Preparation and in vitro investigations of triphenyl[ω-(tetrahydro-2H-pyran-2-yloxy)alkyl]tin(IV) compounds

Article abstract of DOI:10.1002/aoc.3630

The reaction of SnPh₃Li with X(CH₂)_nO–THP (THP?=?tetrahydro-2H-pyran-2-yl; n?=?3, 4, 6, 8, 11; X?=?Cl, Br) afforded organotin(IV) compounds with the general formula Ph₃Sn(CH₂)_nO–THP (1–5). The tetraorganotin(IV) compounds were characterized using multinuclear NMR and infrared spectroscopies and high-resolution mass spectrometry. Anticancer activity of the synthesized compounds was tested in vitro against the A2780 (ovarian), A549 (lung), HeLa (adenocarcinoma) and SW480 (colon) tumour cell lines with SRB assay. The in vitro investigations revealed that when a shorter chain was present a higher activity was achieved; however compounds 1–5 were found to be less active than cisplatin. In addition, the most active compound, 1, enters A2780 cells and causes apoptosis by triggering both intrinsic and extrinsic caspase pathways.

Full text of DOI:10.1002/aoc.3630

Received: 20 May 2016
DOI 10.1002/aoc.3630
Revised: 17 August 2016
Accepted: 21 August 2016
F U L L PA P E R
Preparation and in vitro investigations of triphenyl[ω‐(tetrahydro‐
2H‐pyran‐2‐yloxy)alkyl]tin(IV) compounds
2
1
David Edeler1,2† | Christian Bensing2† | Harry Schmidt | Goran N. Kaluđerović
*
1 Department of Bioorganic Chemistry, Leibniz
The reaction of SnPh3Li with X(CH2)nO–THP (THP = tetrahydro‐2H‐pyran‐2‐yl;
n = 3, 4, 6, 8, 11; X = Cl, Br) afforded organotin(IV) compounds with the general
formula Ph3Sn(CH2)nO–THP (1–5). The tetraorganotin(IV) compounds were char-
acterized using multinuclear NMR and infrared spectroscopies and high‐resolution
mass spectrometry. Anticancer activity of the synthesized compounds was tested
in vitro against the A2780 (ovarian), A549 (lung), HeLa (adenocarcinoma) and
SW480 (colon) tumour cell lines with SRB assay. The in vitro investigations
revealed that when a shorter chain was present a higher activity was achieved; how-
ever compounds 1–5 were found to be less active than cisplatin. In addition, the
most active compound, 1, enters A2780 cells and causes apoptosis by triggering
both intrinsic and extrinsic caspase pathways.
Institute of Plant Biochemistry, Weinberg 3,
D‐06120 Halle (Saale), Germany
2 Institute of Chemistry, Martin Luther University
Halle‐Wittenberg, Kurt‐Mothes‐Straße 2, D‐06120
Halle (Saale), Germany
Correspondence
Goran N. Kaluđerović, Department of Bioorganic
Chemistry, Leibniz Institute of Plant Biochemistry,
Weinberg 3, D‐06120 Halle (Saale), Germany.
Email: goran.kaluderovic@ipb‐halle.de
†These authors equally contributed.
