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Artikel Sintesis dan Karakterisasi Senyawa Logam-Fenolik
Artikel Sintesis dan Karakterisasi Senyawa Logam-Fenolik
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
Received 29 October 2013 formation of triorganotin(IV) hydroxycarboxylates, R3Sn(L) [R = Me, n-Bu and Ph; L = anion of glucuronic
Received in revised form 17 January 2014 (HGlu), gallic (HGal) and mandelic (HMal) acid]. They exhibit trigonal–bipyramidal geometry which is
Accepted 22 February 2014
well supported by elemental analysis, IR, 1H, 13C, 119Sn NMR and ESI-MS spectral data. Triorganotin(IV)
Available online xxxx
hydroxycarboxylates have been screened in vitro against five cancer cell lines of human origin viz.
Antitumor Active Organotin Compounds 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 lg/mL. The results of enzyme assays viz. glutathione reduc-
Keywords: tase, glutathione peroxidase, total glutathione content and lipid peroxidase assay on MCF-7 cells indicate
Anti-cancer activity that the reactive oxygen species generated in the cancer cells by triorganotin(IV) hydroxycarboxylates is
Apoptosis responsible for cell death. Marginal increase of lactate dehydrogenase suggests that necrosis is also
DNA cleavage occurring to a small extent. DNA (deoxyribonucleic acid) fragmentation assay, acridine orange assay
Anti-inflammatory activity and comet assay clearly support that the cell death is mainly due to apoptosis. The results obtained
Triorganotin(IV) hydroxycarboxylate 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.
Ó 2014 Elsevier B.V. All rights reserved.
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 (±)-mandelic acid (HMal) and gallic acid (HGal) (Fig. 1), and inves-
[9–23]. Many organotin compounds have been established as tigate their biological activities, viz. in vitro anti-tumor activity
potential cancer chemotherapeutic agents because of their apopto- (against MCF-7, HEK-293, PC-3, HCT-15 and HepG-2), in vivo
tic inducing character [22–26], and they occupy an important place anti-inflammatory activity and toxicity studies. Furthermore, to
in cancer chemotherapy reports [11,12,17b,24]. Furthermore, the view insight the mode of action of the studied complexes various
National Cancer Institute has tested about 2000 tin-based assays, such as lactate dehydrogenase, glutathione reductase, glu-
compounds, the largest number ever tested among metal-based tathione peroxidase, total glutathione content, lipid peroxidase,
drug candidates [27], and recently it has been reported that DNA fragmentation, acridine orange and comet assay have also
four out of thirty interesting inorganic pharmaceuticals are tin been conducted and discussed in this manuscript.
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 2. Material and methods
class of compounds owing to their rich structural chemistry rang-
ing from monomeric, dimeric, tetrameric, oligomeric to polymeric 2.1. Materials
motifs [13,22,23,29–31]. The structures can be easily controlled by
adjusting the carboxylato ligands, tin–R groups and metal-to- The specifications and makes of triorganotin(IV) compounds,
ligand molar ratio [29,32,33]. In addition to their structural diver- and methods to obtain various dried solvents are same as reported
sity, the organotin(IV) carboxylates also present very promising previously [22]. Gallic acid, b-D-glucuronic acid and (±)-mandelic
antitumor activities [11,12,22,23,27,34–42]. Among these, several acid were procured from Alfa Aesar and used as received. Further,
di-n-butyltin(IV) carboxylates have been found to be the most a large number of chemicals used for biological activities are also
active, and in some cases they exhibit much higher activity than same as reported in our previous manuscript [22]. Five cancer cell
clinically used reference compounds such as cis-platin, doxorubi- lines of human origin, viz. MCF-7 mammary cancer, HEK-293 kid-
cine and methotrexate [11,12,27,37]. ney cancer, PC-3 prostate cancer, HCT-15 colon cancer and
The systemic toxicity of chemotherapeutic drugs, their lack of HepG-2 liver cancer were purchased from National Center for Cell
tumor localization and an even distribution throughout the body Science (NCCS) Pune, India. For anti-inflammatory activity and
including tumor tissues are the most significant challenges facing acute toxicity Swiss albino mice were procured from All India
effective cancer chemotherapy. Therefore, a rational drug design Institute of Medical Science (AIIMS) Delhi, India.
is essentially required to optimize specific targeting features and
to control toxicity (side effects), by controlling thermodynamic
2.2. Physical measurements
and kinetic processes of metal complexes viz., choice of metal ion
and its oxidation state [43] or tailoring of ligand scaffold. It is also
The melting points, microanalyses (C, H and N) and tin content
well known that the cytotoxicity is related to some extent to lipo-
were determined as described previously [22]. Molar conductance
philicity of the drugs i.e., the most lipophilic compounds are the
measurements, infrared and far-infrared, nuclear magnetic reso-
most cytotoxic [44]. Further, the ligands having biologically active
nance (1H, 13C and 119Sn) spectra were recorded on the same
pharmacophore with biocompatible properties are tethered to
instruments as reported previously [22]. ESI-MS spectra were
others having multifunctional N and O donors to obtain modulated
recorded on Bruker MicroTOF-Q II mass spectrometer at a flow rate
ligand scaffold which can mute the potential toxicity of metallo-
of 180 lL/h on positive mode in water–acetonitrile mixture.
pharmaceuticals drugs [8].
