Anticancer Properties of Novel 4-methylene-1,2-diphenylpyrazolidin-3-ones

Article abstract of DOI:10.1111/cbdd.12565

The limited success of the currently used antitumor therapies is the driving force for organic chemists to seek new lead structures with anticancer potential. Two α-methylene-γ-lactams with an additional nitrogen atom in the lactam ring, 5-vinyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2b) have been synthesized. Their anticancer activity was assessed in MCF-7 cells. Both compounds inhibited cell proliferation and induced DNA damage and apoptosis, with 2a being the more potent analog. Synergistic effects of 2a used in combination with known anticancer drugs, 5-fluorouracil, taxol, and oxaliplatin were evaluated. Compound 2a significantly enhanced the antitumor action of oxaliplatin and 5-fluorouracil, but not taxol. Two α-methylene-γ-lactams with an additional nitrogen atom in the lactam ring, 5-vinyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4 methylenepyrazolidin-3-one (2b) were synthesized and tested for their anti-cancer activity in MCF-7 cells. Both compounds inhibited cell proliferation and induced DNA damage and apoptosis. Additionally, 2a showed synergistic effect in co-treatment with anti-caner drugs, oxaliplatin and 5-fluorouracil.

Full text of DOI:10.1111/cbdd.12565

Chem Biol Drug Des 2015; 86: 961–968
Research Article
Anticancer Properties of Novel 4-methylene-1,2-
diphenylpyrazolidin-3-ones
1
2
ꢀ
carbonyl function. The great progress in the development
of new methodologies for the synthesis of compounds
with such motif can be noticed, especially in the last
decade (7).
Katarzyna Gach , Jacek Szymanski ,
Dorota Pomorska1, Angelika Długosz1,
Jakub Modranka3, Marlena Michalak3,
Tomasz Janecki3 and Anna Janecka1,*
1Department of Biomolecular Chemistry, Faculty of
Medicine, Medical University of Lodz, Mazowiecka 6/8,
92-215 Lodz, Poland
Also in our laboratory, we have developed synthetic proce-
dures for introducing an exo-methylene group to various
classes of cyclic skeletons, including lactones (8–11) and
lactams (9,10,12). Many of the obtained compounds
turned out to be highly cytotoxic against several cancer
cell lines (7,10,12–14). Recently, we designed a series of
4-methyleneisoxazolidin-5-ones 1 containing an additional
nitrogen atom in the c-lactone ring (15) (Figure 1). Several
of these compounds showed strong cytotoxicity against
leukemia (HL-60 and NALM-6) and breast cancer (MCF-7
and MDA-MB-231) cells (15). The most promising com-
pound 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one
(designated MZ-6) has been subjected to extended biolog-
ical studies which have shed light on its mode of action at
the molecular level. In particular, in MCF-7 cells MZ-6
induced early apoptosis and cell cycle arrest in G0/G1
phase and suppressed migration of cancer cells by
down-regulation of metalloproteinase-9 and urokinase
plasminogen activator, the key proteases involved in the
degradation of extracellular matrix and dissemination of
cancer cells (16,17).
2Central Scientific Laboratory, Division of Public Health,
Faculty of Health Sciences, Medical University of Lodz,
Mazowiecka 6/8, 92-215 Lodz, Poland
3Institute of Organic Chemistry, Lodz University of
_
Technology, Zeromskiego 116, 90-924 Lodz, Poland
*Corresponding author: Anna Janecka,
anna.janecka@umed.lodz.pl
The limited success of the currently used antitumor
therapies is the driving force for organic chemists to
seek new lead structures with anticancer potential. Two
a-methylene-c-lactams with an additional nitrogen atom
in the lactam ring, 5-vinyl-1,2-diphenyl-4-methylenepy-
razolidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4-methy-
lenepyrazolidin-3-one (2b) have been synthesized. Their
anticancer activity was assessed in MCF-7 cells. Both
compounds inhibited cell proliferation and induced DNA
damage and apoptosis, with 2a being the more potent
analog. Synergistic effects of 2a used in combination
with known anticancer drugs, 5-fluorouracil, taxol, and
oxaliplatin were evaluated. Compound 2a significantly
enhanced the antitumor action of oxaliplatin and 5-fluo-
rouracil, but not taxol.
