid coordinates through carboxylate group and N atom [20, 21] and even through N
4
and O sites in some cases [22]. However, in Ca(II) orotate system, orotic acid behaves as
1
tridentate chelating ligand in which N and two oxygen atoms of carboxylato group chelate
the metal [14].
Metal orotates can participate in hydrogen bonding through the donor and acceptor
atoms available even after metal-ligand coordination. Due to this property of orotate, it
becomes an important candidate for designing supramolecular structures [23−25]. These
types of supramolecular structures play a significant role in metallodrug design as well as in
materials chemistry [26].
Organotin carboxylates exhibit structural diversity: from simple monomers [27],
ladder [28], clusters [29], polymer [30] to much more complex structures. Our interest in the
chemistry of organotin carboxylates is not only due to their structural features but also due to
their pharmacological properties [31, 32]. Because of the interesting structural and biological
behaviour of organotin carboxylates as well as of orotic acid, synthesis and biological studies
of organotin(IV) orotates become indispensable. In this present work, we have synthesised
and characterized di- and triorganotin(IV) orotates. However, Me Sn(HOr),
2
Bu Sn(HOr)ꢀH O, Me Sn(H Or) , Bu Sn(H Or) ꢀH O, Me Sn(H Or)ꢀH O, Bu Sn(H Or) have
2
2
2
2
2
2
2
2
2
3
2
2
3
2
been previously synthesised [33], though by different synthetic route. In vitro anti-cancer
3

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activities, enzyme assays, DNA fragmentation assay along with in vivo anti-inflammatory
activity and toxicity of the synthesized complexes have also been discussed.
Insert fig. 1
2
2
. Results and discussion
.1 Chemistry
The reaction of triorganotin chloride(s)/diorganotin dichloride(s) with sodium salt of
orotic acid (formed according to Eq. (1)) in a 1:1/1:2 molar ratio led to the formation of the
complexes according to Eq. (2) and (3).
MeOH
H
3
Or + MeONa
SnCl + Na(H
R = Me, n-Bu and Ph
SnCl + 2Na(H Or)
R = Me and Ph
Na(H
2
Or) + MeOH
Sn(H Or) + NaCl
(1)
(2)
1
:1
R
3
2
Or)
R
3
2
MeOH
1
:2
R
2
2
2
R
2
Sn(H
2
Or)
2
+ 2NaCl
(3)
MeOH
The reactions of diorganotin oxide with orotic acid in 1:2 and 1:1 molar ratio led to
the formation of the complexes according to Eq. (4) and Eq. (5), respectively.
1
:2
R SnO + H Or
R Sn(H Or) + H O
(4)
(5)
2
3
2
2
2
2
MeOH
R = n-Bu and n-Oct
1
:1
R SnO + H Or
R Sn(HOr) + H O
2 2
2
3
MeOH
R = n-Bu
The reactions in Eq. (1)–(3) required refluxing for 18−24 h and Eq. (4)/Eq. (5)
required 14−16 h of refluxing. The resulting solids were obtained in 50−62% yield. From the
analytical data it can be inferred that the resulting triorganotin(IV) orotates crystallized with
4

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1
:1 stoichiometry and diorganotin(IV) orotates with 1:2 stoichiometry. The complexes:
Me
Bu
2
Sn(HOr), Bu
2 2 2 2 2 2 2 2 2 3 2 2
Sn(HOr)ꢀH O, Me Sn(H Or) , Bu Sn(H Or) ꢀH O, Me Sn(H Or)ꢀH O and
3
Sn(H Or) have already been synthesized [33], although by different synthetic route. The
2
synthesized complexes are stable towards air and moisture for more than six months, and can
be stored in vacuum desiccators. They are soluble in methanol and DMSO, but sparingly
soluble in chloroform and other organic solvents. Very low molar conductance values (0.5–
-
1
2
-1
−3
0
.8 ohm cm mol ) of 10 M solutions of the complexes in methanol indicate their non-
electrolytic nature.
IR stretching frequencies, ν (OCO) and ν (OCO) for carboxylates have been
as
s
utilized as a characteristic tool to confirm the mode of coordination through carboxylate
oxygen, and also to identify the nature (monodentate, bidentate or bridging/chelating) of
bonding of the carboxylate group. Unless the carboxylate group is branched at the α- carbon
or the organic substituents at the tin atom are bulky, carboxylate groups in organotin(IV)
carboxylates generally adopt a bridge structure in the solid state [34(a)]. The IR absorption
-1
spectra of organotin(IV) orotates indicate that the νas(OCO) values (1640−1615 cm ) get
-1
shifted to lower frequencies and the ν (OCO) frequencies (1465−1439 cm ) either remain at
s
the same value or move to lower frequencies in comparison with orotic acid. The
s
(νas−ν )(OCO) (ꢁν) is useful in identifying structural features [34]. For the organotin(IV)
-1
-1
orotates the ꢁν is larger (181−174 cm < 200 cm ) in comparison with sodium orotate (ꢁν =
-1
1
50 cm ), which indicates asymmetric bidentate coordination mode of carboxylate group;
-1
2 2 2
except for n-Bu Sn(H Or) , where two ꢁν values (183, 137 cm ) are observed, suggesting
symmetric as well as asymmetric bidentate coordination mode of carboxylate group [35].
-1
Furthermore, a broad and weak intensity band in the region 2800−2900 cm which
was present in the IR spectrum of orotic acid, disappeared in the studied complexes,
suggesting the deprotonation of the COOH group upon complexation [34 (a)]. Two bands in
5

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-1
1
the region 3100−3300 cm due to ν(N−H) and the deformation frequencies of N H (1486 ± 9
-1
3
-1
cm ) and N H (1375 ± 15 cm ) are present in all the tri- and diorganotin(IV) orotates except
n-Bu Sn(H Or) and n-Bu Sn(HOr). This suggests non-participation of both the NH groups of
the pyrimidine ring of the orotic acid in the complex formation. As far as n-Bu Sn(H Or) and
2
2
2
2
2
2
2
2
n-Bu Sn(HOr) are concerned, the presence of only one NH deformation band suggests that
one of the two NH groups of orotic acid is involved in coordination with tin. Therefore, in
dibutylytin(IV) orotates, orotic acid behaves either as monoprotic or as diprotic ligand, viz. n-
1
Bu
2
Sn(HOr) in which a doubly deprotonated ligand linked through N and OCO groups to tin
atom, and in n-Bu Sn(H Or) , monoanion of orotic acid binds to tin, presumably with one of
2
2
2
the two orotate units coordinating the tin atom through the OCO group and the other through
the deprotonated nitrogen atom of an NH group [33]. Appearance of new band in the region
−1
5
72−528 cm assignable to the ν(Sn−O) further supports the bonding through O atom in
triorganotin and diorganotin orotates, except n-Bu Sn(H Or) and n-Bu Sn(HOr) in which
2
2
2
2
along with Sn−O bond, Sn−N bond is formed which is supported by the presence of ν(Sn−N)
-1
in the region 428−445 cm . The ν (Sn−C) and ν (Sn−C) in the studied complexes are also
as
s
observed which are consistent with the literature values [34].
On the basis of IR spectral studies, it may be concluded that orotic acid behaves as:
1
(
a)
b)
a dianion, doubly deprotonated ligand linked through N and OCO in R Sn(HOr)
2
(R = n-Bu).
(
a monoanion in R
2
2 2
Sn(H Or) (R = n-Bu), one of the orotate unit binds the tin atom via
carboxylate oxygen and the other via the deprotonated nitrogen atom of the
1
N H group.
(
c)
d)
a monoanion in R Sn(H Or) (R = Ph, n-Bu and Me), coordination with the tin atom is
3
2
only through OCO.
(
a monoanion in R
2
Sn(H
2
Or)
2
(R = Ph, Me and n-Oct), bonding to tin is only through
6

