Synthesis and structure–activity relationship study of novel 3-diethoxyphosphorylfuroquinoline-4,9-diones with potent antitumor efficacy

Article abstract of DOI:10.1016/j.ejmech.2021.113429

Herein we report an efficient synthesis of a series of regioisomeric N,O-syn and N,O-anti 3-diethoxyphosphorylfuroquinoline-4,9-diones combining furoquinoline-5,8-dione skeleton, present in several highly cytotoxic compounds, with diethoxyphosphoryl moiet

Full text of DOI:10.1016/j.ejmech.2021.113429

European Journal of Medicinal Chemistry 219 (2021) 113429
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and structureeactivity relationship study of novel 3-
diethoxyphosphorylfuroquinoline-4,9-diones with potent antitumor
efficacy
,
,
, **
Jakub Modranka a 1, Joanna Drogosz-Stachowicz b 1, Anna Pietrzak c, Anna Janecka b
,
,
*
Tomasz Janecki a
a
_
ꢀ ꢀ
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Łodz, Poland
Department of Biomolecular Chemistry, Medical University of Łodz, Mazowiecka 6/8, 92-215, Łodz, Poland
Institute of General and Ecological Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Łodz, Poland
b
c
ꢀ ꢀ
ꢀ ꢀ
_
ꢀ ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report an efficient synthesis of
a series of regioisomeric N,O-syn and N,O-anti 3-
Received 19 February 2021
Received in revised form
27 March 2021
Accepted 27 March 2021
Available online 3 April 2021
diethoxyphosphorylfuroquinoline-4,9-diones combining furoquinoline-5,8-dione skeleton, present in
several highly cytotoxic compounds, with diethoxyphosphoryl moiety. The cytotoxic activity of the ob-
tained analogs was tested against two human cancer cell lines: promyelocytic leukemia HL-60 and breast
cancer adenocarcinoma MCF-7 and for comparison on human umbilical vein endothelial cells HUVEC and
mammary gland/breast MCF-10 A cells. Several diethoxyphosphorylfuroquinoline-4,9-diones proved to
be highly cytotoxic for cancer cells with IC50 values even below 0.1
mM. Interestingly, N,O-syn 3-
Keywords:
diethoxyphosphorylfuroquinoline-4,9-diones were 3- to 7-fold more active against HL-60 cells than
the respective N,O-anti regioisomers. The most promising analogs 9c and 9i, with the highest cancer/
healthy cells cytotoxicity ratio, were further evaluated to establish their mode of action. In HL-60 cells
these analogs enhanced intracellular ROS generation and NAD(P)H:quinone oxidoreductase 1 (NQO1)
depletion which led to the cell cycle arrest in the S-phase, reduced cell proliferation, DNA damage and
apoptosis.
Quinolinediones
Phosphonates
Hybrid molecules
Cytotoxic activity
Apoptosis
DNA damage
© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
of some parasitic protozoa, e.g. Trypanosoma brucei rhodesiense [8]
and benzofuroquinoline-5,8-diones 3 were identified as topo-
It is well established that nitrogen-containing heterocycles are
found in various natural products, biologically active structures,
and medicinally relevant compounds [1e3]. Among them, quino-
line-5,8-diones have gained considerable attention in medicinal
chemistry, due to the fact that compounds containing such skeleton
possess multiple biological properties, including antimicrobial,
antiprotozoan and anticancer activity [4e6]. This structural motif
can be found in antitumor antibiotics, such as streptonigrin 1 and
its derivatives [7] (Fig. 1). Natural ascidiathiazone A 2, obtained
from a New Zealand ascidian Aplidium species, is a growth inhibitor
isomerase II inhibitors and their cytotoxicity against several types
of human cancer cell lines was similar or higher than that of
doxorubicin [9]. Also, furoquinoline-5,8-diones 4 were reported as
compounds having high cytotoxic activity against cancer cells, as
well as cancer stem cells [10]. It is worth to stress that the authors
didn’t notice major differences in the activity between N,O-anti and
N,O-syn isomers. Recently, furoquinoline-5,8-diones 5 have been
identified as effective tyrosyl-DNA phosphodiesterase 2 (TDP2)
inhibitors [11] and many analogs of 5 were designed to probe
various substituents at position 3 and alternations in the furan ring
motif.
On the other hand, phosphorus-substituted heterocycles have
received considerable attention mainly due to their synthetic ap-
plications and growing usefulness in drug design [12e14]. There
are numerous reports claiming that the presence of phosphoryl
group can enhance biological activity. It can mimic the tetrahedral
intermediates formed in enzymatic reactions involved in the
* Corresponding author.
** Corresponding author.
E-mail addresses: anna.janecka@umed.lodz.pl (A. Janecka), tomasz.janecki@p.
lodz.pl (T. Janecki).
1
J.M and J. D-S. contributed equally.
https://doi.org/10.1016/j.ejmech.2021.113429
0223-5234/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Fig. 1. Structures of some natural and synthetic quinoline-5,8-diones.
carboxyl group metabolism [13,15,16] and this rationale has resul-
ted in the design of some important drugs [17], such as N-phos-
phonoacetyl-L-aspartic acid (PALA) with anticancer activity or
10, respectively. These mixtures were successfully separated by
column chromatography to give pure 9 and 10 in yields given in
Table 1. Structures of all obtained compounds were confirmed by
1H, 13C and 31P NMR spectra. Distinction between N,O-syn and N,O-
anti regioisomers 9i and 10i was made using X-ray analysis.
Yellow crystals of two isomers of 9i and 10i were obtained by
slow evaporation of ethyl acetate. Their crystal structures were
determined by low temperature (100 K) single crystal X-ray anal-
ysis. Molecular conformations of 9i and 10i are shown in Fig. 2.
Regiochemistry of the remaining 3-diethoxyphosphorylfuro
quinoline-4,9-diones 9a-h and 10a-h was assigned on the basis of
the similarities of their 13C and 31P NMR spectra to the spectra of 9i
and 10i, respectively. For example, in 13C NMR spectrum of 9i, C-4a
atom (Fig. 2) has chemical shift of 130.48 and chemical shifts of this
atom in all syn-regioisomers 9a-h are very similar and fall within the
range from 130.37 to 130.64. Accordingly, chemical shifts of C-8a
atoms in 10a-h fall within the range from 128.74 to 128.85 and the
chemical shift of this atom in 10i is 128.85. In 31P NMR spectra syn-
regioisomer 9i has greater chemical shift value than anti-regioisomer
10i (6.16 and 5.84, respectively) therefore syn-regiochemistry can be
assigned to all these regioisomers in pairs, which had greater chem-
ical shift value in 31P NMR spectrum.
fosinopril which is a clinically useful antihypertensive agent.