Recently, we have reported the synthesis and the cyto-
toxic activity of a large series of, so far unknown, 4-methy-
lenepyrazolidin-3-ones 2 (Figure 1) containing an additional
nitrogen atom in the c-lactam ring (18). Such compounds
can be viewed as a modification of a-methylene-c-lactams
which are believed to be less toxic for normal cells than
c-lactones (19,20) but usually are also less active as anti-
cancer agents (10,12,13). Two of these novel compounds,
5-vinyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2a) and
Key words: anticancer drugs, apoptosis, breast cancer, cell
proliferation, DNA damage, a-Methylene-c-lactams
Received 12 November 2014, revised 11 February 2015 and
accepted for publication 25 March 2015
Advances in cancer treatment require the continued devel-
opment of novel and improved chemotherapeutic agents.
Throughout history, natural products have been a rich
source of anticancer drugs with diverse chemical struc-
tures and bioactivities. Many plant-derived natural com-
pounds are characterized by an a-methylene-c-lactone or
-lactam scaffold (1,2). Such compounds can alkylate cellu-
lar thiols disrupting many major processes in the cells
(3–6). The structural fragment responsible for such alkyl-
ation is the exocyclic methylene group conjugated with a
Figure 1: General structures of 4-methyleneisoxazolidin-5-one 1
and 4-methylenepyrazolidin-3-one 2.
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12565
961

Gach et al.
5-phenyl-1,2-diphenyl-4-methylenepyrazolidin-3-one (2b),
showed an exceptional cytotoxicity (the IC50 values in low
mM range) against MCF-7 cells. This remarkable activity,
unprecedented among the a-methylene-c-lactams tested
so far (10,21), prompted us to perform further biological
tests. The effects of pyrazolidinones 2a and 2b on cell
proliferation, DNA damage, apoptosis induction, reactive
oxygen species (ROS) generation, and cell cycle were
evaluated when these compounds were incubated with
MCF-7 cells alone or in combination with well-known anti-
cancer drugs, 5-fluorouracil (5-FU), taxol (Tx), and oxalipla-
tin (Ox). The purpose of the simultaneous administration of
two agents with different mechanisms of action was to
discover if novel synthetic compounds would enhance the
anticancer potential of commonly used chemotherapeu-
tics.
Cell viability assay (MTT)
The MTT assay was performed according to the known
procedure (22). The cells were grown to subconfluent lev-
els in the culture medium and then plated onto 24-well
plates (104 cells/well) in the final volume of 1 mL of the
culture medium. After 24 h, various concentrations of the
tested compounds were added and the plates were incu-
bated for 24 h. Then, the cells were incubated for 2 h at
37 °C with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide [MTT, 5 mg/mL in phosphate buffered saline
(PBS, Gibco, Invitrogen, Carlsbad, CA, USA)]. The absor-
bance of the blue formazan product was measured at
540 nm using an automated plate reader (iMark Bio-Rad,
Hercules, CA, USA) and compared with control (untreated
cells). The IC50 values, which represent 50% inhibitory
concentration of a compound, were assessed. All experi-
ments were performed in triplicate.
Materials and Methods
Analysis of cell proliferation, apoptosis and DNA
damage by flow cytometry
General
Ox and Tx were purchased from Tocris Bioscience (Bristol,
UK) and 5-FU and menadione from Sigma-Aldrich (St.
Louis, MO, USA). Ox and menadione were dissolved in
culture medium. 5-FU, Tx, and synthetic 4-methylenepy-
razolidin-3-ones were dissolved in DMSO and further
diluted in culture medium to obtain <0.1% DMSO concen-
tration. In each experiment, controls without and with
0.1% DMSO were performed. 0.1% DMSO had no effects
on the observed parameters.