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OCO.
Finally, as already mentioned, in the studied complexes the carboxylato fragment of
the orotate ligand behaves as a bidentate group. These bonding characteristics and consequent
1
13
geometries at the tin in the organotin(IV) orotates have been further investigated by H, C
119
and
Sn NMR spectroscopic studies.
1
13
6
The H and C NMR spectra of organotin(IV) orotates were recorded in DMSO-d .
119
The Sn NMR spectral data was recorded in CD
3
OD. The number of protons of various
groups calculated from integration curves and those calculated for the expected molecular
1
formula agrees with each other. In the H NMR spectra of all the studied complexes, the
COOH signal (δ ~12.94 ppm) of free orotic acid is missing which suggests the deprotonation
of carboxylate group of the acid on complexation. In triorganotin(IV) orotates, the presence
1
3
of N (H) and N (H) proton resonances shows the non-involvement of nitrogen atoms in
1
13
coordination. These complexes exhibit only one set of H and C resonances, indicating the
2
1
119
existence of single tin species. The observed coupling constant J( H− Sn) values for
triorganotin(IV) orotates are in the range 65.20−71.03 Hz, which correspond to penta-
coordinated, trigonal-bipyramidal geometry [36]. The three alkyl/phenyl groups are in
equatorial plane of trigonal-bipyramid with tin atom in centre and the most electronegative
atoms (i.e. oxygen) occupying axial positions. The ∠ C−Sn−C calculated with the help of
2
2
2
Lockhart and Manders equation [36] (θ = 0.0161| J| −1.32| J| + 133.4) lies in the range
15.74˚−120.86˚, which also suggests equatorial disposition of organic groups in a distorted
trigonal-bipyramidal geometry.
In Ph Sn(H Or) , Me Sn(H
resonances are observed and their integrated intensities indicate their non-involvement in
1
1
3
2
2
2
2
2 2 2 2 2
Or) and n-Oct Sn(H Or) , both N (H) and N (H) proton
1
13
coordination with tin. These complexes also exhibit only one set of H and C resonances,
2
1
indicating the existence of single tin species. Further, for diorganotin(IV) complexes, J( H−
7

ACCEPTED MANUSCRIPT
1
19
Sn) value has only been determined for Me
2
Sn(H
2
Or)
2
(85.51 Hz) which corresponds to an
o
octahedral geometry around tin [36]. The calculated value of ∠ C−Sn−C is 138.25 , which
indicates approximately trans disposition of two organic groups or a distorted linear C−Sn−C
skeleton for highly distorted octahedral n-R Sn(H Or) , which would correspond to the
2
2
2
structure of Fig. 2 (b) with the orotate anion bound through bidentate carboxylato groups.
All the magnetically non-equivalent carbons of alkyl or phenyl groups attached to the
tin atom have also been identified and their chemical shift values are in close agreement with
1
13
119
the reported values [34 (a)]. The heteronuclear coupling constant J( C− Sn) has been
calculated for most of the complexes having well resolved satellites. The observed
1
13
119
J( C− Sn) values are 465.60−485.00 Hz and 703.27−778.01 Hz for R
3 2
Sn(H Or) and
R
2
Sn(H Or) , respectively. These values lie in the range of penta-coordinated trigonal-
2
2
bipyramidal and hexa-coordinated octahedral environment around tin atom [37 − 39].
Moreover, the ∠ C−Sn−C values calculated with the help of Lockhart and Manders equation
1
13
119
o
[
37] [(| J( C − Sn|) = 11.4θ–875] are in the range 117.60 −119.30˚ and 138.44˚−145.00˚
for R Sn(H Or) and R Sn(H Or) , respectively. These values also support a distorted trigonal
3
2
2
2
2
bipyramidal geometry with equatorial three organic groups and distorted octahedral geometry
with the two trans organic groups for tri- and diorganotin(IV) orotates, respectively.
1
19
Sn NMR chemical shifts are very sensitive to coordination number of tin and are
119
greatly shifted up field on complexation with Lewis base.
Sn NMR shifts for tri-n-
butyltin(IV)-, trimethyltin(IV)- and triphenyltin(IV) orotates are δ −120.25 ppm, −138.41
ppm and −180.00 ppm, respectively, which clearly suggest the trigonal-bipyramidal
1
19
geometry [39] for the complexes (as shown in Fig. 2 (a)). The
Sn NMR shifts for
dimethyltin(IV)-, diphenyltin(IV)- and di-n-octyltin(IV) orotates are −330.14 ppm, −540.76
ppm and −352.02 ppm, respectively, suggesting an octahedral geometry [39] for the
2 2
dialkyltin(IV) orotates (except n-Bu Sn(H Or)) (Fig. 2 (b)).
8

ACCEPTED MANUSCRIPT
1
2
The N (H) proton resonance is absent in n-Bu Sn(HOr), suggesting the deprotonation
1
1
of N H group and the subsequent involvement of N in bonding with tin. The precise values
2
1
119
of J( H− Sn) cannot be obtained due to overlapping of broad multiplets, where as
1
13
119
119
J( C− Sn) value (778.01 Hz) gives ∠ C−Sn−C = 145˚. Sn NMR is highly sensitive and
hence able to detect two resonances in 1:5 intensity ratio, one corresponds to trigonal
bypiramidal geometry (δ = −123.10 ppm) and the other to octahedral geometry (δ = −368.00
ppm). This may be rationalized as: in solution coordination number of tin is frequently
changing between five and six, involving a solvent molecule as sixth ligand (Fig. 2 (c) and
Fig. 2 (d)) giving rise to equilibrium between penta- (trigonal-bipyramidal) and hexa-
coordinate (octahedral) species.
1
13
H and C NMR chemical shifts for the orotato moiety in n-Bu
2
2 2
Sn(H Or) suggest
1
that the ligand is present partly as carboxylato monoanion, and partly as the N deprotonated
2 2 2
monoanion [33]. In IR spectrum of n-Bu Sn(H Or) only one δ(N−H) is present, and hence it
1
can be inferred that N is probably involved in bond formation with tin atom. As it is well
depicted by stoichiometric ratio of the complex, tin is attached to two orotato moieties.
3
1
1
Further, two N H and only one N H proton resonances are observed in H NMR spectrum of
n-Bu
2
2 2
Sn(H Or) . Only one set of resonances is found for the tin-bound butyl groups both in
1
13
H and C NMR spectra. These results suggest that one of the orotate units may be
1
13
coordinated to tin atom via deprotonated N , while other unit via carboxylate group. In C
NMR spectrum of the complex two sets of resonances of orotate anion are also observed,
supporting two different coordinating environments of both orotate anions around tin atom.
1
19
Only one Sn chemical shift is observed at −350.00 ppm, which suggests a single tin species
in solution having an octahedral geometry around tin (Fig. 2 (e)).
2
.2. Biological screening
2
.4.1. Anti-cancer screening
9