KEYWORDS
apoptosis, caspase, cytotoxicity, organotin(IV) compounds
1
|
INTRODUCTION
and 1,2,4‐benzenetricarboxylato‐1,2‐anhydride).[21] Mostly,
organotin(IV) compounds induce apoptosis in various cancer
cell lines.[17]
Platinum‐based compounds have been in focus for many
years as potential antitumor agents since the discovery of cis-
platin in the 1960s.[1–8] In the meantime, in the same field
research is also directed to the development of non‐platinum
metal‐based compounds (e.g. Ru(II), Ti(IV), Au(III), Ga(III),
Sn(IV))[9–13] because they can have different targets in cells
and might exhibit mechanisms of action in cancer cells other
than those of platinum‐based ones. Tin(IV) compounds are
interesting because of well‐known multiple applications:
industrial and agricultural applications as well as biological
(biocides, antimicrobials and potential antiviral and antican-
cer agents).[14–17] In the last few decades the anti‐tumour
activity of organotin(IV) compounds has been quite well
investigated.[9,18,19] Some of the organotin(IV) compounds,
e.g. tricyclohexyltin(IV) derivatives, are capable of overcom-
ing the multidrug resistance noticed for other commercial
metal‐containing drugs,[20] or are more selective towards
tumour cells than cisplatin, such as [NHEt3][SnPh3ClL] com-
pounds (L = N‐phthaloylglycinato, N‐phthaloyl‐L‐alaninato
Various mono‐ and polynuclear organotin(IV) com-
pounds comprised of di‐ or triorganyltin(IV) (organyl =
methyl, phenyl, etc.) and carboxylato or thiolato ligands
showed high in vitro anticancer activity.[22–28] Generally,
the most active are found to be triphenyltin(IV) compounds
(mostly in nM concentration), much more active than cis-
platin. On the other hand, less active are found to be tin(IV)
compounds bearing Me groups, as well being less active than
cisplatin. However, in the literature are described some com-
pounds which do not follow such structure–activity
relationship.
Herein is reported the synthesis and structural
characterization of triphenyltin(IV) compounds containing
ω‐(tetrahydro‐2H‐pyran‐2‐yloxy)alkyl moieties (alkyl
n‐propyl (1), n‐butyl (2), n‐hexyl (3), n‐octyl (4), n‐undecyl
(5)). Furthermore, the cytotoxic activity of 1–5 against ovar-
ian A2780, lung A549, adenocarcinoma HeLa and colon
SW480 tumour cell lines was investigated. Accumulation
in cells, induction of apoptosis and caspase activation of
the most active compound, 1, were tested against the
A2780 cell line.
=
*Dedicated to Prof. Dr Dirk Steinborn on the occasion of his 70th birthday.
Appl Organometal Chem 2016; 1–6
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
EDELER ET AL.
2
| EXPERIMENTAL
2.1 | Materials and methods
Synthesis of 1–5 was performed under an atmosphere of dry
argon using standard Schlenk techniques. Before use,
solvents were distilled from the appropriate drying agents
and degassed. NMR spectra were recorded on a Varian
FIGURE 1 Numeration of C atoms.
(m), 668 (w), 380 (m), 331 (s). 1H NMR (CDCl3,
400 MHz, δ, ppm): 1.49–1.90 (m, 8H; Cβ,γ,δ,1H2),
2.09–2.19 (m, 2H; C2H2), 3.44–3.53 and 3.80–3.91 (m, 4H;