Cell proliferation, DNA damage and apoptotic cell death
were determined using the ‘Apoptosis, DNA Damage, and
Cell Proliferation Kit’ (BD Bioscience, San Jose, CA, USA),
according to the manufacturer guidelines. The test kit
included all the necessary components, such as BD Cyto-
fix/CytopermTM Fixation/Permeabilization Solution; BD
Perm/WashTM Buffer; BD Cytofix/CytopermTM Plus Permea-
bilization Buffer, DAPI, BrdU, DNase and antibodies
(PerCP-CyTM5.5 Mouse Anti-BrdU Alexa Fluorꢀ 647 Mouse
Anti-H2AX (pS139); PE Mouse Anti-Cleaved PARP
(Asp214)). Briefly, MCF-7 cells were seeded in 25 cm2 cell
culture flasks at a density 0.1 9 106/mL in 10 mL of stan-
dard growth medium. After 24 h, the growth medium was
replaced by a fresh growth medium supplemented with
the tested compounds in the desired concentrations. The
cells incubated without tested compounds were used as
controls. After 24-h incubation, the culture medium was
collected, and the floating cells were counted and dis-
carded. The number of discarded cells was never greater
that 15% of the cell population. The fresh growth medium
supplemented with BrdU (final concentration of 10 lM)
was added to each flask. After 8-h incubation, culture
medium was collected, and cells were washed twice with
phosphate buffered saline (PBS, Gibco, Invitrogen) and
harvested by trypsinization. Cells detached from a culture
flask and cells floating in culture medium were combined
into one tube and centrifuged (200 9 g, 5 min). The total
number of cells was determined. Cells were resuspended
in BD Cytofix/Cytoperm Fixation/Permeabilization Solution
(100 lL per tube) and incubated for 30 min on ice. Then,
cells were washed once with 1 ml of 1 9 BD Perm/Wash
Buffer and centrifuged (200 9 g, 5 min). Supernatant was
discarded. Cells were stored in 4 °C overnight in 500 lL
of staining buffer. After washing (as previously described),
cells were fixed and permeabilized according to the manu-
facturer protocol. In the next step, cells were treated with
Chemical synthesis
Pyrazolidinones 2a and 2b were synthesized as reported (18),
starting from 3-methoxy-2-diethoxyphosphorylacrylate and
1,2-diphenylhydrazine. Thus, obtained 4-diethoxyphosphoryl-
1,2-diphenyl-1,2-dihydro-3H-pyrazol-3-one was used as a
Michael acceptor in the reaction with vinylmagnesium or phe-
nylmagnesium chloride to give 5-vinyl or 5-phenyl-4-dieth-
oxyphosphoryl-1,2-diphenylpyrazolidin-3-one, respectively.
These pyrazolidinones were next used as Horner–Wads-
worth–Emmons reagents for the olefination of formaldehyde
to furnish the final 4-methylene-1,2-diphenylpyrazolidin-3-
ones 2a,b in 85% and 89% yield, respectively.
Cell culture
The MCF-7 cell line was purchased from the European
Collection of Cell Cultures (ECACC). The MCF-7 cells
were cultured in Minimum Essential Medium Eagle
(MEME, Sigma-Aldrich) with glutamine (2 mM) (Sigma-
Aldrich), MEM Non-essential amino acid solution 100x
(Sigma-Aldrich), gentamycin (5 lg/mL) and 10% heat-
inactivated fetal bovine serum (FBS) (both from Biological
Industries, Beit-Haemek, Israel). Cells were maintained at
37 °C in a 5% CO2 atmosphere and were grown until
80% confluent.
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Chem Biol Drug Des 2015; 86: 961–968

Anticancer Properties of Pyrazolidinones
100 lL of diluted DNase (1 mg/mL in DPBS) per tube and
incubated for 1 h at 37 °C to expose BrdU epitopes.
