Synthesis, characterization, anticancer evaluation and mechanisms of cytotoxic activity of novel 3-hydroxy-3-pyrrolin-2-ones bearing thenoyl fragment: DNA, BSA interactions and molecular docking study

Article abstract of DOI:10.1016/j.bioorg.2019.102954

In order to make a progress in discovering a new agents for chemotherapy with improved properties and bearing in mind the fact that substituted 3-hydroxy-3-pyrrolin-2-ones belong to a class of biologically active compounds, series of novel 1,5-diaryl-4-(2

Full text of DOI:10.1016/j.bioorg.2019.102954

Accepted Manuscript
Synthesis, characterization, anticancer evaluation and mechanisms of cytotoxic
activity of novel 3-hydroxy-3-pyrrolin-2-ones bearing thenoyl fragment: DNA,
BSA interactions and molecular docking study
Nenad Joksimović, Jelena Petronijević, Nenad Janković, Dejan Baskić, Suzana
Popović, Danijela Todorović, Sanja Matić, Goran A. Bogdanović, Milan
Vraneš, Aleksandar Tot, Zorica Bugarčić
PII:
S0045-2068(19)30140-3
https://doi.org/10.1016/j.bioorg.2019.102954
102954
DOI:
Article Number:
Reference:
YBIOO 102954
Bioorganic Chemistry
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Accepted Date:
28 January 2019
3 April 2019
25 April 2019
Please cite this article as: N. Joksimović, J. Petronijević, N. Janković, D. Baskić, S. Popović, D. Todorović, S.
Matić, G.A. Bogdanović, M. Vraneš, A. Tot, Z. Bugarčić, Synthesis, characterization, anticancer evaluation and
mechanisms of cytotoxic activity of novel 3-hydroxy-3-pyrrolin-2-ones bearing thenoyl fragment: DNA, BSA
interactions and molecular docking study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.102954
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Synthesis, characterization, anticancer evaluation and mechanisms of cytotoxic
activity of novel 3-hydroxy-3-pyrrolin-2-ones bearing thenoyl fragment: DNA,
BSA interactions and molecular docking study
Nenad Joksimović,a Jelena Petronijević,a Nenad Janković,a Dejan Baskić,b Suzana Popović,b
Danijela Todorović,c Sanja Matić,d Goran A. Bogdanović,e Milan Vraneš,f Aleksandar Totf and
Zorica Bugarčić*a
aUniversity of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanovića
12, 34000 Kragujevac, Serbia. E-mail: zoricab@kg.ac.rs;
bUniversity of Kragujevac, Faculty of Medical Sciences, Centre for Molecular Medicine and
Stem Cell Research, Svetozara Markovića 69, 34000 Kragujevac, Serbia;
cUniversity of Kragujevac, Faculty of Medical Sciences, Department of Genetics, Svetozara
Markovića 69, 34000 Kragujevac, Serbia;
dDoctoral academic study, Faculty of Medical Sciences, University of Kragujevac, Svetozara
Markovica 69, 34000 Kragujevac, Serbia;
eVinča Institute of Nuclear Science University of Belgrade, P.O. Box 522, 11001, Belgrade,
Serbia
fUniversity of Novi Sad, Department of Chemistry, Biochemistry and Environmental Protection,
Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia;
Keywords: 3-hydroxy-3-pyrrolin-2-ones, mechanisms of cytotoxic activity, antitumor activity,
DNA binding study, BSA binding study, molecular docking.
Abstract: In order to make a progress in discovering a new agents for chemotherapy with
improved properties and bearing in mind the fact that substituted 3-hydroxy-3-pyrrolin-2-ones
belong to a class of biologically active compounds, series of novel 1,5-diaryl-4-(2-
thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones were synthesized and characterized by spectral
(UV-Vis, IR, NMR, ESI-MS), X-ray and elemental analysis. All compounds were examined for
their cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts

(MRC-5). Four compounds, 3-hydroxy-1-(p-tolyl)-4-(2-thienylcarbonyl)-5-(4-chlorophenyl)-2,5-
dihydro-1H-pyrrol-2-one (D10), 3-hydroxy-1-(3-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-
(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D13), 3-hydroxy-1-(4-nitrophenyl)-4-(2-
thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one (D14), and 3-hydroxy-1-
(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-pyrrol-2-one
(D15), that showed the highest cytotoxicity against malignant cells and the best selectivity
towards normal cells were selected for further experiments. Results obtained by investigating
mechanisms of cytotoxic activity suggest that selected 3-hydroxy-3-pyrrolin-2-one derivatives in
HeLa cells induce apoptosis that is associated with S phase arrest (D13, D15, and D10) or
unrelated to cell cycle distribution (D14). Additionally, to better understand their suitability for
potential use as anticancer medicaments we studied the interactions between biomacromolecules
(DNA or BSA) and D13 and D15. The results indicated that D13 and D15 have great affinity to
displace EB from the EB-DNA complex through intercalation [Ksv = (3.7 ± 0.1) and (3.4 ± 0.1) ×
103 M-1, respectively], an intercalative mode also confirmed through viscosity measurements. Ka
values, obtained as result of fluorescence titration of BSA with D13 and D15 [Ka = (4.2 ± 0.2)
and (2.6 ± 0.2) × 105 M, respectively], support the fact that a significant amount of the tested
compounds could be transported and distributed through the cells. In addition, by DNA and
BSA molecular docking study for D13, D14 and D15 is determined and predicted the binding
mode and the interaction region.
INTRODUCTION
One of the biggest health problem in our society and one of the primary targets in
medicinal chemistry nowadays is cancer. Since the discovery of cisplatin and its use in clinical
therapy, scientists have made a lot of effort to synthesize many platinum-based drugs that could
be less toxic to healthy tissue [1,2,3] and to overcome the resistance of some tumors to cisplatin
[4,5]. The main goal for developing non-platinum anticancer drugs is to overcome the limits of
platinum drugs: severe toxicity on healthy tissue, narrow range of activity and after treatment
acquired resistance [1–5]. The discovery of new nonplatinum drugs with less side effects is of
the major interest in pharmaceutical chemistry [6]. Substituted 3-hydroxy-3-pyrrolin-2-ones
belong to a class of biologically active compounds. They can be obtained in the reaction between

ethyl-2-hydroxy-4-thienyl-4-oxo-2-butenoate with a mixture of aryl(alkyl)amine and an aldehyde
by short-term heating of a mixture of starting reagents [7]. These compounds possess different
types of pharmacological activities [8]. For example, some investigations showed that they have
good antimicrobial activity [9-12]. Also, these compounds have many other biological activities
such as anti-inflammatory, antiviral, antifungal, and nootropic [13-15]. To sum up, various
biologically active natural products containing either 3-acyl-5-hydroxy-3-pyrrolin-2-one or 3-
acyl-3,4-epoxy-5-hydroxypyrrolidin-2-one are isolated, suggesting that the pyrrolidinone moiety
is mostly responsible for the biological activity of these compounds.
In order to discover new anticancer agents with improved properties and bearing in mind
previous facts, we studied three-component, the one-pot reaction of acylpyruvic acid ethyl ester
with a mixture of an aromatic aldehyde and aromatic amine to produce novel potential antitumor
drugs. It was found that reaction between above-mentioned substrates, in glacial acetic acid as
solvent, lead to the formation of 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones
(Scheme 1). The fifteen 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones were
exposed to cytotoxic evaluation, fluorescence measurements and molecular docking studies. In
addition, the potential mechanism of cytotoxic activity of some 3-hydroxy-3-pyrrolin-2-one
derivates was investigated.
Scheme 1 General synthetic pathway towards 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-
pyrrolin-2-ones (D1-D15).