Optical density (OD) in MTT assay, gel electrophoresis, enzymes
Anti-tumor activity of several organotin compounds has been
assays, in vivo anti-inflammatory activity and toxicity were done
reported by various workers, but their solubility has always
according to the reported procedures with slight modifications as
remained a problem. This can be solved by choosing ligands having
described in our previous manuscript [22].
polar groups such as hydroxy groups, or other oxygen or nitrogen
containing groups or substituted fluorine which may enhance their 0,34024 g
solubility. Glucuronic acid (HGlu), mandelic acid (HMal) and gallic 2.3. Synthesis
acid (HGal) are such hydroxycarboxylic acids which are highly sol-
uble in water, methanol, ethanol and other organic solvents and 2.3.1. Synthesis of sodium salt of hydroxycarboxylic acids
possess various medicinal properties. Glucuronic acid (uronic acid) Hydroxycarboxylic acid (HGlu/HMal/HGal, 2.0 mmol) was
exhibits anti-oxidant activity [45]. Mandelic acid (a-hydroxyl car- dissolved in 10 mL of specially dried ethanol under dry nitrogen
boxylic acid) has been used for the treatment of skin problems, and and added to sodium ethoxide, prepared by reacting sodium
also possess anti-bacterial activity [46]. Gallic acid is a naturally (0.058 g, 2.5 mmol) with dry ethanol (25 mL). The resulting mix-
occurring plant phenol obtained by the hydrolysis of tannins and ture was refluxed giving a clear solution of sodium salt of the acid
is known to display some pharmacological activities such as anti- within half an hour. Refluxing was continued for another 1–2 h
cancer, fungicidal/fungi-static, antiviral, anti-inflammatory and with constant stirring. The solution was concentrated and solid
anti-oxidant properties [47]. was dried in vacuum.
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 (cm1): mas(OCO) 1616s, ms(OCO) 1429m, Dm
NMR spectral analysis [49], di-n-butyltin(IV) and diethyltin(IV) 187, mas(COC) 1245m, ms(COC) 1110m, m(OH-1) 3533s, m(OH-2)
complexes of methoxy analog of gallic acid [50], reaction of ethyl- 3400s, m(OH-3) 3488s, m(OH-4) 3509s. 1H NMR (500.13 MHz,
trichlorostannane with N,N-dimethylamide of mandelic acid [51] Bz-d6, ppm): d 4.63 (d, 1H, H-1, J1,2 = 8.0 Hz), 3.25 (dd, 1H, H-2,
and synthesis of trimethyltin derivative of mandelic acid [52] are J1,2 = 8.0 Hz, J2,3 = 9.1 Hz), 3.50 (dd, 1H, H-3, J2,3 = 9.1 Hz,
few reports available in literature. Moreover, the functional groups J3,4 = 9.2 Hz), 3.96 (dd, 1H, H-4, J3,4 = 9.2 Hz, J4,5 = 9.6 Hz), 3.93 (d,
attached to carboxylic acid may have pronounced influence 1H, H-5, J4,5 = 9.6 Hz), 4.81 (s, 1H, OH-1), 3.55 (s, 1H, OH-2), 3.72
(increase/decrease) on the biological properties of organotin(IV) (s, 1H, OH-3), 3.86 (s, 1H, OH-4). 13C NMR (125.75 MHz, Bz-d6,
carobxylates [22,34–37]. Therefore, it becomes indispensable to ppm): d 95.86 (C-1), 74.53 (C-2), 72.43 (C-3), 72.74 (C-4), 76.48
synthesize organotin(IV) derivatives of b-D-glucuronic acid (HGlu), (C-5), 175.31 (C-6).
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
2.3.1.2. HGlu. IR (cm1): mas(OCO) 1596s, ms(OCO) 1423s, Dm 173, (s, 2H, H-3, H-7), 3.71 (s, 1H, OH-4), 3.82 (s, 1H, OH-5), 3.76 (s,
mas(COC) 1249m, ms(COC) 1115m, m(OH-1) 3530s, m(OH-2) 3405s, 1H, OH-6). 13C NMR (125.75 MHz, CD3OD, ppm): d 167.96 (C-1),
m(OH-3) 3486s, m(OH-4) 3511s. 1H NMR (500.13 MHz, Bz-d6, 128.48 (C-2), 129.84 (C-3, C-7), 137.60 (C-4, C-6), 140.05 (C-5).
