In vitro evaluation of antiproliferative properties of novel organotin(IV) carboxylate compounds with propanoic acid derivatives on a panel of human cancer cell lines

Article abstract of DOI:10.3390/molecules26113199

The synthesis of novel triphenyltin(IV) compounds, Ph₃SnLn (n = 1–3), with oxaprozin (3-(4,5-diphenyloxazol-2-yl)propanoic acid), HL1, and the new propanoic acid derivatives 3-(4,5bis(4-methoxylphenyl)oxazol-2-yl)propanoic acid, HL2, and 3-(2,5-dioxo-4,4-diphenylimidazolidin1-yl)propanoic acid, HL3, has been performed. The ligands represent commercial drugs or their derivatives and the tin complexes have been characterized by standard analytical methods. The in vitro antiproliferative activity of both ligands and organotin(IV) compounds has been evaluated on the following tumour cell lines: human prostate cancer (PC-3), human colorectal adenocarcinoma (HT29), breast cancer (MCF-7), and hepatocellular cancer (HepG2), as well as on normal mouse embryonic fibroblast cells (NIH3T3) with the aid of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-12 diphenyltetrazolium bromide) and CV (crystal violet) assays. Contrary to the inactive ligand precursors, all organotin(IV) carboxylates showed very good activity with IC₅₀ values ranging from 0.100 to 0.758 μM. According to the CV assay (IC₅₀ = 0.218 ± 0.025 μM), complex Ph₃SnL1 demonstrated the highest cytotoxicity against the caspase 3 deficient MCF-7 cell line. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated a two-fold lower concentration of tin in MCF-7 cells in comparison to platinum. To investigate the mechanism of action of the compound Ph₃SnL1 on MCF-7 cells, morphological, autophagy and cell cycle analysis, as well as the activation of caspase and ROS/RNS and NO production, has been performed. Results suggest that Ph₃SnL1 induces caspase-independent apoptosis in MCF-7 cells.

Full text of DOI:10.3390/molecules26113199

molecules
Article
In Vitro Evaluation of Antiproliferative Properties of Novel
Organotin(IV) Carboxylate Compounds with Propanoic Acid
Derivatives on a Panel of Human Cancer Cell Lines
1,2,3,
4
5
Nebojša Ð. Pantelic´
*
, Bojan Božic´
, Bojana B. Zmejkovski 5, Nebojša R. Banjac 2, Biljana Dojcˇinovic´ ,
Ludger A. Wessjohann 3 and Goran N. Kalud¯erovic´
*
1,3,
1
Department of Engineering and Natural Sciences, University of Applied Sciences Merseburg,
Eberhard-Leibnitz-Straße 2, DE-06217 Merseburg, Germany
2
3
4
5
Department of Chemistry and Biochemistry, Faculty of Agriculture, University of Belgrade, Nemanjina 6,
11080 Zemun, Belgrade, Serbia; nbanjac@agrif.bg.ac.rs
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3,
06120 Halle (Saale), Germany; Ludger.Wessjohann@ipb-halle.de
Faculty of Biology, Institute of Physiology and Biochemistry “Ivan Djaja”, University of Belgrade,
Studentski trg 16, 11000 Belgrade, Serbia; bbozic@bio.bg.ac.rs
Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade,
Studentski trg 14, 11000 Belgrade, Serbia; bojana.zmejkovski@ihtm.bg.ac.rs (B.B.Z.);
bmatic@chem.bg.ac.rs (B.D.)
*
Correspondence: pantelic@agrif.bg.ac.rs (N.Ð.P.); goran.kaluderovic@hs-merseburg.de (G.N.K.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The synthesis of novel triphenyltin(IV) compounds, Ph3SnLn (n = 1–3), with oxaprozin
(3-(4,5-diphenyloxazol-2-yl)propanoic acid), HL1, and the new propanoic acid derivatives 3-(4,5-
bis(4-methoxylphenyl)oxazol-2-yl)propanoic acid, HL2, and 3-(2,5-dioxo-4,4-diphenylimidazolidin-
1-yl)propanoic acid, HL3, has been performed. The ligands represent commercial drugs or their
derivatives and the tin complexes have been characterized by standard analytical methods. The
in vitro antiproliferative activity of both ligands and organotin(IV) compounds has been evaluated on
the following tumour cell lines: human prostate cancer (PC-3), human colorectal adenocarcinoma (HT-
29), breast cancer (MCF-7), and hepatocellular cancer (HepG2), as well as on normal mouse embryonic
fibroblast cells (NIH3T3) with the aid of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-12 diphenyltetrazolium
bromide) and CV (crystal violet) assays. Contrary to the inactive ligand precursors, all organotin(IV)
Citation: Pantelic´, N.Ð.; Božic´, B.;
Zmejkovski, B.B.; Banjac, N.R.;
Dojcˇinovic´, B.; Wessjohann, L.A.;
Kalud¯erovic´, G.N. In Vitro Evaluation
of Antiproliferative Properties of
Novel Organotin(IV) Carboxylate
Compounds with Propanoic Acid
Derivatives on a Panel of Human
Cancer Cell Lines. Molecules 2021, 26,
3199. https://doi.org/10.3390/
molecules26113199
carboxylates showed very good activity with IC50 values ranging from 0.100 to 0.758
µM. According
to the CV assay (IC50 = 0.218 0.025 M), complex Ph3SnL1 demonstrated the highest cytotoxicity
±
µ
Academic Editor: Fabio Marchetti
against the caspase 3 deficient MCF-7 cell line. Inductively coupled plasma mass spectrometry
(ICP-MS) analysis indicated a two-fold lower concentration of tin in MCF-7 cells in comparison
to platinum. To investigate the mechanism of action of the compound Ph3SnL1 on MCF-7 cells,
morphological, autophagy and cell cycle analysis, as well as the activation of caspase and ROS/RNS
and NO production, has been performed. Results suggest that Ph3SnL1 induces caspase-independent
apoptosis in MCF-7 cells.
Received: 28 April 2021
Accepted: 24 May 2021
Published: 27 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: triphenyltin(IV); breast cancer; cytotoxicity; apoptosis; ICP-MS
1. Introduction
Copyright:
© 2021 by the authors.
Carcinogenic diseases represent a spectrum of related disorders, characterized by
uncontrolled cell division in individual organs, with a tendency to invade other parts of
the body. Viruses appear as causative agents of about 15 percent of all human cancers
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
worldwide [
1
]. Additionally, a large fraction of cancerous diseases arise spontaneously,
]. Food enriched
while a significant share is caused by exposure to ionising radiation [
2
with mycotoxins, different hormones and other compounds represent another important
source of cancer-causing agents [3,4].
Molecules 2021, 26, 3199. https://doi.org/10.3390/molecules26113199
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3199
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The discovery of cisplatin paved the way for the treatment of oncological diseases
with metal-based compounds. Unfortunately, the use of cisplatin and other platinum
derivatives for the treatment of malignant tumours causes a series of side effects such as
nausea, vomiting, neurotoxicity, nephrotoxicity, and ototoxicity [
effects of metal-based cytotoxins, the focus of novel investigations shifted to non-platinum
candidates [ ]. Organotin compounds exhibit an excellent potential for varying the organic
5,6]. To reduce these side
7
moieties of the donor ligand linked to the metal, which enables fine-tuning of the physico-
chemical properties of such novel coordination compounds. Consequently, a variety
of diorganotin and triorganotin(IV) complexes with significant in vitro antiproliferative
activity against solid and hematologic cancers have been synthesised [
the tin complexes ((carboxylato)triphenyltin(IV) and tetraorganotin(IV)) have been found
to display even higher cytotoxic activity than cisplatin [ 10]. Compared to platinum drugs,
8]. More recently,
9
,
organotin(IV) compounds benefit from better water solubility, lower toxicity and reduced
side effects [11]. Noteworthily, these types of complexes display a strong apoptotic effect
on tumour cells, via p53 tumour suppression [11]. Organotin(IV) carboxylates, compared
to organotin(IV) compounds with thiolato and ditiocarbamato ligands, usually display
higher cytotoxic activity against human tumour cell lines [12].
Investigation of the triphenyltin(IV) complex with 2,5-dimethyl-3-furoic acid as a
ligand showed high activity, especially against K562 and Fem-x cell lines, with IC50 values
30 to 112 times lower than cisplatin. Additionally, a relatively high selectivity vs. non-
tumour immortalized cells was demonstrated for this complex [13]. Other triphenyltin(IV)
carboxylates also show very high activity against different cell lines, displaying even
greater cytotoxic activity compared to cisplatin [14]. Recent studies of the triphenyltin(IV)
compounds of 1-(4-carboxyphenyl)-3-ethyl-3-methysuccinimide demonstrated their sig-
nificant activity against the HeLa cells [15]. Furthermore, organotin(IV) complexes of
2-(5-arylidene-2,4-dioxothiazolidin-3-yl)propanoic acid yielded excellent improvements in
antiproliferative drug development against PC-3 cells [16].
In line with our continuing efforts for developing novel organotin compounds as
potential antiproliferative agents, we herein report the synthesis of novel organotin(IV)
complexes with the commercially available drug Oxaprozin as well as two novel propanoic
acid derivatives. Oxaprozin represents a non-steroid anti-inflammatory drug (NSAID),
whose metal complexes previously displayed antiproliferative properties. All compounds,
ligand precursors and complexes synthesised in this study have been characterized by
standard analytical methods, and the corresponding in vitro anticancer activities were
determined on four tumour cell lines: human prostate cancer (PC-3), human colorectal
adenocarcinoma (HT-29), breast cancer (MCF-7), and hepatocellular cancer (HepG2), with
the aid of MTT and CV assays. The mode of action of the novel organotin(IV) complex
Ph3SnL1 on MCF-7 cell lines has been further evaluated via morphological, autophagy
and cell cycle analysis, showing the activation of caspase as well as ROS/RNS and NO
production. In addition, the accumulations of tin and platinum in MCF-7 cells have
been compared.