Furthermore, it was shown that the presence of dialkoxy or dia-
ryloxyphosphoryl group may improve bioavailability of drugs [18]
and also influence cancer progression through the inhibition of
farnesyl protein transferase [19,20] or purine nucleoside phos-
phorylase [21,22].
From some time in our laboratory we have been developing
methods for the synthesis of phosphorylated oxa- and azahetero-
cycles, as a valuable group of synthetically and biologically impor-
tant compounds [23,24]. Recently, we have designed a series of 3-
diethoxyphosphorylnaphthofuran-4,9-diones 6, which integrated
the merits of both, a phosphoryl group and naphthofuran-4,9-
dione skeleton. These compounds showed high cytotoxic activity
against leukemia HL-60, NALM-6 and breast cancer MCF-7 cell
lines, with IC50 values below 10
mM [25]. Now, we decided to
examine, whether the incorporation of the nitrogen atom into the
naphthofuran-4,9-dione moiety might enhance the cytotoxic ac-
tivity of these compounds. Therefore, in this paper we report the
synthesis and anticancer evaluation of a series of N,O-syn and N,O-
anti 3-diethoxyphosphorylfuroquinoline-4,9-diones
9 and 10,
respectively, containing a variety of substituents in position 2.
2.2. Biology
2. Results and discussion
2.2.1. Cytotoxic activity of novel hybrids against cancer and normal
cell lines
2.1. Chemistry
The synthesized N,O-syn and N,O-anti 3-diethoxyphosph
orylfuroquinoline-4,9-diones 9 and 10 were tested in vitro against
two human cancer cell lines: promyelocytic leukemia HL-60 and
breast cancer adenocarcinoma MCF-7 and the most cytotoxic analogs
also against human umbilical vein endothelial cells HUVEC and
mammary gland/breast MCF-10 A cells for 48 h. The obtained results,
which are shown inTable 2, were compared with doxorubicin used as
a positive control. All tested compounds were highlycytotoxic for HL-
The 3-diethoxyphosphorylfuroquinoline-4,9-diones 9a-i and
10a-i were obtained from 6,7-dichloroquinoline-5,8-dione (7) and
b
-ketophosphonates 8, applying the procedure previously
described for the synthesis of 3-diethoxyphosphorylnaphthofuran-
4,9-diones 6 [25] (Scheme 1). 6,7-Dichloroquinoline-5,8-dione (7)
was synthesized by oxidizing 8-hydroxyquinoline with sodium
chlorate in the concentrated HCl solution [26], phosphonates 8a-b
were commercially available and phosphonates 8c-i were prepared
using literature procedure [25]. All reactions proceeded smoothly
and yielded mixtures of N,O-syn and N,O-anti regioisomers 9 and
60 cells, with IC50 values below 1
even below 0.1
M (IC50 for doxorubicin ¼ 0.11
obtained in MCF-7 cells were also very low and varied from below
0.1 Mfor9c,g,hto 1.59 M). All
Mfor9b(IC50 fordoxorubicin¼ 0.89
mM, and in case of analogs 9c,f,h
m
mM). The IC50 values
m
m
m
2

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Scheme 1. Synthesis of 3-diethoxyphosphorylfuroquinolinedione isomers 9a-i and 10a-i.
Table 1
the most active compounds with IC50 below 0.1 mM had phenyl sub-
Yields of N,O-syn and N,O-anti 3-dietoxyphosphorylfuroquinoline-4,9-
diones 9 and 10.
stituent containing either electronwithdrawing groups (p-NO2, 9f; p-
F, 9g; p-Cl, 9h) or electron donating group (p-OMe, 9c). Apparently,
there is no simple correlation between electron density of the phenyl
ring and cytotoxic activity. On the other hand, syn or anti relationship
between furan oxygen and quinoline nitrogen atoms is important for
the activity. N,O-syn 3-diethoxyphosphorylfuroquinoline-4,9-diones
9b-h were 3- to 7-fold more active in HL-60 cells than the respec-
tive N,O-anti regioisomers 10b-h. Only 9a,i were equipotent with
10a,i. This relationship can be also observed in MCF-7 cell line. The
most active N,O-syn diethoxyphosphorylfuroquinoline-4,9-diones
9c,g,h were 4- to 12-fold more active against MCF-7 cells than their
N,O-anti regioisomers 10c,g,h. Although the reason for the enhanced
activityofN,O-syn regioisomersisnotclearitmightbespeculatedthat
they may better fit into the binding site of the protein.
9 or 10
R
Yield [%]a
9
10
a
b
c
Me
Ph
12
32
21
18
28
22
d
25
25
Selected 3-diethoxyphosphorylfuroquinoline-4,9-diones were
also tested on HUVEC and MCF-10 A, to evaluate their influence on
normal cells. The most pronounced difference in cytotoxicity be-
tween cancer and normal cells was observed for 2-(4-
methoxyphenyl)-3-diethoxyphosphorylfuroquinoline-4,9-dione
9c which was 12-fold less toxic for HUVEC than HL-60 cells. The
same compound was also 5-fold less toxic for normal MCF-10 A
than cancer MCF-7 cells.
The presented data clearly showed that the incorporation of a
nitrogen atom into the naphthofuran-4,9-dione moietyenhanced the
cytotoxicity. Comparison of the cytotoxic activity of formerly pub-
lished 3-diethoxyphosphorylnaphthofuran-4,9-diones 6a-c (6a, R ¼
Me; 6b, R ¼ Ph; 6c, R ¼ 2-chlorophenyl) [25] (Table 2) with 3-
diethoxyphosphorylfuroquinoline-4,9-diones 9 or 10 showed that
the latterones were 15- to 30-fold more active in HL-60 cells and 2- to
5-fold more active in MCF-7 cells. For example, 2-phenyl-3-
e
23
26
f
19
26
26
22
28
26
26
27
g
h
i
diethoxyphosporylfuroquinoline-4,6-dione 9b (IC50 ¼ 0.20
30-fold more active than 2-phenyl-3-diethoxyphospory
lnaphthalene-4,9-dione 6b (IC50 ¼ 6.01 M) in HL-60 cells. Also
mM) was
a
Yields of pure, separated products based on 7.
m
Fig. 2. Molecular structures of isomers 9i and 10i.
