Bradykinin antagonists and thiazolidinone derivatives as new potential anti-cancer compounds

Article abstract of DOI:10.1016/j.bmc.2014.06.046

Glioblastoma (GB), the most aggressive brain tumour, and mantle cell lymphoma (MCL), a rare but very aggressive type of lymphoma, are highly resistant to chemotherapy. GB and MCL chemotherapy gives very modest results, the vast majority of patients experience recurrent disease. To find out the new treatment modality for drug-resistant GB and MCL cells, combining of bradykinin (BK) antagonists with conventional temozolomide (TMZ) treatment, and screening of thiazolidinones derivatives were the main objectives of this work. As it was revealed here, BKM-570 was the lead compound among BK antagonists under investigation (IC₅₀ was 3.3 μM) in human GB cells. It strongly suppressed extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation. BK antagonists did not decrease the viability of MCL cells, thus showing the cell-specific mode, while thiazolidinone derivatives, a novel group of promising anti-tumour compounds inhibited proliferation of MCL cells: IC₅₀ of ID 4526 and ID 4527 compounds were 0.27 μM and 0.16 μM, correspondingly. However, single agents are often not effective in clinic due to activation of collateral pathways in tumour cells. We demonstrated a strong synergistic effect after combinatorial treatment by BKM-570 together with TMZ that drastically increased cytotoxic action of this drug in rat and human glioma cells. Small proportion of cells was still viable after such treatment that could be explained by presence of TMZ-resistant cells in the population. It is possible to expect that the combined therapy aimed simultaneously at different elements of tumourigenesis will be more effective with lower drug concentrations than the first-line drug temozolomide used alone in clinics.

Full text of DOI:10.1016/j.bmc.2014.06.046

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bradykinin antagonists and thiazolidinone derivatives as new
potential anti-cancer compounds
b
c
b
c
Stanislav Avdieiev a, , Lajos Gera , Dmytro Havrylyuk , Robert S. Hodges , Roman Lesyk ,
⇑
Vincent Ribrag d,e, Yegor Vassetzky f, Vadym Kavsan a
a Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, 150 Zabolotnogo Str., Kyiv 03680, Ukraine
b Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO 80045, USA
c Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, 69 Pekarska Str., Lviv 79010, Ukraine
d Gustave Roussy Institute, 39 Camille-Desmoulins Str., Villejuif 94805, France
e INSERM U1009, The University of Paris-Sud 11, Gustave Roussy Institute, 39 Camille-Desmoulins Str., Villejuif 94805, France
f CNRS UMR8126, The University of Paris-Sud 11, Gustave Roussy Institute, 39 Camille-Desmoulins Str., Villejuif 94805, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Glioblastoma (GB), the most aggressive brain tumour, and mantle cell lymphoma (MCL), a rare but very
aggressive type of lymphoma, are highly resistant to chemotherapy. GB and MCL chemotherapy gives
very modest results, the vast majority of patients experience recurrent disease. To find out the new treat-
ment modality for drug-resistant GB and MCL cells, combining of bradykinin (BK) antagonists with con-
ventional temozolomide (TMZ) treatment, and screening of thiazolidinones derivatives were the main
objectives of this work. As it was revealed here, BKM-570 was the lead compound among BK antagonists
Received 18 February 2014
Revised 28 May 2014
Accepted 23 June 2014
Available online xxxx
Keywords:
Multitargeted complex therapy
Glioblastoma
Mantle cell lymphoma
Bradykinin antagonists
Thiazolidinones
under investigation (IC50 was 3.3 lM) in human GB cells. It strongly suppressed extracellular signal-reg-
ulated kinases 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation. BK antagonists did not decrease
the viability of MCL cells, thus showing the cell-specific mode, while thiazolidinone derivatives, a novel
group of promising anti-tumour compounds inhibited proliferation of MCL cells: IC50 of ID 4526 and ID
4527 compounds were 0.27 lM and 0.16 lM, correspondingly. However, single agents are often not
effective in clinic due to activation of collateral pathways in tumour cells. We demonstrated a strong syn-
ergistic effect after combinatorial treatment by BKM-570 together with TMZ that drastically increased
cytotoxic action of this drug in rat and human glioma cells. Small proportion of cells was still viable after
such treatment that could be explained by presence of TMZ-resistant cells in the population.
It is possible to expect that the combined therapy aimed simultaneously at different elements of tumo-
urigenesis will be more effective with lower drug concentrations than the first-line drug temozolomide
used alone in clinics.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
resistant and have the poor prognosis. The development of new
treatment modalities, especially those based on multitargeted
The tumours of central nervous system constitute from 2% to 5%
of all human cancers, about half of them are the brain glial tumours
of different malignancy grades of which glioblastoma (GB), the
most malignant glioma, occupies up to 50%.1 Mantle cell lym-
phoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma char-
acterized cytogenetically by the t(11;14)(q13;32) resulting in
overexpression of cyclin D1.2 GB and MCL patients exhibit an
aggressive clinical course however, first-line cytotoxic chemother-
apy is unable to induce long-term remission in the majority of
patients, both GB and MCL are known to be highly therapy
therapy, is desperately needed for these diseases.