Molecules 2021, 26, 3199
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2. Results and Discussion
2.1. Synthesis and Characterization
In the reaction of ligand precursors (HL1
,
HL2, or HL3) which were deprotonated
with LiOH and reacted with equimolar amounts of Ph3SnCl, the expected compounds
Ph3SnL1 Ph3SnL2 or Ph3SnL3 were obtained as white solids in good yields (Scheme 1a–c).
,
The complexes were soluble in dimethyl-sulfoxide, chloroform, dichloromethane, and
acetonitrile. They were characterized via elemental microanalysis, IR and NMR (1H, 13C,
119Sn) spectroscopy and mass spectrometry. The numbering of atoms in Scheme 1a–c was
only used for NMR data assignation.
(a) Synthesis of compound Ph3SnL1
(b) Synthesis of compounds HL2 and Ph3SnL2
Scheme 1. Cont.

Molecules 2021, 26, 3199
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(c) Synthesis of compounds HL3 and Ph3SnL3
Scheme 1. Synthesis of compounds Ph3SnL1, HL2, Ph3SnL2, HL3 and Ph3SnL3.
The elemental analysis corresponds to the predicted formulae. Asymmetric
ν
(–CH3/–
CH2/–CH) vibrations of moderate intensities were found in the range of 3200–2800,
corresponding to unsaturated CH bonds (above 3000) and saturated CH bonds (below
3000) cm−1. Further, in the known aromatic vibration region (2000–1600 cm−1) of com-
plexes compared to ligand precursors, a pattern change was observed which indicates the
additional presence of aromatic moieties in the novel compounds. Strong
(C–O) absorption stretching bands are present in all three complexes, at 1648, 1432 cm−1
Ph3SnL1); 1713, 1452 cm−1 Ph3SnL2) and 1710, 1419 cm−1
Ph3SnL3). Noteworthy, in
the spectra of ligand precursors, these bands were found at 1720, 1443 cm−1
HL1), 1708,
1436 cm−1 HL2), and 1701, 1419 (HL3) cm−1. This suggests a monodentate coordination
ν(C=O) and
ν
(
(
(
(
(
of the carboxylate oxygen atom as carbon–oxygen bond lengths are affected, becoming
unequal. The C=O bond vibrates at higher wavelength values which is most visible in
the case of HL1/Ph3SnL1
[17]. Differences between asymmetric and symmetric vibrations
were more than 200 cm−1, confirming a monodentate coordination to the tin centre [18].
Additionally, medium ν
(C=O) bands at 1777 (HL3) and 1770 (Ph3SnL3) cm−1 confirm the
presence of hydantoin rings. Moreover, a weak band at 454, 447 and 449 cm−1, observed for
all three compounds, respectively, is due to Sn–O vibrations, strongly suggesting the for-
mation of organotin(IV) carboxylates [15,16]. The ESI-MS of all synthesised organotin(IV)
compounds has been recorded in positive ion mode and in all cases the [M + H]+ mass
peak was found.
In 1H NMR spectra, for all compounds the characteristic pattern for the –CH2CH2–
group from the propanoic acid moiety can be observed at around 2.8–3.9 ppm. The chemical
shifts at 3.76, 3.77 (HL2) and 3.83, 3.87 ppm (Ph3SnL2) belong to metoxyl hydrogen atoms.
As expected, aromatic protons resonated between 6 and 8 ppm. Coupling of hydrogen
atoms with the tin centre can be observed as satellite signals in all organotin compounds,