Then, cells were simultaneously stained with fluoro-
chrome-labeled anti-BrdU, H2AX (pS139) and cleaved
PARP (Asp214) antibodies for 20 min at room tempera-
ture. After washing, DNA staining for cell cycle analysis
using DAPI solution (1 lg/mL of staining buffer) was per-
formed. Cells were resuspended in staining buffer and
analyzed by flow cytometry using Becton Dickinson Canto
II. The data were visualized and quantified by constructing
a dot-plot using the BD FACSDIVA software version 6.1.3. (BD
Bioscience).
were expressed as means ꢀ SEM. Statistical comparisons
were assessed by a one-way ANOVA followed by a post
hoc multiple comparison Student–Newman–Keuls test. A
probability level of 0.05 or lower was considered statisti-
cally significant.
Results and Discussion
Chemistry
Synthetic compounds, 5-vinyl-1,2-diphenyl-4-methylenepy-
razolidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4-methyle-
nepyrazolidin-3-one (2b), were obtained according to the
reaction sequence outlined in Scheme 1. The structures
and purity of compounds 2a,b were confirmed by IR, 1H
NMR, 13C NMR, ESI MS, and elemental analyses.
Analysis of reactive oxygen species induction by
flow cytometry
Induction of ROS generation was determined by flow
cytometry, based on the staining with CellROXꢀ Oxidative
Stress Reagents (Molecular Probes; Life Technologies,
Carlsbad, CA, USA). CellROXꢀ Oxidative Stress Reagents
are fluorogenic probes designed to reliably measure ROS
in live cells. The cell-permeable reagents are non-fluores-
cent or very weakly fluorescent, while in a reduced state
and upon oxidation they exhibit strong fluorogenic signal.
CellROXꢀ Green Reagent is a DNA dye, and upon oxida-
tion, it binds to DNA; thus, its signal is localized primarily
in the nucleus and mitochondria.
Biology
The search for effective plant-derived anticancer agents or
their synthetic analogs has continued to be of interest to
biologists and chemists for a long time. In this report, cyto-
toxicity and anticancer activity of synthetic, so far
unknown, pyrazolidinones 2a and 2b was tested in MCF-7
cells. The cytotoxic effect of these compounds was stud-
ied using the MTT assay. Cells were exposed to a board
range of compound concentrations for 24 h. Both tested
pyrazolidinones showed similar high cytotoxic activity with
IC50 values of 5.1 ꢀ 0.2 lM and 4.5 ꢀ 0.3 lM, for 2a and
2b, respectively. Furthermore, the ability of these com-
pounds to inhibit cell proliferation, generate DNA damage,
and induce apoptotic cell death was examined by flow
cytometry using the ‘Apoptosis, DNA Damage, and Cell
Proliferation Kit’ (BD Bioscience).
Briefly, MCF-7 cells were seeded in 6-well cell culture
plate at a density 0.16 9 106/mL in 3 mL of standard
growth medium. After 24 h, the growth medium was
replaced by a fresh growth medium supplemented with
the tested compounds in the desired concentrations.
MCF-7 cells treated with the tested compounds for 24 h
were incubated with 5 lM CellROXꢀ Green Reagent at
37 °C for 30 min in the dark. Then, cells were harvested by
trypsinization, centrifuged (200 9 g, 5 min), and washed
twice with PBS. After washing, cells were resuspended in
PBS and analyzed by flow cytometry (Becton Dickinson
Canto II). Menadione was used as a positive control.
MCF-7 cells were incubated for 24 h with increasing con-
centrations: 0.5 9 IC50, 1 9 IC50, and 2 9 IC50 of pyrazo-
lidinones 2a or 2b. Then, culture medium was changed
and cells were incubated for additional 8 h with bromode-
oxyuridine (BrdU). Cells were simultaneously stained with
fluorochrome-labeled anti-BrdU, H2AX (pS139), and
cleaved PARP (Asp214) antibodies. BrdU incorporation
into DNA was used as an index of cell proliferation. The
DNA damage was evaluated using Alexa Fluor 647 Mouse
Anti-H2AX (pS139) antibodies, directed against the phos-
phorylated form of human H2AX protein at the pS139 resi-
Statistical analysis
Statistical analyses were performed using PRISM 4.0
(GraphPad Software Inc., San Diego, CA, USA). The data
O
O
O
NaH,
R
(CH2O)n
THF, r.t.,
RMgCl, THF
toluene,
R
P(OEt)2
P(OEt)2
P
COOEt
(EtO)2
2 h
Ph
reflux,
2 h
reflux, 80 h
+
PhNHNHPh
N
N
N
O
Ph
Ph
N
O
O
N
OMe
N
Ph
Ph
Ph
83%
5a R vinyl,
=
,
vinyl, 85%
R
=
2a
3
4
,
, 83%
5b
2b R Ph, 89%
=
,
R = Ph, 35%
,
Scheme 1: Synthesis of pyrazolidinones 2a,b.