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All of the studied organotin(IV) orotates along with standard drugs, viz. cis-platin, 5-
fluorouracil and methotrexate have been screened in vitro against five cancer cell lines of
human origin, viz. MCF-7 mammary cancer, HEK-293 kidney cancer, PC-3 prostate cancer,
HCT-15 colon cancer and HepG-2 liver cancer, and their average IC50 values for three
experiments (runs) are presented in Table 1. Among the studied complexes n-Oct Sn(H Or)
2
2
2
is the most active complex against all the cell lines, followed by Ph
3
Sn(H
are active against HCT-15. Me Sn(H
are also active against PC-3, and HepG-2, respectively. n-Bu Sn(H
2
Or). Further,
Or) and
Or) and
Me
Me
3
2
Sn(H
2
2
Or) and Me
Or)
2
Sn(H
2
Or)
2
3
2
Sn(H
2
2
2
2
n-Bu Sn(HOr) are the least active complexes among all the studied complexes.
2
All the studied complexes are less active in comparison to cis-platin, 5-fluorouracil
and methotrexate, except n-Oct Sn(H Or) which exhibits slightly less activity (or almost
comparable) against HCT-15 in comparison to 5-fluorouracil and Me Sn(H Or) which is also
2
2
2
3
2
slightly less active (or almost comparable) against PC-3 in comparison to methotrexate. It has
been reported [40] that compounds exhibiting IC50 < 1.0 ꢂg/ml are considered as highly
cytotoxic; IC 1.0−10.0 ꢂg/ml− cytotoxic; IC 10.0−30.0 ꢂg/ml− moderately cytotoxic; IC
50
50
50
>
30.0 < 100.0 ꢂg/ml− mildly cytotoxic; and IC50 > 10.0 ꢂg/ml− as non-cytotoxic. Therefore,
Ph Sn(H Or), Me Sn(H Or), n-Oct Sn(H Or) and Me Sn(H Or) are considered as
moderately/mildly cytotoxic selectively against a few cell lines, whereas n-Oct Sn(H Or) is
toxic (IC 9.14 ꢂg/ml) against HCT-15. Fig. 3 and Fig. 4 represent MCF-7 and PC-3 cancer
3
2
3
2
2
2
2
2
2
2
2
2
2
50
cells (for control and treated), respectively, before and after the treatment with IC value of
5
0
2 2 2
n-Oct Sn(H Or) .
Insert fig. 2 (a), Insert fig 2 (b), Insert fig. 2 (c), and 2 (d)
Insert fig. 2 (e), Insert fig. 2 (f) and Insert Table 1
Insert fig. 3 (a) and 3 (b), Insert fig. 4 (a) and 4 (b)
1
0

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The low to moderate cytotoxicity of the studied organotin(IV) orotates may be
explained on the basis of structure-activity relationship. The activity of the organotin
complexes mainly depends on the hydrolytic stability of Sn − X (X = O, N, Cl, F and S)
whereas Sn − C bond is hydrolytically more stable. It has been reported [34 (a), 41, 42] that
+
2+
either organotin(IV) compounds as whole or R Sn and R Sn
produced on
3
2
hydrolysis/dissociation may be the final active species responsible for the anticancer activity
of tri- and diorganotin compounds. Since the studied organotin(IV) orotates are very stable to
air and moisture, and may have relatively strong Sn−X (O/N) bond; this may be correlated to
the observed low to moderate activity of these studied organotin(IV) orotates.
4
.2. Enzymatic assays
The observed data of the enzymatic assays of n-Oct
2
2 2
Sn(H Or) against MCF-7 and
PC-3 are presented in Table 2 and Table 3, respectively (graphical representation in Fig. 5 to
Fig. 8). Necrosis could be excluded by considering the marginal increase in extracellular
lactate dehydrogenase (LDH) concentration ratio of treated and untreated. The non-specific
necrosis mediated through membrane toxicity is observed with in short periods of time,
which may be responsible for the marginal increase in LDH concentration. Substantial folds
increase in induction of other antioxidant enzymes’ activity suggests that reactive oxygen
species (ROS) have been produced in the cells, resulting oxidative stress which is responsible
for causing cell death.
Lipid peroxidation is one of the most accepted assay to measure oxidative damage.
High lipid peroxidation has been observed which suggests that the dioctyltin(IV) orotate
stimulates the oxidative reaction leading to the generation of reactive oxygen species (ROS).
It is well known that enzymes like glutathione peroxidase along with glutathione
reductase are important for regulating the redox system of cells [43]. Any change or
alteration in activity of these enzymes affects the oxidant status of cells and hence subjecting
1
1

ACCEPTED MANUSCRIPT
the cells to oxidative stress [44]. There have been many evidences where oxidative stress has
been shown to play a vital role in apoptosis [45, 46].
Insert Table 2 and Table 3
Elevation in glutathione peroxidase activity has been observed in cells after the
2 2 2
treatment with n-Oct Sn(H Or) . The enzyme converts the pool reduced glutathione (GSH)
to oxidised glutathione, thereby decreasing the peroxides concentration. Increase in
glutathione peroxidase activity was also observed by Han et. al, where ellagric acid induces
apoptosis [47]. We have also observed a significant depletion in glutathione reductase (GR)
activity and marginal increase in total glutathione (GSSG + GSH).
The cell death may be induced by various mechanisms such as cell membrane
damage, as observed by an increase in LDH activity (though the increase is marginal and
insignificant with respect to control at p < 0.05) resulting in leakage of cell enzymes like
GST, LDH and GSH. The other possible way may be the oxidative stress which affects cell
survival and hence inhibiting cell growth.
Alternatively, the complex induced peroxides generation, metabolized by glutathione
peroxidase (GPx), resulting in the formation of oxidized glutathione (GSSG) from reduced
form of glutathione (GSH). This leads to reduction of GSH concentration and oxidation of
NADPH (Eq. 6 and 7). Depletion of GSH and NADPH oxidation are associated with altered
2
+
Ca homeostasis, which is considered a critical event in peroxide induced loss of cell
2
+
viability [48]. Organotins also induce apoptosis by disturbing [Ca ] and mitochondrial
activity, resulting oxidative stress in tumour cells [49].
GPx
R-OOH + 2 GSH
R-OH + GSSH + H O
(6)
(7)
2
GR
GSSH + NADPH + H+
2
GSH + NADP+
1
2