Molecules 2021, 26, 3199
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with ortho hydrogen atoms near the positions given in the data (experimental section),
but due to overlap with resonances of hydrogen atoms from the five phenyl moieties this
cannot be easily distinguished for characterization. Ligand precursors have less shielded
hydrogen atoms (between 11–12 ppm) belonging to their carboxylate groups. Expectedly,
this resonance is not present in the organotin compounds, confirming coordination by the
carboxylate group to tin(IV).
13C NMR spectra showed all expected signals for these types of compounds. Carbon
atoms C2 and C3 from –CH2CH2– groups resonated in both ligand precursors and organ-
otin(IV) compounds at 23–38 ppm. Metoxyl carbon atoms in HL2/Ph3SnL2 resonated at
55.5 ppm. In the area 110–160 ppm, all resonances of aromatic carbon atoms can be found,
nevertheless, overlapping was observed. Chemical shifts of two carbonyl carbon atoms
from the hydantoin rings in HL3/Ph3SnL3 can be allocated at around 156 and 173 ppm.
The carbon atoms from the carboxylate moieties in organotin(IV) compounds as well as the
ligand precursors had the highest ppm values, as presumed. Furthermore, 119Sn NMR spec-
tra showed a singlet at −96.1, −95.0 and −89.3 ppm for Ph3SnL1, Ph3SnL2, and Ph3SnL3,
respectively, suggesting the tetrahedral geometry of triphenyltin(IV) compounds [19].
In order to investigate the stability of the complexes, time-dependent 13C NMR
spectroscopy was performed. The recorded 13C NMR spectra were compared immediately
after dissolving the investigated compounds, and again after 2 and 24 h in CDCl3. The
results show that at the examined time points there were no changes in the chemical shift
resonances of carbon atoms, nor the appearance of new signals that can be attributed to
decomposition products in the applied medium.
2.2. Cytotoxic Activity
The in vitro anticancer activity of ligand precursors, HL1–HL3 and its organotin(IV)
compounds was tested in a panel of four tumour cell lines: PC3 (prostate cancer cells),
HT-29 (colon cancer cells), MCF-7 (breast cancer cells), and HepG2 (hepatic cancer cells), as
well as on normal NIH3T3 cells (mouse embryonic fibroblast) using MTT and CV assays
after 48 h of incubation. Results are presented in Table 1.
Table 1. IC50 values (mean
assays (48 h incubation).
±
SD, µM) of synthesised compounds and cisplatin using MTT and CV
PC-3
HT-29
MCF-7
HepG2
NIH3T3
HL1-HL3 (MTT/CV)
>100
>100
>100
>100
>100
Ph3SnL1 (MTT)
Ph3SnL1 (CV)
0.181
0.556
±
0.027
0.038
0.237
0.465
±
0.012
0.046
0.132
0.218
±
0.021
0.025
0.146
0.366
±
0.028
0.051
2.17 ± 0.53
±
±
±
±
2.66 ± 0.52
Ph2SnL2 (MTT)
Ph3SnL2 (CV)
0.575
0.785
±
±
0.043
0.018
0.453
0.727
±
±
0.063
0.018
0.309
0.543
±
±
0.059
0.026
0.223
0.628
±
±
0.084
0.165
1.99 ± 1.12
2.61 ± 0.52
Ph3SnL3 (MTT)
Ph3SnL3 (CV)
0.211
0.458
±
0.042
0.029
0.218
0.471
±
0.015
0.048
0.131
0.219
±
0.017
0.016
0.100
0.255
±
0.030
0.085
1.82 ± 0.99
±
±
±
±
2.09 ± 1.03
cisplatin (MTT)
cisplatin (CV)
14.30 ± 0.26
12.95 ± 0.45
14.18 ± 0.73
15.97 ± 0.95
15.16 ± 1.04
15.83 ± 0.55
3.47 ± 0.46
3.65 ± 0.35
8.81 ± 0.51
14.21 ± 1.95
All investigated organotin(IV) compounds demonstrated outstanding antiproliferative
activity versus all tested tumour cell lines with IC50 values ranging from 0.100–0.785 M.
µ
Generally, compounds Ph3SnL1 and Ph3SnL3 exhibited a significantly higher cytotoxicity
against all tumour cells compared to Ph3SnL2. Compound Ph3SnL3 showed the strongest
inhibitory effect on cell survival in HepG2 hepatic tumour cells, while compound Ph3SnL1
was found to be the most active against MCF-7 breast cells (Table 1). Unfortunately, novel
organotin(IV) compounds show high toxicity against normal NIH3T3 cells. However,
obtained IC50 values are significantly higher toward tumour cell lines, thus, investigated
organotin(IV) compounds showed 3 to 12 times higher ca. selectivity (CV assay) towards
tumour cells. On the other hand, the ligand precursors HL1–HL3 did not show any
influences on cell proliferation at the applied concentrations.