Chem Biol Drug Des 2015; 86: 961–968
963

Gach et al.
A
Figure 3: Effect of 2a and 2b, at IC50 concentrations, on cell
cycle distribution in MCF-7 cells. The percentage of cells at each
stage of the cycle was analyzed by flow cytometry, following DNA
staining with DAPI and compared with untreated cells regarded as
control, C. The results are representative of three independent
experiments.
B
apoptosis induction experiments, 2a worked in a dose-
dependent manner, while for 2b, a significant effect was
observed only at the highest concentration (2 9 IC50) (Fig-
ure 2C). In the performed tests, the effects exerted by 2a
were much stronger than for 2b. At IC50 concentration, 2a
inhibited MCF-7 cell proliferation in about 74% and
induced DNA damage and apoptosis in about 15% of cell
population.
C
Cell cycle analysis revealed that in MCF-7 cells both tested
compounds caused G0/G1 cell cycle arrest. In untreated
cells, used as control, 55.6% of cells was in the G0/G1
phase, while in cells treated with 2a or 2b, these values
increased up to 61.7% and 70.2%, respectively (Figure 3).
We also observed an increase in the percentage of cells in
sub-G0/G1-phase (corresponding to apoptotic cells), from
0.3% for control to about 2% for cells treated with the
tested compounds.
Figure 2: Inhibition of cell proliferation (BrdU test) (A), induction
of DNA damage (H2AX test) (B), and induction of apoptosis
(PAPR test) (C) by 2a and 2b in MCF-7 cells. After 24-h
incubation with tested compounds, medium was changed, fresh
medium with BrdU was added, and cells were incubated for
additional 8 h. The cells were stained with fluorochrome-labeled
anti-BrdU, anticleaved PARP (Asp214) and anti-H2AX (pS139)
antibodies for 20 min at room temperature and analyzed by flow
cytometry. Data represent mean ꢀ SEM of three independent
experiments. Statistical significance was assessed using one-way
ANOVA and a post hoc multiple comparison Student–Newman–
Keuls test. **p < 0.01 and ***p < 0.001 were considered as
significantly different from untreated cells regarded as control, C.
A new promising paradigm for cancer treatment is a com-
bination chemotherapy, involving at least two drugs that
work by different mechanisms, thereby decreasing the
likelihood that resistant cancer cells will develop (23) and
increasing anticancer effects by synergistic action. The
high anticancer activity of our synthetic pyrazolidinones
prompted us to perform combination experiments with
known anticancer drugs. We investigated possible syner-
gistic effects of 2a used in combination with 5-FU, Tx, and
Ox. On the basis of their mode of action, these drugs can
be classified as an antimetabolite (24), mitotic inhibitor
(25,26), and alkylating agent (27), respectively. Synergism
in drugs is defined as joint action of two drugs in such a
manner that one enhances the action of the other to pro-
duce an effect greater than that which may be obtained
with either one alone. MCF-7 cells were co-incubated with
2a (at IC50 concentration) and each of the mentioned
above anticancer drugs for 24 h. The concentrations of
anticancer drugs were chosen based on literature data
and were 25, 30 and 35 lM for 5-FU, Tx, and Ox, respec-
tively (24–28). The effects of the combination treatment of
MCF-7 cells on inhibition of cell proliferation, induction of
due that is considered a biomarker for DNA double-strand
breaks (DSB). The induction of apoptosis was assessed
using fluorochrome-labeled PE anticleaved PARP (Asp214)
antibodies recognizing 89 kDa-cleaved fragment of PARP
that can serve as a marker of cellular apoptosis.