Cε,3H2), 4.52–4.56 (m, 1H; CαH), 7.40–7.45 (m, 9H; Ar‐Hm
and Ar‐Hp), 7.60–7.66 (m, 6H; Ar‐Ho). 13C NMR (CDCl3,
1
INOVA UNITY 500 NMR spectrometer (11.74 T, H NMR
(499.75 MHz), internal standard solvent; 13C NMR
(125.7 MHz), internal standard solvent; 119Sn NMR
(186.3 MHz), external standard (CH3)4Sn (0.0 ppm)) and
1
on a Varian VNMRS 400 NMR spectrometer (9.35 T, H
1
101 MHz, δ, ppm): 7.2 (C1, J(119Sn,C) = 396.7 Hz), 19.7
NMR (399.96 MHz), internal standard solvent; 13C NMR
(100.6 MHz), internal standard solvent; 119Sn NMR
(149.1 MHz), external standard (CH3)4Sn). Electrospray
high‐resolution mass spectra (HR‐ESI‐MS) were obtained
in positive‐ and negative‐ion modes on a Bruker Apex III
(FTR‐ICR) mass spectrometer. Infrared (IR) spectra were
measured from 4000 to 250 cm−1 on a Bruker Tensor 27
FT‐IR spectrometer with diamond ATR. Commercially
available reagents Ph3SnCl and 3,4‐dihydro‐2H‐pyran
(THP) were purchased from Acros and used without further
purification, while 2‐[(3‐chloropropyl)oxy]tetrahydropyrane,
2‐[(4‐chlorobutyl)oxy]tetrahydropyrane, 2‐[(6‐chlorohexyl)-
oxy]tetrahydropyrane, 2‐[(8‐bromoctyl)oxy]tetrahydropyrene
(Cγ), 25.5 (Cδ), 26.7 (C2, 2J(119Sn,C) = 20.0 Hz), 30.7
(Cβ), 62.4 (Cε), 70.4 (C3, 3J(119Sn,C) = 72.1 Hz), 99.0
(Cα), 128.5 (Cx, 3J(119Sn,C) = 48.9 Hz), 128.9 (Cp, 4J
2
(
119Sn,C) = 11.0 Hz), 137.1 (Co, J(119Sn,C) = 36.1 Hz),
1
138.9 (Ci, J(119Sn,C) = 489.4 Hz). 119Sn NMR (CDCl3,
149 MHz, δ, ppm) = −99.0 (s, Ph3Sn).
Triphenyl[4‐(tetrahydro‐2H‐pyran‐2‐yloxy)butyl]-
stannane (2). Purification: n‐hexane/ethyl acetate/acetic acid
(8/2/0.01). Yield 0.375 g (18%); pale yellow, highly viscous
oil, odourless. HR‐ESI‐MS: m/z for [C27H32O2NaSn]+ calcd:
531.1316, found: 531.1316. IR (ATR, cm−1): 3445 (b), 2997
(w), 2914 (w), 1661 (b), 1436 (w), 1407 (w), 1312 (w), 1020
1
(vs), 953 (m), 698 (m), 668 (w), 382 (m), 332(s). H NMR
and
2‐[(11‐bromoundecyl)oxy]tetrahydropyrane
were
(CDCl3, 400 MHz, δ, ppm): 1.49–1.93 (m, 12H; Cβ,γ,δ,1–
3H2), 3.39–3.46 and 3.74–3.79 (m, 2H; CεH2), 3.47–3.53
and 3.80–3.87 (m, 2H; C4H2), 4.55–4.59 (m, 1H; CαH),
7.38–7.44 (m, 9H; Ar‐Hm and Ar‐Hp), 7.57–7.63 (m, 6H;
synthesized using procedures from the literature.[29]
2.2 | General procedure for synthesis of triphenyl[ω‐
(tetrahydro‐2H‐pyrane‐2‐yl‐oxy)alkyl]stannanes (1–5)
Ar‐Ho). 13C NMR (CDCl3, 101 MHz, δ, ppm): 10.8 (C1, J
1
(
119Sn,C) = 394.5 Hz), 19.6 (Cγ), 23.4 (C2, 2J(119Sn,
3
The synthesis was carried out following known literature pro-
cedures.[30–33] Elemental lithium (2.5 eq, 180 mg) was added
toTHF (20 ml) and refluxed for 30 min. Triphenyltin chloride
(1 equation 4.2 g) was added to the reaction mixture, which
turned to dark green, and refluxed for 6 h with vigorous
stirring. After completion of the reaction, the mixture was fil-
tered through glass wool. The particular 2‐[(ω‐halogenalkyl)
oxy]tetrahydropyrane (0.5 eq.) was added via a syringe and
the reaction mixture was stirred overnight at room tempera-
ture. To complete the conversion, necessary for 3–5, the reac-
tion mixture was heated for 6 h under reflux. The dark green
solution was quenched with HCl (2.5 ml), the reaction
mixture turning orange. To complete hydrolysis, the solution
was stirred for 30 min at room temperature. The solvent was
distilled off via rotary evaporation. Colum chromatography
yielded pure compounds. Figure 1 shows the numeration of
C atoms.
