Simple 2(5H)-furanone derivatives with selective cytotoxicity towards non-small cell lung cancer cell line A549 – Synthesis, structure-activity relationship and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.03.021

A series of 5-alkoxy derivatives of 3,4-dichloro-5-hydroxyfuran-2-(5H)-one (mucochloric acid, MCA) were obtained and subsequently subjected to modification in the C-4 position of 2(5H)-furanone ring. The cytotoxicity of newly synthesized compounds was evaluated in MTT assay against non-small cell lung cancer (A549) and healthy lung epithelial cell line (BEAS-2B). The derivatives containing a branched alkoxy substituent in the C-5 position demonstrated the highest anticancer properties, whereas modification of compounds in the C-4 position of 2(5H)-furanone ring only slightly improve their antiproliferative properties. Compounds 12 and 15 exhibited the best selectivity towards A549 cells and were also evaluated in a panel of cancer cell lines of different origin. Further investigation revealed that treatment of A549 cell line with compounds 12 and 15 led to G2 phase cell cycle arrest and induction of caspase-independent cell death. Moreover, compound 12 was found to act synergistically with erlotinib.

Full text of DOI:10.1016/j.ejmech.2018.03.021

Accepted Manuscript
Simple 2(5H)-furanone derivatives with selective cytotoxicity towards non-small cell
lung cancer cell line A549 – Synthesis, structure-activity relationship and biological
evaluation
Anna Byczek-Wyrostek, Radosław Kitel, Klaudia Rumak, Magdalena Skonieczna,
Anna Kasprzycka, Krzysztof Walczak
PII:
S0223-5234(18)30262-9
10.1016/j.ejmech.2018.03.021
EJMECH 10286
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 May 2017
Revised Date: 5 March 2018
Accepted Date: 7 March 2018
Please cite this article as: A. Byczek-Wyrostek, Radosł. Kitel, K. Rumak, M. Skonieczna, A. Kasprzycka,
K. Walczak, Simple 2(5H)-furanone derivatives with selective cytotoxicity towards non-small cell lung
cancer cell line A549 – Synthesis, structure-activity relationship and biological evaluation, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Simple 2(5H)-furanone derivatives with selective cytotoxicity towards non-
small cell lung cancer cell line A549 – synthesis, structure-activity
relationship and biological evaluation
a,b*
a,b,c,d
a
b,e
Anna Byczek-Wyrostek , Radosław Kitel
, Klaudia Rumak , Magdalena Skonieczna ,
a,b
a,b
Anna Kasprzycka , Krzysztof Walczak
a
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of
Chemistry, Silesian University of Technology, Krzywoustego Street 4, 44-100 Gliwice, Poland
b
Centre of Biotechnology, Silesian University of Technology, Krzywoustego Street 8, 44-100
Gliwice, Poland
c
Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa Street 7a, 30-
3
87 Krakow, Poland
d
Department of Organic Chemistry, Jagiellonian University, Ingardena Street 3, 30-060
Krakow, Poland
e
Biosystems Group, Institute of Automatic Control, Silesian University of Technology,
Akademicka Street 16, 44-100 Gliwice, Poland
*
Corresponding author. Tel.: +48 32 237 1308, Fax: +48 32 237 2094.
E-mail address: anna.kasprzycka@polsl.pl (A. K.)
ABSTRACT
A series of 5-alkoxy derivatives of 3,4-dichloro-5-hydroxyfuran-2-(5H)-one (mucochloric
acid, MCA) were obtained and subsequently subjected to modification in the C-4 position of
2
(5H)-furanone ring. The cytotoxicity of newly synthesized compounds was evaluated in
MTT assay against non-small cell lung cancer (A549) and healthy lung epithelial cell line
BEAS-2B). The derivatives containing a branched alkoxy substituent in the C-5 position
demonstrated the highest anticancer properties, whereas modification of compounds in the C-
position of 2(5H)-furanone ring only slightly improve their antiproliferative properties.
(
4
Compounds 12 and 15 exhibited the best selectivity towards A549 cells and were also
evaluated in a panel of cancer cell lines of different origin. Further investigation revealed that
treatment of A549 cell line with compounds 12 and 15 led to G2 phase cell cycle arrest and

ACCEPTED MANUSCRIPT
induction of caspase-independent cell death. Moreover, compound 12 was found to act
synergistically with erlotinib.
Keywords: 2(5H)-Furanone, Nucleophilic substitution, Anticancer activity, Apoptosis
Abbreviations
DAPI (4',6-diamidino-2-phenylindole), dH O (deionized water), DMSO (dimethyl sulfoxide),
2
ERB (erlotinib), ESI-MS (high-resolution electrospray ionization mass spectroscopy), FBS
(
fetal bovine serum), FITC (fluorescein isothiocyanate), IC50 (half maximal inhibitory
concentration), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), NMR
nuclear magnetic resonance), NSCLC (non-small-cell lung carcinoma), PI (propidium
iodide), SI (selectivity index), TLC (thin layer chromatography), TMS (tetramethylsilane).
(
1
. Introduction
2
(5H)-Furanone (2H-furan-5-on, γ-crotonolactone, Figure 1) is an organic compound
that belongs to a group of α,β-unsaturated lactones, which are widely distributed in many
natural products. [1-3] Compounds having 2(5H)-furanone skeleton in their structure are
known to exhibit a wide range of biological activity, including antitumor, [3-8] antibacterial,
[
9-12] antifungal, [13,14] antiviral, [15] anti-inflammatory [16-19] and antioxidant. [17,20].
Several previous reports have confirmed the anticancer activity of compounds
bearing a 2(5H)-furanone ring in their structure. [6,7] 5-Butoxy-3,4-dichloro-2(5H)-furanone
Figure 1a) is cytotoxic at high millimolar concentration in MAC 13 and MAC 16 murine
colon cancer cell lines. [6] Further modifications of parent compound led to 3,4-dichloro-5-
oxirane-2-ylmethoxy)-2(5H)-furanone derivative (Figure 1b) with cytotoxicity in nanomolar
range in the same cancer cell lines. [7] Peng and co-workers [21] showed that the compound
E)-3-(4-fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-yl)methyl)phenyl)
(
(
(
acrylamide (Figure 1c) inhibits the formation of complex DNA-topoisomerase I. Also natural
compounds known as rubrolides (Figure 1d) bearing 2(5H)-furanone moiety, possess anti-
cancer activity in HT-29, MEL-28, P-388 and A549 cell lines. [22]
[
Figure1]

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These findings prompted us to investigate how simple modifications of 2(5H)-
furanone scaffold affect their cytotoxic properties. Herein, we report the synthesis of mono-
and disubstituted 2(5H)-furanone derivatives with the aim of establishment of structure-
activity relationship (SAR) and exploration of their mechanism of action in non-small-cell
lung cancer cell line.
2
. Chemistry
The synthetic pathway leading to mono- and disubstituted 2(5H)-furanone derivatives
is outlined in the Scheme 1. Our synthetic efforts started from mucochloric acid (MCA, 1),
which contains 2-(5H)-furanone and is readily available from commercial sources. Given the
multiple functional groups in the different chemical environment, MCA offers accessibility to
regioselective functionalization and thus rapid generation of structurally diverse compound
libraries. First set of compounds, monosubstituted 5-alkoxy derivatives (9-15) were obtained
in the reaction of MCA (1) with various alcohols (2-8) in toluene. The second library of
compounds was obtained from 12, 13 and 15 in the reaction with aromatic amines (16-19) or
piperidine (20) in DMSO. This synthetic strategy afforded disubstituted compounds (21-35)
in moderate to excellent yields ranging from 29 to 92%.
[
Scheme 1]
1
13
The structure of all compounds was confirmed by H NMR, C NMR, high-resolution
electrospray ionization mass spectroscopy (ESI-MS) and elemental analysis.
3
. Results and Discussion
3
.1. Cytotoxicity
Compound 1 and all obtained derivatives were evaluated for their in vitro cytotoxicity
(Table 1) against non-small cell lung cancer (A549) and healthy lung epithelial cells (BEAS-
2
B). The IC50 values, defined as 50% cell growth inhibition in comparison to untreated
control, were determined using MTT assay. [24] With exception of 21, 24-26 and 35, all
compounds displayed significant improvement in the antiproliferative activity in the A549
cell line. Compounds having substituent only in the C-5 position of 2(5H)-furanone ring
showed higher cytotoxicity than their counterparts bearing a modification on both C-4 and C-
5
carbons, highlighting the importance of two halogens for the cytotoxic effect. The most
active monosubstituted derivative 11 bearing propyl group, displayed IC value 3.6 µM and
5
0