Molecules 2021, 26, 3199
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Additionally, it should be noted that all analysed organotin(IV) compounds were
several times more active compared to cisplatin, a clinically used drug (Table 1), especially
compounds Ph3SnL1 and Ph3SnL3, which displayed up to 120-fold higher activity against
MCF-7. Despite the high toxicity, the selectivity between normal and cancer cells of novel
compounds is higher or comparable to cisplatin.
Moreover, the triphenyltin(IV) compounds expressed significantly higher antiprolifer-
ative activity compared to Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes with oxaprozin
as a ligand [20]. According to the results obtained by MTT assay, it was observed that com-
pound Ph3SnL3 had its highest activity against HepG2 cells, while compound Ph3SnL1
shows the lowest IC50 value versus MCF-7 in the CV assay (Table 1). A discrepancy in
MTT and CV assays should be noted, possibly pointing out that investigated organotin(IV)
compounds affect cell metabolism pathways. Such effects have already been described in
literature, e.g., as being caused by 2-oxoheptyl ITC [21]. To examine the mode of action, the
compound Ph3SnL1 and the MCF-7 tumour cell line were selected for further investigation.
2.3. Drug Uptake
The cellular uptake features of metal-based cytotoxic drugs strongly impact the under-
lying anticancer performance [22]. Intracellular tin and platinum uptake in MCF-7 cells has
been investigated with the aid of inductively coupled plasma mass spectrometry (ICP-MS)
analysis. The treatment of cells with Ph3SnL1 and cisplatin for 24 h in concentrations
corresponding to their IC50 values, as obtained by the CV assay, was performed.
The results (Figure 1) point to significantly different metal uptake, with emphasis on
the approximately twofold higher platinum concentration in MCF-7 cells compared to
tin. Interestingly, the results indicate that a minor amount of tin causes significantly more
potent cytotoxic effect against MCF-7 cells, compared to the clinically used anticancer drug
cisplatin. Considering the uptake, it should be emphasized that half the concentration
of tin (compared to cisplatin) in MCF-7 cells results in up to 70 times more powerful
antiproliferative activity.
Figure 1. Platinum and tin uptake in treated MCF-7 cells with cisplatin (at IC50 = 15.83
Ph3SnL1 (at IC50 = 0.218 µM) after 24 h of action.
µM) and
2.4. Morphological Study
The results of morphological changes of MCF-7 cells obtained by AO and DAPI stain-
ing following treatment with Ph3SnL1 and cisplatin in concentrations corresponding to
their IC50 and 2
×
IC50 values after 24 h of incubation, are presented in Figure 2. It can be
observed that the cell treatment triggers morphological changes in MCF-7, indicating the
possible induction of apoptosis, as identified by the permeable dye acridine orange. Com-
pared to untreated cells, notable morphological changes are observed in cells treated by
Ph3SnL1 at both concentrations applied (IC50 or 2
tion is considerably higher in treated compared to untreated control cells. Furthermore, the
results obtained by DAPI staining confirmed nuclear condensation, suggesting apoptotic
×
IC50). The level of nucleus condensa-

Molecules 2021, 26, 3199
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morphological changes in treated MCF-7 cells with Ph3SnL1 (Figure 2). The control cells
remained intact and uniform.
Figure 2. Fluorescent micrographs of MCF-7 cells. (
A
,F)—control (untreated cells), (B,G)—cisplatin
(IC50), (
C
,
H
)—cisplatin (2
×
IC50), (
D
,
I
)—Ph3SnL1 (IC50), ( )—Ph3SnL1 (2 IC50); AO = acridine
E
,
J
×
0
orange; DAPI = 4 ,6-diamidino-2-phenylindole.
2.5. Flow Cytometry Analysis
The activation of caspase is closely linked to cell apoptosis, the controlled cell death,
via the mitochondrial pathway or cell death receptor [23 24]. To evaluate whether caspases
,
get activated to trigger apoptosis, MCF-7 cells were treated for 48 h with Ph3SnL1, and
cisplatin as a reference substance, in the concentrations corresponding to their IC50 values,
stained with apostat, and analysed using flow cytometry. As shown in Figure 3a, Ph3SnL1
did not induce the activation of caspases, moreover, Ph3SnL1 induced a down-regulation of
caspase expression. Due to this, it can be concluded that cell death is accomplished through
caspase-independent apoptosis. Moreover, as MCF-7 cell line is caspase-3 deficient [25,26],
this was an anticipated result. Besides, the accomplishment of apoptosis induced with
Ph3SnL1 in MCF-7 cells (confirmed with AO and DAPI assays) was not disturbed as this
type of cell death may be activated through other death signals not involving caspase
activation [27].
(a)
(b)
Figure 3. Flow cytometry results: (a)—caspase activation (apostat), (b)—autophagy (AO).

Molecules 2021, 26, 3199
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The literature data show that autophagy in cancer cells plays a dual role in cell survival
and cell death [28,29]. To investigate whether the test compounds could induce autophagy,
MCF-7 cells were stained with AO after treatment at IC50 concentrations of Ph3SnL1 and
cisplatin for 48 h. The results obtained by flow cytometry analysis show that the autophagy
process is not triggered by Ph3SnL1. On the contrary, cisplatin induces an autophagic
response in MCF-7 cells at the applied concentration (Figure 3b).
The mode of action was further investigated by analysing the effects of Ph3SnL1
and cisplatin on the cell cycle phase distribution of MCF-7 cells. This was evaluated
by cytofluorimetric analysis using DAPI staining. The obtained results show that the
incubation with Ph3SnL1 for 48 h does not lead to any significant changes in the cell cycle
distribution (Figure 4). The analogous treatment with cisplatin also did not lead to any
observable changes in the population of cells in the sub-G1 phase, while a considerable
decrease was noted for the G1/G0 phase, which was coupled with an increase in the
cell population in S and G2/M phases (Figure 4). The exhibited results suggest that
the investigated organotin(IV) compound, Ph3SnL1, has a mode of action different to
cisplatin. While cisplatin acts on DNA (among other effects), the lipophilic cationic nature
of triphenyltin(IV) may guide it preferentially to mitochondria to intervene with cells’
energy metabolism.
Figure 4. Effects of Ph3SnL1 and cisplatin on cell cycle phase distribution: MCF-7 cells were exposed
to IC50 doses for 48 h.
Reactive oxygen (ROS) and nitrogen species (RNS) are natural by-products of the
aerobic metabolism in cells [30,31]. At physiological concentrations, they play a significant
role in regulating numerous cellular processes. On the other hand, it is well-known that
the reactive oxygen (O2•−, H2O2, OH•−) and nitrogen species (NO) are highly reactive
molecules that easily damage cells by interacting with biomolecules such as proteins,
lipids, carbohydrates, and nucleic acids, thus triggering different pathological conditions
(inflammation, cardiovascular disorders, neurodegenerative diseases, and the formation
of cancer cells) [32–35]. The influence of Ph3SnL1 and cisplatin on the production of
ROS/RNS species in MCF-7 cells was evaluated by flow cytometry treatment of the cells
with tested compounds in IC50 concentrations for 48 h. The obtained results demonstrate
that Ph3SnL1 does not affect the production of ROS and RNS within MCF-7 cells, while
cisplatin elevates the production of both reactive oxygen and nitrogen species in cancer
cells (Figure 5a,b).