C) = 20.9 Hz), 25.6 (Cδ), 30.7 (Cβ), 34.1 (C3, J(119Sn,
C) = 64.1 Hz), 62.1 (Cε), 66.7 (C4), 98.7 (Cα), 128.5 (Cx,
3J(119Sn,C) = 48.9 Hz), 128.9 (Cp, J(119Sn,C) = 11.0 Hz),
4
137.1 (Co, 2J(119Sn,C) = 36.1 Hz), 138.9 (Ci, 1J(119Sn,
C) = 489.4 Hz). 119Sn NMR (CDCl3, 149 MHz, δ, ppm):
−100.1 (s, Ph3Sn).
Triphenyl[6‐(tetrahydro‐2H‐pyran‐2‐yloxy)hexyl]-
stannane (3). Purification: n‐hexane/ethyl acetate/acetic acid
(9/1/0.01). Yield 1.166 g (47%); pale yellow, highly viscous
oil, odourless. HR‐ESI‐MS: m/z for [C29H36O2NaSn]+ calcd:
559.1629, found: 559.1631. IR (ATR, cm−1): 2996 (w), 2912
(w), 1435 (w), 1405 (w), 1311 (w), 1042 (vs), 952 (m), 700
(m), 668 (w), 382 (m), 331 (s). 1H NMR (CDCl3,
400 MHz, δ, ppm): 1.33–1.91 (m, 16H; Cβ,γ,δ,1–5H2),
3.38–3.42 and 3.70–3.77 (m, 2H; CεH2), 3.50–3.57 and
3.87–3.94 (m, 2H; C6H2), 4.58–4.61 (m, 1H; CαH),
7.39–7.45 (m, 9H; Ar‐Hm and Ar‐Hp), 7.57–7.63 (m, 6H;
1
Triphenyl[3‐(tetrahydro‐2H‐pyran‐2‐yloxy)propyl]-
stannane (1). Purification: n‐hexane/ethyl acetate/acetic acid
(7/3/0.01). Yield 1.536 g (68%); pale yellow, highly viscous
oil, odourless. HR‐ESI‐MS: m/z for [C26H30O2NaSn]+ calcd:
517.1160, found: 517.1160. IR (ATR, cm−1): 2995 (w), 2912
(w), 1436 (w), 1407 (w), 1309 (w), 1042 (vs), 954 (m), 697
Ar‐Ho). 13C NMR (CDCl3, 101 MHz, δ, ppm): 11.1 (C1, J
(
119Sn,C) = 396.8 Hz), 19.8 (Cγ), 25.6 (Cδ), 25.8 (C4), 26.6
2
(C2, J(119Sn,C) = 22.1 Hz), 29.7 (C5), 30.8 (Cβ), 34.1 (C3,
3J(119Sn,C) = 62.2 Hz), 62.3 (Cε), 66.6 (C6), 99.8 (Cα),
128.5 (Cx, 3J(119Sn,C) = 48.3 Hz), 128.8 (Cp, 4J(119Sn,
C) = 10.9 Hz), 137.1 (Co, J(119Sn,C) = 35.1 Hz), 139.1
2

EDELER ET AL.
3
(Ci, 1J(119Sn,C) = 479.4 Hz). 119Sn NMR (CDCl3, 149 MHz,
δ, ppm): −99.7 (s, Ph3Sn).
foetal calf serum and 1% antibiotics (culture medium) at 37°
C in a humidified atmosphere with 5% CO2. Stock solutions
of investigated compounds (20 mM) were freshly prepared
in dimethylsulfoxide and diluted to various working concen-
trations with medium.