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was 34-times more potent than parent compound (IC value 122.9 µM). Among disubstituted
5
0
derivatives, compound 33 appeared to be most active with IC value 13.3 µM, showing 9-
5
0
fold improvement in anticancer activity, when compared the parent compound.
The major challenge in the development of novel anticancer compounds is their
selectivity towards cancer cells. An optimal agent should affect only cancer cells and spare
the healthy ones. Keeping this in mind, we assessed the cytotoxicity of all compounds also in
healthy bronchial cell line BEAS-2B. The most potent compound from the series of
monosubstituted derivatives 11 had almost the same IC50 values in both cancer and non-
cancerous cell lines, indicating no selectivity towards cancer cells. Noteworthy, compounds
1
2, 14 and 15, having branched aliphatic chains, were more cytotoxic in A549 cell line with
selectivity index (SI) values [25] 0.61, 0.22 and 0.88, respectively (Table 1). Based on these
results, compounds 12 and 15 along with parent compound 1 were selected for further
evaluation.
[
Table 1]
Compounds 1, 12 and 15 were also investigated in a panel of 12 human cancer cell
lines of different origin (Table 2). In most of the tested cell lines, 5-substituted derivatives (12
and 15) show higher cytotoxicity than the parent compound. Only in HCT 116 wt cell line
activities of novel derivatives were similar to the parent compound (8.3 µM vs. 7.2 µM). The
most prominent improvement in the antiproliferative activity of both 12 and 15 was observed
for hepatocellular carcinoma HepG2 cell line (6.5-fold) and in androgen-independent prostate
cancer cell line DU 145 (5-fold).
[
Table 2]
3
.2. Cell cycle analysis
To test whether selected compounds (1, 12 and 15) perturb the cell cycle progression
of A549 cells, we evaluated their effects after continuous exposure in the two time regimes
24 and 72 h). All compounds were used at 10 µM concentration. Treatment of cells with 12
(
after 24 hours caused a slight increase in the percentage of cells in the sub-G1 (11.2%) and
G2/M (23.4%) phase when compared to untreated control (Figure 2A, 2C). No significant
changes in cell cycle were observed when the cells were treated with compounds 1 and 15. To
elucidate whether compounds trigger G2 phase arrest or cell cycle arrest in mitosis, we

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counted mitotic nuclei. We found that all tested compounds did not induce the accumulation
of A549 mitotic cells after 24 hours of treatment, indicating that cells are arrested in G2 phase
(Figure 2B). To confirm cell cycle arrest in G2 phase at the molecular level, we analyzed the
expression of cyclin B1 which governs transition of the cell cycle through mitosis. During G2
phase the level of cyclin B1 increases ensuring the progression of the cell cycle to mitosis
[
26-28]. We found that none of the compounds increased the expression level of cyclin B1
and compound 12 downregulated the cyclin B1 (Figure 2D). This clearly indicates that most
active compounds arrest the cell cycle in early G2 phase rather than in mitosis.
[
Figure 2]
3
.3. Apoptosis induction
The increase of sub-G1 population in the cell cycle analysis after treatment with
compound 12, indicates DNA fragmentation and suggest that this compound trigger apoptosis
in A549 cells. To investigate this first, we checked the percentage of the sub-G1 fraction after
7
2 h treatment of A549 cells with high doses of evaluated compounds (20 µM). As depicted
in Figure 3A, B, 1 did not produce any significant changes in the cell cycle progression, nor
in the sub-G1 content. Notably, a massive increase in the percentage of sub-G1 cells (~40%)
was observed after treatment with compound 12. Interestingly, a higher dose of compound 15
did not lead to any changes in the sub-G1 fraction. This strongly supports our hypothesis that
compound 12 induced apoptosis in A549 cells.
To confirm that compound 12 trigger apoptosis in A549 cells, we employed an
Annexin V/propidium iodide double staining assay using flow cytometry analysis (Figure
3
C, D). [29] Continuous exposure of A549 cells with 12 at 20 µM concentration for 72 hours
induced an almost 36-fold increase in the percentage of both early and late apoptotic cells
32.2%) compared to the untreated control (0.9%). Surprisingly, treatment of A549 cells with
5 at the same concentration resulted also in the increase of Annexin-positive cells (20.3%).
(
1
As expected, compound 1 displayed no effects on apoptosis.
To elucidate the molecular mechanism of apoptosis triggered by compound 12 we
determined the expression of 35 apoptosis-related proteins using commercially available
Proteome Profiler Human Apoptosis Array Kit. On the basis of the densitometric analysis, we
failed to observe any significant changes in the expression levels of both pro- and
antiapoptotic proteins after 72 h treatment of A549 cells with 1, 12 and 15 (Figure 3E, F).
Additionally, none of the compounds triggered significant caspase-3 cleavage, which activity

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is a hallmark of apoptotic cell death. Furthermore, we confirmed the expression of pro-
caspase-3 using specific antibodies in western blot assay. Consistent with the previous results,
the level of pro-caspase-3 did not change significantly for all tested compounds. Taking this
into account we speculate, that A549 cells upon treatment of 12 underwent caspase-
independent cell death which is more likely through observed cell cycle perturbations (G2
arrest) than regulation of apoptosis-related proteins.
[
Figure 3]
3
.4. Clonogenic potential
Next, we sought to test whether cells treated once with most active compounds retain
their clonogenic potential [31]. Results indicate that, in contrast to compound 1, both
derivatives 12 and 15 were able to decrease the clonogenic potential of A549 cells, even at
low micromolar concentration. The most prominent effect was observed for compound 12.
After 72 hours of treatment with 12, only 40% and 15% of cells formed colonies at a
concentration of 5 µM and 10 µM, respectively (Figure 4). This clearly indicates that
compound 12 significantly reduced the clonogenic potential of A549 cells.
[
Figure 4]
3
.5.Combination of 12 with erlotinib
Erlotinib (Tarceva), an EGFR inhibitor, is a FDA-approved targeted drug for the
treatment of NSCLC (non-small-cell lung carcinoma). However, previous reports have shown
that A549 cell line, which harbors KRAS mutation, is highly resistant to erlotinib [32-34].
This is in agreement with our results that confirmed that A549 cells are insensitive to this
agent. Taking into account that our best compound 12 decreased cell viability of A549 cells at
low-micromolar concentrations, we reasoned to test the combination of compounds 12 with
erlotinib.
First, we assessed the effect of the combination of different doses of erlotinib and
compound 12 (10 µM) on the viability of A549 cells after 72 hours of treatment. We observed
a slight synergistic effect but only when erlotinib was tested at relatively high concentrations
(Figure 5A). Since increasing erlotinib dose did not result in any significant decrease in cell
viability, for further evaluation we choose a combination of 12 and erlotinib at equimolar
concentrations (10 µM). This combination decreased the number of colonies of A549 cells for