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, 2.3.2. Synthesis of triorganotin(IV) derivatives of hydroxycarboxylic
J2,3 = 9.0 Hz, J3,4 = 9.1 Hz), 3.85 (dd, 1H, H-4, J3,4 = 9.1 Hz, acids
J4,5 = 9.5 Hz), 3.76 (d, 1H, H-5, J4,5 = 9.5 Hz), 4.77 (s, 1H, OH-1), A hot ethanolic solution (20 mL) of trimethyltin(IV) chloride
3.49 (s, 1H, OH-2), 3.62 (s, 1H, OH-3), 3.90 (s, 1H, OH-4). 13C (0.398 g, 2.0 mmol) or tri-n-butyltin(IV) chloride (0.651 g,
NMR (125.75 MHz, Bz-d6, ppm): d 95.61 (C-1), 74.49 (C-2), 72.32 2.0 mmol) or triphenyltin(IV) chloride (0.771 g, 2.0 mmol) was
(C-3), 72.68 (C-4), 76.39 (C-5), 173.22 (C-6). 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.
2.3.1.3. Na(Mal). IR (cm1): mas(OCO) 1597w, ms(OCO) 1420s, Dm The resulting solution was refluxed with constant stirring for an-
177, m(OH-2) 3610s. 1H NMR (500.13 MHz, CD3OD, ppm): d 5.11 other 5–6 h under dry nitrogen atmosphere. The resulting solution
(s, 1H, H-2), 7.73 (m, 1H, H-4), 7.31 (m, 2H, H-5 + H-7), 7.21 (m, was then centrifuged and filtered in order to remove the sodium
1H, H-6), 7.52 (m, 1H, H-8), 3.30 (s, 1H, OH-2). 13C NMR chloride formed during the reaction. The excess of solvent was re-
(125.75 MHz, CD3OD, ppm): d 175.33 (C-1), 75.22 (C-2), 136.19 moved under reduced pressure and the products thus obtained
(C-3), 127.21 (C-4), 127.41 (C-5), 126.21 (C-6), 127.33 (C-7), were washed with either ethanol–hexane or ethanol–petroleum
126.06 (C-8). ether (b.p. 40–60 °C) mixture (1:3 v/v).
2.3.1.4. HMal. IR (cm1): 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(OH-2) 3621m. 1H NMR (500.13 MHz, CD3OD, ppm): d 11.81 (s, for C24H24O7Sn: Sn, 22.92; H, 4.42; C, 53.07. Found: Sn, 22.99; H,
1H, (OCO)–H), 5.01 (s, 1H, H-2), 7.70 (m, 1H, H-4), 7.23 (m, 2H, 4.40; C, 53.01%. Mw: 543. IR (cm1): mas(OCO) 1626s, ms(OCO)
H-5 + H-7), 7.19 (m, 1H, H-6), 7.49 (m, 1H, H-8), 3.50 (s, 1H, OH- 1422s, Dm 204, mas(Sn–C) 277s, ms(Sn–C) 240s, mas(COC) 1150m, ms
2). 13C NMR (125.75 MHz, CD3OD, ppm): d 173.33 (C-1), 75.12 (COC) 1005m, m(Sn–O) 546w, m(OH-1) 3480br, m(OH-2) 3379w,
(C-2), 136.12 (C-3), 127.13 (C-4), 127.36 (C-5), 126.16 (C-6), m(OH-3) 3360br, m(OH-4) 3410m. 1H NMR (500.13 MHz, CD3OD,
127.29 (C-7), 125.96 (C-8). 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
2.3.1.5. Na(Gal). IR (cm1): mas(OCO) 1629s, ms(OCO) 1403s, Dm 226, (s, 1H, OH-1), 3.94 (s, 1H, OH-2)a, 3.95 (s, 1H, OH-3), 3.99 (s, 1H,
(OH-4) 3533br, m(OH-5) 3440br, 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,
(500.13 MHz, CD3OD, ppm): d 7.30, 7.89 (s, 2H, H-3, H-7), 3.81 (s, H-d, J = 6.5 Hz). 13C NMR (125.75 MHz, CD3OD, ppm): d 97.24 (C-
1H, OH-4), 3.71 (s, 1H, OH-5), 3.90 (s, 1H, OH-6). 13C NMR 1), 74.42 (C-2), 72.65 (C-3); 73.93b (C-4), 77.51 (C-5), 178.00 (C-
(125.75 MHz, CD3OD, ppm): d 169.68 (C-1), 127.98 (C-2), 130.01 6), 129.93 (C-a,1J(13C–119Sn) = 466.21 Hz, h = 117.65°), 138.12 (C-
(C-3, C-7), 137.89 (C-4, C-6), 140.13 (C-5). b, 2J(13C–119Sn) = 44.33 Hz), 129.52 (C-c, 3J(13C–119Sn) = 68.00 Hz),
130.24 (C-d). 119Sn NMR (186.50 MHz, CD3OD, ppm): d 141.44.