3

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Table 2
In vitro cytotoxic activity of 3-diethoxyphosphorylfuroquinoline-4,9-diones 9a-i, 10a-i and 3-diethoxyphosphorylnaphthofuran-4,9-diones 6a-c tested on two cancer and two
normal cell lines.
a
No
IC50
(
mM)
HL-60
MCF-7
HUVEC
MCF-10 A
HUVEC/HL-60
MCF-10 A/MCF-7
9a
10a
9b
10b
9c
10c
9d
10d
9e
10e
9f
10f
9g
10g
9h
0.84 0.01
0.94 0.01
0.20 0.01
0.76 0.01
0.09 0.00
0.63 0.01
0.30 0.02
0.72 0.01
0.16 0.01
0.65 0.01
0.07 0.01
0.41 0.02
0.13 0.01
0.39 0.01
0.09 0.01
0.37 0.01
0.37 0.01
0.38 0.01
8.13 0.16
6.01 0.18
6.35 0.46
0.11 0.01
1.59 0.05
5.94 0.01
1.59 0.01
1.10 0.01
0.10 0.01
1.21 0.04
1.50 0.16
0.98 0.02
0.95 0.03
1.15 0.04
1.32 0.01
0.76 0.01
0.08 0.01
0.74 0.01
0.09 0.01
0.35 0.01
0.48 0.01
0.54 0.02
5.70 0.21
2.40 0.30
2.34 0.18
0.89 0.12
2.88 0.12
3.07 0.11
1.68 0.01
0.38 0.01
1.05 0.045
0.34 0.01
0.42 0.01
1.82 0.08
0.10 0.01
0.39 0.01
0.09 0.01
0.33 0.01
0.55 0.02
1.00 0.001
0.11 0.01
0.27 0.01
1.11 0.03
0.33 0.01
8.28 0.19
11.10 0.57
3.4
3.3
8.4
0.5
12
0.5
1.4
2.5
0.6
0.6
1.3
0.8
4.2
2.6
1.1
0.7
3
5.2
1.9
0.50 0.01
5.37 0.11
5
5.5
0.33 0.01
3.42 0.01
0.27 0.03
4
4.6
3
10h
9i
3.29 0.09
2.45 0.12
6.9
4.5
10i
6ab
6bb
6cb
Dox
0.86
3.73 0.18
3.8 0.45
10.03 0.43
1.37 0.28
2
34
4
1.5
a
Compound concentration required to inhibit cell proliferation by 50% after 48 h treatment. Data are expressed as the mean SEM from the concentration-response curves
of at least three experiments.
b
Data taken from the reference [25].
cancer/healthy cells IC50 ratio was in favor of 3-diethoxyphos
porylfuroquinoline-4,6-diones. When 2-(2-chlorophenyl)-3-dietho
xyphosporylfuroquinoline-4,6-dione 9i was compared with 2-(2-
chlorophenyl)-3-diethoxyphosporylnaphthalene-4,9-dione 6c the
first one was 3-fold less toxic in HUVEC than HL-60 cells and 7-fold
less toxic in MCF-7A than MCF-7 cells. Corresponding values for 2-
also a significant subG0/G1 cell cycle arrest in 6.6 0.4%,14.3 0.9%
and 6.7 0.8%, 16.9 0.4% cells, respectively (Fig. 3 D, E).
2.2.3. Antiproliferative activity
The S-phase of the cell cycle is a part of the interphase in which
replication of DNA takes place [27]. However, considering that the
tested compounds decreased the cells’ metabolic activity, the in-
crease of number of cells in the S phase was most likely not caused
by the increase in cell population actively replicating DNA, but
rather due to accumulation of cells with arrested progress in this
phase. To confirm this hypothesis, the ability of 9c and 9i to inhibit
HL-60 cell proliferation was evaluated by exposure of cells to 5-
bromodeoxyuridine (BrdU), a thymidine analog that incorporates
into newly synthesized DNA during the S-phase of the cell cycle,
followed by flow cytometry analysis. The results showed that both
compounds significantly decreased the population of actively
cycling cells that incorporated BrdU. As presented in Fig. 3 B, C,
treatment with 9i at IC50 and 2IC50 concentrations for 24 h
(2-chlorophenyl)-3-diethoxyphosporylnaphthalene-4,9-dione
were 2 and 4, respectively.
6c
The most promising analogs 9c and 9i, with the highest cancer/
healthy cells cytotoxicity ratio, were chosen for further evaluation
in order to establish their mode of action. Cytotoxicity of these two
compounds in HL-60 cells was then assessed after 24 h incubation.
Both compounds showed high cytotoxic activity with IC50 ¼ 0.11
m
M for 9c and IC50 ¼ 0.11
mM for 9i (Fig. 3 A).
2.2.2. Cell cycle arrest
The cell cycle arrest is a mechanism by which anticancer drugs
may exert their cytotoxic action. The cell cycle distribution was
assessed by DNA staining with DAPI followed by flow cytometry
analysis. HL-60 cells were exposed to 9c (at IC50, 2IC50 and 4IC50
concentrations) and 9i (at IC50 and 2IC50 concentrations). Both
compounds significantly induced the cell cycle arrest in S phase
(47.9 0.71%, 50.8 0.5%, 60.0 0.9% and 40.6 0.6%, 47.5 2.27%,
respectively), while population of cells in G0/G1 and G2/M phases
was diminished. Moreover, treatment with 9c (at 2IC50 and 4IC50
concentrations) and 9i (at IC50 and 2IC50concentrations) caused
decreased proliferating cell population to 29.6
1.4 0.3%, respectively, whereas 9c at IC50, 2IC50 and 4IC50 con-
centrations caused reduction to 49.4 2.7%, 20.4 0.6% and
2.4% and
1.9 0.2%, respectively, in comparison to control (86.0 0.01%). The
results indicated, that accumulation of cells in the S-phase caused
by 9c and 9i treatment was simultaneous with decreased DNA
synthesis and cell proliferation, hence cells’ progression was
arrested in the S-phase.