The nonapeptide bradykinin (BK) has been shown to act as a
growth factor in human lung, prostate, gastrointestinal, breast,
and ovarian cancers.3,4 Previously, Gera et al.5,6 developed and syn-
thesized a series of potent and metabolism-resistant BK antago-
nists, among which BKM-570 caused an impressive inhibition of
human small cell lung cancer (SCLC) cell growth in vitro. Injection
of this compound in nude mice displayed even higher inhibitory
effects than the vascular endothelial growth factor receptor (VEG-
FR) inhibitor Semaxanib (SU5416) or the conventional chemother-
apeutic drug cisplatin.7 Among other BK antagonists, the
inexpensive BKM-1800 derivative of BKM-570 had also valuable
anti-cancer properties.7 Interestingly, that orally active antagonist
⇑
Corresponding author. Tel.: +380 44 5263498; fax: +380 91 9275155.
E-mail address: stasavdieiev@gmail.com (S. Avdieiev).
http://dx.doi.org/10.1016/j.bmc.2014.06.046
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Avdieiev, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.046

2
S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
B10238 showed poor potency against cancer in vitro, however, was
highly active in vivo.5
Ser-
D
-Igl-Oic-Arg, where F5c is (E)-1,2,3,4,5,6-penta-fluo-rocin-
namoyl, Hyp is trans-4-hydroxyprolyl, Igl is
and Oic is octahydroindole-2-carbonyl); BKM-570 is N -[(E)-
2,3,4,5,6-penta-fluorocin-namoyl]-N-(2,2,6,6-tetramethylpiperi-
din-4-yl)-O-(2,6-dichlorbenzyl)-L-tyrosin-amide; BKM-1800 is
a
-(2-indanyl)glycyl,
a
Last years, evaluation of anti-tumour activity appeared to be
actual and promising for the thiazolidinone derivatives.8,9 Several
subgroups of these compounds, namely 5-arylidene-2-amino-4-
thiazolones and pyrazoline substituted thiazolones, were evalu-
ated for anti-cancer activity in vitro in a standard National Cancer
Institute 60 cancer cell line assay. The majority of the tested sub-
stances have shown significant anti-tumour effects at the micro-
molar and submicromolar level.10,11
However, most tumours are driven by multiple molecular aber-
rations that cannot be controlled by a single targeted agent. The
creation of smart schemes of multitargeted therapy aimed simulta-
neously at different elements of tumour formation mechanisms
should be an effective strategy for cancer treatment.12 The main
purpose of this manuscript is to identify novel anti-cancer com-
pounds and to develop modern approaches to complex glioma
therapy based on the newly developed compounds and conven-
tional chemotherapeutics used in clinics.
In this paper, we demonstrated that BKM-570 is the lead cyto-
toxic substance for glioma cells, while thiazolidinones ID 4526 and
ID 4664 appeared to be efficient suppressors of MCL cell viability.
Decreased viability of cells after BKM-570 treatment was probably
associated with inhibition of ERK1/2 and AKT1 phosphorylation.
Combination of BKM-570 with first-line drug temozolomide
(TMZ) was much more effective even in much less concentration
than anti-GB TMZ used alone.
Fmoc-OC2Y-Atmp, where Fmoc is N-(fluorenyl-9-methoxycar-
bonyl), OC2Y is (O-2,6-dichlorobenzyl)tyrosyl, and Atmp is
4-amino-2,2,6,6-tetramethyl-piperidine. Compounds were pre-
pared using solution or solid phase methods, purified by HPLC
and characterized by thin layer chromatography (TLC) and laser
desorption mass spectrometry (LDMS).
ID 28 and ID 3166 (Scheme 1) were synthesized starting from
2-(4-hydroxyphenylamino)thiazol-4-one 1 and 3-(benzothiazol-
2-ylamino)-2-thioxo-thiazolidin-4-one 2, respectively, using
standard Knoevenagel reaction procedure (medium: acetic acid,
catalyst: fused sodium acetate) and structurally analyzed as
described elsewhere.10,13
The pyrazoline–thiazolidinone–isatin conjugates ID 3643, ID
4522, ID 4523, ID 4524, ID 4525, ID 4526, ID 4527, and ID 4664
were synthesized as described here by one-pot methodology
involving reaction of 3,5-diaryl-1-thiocarbamoyl-2-pyrazolines 3
with chloroacetic acid and isatins in the presence of fused sodium
acetate in refluxing acetic acid (Scheme 2).14
The oxadiazole–thiazolidinone hybrid ID 4132 (Scheme 3) was
synthesized following the reaction of generated in situ potassium
salts of 5-(4-dimethylamino-benzylidene)-thiazolidine-2,4-dione
4
with 2-chloro-N-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-yl]-
acetamide.15
Synthesized compounds were characterized by NMR spectra,
which are presented in the experimental part. 1H NMR spectra of
compounds ID 4522, ID 4523, ID 4524, ID 4525, ID 4526, ID
4527, and ID 4664 show characteristic patterns of the AMX system
for CH2–CH protons of pyrazoline fragment. The chemical shifts of
the protons HA, HM, and HX have been assigned to about d ꢀ3.47–
3.52, d ꢀ4.18–4.20, and d ꢀ5.85–6.03 with coupling constants of
JAM = 18.0–18.6, JMX = 11.0–11.5, and JAX = 3.5–3.9 Hz, respectively.