Triphenyl[8‐(tetrahydro‐2H‐pyran‐2‐yloxy)octyl]-
stannane (4). Purification: n‐hexane/ethyl acetate/acetic acid
(9/1/0.01). Yield 0.704 g (32%); pale yellow, highly viscous
oil, odourless. HR‐ESI‐MS: m/z for [C31H40O2NaSn]+ calcd:
587.1942, found: 587.1949. IR (ATR, cm−1): 2925 (w), 2850
(w), 1428 (m), 1117 (w), 1074 (m), 1023 (s), 904 (w), 868
(w), 813 (w), 727 (s), 698 (vs), 658 (w), 445 (m), 380 (w),
2.3.1
| SRB assay
The viability of adherent viable cells was measured by SRB
assay.[34,35] Cells were exposed to a wide range of doses of
the compounds for 96 h and then fixed with 10% trichloro-
acetic acid for 2 h at 4°C. After fixation, cells were washed
in distilled water, stained with 0.4% SRB solution for
30 min at room temperature, washed and dried overnight.
The dye was dissolved in 10 mM Tris buffer and the absor-
bance was measured at 570 nm. IC50 values, defined as the
concentrations of the compounds at which 50% cell inhibi-
tion occurs, were calculated using four‐parameter logistic
function and presented as the mean from three independent
experiments.
1
332 (w), 261 (s). H NMR (CDCl3, 500 MHz, δ, ppm):
1.25–1.91 (m, 20H; Cβ,γ,δ,1–7H2), 3.37–3.44 and 3.73–3.79
(m, 2H; CεH2), 3.50–3.57 and 3.88–3.94 (m, 2H; C8H2),
4.62 (br, 1H; CαH), 7.40 (br, 9H; Ar‐Hm and Ar‐Hp),
7.56–7.62 (m, 6H; Ar‐Ho). 13C NMR (CDCl3, 126 MHz, δ,
1
ppm): 11.1 (C1, J(119Sn,C) = 398.5 Hz), 19.6 (Cγ), 25.5
(Cδ), 26.1 (C4), 26.5 (C2, 2J(119Sn,C) = 23.3 Hz), 28.9,
3
29.3 (C5 and C6), 29.7 (C7), 30.8 (Cβ), 34.1 (C3, J(119Sn,
C) = 61.8 Hz), 62.2 (Cε), 67.6 (C8), 98.7 (Cα), 128.4 (Cx,
4
3J(119Sn,C) = 48.1 Hz), 128.7 (Cp, J(119Sn,C) = 11.1 Hz),
137.0 (Co, 2J(119Sn,C) = 35.8 Hz), 139.1 (Ci, 1J(119Sn,
C) = 479.7 Hz). 119Sn NMR (CDCl3, 186 MHz, δ, ppm):
−99.5 (s, Ph3Sn).
2.3.2
| Inductively coupled plasma (ICP) MS analysis
A2780 cells (1.2 × 106) were seeded in a flask. After 24 h,
cells were treated with IC50 dose of 1 (10 ml) for 24 h. After-
wards the cells were washed with PBS (4 ml) and trypsinized
(1 ml). Cells were resuspended in PBS (4 ml) and centrifuged
(5000 U min−1, 5 min). Cell pallet was washed twice with
PBS (1 ml). Lyophilized cells were analysed using ICP MS
(Wessling Laboratorien GmbH, Oppin, Germany).
Triphenyl[11‐(tetrahydro‐2H‐pyran‐2‐yloxy)undecyl]-
stannane (5). Purification: n‐hexane/ethyl acetate/acetic acid
(7/3/0.01). Yield 1.020 g (57%); pale yellow, highly viscous
oil, odourless. HR‐ESI‐MS: m/z for [C34H46O2NaSn]+ calcd:
629.2412, found: 629.2413. IR (ATR, cm−1): 3445 (b), 3000
(w), 2915 (w), 1661 (b), 1434 (w), 1405 (w), 1310 (w), 1023
1
(vs), 952 (m), 698 (m), 670 (w), 381 (m), 332 (s). H NMR
(CDCl3, 400 MHz, δ, ppm): 1.19–1.93 (m, 31H; Cβ,γ,δ,1–
10H2), 3.39–3.46 and 3.74–3.82 (m, 2H; CεH2), 3.50–3.58
and 3.88–3.96 (m, 2H; C11H2), 4.60–4.65 (m, 1H; CαH),
7.37–7.43 (m, 9H; Ar‐Hm and Ar‐Hp), 7.55–7.62 (m, 6H;
2.3.3
| Morphological analysis (acridine orange (AO)/
ethidium bromide (EB) double staining)
Compound 1‐ and cisplatin‐induced cell death in A2780
ovarian tumour cells was determined using AO and EB
double staining according to standard procedures and exam-
ined under a fluorescence microscope.[36] A2780 cells were
seeded overnight on eight‐chamber slides (6000 cells per
chamber) in 400 μl of complete medium. The next day, cells
were treated with 2 × IC50 of the investigated compounds for
24 h. After this period, cells on the slides were stained with
AO/EB mixture (3 μg ml−1 AO and 10 μg ml−1 EB in
PBS), and visualized under a fluorescence microscope
(Carl Zeiss).