4

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Fig. 3. Effect of 9c and 9i treatment on metabolic activity (A) cell proliferation (B, C) and cell cycle distribution (D, E) in HL-60 cells. A: Metabolic activity of HL-60 cells treated with
9c and 9i for 24 h assessed by MTT assay. The error bars represent mean of two independent experiments each preformed in triplicate SEM. BeE: Cells were exposed to 9c (at IC50
,
2IC50 and 4IC50 concentrations) or to 9i (at IC50 and 2IC50 concentrations) for 24 h, stained with BrdU and DAPI and analyzed by flow cytometry. The error bars represent mean of
three replicates SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant in comparison to control.
2.2.4. Induction of apoptosis and DNA damage
less genotoxic, leading to H2AX phosphorylation only in 3.6 0.1%
and 8.4 0.1% cell population after treatment with 9c at 2IC50 and
4IC50 concentrations, respectively, compared to control (2.2 0.1%).
The appearance of cells in sub-G0/G1 phase in the cell cycle
analysis suggested induction of apoptosis in HL-60 cells by treat-
ment with 9c and 9i. Hence, occurrence of 89 kDa fragment of
cleaved-PARP, generated by cleavage of PARP1 by caspase 3 and 7
during caspase-mediated apoptosis [28], was investigated using
Anti-Cleaved PARP (Asp214) antibodies and flow cytometry anal-
ysis (Fig. 4 D, E). 9c at IC50, 2IC50 and 4IC50 concentrations, dose-
dependently increased the population of cells with cleaved PARP
to 24.7 3.2%, 46.6 1.2% and 77.5 1.8%, respectively. Treatment
with 9i at IC50 and 2IC50 concentrations also led to the significant
PARP cleavage in 33.8 2.1% and 81.0 1.0% of cell population,
2.2.5. Induction of mitochondrial dysfunction
Mitochondrial transmembrane potential (DJm) is considered
a vital indicator of mitochondrial function and is usually used to
assess the health of cells. Loss of DJm is often observed in un-
healthy or apoptotic cells. Detection of DJm may be performed
using JC-1 dye, which has an ability to enter the healthy cells’
mitochondria, accumulate and form aggregates exhibiting red
fluorescence. JC-1 dye does not form aggregates in unhealthy
cells with low DJm, which consequently show lower fluores-
cence [31]. To examine the influence of 9c and 9i treatment on
DJm status, HL-60 cells were stained with JC-1 fluorochrome
and analyzed using flow cytometry. In the healthy, untreated HL-
respectively, compared to control (2.1
0.4%). Moreover, pro-
apoptotic activity of 9c and 9i was confirmed by Annexin V and
Propidium iodide staining (Fig. 4AeC). In the control group,
91.1 0.1% of HL-60 cells were healthy, 2.5 0.1% were in early
stage apoptosis (Annexin V-positive and PI-negative) and 6.1 0.1%
were in late stage apoptosis (both Annexin V and PI-positive). 9c
induced a small increase in the early apoptotic cells amount but the
number of late apoptotic cells after exposure to IC50, 1.5IC50 and
60 cells only 4.7
Treatment with 9c at IC50
significantly increased the number of cells showing low DJm to
43.4 1.0%, 62.8 0.7% and 85.7 0.4% cell population,
respectively. Similarly, 9i (at IC50, 2IC50 concentration) also
0.5% of cells showed low red fluorescence.
,
2IC50 and 4IC50 concentrations
2IC50 concentrations of 9c was more prominent (16.3
0.9%,
22.5 0.8%, 27.4 2.0%, respectively). 9i at IC50, 1.5IC50 and 2IC50
concentrations significantly induced both, early stage apoptosis in
18.3 0.6%, 21.2 0.4% and 17.2 1.0% cells, respectively, and late
stage apoptosis in 37.4 3.8%, 63.6 0.2% and 68.4 1.1% cells,
respectively. The results indicate, that 9c and 9i significantly
induced caspase-dependent apoptosis in HL-60 cells.
An early cells’ response to DNA damage include the histone
variant H2AX phosphorylation on Ser139, producing gH2AX, which
is often used as a biomarker for DNA double-strand breaks (DSBs)
detection [29,30]. Therefore, induction of DSBs was assessed by
induced mitochondrial depolarization in 47.2
74.9 0.6% of cells, respectively (Fig. 5 A, B).
1.8% and
2.2.6. Reactive oxygen species generation
Unlike normal cells, cancer cells exhibit increased ROS production
due to elevated metabolic rate. However, cancer cells also demon-
strate over-activated antioxidant systems that enable cell adaptation
and ROS amount maintenance below toxic levels [32]. Excessive ROS
generation or reduced antioxidant activity may cause oxidative
stress leading to DNA, protein and lipid damage and eventually to
cell death [33]. Many chemotherapeutic agents have been reported
to cause apoptosis of cancer cells by substantial ROS increase. Thus,
augmenting ROS production by exogenous compounds or inhibitors
of ROS scavengers may be used as a therapeutic target in anticancer
staining the HL-60 cells with
cytometry analysis (Fig. 4 F, G). Exposure of cells to 9i at IC50 and
2IC50 concentrations induced significant H2AX generation in
13.8 0.7% and 35.2 0.6% of cells, respectively. 9c was noticeably
gH2AX fluorescent antibody and flow
g
5

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Fig. 4. Induction of apoptosis and DNA damage. A, B, C: HL-60 cells were exposed to 9c and 9i at IC50, 1.5IC50 and 2IC50 concentrations for 24 h, stained with Annexin V and PI and
analyzed by flow cytometry. DeG: Cells were treated with 9c (IC50, 2IC50 and 4IC50 concentrations) and 9i (IC50 and 2IC50 concentrations) for 24 h, stained with PE Anti-Cleaved
PARP (Asp214) and Anti-H2AX (pS139) antibodies and analyzed by flow cytometry. The error bars represent mean of three replicates SEM. *p < 0.05, **p < 0.01, and ***p < 0.001
were considered significant in comparison to untreated control.
drug development [34]. The influence of 9c and 9i on ROS generation
in HL-60 cells was assessed by flow cytometry using CellROX
Oxidative Stress Green Reagent (LifeTechnologies, Carlsbad, CA, USA)
(Fig. 5C). Treatment with 9c and 9i at IC50 concentrations for 24 h
significantly increased intracellular ROS production in 37.0 4.7%
and 72.5 0.9% of cells, respectively. The results suggest that the
investigated compounds induce oxidative stress in HL-60 cells which
may cause the observed cytotoxic activity.
treatment development [37]. Additionally, recent studies imply
that some quinolinediones are substrates of NQO1 and show se-
lective cytotoxicity in cancer cell lines with upregulated NQO1 [38].