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about 89% (relative to the vehicle). Compound tested alone at 10 µM decreased cell colony
formation for 86% and 75% for compound 12 and erlotinib, respectively.
Further, we checked influence combination erlotinib and 12 on cell cycle (Figure 5B).
After 72 hours treatment of A549 cells with combination 10 µM of 12 and 10 µM of erlotinib,
we observed an increase of sub-G1 cells to 28.0% (Figure 5C). When tested alone, erlotinib
caused only slight increase in the percentage of apoptotic cells (8%).
Finally, we assessed clonogenic potential after treatment of A549 cells with above
compound combination (Figure 5D, 5E). This led to significant reduction of clonogenic
potential of cells when compared to single-agent treatment.
[
Figure 5]
4
. Conclusion
In summary, simple alkylation of mucochloric acid 1 to compounds 12 and 15,
resulted in novel potent derivatives with increased cytotoxic potency and better selectivity
towards cancer cells in comparison to lead compound 1. Observed cytotoxic effect resulted
mainly from G2 phase cell cycle arrest in case of compound 12, which was also able to trigger
caspase-independent apoptosis in A549 cells. Moreover, after treatment A549 cells with
compound 12, a significant decrease in the clonogenic potential was also observed. The
combination of 12 with erlotinib showed synergistic anticancer activity in comparison to
treatment with each single agent in A549 cells. To summarise, our data indicate that simple
compounds bearing 2(5H)-furanone scaffold might be valuable starting points for further
development.
Although direct molecular targets of the most potent compound are not known yet,
some clues about their potential mechanism of action may come from the recent paper of Wu
and co-workers, who showed that compounds having biphenyl substituents on C-4 of 2(5H)-
furanone moiety are able to bind to c-myc G4 DNA quadruplexes [8]. In this context, it will
be interesting to evaluate our compounds for their ability to bind such DNA structures.
5
. Experimental section
5
.1. Chemistry
All reagents were obtained from commercially available sources (Merck, Acros
Organics, Alpha Aesar, Sigma-Aldrich) and were used without purification. The synthetic
pathways were shown in Scheme 1. NMR spectra were recorded on Agilent spectrometer 400

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MHz and Varian spectrometer 600 MHz in DMSO-d solution using tetramethylsilane (TMS)
6
as an internal standard. Chemical shifts are reported as δ values (ppm). NMR solvent was
purchased from ACROS Organics. Melting point measurements were performed in an open
capillary using Stuart® SMP30 apparatus and are uncorrected. High-resolution electrospray
ionization mass spectroscopy (ESI-MS) experiments were performed using a Waters Xevo G2
QTOF instrument equipped with an injection system (cone voltage 50 V; source 120°C).
Elemental analysis experiments were performed using a Perkin Elmer 2400 Series II CHNS/O
System. Analyses indicated by the symbols of the elements or functions were within ± 0.4 %
of the theoretical values
5
.1.1. 5-Substituted of 3,4-dichloro-2(5H)-furanone derivatives
3
,4-Dichloro-5-hydroxy-2(5H)-furanone (1, 1eq.) was dissolved in anhydrous toluene (10:1, v
toluene:mmol 1) contained concentrated sulfuric acid (0.025 eq.). Next alcohol 2-8 (15 eq.)
was added. The reaction mixture was refluxed until TLC indicated the total decay of the
starting material (8-108 h). The volatiles were removed under diminished pressure. The
residue was purified by silica gel column chromatography (toluene:ethyl acetate, 10:1 v/v) to
give 9-15.
5
.1.1.1. 3,4-Dichloro-5-methoxy-2(5H)-furanone (9)
1
Colorless oil; yield: 81%; H NMR (400 MHz, DMSO-d ) δ: 3.51 (s, 3H, -OCH ), 6.23 (s,
6
3
1
3
1
1
H, H-5); C NMR (100 MHz, DMSO-d ) δ: 55.77 (C-1’), 101.44 (C-5), 123.20 (C-3),
6
+
47.44 (C-4), 162.93 (C-2); ESI-MS (CH OH): m/z 182.9616 [M] (m/z calcd. 182.9616
3
+
[
M] ); Anal. Calcd. for C H Cl O : C, 32.82; H, 2.20. Found: C, 32.41; H, 1.95.
5
4
2
3
5
.1.1.2. 3,4-Dichloro-5-ethoxy-2(5H)-furanone (10)
1
Colorless oil; yield 77%; H NMR (400 MHz, DMSO-d ) δ: 1.21 (t, 3H, J = 7.2 Hz, -CH ),
6
3
1
3
3
.82 (q, 2H, J = 7.2 Hz, -OCH -), 6.23 (s, 1H, H-5); C NMR (100 MHz, DMSO-d ) δ: 14.81
2 6
(C-2’), 65.27 (C-1’), 100.89 (C-5), 122.86 (C-3), 147.73 (C-4), 163.02 (C-2); ESI-MS
+
+
(
CH OH): m/z 218.9594 [M+Na] (m/z calcd. 218.9592 [M+Na] ) ; Anal. Calcd. for
3
C H Cl O : C, 36.58; H, 3.07. Found: C, 36.25; H, 2.74.
6
6
2
3
5
.1.1.3. 3,4-Dichloro-5-propyloxy-2(5H)-furanone (11)
1
Yellow oil; yield 80%; H NMR (400 MHz, DMSO-d ) δ: 0.90 (t, 3H, J = 7.4 Hz, -CH ),
6
3
1
3
1
.57-1.63 (m, 2H, -CH -CH ), 3.72 (t, 2H, J = 6.5 Hz, -OCH -), 6.28 (s, 1H, H-5); C NMR
2 3 2

ACCEPTED MANUSCRIPT
(
100 MHz, DMSO-d ) δ: 10.03 (C-3’), 22.15 (C-2’), 70.86 (C1’), 100.99 (C-5), 122.87 (C-3),
6
+
1
47.74 (C-4), 163.02 (C-2); ESI-MS (CH OH): m/z 210.9932 [M] (m/z calcd. 210.9932
3
+
[
M] ); Anal. Calcd. for C H Cl O : C, 39.84; H, 3.82. Found: C, 40.21; H, 3.63.
7
8
2
3
5
.1.1.4. 3,4-Dichloro-5-isopropyloxy-2(5H)-furanone (12)
1
White syrup; yield: 82%; H NMR (400 MHz, DMSO-d ) δ: 1.22-1.24 (m, 6H, 2x-CH ),
6
3
1
3
4
2
.11-4.21 (m, 1H, -OCH-), 6.34 (s, H, H-5); C NMR (100 MHz, DMSO-d ) δ: 22.16 (C-2’),
6
3.10 (C-2’), 75.13 (C-1’), 100.31 (C-5), 124.17 (C-3), 147.86 (C-4), 163.55 (C-2); ESI-MS
+
+
(
CH OH): m/z 210.9929 [M] (m/z calcd. 210.9929 [M] ); Anal. Calcd. for C H Cl O : C,
3
7
8
2
3
3
9.84; H, 3.82. Found: C, 39.92; H, 3.54.
5
.1.1.5. 5-n-Butyloxy-3,4-dichloro-2(5H)-furanone (13)
1
Colorless oil; yield: 84%; H NMR (400 MHz, DMSO-d ) δ: 0.89 (t, 3H, J = 7.2 Hz, -CH ),
6
3
1
.31-1.40 (m, 2H, -CH -CH ), 1.53-1.60 (m, 2H, -OCH -CH ), 3.77 (t, 2H, J = 6.4 Hz, -
2 3 2 2
1
3
OCH -), 6.24 (s, 1H, H-5); C NMR (100 MHz, DMSO-d ) δ: 13.44 (C-4’), 18.28 (C-3’),
2
6
3
0.75 (C-2’), 68.68 (C-1’), 103.22 (C-5), 117.51 (C-3), 144.56 (C-4), 164.17 (C-2); ESI-MS
+
+
(
CH OH): m/z 225.0085 [M] (m/z calcd. 225.0085 [M] ); Anal. Calcd. for C H Cl O : C,
3
8
10
2
3
4
2.69; H, 4.48. Found: C, 42.84; H, 4.67.
5
.1.1.6. 3,4-Dichloro-5-tert-butyloxy-2(5H)-furanone (14)
1
White syrup; yield: 45%; H NMR (400 MHz, DMSO-d ) δ: 1.31 (s, 9H, 3x-CH ), 6.49 (s,
6
3
1
3
1
1
H, H-5); C NMR (100 MHz, DMSO-d ) δ: 27.70 (3 x C-2’), 78.68 (C-1’), 97.15 (C-5),
6
+
21.76 (C-3), 148.97 (C-4), 163.33 (C-2); ESI-MS (CH OH): m/z 246.9905 [M+Na] (m/z
3
+
calcd. 246.9905 [M+Na] ); Anal. Calcd. for C H Cl O : C, 42.69; H, 4.48. Found: C, 43.09;
8
10
2
3
H, 4.36.
5
.1.1.7. 3,4-Dichloro-5-neopentyloxy-2(5H)-furanone (15)
1
Colorless oil; yield: 74%; H NMR (400 MHz, DMSO-d ) δ: 0.91 (s, 9H, 3x-CH ), 3.43 (s,
6
3
1
3
2
H, -OCH -), 6.28 (s, 1H, H-5); C NMR (100 MHz, DMSO-d ) δ: 25.96 (3 x C-3’), 31.47
2 6
(C-2’), 78.39 (C-1’), 101.16 (C-5), 122.94 (C-3), 147.70 (C-4), 162.97 (C-2); ESI-MS
+
+
(
CH OH): m/z 239.0242 [M] (m/z calcd. 239.0242 [M] ); Anal. Calcd. for C H Cl O : C,
3
9
12
2
3
4
5.21; H, 5.06. Found: C, 45.59; H, 5.22
.
5
.1.2. 4- and 5-Substituted of 3,4-dichloro-2(5H)-furanone derivatives