1
2.3.1.6. HGal. IR (cm1): mas(OCO) 1607m, ms(OCO) 1425 s, Dm 182, H NMR (500.13 MHz, CDCl3, ppm): d 4.82 (d, 1H, H-1,
(OH-4) 3540m, m(OH-5) 3509 w, 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,
(500.13 MHz, CD3OD, ppm): d 12.23 (s, 1H, (OCO)–H), 7.28, 7.85 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
4 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,
(C-4), 77.32 (C-5), 176.45 (C-6), 129.80 (C-a, 1J(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 (186.50 MHz, CD3OD, ppm): d 182.51. ESI-MS: 502 [M+H]+, 524
(ML)+, 274 [HSnPh2]+, 198 [H2SnPh]+. [M+Na]+ (100%), 350 (ML)+, 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 (cm1): mas(OCO) 1625m, ms(OCO) Calc. for C20H34O3Sn: Sn, 26.98; H, 7.71; C, 54.42. Found: Sn,
1415s, Dm 210, mas(Sn–C) 557sh, ms(Sn–C) 510s, mas(COC) 1161m, 26.93; H, 7.76; C, 54.49%; Mw: 441. IR (cm1): mas(OCO) 1609s,
ms(COC) 1015m, m(Sn–O) 560w, m(OH-1) 3494br, m(OH-2) 3373br, ms(OCO) 1396s, Dm 213, mas(Sn–C) 546s, ms(Sn–C) 515 m, m(Sn–O)
m(OH-3) 3436 w, m(OH-4) 3419br. 1H NMR (500.13 MHz, CD3OD, 562w, m(OH-2) 3271br. 1H NMR (500.13 MHz, CD3OD, ppm): d
ppm): d 5.01 (d, 1H, H-1, J1,2 = 8.1 Hz), 3.40 (m, 1H, H-2); 3.43 5.90 (s, 1H, H-2), 7.71 (m, 1H, H-4), 7.30 (m, 2H, H-5 + H-7), 7.22
(m, 1H, H-3), 3.89 (m, 1H, H-4), 3.79 (d, 1H, H-5, J4,5 = 9.0 Hz), (mbr, 1H, H-6), 7.60 (m, 1H, H-8), 3.88 (s, 1H, OH-2), 1.35 (t, 6H,
4.82 (s, 1H, OH-1), 3.83 (s, 1H, OH-2), 3.92 (s, 1H, OH-3), 3.98 (s, 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-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
(m, 6H, H-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
CD3OD, ppm): d 97.89 (C-1), 74.41 (C-2), 72.65 (C-3), 73.16b (C- (C-5), 126.46 (C-6), 127.86 (C-7), 126.37 (C-8), 19.71 (C-a, 1J
4), 77.44 (C-5), 176.61 (C-6), 20.26 (C-a, 1J(13C–119Sn) = 454.43 Hz, (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.
14.21 (C-d). 119Sn NMR (186.50 MHz, CD3OD, ppm): d 168.10. ESI-MS: 442 [M+H]+, 464 [M+Na]+, 290 (ML)+ (100%), 731
1
H NMR (500.13 MHz, CDCl3, ppm): d 4.93 (d, 1H, H-1, [M+SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+.
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, 2.3.2.6. Me3Sn(Mal). White solid. M.P.: (123)d 118–120 °C; Yield:
1H, OH-2), 3.92 (s, 1H, OH-3), 3.97 (s, 1H, OH-4), 1.83 (t, 6H, H-a, 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 (cm1): mas(OCO) 1628s,
J = 6.8 Hz). 13C NMR (125.75 MHz, CDCl3, ppm): d 97.06 (C-1), ms(OCO) 1417m, Dm 211, mas(Sn–C) 601s, ms(Sn–C) 566s, m(Sn–O)
74.32 (C-2), 72.38 (C-3), 72.89b (C-4), 77.10 (C-5), 176.40 (C-6), 557w, m(OH-2) 3265br. 1H NMR (500.13 MHz, CD3OD, ppm): d
22.20 (C-a, 1J(13C–119Sn) = 334.65 Hz, h = 106.11°), 27.33 (C-b), 6.62 (s, 1H, H-2), 7.77 (m, 1H, H-4), 7.32 (m, 2H, H-5 + H-7), 7.25
26.82 (C-c, 3J(13C–119Sn) = 81.11 Hz), 12.11 (C-d). 119Sn NMR (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 H-a, 2J(1H–119Sn) = 64.10 Hz, h = 114.93). 13C NMR (125.75 MHz,
[M+Na]+ (100%), 290 (ML)+, 234 [HSnBu2]+, 178 [H2SnBu]+. 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
5.08; C, 30.28%. Mw: 357. IR (cm1): mas(OCO) 1626s, s(OCO) [M+Na]+, 164 (ML)+ (100%), 479 [M+SnMe3]+, 150 [HSnMe2]+,
1413m, Dm 213, mas(Sn–C) 590s, ms(Sn–C) 575s, mas(COC) 1154m, 136 [H2SnMe]+.