The chemical shift for the CH(4) of indoline cycle is insignificantly
displaced in weak magnetic field (d = 8.95–9.14) and clearly indi-
cated that only Z-isomers were obtained. The protons of the meth-
ylene group (CH2CO) of ID 4132 appear as broad singlet at
d = 4.60 ppm.
2. Results and discussion
2.1. Compounds under investigation
Compounds synthesized for the first time for this investigation
are described in the experimental section. Cytotoxic properties of
tested substances are summarized in the Table 1. Chemical struc-
tures of compounds used in this study are depicted in Figure 1.
Design and synthesis of peptide B10238, non-peptide small
mimetic BKM-570 and non-peptide small mimetic BKM-1800 were
described previously.5–7 B10238 is F5c-
D-Arg-Arg-Pro-Hyp-Gly-Igl-
Compounds ID 28 and ID 4523, immobilized on a PEGylated
carrier (PEGylated olygoelectrolyte polymers),16 were kindly pro-
vided by Dr. A. Zaichenko (Lviv Polytechnic National University).
Table 1
Compounds under investigation
IC50 (lM)
U373
C6
293_CHI3L1 Granta Jeko
UPN1 Mino
2.2. Cytotoxicity of synthesized compounds in cancer cell lines
Bradykinin antagonists
b
BKM-570
BKM-1800
B10238
3.3
20
4
15
3.8
25.8
67.9
63.3
41.2
>100
49.3
52.3
50.5
>100
>100
>100
—
—
—
Increased proliferation rate is a common feature of cancer cells.
To analyse the anti-viability effects of synthesized compounds, we
used several different cancer cell lines, namely rat glioma C6 cells,
human GB U373 cells, 293 cells, overexpressing CHI3L1 oncogene
(293_CHI3L1),17 MCL cell lines Granta, Jeko, UPN1, and Mino.
b
b
>100a 37
Thiazolidinones
ID 11
b
b
b
b
b
b
—
—
—
—
4
—
—
—
—
—
—
>100
>100
>100 >100
>100 >100
—
b
ID 28
61.2
ID 28car*
ID 3166
15
0.13
—
—
—
—
—
—
—
b
b
b
b
b
b
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
b
b
b
b
b
b
b
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
55.3
bromide-based) cell viability assay revealed significant dose-
dependent cytotoxicity of BKM-570 and BKM-1800 in 293_CHI3L1
and U373 cells 72 h after addition to the culture medium (Fig. 2,
panels A and B). IC50 (molar concentration of compound leading
to 50% cell death compared to control group treated with DMSO
alone) for BKM-570 and BKM-1800 in 293_CHI3L1 cells was
3.8 lM and 25.8 lM, respectively, and 3.3 lM and 20 lM, respec-
tively, in U373 cells. B10238 did not suppress U373 cell viability
at all and had only a partial cytotoxic activity in 293_CHI3L1 cells,
b
b
b
ID 3643
>100
>100
>100 >100
>100 >100
—
—
ID 4132*
ID 4522*
ID 4523*
ID 4523car.*
ID 4524*
ID 4525*
ID 4526*
ID 4527*
ID 4664*
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
68.2
80.7
1.58
0.73
10
0.27
7.4
0.16
b
b
b
1.22
0.13
b
b
b
b
b
b
b
—
—
—
—
b
b
b
b
b
b
b
b
—
—
—
—
—
—
b
b
b
b
b
b
b
b
—
with IC50 100
effective of BK antagonists under study, with IC50
values for BKM-1800 and B10238 in C6 cells were 15
37 M, respectively (Fig. 2, panel C). In our study we revealed very
l
M. In C6 rat glioma cells, BKM-570 was the most
M. The IC50
M and
*
Newly synthesized compounds are marked by asterisk. Lead compounds are
4
l
given in italics and underlined.
—a
Compound did not reach the IC50 value in corresponding cell line.
Compound was not tested in corresponding cell line.
l
—b
l
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S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Figure 1. Structure of BKM-570, TMZ, ID 28, ID 3166, ID 3643, ID 4132, ID 4522, ID 4523, ID 4524, ID 4525, ID 4526, ID 4527, and ID 4664.