ACCEPTED MANUSCRIPT
In many anticancer drugs necrosis may occur along with apoptosis, like cladribine,
cis-platin, doxorubicin and 5-fluorouracil, which possess both apoptotic and necrotic effects
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012644 - pone.0012644-
Guchelaar1[50, 51]. To determine where apoptosis is the main cause of cell death, DNA
fragmentation assay, a land mark for apoptosis, has also been performed.
Insert fig. 5, fig. 6, fig. 7 and fig. 8
2
.4.3. DNA Fragmentation Assay
DNA extracted from MCF-7 (lane 1) and PC-3 (lane 6) was taken as control. The
cells were treated with di-n-octyltin(IV) orotate and DNA extracted from, MCF-7 (lane 3)
and PC-3 (lane 8) was taken as sample. The DNA extracted from cis-platin treated MCF- 7
(
lane 4) and PC-3 (lane 7) cells, was taken as positive control. DNA ladder of 100 base pair
lane 2, 5) was taken as DNA marker. Gel electrophoresis assay was conducted and recorded
(
on Bio-rad UV transilluminator as already described in experimental section after addition of
ethidium bromide. The results are shown in Fig. 9. DNA fragmentation has long been used to
distinguish apoptosis from necrosis, and it is among the most reliable method for detection of
apoptotic cells. Fig. 9 clearly shows the formation of DNA–ladder (trailing) which indicates
that the DNA breakage or fragmentation has occurred, this can only happen when cell
apoptosis takes place. Therefore, the cytotoxic effect of the organotin(IV) orotates is
selectively mediated through the induction of apoptosis.
Insert fig. 9
2
.4.4. Anti-inflammatory activity
The anti-inflammatory activity data (% inhibition) of organotin(IV) orotates are
presented in Table 4. The results obtained indicate the order of the anti-inflammatory activity
of the studied complexes as: Ph Sn(H Or) > n-Oct Sn(H Or) > Ph Sn(H Or)
>
3
2
2
2
2
2
2
2
Bu
2
Sn(H
2
Or)
2
> n-Bu
3
Sn(H
2
Or) > n-Bu
2
Sn(HOr) > Me
2
Sn(H
2
Or)
2
> Me
3
2
Sn(H Or).
1
3

ACCEPTED MANUSCRIPT
Ph
3
Sn(H
2
Or) exhibited potent anti-inflammatory activity ( >39.18% inhibition), which was
significantly greater than that of ibuprofen (36.08% inhibition), however it was less than that
of indomethacein (60.82% inhibition). Ph Sn(H Or) and n-Oct Sn(H Or) showed the good
anti-inflammatory activity (21.13 and 23.71 % inhibition, respectively), Bu Sn(H Or) and n-
2
2
2
2
2
2
2
2
2
Bu Sn(H Or) exhibited low activity (18.56 and 14.95 % inhibition, respectively), whereas
3
2
Me
2 2 2 3 2 2
Sn(H Or) , Me Sn(H Or) and n-Bu Sn(HOr) exhibited very low anti-inflammatory
activity (~12.37 – 8.25% inhibition).
It can be concluded from the observed anti-inflammatory activity that diorganotin(IV)
orotates are more active than triorganotin(IV) orotates except Ph Sn(H Or). The highest
3
2
activity of Ph Sn(H Or) may be due to the formation and frequent transportation of
3
2
+
3
Ph Sn(IV) moiety across the cellular membrane as a part of the mechanism [34].
2
.4.5. Toxicity
For the commercial and pharmacological aspects a drug with a better patient
compatibility, low toxicity and high anti-inflammatory activity with minimum adverse side
effects and wider safety margin is highly desirable. The LD50 values of the studied
-1
organotin(IV) orotates are found to be > 600 mg kg in albino mice . Further, the studied
derivatives are less toxic than the organotin derivatives of the simple α-amino acids (LD50
-1
-1
<
50 mg kg ) and dipeptides (>400 mg kg ) [34b, 52] indicating that the bigger biomolecules
lower the toxicities.
Insert Table 4
3
. Conclusions
Organotin(IV) orotates have been synthesized through the sodium salt method as well
1
13
119
as azeotropic removal of water. IR, H, C and
Sn NMR spectral studies of all the
synthesized triorganotin(IV) orotates suggest a distorted trigonal–bipyramidal geometry,
whereas a distorted trans-octahedral structure has been proposed for diorganotin(IV) orotates
1
4

ACCEPTED MANUSCRIPT
except n-Bu
2
Sn(HOr). In n-Bu
2
Sn(HOr), two sets of resonances have been observed in its
1
19
Sn NMR spectrum, indicating existence of two tin species, one having trigonal–
bipyramidal structure and the other octahedral structure involving the solvent as the sixth
ligand.
In vitro anti-cancer screening of these carboxylates suggests that di-n-octyltin(IV)
orotate is the most active complex among the studied complexes in this manuscript, which
exhibits potent anti-cancer activity against all the cell lines. Significant increase in
glutathione-peroxidase activity and lipid peroxidation assay support that cells undergo
oxidative stress. Marginal increase in LDH suggests that necrosis may also occur to a small
extent. Furthermore, DNA ladder formation indicates that these complexes inhibit the cancer
cell growth through apoptosis. Taken together; the results indicate that the organotin orotates
possesses dual-capability of cell death: necrosis and apoptosis. However, further experiments
are needed to establish the fact more strongly.
An anticancer agent is expected to possess an anti-inflammatory activity. It has been
observed that diorganotin(IV) orotates show better anti-inflammatory activity as compared
with triorganotin(IV) analogs except triphenyltin(IV) orotate. Among the studied complexes,
triphenyltin(IV) orotate exhibits the highest anti-inflammatory activity followed by di-n-
octyltin(IV) orotate. In vitro cytotoxicity assays, in vivo anti-inflammatory activity and
toxicity results of the studied complexes predict their potency as metal based anti-cancer
drugs.
5
. Experimental
5
.1.1. Materials
Solvents, viz. acetone (E. Merck), benzene (E. Merck), DMSO (E. Merck-HPLC
grade), n-hexane (60−80 ºC fraction from petroleum) (E. Merck), petroleum benzene (60-80
1
5

ACCEPTED MANUSCRIPT
ºC) (E. Merck) and petroleum ether (40-60 ºC) (E. Merck) were dried before use. Specially
dried absolute methanol (s.d. fine-chemicals limited) was employed for the synthesis work.
Organotin compounds, viz. trimethyltin(IV) chloride(TMTC) (Sigma Aldrich), tri-n-
butyltin(IV) chloride(TBTC) (Merck-Schuchardt), triphenyltin(IV) chloride(TPTC) (E.
Merck), dimethyltin(IV) dichloride (DMTC) (Sigma Aldrich), di-n-butyltin(IV) oxide
(DBTO) (E. Merck) and di-n-octyltin(IV) oxide (DOTO) (E. Merck), diphenyltin(IV)
dichloride (Aldrich Chemicals)were used as received.. Orotic acid (H Or) (Sigma Aldrich)
3
was used as received. For biological activities, following chemicals were used, MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma), sorensen’s buffer, PMSF,
phosphate buffer saline, agarose gel, buffer solution (10 mM tris pH 7.5, 1 mM EDTA, 0.2%
triton X-100), lysis buffer (10 mM tris pH 7.5, 1 mM EDTA, 0.2% triton X-100), phenol (E.
Merck), chloroform (E. Merck), isoamyl alcohol (E. Merck), EDTA, RNase, 50X TAE
buffer, glacial acetic acid (s.d. fine-chemicals limited) , 6X gel loading dye, bromophenol
blue, xylene cyanol FF, glycerol, Na-phosphate buffer, NADH, NADPH, Na-pyruvate,
Ellman’s reagent, glutathione reductase, CDNB (chlorodinitro benzene), GSH (reduced
glutathione), SDS, thiobarbituric acid and carrageenan (Renkem). Five cancer cell lines of
human origin, viz. MCF-7 mammary cancer, HEK-293 kidney 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.
5
.1.2. General Methods
The melting points of synthesized complexes were determined with a Toshniwal
capillary melting point apparatus and are uncorrected. Carbon, hydrogen and nitrogen
analyses of the synthesized complexes were carried out on a Perkin-Elmer, CHNS-rapid
elemental analyzer. The tin content in the synthesized complexes was determined
1
6