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Derivatives 12, 13 and 15 (1eq.) were dissolved in anhydrous DMSO (5ml to 1eq.), next
amine 16-20 (2eq.) was added. The reaction mixture was stirred at room temperature until
TLC indicated the consumption of all the starting material (2-24h). The reaction mixture was
quenched with cold water (10 ml) and extracted with dichloromethane (2x10 ml). The organic
layer was dried (anhydrous MgSO ). The solvent was removed under reduced pressure and
4
the crude product was purified by column chromatography (toluene:ethyl acetate, 7:1 v/v) to
give compounds 21-35.
5
.1.2.1. 3-Chloro-4-phenylamino-5-isopropyloxy-2(5H)-furanone (21)
o
1
Yellow solid; yield: 62%; m.p.=114-115 C; H NMR (400 MHz, DMSO-d ) δ: 0.77 (d, 3H, J
6
=
6.2 Hz, -CH ), 1.08 (d, 3H, J = 6.2 Hz, -CH ), 3.72-3.82 (m, 1H, -OCH-), 6.36 (s, 1H, H-5),
3 3
7
.20-7.25 (m, 3H, Ar-3’’, Ar-4’’, Ar-5’’), 7.35-7.39 (m, 2H, Ar-2’’, Ar-6’’), 9.65 (s, 1H, -
1
3
NH); C NMR (100 MHz, DMSO-d ) δ: 21.48 (C-2a’), 22.83 (C-2b’), 73.34 (C-1’), 87.84
6
(C-5), 97.04 (C-3), 123.43 (C-2’’, C-6’’), 125.12 (C-1’’), 128.71 (C3’’, C-5’’), 137.87 (C-
+
4
’’), 155.48 (C-4), 166.79 (C-2); ESI-MS (CH OH): m/z 268.0740 [M] (m/z calcd. 268.0740
3
+
[
M] ); Anal. Calcd. for C H ClNO : C, 58.32; H, 5.27; N, 5.23. Found: C, 58.02; H, 5.03;
13
14
3
N, 4.94.
5
.1.2.2. 4-(4’-Bromophenylamino)-3-chloro-5-isopropyloxy-2(5H)-furanone (22)
o
1
Yellow solid; yield: 44%; m.p.=163-164 C; H NMR (400 MHz, DMSO-d ) δ: 0.84 (d, 3H, J
6
=
6.2 Hz, -CH ), 1.09 (d, 3H, J = 6.2 Hz, -CH ), 3.83 (sep, 1H, J = 6.2 Hz, -OCH-), 6.36 (s,
3 3
1
H, H-5), 7.18-7.21 (m, 2H, Ar-2’’, Ar-6’’), 7.52-7.56 (m, 2H, Ar-3’’, Ar-5’’), 9.69 (s, 1H, -
1
3
NH); C NMR (100 MHz, DMSO-d ) δ: 21.55 (C-2a’), 22.83 (C-2b’), 73.34 (C-1’), 88.78
6
(C-5), 97.02 (C-3), 117.16 (C-4’’), 125.22 (C-2’’, C-6’’), 131.51 (C-3’’, C-5’’), 137.32 (C-
+
1
’’), 154.96 (C-4), 166.63 (C-2); ESI-MS (CH OH): m/z 345.9846 [M] (m/z calcd. 345.9845
3
+
[
M] ); Anal. Calcd. for C H BrClNO : C, 45.05; H, 3.78; N, 4.04. Found: C, 44.87; H, 3.51;
1
3
13
3
N, 3.67.
5
.1.2.3. 3-Chloro-4-(4’-chlorophenylamino)-5-isopropyloxy-2(5H)-furanone (23)
o
1
Yellow solid; yield: 66%; m.p.=158-159 C; H NMR (400 MHz, DMSO-d ) δ: 0.84 (d, 3H, J
6
=
4.0 Hz, -CH ), 1.09 (d, 3H, J = 4.0 Hz, -CH ), 3.79-3.85 (m, 1H, -OCH-), 6.37 (s, 1H, H-5),
3 3
1
3
7
.25-7.27 (m, 2H, Ar-2’’, Ar-6’’), 7.41-7.43 (m, 2H, Ar-3’’, Ar-5’’), 9.71 (s, 1H, -NH); C
NMR (100 MHz, DMSO-d ) δ: 22.96 (C-2a’, C-2b’), 73.42 (C-1’), 88.72 (C-3), 97.17 (C-5),
6
1
24.91 (C-2’’, C-6’’), 129.16 (C-3’’, C-5’’), 129.20 (C-4’’), 136.96 (C-1’’), 155.15 (C-4),