Molecules 2021, 26, 3199
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(a)
(b)
Figure 5. Flow cytometry results: (a)—ROS/RNS activation (DHR), (b)—NO activation (DAF-FM).
3. Materials and Methods
3.1. Measurements
Elemental analyses were performed on an Elemental Vario EL III microanalyzer. A
Nicolet 6700 FT–IR spectrometer (THERMO Scientific, Waltham, MA, USA) and ATR
technique were used for recording mid-infrared spectra (4000–400 cm−1). 1H and 13C
NMR spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker, Billerica,
MA, USA). 119Sn NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer.
Biological investigations were performed on a microplate reader (Spectramax, Molecular
Devices USA, Silicon Valley, CA, USA), a laminar flow cabinet (Herasafe KS, Thermofischer
Scientific, Waltham, MA, USA) and FACSAria III, BD Biosciences, DB Biosciences, (Basel,
Switzerland). Mass spectrometry was performed using API-3200 triple quadrupole MS
(AB Sciex) Agilent 1200 with autosampler (Applied Biosystem, Framingham, MA, USA)
with electrospray positive ionisation mode. Regents and solvents were purchased from
Merck (Belgrade, Serbia) and used without further purification.
3.2. Synthesis of Ligands Precursors
The ligands, 3-(4,5-diphenyloxazol-2-yl)propanoate, L1, 3-(4,5-bis(4-methoxylphenyl)
oxazol-2-yl)propanoate, L2, and 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)propanoate,
L3, were synthesised by the neutralisation of previously prepared propanoic acid deriva-
tives (HL1, HL2 and HL3). HL1, the commercially available drug oxaprozin, was syn-
thesised according to a previously published method and its chemical characterization
1
(FT-IR, H-NMR and 13C-NMR spectra) fully matched previous spectra [20]. HL2, the
para-methoxyl derivative of Oxaprozin, was been synthesised according to a previously
published method [20] whereby the starting reagent 2-hydroxyl-1,2-bis(4-methoxylphenyl)
ethanone (para-methoxyl derivative of benzoine) was used for HL2, instead 2-hydroxyl-
1,2-diphenylethanone (benzoine) for HL1. Further, HL3 is a propanoic acid derivative of
the commercially available drug Phenytoin and was synthesised according to a previously
published method [36].
3-(4,5-bis(4-Methoxylphenyl)oxazol-2-yl)propanoic acid, HL2: White solid; 1H NMR
(500 MHz, DMSO-d6):
3.76 (s, 3H, CH3O–Ph), 3.77 (s, 3H, CH3O–Ph), 6.90–7.20 (m, 4H, C8H, C10H,C15H, C17H),
7.30–7.60 (m, 2H, C7H, C11H), 7.80–8.00 (m, 2H, C14H, C18H), 10.86 (s, 1H, COOH) ppm.13
NMR (125 MHz, DMSO-d6):
23.4 (C3), 30.4 (C2), 55.5 (CH3O–Ph), 114.7 (C8, C10, C15,
C17), 125.6 (C13), 128.0 (C14, C18), 129.0 (C7, C11), 132.0 (C6), 133.9 (C5), 144.2 (C12), 159.8
(C4), 162.4 (C9, C16), 173.6 (C1) ppm. Selected FT-IR data (KBr) cm−1: 3006 [
(CH)], 2928
(CH)], 2638 [ (CH2)], 1708 [ (C=O)], 1586 [ (CC)], 1517 [ (CC)], 1436 [ (CO), (CH2)],
1246 [ω(CH2), δ(COH)], 1176, 1030, 835, 683, 609, 534.
3-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)propanoic acid, HL3: White solid; 1H
NMR (500 MHz, DMSO-d6)
2.47 (t, 2H, 3J(H,H) = 7.5 Hz, C2H), 3.56 (t, 2H, 3J(H,H) = 7.5 Hz,
δ
2.77 (t, 2H, 3J(H,H) = 7.5 Hz, C2H), 3.22 (t, 2H, 3J(H,H) = 7.5 Hz, C3H),
C
δ
ν
[ν
ν
ν
ν
ν
δ
δ
δ