ms(COC) 1019m, m(Sn–O) 550w, m(OH-1) 3484br, m(OH-2) 3370br,
m(OH-3) 3429 w, m(OH-4) 3421br. 1H NMR (500.13 MHz, CD3OD, 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, Calc. for C25H20O5Sn: Sn, 22.93; H, 3.85; C, 57.80. Found: Sn, 22.87;
1H, H-3), 3.92 (m, 1H, H-4), 3.75 (d, 1H, H-5, J4,5 = 9.2 Hz,), 4.81 H, 3.89; C, 57.79%. Mw: 519. IR (cm1): mas(OCO) 1642 m, ms(OCO)
(s, 1H, OH-1), 3.89 (s, 1H, OH-2), 3.90 (s, 1H, OH-3), 3.91 (s, 1H, 1413s, Dm 229, mas(Sn–C) 288s, ms(Sn–C) 225m, m(Sn–O) 556w,
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,
1 13
4.3 (C-a, J( C–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, NMR (125.75 MHz, CD3OD, ppm): d 172.43 (C-1), 123.72 (C-2),
CDCl3, ppm): d 4.94 (d, 1H, H-1, J1,2 = 8.1 Hz), 3.37 (m, 1H, H-2), 110.02 (C-3), 144.04 (C-4), 141.41 (C-5), 137.87 (C-6), 109.00 (C-
3.39 (m, 1H, H-3), 3.90 (m, 1H, H-4), 3.70 (d, 1H, H-5, 7), 130.15 (C-a, 1J(13C–119Sn) = 484.57 Hz, h = 119.21°), 137.54 (C-
J4,5 = 9.3 Hz), 4.98 (s, 1H, OH-1), 3.82 (s, 1H, OH-2), 3.92 (s, 1H, b), 129.66 (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 (ML)+
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.
119
Sn NMR (186.50 MHz, CDCl3, ppm): d 49.21. ESI-MS: 358 Calc. for C19H32O5Sn: Sn, 25.93; H, 6.97; C, 49.67. Found: Sn, 25.90;
[M+H]+, 380 [M+Na]+, 164 (ML)+ (100%), 150 [HSnMe2]+, 136 H, 6.92; C, 49.63%. Mw: 459. IR (cm1): mas(OCO) 1637s, ms(OCO)
[H2SnMe]+. 1410s, Dm 227, mas(Sn–C) 550s, ms(Sn–C) 521s, m(Sn–O) 565w,
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 (cm1): mas(OCO) 1650w, ms(OCO) H-a, 2J(1H–119Sn) = 56.17 Hz, h = 110.05°), 1.56 (m, 6H, H-b), 1.21
1427s, Dm 223, mas(Sn–C) 291s, ms(Sn–C) 230s, m(Sn–O) 555w, (m, 6H, H-c), 0.87 (t, 9H, H-d, J = 6.7 Hz). 13C NMR (125.75 MHz,
m(OH-2) 3268br. 1H NMR (500.13 MHz, CD3OD, ppm): d 4.81 CD3OD, ppm): d 173.51 (C-1), 124.02 (C-2), 109.72 (C-3), 143.30
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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 lg/mL of RNAase for
(C-b), 27.35 (C-c:), 13.17 (C-d). 119Sn NMR (186.50 MHz, CD3OD, 1 h at 37 °C.
ppm): d 161.43. ESI-MS: 460 [M+H]+, 482 [M+Na]+, 290 (M-L)+ For agarose gel electrophoresis, 0.4 g of agarose was added to
(100%), 749 [M+SnBu3]+, 234 [HSnBu2]+, 178 [H2SnBu]+. 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
2.3.2.9. Me3Sn(Gal). White solid. M.P.: 230–235 °C. Yield: 68%. Anal. was completely dissolved. When the solution becomes lukewarm,
Calc. for C10H14O5Sn: Sn, 35.74; H, 4.20; C, 36.04. Found: Sn, 35.77; 2 lL of ethidium bromide (10 mg/mL stock) was added. This solu-
H, 4.26; C, 36.09%. Mw: 333. IR (cm1): mas(OCO) 1643s, ms(OCO) tion was then poured into the gel casting tray fitted with appropri-
1421m, Dm 222, mas(Sn–C) 587m, ms(Sn–C) 568 s, m(Sn–O) 551w, ate comb and allowed to solidify. This gel was used for the
m(OH-4) 3441br, m(OH-5) 3432br, m(OH-6) 3415br. 1H NMR submerged electrophoresis for separating the DNA samples using
(500.13 MHz, CD3OD, ppm): d 7.85, 7.92 (s, 2H, H-3, H-7)c, 4.17 suitably 1 or 0.5 TAE as the running buffer. DNA samples were
(s, 1H, OH-4), 3.75 (s, 1H, OH-5), 4.73 (s, 1H, OH-6), 0.04, 0.08 (s, mixed with gel loading dye and loaded into the wells along with
9H, H-a). 13C NMR (125.75 MHz, CD3OD, ppm): d 174.10 (C-1), the controls. Gel was run at 80 V for 1–2 h till the dye front reached
123.91 (C-2), 110.18 (C-3), 144.52 (C-4), 142.31 (C-5), 137.28 the last one-third of the gel and was observed in a UV illuminator.