In our study, inhibitory activity against NQO1 was investigated
using NQO1 activity assay kit (Abcam, Cambridge, MA, USA).
Treatment with 9c and 9i at 0.1 mM, 0.5 mM and 1 mM for 24 h
induced significant dose-dependent inhibition of NQO1 activity. 9c
was a more potent NQO1 inhibitor than 9i (Fig. 5D). Therefore
NQO1 depletion may sensitize cancer cells to oxidative stress,
finally leading to reduced cell proliferation and apoptosis.
2.2.7. NQO1 activity alteration
The results of several studies indicate, that inhibition of anti-
oxidant enzymes leads to apoptosis of cancer cells [35]. One of the
cellular antioxidant systems, which is often overexpressed in
3. Conclusions
several cancer tissues, is NAD(P)H:quinone oxidoreductase
(NQO1) [36]. Therefore, NQO1 may pose as a target for anticancer
1
In this report, we described an efficient synthesis of a series of
regioisomeric N,O-syn and N,O-anti 3-diethoxyphosphorylfuroq
6

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
Fig. 5. A, B: Effect of 9c and 9i 24 h treatment on mitochondrial membrane potential in HL-60 cells. Cells were treated with 9c (at IC50, 2IC50 and 4IC50 concentrations) or 9i (at IC50
and 2IC50 concentrations), stained with JC-1 dye and analyzed by flow cytometry. C: Effect of 9c and 9i on ROS formation in HL-60 cells. Cells were treated with IC50 concentrations
of 9c and 9i for 24 h, stained with CellROX Green Reagent and analyzed by flow cytometry. D: NQO1 activity in HL-60 cells after compound treatment. Cells were treated with 9c or
9i (0.1 mM, 0.5 mM and 1 mM) for 24 h. NQO1 activity was assessed by the NQO1 activity assay kit (Abcam). The error bars represent mean of three replicates SEM. *p < 0.05,
**p < 0.01, and ***p < 0.001 were considered significant in comparison to control.
uinoline-4,9-diones, combining furoquinoline-5,8-dione skeleton
with diethoxyphosphoryl moiety. The in vitro screening of the novel
compounds in the MTT test revealed their very high cytotoxic ac-
tivity in the low micromolar range in HL-60 and MCF-7 cells. An
interesting observation was that in both tested cancer cell lines N,O-
syn 3-diethoxyphosphorylfuroquinoline-4,9-diones were more
cytotoxic than the respective N,O-anti regioisomers. The compari-
son of the cytotoxic activity of 3-diethoxyphosphorylfuroquin
4. Experimental
4.1. General methods
Analytical grade solvents and commercially available reagents
were used without further purification. NMR spectra were recorded
on a Bruker UltraShield 700 instrument, running at 700 MHz for 1H,
and 176 MHz for 13C and 283 MHz for 31P. Chemical shifts (
d) are
oline-4,9-diones
9
and 10 with formerly published 3-
reported in ppm relative to residual solvent signals (CDCl3:
7.26 ppm for 1H NMR, 77.16 ppm for 13C NMR). Mass spectra were
obtained on a Bruker Maxis Impact spectrometer using electro-
spray (ESþ) ionization (referenced to the mass of the charged
species). Thin-layer chromatography was performed with pre-
coated TLC sheets of silica gel 60 F254 (Aldrich®) and visualized by
ultraviolet irradiation. Silica gel (silica gel 60, 230e400 mesh,
Aldrich®) was used for column chromatography.
diethoxyphosphorylnaphthofuran-4,9-diones showed that ana-
logs containing a nitrogen atom were 15- to 30-fold more active in
HL-60 cells and 2- to 5-fold more active in MCF-7 cells.
Among novel analogs, compounds 9c and 9i had the most
favorable cancer/healthy cells cytotoxicity ratio and their activity in
HL-60 cells was further evaluated. Both these analogs showed
similar mode of action, significantly inducing the S-phase cell cycle
arrest, reducing cell proliferation, promoting DNA damage and
apoptosis. It could be speculated that the decreased proliferation
and mitochondria-dependent apoptosis in HL-60 cells induced by
9c and 9i were associated with the enhanced intracellular ROS
generation and NQO1 depletion. The results suggest, that the novel
furoquinoline-4,9-dione analogs may be further developed as a
potential anti-cancer treatment.
4.2. General procedure for the synthesis of 3-
diethoxyphosphorylfuroquinoline-4,9-diones 9a-i and 10a-i
A
mixture of 6,7-dichloroquinoline-5,8-dione 7 (0.91 g,
4.0 mmol) K2CO3 (1.66 g, 12.0 mmol) and corresponding diethyl
acylmethylphosphonate 8 (4.0 mmol) in MeCN (50 mL) was stirred
7

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
at 65 ꢀC for 18 h. The reaction mixture was poured into cold water
(50 mL). Extraction with EtOAc (3 ꢁ 50 mL), drying (MgSO4) and
evaporation of the solvent gave the crude product, which was pu-
rified by column chromatography [EtOAc].
113.83, 107.29 (d, J ¼ 214.9 Hz), 63.30 (d, J ¼ 6.3 Hz), 55.58, 16.34 (d,
J ¼ 6.5 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 442.1056, observed 442.1066.