Scheme 1. Synthesis of ID 28 and ID 3166 compounds.
low cytotoxicity of B10238, same as it was reported previously,
although it suppressed tumour growth in mice, what may suggest
that other mechanisms, probably inhibition of angiogenesis, could
come into play in vivo.6 BKM-1800, a small non-peptide BK-antag-
onist, possessed higher activity in SCLC cells7 than in 293_CHI3L1,
U373, and C6 cells in this investigation (Fig. 2, panels A–C), while
BKM-570 was shown to be the lead compound both in 293_CHI3L1,
probably was due to some proportion of cells in the population,
more sensitive to the used compounds.
BKM-570 inhibited ERK1/2 and AKT1 phosphorylation in
293_CHI3L1 and U373 cells in a dose-dependent manner, whereas
B10238 treatment did not decrease their level of phosphorylation.
In 293_CHI3L1 cells 10
level of phospho-ERK1/2 and 50
abolishment of ERK1/2 and AKT1 phosphorylation (Fig. 3, panel A).
Same correlation was observed in MTT-test: 50 M BKM-570
reduced number of viable cells to basal level. As it concerns AKT1
phosphorylation, even 10 M BKM-570 was sufficient to strongly
suppress the phospho-AKT1 signal (Fig. 3, panel A). Since
lM BKM-570 suppressed efficiently the
l
M BKM-570 led to the complete
U373, and C6 (Fig. 2, panels A–C) and in SCLC cells (IC50 1.8 lM), as
well as in vivo (91% inhibition of tumour growth) in xenograft
l
mice.6
Points in the graphs A and C, corresponding to the lowest con-
centration of the compound used varied from 90% to 70% that
l
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S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 2. Synthesis of ID 3643, ID 4522, ID 4523, ID 4524, ID 4525, ID 4526, ID 4527, and ID 4664 compounds. Reagents, conditions and yields: ClCH2COOH (1.0 equiv),
AcONa (2.0 equiv), AcOH, reflux 5 h, 67–85%.
Scheme 3. Synthesis of ID 4132 compound.
Figure 2. Cell viability after treatment with BK antagonists (MTT assay). 293_CHI3L1 (A), U373 (B), and C6 (C) cells were cultured in the absence (vehicle control) or in the
presence of increasing doses of BKM-570, BKM-1800, or B10238 for 72 h. Data are expressed as the percentage of viable cells compared to vehicle control (100%). Each point
represents the mean of three experiments performed in triplicate.
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S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 3. Impact of BK antagonists on AKT1 and ERK1/2 phosphorylation. Western
blot analysis of phospho-AKT1 (pAKT1) and phospho-ERK1/2 (pERK1/2) in
293_CHI3L1 (A) and U373 (B) cells after treatment with BKM-570 or B10238.
non-stimulated U373 cells have much lower levels of ERK1/2 and
AKT1 phosphorylation compared to 293_CHI3L1 cells, we used
10% FBS to induce their phosphorylation before incubating cells
with BK antagonists. Under such conditions, 10
nificantly suppressed the phospho-AKT1 signals. However, in con-
trast to AKT1, even 50 M BKM-570 did not inhibit completely
lM BKM-570 sig-
l
Figure 4. Effect of BK antagonists combination with temozolomide (TMZ) on cell
phosphorylation of ERK1/2 (Fig. 3, panel B) that could be explained
by the absence of phosphatase PTEN in these cells18,19 which reg-
ulates ERK1/2 phosphorylation.20 The inhibition of AKT1 and
ERK1/2 phosphorylation by BKM-570 is probably involved in the
mechanisms underlying the anti-proliferative activity of this com-
pound, as it has been shown for other anti-cancer reagents.21,22
The first-line GB chemotherapy is the cytotoxic drug temozolo-
mide (TMZ) but its effectiveness is temporary: the average survival
expectancy is 14.6 months and the overall 5-year survival rate for
GBM is only 9.8%.23 Moreover, the high TMZ doses (75–200 mg per
square meter of body-surface area per day) used for GB treatment
are known to induce neurotoxicity and cognitive dysfunction as
treatment-related side-effects.24,25 Attempts to increase the anti-
tumour TMZ efficacy by combining with different substances
reported previously were not very successful.26,27
viability (MTT assay). U373 (A) or C6 (B) cells were cultured in the medium with
TMZ alone either TMZ with 1 lM of BKM-570, or TMZ with 1 lM of BKM-1800 for
72 h. Data are expressed as the percentage of viable cells compared to vehicle
control (100%). Each point represents the mean of two experiments performed in
triplicate.
down regulation of TMZ-induced checkpoint kinase 2 (Chk2)
activity and G2 arrest, thus protecting cells from TMZ-mediated
cytotoxicity.28
It was revealed that the cytotoxic activity of BK antagonists was
restricted to the 293_CHI3L1, C6, and U373 cell lines and did not
exist in MCL cell lines Granta, JeKo, and UPN1 (Fig. 5). BKM-570
and BKM-1800 produced only 50% inhibition of Granta and JeKo
cells viability in concentrations range of 40–65 lM (Fig. 5, panels
A and B), and did not suppress at all UPN1 cells viability (Fig. 5,
panel C). B10238 had the lowest activity of BK antagonists under
experiment. Cell specificity of BK antagonist cytotoxicity may
reflect differences in BK pathways and innate sensitivity to BK
antagonists between blood (MCL) cells and cells of other origin
(293_CHI3L1, U373, and C6 cells).