(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 (ML)+ (100%), 497 2.4.2. Acridine orange assay
[M+SnMe3]+, 150 [HSnMe2]+, 136 [H2SnMe]+. Acridine orange staining is one of the acceptable assays for
Mw: molecular weight; IR: s, strong; m, medium; w, weak; sh, evaluating the apoptosis by analysing the plasma-membrane
shoulder; br, broad; Dm = mas(OCO) ms(OCO); permeability, nuclear morphology as well as the chromatin con-
NMR: hydrogen and carbon number according to Fig. 1; s, singlet; densation [55,56]. In the assay, the cancer cells were stained with
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; angle h acridine orange (AO)/ethidium bromide (EB) dye mixture (100 lg/
calculated from Eq.: \C–Sn–C = 0.0161|2J|2 1.32|2J| + 133.4 [32] mL of AO and 100 lg/mL of EB) and assay was performed according
and |(1J(13C119Sn)| = 11.4h 875; avery weak intensity signal; bbroad to the method described by Takahashi et al. [56]. Briefly, 0.5 106
signal; c the signals of the two hydrogens may interexchange; cancer (MCF-7) cells were seeded in a 6-well plate and incubated
d
According to Ref. [52]; with the respective IC50 value of n-Bu3Sn(Gal) and cis-platin
(positive control) for 24 h and then washed with PBS. After
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).
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6 M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
3. Results and discussion found to be comparable with that of sodium salt of the gallic acid,
which indicates that the carboxylate group is bidentate and
3.1. Synthetic aspects bridging [22,59,60]. The disappearance of a broad band (2833–
2957 cm1) in the spectra of the triorganotin(IV) hydroxycarboxy-
The reaction of triorganotin chloride with sodium salt of lates (which was present in the free acids as a weak intensity
hydroxycarboxylic acid (formed according to Eq. (1)) in a 1:1 M band), suggests the deprotonation of the free (COOH) group upon
ratio led to the formation of complexes according to Eq. (2). complexation. Furthermore, appearance of a m(Sn–O) at
556 ± 10 cm1 supports the bonding of carboxylate oxygen with
HL þ NaOEt !NaL þ EtOH ð1Þ
the tin atom. The ms(Sn–C) and mas(Sn–C) have been observed at
their corresponding frequencies and are in accordance with the lit-
HL ¼ HGlu; HMal and HGal
erature values [21–23]. The m(O–H) of complexes broadens and
shifts towards lower frequency, indicating the involvement of hy-
1:1
droxy (OH) group in intra-/intermolecular hydrogen bonding.
It has been observed from the IR spectra of R3Sn(Glu) that the
R3 SnCl þ NaL!R3 SnðLÞ þ NaCl 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
R ¼ Me; n-Bu and Ph
oxygen may also be participating in weak interaction with organo-
The reaction in Eq. (1) required 1–2 h and Eq. (2) required 5–6 h tin(IV) moiety. The carboxylate groups in organotin(IV) carboxyl-
of refluxing. The resulting products were obtained in good yield ates generally adopt a bridge structure in the solid state, unless
(51–68%). From the analytical data it can be inferred that the the organic substituent at the tin atom is bulky or carboxylate
resulting triorganotin(IV) hydroxycarboxylates crystallized with group is branched at the a-carbon [29]. Mandelic acid (a-hydroxy-
1:1 stoichiometry. R3Sn(Glu) are soluble in water/DMSO mixture, carboxylic acid) is branched at a-carbon, therefore, due to its
methanol, ethanol, ether, DMSO, benzene and chloroform, whereas expected steric property; the bridging through carboxylate group
R3Sn(Mal) and R3Sn(Gal) are soluble in DMSO and water/DMSO can be excluded [29]. But there is a possibility of hydroxy (OH)
mixture, show low solubility in ethanol, methanol, and insoluble group participation in bridging or interaction with tin, which has
in non-polar solvents. The synthesized complexes are found been supported by the relative lowering of m(OH) of the complexes
to be stable towards air and moisture. Diorganotin(IV) derivatives (3265–3271 cm1) in comparison with free mandelic acid
of these hydroxycarboxylic acids have not been obtained (3621 cm1) [52].
in pure form under the conditions used for triorganotin(IV)
hydroxycarboxylates. 3.4. Multinuclear (1H, 13
C and 119
Sn) NMR spectral studies
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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 midal geometry about tin. The proposed structures for the studied
five-coordinated geometry in coordinating solvent. The \CSnC triorganotin(IV) hydroxycarboxylates have been presented in
calculated for Me3Sn(Glu) in CDCl3 (106.47°) and CD3OD Fig. 2.