d 7.63. HRMS m/z, calcd
Diethyl
(4,9-dioxo-2-(3,4,5-trimethoxyphenyl)-4,9-dihydrofuro
Diethyl (2-methyl-4,9-dioxo-4,9-dihydrofuro[3,2-g]quinolin-3-yl)
[3,2-g]quinolin-3-yl)phosphonate (9d): red solid, mp 186e188 ꢀC;
phosphonate (9a): yellow solid, mp 190e192 ꢀC; 1H NMR (700 MHz,
1H NMR (700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.54 (dd,
CDCl3)
d
9.02 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.50 (dd, J ¼ 7.9, 1.7 Hz, 1H),
J ¼ 7.9, 1.7 Hz, 1H), 7.71 (dd, J ¼ 7.9, 4.6 Hz, 1H), 7.24 (s, 2H),
4.26e4.18 (m, 2H), 4.18e4.11 (m, 2H), 3.94 (s, 6H), 3.91 (s, 3H), 1.23
7.69 (dd, J ¼ 7.9, 4.6 Hz, 1H), 4.29 (ddq, J ¼ 10.1, 8.4, 7.1 Hz, 2H), 4.21
(ddq, J ¼ 10.1, 8.6, 7.1 Hz, 2H), 2.80 (d, J ¼ 2.0 Hz, 3H), 1.35 (t,
(t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 177.69, 171.55, 165.87
J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d
177.79, 171.47, 168.85 (d,
(d, J ¼ 23.6 Hz),154.50,153.00,152.35 (d, J ¼ 12.1 Hz),147.86,141.24,
135.50, 132.15 (d, J ¼ 10.1 Hz), 130.64, 127.77, 122.59, 107.86, 107.62
(d, J ¼ 213.0 Hz), 63.16 (d, J ¼ 5.8 Hz), 61.13, 56.55, 16.28 (d,
J ¼ 26.1 Hz), 154.47, 152.41 (d, J ¼ 11.8 Hz), 147.89, 135.37, 130.50 (d,
J ¼ 9.6 Hz), 130.37, 127.67, 107.96 (d, J ¼ 214.7 Hz), 63.01 (d,
J ¼ 5.7 Hz), 16.45 (d, J ¼ 6.4 Hz), 14.60. 31P NMR (283 MHz, CDCl3)
J ¼ 6.6 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 502.1267, observed 502.1288.
d 7.86. HRMS m/z, calcd
d
8.41; HRMS m/z, calcd [MþH]þ 350.0793, observed 350.0796.
Diethyl (2-methyl-4,9-dioxo-4,9-dihydrofuro[2,3-g]quinolin-3-yl)
Diethyl
(4,9-dioxo-2-(3,4,5-trimethoxyphenyl)-4,9-dihydrofuro
phosphonate (10a): yellow solid, mp 110e112 ꢀC; 1H NMR
[2,3-g]quinolin-3-yl)phosphonate (10d): orange solid, mp
(700 MHz, CDCl3)
d
9.03 (dd, J ¼ 4.7, 1.8 Hz, 1H), 8.52 (dd, J ¼ 7.9,
168e170 ꢀC; 1H NMR (700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.6, 1.7 Hz,
1.8 Hz, 1H), 7.69 (dd, J ¼ 7.9, 4.7 Hz, 1H), 4.37e4.29 (m, 2H),
1H), 8.54 (dd, J ¼ 7.8,1.7 Hz,1H), 7.70 (dd, J ¼ 7.8, 4.6 Hz, 1H), 7.28 (s,
2H), 4.30e4.24 (m, 2H), 4.24e4.18 (m, 2H), 3.94 (s, 6H), 3.91 (s, 3H),
4.29e4.21 (m, 2H), 2.82 (d, J ¼ 2.1 Hz, 3H), 1.35 (t, J ¼ 7.1 Hz, 6H). 13
C
NMR (176 MHz, CDCl3)
d
177.01, 172.35, 168.90 (d, J ¼ 26.6 Hz),
1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 176.90, 172.43,
154.47, 151.43 (d, J ¼ 11.7 Hz), 148.91, 134.84, 131.20 (d, J ¼ 9.7 Hz),
128.77,127.49,108.49 (d, J ¼ 215.2 Hz), 63.25 (d, J ¼ 6.1 Hz),16.46 (d,
165.91 (d, J ¼ 24.1 Hz), 154.57, 153.02, 151.33 (d, J ¼ 12.1 Hz), 149.11,
141.19, 134.78, 132.65 (d, J ¼ 9.6 Hz), 128.75, 127.51, 122.71, 108.27
(d, J ¼ 214.2 Hz), 107.82, 63.37 (d, J ¼ 6.3 Hz), 61.11, 56.56, 16.33 (d,
J ¼ 6.6 Hz),14.60. 31P NMR (283 MHz, CDCl3)
[MþH]þ 350.0793, observed 350.0797.
d 7.67. HRMS m/z, calcd
J ¼ 6.9 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 502.1267, observed 502.1268.
d 7.64. HRMS m/z, calcd
Diethyl (4,9-dioxo-2-phenyl-4,9-dihydrofuro[3,2-g]quinolin-3-yl)
phosphonate (9b): orange solid, mp 146e148 ꢀC; 1H NMR (700 MHz,
Diethyl (2-(benzo[d] [1,3]dioxol-5-yl)-4,9-dioxo-4,9-dihydrofuro
[3,2-g]quinolin-3-yl)phosphonate (9e): red solid, mp 122e124 ꢀC;
CDCl3)
d
9.03 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.54 (dd, J ¼ 7.9, 1.7 Hz, 1H),
7.88e7.84 (m, 2H), 7.71 (dd, J ¼ 7.9, 4.6 Hz, 1H), 7.55e7.50 (m, 1H),
7.50e7.45 (m, 2H), 4.19 (ddq, J ¼ 10.1, 7.2, 7.1 Hz, 2H), 4.10 (ddq,
J ¼ 10.1, 8.7, 7.1 Hz, 2H), 1.18 (t, J ¼ 7.1 Hz, 6H).; 13C NMR (176 MHz,
1H NMR (700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.54 (dd,
J ¼ 7.9, 1.7 Hz, 1H), 7.71 (dd, J ¼ 7.9, 4.6 Hz, 1H), 7.49 (dd, J ¼ 8.1,
1.8 Hz, 1H), 7.40 (d, J ¼ 1.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 1H), 6.06 (s,
2H), 4.24 (ddq, J ¼ 10.1, 7.8, 7.1 Hz, 2H), 4.16 (ddq, J ¼ 10.1, 8.8, 7.1 Hz,
CDCl3)
d
177.74, 171.60, 166.00 (d, J ¼ 23.5 Hz), 154.47, 152.68 (d,
J ¼ 12.5 Hz), 147.85, 135.47, 131.87 (d, J ¼ 10.1 Hz), 131.56, 130.58,
130.35, 128.25, 127.78, 127.76, 108.20 (d, J ¼ 213.8 Hz), 63.14 (d,
2H), 1.26 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 177.77,
171.54,165.84 (d, J ¼ 24.0 Hz),154.48,152.32 (d, J ¼ 12.2 Hz),150.65,
147.92, 147.68, 135.49, 132.04 (d, J ¼ 10.1 Hz), 130.63, 127.73, 125.91,
121.34, 110.35, 108.32, 107.14 (d, J ¼ 213.6 Hz), 101.98, 63.16 (d,
J ¼ 6.0 Hz), 16.16 (d, J ¼ 6.8 Hz). 31P NMR (283 MHz, CDCl3)
HRMS m/z, calcd [MþH]þ 412.0950, observed 412.0965.
d 7.40;
Diethyl (4,9-dioxo-2-phenyl-4,9-dihydrofuro[2,3-g]quinolin-3-yl)
phosphonate (10b): yellow solid, mp 134e136 ꢀC; 1H NMR
J ¼ 5.8 Hz), 16.32 (d, J ¼ 6.8 Hz). 31P NMR (283 MHz, CDCl3)
HRMS m/z, calcd [MþH]þ 456.0848, observed 456.0863.
d 7.71.