The obtained results demonstrated that both tested com-
pounds dose dependently inhibited cell proliferation (Fig-
ure 2A) and generated DNA damage (Figure 2B). In
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Chem Biol Drug Des 2015; 86: 961–968

Anticancer Properties of Pyrazolidinones
A
Figure 4: Combined effect of 2a used at IC50 concentration and
5-FU (25 ll), Tx (30 nM), or Ox (35 ll) on inhibition of cell
proliferation (BrdU test) (A), induction of DNA damage (H2AX test)
(B) and induction of apoptosis (PAPR test) (C) in MCF-7 cells. Cells
were co-incubated with 2a and an anticancer drug for 24 h. Then,
medium was changed, fresh medium with BrdU was added, and
cells were incubated for additional 8 h. The cells were stained with
fluorochrome-labeled anti-BrdU anticleaved PARP (Asp214) and
anti-H2AX (pS139) antibodies for 20 min at room temperature and
analyzed by flow cytometry. Data represent mean ꢀ SEM of three
independent experiments. Statistical significance was assessed
using one-way ANOVA and a post hoc multiple comparison Student–
Newman–Keuls test. *p < 0.05, **p < 0.01, and ***p < 0.001 in
comparison with respective anticancer drug alone; #p < 0.05,
##p < 0.01, ###p < 0.001, in comparison with 2a.
B
Figure 5: Combined effect of 2a (IC50 concentration) and Ox
(35 ll) on cell cycle distribution in MCF-7 cells. The percentage
of cells at each stage of the cycle was analyzed by flow
cytometry, following DNA staining with DAPI and compared with
untreated cells regarded as control. The results are representative
of three independent experiments.
C
(Figure 4A) and enhanced DNA damage (Figure 4B). On
the other hand, incubation of the cells with 2a and 5-FU
did not influence caspase-3 activity and induction of apop-
tosis (Figure 4C).
The action of Ox was strongly enhanced by 2a. In particu-
lar, inhibition of cell proliferation produced by Ox in combi-
nation with 2a was about four times stronger than
observed for Ox alone. Similarly, the apoptosis induction
caused by Ox was synergistically enhanced by 2a (Fig-
ure 4C). The analysis of H2AX phosphorylation revealed
that Ox alone in 35 lM concentration was able to induce
DNA damage in about 27% of cell population. However,
this high value was not increased but rather decreased in
the combination experiments and was similar to the value
obtained for 2a alone (Figure 4B). It is likely that the action
of Ox is masked by 2a. The same masking effect was
observed when cell cycle distribution was analyzed. The
percentage of subG0/G1 cell population in the combina-
tion experiment (Ox and 2a) decreased from 17% for Ox
alone to 10% (Figure 5). The representative multiparameter
flow cytometry analyses for the combination of Ox and 2a
are shown in Figure 6. The action of Tx was not enhanced
by 2a in any of the performed tests (Figure 4A–C).
DNA damage and apoptosis were evaluated and com-
pared with the effects produced by these substances
alone. We considered the effect of synergistic only when
there was a statistically significant change vs both the anti-
cancer drug and 2a. The statistical significance of com-
bined treatment is shown in Figure 4A–C.
We have shown that the combined treatment with 5-FU
and 2a effectively decreased the MCF-7 cell proliferation
Chem Biol Drug Des 2015; 86: 961–968
965

Gach et al.
Control
Control
A
B
26%
1.3%
98%
0.2%
0.5%
2a + Ox
2a + Ox
Figure 6: Multiparameter analysis
(proliferation, DNA damage and apoptosis)
of the synergistic effect of Ox (35 ll) in
combination with 2a (IC50 concentration) in
MCF-7 cells. Panel A: DAPI vs BrdU PerCP-
CyTM5.5 staining profile. BrdU positive cells
are in the inside frame. Panel B: cleaved
PARP (Asp214) PE vs H2AX (pS139) Alexa
profile. Squares Q1 + Q2 represent H2AX
(pS139) Alexa positive cells, squares
Q2 + Q4 represent cleaved PARP (Asp214)
PE positive cells, whereas square Q3
represents H2AX (pS139) Alexa and cleaved
PARP (Asp214) PE negative cells.