Molecules 2021, 26, 3199
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C3H), 7.24–7.46 (m, 10H, C8H–C18H), 9.60 (s, 1H, NH), 12.36 (s, 1H, COOH) ppm.13
C
NMR (125 MHz, DMSO-d6)
δ
32.6 (C2), 35.3 (C3), 70.0 (C6), 127.6 (C8, C12, C14, C18), 128.7
(C10, C16), 129.1 (C9, C11, C15, C17), 129.6 (C7, C13), 155.7 (C5), 172.6 (C4), 173.6 (C1), ppm.
Selected FT-IR data (KBr) cm−1: 3232 [
1777 [ (C=O), hydantoin ring], 1701 [
ν
(N
−
H)], 3064 [
ν
(CH)], 2945 [
ν
(CH)], 2635 [
(C=C)], 1419 [δ
ν
(CH2)],
(CO),
ν
ν
(C=O), carboxyl group], 1451 [
ν
δ(CH2)], 1227 [ω(CH2), δ(COH)], 1141, 946, 696, 598.
3.3. Synthesis of Organotin(IV) Compounds
Triphenyltin(IV) compounds were synthesised as follows: a suspension of the ligand
precursors, 0.5 mmol of HL1 (147 mg), HL2 (177 mg), and HL3 (162 mg), in 5 mL of distilled
water was treated with a solution of 1 M LiOH (0.5 mL, 0.5 mmol) and a clear solution
◦
formed after 3 h of stirring at 40 C. Then 5 mL of a methanolic solution of Ph3SnCl (0.193 g,
0.5 mmol) was added. The mixture was stirred for 6 h while a white precipitate formed. The
solvent was then filtered off, and the precipitate (product) washed with 3 mL of distilled
cold water and dried in vacuum over silica gel.
(3-(4,5-Diphenyloxazol-2-yl)propanoato)triphenyltin(IV), Ph3SnL1: White solid; Yield:
58%. Anal. calcd. for C36H29NO3Sn: C, 67.32; H, 4.55; N, 2.18%. Found: C, 67.28; H, 4.60;
1
N, 2.15%. H NMR (500 MHz, CDCl3):
δ
3.00 (t, 2H, 3J(H,H) = 7.5 Hz, C2H), 3.20 (t, 2H,
3J(H,H) = 7.5 Hz, C3H), 7.27–7.35 (m, 6H, C8H, C9H, C10H, C15H, C16H, C17H), 7.35–7.48 (m,
0
0
0
0
9H, C3 H, C4 H, C5 H), 7.49–7.61 (m, 4H, C7H, C11H, C14H, C18H), 7.60–7.80 (m, 6H, C2 H,
0
C6 H) ppm. 13C NMR (125 MHz, CDCl3):
δ
24.6 0(C3), 31.1 (C2), 126.6 (C7, C11, C14, C18),
0
0
127.9 (C4 ), 128.4 ( C80 , C10, C15, C17), 0128.9 (C3 , C5 ), 130.0 (C9,C16), 132.8 (C13), 134.6 (C12),
0
135.1 (C6), 136.8 (C2 , C6 ), 138.0 (C1 ), 145.2 (C5), 162.1 (C4), 178.3 (C1) ppm. 119Sn NMR
(149.2 MHz, CDCl3): δ −96.1 ppm. Selected FT-IR data (ATR) cm−1: 3049 [
ν
(CH)], 2930
(CC)], 1432 [ (CO), (CH2)],
(CH)], 965 [τ(HCCH)], 732
[
ν
(CH)], 2611 [
1400 [ (CH),
[δ(CH2), γ(COC)], 454 [ν(Sn–O)]. ESI-MS (MeCN): m/z 643.32 [M + H]+.
ν
(CH2)], 1648 [
ν
(C=O)], 1575 [
ν
(CC)], 1537 [
ν
δ
δ
ω
ν
(CC)], 1313 [
ω
(CH2),
δ
(COH)], 1076 [
δ
(CCC),
ω
(3-(4,5-bis(4-Methoxylphenyl)oxazol-2-yl)propanoato)triphenyltin(IV), Ph3SnL2: White
solid; Yield: 60%. Anal. calcd. for C38H33NO5Sn: C, 64.98; H, 4.74; N, 1.99%. Found:
3
C, 64.88; H, 4.79; N, 2.01%. 1H NMR (500 MHz, CDCl3):
δ
2.92 (t, 2H, J(H,H) = 7.5 Hz,
C2H), 3.21 (t, 2H, 3J(H,H) = 7.5 Hz, C3H), 3.83 (s, 3H, CH3O–Ph), 3.87 (s, 3H, CH3O–Ph),
0
0
0
6.60–7.10 (m, 4H, C8H, C10H, C15H, C17H), 07.30–7.50 (m, 9H, C3 H, C4 H, C5 H), 7.69–7.90
0
(m, 10H, C7H, C11H, C14H, C18H, C2 H, C6 H) ppm. 13C NMR (125 MHz, CDCl3): 0
δ
25.9
0
0
(C3), 32.9 (C2), 55.5 (CH3O–Ph), 114.0 (C8, C10), 127.0 (C14, C18), 128.0 (C4 ), 128.3 (C2 , C6 ),
0
0
0
128.4 (C13), 129.1 (C7, C11), 132.6 (C6), 135.2 (C5), 135.9 (C3 , C5 ), 137.5 (C1 ), 145.3 (C12),
157.1 (C4), 160.4 (C9, C16), 169,1 (C1), ppm. 119Sn NMR (149.2 MHz, CDCl3): δ −95.0 ppm.
Selected FT-IR data (ATR) cm−1: 3050 [
ν
(CH)], 2969 [
ν
(CH)], 2928 [
ν
(CH2)], 2836 [ (CH2)],
ν
1713 [
ν
(C=O)], 1591 [
ν
(CC)], 1514[
ν
(CC)], 1452 [ (CO),
δ
δ
(CH2)], 1242 [
ω
(CH2), δ(COH)],
1169, 1034, 840, 683, 611, 529, 447 [ν(Sn–O)]. ESI-MS (MeCN): m/z 703.41 [M + H]+.
(3-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)propanoato)triphenyltin(IV), Ph3SnL3
:
White solid; Yield: 65%. Anal. calcd. for C36H30N2O4Sn: C, 64.21; H, 4.49; N, 4.16%. Found:
3
C, 64.31; H, 4.42; N, 4.07%. 1H NMR (500 MHz, CDCl3):
δ
2.79 (t, 2H, J(H,H) = 7.5 0Hz,
C2H), 3.89 (t, 2H, 3J(H,H) = 7.5 Hz, C3H), 7.10–7.27 (m, 10H, C8H–C18H), 7.30 (s, 3H, C4 H),
0
0
0
0
7.41 (s, 6H, C3 H, C5 H), 7.68 (s, 6H, C2 H, C6 H) ppm. 13C NMR (125 MHz, CDCl3) δ 32.5
0
0
(C2), 38.1 (C3), 70.1 (C6), 1026.5 (C8, C12, C14, C18), 128.3 (C2 , C6 ), 129.0 (C9, C11, C15, C17),
0
0
0
129.4 (C10, C16), 131.9 (C4 ), 137.0 (C3 , C5 ), 138.1 (C7, C13), 140.0 (C1 ), 156.2 (C5), 173.2
(C4), 173.7 (C1) ppm
.
119Sn NMR (149.2 MHz, CDCl3): δ −89.3 ppm. Selected FT-IR data
(ATR) cm−1: 3500 [
ν
(NH)], 3051 [
ν
(CH)], 2940 [
ν
(CH)], 1770 [
ν(C=O), hydantoin ring], 1710
[
ν
(C=O), carboxyl group], 1430 [
ν
(C=C)], 1419 [
δ
(CO), δ(CH2)], 1225 [ω(CH2), δ(COH)],
1140, 946, 696, 598, 449 [ν(Sn–O)]. ESI-MS (MeCN): m/z 674.38 [M + H]+.
3.4. Cell Lines, General Conditions and IC50 Determination
The cell proliferations of ligand precursors and organotin(IV) compounds were evalu-
ated with the aid of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-12 diphenyltetrazolium bromide)