ACCEPTED MANUSCRIPT
gravimetrically as SnO
2
[34 (a)]. Molar conductance measurements were carried out on the
Cyberscan 510-Eutech Instruments [34 (a)]. Infrared and far-infrared spectra were recorded
-1
on a Themo Nikolet Nexus FTIR spectrophotometer in KBr discs in the range 4000–400 cm
-1
and Thermo Scientific Nikolet 6700 FTIR in CsI discs in the range 600–200 cm ,
1
13
119
respectively. Nuclear magnetic resonance ( H, C and Sn) spectra were recorded on
Bruker AVANCE (500.13 MHz) spectrometer at the Institute Instrumentation Centre (IIC),
IIT Roorkee using tetramethylsilane (TMS) as internal standard. Optical density (OD) was
recorded on a Dynatech MR 5000 microplate reader at the Department of Biotechnology, IIT
Roorkee. Gel electrophoresis was carried out by using Bio-rad UV Transilluminator. The
average IC50 estimated via MTT assay, enzymes assays, toxicity and in vivo anti-
inflammatory activity were done according to the procedures as described in section 5.4, 5.5,
5
5
.6, 5.7 and 5.8.
.2. Synthesis of triorganotin(IV)/diorganotin(IV) complexes of orotic acid by sodium salt
method
Orotic acid (0.32 g, 2.0 mmol) was dissolved in the minimum amount (20 mL) of
specially dried methanol under dry nitrogen and added to sodium methoxide, prepared by
reacting sodium (0.058 g, 2.5 mmol) with dry methanol (25 mL). The resulting mixture was
first stirred at room temperature for half an hour and then refluxed giving a clear solution of
sodium salt of orotic acid within half an hour. Refluxing was continued for another 4−6 h
with constant stirring. A hot methanol solution (20 mL) of trimethyltin(IV) chloride (0.398 g,
2
2
.0 mmol)/tri-n-butyltin(IV) chloride (0.651 g, 2.0 mmol)/triphenyltin(IV) chloride (0.771 g,
.0 mmol)/dimethyltin dichloride (0.220 g, 1.0 mmol)/diphenyltin dichloride (0.344 g, 1.0
mmol) was added to the solution of the preformed sodium salt of the acid. The resulting
solution was further refluxed with constant stirring for another 14−18 h under dry nitrogen
atmosphere. It was then centrifuged and filtered in order to remove the sodium chloride
1
7

ACCEPTED MANUSCRIPT
formed. The excess of solvent was removed under reduced pressure and the solid product
thus obtained was recrystallized from either methanol-hexane or methanol-petroleum ether
(
b.p. 40−60 °C) mixture (1:3 v/v).
.2.1. Ph Sn(H Sn]. Yield (%): 50; m. p. (°C): > 300; Colour and Physical
State: Yellow solid; Analysis (%) Found (calculated) Sn: 23.62 (23.56), C: 54.30 (54.71), N:
5
3
2 18 4 2
Or) [C23H O N
-1
1
3
5
.61(5.54), H: 3.20 (3.56); IR (cm ): ν(NH) 3156 br, 3115 s; δ(N H) 1488 s, δ(N H) 1378 s;
4
2
s
ν (OCO) 1439 s, νas(OCO) 1615 s, ∆ν 176; ν(C =O) 1676 s, ν(C =O) 1724 s, ν(Sn-O) 572 w,
1
1
ν (Sn-C) 288 s, ν (Sn-C) 225 s; H NMR δ (ppm) (DMSO-d ): 11.52 (s, 1H, N -H); 10.88 (s,
as
s
6
3
5
1
3
1
H, N -H); 5.79 (s, 1H, C -H); 7.75 (d, 6H, 3.6 Hz, H-β); 7.55−7.54 (mbr, 6H, H-γ); 7.44 (t,
13
2
4
6
H, 7.1 Hz, H-δ); C NMR δ (ppm) (DMSO-d ): 163.11 (OCO); 157.10 (C ); 167.39 (C );
5
6
1
13 119/117
07.20 (C ); 151.00 (C ); 139.03 (C-α), [ J( C
Sn) = 485.00 Hz]; 137.11 (C-β); 128.20
OD): −180.00.
Sn]. Yield (%): 62; m. p. (°C): 140−142 (140 [33]);
Colour and Physical State: Pale-Yellow solid; Analysis (%) Found (calculated) Sn: 26.75
b
119
(
C-γ); 130.34 (C-δ); [θ = 119.30˚] ; Sn NMR δ (ppm) (CD
3
5
.2.2. n-Bu Sn(H
3
2 30 4 2
Or) [C17H O N
-1
(
26.74), C: 45.51(45.84), N: 6.00 (6.29), H: 6.97(6.74); IR (cm ): ν(NH) 3168 br, 3120 br;
1
3
4
δ(N H) 1490 s, δ(N H) 1360 s; ν
s
(OCO) 1460 s, νas(OCO) 1640 s, ∆ν 180; ν(C =O) 1669 s,
2
1
ν(C =O) 1729 s, ν(Sn-O) 555 w, ν (Sn-C) 607 m, ν (Sn-C) 510 s; H NMR δ (ppm) (DMSO-
as
s
1
3
5
6
d ): 11.10 (s, 1H, N -H); 11.01 (s, 1H, N -H); 5.56 (s, 1H, C -H); 1.52 (t, 6H, 5.7 Hz, H-α),
2
1
119/117
[
J( H−
Sn) = 65.20 Hz]; 1.50 (m, 6H, H-β); 1.32 (m, 6H, H-γ); 0.92 (t, 9H, 6.0 Hz, H-
a
13
2
4
δ); [θ = 115.74˚] ; C NMR δ (ppm) (DMSO-d
6
): 164.11 (OCO); 157.15 (C ); 167.66 (C );
5
6
1
13 119/117
1
06.50 (C ); 150.08 (C ); 20.03 (C-α), [ J( C
Sn) = 480.04 Hz]; 28.01 (C-β); 27.60 (C-
b
119
γ); 13.89 (C-δ); [θ = 118.86˚] ; Sn NMR δ (ppm) (CD OD): −120.25.
3
5
.2.3. Me
3 2 8 12 4 2
Sn(H Or) [C H O N Sn]. Yield (%): 55; m. p. (°C): 174; Colour and Physical
State: White solid; Analysis (%) Found (calculated) Sn: 37.00 (37.30), C: 30.00 (30.09), N:
-1
1
3
8
.23 (8.77), H: 4.00 (3.76); IR (cm ): ν(NH) 3166 m, 3129 m; δ(N H) 1494 s, δ(N H) 1365
1
8