(700 MHz, CDCl3)
d
9.04 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.53 (dd, J ¼ 7.8,
Diethyl (2-(benzo[d] [1,3]dioxol-5-yl)-4,9-dioxo-4,9-dihydrofuro
[2,3-g]quinolin-3-yl)phosphonate (10e): orange solid, mp
1.8 Hz, 1H), 7.91e7.87 (m, 2H), 7.69 (dd, J ¼ 7.8, 4.6 Hz, 1H),
7.54e7.50 (m, 1H), 7.50e7.45 (m, 2H), 4.27e4.21 (m, 2H), 4.21e4.14
180e182 ꢀC; 1H NMR (700 MHz, CDCl3)
d
9.06 (dd, J ¼ 4.6, 1.7 Hz,
(m, 2H), 1.20 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d
176.95,
1H), 8.55 (dd, J ¼ 7.8, 1.7 Hz, 1H), 7.70 (dd, J ¼ 7.8, 4.6 Hz, 1H), 7.53
(dd, J ¼ 8.1,1.8 Hz,1H), 7.43 (d, J ¼ 1.8 Hz,1H), 6.92 (d, J ¼ 8.1 Hz,1H),
6.06 (s, 2H), 4.28 (ddq, J ¼ 10.1, 7.8, 7.1 Hz, 2H), 4.22 (ddq, J ¼ 10.1,
8.8, 7.1 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
172.47, 166.06 (d, J ¼ 23.6 Hz), 154.54, 151.69 (d, J ¼ 12.1 Hz), 149.04,
134.78, 132.40 (d, J ¼ 10.0 Hz), 131.51, 130.32, 128.77, 128.27, 127.87,
127.49, 108.81 (d, J ¼ 214.8 Hz), 63.35 (d, J ¼ 6.3 Hz), 16.23 (d,
J ¼ 6.8 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 412.0950, observed 412.0957.
d
7.16. HRMS m/z, calcd
d
176.98, 172.42, 165.93 (d, J ¼ 24.2 Hz), 154.54, 151.32 (d,
J ¼ 12.1 Hz), 150.63, 149.13, 147.71, 134.80, 132.60 (d, J ¼ 9.9 Hz),
Diethyl (2-(4-methoxyphenyl)-4,9-dioxo-4,9-dihydrofuro[3,2-g]
128.83, 127.50, 125.93, 121.44, 110.32, 108.35, 107.77 (d,
quinolin-3-yl)phosphonate (9c): orange solid, mp 208e210 ꢀC; 1H
J ¼ 214.7 Hz), 101.97, 63.39 (d, J ¼ 6.3 Hz), 16.39 (d, J ¼ 6.7 Hz). 31
P
NMR (700 MHz, CDCl3)
d
9.04 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.53 (dd,
NMR (283 MHz, CDCl3)
observed 456.0854.
d
7.40. HRMS m/z, calcd [MþH]þ 456.0848,
J ¼ 7.9, 1.7 Hz, 1H), 7.91e7.87 (m, 2H), 7.70 (dd, J ¼ 7.9, 4.6 Hz, 1H),
7.01e6.96 (m, 2H), 4.26e4.17 (m, 2H), 4.17e4.09 (m, 2H), 3.87 (s,
Diethyl (2-(4-nitrophenyl)-4,9-dioxo-4,9-dihydrofuro[3,2-g]qui-
3H), 1.23 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d
177.82,
nolin-3-yl)phosphonate (9f): yellow solid, mp 162e164 ꢀC; 1H NMR
171.53, 166.34 (d, J ¼ 23.8 Hz), 162.41, 154.43, 152.36 (d, J ¼ 12.2 Hz),
147.97, 135.44, 132.15 (d, J ¼ 10.1 Hz), 132.14, 130.63, 127.66, 120.09,
113.82, 106.69 (d, J ¼ 213.6 Hz), 63.08 (d, J ¼ 5.8 Hz), 55.59, 16.29 (d,
(700 MHz, CDCl3)
d
9.09 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.58 (dd, J ¼ 7.9,
1.7 Hz, 1H), 8.37e8.33 (m, 2H), 8.18e8.14 (m, 2H), 7.75 (dd, J ¼ 7.9,
4.6 Hz, 1H), 4.29 (ddq, J ¼ 10.2, 8.0, 7.1 Hz, 2H), 4.20 (ddq, J ¼ 10.2,
8.9, 7.1 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
J ¼ 6.6 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 442.1056, observed 442.1059.
d 7.94. HRMS m/z, calcd
d
177.42, 171.65, 163.02 (d, J ¼ 23.7 Hz), 154.76, 153.28 (d,
Diethyl (2-(4-methoxyphenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
J ¼ 12.0 Hz), 149.37, 147.76, 135.66, 133.59, 131.56, 131.42 (d,
J ¼ 9.1 Hz), 130.64, 128.03, 123.38, 110.72 (d, J ¼ 212.0 Hz), 63.53 (d,
quinolin-3-yl)phosphonate (10c): yellow solid, mp 156e158 ꢀC; 1H
NMR (700 MHz, CDCl3)
d
9.04 (dd, J ¼ 4.7, 1.7 Hz, 1H), 8.53 (dd,
J ¼ 5.9 Hz), 16.38 (d, J ¼ 6.4 Hz). 31P NMR (283 MHz, CDCl3)
HRMS m/z, calcd [MþH]þ 457.0801, observed 457.0802.
d 7.27.