Fluorescence spectroscopy is an excellent experimental method to study interactions
between biomacromolecules (DNA, carrier proteins, etc.) and small drugs such as presented
compounds. The investigations on the interaction of small drugs with deoxyribonucleic acid are
of great interest due to their potential use as anticancer medicaments [16,17,18]. In general, small
molecules can interact with DNA through covalent interactions (interstrand/intrastrand cross-
linking) [19,20,21] or via noncovalent associations (stacking [22,23,24], groove binding [25,26],
intercalation [27]). Bovine serum albumin (BSA) is the protein that has the ability to transport a
large number of endogenous and exogenous ligands such as amino acids, fatty acids, steroids,
metal ions, and drugs in the bloodstream. Therefore, it is important to investigate drug-protein
interactions due to structural similarities of BSA with human serum albumin (HSA) [28].
RESULTS AND DISCUSSION
Synthesis and characterization
We used the three-component reaction between acylpyruvic acid ethyl ester, (Z)-ethyl 2-
hydroxy-4-oxo-4-(thiophen-2-yl)but-2-enoate (A), and a mixture of differently substituted
aromatic aldehyde (B) and an aromatic amine (C) to synthesize fifteen 1,5-diaryl-4-(2-
thienylcarbonyl)-3-hydroxy-3-pyrrolin-2-ones (D1-D15), fourteen novel and one previously
reported. All the compounds are obtained in glacial acetic acid as catalyst and solvent, by the
procedure described earlier [29] with minor corrections of reaction conditions.
All synthesized novel compounds were characterized by UV-Vis, NMR (1H and 13C
spectra of compounds D1-D15 are presented in the ESI, Figure S1-S30), MS spectroscopy and
elemental analysis. For one, compound D2 that crystallized from DMSO solution, crystal
structure was determined by using single-crystal X-ray diffraction analysis.
Crystal and molecular structures of D2
Single-crystal X-ray diffraction analysis has shown that the D2 compound crystallized
with two molecules of DMSO (Figure 1). The existence of two carbonyl groups (the C1−O1
and C5−O3) and one hydroxyl (the C2−O2H) is clearly confirmed. The central N1 five-
membered ring is almost ideally planar (root-mean-square deviation of fitted atoms is 0.012 Å).
This ring forms a dihedral angle of 16.9(1)° with neighboring C16−C21 phenyl ring. The C5−O3

carbonyl group is directed between the N1 and S1 five-membered rings [the C4−C3−C5−O3 and
O3−C5−C6−S1 torsion angles are −16.1(3) and −18.0(3)° respectively] approximately bisecting
the dihedral angle between these two heterocycles. With a dihedral angle of 89.2(1)° the
C10−C15 phenyl ring is almost perfectly placed in an orthogonal position relating to the central
N1 ring.
Figure 1 Crystal structure and atom-numbering scheme of D2. Displacement ellipsoids are
drawn at the 30% probability level.
The C2−C3 bond with the bond length of 1.341(3) Å (Table S1) is the shortest C−C bond
in the whole molecule while the longest one is C4−C10 [1.522(3) Å]. The N1 is clearly an sp2
hybridized atom since that the sum of bond angles around this atom is 359.9° and all N−C bonds
are coplanar. The N1 forms three N−C bonds with significantly different bond lengths (Table
S1). The shortest N1−C1 bond with an interatomic distance of 1.369(3) Å probably enables
electron π delocalization across the N1−C1−O1 fragment.
The O2−H…O4i is only one classical intermolecular hydrogen bond in the crystal
structure of D2, however, it has quite short H…O distance of 1.78(3) Å [O2−H = 0.80(3) Å,
O2…O3i = 2.538(3) Å, O2−H…O3i = 159(3)°; symmetry code: (i) x+1, y, z]. The C1−O1 and

C5−O3 carbonyl groups form only very weak C−H…O hydrogen bonds with H…O distance
longer than 2.55 Å. Intermolecular C−H…π interactions dominate in the crystal packing by their
frequency. Thus in Figure 1 one can see that the methyl groups from two DMSO molecules are
directed to the centers of neighboring π systems.
Biological evaluation
Cytotoxic activity of 3-hydroxy-3-pyrrolin-2-on derivates
In the first screening stage, cytotoxic activity of newly synthesized compounds (D1-D15)
against HeLa, MDA-MB 231 and MRC-5 was evaluated by MTT assay. Cells were treated with
single dose (100 μM) concentration of tested compounds. Four compounds: D10, D13, D14, and
D15 that showed the highest cytotoxicity against malignant cells and the best selectivity towards
normal cells, were selected for further experiments (Table S3).
Cytotoxic effect of compounds D10, D13, D14, and D15 was further examined in a range
of five concentrations on all three cell lines during 24h and 48h using MTT and SRB assays. The
main difference between these two assays is that MTT measures metabolic activity, while SRB is
based on measurement of cellular protein content and indirectly measures cell number, so we
performed both tests to define whether tested compounds kill or affect metabolic activity of cells
[30,31]. Generally, both MTT and SRB assay showed comparable values of cytotoxic effect for
all tested compounds on all three designated cell lines. Results of both assays are presented in the
form of comparative dose-response curves (Figure 2).

Figure 2 Comparative dose-response curves of MTT and SRB assay after 48h treatment of
HeLa, MDA-MB 231 and MRC-5 with compounds D10, D13, D14, and D15. Results are
presented as mean ±SD

Compounds D10, D13, D14, and D15 showed the cytotoxic effect on subjected tumor
cell lines, being more potent for HeLa, than for MDA-MB 231. Calculated IC50 values for MDA-
MB 231 and non-transformed fibroblasts MRC-5 were above the highest concentration used in
experiments (IC50 > 100 μM). It was shown that all treatments on HeLa cells induced growth
inhibition activity in dose and time-dependent manner. HeLa cells were more susceptible to
tested compounds with moderate cytotoxic activity. Compound D13 exhibited the strongest
cytotoxic effect after 48h of exposure against HeLa cells with IC50 values of 40.9 ± 5.9 μM and
54.35 ± 8.56 μM for SRB and MTT assays, respectively (Table S4).
From the above mentioned analysis, it can be noticed that when R1 is benzyloxy group,
activity of the compounds has been significantly improved as can be seen for three compounds
(D13, D14, and D15) containing this fragment. Another significant influence of the substituents
at -R1 was not observed except in case D10 (R1 = Cl) where there is also improved antitumour
activity, but to some lesser extent compered to compounds with benzyloxy group. Also, it was
observed that the shift of the nitro group from meta- to para-position, as can be seen in
compounds D13 and D14, had some influence to the cell growth inhibition. Some better
antitumor activity is achieved when NO2 group is in meta-position, as observed for D13 in both
MTT and SRB assay. This different activity was also observed for some organic compounds in a
recent study [32], and is most likely related to the steric effect of the compounds. Slight
difference in the activity was observed in D15, when Cl is replaced in R4, showing the positive
change in antitumour activity compering to D14.
There is no large amount of data on the antitumor effects of these types of compounds in
the literature. Several studies indicate that their biological activity depends on the modifications
of the heterocyclic ring. The 3-hydroxy-pyrrolin-2-one derivatives exhibited antitumor activity in
vitro on three mouse tumor cell lines hepatoma (MG-22A), melanoma (V16) and neuroblastoma
(Neuro2A) [33]. In vivo studies have shown that some pyrrolin-2-one derivatives had genotoxic
effects on the mouse bone marrow [34]. There are also data on the antitumor effects of some
diaryl-3-pyrroline-2-on derivatives on human malignant cells, such as U-937 histiocytic
lymphoma cells [35]. Almost no data are available on the mechanism of action of these organic
compounds, as well as the type of cell death they induce, so we examined whether cytotoxicity
induced by tested compounds was due to apoptosis or cell cycle arrest.