ACCEPTED MANUSCRIPT
4
2
s; ν
s
(OCO) 1452 s, νas(OCO) 1628 m, ∆ν 176; ν(C =O) 1681 s, ν(C =O) 1710 s, ν(Sn-O): 528
1
1
s 6
w, νas(Sn-C) 616 s, ν (Sn-C) 514 m; H NMR δ (ppm) (DMSO-d ): 11.11 (s, 1H, N -H);
3
5
2
1
119/117
1
1
0.81 (s, 1H, N -H); 5.68 (s, 1H, C -H); 0.59 (s, 9H, H-α), [ J( H−
Sn) = 71.03 Hz]; [θ =
a
13
2
4
20.86˚] ; C NMR δ (ppm) (DMSO-d ): 163.57 (OCO); 157.03 (C ); 167.27 (C ); 107.10
6
5
6
1
13 119/117
b 119
(
C ); 151.01 (C ); –4.41 (C-α), [ J( C
ppm) (CD OD): −138.41.
.2.4. Ph Sn(H Or)
Sn) = 465.60 Hz]; [θ = 117.60˚] ; Sn NMR δ
(
3
5
2
2
2
[C22H
16
O
8
N
4
Sn]. Yield (%): 52; m. p. (°C): > 300; Colour and Physical
State: Pale-Yellow solid; Analysis (%) Found (calculated) Sn: 20.32 (20.41), C: 45.25
-1
1
(
45.28), N: 9.12 (9.61), H: 3.00 (2.74); IR (cm ): ν(NH) 3156 br, 3122 s; δ(N H) 1486 m,
3
4
2
δ(N H) 1360 s; ν
s
(OCO) 1440 s, νas(OCO) 1624 s, ∆ν 184; ν(C =O) 1675 s, ν(C =O) 1720 s,
1
ν(Sn-O) 561 w, νas(Sn-C) 285 s, ν
s
(Sn-C) 220 s; H NMR δ (ppm) (DMSO-d
6
): 11.57 (s, 2H,
1
3
5
N -H); 10.88 (s, 2H, N -H); 5.79 (s, 2H, C -H); 7.95 (d, 4H, 3.2 Hz, H-β); 7.52 − 7.44 (mbr,
1
3
4
1
H, H-γ); 7.39 − 7.37 (t, 2H, 7.1 Hz, H-δ); C NMR δ (ppm) (DMSO-d ): 164.00 (OCO);
6
2
4
5
6
1
13 119/117
57.20 (C ); 167.58 (C ); 106.87 (C ); 149.58 (C ); 136.83 (C-α), [ J( C
Sn) = 703.27
OD):
b
119
Hz]; 128.61 (C-β); 127.43 (C-γ); 128.34 (C-δ); [θ = 138.44˚] ; Sn NMR δ (ppm) (CD
540.76.
.2.5. Me
Colour and Physical State: White solid; Analysis (%) Found (calculated) Sn: 25.12 (25.92),
3
−
5
2 2 2 12 8 4
Sn(H Or) [C12H O N Sn]. Yield (%): 51; m. p. (°C): > 300 (300 dec. [33]);
-1
1
C: 31.56 (31.37), N: 11.98 (12.20), H: 2.77 (2.61); IR (cm ): ν(NH) 3167 br, 3128 m; δ(N H)
3
4
1
477 m, δ(N H) 1389 s; ν (OCO) 1450 s, ν (OCO) 1631 s, ∆ν 181; ν(C =O) 1682 s,
s as
2
1
ν(C =O) 1726 s; ν(Sn-O) 560 w, νas(Sn-C) 620 s, ν
s
(Sn-C) 590 m; H NMR δ (ppm) (DMSO-
1
3
5
d ): 11.01 (s, 2H, N -H); 10.71 (s, 2H, N -H); 5.79 (s, 2H, C -H); 0.88 (s, 6H, H-α),
6
2
1
119/117
a 13
[
J( H−
Sn) = 85.51 Hz]; [θ = 138.25˚] ; C NMR δ (ppm) (DMSO-d
6
): 164.00 (OCO);
2
4
5
6
119
1
57.05 (C ); 167.10 (C ); 107.66 (C ); 150.22 (C ); 0.51 (C-α);
Sn NMR δ (ppm)
3
(CD OD): −330.14.
1
9

ACCEPTED MANUSCRIPT
5
.3. Synthesis of di-n-butyltin(IV)/di-n-octyltin(IV) complexes of orotic acid by the
azeotropic removal of water method
The complexes were prepared under anhydrous nitrogen atmosphere by dropwise
addition of dry, hot methanol (20 mL) solution of di-n-butyltin oxide (0.498 g, 2.0 mmol) or
di-n-octyltin oxide (0.722 g for 2.0 mmol) to a hot methanol solution (15−20 mL) of orotic
acid (0.64 g, 4.0 mmol). When a methanolic solution of di-n-butyltin oxide (2 mmol) was
2
added to 2 mmol of orotic acid, then n-Bu Sn(HOr) was formed. The reaction mixture
obtained was refluxed with constant stirring for at least 14−16 h with azeotropic removal of
water. The solution was filtered, and the excess of solvent was removed under reduced
pressure and allowed to cool. The solid product thus obtained was recrystallized by either
methanol-hexane or methanol-petroleum ether (b.p. 40−60 ºC) mixture (1:3 v/v).
5
2 2 2 40 8 4
.3.1. n-Oct Sn(H Or) [C26H O N Sn]. Yield (%): 57; m. p. (°C): 250; Colour and Physical
State: Yellow solid; Analysis (%) Found (calculated) Sn: 18.23 (18.16), C: 47.55 (47.63), N:
-1
1
3
8
.31 (8.55), H: 6.01 (6.11); IR (cm ): ν(NH) 3278 br, 3138 br; δ(N H) 1491 m, δ(N H) 1366
4
2
s; ν (OCO) 1465 s, ν (OCO) 1639 s, ∆ν 174; ν(C =O) 1677 s, ν(C =O) 1732 s, ν(Sn-O) 534
s
as
1
1
w, νas(Sn-C) 623 s, ν
s
(Sn-C) 490 m; H NMR δ (ppm) (DMSO-d
6
): 11.56 (s, 2H, N -H);
3
5
1
1
0.82 (s, 2H, N -H); 5.70 (s, 2H, C -H); 1.41 (t, 4H, 3.3 Hz, H-α) 1.47 (m, 4H, H-β); 1.50 −
.10 (mbr, 12H, H-γ, H-δ, H-θ); 0.98−0.88 (mbr, 8H, H-λ, H-ꢂ); 0.88 (t, 6H, 6.1 Hz, H-ω);
1
3
2
4
5
6
C NMR δ (ppm) (DMSO-d ): 163.93 (OCO); 157.30 (C ); 167.95 (C ); 107.09 (C ); 150.22
6
(
C ); 26.10 (C-α); 31.56 (C-β); 29.11 (C-γ); 32.23 (C-δ); 28.92 (C-θ); 28.07 (C-λ); 22.56 (C-
1
19
ꢂ); 14.02 (C-ω); Sn NMR δ (ppm) (CD
.3.2. n-Bu Sn(H Or) Sn].Yield (%): 57; m. p. (°C): 135−137; Colour and
Physical State: Creamish solid; Analysis (%) Found (calculated) Sn: 21.00 (21.90), C: 39.28
3
OD): −352.02.
5
2
2
2 24 8 4
[C18H O N
-1
3
(
39.78), N: 10.10 (10.31), H: 4.21 (4.41); IR (cm ): ν(NH) 3161 m, 3111 m; δ(N H) 1377 s;
4
2
ν
s
(OCO) 1456 s, νas(OCO) 1639 m, 1593 m, ∆ν 183, 137; ν(C =O) 1669 s, ν(C =O) 1728 s,
2
0