To find out compounds suitable for inhibition of viability in MCL
cells, we have screened a set of new promising compounds belong-
ing to thiazolidinones family. Previously it was reported that in sil-
ico molecular modeling of thiazolidinone based conjugates
revealed their increased affinity towards antiapoptotic proteins
of the Bcl-2 family which are often overexpressed in leukemia
and lymphoma cells.14 These compounds were tested at the
National Cancer Institute (NCI, USA) on a panel of 60 tumour cell
In our study, the addition of BKM-570 in non-cytotoxic concen-
tration of 1 lM to as little as 10 lM TMZ-containing culture med-
ium gave about 80% reduction of viable rat C6 and human U373
glioma cells compared to TMZ used alone (Fig. 4). The observed
combinatorial effect of BKM-570 and TMZ was synergistic, rather
than additive: it was much higher than simple arithmetic sum of
their individual effects. However, there was no complete suppres-
sion of cell viability that could be explained by the presence of
TMZ-resistant cells in the population. On the other hand, such
combination with BKM-1800 did not induce increased cytotoxicity,
and even reduced the effect of TMZ in U373 cells (Fig. 4, panel B).
Suppression of AKT1 phosphorylation may contribute to the
improved cytotoxicity seen after combining by BKM-570 with
TMZ, since AKT1 activation has been shown to play a role in the
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S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Effect of BK antagonists on MCL cells viability (MTT assay). Granta (A), JeKo (B), and UPN1 (C) cells were cultured in the absence (vehicle control) or in the presence
of increasing doses of BKM-570, BKM-1800, or B10238 for 72 h. Data are expressed as the percentage of viable cells compared to vehicle control (100%). Each point represents
the mean of three experiments performed in triplicate.
Figure 6. Effect of thiazolidinones on MCL cells viability (MTT assay). Granta (A), JeKo (B), and UPN1 (C) cells were cultured in the absence (vehicle control) or in the presence
of increasing doses of thiazolidinones for 72 h. Data are expressed as the percentage of viable cells compared to vehicle control (100%). Each point represents the mean of
three experiments performed in triplicate.
lines, and their high toxicity towards leukemia cell lines was
revealed.10 Twelve compounds of the thiazolidinone family were
involved in our study (Table 1). Among them, ID 11, ID 28, ID
3166, and ID 3643 were already published,10 while ID 4132, ID
4522, ID 4523, ID 4524, ID 4525, ID 4526, ID 4527, and ID 4664
were newly developed substances.
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S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 7. Inhibition of Mino cells viability by ID 4523 (A) and ID 4523-related
thiazolidinones (B) (MTT assay). Cells were cultured in the absence (vehicle control)
or in the presence of increasing doses of thiazolidinones for 72 h. Data are
expressed as the percentage of viable cells compared to vehicle control. Each point
represents the mean of three experiments performed in triplicate.
Figure 8. Cytotoxicity of thiazolidinones in 293_CHI3L1, U373, and C6 cells (MTT
assay). (A) 293_CHI3L1 or U373 cells were cultured in the absence (vehicle control)
or in the presence of increasing doses of ID 28car for 72 h. (B) C6 cells were cultured
as described above with ID28_car., ID 4523 and ID 4523car. Data are expressed as
the percentage of viable cells compared to vehicle control (100%). Each point
represents the mean of three experiments performed in triplicate.
Based on the NCI data, compounds ID 11, ID 28, ID 3166, ID
3643, and ID 4132 were selected as those with prominent activity.
Nevertheless, we observed only slight decreased viability of treated
cells, even with 100 lM concentrations of these substances, as well
strongest cytotoxic properties (IC50 0.13
significantly higher in comparison with C6 rat glioma cells (LC50
M) and human U373 cells (IC50 15 M) (Fig. 8, panel A). In C6
cells ID 4523 had IC50 1.22 M, while immobilization on a PEGylat-
ed carrier enhanced the growth inhibitory properties of ID 4523
(LC50 0.13 M) (Fig. 8, panel B). Thus, the cell specific modes of
action were observed for certain members of bradykinin antago-
nists and thiazolidinone derivatives, indicating the involvement
of different mechanisms to implement the cytotoxic properties of
these compounds.
lM) in 293_CHI3L1 cells,
as with novel compound ID 4522 (Figs. 6 and 7, panel A). Although
we did not observe valuable cytotoxicity of BK antagonists and
thiazolidinone derivatives in the most MCL cells, same 60–70% of
viable cells remained at the lowest and the highest concentration
of compounds used. It is possible to suppose that treated cell lines
contained a small subpopulation of cells, which were more sensi-
tive to the applied compounds.