1
Ar‐Ho). 13C NMR (CDCl3, 101 MHz, δ, ppm): 11.2 (C1, J
(
119Sn,C) = 398.0 Hz), 19.7 (Cγ), 25.6 (Cδ), 26.3 (C4), 26.6
2
(C2, J(119Sn,C) = 22.3 Hz), 29.1, 29.5, 29.5, 29.6, 29.6,
(C5–C9), 29.9 (C10), 30.8 (Cβ), 34.3 (C3, 3J(119Sn,
C) = 60.1 Hz), 62.3 (Cε), 67.7 (C11), 98.9 (Cα), 128.4 (Cx,
3J(119Sn,C) = 48.0 Hz), 128.8 (Cp, J(119Sn,C) = 11.0 Hz),
4
137.1 (Co, 2J(119Sn,C) = 35.1 Hz), 139.2 (Ci, 1J(119Sn,
C) = 480.3 Hz). 119Sn NMR (CDCl3, 149 MHz, δ, ppm):
−99.6 (s, Ph3Sn).
2.3 | In vitro studies
2.3.4
| Caspase 2, 3, 8 and 9 enzyme activity assays
RPMI‐1640, phosphate‐buffered saline (PBS), penicillin/
Activity of caspases 2, 3, 8 and 9 was measured using the
caspase substrate cleavage assay. A2780 cells were seeded
at a density of 1 × 106 per well in a six‐well plate. After expo-
sure to IC50 concentration of 1, cells were sampled after 2 h
for cleavage of caspases. Adherent cells were washed with
cold PBS, collected with a cell scraper and suspended in cell
lyses buffer (50 mM Hepes, pH = 7.4, 1% Triton X100, all
from Sigma‐Aldrich). After incubation for 10 min on ice
and centrifugation, protein concentrations of the supernatants
were measured according to a method of Bradford (Bio‐Rad
streptomycin, 10× Dulbecco’s PBS without Ca2+ and Mg2+
,
trypsin–EDTA 0.05%/0.02% (PAA Laboratories GmbH),
proteinase K 20 mg ml−1 in PBS, RNAse A (Roche Diag-
nostics GmbH) and specific caspase substrates (2, Ac‐
VDVAD‐pNA; 3, Ac‐DEVD‐pNA; 8, Ac‐IETDpNA; 9,
Ac‐LEHD‐pNA; Axxora, Loerrach, Germany) were used.
Human tumour cell lines A2780 (ovarian), A549 (lung),
HeLa (adenocarcinoma) and SW480 (colon) were routinely
maintained in RPMI‐1640 medium supplemented with 10%

4
EDELER ET AL.