The mechanisms of cytotoxic activity
To determine the type of cell death induced by selected 3-hydroxy-3-pyrrolin-2-one
derivatives, Annexin V-FITC/7-AAD assay was performed. Flow cytometric analysis showed
that all tested compounds induced apoptosis in HeLa cells, while the percent of necrotic cells
was negligible. Considering that apoptosis, as opposed to necrosis, does not induce inflammation
and injury of surrounding tissue, agents that induce this type of cell death can be envisaged as
potential antitumor drugs [36]. In accordance with the results of MTT and SRB assay, flow
cytometry showed that treatment of MDA-MB cells with 100 μM concentration of all tested
compounds resulted in the small percent of dead, predominantly apoptotic cells (Figure 3).
Figure 3 Flow cytometric analysis of Annexin V-FITC/7-AAD staining. Graphs showing the
percent of early apoptotic (EA), late apoptotic (LA) and necrotic cells (N) in untreated (control)
and treated HeLa and MDA-MB 231 cells. Results are presented as average ± SD of three
independent experiments.
Likewise, the effect of 3-hydroxy-3-pyrrolin-2-one derivates on autophagy was different
in these cell lines. While in HeLa cells compounds D13 and D15 induced marked and compound
D10 moderate increases in the intensity of red fluorescence. Contrary, in MDA-MB 231 cells not
only that compounds D13 and D15 had no influence on autophagy induction but compounds D10
and D14 even inhibited autophagy in certain extent (Figure 4).

Figure 4 Quantification of autophagy. Graphs showing the change in red/green (FL3/FL1) mean
fluorescence intensity in untreated (control) and treated HeLa and MDA-MB 231 cells. Results
are presented as average ± SD from three independent experiments.
To find out the role which autophagy plays in 3-hydroxy-3-pyrrolin-2-one derivates
derivatives-induced cytotoxicity, cells were cotreated with autophagy inhibitor chloroquine
(CQ). Results showed opposite effect of compounds D13 and D15 in HeLa and MDA-MB 231
cells (Figure 5). While in HeLa cells inhibition of autophagy enhanced apoptosis, in MDA-MB
231 cells resulted in the decrease of Annexin V-positive cells. In compounds D10 and D14,
treated both HeLa and MDA-MB 231 cells autophagy inhibition had no effect on the induction
of apoptosis.
Figure 5 Influence of autophagy inhibition on apoptosis. Percent of apoptotic cells with (CQ+)
or without (CQ-) cotreatment with autophagy inhibitor chloroquine determined by flow
cytometric analysis of Annexin V/7-AAD double stained cells. Results are presented as average
± SD from three independent experiments.

Dual role of autophagy, a catabolic process by which cells degrade and recycle their own
constituents, is well documented. Autophagy was first described as a mechanism for cell survival
that maintains normal cellular function under conditions of intra- and extracellular stresses.
However, autophagy may also activate the apoptotic program and promote cell death. For this
reason, agents targeting autophagy may serve as a therapeutic advantage [37]. Our results
showed that in HeLa cells autophagy induced by D13 and D15 has a cytoprotective role, since
its’ inhibition enhanced apoptosis. In D13 and D15 treated MDA-MB 231 cells autophagy stayed
on the basal level, therefore inhibition of autophagy, as a homeostatic mechanism, resulted in
alleviation of cell death. Although compound D10 enhanced autophagy in HeLa cells,
cotreatment with CQ had no effect on apoptosis, showing that autophagy is not implicated in the
cell death pathway. These results demonstrated that autophagy is not a part of the mechanisms of
cytotoxicity induced by either tested 3-hydroxy-3-pyrrolin-2-one derivates.
To further reveal mechanisms underlying cytotoxicity induced by the tested compounds,
cell cycle distribution was analyzed by flow cytometry. In multicellular organisms both
apoptosis and process of cell cycle progression are essential for maintaining tissue homeostasis.
Accumulated evidence revealed that these two processes share a set of regulatory molecules.
Manipulation with cell cycle may prevent or induce apoptosis. Our results demonstrated that in
HeLa cells treated with compounds D13, D15 and D10 arrested cells in S phase, indicating
inhibition of DNA synthesis, while compound D14 had no effect on cell cycle progression. As
expected, 100 μM concentration of the 3-hydroxy-3-pyrrolin-2-one derivates didn’t affect cell
cycle progression in MDA-MB 231 cells (Figure 6).
Figure 6 Cell cycle analysis. Graphs showing cell cycle distribution in untreated (control) and
treated HeLa and MDA-MB cells. Results are presented as average ± SD of three independent
experiments.

In conclusion, HeLa and MDA-MB 231 cell lines differ not only in histological type, but
are also highly heterogeneous regarding mutations related to genes that control programmed cell
death and cell cycle progression. This fact explains the different sensitivity of these cell lines to
the tested compounds. Tested compounds apparently target molecules that are diversely
expressed in HeLa and MDA-MB 231 cells. The data presented suggest that selected 3-hydroxy-
3-pyrrolin-2-one derivatives in HeLa cells induce apoptosis that is associated with S phase arrest
(D13, D15, and D10) or unrelated to cell cycle distribution (D14).
Fluorescence measurements
Fluorescence quenching on EB–DNA
Ethidium bromide (EB) is compound known by intense fluorescence emission in the
presence of DNA. Fluorescence emission, the consequence of strong intercalation between the
base pairs of DNA and EB, can be quenched by the addition of the drug which can compete with
EB to bind with DNA confirming that drug intercalates to base pairs of DNA [38,39].
Fluorescence quenching spectra (Figure 7) of titration EB–DNA with D13 and D15, recorded in
the range of 550–750 nm, show a decreasing trend with the increasing amounts of the
compounds at 610 nm indicating that EB was partially replaced by compounds D13 and D15.
The molar ratios of EB-DNA : D13 or D15 followed the order: 1 : 0 (control), 1 : 0.2, 1 : 0.4, 1 :
0.6, 1 : 0.8, 1 : 1, 1 : 1.2, 1 : 1.4, 1 : 1.6, 1 : 8, 1 : 2, in a total volume of 5.0 mL, pH 7.4, at 25 °C
with an incubation time of 6 h. Obtained indicated that both compounds bind to DNA by an
intercalative mode.