4
l
l
l
l
However, treatment of MCL cells Mino with ID 4523, immobi-
lized on a PEGylated carrier (ID 4523car) was highly cytotoxic
(IC50 1.58
ID 4524, ID 4525, ID 4526, ID 4527, and ID 4664 behaved even
much better: IC50 was 0.73 M for ID 4524, 10 M for ID 4525,
0.27 M for ID 4526, 7.4 M for ID 4527, and 0.16 M for ID
lM) (Fig. 7, panel A). Chemical analogues of ID 4523:
l
l
3. Conclusions
l
l
l
4664 (Fig. 7, panel B). The IC50 values for the compounds ID 4526
and ID 4664 were significantly better than that of doxorubicin, a
conventional chemotherapeutic drug in clinical use, with IC50 of
Here we described the cytotoxic activity for representatives of
two chemically different classes of molecules, BK antagonists and
thiazolidinone derivatives, using several lines of malignantly trans-
formed cells. Among BK antagonists under experiment, BKM-570
appeared to be the lead compound in the 293_CHI3L1, U373, and
C6 cell lines. BKM-570 strongly inhibited ERK1/2 and AKT phos-
phorylation in 293_CHI3L1 and U373 cells, thus, the cytotoxic
effect of BKM-570 could be mediated by modulation of MAPK-
and PI3K-signaling cascades.
0.37 l
M.29 Thus, such thiazolidinone derivatives possess a valuable
cytotoxic activity in some MCL cells. Optimizing the effectiveness
of these agents is a focus of ongoing research and could be a prom-
ising tool for future anti-MCL drug development.
To address the question whether thiazolidinones have cytotoxic
properties against glioma cells, we tested compounds ID 28, immo-
bilized on a PEGylated carrier (ID 28car), as well as ID 4523 and ID
4523car in C6 and U373 cell lines. ID 28car demonstrated the
BK antagonists, prominently active in 293_CHI3L1, U373, and C6
cell lines did not affect MCL cells. Among five ID 4523-related
Please cite this article in press as: Avdieiev, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.046

8
S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
substances, ID 4524 and ID 4664 were shown to be the most effec-
tive in MCL cells. A significant cytotoxicity was observed also after
treatment of human and rat glioma cells with thiazolidinones ID
28car and ID 4523car. This could indicate the cellular specificity
of cytotoxic properties of BK antagonists and thiazolidinone
derivatives.
Especially important result was the finding that simultaneous
application of bradykinin antagonist BKM-570 drastically
improved the effectiveness of TMZ in glial cells, which alone has
only temporary therapeutic benefit and severe side-effects in
clinics. In this paper we made a pilot attempt to create the smart
drug combinations of newly synthesized compounds and conven-
tional chemotherapeutics that will enable the development of
therapeutic regimens with improved effectiveness and less
toxicity. Further investigations of anti-cancer properties of BK
antagonists, azolidinone derivatives, and their combinations with
each other and with conventional therapeutic agents using animal
models are needed for the evaluation of these compounds in a
pre-clinical study.
4.1.2.3. 3-{2-[3,5-Bis-(4-chlorophenyl)-4,5-dihydropyrazol-1-
yl]-4-oxo-4H-thiazol-5-ylidene}-5-bromo-1,3-dihydroindol-
2-one (ID 4524).
Yield 71%, mp >300 °C. 1H NMR (400 MHz,
DMSO-d6+CCl4): 10.95 (s, 1H), 9.13 (br s, 1H), 7.85 (d, J = 8.1 Hz,
2H), 7.68 (d, J = 8.1 Hz, 2H), 7.36–7.40 (m, 4H), 7.28 (t, J = 7.0 Hz,
1H), 6.98 (t, J = 7.0 Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 6.00–6.04 (m,
1H), 4.19 (dd, J = 18.2, 11.5 Hz, 1H), 3.46 (dd, J = 18.2, 3.8 Hz, 1H).
Calcd for C26H15BrCl2N4O2S: C, 52.20; H, 2.53; N, 9.36; Found: C,
52.11; H, 2.64; N, 9.50.
4.1.2.4. 3-{2-[3-(4-Bromophenyl)-5-(4-chlorophenyl)-4,5-dihy-
dropyrazol-1-yl]-4-oxo-4H-thiazol-5-ylidene}-1,3-dihydroin-
dol-2-one (ID 4525).
Yield 73%, mp >300 °C. 1H NMR (400 MHz,
DMSO-d6+CCl4): 10.84 (s, 1H), 8.95 (d, J = 7.7 Hz, 1H), 7.34–7.85
(m, 8H), 6.92–7.28 (m, 3H), 6.00 (dd, J = 11.4, 3.8 Hz, 1H), 4.20
(dd, J = 18.1, 11.4 Hz, 1H), 3.46 (dd, J = 18.1, 3.8 Hz, 1H). Calcd for
C26H16BrClN4O2S: C, 55.38; H, 2.86; N, 9.94; Found: C, 55.45; H,
2.72; N, 10.05.
4.1.2.5. 5-Bromo-3-{2-[3-(4-bromophenyl)-5-(4-chlorophenyl)-
4,5-dihydro-pyrazol-1-yl]-4-oxo-4H-thiazol-5-ylidene}-1,3-
dihydroindol-2-one (ID 4526).