ACCEPTED MANUSCRIPT
1
s
ν(Sn-O) 549 w, νas(Sn-C) 604 s, ν (Sn-C) 550 m, ν(Sn-N) 445 w; H NMR δ (ppm) (DMSO-
1
3
5
d
6
): 11.31 (s, 1H, N -H); 11.07, 10.76, (s, 2H, N -H); 5.80, 5.69 (s, 2H, C -H); 1.55 (t, 4H,
1
3
6
.3 Hz, H-α); 1.5 (m, 4H, H-β); 1.32 (m, 4H, H-γ); 0.88 (t, 6H, 6.0 Hz, H-δ); C NMR δ
2
4
(
ppm) (DMSO-d ): 163.23, 165.00 (OCO); 157.50, 160.04 (C ); 167.08, 169.13 (C ); 107.06,
6
5
6
119
1
03.24, (C ); 148.08, 151.57 (C ); 29.03 (C-α); 28.26 (C-β); 27.84 (C-γ); 13.62 (C-δ); Sn
OD): −350.00.
.3.3. n-Bu Sn(H Or) [C H O N Sn]. Yield (%): 52; m. p. (°C): 154; Colour and Physical
NMR δ (ppm) (CD
3
5
2
2
13 20
4
2
State: White solid; Analysis (%) Found (calculated) Sn: 30.15 (30.78), C: 37.12 (37.26), N:
-1
3
*
7
.57 (7.69), H: 5.24 (5.76); IR (cm ): ν(NH) 3165 s, 3110 br; δ(N H) 1491 s; ν (OCO) 1459
s
4
2
s, νas(OCO) 1640 m, ∆ν 181; ν(C =O) 1674 s, ν(C =O) 1716 s, ν(Sn-O) 540 w, νas(Sn-C) 630
1
3
5
s, ν
s 6
(Sn-C) 574 s, ν(Sn-N) 428; H NMR (DMSO-d ): 11.11 (s, 1H, N -H); 5.79 (s, 1H, C -
H); 1.22 (t, 4H, 5.6 Hz, H-α); 1.50 (m, 4H, H-β); 1.35 (m, 4H, H-γ); 0.89 (t, 6H, 6.1 Hz, H-δ);
1
3
2
4
5
C NMR δ (ppm) (DMSO-d ): 163.75 (OCO); 157.00 (C ); 170.08 (C ); 102.86 (C ); 149.08
6
6
1
13 119/117
(
C ); 27.33 (C-α), [ J( C
Sn) = 778.01 Hz]; 26.47 (C-β); 28.00 (C -γ); 13.91 (C-δ); [θ
b
119
c
=
145˚] ; Sn NMR δ (ppm) (CD
3
OD) : −123.10 (vw), −368.00.
-1
1
3
5
.4.1. Orotic Acid (H
3
Or). IR (cm ): ν(NH) 3152 br, 3110 s; δ(N H) 1515 s, δ(N H) 1406 s;
4
2
1
ν
s
(OCO) 1481 s, νas(OCO) 1675 sh, ∆ν 194; ν(C =O) 1652 s, ν(C =O) 1733 s; H NMR
1
3
5
(
(
(
DMSO-d ): 11.60 (s, 1H, N -H); 11.2 (s, 1H, N -H); 5.86 (s, 1H, C -H); 12.94 (s, 1H,
6
1
3
2
4
6
COOH); C NMR δ (ppm) (DMSO-d ): 162.00 (OCO); 157.00(C ); 167.08(C ); 107.00
5
6
C ); 146.08(C ).
-1
1
5
.4.2. Sodium salt of orotic Acid (NaH
2
Or). IR (cm ): ν(NH) 3142 m , 3058 m; δ(N H) 1526
3
4
w, δ(N H) 1426 s; ν
s
(OCO) 1483 m, νas(OCO) 1633 vs, ∆ν 150; ν(C =O) 1567 vs, 1595 sh,
2
1
1
3
ν(C =O) 1742 w; H NMR (DMSO-d ): 11.50 (s, 1H, N -H); 10.89 (s, 1H, N -H); 5.76 (s,
6
5
13
2
4
1
6
H, C -H); C NMR δ (ppm) (DMSO-d ): 163.03 (OCO); 157.39 (C ); 169.03 (C ); 105.00
5
6
(
C ); 147.17 (C ).
2
1

ACCEPTED MANUSCRIPT
*
3
For IR: s: strong, m: medium, w: weak, sh: shoulder;
ν
s
(OCO) merged with δ (N H); ∆ν = νas(OCO) −
νs(OCO).
a
For NMR: s: singlet, d: doublet, t: triplet m; multiplet, br: broad, vw: very weak; angle θ calculated from Eq.:
2
2
2
b
1
13
119
c
∠
C−Sn−C = 0.0161| J| - 1.32| J| + 133.4; angle calculated using Eq.: | J( C− Sn)| = 11.4θ –875; trigonal-
bipyramidal to octahedral ratio = 1:5; Carbon and nitrogen numbering is according to Fig. 1;
β
β
γ
α
α
CH2
β
CH2
γ
α
β
γ
δ
Θ
λ
µ
ω
α
δ
δ
;
CH3 ;
CH2
CH3
;
Sn
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
γ
5
.5. In vitro anti-cancer screening and cell viability assays in human cancer cell lines
The studied complexes were screened in vitro against five human cancer cell lines
[
53]. Test and reference compounds were dissolved in dimethyl sulfoxide (DMSO) to form 4
mM stock solutions. These derivatives in DMSO are stable even for more than 15 days. Cell
viability was evaluated by using a colorimetric method based on the tetrazonium salt MTT,
which is reduced in cells to yield purpule formazan crystals. The experiment was started on
3
day 0. On day 0, 5 × 10 cells in 200 ꢂL in medium were seeded in 96-wells flat-bottom
microtitre plates (Griener, Germany). The plates were preincubated for 48 h at 37 °C, 8.5%
2
CO , to allow the cells to adhere. On day 2, serial dilutions (in DMSO) using the stock
solution were prepared. On day 3, DMSO (same volume as used for all dilution) was used as
vehicle control and every dilution (test and reference) was used in triplicate by adding 50 ꢂL
to a column of three wells. Cultures were incubated for 24 h at 37 °C. On day 4, cultures
were assayed by the addition of 25 ꢂL of 5 mg/mL MTT and incubated for 4 h at 37 °C. MTT
containing media was aspirated, 200 ꢂL of DMSO and 25 ꢂL of Sorensen glycine buffer (0.1
M glycine and 0.1 M NaCl, pH 10.5) were added to lyse the cells and solubilize water
insoluble formazon. The absorbance values (OD) of the lysates were determined on a
Dynatech MR 5000 microplate reader at 570 nm. The whole experiment was repeated three
times and the average of three experiments (runs) has been recorded. Data were used for the
2
2