ACCEPTED MANUSCRIPT
+
+
1
66.73 (C-2); ESI-MS (CH OH): m/z 302.0351 [M] (m/z calcd. 302.0350 [M] ); Anal.
3
Calcd. for C H Cl NO : C, 52.68; H, 4.34; N, 4.64. Found: C, 51.95; H, 4.00; N, 4.54..
1
3
13
2
3
5
.1.2.4. 3-Chloro-4-(4’-fluorophenylamino)-5-isopropyloxy-2(5H)-furanone (24)
o
1
Brown solid; yield: 56%; m.p.=128-130 C; H NMR (400 MHz, DMSO-d ) δ: 0.81 (d, 3H, J
6
=
6.2 Hz, -CH ), 1.08 (d, 3H, J = 6.2 Hz, -CH ), 3.78 (sep, 1H, J = 6.2 Hz, -OCH-), 6.30 (s,
3 3
1
H, H-5), 7.18-7.24 (m, 2H, Ar-2’’, Ar-6’’), 7.26-7.31 (m, 2H, Ar-3’’, Ar-5’’), 9.62 (s, 1H, -
1
3
NH); C NMR (100 MHz, DMSO-d ) δ: 21.53 (C-2a’), 22.87 (C-2b’), 73.31 (C-1’), 87.67
6
2
3
(
C-3), 96.90 (C-5), 115.46 (d, J = 22.6 Hz, C-2’’, C-6’’), 125.91 (d, J = 12.8 Hz, C-3’’,
C-F
4
1
C-5’’), 134.23 (d, J = 2.7 Hz, C-1’’), 155.69 (C-4), 159.69 (d, J = 240.9 Hz, C-4’’),
C-F
C-F
+
+
1
66.80 (C-2); ESI-MS (CH OH): m/z 286.0646 [M] (m/z calcd. 286.0646 [M] ); Anal.
3
Calcd. for C H ClFNO : C, 54.65; H, 4.59; N, 4.90. Found: C, 54.75; H, 4.39; N, 4.83.
1
3
13
3
5
.1.2.5. 3-Chloro-5-isopropyloxy-4-(piperidin-1-yl)-2(5H)-furanone (25)
o
1
Brown solid; yield: 29%; m.p.=84-85 C; H NMR (600 MHz, DMSO-d ) δ: 1.20 (t, 6H, J =
6
6
.0 Hz, 2x-CH ), 1.59-1.63 (m, 6H, -N(CH ) (CH ) ), 3.58 (br.s., 4H, -N(CH ) (CH ) ), 4.07
3 2 2 2 3 2 2 2 3
1
3
(
sep, 1H, J = 6.0 Hz, -OCH-), 6.18 (s, 1H, H-5); C NMR (150 MHz, DMSO-d ) δ: 21.89 (C-
6
4
8
’’), 23.20 (C-2a’), 23.30 (C-2b’), 25.82 (C-3’’, C-5’’), 48.34 (C-2’’, C-6’’), 72.52 (C-1’),
+
2.96 (C-3), 96.14 (C-5), 155.28 (C-4), 167.73 (C-2); ESI-MS (CH OH): m/z 260.1053 [M]
3
+
(
m/z calcd. 260.1053 [M] ); Anal. Calcd. for C H ClNO : C, 55.49; H, 6.99; N, 5.39. Found:
1
2
18
3
C, 55.64; H, 7.15; N, 5.24.
5
.1.2.6. 5-n-Butyloxy-3-chloro-4-phenylamino-2(5H)-furanone (26)
o
1
Pink solid; yield: 60%; m.p. = 54-56 C; H NMR (400 MHz, DMSO-d ) δ: 0.74 (t, 3H, J =
6
7
.4 Hz, -CH ), 1.04-1.09 (m, 2H, -CH CH ), 1.29-1.33 (m, 2H, -OCH CH ), 3.40-3.46 (m,
3 2- 3 2- 2-
1
H, H , -OCH ), 3.57-3.32 (m, 1H, H , -OCH ), 6.32 (s, 1H, H-5), 7.17-7.24 (m, 3H, Ar-2’’,
a
2-
b
2-
1
3
Ar-4’’, Ar-6’’), 7.34-7.38 (m, 2H, Ar-3’’, Ar-5’’), 9.73 (s, 1H, -NH); C NMR (100 MHz,
DMSO-d ) δ: 13.43 (C-4’), 18.16 (C-3’), 30.66 (C-2’), 68.13 (C-1’), 87.94 (C-3), 97.52 (C-5),
6
1
1
23.03 (C-2’, C-6’’’), 125.00 (C-4’’), 128.69 (C-3’’, C-5’’), 137.75 (C-1’’), 154.76 (C-4),
+
+
66.70 (C-2); ESI-MS (CH OH): m/z 282.0897 [M] (m/z calcd. 282.0897 [M] ); Anal.
3
Calcd. for C H ClNO : C, 59.68; H, 5.72; N, 497. Found: C, 59.78; H, 5.52; N, 5.01.
1
4
16
3
5
.1.2.7. 4-(4’-Bromophenylamino)-5-n-butyloxy-3-chloro-2(5H)-furanone (27)

ACCEPTED MANUSCRIPT
o
1
Yellow solid; yield: 55%; m.p.=114-115 C; H NMR (400 MHz, DMSO-d ) δ: 0.75 (t, 3H, J
6
=
7.4 Hz, -CH ), 1.02-1.10 (m, 2H, -CH CH ), 1.29-1.36 (m, 2H, -OCH CH ), 3.43-3.49 (m,
3 2- 3 2- 2-
1
H, H , -OCH ), 3.59-3.64 (m, 1H, H , -OCH ), 6.33 (s, 1H, H-5), 7.17-7.22 (m, 2H, Ar-2’’,
a 2- b 2-
1
3
Ar-6’’), 7.55-7.56 (m, 2H, Ar-3’’, Ar-5’’), 9.78 (s, 1H, -NH); C NMR (100 MHz, DMSO-
d6) δ: 13.46 (C-4’), 18.20 (C-3’), 30.70 (C-2’), 68.31 (C-1’), 88.95 (C-3), 97.56 (C-5), 117.21
(C-4’’), 124.92 (C-2’’,C-6’’), 131.61 (C-3’’, C-5’’), 137.35 (C-1’’), 154.51 (C-4), 166.59 (C-
+
+
2
); ESI-MS (CH OH): m/z 360.0002 [M] (m/z calcd. 360.0002 [M] ); Anal. Calcd. for
3
C H BrClNO : C, 46.63; H, 4.19; N, 3.88. Found: C, 46.84; H, 4.00; N, 3.72.
14
15
3
5
.1.2.8. 5-n-Butyloxy-3-chloro-4-(4’-chlorophenylamino)-2(5H)-furanone (28)
o
1
Yellow solid; yield: 20%; m.p.=107-109 C; H NMR (400 MHz, DMSO-d ) δ: 0.75 (t, 3H, J
6
=
7.4 Hz, -CH ), 1.02-1.11 (m, 2H, -CH CH ), 1.29-1.36 (m, 2H, -OCH CH ), 3.43-3.49 (m,
3 2- 3 2- 2-
1
H, H , -OCH ), 3.59-3.64 (m, 1H, H , -OCH ), 6.33 (s, 1H, H-5), 7.24-7.27 (m, 2H, Ar-2’’,
a 2- b 2-
1
3
Ar-6’’), 7.40-7.43 (m, 2H, Ar-3’’, Ar-5’’), 9.79 (s, 1H, -NH); C NMR (100 MHz, DMSO-
d6) δ: 13.48 (C-4’), 18.21 (C-3’), 30.72 (C-2’), 68.26 (C-1’), 88.82 (C-3), 97.56 (C-5), 124.65
(C-2’’, C-6’’), 128.71 (C-3’’, C-5’’), 129.12 (C-4’’), 136.92 (C-1’’), 154.62 (C-4), 166.63 (C-
+
+
2
); ESI-MS (CH OH): m/z 316.0507 [M] (m/z calcd. 316.0507 [M] ); Anal. Calcd. for
3
C H C FNO : C, 53.18; H, 4.78; N, 4.43. Found: C, 53.58; H, 4.79; N, 4.36.
14
15 l2
3
5
.1.2.9. 5-n-Butyloxy-3-chloro-4-(4’-fluorophenylamino)-2(5H)-furanone (29)
o
1
Yellow solid; yield: 60%; m.p.=77-78 C; H NMR (400 MHz, DMSO-d ) δ: 0.75 (t, 3H, J =
6
7
.4 Hz, -CH ), 1.06-1.11 (m, 2H, -CH CH ), 1.29-1.36 (m, 2H, -OCH CH ), 3.40-3.45 (m,
3 2- 3 2- 2-
1
H, H , -OCH ), 3.57-3.62 (m, 1H, H , -OCH ), 6.27 (s, 1H, H-5), 7.18-7.22 (m, 2H, Ar-2’’,
a
2-
b
2-
1
3
Ar-6’’), 7.26-7.30 (m, 2H, Ar-3’’, Ar-5’’), 9.71 (s, 1H, -NH); C NMR (100 MHz, DMSO-
d6) δ: 13.46 (C-4’), 18.23 (C-3’), 30.72 (C-2’), 68.17 (C-1’), 87.77 (C-3), 97.45 (C-5), 115.48
2
3
4
(
d, J = 18 Hz, C-2’’, C-6’’), 125.49 (d, J = 8.4 Hz, C-3’’, C-5’’), 134.15 (d, J = 2.7
C-F C-F C-F
1
Hz, C-1’’), 155.05 (C-4), 159.70 (d, J
= 238.2 Hz, C-4’’), 166.73 (C-2); ESI-MS
C-F
+
+
(
CH OH): m/z 286.0646 [M] (m/z calcd. 286.0646 [M] ); Anal. Calcd. for C H ClFO : C,
3
14 15
3
5
6.10; H, 5.04; N, 4.67. Found: C, 55.75; H, 4.88; N, 4.57.
5
.1.2.10. 5-n-Butyloxy-3-chloro-4-(piperidin-1-yl)-2(5H)-furanone (30)
1
Yellow oil; yield: 64%; oil; H NMR (400 MHz, DMSO-d ) δ: 0.88 (t, 3H, J = 7.4 Hz, -CH ),
6
3
1
.31-1.38 (m, 2H, -CH CH ), 1.51-1.62 (m, 8H, -OCH -CH , -N(CH ) (CH ) ), 3.60-3.71
2
-
3
2
2-
2 2
2 3
1
3
(
m, 6H, -OCH , -N(CH ) (CH ) ), 6.11 (s, 1H, H-5); C NMR (100 MHz, DMSO-d ) δ:
2
-
2 2
2 3
6