Molecules 2021, 26, 3199
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and CV (crystal violet) assays toward prostate (PC-3), colon (HT-29), breast (MCF-7) and
hepatic (HepG2) cancer cell lines, and normal mouse embryonic fibroblast cells (NIH 3T3).
The cells were cultivated in T75 flasks containing 10 mL of complete medium and kept at
37 ◦C and 5% CO2 with the aim of detaching the cells. The nutrient medium was RPMI-
1640 (PAA Laboratories) supplemented with 10% foetal bovine serum, L-glutamine, and
penicillin/streptomycin (Sigma). Afterward, 5.000 cells of PC-3 and MCF-7 and 1.500 cells
of HT-29 and HepG2 were transferred into 96 well-plates and used for MTT and CV as-
says. For microscopy investigations as well as FACS measurements (except for ROS/RNS
measurements), MCF-7 cells were seeded overnight in 6-well plates (150,000 cells/well) in
10 mL of complete medium. After 24 h, cells were treated with Ph3SnL1 or cisplatin for 24
and 48 h in the concentrations corresponding to their IC50 and 2
×
IC50 values and used in
various assays as indicated below. In all assays, control cells were untreated MCF-7 cells
incubated for the same time period as treated cells and treated with appropriate reagents
in various assays. BD FACS Aria III was used for flow cytometry experiments and results
were analysed using BD FACSDiva software (BD Biosciences, Heidelberg, Germany) and
Cyflogic (V 1.2.1, CyFlo Ltd., Turku, Finland). A fluorescence microscope Invitrogen EVOS
FL 2 Auto 2 Cell Imaging System was used for visualization of the cells as well as for
detection of fluorescence in the cells.
Ligands HL1
-
HL3, organotin(IV) compounds, and cisplatin were investigated in
M). Using the complete
9 different concentrations (100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01
µ
medium, each stock solution of the test compounds (20 mM in DMSO) was diluted to obtain
the working solution. The final concentration of DMSO never overstepped 0.5%, which is
non-toxic to the cells [37]. Three technical replicates were performed for each concentration.
◦
The treated cells were incubated for 48 h at 37 C under 5% CO2 and the viability of the cells
was determined using MTT and CV assays according to the literature procedure [28,38,39].
The IC50 values, defined as 50% inhibitory concentration, were calculated with a four-
parameter logistic function and presented in a mean. All experiments were performed
in triplicate.
3.5. Metal Uptake
The tin and platinum uptake analysis was performed on MCF-7 cells with the aid of
inductively coupled plasma mass spectrometry (ICP-MS). The cells were seeded in T25
flasks and allowed to grow. After 24 h, the exponentially growing cells were treated with
10 mL of Ph3SnL1 and cisplatin in concentrations corresponding to their IC50 values for
24 h, and thereafter trypsinised and collected by centrifugation at 1200 rpm for 8 min.
Using 5 mL of ice-cold PBS the cells were washed and the metal uptake concentration in
prepared samples was determined according to the previously described procedure [40].
A Thermo Scientific iCAP Qc ICP-MS (Thermo Scientific, Bremen, Germany) instrument
using the supplied autotune protocols and operational software Qtegra was employed for
metal uptake determination. The measurements were performed on isotopes 119Sn, 120Sn,
195Pt, and 198Pt and the concentrations of metals were expressed as ppb (µg/L).
3.6. Morphological Analysis (AO and DAPI Staining)
The morphological changes of MCF-7 cell death induced by Ph3SnL1 and cisplatin
were studied by AO (acridine orange) [41] and DAPI (40,6-diamidino-2-phenylindole) [42
]
staining, and examined under a fluorescence microscope. Acridine orange can cross the cell
membrane and viable and early apoptotic cells can be detected. Chromatin condensation,
seen as dense green areas, or membrane blebbing, both appearing in apoptosis, is easily
proven by AO staining [40]. After 24 h of treatment (IC50 and 2
×
IC50 concentrations)
the cells were stained with acridine orange (3 g/mL AO in PBS), and observed with
µ
a fluorescence microscope (Fluorescence microscope-Invitrogen EVOS FL 2 Auto 2 Cell
Imaging System). On the other hand, for the DAPI assay 0.5 mL of Triton (0.1% in PBS) was
added to the treated cells after the fixing the cells with 0.5 mL of 4% paraformaldehyde
(PFA) for 8 min. Thereafter, 0.5 mL of DAPI solution was used to stain the cells for 5 min