Figure 7 Top: Emission spectra of EB-DNA in the absence (black lines) and in the presence of
compounds D13 and D15. [EB] = 50 μM, [DNA] = 50 μM; [D13] and [D15]= 0–100 μM; pH =
7.4; λex = 500 nm. Bottom: Plots of F0/F versus [Q].
To better understand quantitatively the magnitude of the binding strength of the
compounds with CT-DNA, the quenching constant (Kq) was calculated using Sterne-Volmer
equation [40] by examining the dependence of F0/F on [Q] (Figure 7). Quenching constant for
D13 and D15, presented in Table 1, indicate that both compounds have the capability to
displaced EB from the EB–DNA complex by binding to DNA through intercalation [41].
Table 1 The bimolecular quenching rate constant (kq), Stern–Volmer constant (Ksv), and
correlation coefficient (R) for compounds D13 and D15.
Compound
D13
kq [M-1 s-1]
(3.7 ± 0.1) × 1011 (3.7 ± 0.1) × 103
(3.4 ± 0.1) × 1011 (3.4 ± 0.1) × 103
Ksv [M-1]
R
0.989
0.998
D15

Protein binding experiments
Based on the fact that the efficiency of drugs depends on their ability to bind to a carrier
protein and knowing that bovine serum albumin (BSA) is the protein that has the ability to
transport drugs in bloodstream we investigated the binding affinity of D13 and D15 to BSA. In
the fluorescence quenching experiment, the fluorescence emission titration of BSA with
compounds D13 and D15 was used for the investigation of binding properties by increasing the
concentration of the quenchers, in the wavelength range of 300–500 nm (Figure 8).
Figure 8 Top: emission spectra of BSA in the absence and in the presence of compounds
D13 and D15. [BSA] = 10.0 µM; [D13] or [D15] = 0, 10, 20, 30, 40, 50, 60, 70, 80, 90
and 100 µM; pH = 7.4; λex = 280 nm. Bottom: plots of log(F0-F/F) versus log[Q].
The molar ratios of BSA : D13 or D15 followed the order: 1 : 0 (control), 1 : 1, 1 : 2, 1 : 3, 1 :
4, 1 : 5, 1 : 6, 1 : 7, 1 : 8, 1 : 9, 1 : 10, in total volume of 5.0 mL, pH 7.4, at 25 °C with the
incubation time of 6 h. The fluorescence quenching data such as binding constant (Ka) and
number of binding sites per BSA molecule (n) were described by using eqn [42]:
log(F0-F/F) = logKa + n log[Q]

Where, F0 is the emission intensity in the absence of a quencher, F is the emission
intensity in the presence of a quencher for the complexes D13 or D15–BSA and [Q] is the
concentration of D13 or D15. The Ka value and n were obtained investigating dependence of the
log [(F0 − F)/F] versus log[Q] (Figure 8). The obtained values of the Ka, given in Table 2, that
are in the optimum range which is considered to be 104–106 M−1 [43], indicate that both
compounds have the appropriate ability for binding to BSA. The number of binding sites of D13
or D15 (n ≈ 1.5, Table 2) point that both D13 and D15 bind to BSA in the molar ratio of 1.5 : 1.
Table 2 Binding parameters (Ka and n) and the correlation coefficient (R) for interactions
of D13 and D15 with BSA.
compound
D13
Ka [M-1]
(4.2 ± 0.2) × 105
(2.6 ± 0.2) × 105
n
R
1.47
1.69
0.998
0.998
D15
Viscosity measurements
The viscosity measurement of DNA after treatment with a varying concentration of
ligands provides a reliable indication for the DNA binding mode. In general, this technique
provides more definitive evidence for intercalative binding of particular substances to DNA,
since changes in the DNA chain length are manifested by changes in the viscosity of the
medium.
It is generally suggested that a classical intercalative binding mode causes a significant
increase of DNA viscosity because the intercalative interaction requires the space of adjacent
base pairs to be large enough to accommodate the bound small molecules and elongates the
double helix. When electrostatic, groove or hydrophobic binding occurs in the binding process,
no obvious changes of DNA viscosity are typically caused in the solution.
The relative specific viscosity (η / ηo) of DNA is firmly dependent on the length changes
that may associate with the separation of DNA base pairs caused by intercalative interaction
between DNA’s double helix and a small molecule. The influences of investigated ligands D13

and D15 on the viscosity of DNA are shown in Figure 9. In our case, relative viscosity increase
with a slope value of 0.52 (ligand D13) and 0.55 (ligand D15).
Upon addition of both ligands to DNA, the relative viscosity raises steadily, as in the case
of the classical intercalator. The results indicated that the ligands bind with DNA through the
intercalative mode since the viscosity increase of DNA is ascribed to the intercalative binding
mode, causing the effective lengthening of the DNA. The slope of relative viscosity was similar
for both ligands, pointing out to similar binding affinity towards DNA for investigated
compounds.
1.8
1.6
1.4


1.2
1.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
R = [ligand] / [DNA]
Figure 9 Relative viscosity (η/ηo)1/3 of DNA (0.01 mM) in buffer solution (50 mM NaCl and 5
mM Tris-HCl at pH 7.4) in the presence of the complex D15 (black square) and D13 (red circle)
at increasing amounts (R). Solid lines represent the fitted linear regression curve.
DNA and BSA docking study
The method of molecule simulation is an efficient way to determine and predict the
binding mode and the interaction region. To elucidate the mode of interaction and binding

affinity, docking studies have been performed on B-DNA and BSA in presence of D13, D14 and
D15. The best-docked poses of the compounds with DNA dodecamer are displayed in Figure 10,
and calculated results for binding energies and docked inhibition constant are summarised in
Table 3. The study revealed that ligands under investigation interact with DNA via an
intercalation mode involving outside edge π−π stacking interactions between guanine and benzyl
ring of the ligands. From the ensuing docked structures it is clear that all ligands fits well into the
minor groove of the targeted DNA and it is mainly stabilized by van der Waals interaction as can
be seen from Figure 10. The relative binding energy of the docked structures was found to be
−51.035, –49.814 and –52.312 kJmol–1 for D13, D14 and D15 respectively. The slightly lower
binding energy for D14 can be consequence of one less electrostatic interaction of –NO2 group.
From Figure 10 it can be noted the different orientation of D13 and D14 towards DNA during
docking, compared to D15. Namely, for D13 and D14 docking is additionally stabilized by
electrostatic interactions between –NO2 and adenine, which cause different conformation after
docking in contrast to D15. On the other hand, chlorobenzyl ring for D15 is outside of DNA
helixes and not contribute to the binding affinity of ligand. The slightly higher binding energy for
D15 can be attributed to one more π−π stacking interaction of the ligand with DNA.