ACCEPTED MANUSCRIPT
1
6
3.50 (C-4’), 18.58 (C-3’), 23.23 (C-4’’), 25.83 (C-3’’, C-5’’), 30.90 (C-2’), 48.38 (C-2’’, C-
’’), 67.51 (C-1’), 82.97 (C-3), 96.72 (C-5), 154.84 (C-4), 167.60 (C-2); ESI-MS (CH OH):
3
+
+
m/z 274.1210 [M] (m/z calcd. 274.1210 [M] ); Anal. Calcd. for C H ClNO : C, 57.04; H,
1
3
20
3
7
.36; N, 5.12. Found: C, 57.44; H, 7.63; N, 5.11.
5
.1.2.11. 3-Chloro-4-phenylamino-5-neopentyloxy-2(5H)-furanone (31)
o
1
Yellow solid; yield: 76%; m.p.=166-168 C; H NMR (400 MHz, DMSO-d ) δ: 0.63 (s, 9H,
6
3
1
3
2
3
x-CH ), 3.03 (d, 1H, J = 8.4 Hz, H , -OCH ), 3.25 (d, 1H, J = 8.4Hz, H , -OCH ), 6.42 (s,
3 a 2- b 2-
H, H-5), 7.14-7.18 (m, 1H, Ar-4’’), 7.24-7.25 (m, 2H, Ar-2’’, Ar-6’’), 7.33-7.37 (m, 2H, Ar-
1
3
’’, Ar-5’’), 9.71 (s, 1H, -NH); C NMR (100 MHz, DMSO-d ) δ: 25.89 (3xC-3’), 30.90 (C-
6
’), 77.48 (C-1’), 88.20 (C-3), 97.24 (C-5), 122.79 (C-4’’), 124.94 (C-2’, C-6’’’), 128.82 (C-
’’, C-5’’), 138.04 (C-1’’), 155.08 (C-4), 166.55 (C-2); ESI-MS (CH OH): m/z 296.1053
3
+
+
[
M] (m/z calcd. 296.1053 [M] ); Anal. Calcd. for C H ClNO : C, 60.91; H, 6.13; N, 4.74.
15 18 3
Found: C, 60.73; H, 6.32; N, 4.61.
5
.1.2.12. 4-(4’-Bromophenylamino)-3-chloro-5-neopentyloxy-2(5H)-furanone (32)
o
1
Yellow solid; yield: 50%; m.p.=143-145 C; H NMR (400 MHz, DMSO-d ) δ: 0.65 (s, 9H,
6
3
x-CH ), 3.07 (d, 1H, J = 8.4 Hz, H , -OCH ) 3.25 (d, 1H, J = 8.4Hz, H , -OCH ), 6.42 (s,
3 a 2- b 2-
1
H, H-5), 7.20-7.24 (m, 2H, Ar-2’’, Ar-6’’), 7.51-7.55 (m, 2H, Ar-3’’, Ar-5’’), 9.77 (s, 1H, -
1
3
NH); C NMR (100 MHz, DMSO-d ) δ: 25.83 (3xC-3’), 30.92 (C-2’), 77.49 (C-1’), 89.10
6
(C-3), 97.21 (C-5), 117.04 (C-4’’), 124.61 (C-2’’, C-6’’), 131.64 (C-3’’, C-5’’), 137.53 (C-
+
1
’’), 154.70 (C-4), 166.38 (C-2); ESI-MS (CH OH): m/z 374.0159 [M] (m/z calcd. 374.0158
3
+
[
M] ); Anal. Calcd. for C H BrClNO : C, 48.09; H, 4.57; N, 3.74. Found: C, 48.36; H, 4.49;
1
5
17
3
N, 3.65.
5
.1.2.13. 5-Chloro-4-(4’-chlorophenylamino)-5-neopentyloxy-2(5H)-furanone (33)
o
1
White solid; yield: 60%; m.p.=111-113 C; H NMR (400 MHz, DMSO-d ) δ: 0.65 (s, 9H, 3x-
6
CH ), 3.07 (d, 1H, J = 8.4 Hz, H , -OCH ), 3.25 (d, 1H, J = 8.4 Hz, H , -OCH ), 6.42 (s, 1H,
3
a
2-
b
2-
H-5), 7.27-7.30 (m, 2H, Ar-2’’, Ar-6’’), 7.38-7.42 (m, 2H, Ar-3’’, Ar-5’’), 9.78 (s, 1H, -NH);
1
3
C NMR (100 MHz, DMSO-d ) δ: 25.90 (3xC-3’), 30.99 (C-2’), 77.55 (C-1’), 89.05 (C-3),
6
9
1
7.27 (C-5), 124.38 (C-2’’, C-6’’), 128.79 (C-3’’, C-5’’), 129.04 (C-4’’), 137.17 (C-1’’),
+
54.86 (C-4), 166.47 (C-2); ESI-MS (CH OH): m/z 330.0663 [M] (m/z calcd. 330.0663
3
+
[
M] ); Anal. Calcd. for C H Cl NO : C, 54.56; H, 5.19; N, 4.24. Found: C, 54.87; H, 5.22;
15
17
2
3
N, 4.06.

ACCEPTED MANUSCRIPT
5
.1.2.14. 3-Chloro-4-(4’-fluorophenylamino)-5-neopentyloxy-2(5H)-furanone (34)
o
1
White solid; yield: 60%; m.p.=113-115 C; H NMR (600 MHz, DMSO-d ) δ: 0.65 (s, 9H, 3x-
6
CH ), 3.03 (d, 1H, J = 8.4 Hz, H , -OCH ), 3.25 (d, 1H, J = 8.4 Hz, H , -OCH ), 6.38 (s, 1H,
3
a
2-
b
2-
H-5), 7.17-7.21 (m, 2H, Ar-2’’, Ar-6’’), 7.29-7.32 (m, 2H, Ar-3’’, Ar-5’’), 9.72 (s, 1H, -NH);
1
3
C NMR (150 MHz, DMSO-d ) δ: 25.88 (3xC-3’), 30.94 (C-2’), 77.46 (C-1’), 88.00 (C-3),
6
2
3
9
1
7.13 (C-5), 115.54 (d, J = 22.6 Hz, C-2’’, C-6’’), 125.13 (d, J = 8.4 Hz, C-3’’, C-5’’),
C-F C-F
1
34.39 (C-1’’), 155.27 (C-4), 159.56 (d, J = 240.9 Hz, C-4’’), 166.54 (C-2); ESI-MS
C-F
+
+
(
CH OH): m/z 314.0959 [M] (m/z calcd. 314.0959 [M] ); Anal. Calcd. for C H ClFNO : C,
3
15 17
3
5
7.42; H, 5.46; N, 4.46. Found: C, 57.66; H, 5.50; N, 4.40.
5
.1.2.15. 3-Chloro-5-neopentyloxy-4-(piperidin-1-yl)-2(5H)-furanone (35)
o
1
White solid; yield: 92%; m.p.=95-97 C; H NMR (400 MHz, DMSO-d ) δ: 0.89 (s, 9H,
6
3
4
2
3
xCH ), 1.60-1.62 (m, 6H, -N(CH ) (CH ) ), 3.23 (d, 2H, J = 5.2 Hz, -OCH -), 3.61 (br.s.,
3 2 2 2 3 2
1
3
H, -N(CH ) (CH ) ), 6.11 (s, 1H, H-5); C NMR (100 MHz, DMSO-d ) δ: 23.20 (3xC-3’),
2
2
2 3
6
5.82 (C-3’’, C-5’’), 26.22 (C-2’’, C-6’’), 31.16 (C-2’), 48.30 (C-4’’), 77.20 (C-1’), 83.10 (C-
+
), 96.60 (C-5), 154.65 (C-4), 167.46 (C-2); ESI-MS (CH OH): m/z 288.1368 [M] (m/z
3
+
calcd. 288.1366 [M] ); Anal. Calcd. for C H ClNO : C, 58.43; H, 7.71; N, 4.87. Found: C,
1
4
22
3
5
8.40; H, 7.78; N, 4.81.
5
.2. Cell lines
Human cells lines: non-small lung carcinoma (A549), bronchial epithelial cells
(BEAS-2B), human colon cancer (HCT-116 wt, HT-29, LoVo), human hepatoma cancer
(Hep3B, HepG2), prostate cancer (PC-3, DU 145) breast cancer (MCF-7) and osteosarcoma
(U2OS, SJSA-1) were obtained from American Type Culture Collection (ATCC, Manassas,
VA, USA). HCT 116 -/- p53 (deleted both p53 alleles) were a gift from Dr. Bert Vogelstein.
Cells were grown in DMEM with high glucose medium (Sigma-Aldrich, Germany)
supplemented with 10% (v/v) inactivated fetal bovine serum (FBS) (EURx, Poland) and 1%
antibiotics (10000 µg/ml of streptomycin and 10000 units/ml of penicillin) (Sigma-Aldrich,
Germany) at 37°C in humidified atmosphere with 5% CO . In order to achieve the desired
2
concentrations of the tested compounds, the stock solutions were mixed with fresh medium.
Final DMSO concentration in culture media was less than 0.4%. Control cells in all
experiments were grown in medium containing 0.4% DMSO. This concentration of DMSO
did not alter cell growth rate and cell-cycle, as compared with the vehicle-free medium.