(116.51°) further support pseudo-tetrahedral geometry in CDCl3
and distorted trigonal–bipyramidal geometry in CD3OD. 3.5. ESI-MS spectrometric studies
3.4.2. 13C NMR spectra The ESI-MS spectral data for triorganotin(IV) hydroxycarboxy-
From 13C NMR spectral data of hydroxycarboxylic acids and lates have been recorded in the range m/z 50–1000, using metha-
their triorganotin(IV) derivatives it has been inferred that the nol as solvent and further diluting with acetonitrile and water
signals attributable to –(OCO) show significant downfield shift mixture. In the ESI-MS spectra of triorganotin(IV) hydroxycarboxy-
upon complex formation, which suggests coordination of triorga- lates, the weak molecular ion peak ([M+H]+) appears, which has
notin(IV) moiety with carboxylate group. been used for determining the molecular weight (Mw) of the com-
R3Sn(Glu) shows an interesting coordination behavior. The plexes. Mw for Ph3Sn(Glu), n-Bu3Sn(Glu) and Me3Sn(Glu) have
carbon atom C4 exhibits downfield shift, while C2 remaines unaf- been observed at 543, 483 and 357, respectively; for Ph3Sn(Mal),
fected, and C1 and C3 show only slight change in the chemical n-Bu3Sn(Mal) and Me3Sn(Mal) at 501, 441 and 315, respectively;
shifts. A small downfield shift for the C5 has also been observed, and for Ph3Sn(Gal), n-Bu3Sn(Gal) and Me3Sn(Gal) at 519, 459,
indicating that weak interaction of ring oxygen with triorgano- and 333, respectively. The observed molecular weights (Mw) of tri-
tin(IV) moiety may be possible [49,50]. However, the shifts are organotin(IV) hydroxycarboxylates are in excellent agreement
very small and hence the interactions between ring oxygen and with the calculated values, which verify the proposed molecular
tin atom can be excluded. formula for the complexes. In the ESI-MS spectra of triorgano-
In R3Sn(Mal), the C2 shows a significant downfield shift, which tin(IV) hydroxycarboxylates, [SnR3]+ ion peaks have been observed,
supports the weak interaction between oxygen of hydroxyl group which are due to loss of one mole of ligand from one mole of com-
(OH group) and tin atom. All the other carbon atoms of the acid plex, involving the cleavage of weak Sn–O bond yielding two com-
either remained unaffected or very small change in chemical shifts plementary ions, where the cationic part of the complex is
have been observed for them. A significant change in the chemical measured in positive mode [63]. Triorganotin(IV) hydroxycarboxy-
shift of C4 and C6 in 13C NMR spectra of R3Sn(Gal) has been ob- lates often adopt polymeric structure with five-coordinated tin and
served, which suggests hydrogen bonding through OH groups at the peak corresponding to [M+SnR3]+ provides additional proof for
meta positions of the acid. penta-coordinated linear polymeric motif [64]. The [M+SnR3]+
The 13C NMR chemical shifts of methyl, n-butyl and phenyl peaks have also been observed for R3Sn(Mal) and R3Sn(Gal), which
carbons attached to tin are observed at comparable positions as re- suggest penta-coordinated polymeric nature of the complexes. The
ported previously. The heteronuclear coupling constant subsequent fragment ions [HSnR2]+ and [H2SnR]+ resulted from
1 13
J( C–119Sn) provides useful information about the coordinating [SnR3]+ have been reported earlier in the literature for other
environment and geometry of organotin(IV) complexes. The satel- triorganotin(IV) carboxylates [65], similar peaks have also been ob-
lites have been resolved for most of the complexes and the served in ESI-MS spectra of the synthesised triorganotin(IV)
observed coupling constants 1J(13C–119Sn) are found to be in the hydroxycarboxylates. The ESI-MS spectrometric data do not pro-
range 465.60 ± 20 Hz. These values suggest penta-coordinated vide any crucial information about the structure of the complexes,
organotin moiety for R3Sn(Mal) and R3Sn(Gal). Further, the calcu- except that R3Sn(Mal) and R3Sn(Gal) may be polymeric in nature,
lated values of \CSnC (116.01–119.21°) with the help of Lockhart however the data helped in confirming the Mw and empirical for-
and Manders equation [62] {Eq. 1J(13C–119Sn) = 11.4h 875}, sug- mula of the complexes.
gest distorted trigonal–bipyramidal geometries for the complexes.