Figure 10 Molecular docking of D13 (A), D14 (B) and D15 (C) with DNA and a two-
dimensional representation of interactions
The binding of ligands D13, D14 and D15 to BSA occurs at similar positions in domain I
[44]. The essential driving force of D13, D14 and D15 binding to BSA is hydrophobic and a van

der Waals force. Three hydrogen bonds have been observed in the D13-BSA system: oxygens
from –NO2 group makes two hydrogen bonds with Lys A:136 and Phe A:36 and one is formed
between oxygen (from benzyloxy fragment) and Phe B:36. For ligand D14 interaction with BSA
occurs via 4 hydrogen bonds, three the same like in D13, and one additional between –OH group
and Lys B:136. In the case of D15, only one hydrogen bond is formed with Glu A:38 (Figure
11). The formation of hydrogen bonds stabilizes the complexes of D13 and D14 with BSA
significantly more than D15-BSA which is demonstrated by higher binding energy for D13 and
D14. The interactions of all ligands were additionally intensified by π−π stacking interactions
with Lys amino acids (Lys B:132 and Lys B:136). Because the binding site is located deeply in
the body of BSA instead of being on the surface of the protein, both ligands have to pass through
the amino acid residues on the surface around the binding site to settle at the proper position in
BSA. This process is against the binding process and increases the activation energy of the
binding.

Figure 11 Molecular docking of D13 (A), D14 (B) and D15 (C) with BSA and a two-
dimensional representation of interactions

Table 3 Docking parameters of D13, D14 and D15
Binding energy
/ kJ·mol–1
Docking
docking score
inhibition
compound
constant / μM
DNA
-4.852
-4.551
-5.187
-51.035
-49.814
-52.312
BSA
1.144
1.873
0.684
D13
D14
D15
-4.075
-3.848
-4.253
-55.203
-57.662
-51.643
0.213
0.079
0.895
D13
D14
D15
CONCLUSION
The three-component reaction between acylpyruvic acid ethyl ester and a mixture of
differently substituted aromatic aldehyde and an aromatic amine, in glacial acetic acid as catalyst
and solvent, is used for synthesis of fifteen 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-
pyrrolin-2-ones (D1-D15), fourteen novel and one previously reported. All synthesized
compounds were characterized by UV-Vis, NMR, MS spectroscopy and elemental analysis. In
addition, for compound D2, crystallized from DMSO solution, crystal structure was determined
using single-crystal X-ray diffraction analysis. All the compounds were further tested for their
cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts
(MRC-5). Compounds D10, D13, D14, and D15 showed cytotoxic effect on subjected tumor cell
lines, being more potent for HeLa than for MDA-MB 231, but D13 exhibited the strongest
cytotoxic effect after 48h of exposure against HeLa cells with IC50 values of 40.9 ± 5.9 μM and
54.35 ± 8.56 μM for SRB and MTT assays, respectively. All four compounds that showed the
highest cytotoxicity against malignant cells and the best selectivity towards normal cells were
placed under further experiments. Results obtained by investigating mechanisms of cytotoxic
activity suggest that our tested compounds in HeLa cells induce apoptosis that is associated with
S phase arrest (compounds D13, D15, and D10) or unrelated to cell cycle distribution (compound

D14). In order to better understand their appropriateness for potential use as anticancer
medicaments we studied the interactions between biomacromolecules (DNA or BSA) and D13
and D15. The obtained results indicated that D13 and D15 have great affinity to displace EB
from the EB-DNA complex through intercalation [Ksv = (3.7 ± 0.1) and (3.4 ± 0.1) × 103 M-1,
respectively] that is also confirmed via viscosity measurements experiments. Ka values, obtained
as result of fluorescence titration of BSA with D13 and D15 [Ka = (4.2 ± 0.2) and (2.6 ± 0.2) ×
105 M, respectively], showed that a significant amount of the tested compounds could be
transported and distributed through the cells. In addition, DNA and BSA molecular docking
study for D13, D14 and D15 was further performed to elucidate the mode of interaction and
binding affinity.
EXPERIMENTAL SECTION
Materials
All solvents and substrate were purchased from Sigma. The (Z)-ethyl 2-hydroxy-4-oxo-4-
(thiophen-2-yl)but-2-enoate (A) was synthesized according to the previously described
methodology [45]. Melting-points (Mp) were determined on a Mel-Temp apparatus and are
uncorrected. Thin-layer chromatography (TLC) was carried out on 0.25 mm Sigma-Aldrich
coated silica gel plates (60F-254) using eluent DCM : EtOH (9:1) as a mobile phase and UV
light for visualization. The IR spectra were recorded by a Perkin–Elmer Spectrum One FT-IR
spectrometer on KBr pellet. The NMR spectra of D1-D15 were performed in DMSO-d6 with
TMS as internal standard on a Varian Gemini 200 MHz NMR spectrometer (1H at 200 and 13C at
50 MHz). Mass spectrometry was performed by Waters Micromass Quattro II triple quadrupole
mass spectrometer and MassLynx software for control and data processing, electrospray
ionization in the positive mode was used. The electrospray capillary was set at 3.0 kV and the
cone at 20 V, the ion source temperature was set at 120 °C and the flow rates for nitrogen bath
and spray were 500 l/h and 50 l/h, respectively and the collision energy was 20 eV.
General procedure for synthesis of 1,5-diaryl-4-(2-thienylcarbonyl)-3-hydroxy-3-pyrrolin-
2-ones
A mixture of 1 mmol of an aromatic aldehyde and 1 mmol of an aromatic amine
dissolved in 3 ml of glacial acetic acid was heated to 50 °C. After reaching the temperature, 1

mmol of the (Z)-ethyl 2-hydroxy-4-oxo-4-(thiophen-2-yl)but-2-enoate, dissolved in 2 ml glacial
acetic acid, was added dropwise. After 3-5 h of reaction time precipitation of the product
occurred. The reaction is followed by thin-layer chromatography with CH2Cl2 : CH3CH2OH = 9 :
1 as the eluent. After 8 h of reaction time mixture was cooled, the precipitate was filtered off,
washed with anhydrous diethyl ether, and dried at the room temperature to yield. All the
synthesized compounds were characterized by NMR spectroscopy (1H and 13C spectra are
presented in the ESI, Figure S1-S30).
3-Hydroxy-5-phenyl-1-(o-tolyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one (D1).
hite powder; yield: 85 ; mp = 257 C; IR (KBr): ν 3246, 1710, 1690, 1680, 1612, 1515, 1499,
1405, 1339, 1299, 1248, 1184, 1053, 988, 856, 832, 762, 740 cm-1; 1H NMR (200 MHz, DMSO-
d6): δ = 1.91 (s, 3H, CH3), 6.44 (s, 1H, CH), 7.03-7.30 (m, 4H, CHAr), 7.38-7.43 (m, 2H, CHAr),
13
7.88-8.09 (m, 4H, CHAr), 8.11-8.25 (m, 2H, CHAr) ppm; C NMR (50 MHz, DMSO-d6): δ =
21.2, 61.2, 121.2, 121.9, 124.5, 127.9, 128.4, 128.7, 134.9, 135.6, 135.9, 143.3, 143.7, 143.9,
148.2, 165.5, 172.2 and 180.5 ppm; ESI-MS: m/z (%) = 375 [M]+. Calcd for C22H17NO3S (%): C
70.38, H 4.56, N 3.73; found: C 70.52, H 4.51, N 3.75.
3-Hydroxy-5-phenyl-1-(p-tolyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one (D2).
White powder; yield: 88%; 1H NMR (200 MHz, DMSO-d6): δ = 1.91 (s, 3H, CH3), 6.24 (s, 1H,
CH), 6.99-7.38 (m, 8H, CHAr), 7.47 (d, 2H, J = 8.4 Hz, CHAr), 7.95 (dd, 1H, J = 4.9, 1.1 Hz),
8.05 (dd, 1H, J = 3.8, 1.1 Hz) ppm; 13C NMR (50 MHz, DMSO-d6): δ = 20.6, 21.2, 61.6, 120.2,
122.9, 127.9, 128.1, 128.5, 128.6, 129.3, 133.9, 134.6, 134.9, 135.2, 136.5, 144.1, 149.3 and
164.5 ppm; ESI-MS: m/z (%) = 375 [M]+. Calcd for C22H17NO3S (%): C 70.38, H 4.56, N 3.73;
found: C 70.52, H 4.51, N 3.75.
3-Hydroxy-5-phenyl-1-(4-hydroxyphenyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one
(D3).
ellow powder; yield: 72 ; mp = 246 C; IR (KBr): ν 3468, 3199, 1701, 1677, 1610, 1520, 1455,
1
1409, 1376, 1262, 1239, 1181, 1137, 1125, 996, 836, 824, 740, 714 cm-1; H NMR (200 MHz,
DMSO-d6): δ = 6.16 (s, 1H, CH), 6.61-6.74 (m, 2H, CHAr), 7.07-7.39 (m, 8H, CHAr), 7.95 (dd,
1H, J = 4.9 Hz, 1.1 CHAr), 8.06 (dd, 1H, J = 3.8, 1.1 Hz), 9.47 (s, 1H, OHAr) ppm; 13C NMR (50