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5
.3. MTT assay
Cytotoxicity of compounds was assessed with the MTT (3-[4,5-dimethylthiazol-2-
y]2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Germany) test. Cells were seeded on the
3
9
6-well plates (Nunc, Germany) with 2x10 cells per well. After 24 hours of incubation, the
tested agents were added and incubated for 72 hours. After that the MTT solution (0.5mg/ml)
was added. After 3 hours of incubation, the MTT solution was removed and the formazan
crystals were dissolved in 2-propanol (Avantor, Poland) containing 0.04 M hydrochloric acid
(Avantor, Poland). Finally, the absorbance was measured at the 570 nm wavelength using
multiwall plate reader (BioTek, USA). The experiment was done in at least three independent
replicates. Cell viability was expressed as a percentage versus vehicle control. IC50 was
defined as a concentration of a drug that decreased cell viability by 50% relative to the
untreated control.
5
.4. Cell cycle analysis
4
Cells (5x10 per well) were seeded on 6-well plate (Nunc, Germany) and allowed to
attach for 24ꢀh. Then the medium was replaced with tested compounds and the cells were
incubated for 24 or 72 h. Floating cells were collected and added to adherent cells, harvested
by trypsinization. Cells were washed with PBS (phosphate buffered saline) (Sigma-Aldrich,
Germany) and then fixed in ice-cold ethanol (70%) for 30 min, treated with RNase A (100
µg/mL, EURx, Poland) and stained with propidium iodide (PI) (100 µg/mL, Acros Organics,
Belgium). The DNA content was analyzed using a Becton Dickinson FACS Aria III sorter
(BD Company, USA). Experiments were repeated at least twice. Data were analyzed using
TM
ModFit LT Software (Verity Software House, USA).
5
.5. Annexin V-FITC apoptosis assay
4
5
x10 cells per well were seeded on 6-well plate (Nunc, Germany) and grown for 24ꢀh.
Then the medium was replaced with tested compounds and cells were incubated for 24 or 72
h. Floating cells were collected and added to adherent cells, harvested by trypsinization.
Afterwards, we followed instructions from the manufacturer of the Annexin V-FITC
Apoptosis Detection Kit (Biotool, USA). Fluorescence was measured using a Becton
Dickinson FACS Aria III sorter (BD Company, USA). Experiments were repeated at least
twice. Data were analyzed using Flowing Software (Cell Imaging Core, Turku Centre for
Biotechnology, Turku).

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5
.6. Analysis of the expression of apoptosis-related proteins
The levels of expression of apoptosis-related proteins were evaluated using Human
5
Apoptosis Array Kit (R&D Systems, United Kingdom). 2.5x10 cells were seeded on 100mm
dishes (Nunc, Germany) and allowed to attach for 24ꢀh. Then the medium was replaced with
tested compounds and the cells were incubated for 72 h. Next cells were lysed and the lysates
were clarified by centrifugation for 30 min at 14,000×g. The total protein concentration was
determined using Protein Quantification Kit (Sigma-Aldrich, Germany). Equivalent amounts
of protein were diluted and incubated with the array membranes. The membranes were then
scanned in the G:Box transilluminator (Syngene, United Kingdom) and the protein expression
levels were quantified by densitometric analysis.
5
.7. Western blot analysis
5
2
.5x10 cells were seeded on 100mm dishes (Nunc, Germany) and allowed to attach
for 24ꢀh. Then the medium was replaced with tested compounds and the cells were incubated
for 72 h. Cells were washed with cold PBS (Sigma-Aldrich, Germany) and them 300µl lysis
buffer (50mM Tris pH 7.5, 150mM NaCl, 1% NP40, 50mM PMSF, 1mM DTT, 1x Inhibitor
o
Protease Cocktail) was added to each dish. Cells lysates were rocked for 20 min at 4 C and
centrifuged at 15000xg for 30 min. The concentration of proteins in the supernatants were
determined using Protein Quantification Kit (Sigma-Aldrich, Germany). 60µg of the protein
was loaded into each lane. SDS-polyacrylamide gel electrophoresis was carried out in 12%
gel. After electrophoresis, proteins were transferred to nitrocellulose membranes and blocked
with 5% skim milk in TBST buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween
o
2
0) for 45 minutes at 4 C. The primary antibodies solutions (Cell Signaling Technology,
United States) were added to the membranes and were gently rocked overnight at 4 °C
1:1000 dilutions). Next day, membranes were incubated with appropriate HRP-conjugated
(
secondary antibodies (Cell Signaling Technology, USA) and then ECL substrate was added.
The membranes were then scanned in the G:Box transilluminator (Syngene, United Kingdom)
and the protein expression levels were quantified by densitometric analysis.
5
.8. Mitotic index
4
Cells (5x10 cells per well) were grown on 6-well plate (Nunc, USA) for 24ꢀh. Then
the medium was replaced with the one containing tested agents. Cells were incubated for 24
hours in the cell culture incubator. After washing with cold, PBS cells were fixed in ice-cold

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methanol (−20°C) for 10 minutes, air-dried, washed with PBS, and stained with 2ꢀµg/ml 4′-6′-
diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Germany). Slides were
mounted in Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich, Germany). 1000
DAPI-stained cells were examined in each well by fluorescence microscopy (Zeiss, USA)
using the ×40 objective.
5
.9. Clonogenic assay
4
Cells (5x10 cells per well) were plated in 6-well plate (Nunc, Germany) and treated
with the tested compounds for 24 and 72 h. Adherent cells were harvested by trypsinization.
3
Cells were re-seeded then on the new 6-well plate (Nunc, Germany) with a density of 2x10
cells per well. After 10 days cells were washed with PBS (Sigma-Aldrich, Germany), next
o
cells were fixed in ice-cold ethanol (-20 C) for 3 minutes, air-dried, washed again with PBS
and stained with 0.01% crystal violet (Sigma-Aldrich, Germany) in dH O (deionized water)
2
for 15 minutes. Next plates were washed dH O and allowed to dry. Colonies were counted
2
with a microscope (Zeiss, USA). Aggregates of 30 cells or more were considered colonies.
6
. Statistical analysis
The results were expressed as means ± S.D. and performed by T-student test, p values≤0.05
being considered statistically significant.
Acknowledgments
This work was financially supported by the National Science Centre (Poland) under
the project number 2016/21/B/NZ7/01766.
Cell-based experiments were performed at the Silesian Bio-Farma Center for
Biotechnology, Bioengineering and Bioinformatics project (grant no. POIG.02.01.00-00-
1
66/08), the Operational Programme Innovative Economy for 2007-2013, Priority Axis 2.
1
13
We sincerely thank MSc Katarzyna Hopko for measuring the H and C NMR
spectra, MSc Karol Erfurt for measuring the HR-MS spectra, MSc Pawel Skora for measuring
the elemental analysis and Prof. Joanna Rzeszowska for sharing the cell lines for in vitro
research.