(400 MHz, DMSO-d6+CCl4): 11.00 (s, 1H), 9.14 (s, 1H), 7.38–7.86
(m, 8H), 6.88–7.29 (m, 2H), 6.00–6.04 (m, 1H), 4.18–4.22 (m,
1H), 3.49 (dd, J = 18.0, 3.9 Hz, 1H). Calcd for C26H15Br2ClN4O2S: C,
48.59; H, 2.35; N, 8.72; Found: C, 48.67; H, 2.47; N, 8.58.
4. Experimental section
4.1. Chemistry
Yield 73%, mp >300 °C. 1H NMR
4.1.1. Materials and methods
The starting 3,5-diaryl-1-thiocarbamoyl-2-pyrazoline was
obtained according to the method described previously.30,31
Melting points were measured in open capillary tubes on a
4.1.2.6. 5-Chloro-3-{2-[3-(4-bromophenyl)-5-(4-chlorophenyl)-
4,5-dihydro-pyrazol-1-yl]-4-oxo-4H-thiazol-5-ylidene}-1,3-
dihydroindol-2-one (ID 4527). Yield 73%, mp >300 °C. 1H NMR
(400 MHz, DMSO-d6+CCl4): 10.96 (s, 1H), 9.00 (br s, 1H), 7.37–
7.86 (m, 8H), 6.92–7.29 (m, 2H), 6.01–6.05 (m, 1H), 4.18-4.22 (m,
1H), 3.49 (dd, J = 18.0, 3.6 Hz, 1H). Calcd for C26H15BrCl2N4O2S: C,
52.20; H, 2.53; N, 9.36; Found: C, 52.41; H, 2.68; N, 9.17.
}
BUCHI B-545 melting point apparatus and are uncorrected.
The elemental analyses (C, H, N) were performed using
the Perkin–Elmer 2400 CHN analyzer. Analyses indicated by the
symbols of the elements or functions were within 0.4% of the
theoretical values. The 1H NMR spectra were recorded on Varian
Gemini 400 MHz in DMSO-d6 using tetramethylsilane (TMS) as
an internal standard. Chemical shifts are reported in ppm units
with use of d scale.
4.1.2.7. 5-Bromo-3-{2-[3-(4-chlorophenyl)-5-(4-dimethylamin-
ophenyl)-4,5-di-hydropyrazol-1-yl]-4-oxo-4H-thiazol-5-yli-
dene}-1,3-dihydroindol-2-one (ID 4664).
Yield 67%, mp
4.1.2. General procedure for synthesis of 3-{2-[3,5-bis-(4-
chlorophenyl)-4,5-dihydropyrazol-1-yl]-4-oxo-4H-thiazol-5-
ylidene}-1,3-dihydroindol-2-ones32
>300 °C. 1H NMR (400 MHz, DMSO-d6+CCl4): 11.20 (s, 1H), 9.09
(br s, 1H), 7.91 (d, J = 7.9 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 7.48 (d,
J = 8.5 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.3 Hz, 1H),
6.69 (d, J = 8.6 Hz, 2H), 5.85 (dd, J = 11.1, 3.5 Hz, 1H), 4.10 (dd,
J = 18.6, 11.1 Hz, 1H), 3.50 (dd, J = 18.6, 3.5 Hz, 1H), 2.87 (s, 6H).
Calcd for C28H21BrClN5O2S: C, 55.41; H, 3.49; N, 11.54; Found: C,
55.27; H, 3.38; N, 11.64.
A
mixture of 3,5-diaryl-1-thiocarbamoyl-2-pyrazoline (10
mmol), chloroacetic acid (10 mmol), appropriate isatin (12 mmol),
and anhydrous sodium acetate (10 mmol) was refluxed for 5 h in
glacial acetic acid (10 mL). Precipitate obtained upon cooling was
filtered off, washed with water and methanol and recrystallized
with DMF/ethanol mixtures (1:2 vol).
4.1.3. Synthesis of 2-[5-(4-dimethylaminobenzylidene)-2,4-
dioxo-thiazolidin-3-yl]-N-[5-(4-methoxyphenyl)-
[1,3,4]oxadiazol-2-yl]-acetamide (ID 4132).33
4.1.2.1. 3-{2-[3,5-Bis-(4-chlorophenyl)-4,5-dihydropyrazol-1-
A mixture 5 mmol of N-potassium salt of 5-(4-dimethylamino-
benzylidene)-thiazolidine-2,4-dione and 6 mmol 2-chloro-N-[5-
(4-methoxyphenyl)-[1,3,4]oxadiazol-2-yl]-acetamide as well as
catalytic amount of potassium iodide and carbonate in 15 ml of
mixture DMF–EtOH (vol. 1:1) were refluxed for 4 h. Reaction prod-
uct was filtered off after cooling and pouring into water, washed by
water, ethanol, and diethyl ether. Recrystallized with the mixture
of DMF–ethanol (1:2). Yield 70%, mp 239–240 °C. 1H NMR
(400 MHz, DMSO-d6+CCl4): 7.83–7.85 (m, 3H), 7.48 (d, J = 8.5 Hz,
2H), 7.12 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 4.60 (s, 2H),
3.82 (s, 3H). Calcd for C23H21N5O5S: C, 57.61; H, 4.41; N, 14.60;
Found: C, 57.82; H, 4.52; N, 14.48.
yl]-4-oxo-4H thiazol-5-ylidene}-1,3-dihydroindol-2-one (ID
4522).