ACCEPTED MANUSCRIPT
construction of concentration-percentage inhibition curves and IC50 values were determined
by Origin Pro8 software. The percentage inhibition was calculated as:
Mean OD of vehicle treated cells (negative control) – Mean OD of drug treated cells
_
____________________________________________________________________ × 100
Mean OD of vehicle treated cells (negative control)
5
.6. Enzyme assays: antioxidant enzyme activities
Lipid peroxidation, glutathione peroxidase, glutathione reductase, total glutathione and
lactate dehyrogenase assays were carried out to determine the effect of the synthesized
complexes on cancerous cell lines, compared to control group of cells (non treated). In brief,
tissue lysate were prepared in PBS pH 7.4, containing 2 mM (PMSF) with sonication (3
bursts of 20 seconds each). Equal amounts of protein samples were taken for all samples in
each enzyme assay.
For the estimation of lipid peroxidation 8.1% SDS, 20% acetic acid (pH 3.5) and
thiobarbituric acid were added to the total protein sample, and incubated at 95 °C for 2 h.
After cooling to RT, n-butanol and pyridine mixture (15:1 v/v) was added to the above
incubated contents. It was centrifuged and OD of the organic layer was measured at 532 nm
[
54].
Glutathione reductase assay was performed according to the reported method [55]. 0.01
mL of 10 mM EDTA, 0.06 mL of 10 mM NADPH, 2 µL β-mercaptoethanol, 0.1 mL of 250
mM GSSG (glutathione oxidized) were added to 0.778 mL of potassium phosphate buffer
solution at pH 7.4. To which 0.05 mL of the total homogenate of protein sample was added
and mixed thoroughly. After incubation for 5 min the absorbance of NADPH was measured
at 340 nm.
Glutathione peroxidase activity was measured by the method of Paglia and Valentine
[
3
56]. Reaction mixture contains 0.2 mM NADPH, 4 mM GSH, 4 mM EDTA, 4 mM NaN , 1
2
3

ACCEPTED MANUSCRIPT
unit (U) of glutathione reductase, 0.2 mM H
00 mM phosphate buffer (pH 7.0). The assay was performed at 37 °C and the rate of
reduction of NADPH was followed at 340 nm.
2 2
O and it was treated with the protein sample in
1
Total glutathione was determined according to the protocol described earlier by [57].
Briefly, the cell pellets were washed with ice cold PBS and resuspended in 300 µL of buffer
containing 125 mM potassium dihydrogen phosphate, 6.3 mM EDTA, pH 7.5 and was
subjected to rapid freeze-thaw processing followed by sonication for 1 min. The protein from
the supernatant obtained thereafter was precipitated with 5% trichloroacetic acid for 15 min.
After centrifugation for 15 min the protein free lysate was then analysed for the total
glutathione content. The reduction of glutathione by glutathione reductase was carried out in
a 1 mL cuvette containing NADPH (0.525 mM), 5’,5’- dithiobis-2-nitrobenzoic acid (1.5
mM) and supernatant in the ratio of 2:2:1 and 1 U/mL of glutathione reductase. The
absorbance of the reaction mixture was measured at 405 nm.
For measuring the lactate dehydrogenase, LDH (enzyme involved in glycolytic
pathways) [58], sodium phosphate buffer 0.1 M, 0.13 mM NADH and 34 mM sodium
pyruvate were added to the protein sample. The absorbance of NADH was estimated at 340
nm.
5
.7. DNA fragmentation assay
The DNA cleavage pattern due to n-Oct Sn(H Or) was analyzed by agarose gel
2
2
2
6
electrophoresis [59]. 3 × 10 MCF-7 cells were exposed to the IC value of n-Oct Sn(H Or) .
50
2
2
2
Cells were centrifuged and washed with PBS (phosphate buffer saline), and the pellet was
lysed in 400 µL with hypotonic buffer solution (10 mM tris pH 7.5, 1 mM EDTA and 0.2%
triton X-100) for 15 min at room temperature, and then centrifuged for 15 min at 13000 rpm.
3
1
50 µL of the supernatant was again lysed in 106 µL of the second lysis buffer containing
50 mM NaCl, 10 mM tris–HCl pH 8.0, 40 mM EDTA, 1% SDS and 0.2 mg/mL Proteinase
2
4

ACCEPTED MANUSCRIPT
K (at final concentration) for 4 h at 37 ºC. The DNA was extracted with phenol/chloroform/
isoamyl alcohol (25:25:1 v/v/v), and the pellet obtained was washed with ethanol and
resuspended for RNAase digestion in 15 µL of 10 mM tris, 1 mM EDTA pH 8.5, and 50
µg/mL RNase for 1 h at 37 ºC . The DNA was analysed by running it at 50 V/cm in a 2%
agarose gel in 0.5X TBE (buffer solution containing a mixture of Tris base, boric acid and
EDTA).
5
.8. Anti-inflammatory activity
The in vivo anti-inflammatory activity of the synthesized complexes was evaluated by
applying the carrageenan induced paw oedema bioassay by the method of Winter et al. [60],
using indomethacin and ibuprofen as reference standard drugs. These studies was approved
by institutional animal esthetical committee and performed in compliance with the relevant
laws and institutional guidelines (Reg. No. 563/02/a/CPCSEA).
A freshly prepared suspension of carrageenan (0.2 mL, 1.0% in 0.9% saline) was
injected subcutaneously into the plantar aponeurosis of the hind paw of Swiss albino mice
(pretreated orally with the test compounds via gavage) of both genders (body weight 25−35
g). One group of five mice was kept as a control and the other group of five mice was
pretreated with the complex/standard drugs given orally 30 min before the carrageenan
injection. The paw volume was measured at the time of treatment and after 1−3 h by a water
plethysmometer socrel. The mean increase of paw volume of treated animals was compared
with the animals of control group (five mice treated with carrageenan, but not with test
compounds) and percent anti-inflammatory value (% inhibition) was calculated as given
below:


DT 
×100
DC
%
anti-inflammatory = 1−
2
5

ACCEPTED MANUSCRIPT
where, DT and DC are the volumes of paw oedema in drug treated and control groups,
respectively.
5
.9. Toxicity
LD50 (average lethal dose at 50% survival) of the synthesized complexes were
investigated in Swiss albino mice (body weight 20−25 g). The test compound was injected
intra-peritoneally at various doses in groups of ten mice and percent mortality in the group
-1
was observed after 1 day. The LD50 value (mg kg ) was calculated with the help of method
of Smith [61] from the data obtained.
5
.10. Statistical analysis
Biological results are expressed as mean ± standard error and their significance is
analysed through unpaired two tailed t test (p< 0.05), using GraphPad PRISM 5.0 software.
Acknowledgments
We are thankful to technical staff of the Chemistry Department and Department of
Biotechnology, I. I. T. Roorkee, for spectroscopic studies, elemental analysis and biological
experiments. Thanks are also due to the Head I.I.C. for providing NMR facility. Ms. Monika
Vats (JRF) is thankful to MHRD, New Delhi, for financial Assistance.
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8