Laboratories). Samples (50 μg protein extract) were incu-
bated on a microplate at 37°C overnight in reaction buffer
(50 mM Hepes, pH = 7.4, 0.1% CHAPS, 5 mM EGTA, 5%
glycerol) containing 10 mM DTT (all from Sigma‐Aldrich)
and a specific substrate of caspases. Extinction of released
p‐nitroaniline was measured at 405 nm using a 96‐well plate
reader (Tecan Spectra) and activity of caspases 2, 3, 8 and 9
was evaluated by optical density ratio of treated/untreated
samples.[13,21]
shifts of the carbon atoms C1–C3 in 13C NMR spectra and
coupling constants of 119Sn with C atom, observed in 13C
NMR spectra, remain unchanged for 2–5. Except for 1,
because of a shorter alkyl spacer, the ether oxygen atom
influences the mentioned chemical shifts and coupling con-
stants. The neighbouring hydrogen and carbon atoms of those
ether oxygen atoms show similar trend in chemical shifts in
1H NMR and 13C NMR spectra. Thus, CH2 groups from
pyranyl ring and alkyl chain separate in two distinguishable
multiplets. Tertiary carbon atom from pyranyl ring shows a
chemical shift in lower field. 119Sn NMR spectra of 1–5 con-
firm that they are isolated Ph3Sn(alkyl) compounds.[25,38]
3
| RESULTS AND DISCUSSION
3.1 | Synthesis and characterization
3.2 | In vitro activity
Compounds 1–5 of the general formula Ph3Sn(CH2)nO–THP
(n = 3, 4, 6, 8, 11) have been synthesized in the reaction of
Ph3SnLi with THP‐protected ω‐halogen alkylalcohols (X
(CH2)nO–THP; n = 3, 4, 6, 8, 11; X = Cl, Br; Scheme 1).
Although compounds of this type could be synthesized in
the reaction of Ph3SnCl and α,ω‐halogen(tetrahydro‐2H‐
pyran‐2‐yloxy)alkane via Grignard reagents, additional
deprotection and protection strategy is necessary for the syn-
thesis of pure products in poor yields.[37] After preparative
chromatography using the synthetic procedure presented
herein, the compounds were isolated as colourless, highly
viscous oils in yields of up to 67%.
The synthesized tetraorganotin(IV) compounds were investi-
gated in vitro against four different human tumour cell
lines.[35] Dose‐dependent graphs are presented in Fig. 2 and
IC50 values are presented in Table 2. Cisplatin is used for
comparison.
A general trend can be observed for all cell lines when
treated with compounds 1–5. Namely, as the length of spacer
between tin atom and THP moiety in Ph3Sn(CH2)nO–THP
(n = 3, 4, 6, 8, 11) increases, the activity decreases (IC50:
1 ≈ 2 < 3 < 4 < 5). Thus, the most active compounds are
those with shorter chains without significant difference
HR‐ESI‐MS gave evidence for molecular composition of
the organotin(IV) compounds 1–5. For all compounds,
[M + Na]+ ions in positive mode are formed.
The constitution of compounds 1–5 was determined
using multinuclear NMR spectroscopy (1H, 13C and 119Sn).
Selected data are presented in Table 1. As expected, chemical
FIGURE 2 Survival graphs after 96 h of treatment for A2780, A549, HeLa
and SW480 cells with organotin(IV) compounds 1–5 determined with SRB
assay (SD < 10%, not shown).
SCHEME 1 Synthesis of Ph3Sn(CH2)nO–THP (1–5).
TABLE 1 1H NMR, 13C NMR and 119Sn NMR spectroscopic parameters (δ, ppm; J, Hz) for 1–5
1
2
3
4
5
α
δH
4.52–4.56
7.2
4.55–4.59
10.8
4.58–4.61
11.1
4.62
4.60–4.65
11.2
1
δC
11.1
1J(119Sn,C)
396.7
26.7
394.5
23.4
396.8
26.6
398.5
26.5
23.3
34.1
61.8
62.2
67.6
−99.5
398.0
26.6
2
δC
2J(119Sn,C)
20.0
20.9
22.1
22.3
3
δC
70.4
34.1
34.1
34.3
3J(119Sn,C)
72.1
64.1
62.2
60.1
ε
δC
62.4
see C3
62.1
62.3
62.3
x
δC
66.7
66.6
67.7
δS n
−99.0
−100.1
−99.7
−99.6

EDELER ET AL.