MHz, DMSO-d6): δ = 115.4, 119.9, 125.0, 127.8, 127.9, 128.5, 128.6, 136.6, 144.2, 149.6,
155.4, 164.3 and 180.4 ppm; ESI-MS: m/z (%) = 377 [M]+. Calcd for C21H15NO4S (%): C 66.83,
H 4.01, N 3.71; found: C 67.01, H 4.11, N 3.75.
3-Hydroxy-5-phenyl-1-(4-chorophenyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one
(D4).
hite powder; yield: 81 ; mp = 240 C; IR (KBr): ν 3187, 1709, 1672, 1610, 1495, 1414, 1370,
1298, 1281, 1245, 1187, 1091, 987, 852, 836, 742, 725 cm-1; 1H NMR (200 MHz, DMSO-d6): δ
= 6.32 (s, 1H, CH), 7.09-7.24 (m, 4H, CHAr), 7.29-7.42 (m, 4H, CHAr), 7.61-7.70 (m, 2H, CHAr),
7.96 (dd, 1H, J = 4.9 Hz, 1.1 CHAr), 8.02 (dd, 1H, J = 3.8, 1.1 Hz), 12.0 (s, 1H, OHenol) ppm; 13C
NMR (50 MHz, DMSO-d6): δ = 61.4, 120.5, 124.3, 127.9, 128.6, 128.8, 129.6, 135.4, 136.2,
144.0, 148.9, 164.8 and 180.4 ppm; ESI-MS: m/z (%) = 396 [M]+. Calcd for C21H14ClNO3S (%):
C 63.72, H 3.56, N 3.54; found: C 63.52, H 3.59, N 3.55.
3-Hydroxy-5-phenyl-1-(4-bromophenyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one
(D5).
hite powder; yield: 84 ; mp = 237 C; IR (KBr): ν 3188, 1709, 1684, 1671, 1610, 1492, 1412,
1370, 1297, 1245, 1128, 986, 852, 833, 741, 724 cm-1; 1H NMR (200 MHz, DMSO-d6): δ = 6.31
(s, 1H, CH), 7.02-7.41 (m, 6H, CHAr), 7.41-7.68 (m, 4H, CHAr), 7.96 (dd, 1H, J = 5.0 Hz, 1.1
13
CHAr), 8.05 (dd, 1H, J = 3.8, 1.2 Hz) ppm; C NMR (50 MHz, DMSO-d6): δ = 117.9, 120.5,
124.6, 127.9, 128.6, 131.7, 135.8, 136.2, 144.0, 148.9, 165.8 and 180.4 ppm; ESI-MS: m/z (%) =
440 [M]+. Calcd for C21H14BrNO3S (%): C 57.28, H 3.20, N 3.18; found: C 57.42, H 3.22, N
3.15.
3-Hydroxy-5-phenyl-1-(4-iodophenyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one (D6).
hite powder; yield: 72 ; mp = 239 C; IR (KBr): ν 3178, 1684, 1665, 1608, 1490, 1413, 1383,
1
1292, 1244, 1134, 983, 852, 826, 715, 698 cm-1; H NMR (200 MHz, DMSO-d6): δ = 6.30 (s,
1H, CH), 7.09-7.24 (m, 4H, CHAr), 7.30-7.35 (m, 2H, CHAr), 7.41-7.51 (m, 2H, CHAr), 7.59-7.69
13
(m, 2H, CHAr), 7.96 (dd, 1H, J = 5.0 Hz, 1.1 CHAr), 8.04 (dd, 1H, J = 3.8, 1.2 Hz) ppm; C
NMR (50 MHz, DMSO-d6): δ = 61.2, 90.3, 120.5, 124.7, 127.8, 128.2, 128.6, 134.7, 135.3,

136.2, 137.5, 144.0, 148.8, 165.7 and 180.4 ppm; ESI-MS: m/z (%) = 487 [M]+. Calcd for
C21H14INO3S (%): C 51.76, H 2.90, N 2.87; found: C 51.86, H 2.91, N 2.85.
3-Hydroxy-1-(4-chorophenyl)-4-(2-thienylcarbonyl)-5-(4-nitrophenyl)-2,5-dihydro-1H-pyrrol-2-
one (D7).
ellow powder; yield: 77 ; mp = 225 C; IR (KBr): ν 3102, 1703, 1667, 1599, 1535, 1494, 1411,
1376, 1350, 1264, 1244, 1205, 1092, 987, 836, 745, 728 cm-1; 1H NMR (200 MHz, DMSO-d6): δ
= 6.50 (s, 1H, CH), 7.14-7.29 (m, 1H, CHAr), 7.37 (d, 2H, J = 8.8 Hz, CHAr), 7.65-7.70 (m, 4H,
CHAr), 7.87-8.18 (m, 4H, CHAr) ppm; 13C NMR (50 MHz, DMSO-d6): δ = 21.2, 60.6, 119.4,
123.6, 124.2, 128.6, 128.9, 129.3, 129.9, 134.6, 135.0, 135.3, 143.8, 144.2, 147.3, 149.7, 164.6
and 179.9 ppm; ESI-MS: m/z (%) = 441 [M]+. Calcd for C21H13ClN2O5S (%): C 57.21, H 2.97,
N 6.35; found: C 57.42, H 3.00, N 6.32.
3-Hydroxy-1-(4-bromophenyl)-4-(2-thienylcarbonyl)-5-(4-nitrophenyl)-2,5-dihydro-1H-pyrrol-
2-one (D8).
ellow powder; yield: 79 ; mp = 188 C; IR (KBr): ν 3084, 1702, 1667, 1599, 1534, 1489, 1410,
1375, 1349, 1264, 1243, 1197, 1130, 986, 834, 745, 728 cm-1; 1H NMR (200 MHz, DMSO-d6): δ
= 6.49 (s, 1H, CH), 7.21 (dd, 1H, J = 4.9, 3.9 Hz, CHAr), 7.50 (d, 2H, J = 8.9 Hz, CHAr), 7.57-
13
7.73 (m, 4H, CHAr), 7.90-8.18 (m, 4H, CHAr) ppm; C NMR (50 MHz, DMSO-d6): δ = 21.2,
60.5, 118.1, 119.3, 123.6, 124.5, 128.6, 129.3, 131.8, 134.6, 135.3, 135.4, 143.8, 144.3, 147.3,
149.8, 164.6 and 179.8 ppm; ESI-MS: m/z (%) = 485 [M]+. Calcd for C21H13BrN2O5S (%): C
51.97, H 2.70, N 5.77; found: C 52.02, H 2.71, N 5.75.
3-Hydroxy-1-(4-iodophenyl)-4-(2-thienylcarbonyl)-5-(4-nitrophenyl)-2,5-dihydro-1H-pyrrol-2-
one (D9).
ellow powder; yield: 67 ; mp = 195 C; IR (KBr): ν 3106, 1710, 1669, 1602, 1518, 1490, 1410,
1
1373, 1346, 1267, 1245, 1193, 1129, 1056, 986, 827, 743, 720 cm-1; H NMR (200 MHz,
DMSO-d6): δ = 6.49 (s, 1H, CH), 6.47-6.50 (m, 1H, CH), 7.15-7.27 (m, 1H, CH), 7.47 (d, 2H, J
= 8.8 Hz), 7.60-7.72 (m, 4H, CHAr), 7.95-8.12 (m, 4H, CHAr) ppm; 13C NMR (50 MHz, DMSO-
d6): δ = 21.2, 60.4, 90.7, 119.3, 123.6, 124.5, 128.6, 129.3, 134.6, 135.3, 135.9, 137.7, 143.8,