Yield 85%, mp 340–341 °C. 1H NMR (400 MHz, DMSO-
d6+CCl4): 10.80 (s, 1H), 8.95 (d, J = 7.0 Hz, 1H), 7.92–7.94 (m, 2H),
7.53 (d, J = 8.2 Hz, 2H), 7.37–7.40 (m, 3H), 7.29 (t, J = 7.6 Hz, 1H),
7.00 (t, J = 7.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.00 (dd, J = 11.0,
3.7 Hz, 1H), 4.18 (dd, J = 18.1, 11.0 Hz, 1H), 3.47 (dd, J = 18.1,
3.7 Hz, 1H). Calcd for C26H16Cl2N4O2S: C, 60.12; H, 3.10; N, 10.79;
Found: C, 60.39; H, 3.25; N, 10.61.
4.1.2.2. 3-{2-[3,5-Bis-(4-chlorophenyl)-4,5-dihydropyrazol-1-
yl]-4-oxo-4H-thiazol-5-ylidene}-5-chloro-1,3-dihydroindol-2-
one (ID 4523).
Yield 80%, mp 355–356 °C. 1H NMR (400 MHz,
DMSO-d6+CCl4): 10.96 (s, 1H), 9.00 (br s, 1H), 7.93 (d, J = 8.4 Hz,
2H), 7.53 (d, J = 8.4 Hz, 2H), 7.37–7.39 (m, 4H), 7.29 (d, J = 7.6 Hz,
1H), 6.92 (d, J = 7.5 Hz, 1H), 6.03 (dd, J = 11.2, 3.6 Hz, 1H), 4.20
(dd, J = 18.0, 11.2 Hz, 1H), 3.52 (dd, J = 18.0, 3.6 Hz, 1H). Calcd for
4.2. Biological experiments
4.2.1. Cell cultures
C
26H15Cl3N4O2S: C, 56.38; H, 2.73; N, 10.12; Found: C, 56.22; H,
U373 cells (human glioblastoma–astrocytoma cells) were
kindly provided by Dr. M. Sanson (INSERM, U711, Biologie des
2.84; N, 10.20.
Please cite this article in press as: Avdieiev, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.046

S. Avdieiev et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
Interactions Neurones & Glie, Paris, France), MCL cell lines (Granta,
Acknowledgments
JeKo, Mino, and UPN1) by Dr. V. Ribrag (Departement de Medecine,
Institut Gustave Roussy, Villejuif, France), C6 cells by Dr. V. Baklau-
shev (State Research Center of Forensic and Social Psychiatry, Mos-
cow, Russian Federation). 293_CHI3L1 cells (293 cells stably
produced CHI3L1 oncoprotein) were kindly gifted by Dr. O. Bal-
ynska (Institute of Molecular Biology and Genetics, Kyiv, Ukraine).
293_CHI3L1, U373, and C6 cells were grown in DMEM (Hyclone,
USA), UPN1 cells in alpha-MEM (Gibco, USA), Granta, JeKo, and
Mino cells in RPMI (Gibco, USA). All culture mediums were supple-
mented with FBS at 10%, penicillin at 100 units/ml, and streptomy-
This work was supported in part by National Academy of Sci-
ences of Ukraine in frames of the program ‘Fundamental basis of
molecular and cell biotechnologies’, Project 294932 ‘COMBIOM’,
Grants N2006 008196 from the Fondation de France and ERABL
from the Institut National du Cancer de France (INCa). We
acknowledge support from FEBS for the Collaborative Experimen-
tal Scholarship for Central and Eastern Europe to S.A. and the J.
Stewart Endowed Chair in Peptide Chemistry to R.S.H.
cin at 100 lg/ml final concentration. Cells were cultivated in the
References and notes
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4.2.4. MTT assay
293_CHI3L1, U373, C6, Granta, JeKo, UPN1, or Mino cells were
seeded in triplicates into 96-well plate at density 3 Â 103 cells/
well. Compounds were applied at final concentration 0.01, 0.1, 1,
10, and 100
natorial treatment, TMZ was added to the final concentration of 10,
100, 250, 500, and 1000 M combined with 1 M BKM-570 or
lM, which were reached by serial dilution. For combi-
l
l
BKM-1800. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) (Sigma), which is converted to dark blue, water-
insoluble MTT formazan by mitochondrial dehydrogenases, was
used to determine viable cells according to manufacturer’s proto-
col (Sigma).
Please cite this article in press as: Avdieiev, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.06.046