5
TABLE 2 IC50 values (μM) of 1–5 against A2780, A549, HeLa and SW480 cell lines (96 h)
Compound
A2780
A549
HeLa
SW480
1
9.27 1.13
13.53 2.29
35.44 1.52
57.55 9.13
>100
37.72 4.97
50.84 7.90
51.77 2.30
>100
45.77 1.27
52.86 1.41
66.00 1.73
>100
42.94 1.01
52.82 8.71
72.20 1.06
>100
2
3
4
5
>100
>100
>100
Cisplatin
0.55 0.33
1.54 0.28
4.60 0.34
3.22 0.24
between propyl and butyl moieties. On the other hand,
discrimination between tumour cells upon action of 1–5 is
more than obvious. The highest activity of investigated com-
pounds is observed against ovarian A2780 cells, followed by
lung A253, adenocarcinoma HeLa and colon SW480 cells.
Furthermore, results reported herein are contrary to findings
for [PtCl2{NH2CH2CH2NH(CH2)nO–THP}] or [PtCl2
(NH2CH2CH{(CH2)nO–THP}CH2NH2)] complexes (n = 3,
4, 6, 8, 11), where the most active complexes were found to
be those with longer lipophilic spacers.[29,39] However, the
activity of the organotin compounds is lower than that of cis-
platin (Table 2). Compound 1 as well as the A2780 cell line
were selected for further investigations because of the
greatest activity of this compound against those cells.
The supravital dye AO enters cells independently from
disruption of membrane integrity and might fluoresce from
green to orange. On the other hand, EB can only react with
its cellular targets upon membrane disruptions (red fluores-
cence). Non‐treated A2780 cells (control) as well as cells
treated with 1 or cisplatin were allowed to grow for 24 h
and afterwards investigated using fluorescence microscopy.
As expected, control cells show normal morphology of
A2780 cells, thus flattened vital cells, along with marginal
number of apoptotic cells. Obviously morphological changes
occur upon treatment of A2780 cells with 1 or cisplatin
(Fig. 3A). In both cases cells do not show any sign of neither
secondary necrosis nor necrosis. Cell morphological changes
are seen as rounding of the cells as chromatin condensations
which indicate that 1, as well as cisplatin, causes apoptosis in
A2780 cells.
3.3 | Organotin(IV) compound 1 enters A2780 cells and
In order to understand which mechanism is involved in
triggering apoptosis, as seen in AO/EB double staining assay,
it was analyzed whether caspases are involved as downstream
effectors in induced cell death. Thus, the activities of
upstream caspases 2, 8 and 9 as well as downstream caspase
3 were analysed in the present study. A2780 cells treated with
compound 1 for 2 h show activation of all investigated
caspases (Fig. 3B). Based on these results, organotin(IV)
compound 1 triggers apoptosis by both internal (intrinsic or
mitochondrial pathway, caspase 9 pathway) and external
signals (extrinsic or death receptor pathway).
causes apoptosis
To determine if compound 1 accumulates in the cells, A2780
cells were treated with IC50 dose of 1 for 24 h and tin uptake
was assessed using ICP MS analysis. It is found that 1 is pres-
ent in high concentration, 2500 ppm, in the A2870 cell line.
In order to investigate if apoptosis is involved in A2780 cell
death upon treatment with 1, at first double staining AO/EB
assay was performed and cells were analysed by fluorescence
microscopy (Fig. 3A).
4
| CONCLUSIONS
Five ω‐TPH‐functionalized alkyltriphenyltin(IV) compounds
were prepared and characterized. The cytotoxic activity of
these compounds has been tested against ovarian A2780,
lung A549, adenocarcinoma HeLa and colon SW480 tumour
cell lines. Cytotoxic results pointed to a structure–activity
relationship: for shorter alkyl chains, a higher activity is
achieved. Against the investigated cell lines all compounds
are found to be less active than cisplatin. The most active
compound, 1, enters A2780 cells and causes apoptosis by
activation of both intrinsic and extrinsic caspase pathways.
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