144.3, 147.3, 149.9, 164.6 and 179.8 ppm; ESI-MS: m/z (%) = 532 [M]+. Calcd for
C21H13IN2O5S (%): C 47.38, H 2.46, N 5.26; found: C 47.30, H 2.44, N 5.28.
3-Hydroxy-1-(p-tolyl)-4-(2-thienylcarbonyl)-5-(4-chlorophenyl)-2,5-dihydro-1H-pyrrol-2-one
(D10).
ellow powder; yield: 92 ; mp = 240 C; IR (KBr): ν 3099, 1708, 1666, 1595, 1513, 1407, 1379,
1
1268, 1245, 1089, 988, 826, 751, 729 cm-1; H NMR (200 MHz, DMSO-d6): δ = 1.91 (s, 3H,
CH3), 6.28 (s, 1H, CH), 7.10 (d, 2H, J = 8.3 Hz), 7.16-7.28 (m, 3H, CH), 7.35 (d, 2H, J = 8.5
Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.96 (dd, 1H, J = 4.9, 1.0 Hz), 8.08 (dd, 2H, J = 3.8, 1.0 Hz) ppm;
13C NMR (50 MHz, DMSO-d6): δ = 20.6, 21.2, 60.8, 119.6, 122.8, 128.4, 128.6, 129.3, 129.7,
132.6, 133.6, 134.5, 135.0, 135.2, 135.7, 144.0, 149.5, 164.3 and 180.2 ppm; ESI-MS: m/z (%) =
410 [M]+. Calcd for C22H16ClNO3S (%): C 64.47, H 3.93, N 3.42; found: C 64.53, H 3.91, N
3.40.
3-Hydroxy-1,5-bis(4-chlorophenyl)-4-(2-thienylcarbonyl)-2,5-dihydro-1H-pyrrol-2-one (D11).
hite powder; yield: 85 ; mp = 231 C; IR (KBr): ν 3172, 1711, 1670, 1607, 1494, 1416, 1371,
1
1280, 1248, 1090, 988, 833, 760, 724 cm-1; H NMR (200 MHz, DMSO-d6): δ = 6.34 (s, 1H,
CH), 7.19-7.28 (m, 3H, CH), 7.34-7.43 (m, 4H, CH), 7.66 (d, 2H, J = 9.0 Hz), 7.97 (dd, 1H, J =
4.9, 1.1 Hz), 8.09 (dd, 1H, J = 3.8, 1.1 Hz), 11.87 (br. s, 1H, OHenol) ppm; 13C NMR (50 MHz,
DMSO-d6): δ = 21.5, 60.9, 120.2, 124.6, 128.9, 129.0, 129.2, 130.0, 130.2, 133.1, 135.1, 135.7,
144.3, 149.6, 165.0, 172.5 and 180.6 ppm; ESI-MS: m/z (%) = 430 [M]+. Calcd for
C21H13Cl2NO3S (%): C 58.62, H 3.05, N 3.26; found: C 58.68, H 3.02, N 3.25.
3-Hydroxy-1-(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-bromophenyl)-2,5-dihydro-1H-pyrrol-
2-one (D12).
hite powder; yield: 80 ; mp = 230 C; IR (KBr): ν 3161, 1711, 1670, 1606, 1492, 1412, 1371,
1
1279, 1284, 1092, 988, 831, 759, 722 cm-1; H NMR (200 MHz, DMSO-d6): δ = 6.34 (s, 1H,
CH), 7.18-7.28 (m, 3H, CHAr), 7.35-7.43 (m, 2H, CHAr), 7.47-7.54 (m, 2H, CHAr), 7.56-7.65 (m,
2H, CHAr), 7.96 (dd, 1H, J = 4.9, 1.1 Hz, CHAr), 8.09 (dd, 1H, J = 3.8, 1.1 Hz, CHAr), 11.96 (br.
13
s, 1H, OHenol) ppm; C NMR (50 MHz, DMSO-d6): δ = 21.5, 60.9, 118.3, 120.3, 124.9, 128.9,
129.0, 130.1, 132.1, 133.1, 135.1, 135.6, 135.7, 135.9, 144.3, 149.6, 165.0, 172.5 and 180.6 ppm;