Molecules 2021, 26, 3199
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in the dark [42]. The morphological characteristics were visualized under a fluorescence
microscope (Fluorescence microscope-Invitrogen EVOS FL 2 Auto 2 Cell Imaging System).
3.7. Activation of Caspases
After 48 h of incubation (with IC50 concentrations) the apostat assay was performed
as indicated by the supplier. The cells were resuspended in 1 mL of PBS and analysed with
flow cytometry [27].
3.8. Autophagy Analysis
The MCF cells were incubated at IC50 concentrations of Ph3SnL1 and cisplatin for
◦
48 h at 37 C under 5% CO2. Thereafter, the medium was collected into 15 mL Falcon
tubes, while the cells were washed with 1 mL of PBS. The cells were detached using 0.5 µL
of trypsin. The deactivation of trypsination was performed with the formerly collected
medium, while for the untreated cells, 1 mL of complete medium was added, and the cells
were collected by the pipette tip harvesting technique from each well into a new Falcon
tube, which then was centrifuged for 3 min at 1000 rpm. The supernatant was discarded,
and the cells were washed with 1 mL of PBS, centrifuged for 3 min at 1000 rpm and then
the supernatant was discarded. Afterwards, 500 µL of the acridine orange (AO) working
solution was used to stain the cells which then were incubated for 30 min at 37 ◦C under
5% CO2. The deactivation of the staining process was performed by adding 1 mL of PBS,
and after centrifugation for 3 min at 1000 rpm, the supernatant was discarded. The cells
were resuspended in 1 mL of PBS and analysed by flow cytometry [27].
3.9. Cell Cycle Analysis
Upon 48 h of treatment at IC50 concentrations, the MCF-7 cells were detached using
0.5
natant was discarded. Afterwards, the cells were washed with 1 mL of PBS, centrifuged for
3 min at 1000 rpm and the supernatant was discarded. The cells were resuspended in 300
µL of trypsin. The Falcon tubes were centrifuged for 3 min at 1000 rpm and the super-
µL
of ice-cold PBS buffer and then fixed by adding them dropwise to 700 µL of ice-cold abso-
lute ethanol. The cells were stored for 24 h at 4 ◦C, then centrifuged for 3 min at 1000 rpm,
0
washed with PBS and stained with 1 mL of DAPI (4 ,6-diamidino-2-phenylindole) working
solution [39]. The prepared cells were incubated at room temperature for 10 min and
analysed by flow cytometry.
3.10. Investigation of ROS/RNS Production
For the investigation of ROS/RNS production the cells from the T25 flask were col-
lected by applying 1 mL of trypsin and diluted with 9 mL of complete medium. Using
1 mL of DHR (dihydrorhodamine) working solution the cells were stained and incubated
◦
at 37 C and 5% CO2 for 10 min. The staining process was stopped by applying 4 mL
of complete medium, then the solution was centrifuged for 3 min at 1000 rpm and the
supernatant was discarded. The cells were resuspended in complete medium (1 mL/well)
and seeded in a 6 well plate. After 24 h incubation, the cells were treated with IC50 of the
tested compounds and incubated for 48 h at 37 ◦C and 5% CO2. The cells were detached
using 0.5 µ
L of trypsin and incubated for 3 min at 37 ◦C and 5% CO2. The trypsinisation
was stopped by adding 1 mL of complete medium to each well, and cells were centrifuged
at 1000 rpm for 3 min, washed with 1 mL of PBS, centrifuged, and then resuspended in
1 mL of PBS. The prepared cells were analysed by flow cytometry [27].
3.11. Investigation of NO Production
For NO (nitric oxide) production evaluation, cells were treated at IC50 of the test
compounds and incubated for 48 h at 37 ◦C under 5% CO2. The cells were washed with
0
PBS and then treated with 1 mL of DAF-FM (4-amino-5-methylamino-2,7 -diflurofluorescin
diacetate) working solution. The treated cells were incubated for 1 h at 37 ◦C and 5% CO2.
The stain was deactivated by incubation in a medium for 15 min before the cells were

Molecules 2021, 26, 3199
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washed with 1 mL of PBS. The cells were detached using 0.5
µ
L of trypsin and incubated
for 3 min at 37 ◦C and 5% CO2, centrifuged at 1000 rpm for 3 min, and washed with 1 mL
of PBS. The prepared cells were analysed by flow cytometry [39].
4. Conclusions
Three novel triphenyltin(IV) compounds based on commercial drugs and their deriva-
tives have been synthesised, characterised and tested in vitro for antiproliferative activity on
four different tumour cell lines. The ligand precursor oxaprozin (3-(4,5-diphenyloxazol-2-
yl)propanoic acid), HL1, and the new propanoic acids derivatives 3-(4,5-bis(4-methoxylphenyl)
oxazol-2-yl)propanoic acid, HL2, and 3-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)propanoic
acid, HL3, did not affect cell proliferation in any cancer cell line tested. Conversely, the
IC50 values of the triphenyltin(IV) compounds ranged from 0.100 to 0.758 µM. Complex
Ph3SnL1 emerged as the most potent cytotoxic agent against MCF-7 cells (CV assay:
IC50 = 0.218 ± 0.025 µM). Different biological assays for the investigation of its mechanism
of action suggest that Ph3SnL1 induces caspase-independent apoptosis in MCF-7 cells.
Moreover, ICP-MS analysis indicates that this is achieved with lower intracellular con-
centrations of tin in MCF-7 cells than platinum. Considering the required extracellular
concentrations, the triphenyltin(IV) compounds show a better cell bioavailability and accu-
mulation, a common phenomenon for lipophilic (and cationic) substances. In conclusion,
Ph3SnL1 emerges as a novel and promising potential antiproliferative drug which should
be confirmed by a series of novel experiments. Although neither ROS nor RNS—often
related to the cell’s energy metabolism [43]—are involved, subcellular investigation and
the molecular mode of action of triphenyltin(IV) compounds remain to be studied in
biochemical follow-up research.
Author Contributions: Conceptualization, N.Ð.P. and G.N.K.; methodology, N.Ð.P., B.B. and G.N.K.;
validation, B.D.; formal analysis, N.Ð.P., B.B. and N.R.B.; investigation, N.Ð.P., B.B., B.B.Z. and
B.D.; resources, L.A.W. and G.N.K.; data curation, N.Ð.P., B.B. and B.B.Z.; writing—original draft
preparation, N.Ð.P., B.B., B.B.Z. and N.R.B.; writing—review and editing, N.Ð.P., B.B., L.A.W. and
G.N.K.; visualization, N.R.B. and B.D.; supervision, N.Ð.P. and G.N.K.; project administration, L.A.W.
All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Ministry of Education, Science and Technological De-
velopment of the Republic of Serbia, Contract numbers: 451-03-9/2021-14/200026, 451-03-9/2021-
14/200116 and 451-03-9/2021-14/200178; National Scholarship for Postdoctoral Studies of the Repub-
lic of Serbia (N. Ð. Pantelic´); German Academic Exchange Service (DAAD) Grant number 57448219.
Data Availability Statement: Data supporting obtained results can be obtained from the authors
upon request.
Acknowledgments: We would like to express deep gratitude to M. Lerbs for her support in the
laboratory for cell cultures (IPB, Halle, Germany), and the team of Instrumental analysis of the
Faculty of Chemistry in Belgrade, Serbia, for recording of the spectrum.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Sample Availability: Samples of the compounds reported herein are available from the authors.
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