The 1J(13C–119Sn) values for R3Sn(Glu) have been observed in 3.6. Biological studies
the range 329.07–350.33 Hz in CDCl3, while 454.43–466.21 Hz in
CD3OD, indicating four-coordinated geometry in non-coordinating All the three hydroxycarboxylic acids viz. glucuronic acid,
solvent and five-coordinated geometry in coordinating solvent for mandelic acid and gallic acid are biologically important molecules
the complexes. The \CSnC calculated for R3Sn(Glu) in CDCl3 and individually known for their therapeutic applications. Thus, it
(107.46–105.61°) and CD3OD (117.68–115.97°) further support becomes indispensable to evaluate the biological behavior of syn-
the pseudo-tetrahedral geometry in CDCl3 and distorted trigonal– thesized triorganotin(IV) hydroxycarboxylates especially for their
bipyramidal geometry in CD3OD. anti-cancer and anti-inflammatory activities.
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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)solvent), (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.
moderately cytotoxic in nature. Triorganotin(IV) hydroxycarboxy- xybenzo-18-crown-6 and -15-crown-5 are less active than the
lates exhibit activity in the range 4–30 lg/mL. Therefore, it can corresponding triphenyltin derivatives but they are more active
be concluded that the complexes are cytotoxic to moderately cyto- than di-n-butyltin analogues [12b]. Triphenyltin derivatives of
toxic in nature. R3Sn(Gal) (gallic acid derivatives) are cytotoxic substituted-phenylacetic acid/-furoic acid/1,4-benzodioxane-6-
against MCF-7, HEK-293 and PC-3 and moderately cytotoxic carboxylic acid are more active than diphenyltin analogues [39].
against HCT-15 and HepG-2. R3Sn(Mal) and R3Sn(Glu) (mandelic However, in the present study tri-n-butyltin carboxylates are more
and glucuronic acid derivatives) exhibit moderate activity against active than the corresponding trimethyltin derivatives which are in
all cell lines. It has been observed that the activity increases as turn more active than the corresponding triphenyltin derivatives.
the alkyl carbon chain length increases, and further, the alkyl The highest activity of the tri-n-butyltin carboxylates may also
derivatives are more active than phenyl derivatives, similar results be related to the most lipophilic character of the n-butyl groups
have also been reported earlier [27]. as the lipophilicity of the more stable C-bound groups on tin is
The first systematic structure–activity relationships on several important in controlling their toxicity [27]. Further, the studied tri-
di- and tri-organotin(IV) carboxylates made by Gielen and his organotin derivatives of gallic acid are more active than those of
co-workers [12,27] reveals that di-n-butyltin(IV) carboxylates are mandelic acid which are in turn more active than those of glucu-
the most active compounds, and some of them exhibit much high- ronic acid.
er potency [11,12,27,37]. Further, tri-n-butyltin difluorobenzoates The structure–activity correlation for the studied triorgano-
are less active than the corresponding triphenyltin derivatives tin(IV) hydroxy-carboxylates reveals that the substituent attached
and also less active than the corresponding di-n-butyltin to OAC@O group has pronounced effect on the activity. Moreover,
derivatives [12a]. Similarly, tri-n-butyltin 3,6-dioxaheptanoates/ the activity of organotin compounds also depends on availability of
3,6,9-trioxadecanoates and tri-n-butyltin derivatives of 4-carbo- 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
Table 2
Enzymatic assays of n-Bu3Sn(Gal) against MCF-7.
Enzymes Activity
Untreated Treated Induction fold
a
Lipid peroxidation 4.58 ± 0.65 9.89 ± 0.11 2.16
Glutathione peroxidaseb 8.31 ± 0.03 9.81 ± 0.06 1.18
Total Glutathionec 6.31 ± 0.57 6.52 ± 1.25 1.03
Glutathione reductaseb 6.31 ± 0.027 4.95 ± 0.12 0.78
Lactate dehydrogenaseb 7.82 ± 0.03 8.05 ± 0.18 1.03
a
nmol/mg protein.
b
lmol/mg protein/min.
c
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.
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10 M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
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- under fluorescent microscope. The Fig. 8 represents the single cell
gests that apoptotic pathway is mainly responsible for the cell gel (alkaline) electrophoresis showing DNA fragmentation in MCF-
death. 7 cells treated with n-Bu3Sn(Gal) in comparison to cis-platin and
control. The DNA breaks or fragmentation from individual cell
3.6.4. Acridine orange assay has been observed as comet tails (marked by arrows). However,
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).
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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12 M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx
Table 3
Anti-inflammatory activity and toxicity data of triorganotin(IV) hydroxycarboxylates.
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M. Nath et al. / Inorganica Chimica Acta xxx (2014) xxx–xxx 13
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Gielen, E.R.T. Tiekink (Eds.), Metallotherapeutic Drugs and Metal-Based [44] M. Hanif, S.M. Meier, W. Kandioller, A. Bytzek, M. Hejl, C.G. Hartinger, A.A.
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