ESI-MS: m/z (%) = 475 [M]+. Calcd for C21H13BrClNO3S (%): C 53.13, H 2.76, N 2.95; found:
C 53.22, H 2.74, N 2.96.
3-Hydroxy-1-(3-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-
pyrrol-2-one (D13).
ellow powder; yield: 73 ; mp = 209 C; IR (KBr): ν 3159, 1681, 1666, 1606, 1496, 1414, 1383,
1
1243, 1172, 1091, 984, 846, 755, 715 cm-1; H NMR (200 MHz, DMSO-d6): δ = 4.92 (s, 2H,
OCH2), 6.28 (d, 1H, J = 4.5 Hz, CH), 6.82 (d, 2H, J = 8.5 Hz, CHAr), 7.16-7.52 (m, 10H, CHAr),
7.61-7.73 (m, 2H, CHAr), 7.97-8.07 (m, 2H, CHAr), 11.68 (br. s, 1H, OHenol) ppm; 13C NMR (50
MHz, DMSO-d6): δ = 61.2, 115.0, 124.7, 128.2, 128.3, 128.6, 128.8, 129.0, 129.1, 129.5, 129.6,
129.9, 132.3, 135.1, 135.6, 135.7, 137.3, 144.4, 149.0, 158.6, 165.0 and 180.9 ppm; ESI-MS:
m/z (%) = 513 [M]+. Calcd for C28H20N2O6S (%): C 65.62, H 3.93, N 5.47; found: C 65.71, H
3.91, N 5.44.
3-Hydroxy-1-(4-nitrophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-
pyrrol-2-one (D14).
ellow powder; yield: 77 ; mp = 242 C; IR (KBr): ν 3226, 1689, 1678, 1609, 1512, 1338, 1248,
1172, 988, 859, 753, 722 cm-1; 1H NMR (200 MHz, DMSO-d6): δ = 4.91 (s, 2H, OCH2), 6.38 (s,
1H, CH), 6.83 (d, 2H, J = 8.6 Hz, CHAr), 7.20-7.35 (m, 8H, CHAr), 7.89-8.08 (m, 4H, CHAr),
8.18 (d, 2H, J = 9.2 Hz, CHAr) ppm; 13C NMR (50 MHz, DMSO-d6): δ = 60.8, 69.4, 114.8,
121.2, 122.0, 124.4, 127.5, 127.9, 128.5, 128.7, 129.1, 134.9, 135.5, 136.9, 142.2, 143.6, 143.8,
147.9, 158.2, 165.3 and 180.4 ppm; ESI-MS: m/z (%) = 513 [M]+. Calcd for C28H20N2O6S (%):
C 65.62, H 3.93, N 5.47; found: C 65.70, H 3.92, N 5.49.
3-Hydroxy-1-(4-chlorophenyl)-4-(2-thienylcarbonyl)-5-(4-(benzyloxy)phenyl)-2,5-dihydro-1H-
pyrrol-2-one (D15).
ellow powder; yield: 79 ; mp = 205 C; IR (KBr): ν 3178, 1682, 1667, 1607, 1512, 1496, 1384,
1
1244, 1173, 1092, 985, 847, 756, 716 cm-1; H NMR (200 MHz, DMSO-d6): δ = 4.93 (s, 2H,
OCH2), 6.28 (s, 1H, CH), 6.83 (d, 2H, J = 8.8 Hz, CHAr), 7.21-7.39 (m, 10H, CHAr), 7.67 (d, 2H,
J = 8.9 Hz, CHAr), 7.97 (d, 1H, J = 4.9 Hz, CHAr), 8.07 (d, 1H, J = 3.8 Hz, CHAr), 11.93 (s, 1H,
OHenol) ppm; 13C NMR (50 MHz, DMSO-d6): δ = 61.2, 69.7, 115.0, 115.8, 120.8, 124.7, 128.1,

128.2, 128.3, 128.6, 128.8, 129.0, 129.1, 129.5, 129.8, 132.3, 135.1, 135.6, 135.7, 137.3, 144.4,
149.0, 158.6, 165.0 and 180.8 ppm; ESI-MS: m/z (%) = 502 [M]+. Calcd for C28H20ClNO4S (%):
C 66.99, H 4.02, N 2.79; found: C 67.04, H 3.99, N 2.77.
X-ray crystal structure determination
Single-crystal X-ray diffraction data for D2 were collected at an Oxford Gemini S
diffractometer equipped with a CCD detector, using monochromatized MoKα radiation (λ =
0.71073 Å). Data reduction and empirical absorption correction were performed with
CrysAlisPRO [46]. The structure was solved by direct methods using SHELXS and refined on F2
by full-matrix least-squares using SHELXL [47]. All non-hydrogen atoms were refined
anisotropically. One of two DMSO molecules is disordered over two positions of the S3 atom
with occupation numbers of 0.653 and 0.347.
The H atom bonded to the O2 atom from hydroxyl group was located in difference
Fourier map and refined isotropically. The rest of the H atoms were placed in geometrically
calculated positions and refined using the riding model with Uiso values constrained to 1.2Ueq or
1.5Ueq of the parent C atoms. The PARST [48] and PLATON [49] software were used to
perform the geometrical calculation, while ORTEP3 [50] and was employed for molecular
graphics. Crystallographic details for structure analysis of the D2 compound are summarized in
Table S2.
Cell lines and culture
Healthy human fetal lung fibroblasts (MRC-5) and two human cancer cell lines: cervix
adenocarcinoma cells (HeLa) and breast adenocarcinoma cells (MDA-MB 231) were used for
cytotoxicity screening of fifteen, previously synthesized, organic compounds. All cell lines were
purchased from American Type Culture Collection (ATTC).
Mantainence of HeLa and MRC-5 were carried out using Dulbecco’s modified Eagle’s
medium (DMEM), while MDA-MB 231 was maintained in Roswell Park Memorial Institute
medium (RPMI-1640). Both mediums were supplemented with 10 % heath-inactivated fetal
bovine serum (FBS), L-glutamine (2 mM), non-essential amino acids (0,1 mM), penicillin (100
IU/ml) and streptomycin (100 μg/ml). Cells were kept as a monolayer culture in 25 cm2 plastic
0
flasks (Greiner Bio-One, Austria) under standard culture conditions of 37 C, humidified air and

5 % CO2. The media was changed every three days and cells were subcultured when necessary
(0.05 % trypsin - 0.53 mM EDTA). Depending on the type of experiment, cells were seeded in
96- or 24-well microtirar plates (Thermo Scientific, New York, NY).
Cytotoxicity assays
Effects of 3-hydroxy-3-pyrrolin-2on derivates (D1-D15) on HeLa, MDA-MB 231 and
MRC-5 cells viability were evaluated by two colorimetric assays: MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) and SRB (sulforhodamine B). In all experiments, cells
were plated in 96-well plates at the density of 3x103 cells per well and incubated to adhere
overnight.
MTT assay
The MTT assay, based on the reduction of the yellow tetrazolium salt to a purple
formazan dye by succinate-tetrazolium reductase, which exists in the mitochondrial respiratory
chain and is active only in viable cells, was firstly described by Moshman [30]. After adherence,
in the first stage (screening), medium was removed and cells were treated with synthesized
compounds (D1-D15) in single dose at concentration 100 μM or media alone, and in the second
stage, with selected compounds which exhibited the best inhibitory effect in screening stage, at
concentration 1 μM, 3 μM, 10 μM, 30 μM, 100 μM or media alone. After 24h and 48h
incubation with tested compounds, the medium was replaced with 100 μL of MTT (0.5 mg/mL
PBS) and incubated for at least 2h. Thereafter, colored formazan crystals were dissolved with
150 μL of DMSO. Absorbance was measured at 590 nm with a multiplate reader (Zenyth 3100,
Anthos Labtec Instruments GmbH, Austria). Cytotoxicity was calculated according to the
formula: (1-Atest/Acontrol) x 100. The IC50 values of compounds were calculated using Microsoft
Office
Excel
free
add-in
ED50plus
v1.0
software
downloaded
from:
http://www.sciencegateway.org/protocols/cellbio/drug/data/.
SRB assay
Cytotoxicity was assessed by SRB assay as described by Skehan et al. [31] Effects of the
selected 3-hydroxy-3-pyrrolin-2-on derivatives on cell viability was examined on HeLa, MDA-
MB 231 and MRC-5 in concentration of 1 μM, 3 μM, 10 μM, 30 μM, 100 μM during 24 h and