1.1%
8%
7%
73%
12%
Green Reagent assay was used. Compound 2a slightly
increased ROS production in MCF-7 cells. However, a
strong synergistic effect was observed when cells were
treated with 2a and Ox (Figure 7). The obtained results
indicated that the synergistic cytotoxic effect of 2a and Ox
may be mediated by induction of apoptosis through mito-
chondrial pathway.
Conclusions
In this report, we have described anticancer activity of
two novel a-methylene-c-lactams with an additional nitro-
gen atom in the lactam ring. Compounds with such skel-
eton have been unknown so far. We have demonstrated
that in MCF-7 cell 5-vinyl-1,2-diphenyl-4-methylenepyraz-
olidin-3-one (2a) and 5-phenyl-1,2-diphenyl-4-methyle-
Figure 7: Combined effect of 2a (IC50 concentration) and Ox
(35 ll) on ROS generation in MCF-7 cells. The cells were stained
with 5 ll CellROXꢀ Green Reagent. Menadione (100 ll, 1-h
incubation) was used as a positive control. Data represent mean
ꢀ SEM of three independent experiments. Statistical significance
was assessed using one-way ANOVA and a post hoc multiple
nepyrazolidin-3-one (2b) displayed
a broad range of
anticancer activities including inhibition of cell proliferation,
generation of DNA damage and induction of apoptosis.
Compound 2a with vinyl in position 5 was the more
active analog of the two. Additionally, this analog syner-
gistically enhanced the anticancer activity of known anti-
cancer drugs, 5-FU and Ox. Increased ROS generation
observed predominantly in combined treatment (2a and
Ox) pointed at mitochondrial mechanism of apoptosis
induction. The obtained results show that in the search
for new chemotherapeutics cheap and easy to obtain
synthetic compounds can replace more complex struc-
tures found in nature.
comparison
Student–Newman–Keuls
test.
*p < 0.05,
***p < 0.001 in comparison with control, C; ###p < 0.001 in
comparison with 2a, &&&p < 0.001 in comparison with Ox.
One of the possible mechanisms of the cytotoxic activity
of compounds with exo-methylene motif conjugated with a
carbonyl group is associated with ROS generation in can-
cer cells. To test the effect of 2a alone and in combination
with Ox and 5-FU on the ROS generation, CellROXꢀ
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Chem Biol Drug Des 2015; 86: 961–968

Anticancer Properties of Pyrazolidinones
11. Modranka J., Albrecht A., Janecki T. (2010) A convenient
entry to 3-methylidenechroman-2-ones and 2-methyliden-
edi-hydrobenzochromen-3-ones. Synlett;19:2867–2870.
12. Janecki T., Błaszczyk E., Studzian K., Janecka A., Kra-
Acknowledgments
This work was financed by the Medical University of Lodz
(No 502-14-191 to KG and No 503/1-156-02/503-01) and
by the Ministry of Science and Higher Education (Project
No. N N204 005736).
ꢀ
jewska U., Rozalski M. (2005) Novel synthesis, cyto-
toxic evaluation, and structure-activity relationship
studies of a series of alpha-alkylidene-gamma-lactones
and lactams. J Med Chem;48:3516–3521.
ꢀ
_
13. Albrecht A., Albrecht Ł., Rozalski M., Krajewska U.,
Janecka A., Studzian K., Janecki T. (2010) A conve-
nient synthesis and cytotoxic evaluation of b-aryl-a-
methylidene-c-lactones and b-aryl-a-methylidene-c-lac-
tams. New J Chem;34:750–761.
Conflict of Interest
None declared.
14. Modranka J., Albrecht A., Jakubowski R., Krawczyk
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