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Figure Captions
Figure 1. Structure of compounds bearing 2(5H)-furanone with anticancer activity (a) 5-
butoxy-3,4-dichloro-2(5H)-furanone, (b) 3,4-dichloro-5-(oxiran-2-ylmethoxy)-2(5H)-
furanone, (c) (E)-3-(4-fluorophenyl)-N-(4-((2-methylene-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)acrylamide and (d) rubrolide M. [6, 7, 19, 20]
Figure 2. Compound 12 perturbs cell cycle in A549 cells. (A) Cells cycle after 24 and 72 h
treatment with 1, 12 and 15 against (B) Mitotic index after 24 hours treatment with 1, 12 and
1
5. (C) Percentage of cells in sub-G1 after 24 and 72 h treatment with 1, 12 and 15 (D)
Western blot analysis of cyclin B1 expression after 72 h treatment with 10 µM of 1, 12 and
5. β-actin was used as a loading control.
1
Figure 3. Compound 12 promotes cell death in A549 cells. (A) Cells cycle after 72 h
treatment with 20 µM of 1, 12 and 15, (B) Comparison of the percentage of cells in sub-G1
phase, (C) Induction of apoptosis after 72 h treatment with 20 µM of 1, 12 and 15 detected in
annexin V-FITC/PI assay (D) Comparison of the percentage of cells in early and late
apoptosis. (E) Changes of apoptosis-related proteins found in A549 cell line after 72 hours
treatment with 20 µM of 1, 12 and 15. (F) Pixel density of apoptosis-proteins after 72 hours
treatment 1, 12 and 15 against A549 cell lines. (G) Western blot analysis of procaspase-3
expression in A549 cells treated with 20 µM of 1, 12 and 15 after 72 hours. β-actin was used
as a loading control.
Figure 4. Compound 12 decreases the clonogenic potential of A549 cells. (A) Representative
colony formation images of the non-small cell lung cancer after treatment with compounds
(
B) The fraction of surviving cells after 72 hours treatment with 10 µM 1, 12 and 15 against
A549 cell line. (C) Number of colonies formed after 72 hours treatment with 10 µM 1, 12 and
5 against A549 cell line.
1
Figure 5. Compound 12 act synergistically with erlotinib in A549 cells. (A) Cell viability
after 72 hours treatment of A549 cells with 12, erlotinib (ERB) and their combination. (B)
Cell cycle and (C) percentage of cells in sub-G1 after 72 hours treatment with 12, ERB and
their combination at 10 µM concentration. (D) Representative colony formation images of
A549 cells after treatment with indicated compounds. (E) Number of colonies formed after 72
hours treatment with 10 µM 1, 12, ERB and their combination.
Scheme 1. Synthesis of 5- and 4,5- substituted mucochloric acid (1) derivatives; a) toluene,
conc. H SO , reflux b) DMSO, room temperature. Numbers in brackets indicate starting
2
4
alcohol (2-8) or amine (16-20). [6, 7, 12, 23]

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Table 1. Cytotoxicity and selectivity index (SI) of 3,4-dichloro-5-hydroxy-2(5H)-furanone
derivatives against A549 and BEAS-2B cell lines
a
IC50 for cell lines (µM)
1
2
b
Compounds
R
R
SI
A549
122.9 ± 12.3
5.1 ± 0.1
6.3 ± 0.1
3.6 ± 0.2
6.7 ± 0.7
22.5 ± 4.5
14.0 ± 2.0
7.7 ± 1.6
BEAS-2B
45.0 ± 1.3
4.6 ± 0.5
3.8 ± 0.4
3.5± 0.1
1
9
H
Me
-
-
-
-
-
-
-
-
-0.44
0.05
0.22
-0.02
0.61
0.00
0.22
0.88
10
11
12
13
14
15
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Et
Pr
i-Pr
27.5 ± 3.7
22.3 ± 4.0
23.1 ± 4.9
59.0 ± 9.1
n-Bu
tert-Bu
neopentyl
i-Pr
c
c
d
C
H
-NH-
-NH-
-NH-
-NH-
piperidin-1-yl
-NH-
-NH-
-NH-
-NH-
piperidin-1-yl
-NH-
-NH-
-NH-
-NH-
neopentyl piperidin-1-yl
>400
>400
nc
6
5
i-Pr
4-Br-C
4-Cl-C
4-F-C
H
95.9 ± 3.8
39.6 ± 6.0
185.5 ± 8.9
201.9 ± 8.4
77.0 ± 9.4
0.32
0.29
6
4
i-Pr
H
6 4
c
d
i-Pr
H
>400
nc
6
4
c
c
d
i-Pr
>400
>400
nc
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
neopentyl
C
H
175.4 ± 18.7
46.5 ± 6.7
58.1 ± 1.1
78.5 ± 5.2
93.3 ± 6.3
76.2 ± 0.4
28.0 ± 1.5
13.3 ± 2.0
26.7 ± 3.5
206.1 ± 3.2
42.7 ± 7.0
60.8 ± 11.2
122.4 ± 3.2
172.1 ± 1.8
76.2 ± 1.6
30.9 ± 0.2
30.3 ± 4.5
47.6 ± 1.1
0.07
-0.04
0.02
0.19
0.27
0.00
0.23
0.36
0.25
6
5
4-Br-C
4-Cl-C
4-F-C
H
4
6
H
4
6
H
4
6
C H
6 5
neopentyl 4-Br-C
neopentyl 4-Cl-C
neopentyl 4-F-C
H
4
6
H
4
6
H
6
4
c
c
d
>400
>400
nc
a
IC50 is the concentration of a drug that decreased cell viability by 50% relative to the
untreated control.

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b
SI has been calculated as a logarithm of a ratio of IC value for healthy cells (BEAS-2B)
5
0
and the IC value for cancer cells (A549). [25]
5
0
c
The maximum use of the compound concentration.
d
nc (not calculated) – the SI could not be calculated due to the lack of the IC value for
5
0
healthy/cancer cells.

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Table 2. Cytotoxicity of compounds 1, 12 and 15 against panel of cancer cell lines
IC for compounds (µM)
5
0
Cell lines
1
12
15
HCT 116 wt
HCT 116 p53-/-
HT-29
8.3 ± 1.1
7.2 ± 1.6
6.8 ± 0.3
7.2 ± 0.1
2.8 ± 0.1
19.8 ± 1.5
16.8 ± 1.0
21.1 ± 0.1
7.7 ± 0.1
12.4 ± 2.0
4.8 ± 0.3
25.4 ± 2.8
7.2 ± 0.1
6.2 ± 0.6
7.2 ± 0.1
7.3 ± 0.6
27.8 ± 3.2
17.6 ± 0.6
38.1 ± 3.9
11.1 ± 1.9
11.2 ± 1.5
6.0 ± 0.3
26.8 ± 1.7
10.8 ± 1.0
21.8 ± 0.8
17.2 ± 2.3
85.1 ± 13.5
111.3 ± 18.1
58.5 ± 3.1
39.4 ± 1.8
32.3 ± 4.4
24.7 ± 1.1
129.7 ± 8.0
LoVo
Hep3B
HepG2
U2OS
SJSA-1
MCF-7
PC-3
DU 145

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Highlights
1
. Novel library of mono- and disubstituted 2(5H)-furanone derivatives was successfully
obtained.
2
. Most active compound 12 displayed high selectivity towards malignant cell line A549.
. Compound 12 induced G2 cell cycle arrest, suppressed colony formation and
promoted caspase-independent cell death in A549 cell line.
3
4
. Compound 12 showed slight synergetic effects with erlotinib against A549 cell line.