J ¼ 7.8, 1.7 Hz, 1H), 7.94e7.89 (m, 2H), 7.69 (dd, J ¼ 7.8, 4.7 Hz, 1H),
7.01e6.97 (m, 2H), 4.28e4.22 (m, 2H), 4.22e4.16 (m, 2H), 3.86 (s,
Diethyl (2-(4-nitrophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]qui-
3H), 1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d
177.02,
nolin-3-yl)phosphonate (10f): yellow solid, mp 212e214 ꢀC; 1H NMR
172.38,166.40 (d, J ¼ 24.6 Hz),162.38, 154.45, 151.33 (d, J ¼ 12.2 Hz),
(700 MHz, CDCl3)
1.7 Hz, 1H), 7.96e7.92 (m, 2H), 7.70 (dd, J ¼ 7.8, 4.7 Hz, 1H), 7.17 (dd,
d
9.05 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.53 (dd, J ¼ 7.8,
149.12, 134.74, 132.70 (d, J ¼ 9.7 Hz), 132.10, 128.83, 127.44, 120.17,
8

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
J ¼ 9.5, 7.7 Hz, 2H), 4.30e4.23 (m, 2H), 4.20 (ddq, J ¼ 10.1, 8.7, 7.1 Hz,
quinolin-3-yl)phosphonate (10i): brown solid, mp 170e172 ꢀC; 1H
2H), 1.24 (t, J ¼ 7.0 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d
176.61,
NMR (700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.7, 1.7 Hz, 1H), 8.54 (dd,
172.56,163.07 (d, J ¼ 24.0 Hz),154.89,152.33 (d, J ¼ 12.0 Hz),149.34,
148.98, 134.97, 133.64, 131.94 (d, J ¼ 9.2 Hz), 131.52, 128.75, 127.72,
123.38, 111.25 (d, J ¼ 212.4 Hz), 63.74 (d, J ¼ 6.3 Hz), 16.39 (d,
J ¼ 7.8, 1.7 Hz, 1H), 7.70 (dd, J ¼ 7.8, 4.7 Hz, 1H), 7.53e7.44 (m, 3H),
7.39e7.34 (m, 1H), 4.22 (ddq, J ¼ 10.1, 8.1, 7.1 Hz, 2H), 4.13 (ddq,
J ¼ 10.1, 8.9, 7.1 Hz, 2H), 1.15 (t, J ¼ 6.8 Hz, 6H). 13C NMR (176 MHz,
J ¼ 6.6 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 457.0801, observed 457.0803.
d
5.88. HRMS m/z, calcd
CDCl3)
d
176.95, 172.57, 162.97 (d, J ¼ 22.3 Hz), 154.64, 152.20 (d,
J ¼ 12.1 Hz), 148.90, 134.88, 134.72, 132.75, 132.27, 131.23 (d,
J ¼ 10.2 Hz), 129.68, 128.85, 128.22, 127.55, 126.48, 112.41 (d,
J ¼ 215.7 Hz), 63.30 (d, J ¼ 6.1 Hz), 16.20 (d, J ¼ 6.9 Hz). 31P NMR
Diethyl (2-(4-fluorophenyl)-4,9-dioxo-4,9-dihydrofuro[3,2-g]qui-
nolin-3-yl)phosphonate (9g): yellow solid, mp 148e150 ꢀC; 1H NMR
(700 MHz, CDCl3)
d
9.04 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.54 (dd, J ¼ 7.9,
(283 MHz, CDCl3)
observed 446.0563.
d
5.83. HRMS m/z, calcd [MþH]þ 446.0560,
1.7 Hz, 1H), 7.94e7.89 (m, 2H), 7.71 (dd, J ¼ 7.9, 4.6 Hz, 1H),
7.20e7.14 (m, 2H), 4.22 (ddq, J ¼ 10.1, 8.1, 7.2 Hz, 2H), 4.14 (ddq,
J ¼ 10.1, 8.7, 7.1 Hz, 2H), 1.23 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz,
4.3. Biological tests
CDCl3)
d
177.65, 171.58, 164.99 (d, J ¼ 23.9 Hz), 164.71 (d,
J ¼ 253.7 Hz), 154.52, 152.63 (d, J ¼ 12.1 Hz), 147.81, 135.49, 132.69
(d, J ¼ 8.8 Hz), 131.74 (d, J ¼ 10.0 Hz), 130.57, 127.80, 123.96 (d,
J ¼ 3.6 Hz), 115.60 (d, J ¼ 22.2 Hz), 108.13 (d, J ¼ 213.1 Hz), 63.20 (d,
4.3.1. Cell cultures and sample preparations
HL-60 and MCF-7 cell lines were purchased from the European
Collection of Cell Cultures (ECACC). Leukemia cells were cultured in
RPMI 1640 plus GlutaMax I medium (Invitrogen, Grand Island, NY,
USA), supplemented with 10% heat-inactivated FBS (Biological In-
J ¼ 5.8 Hz), 16.26 (d, J ¼ 6.8 Hz). 31P NMR (283 MHz, CDCl3)
HRMS m/z, calcd [MþH]þ 430.0856, observed 430.0865.
d 7.27.
Diethyl (2-(4-fluorophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]qui-
nolin-3-yl)phosphonate (10g): yellow solid, mp 130e132 ꢀC; 1H
dustries, Beit-Haemek, Israel) and antibiotics (100 mg/mL strepto-
mycin and 100 U/mL penicillin) (Sigma-Aldrich, St. Louis, MO, USA).
MCF-7 cells were cultured in Minimum Essential Medium Eagle
(MEME, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (Biological Industries,
Beit-Haemek, Israel), 2 mM glutamine, Men Non-essential amino
NMR (700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.6, 1.8 Hz, 1H), 8.53 (dd,
J ¼ 7.8, 1.8 Hz, 1H), 7.96e7.92 (m, 2H), 7.70 (dd, J ¼ 7.8, 4.6 Hz, 1H),
7.20e7.15 (m, 2H), 4.30e4.23 (m, 2H), 4.20 (ddq, J ¼ 10.1, 8.7, 7.1 Hz,
2H), 1.24 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 176.87,
172.46, 165.07 (d, J ¼ 24.2 Hz), 164.71 (d, J ¼ 253.6 Hz), 154.60,
151.65 (d, J ¼ 12.2 Hz), 149.01, 134.81, 132.67 (d, J ¼ 8.9 Hz), 132.29
(d, J ¼ 9.6 Hz), 128.74, 127.54, 124.03 (d, J ¼ 3.3 Hz), 115.61 (d,
J ¼ 22.2 Hz), 108.70 (d, J ¼ 214.2 Hz), 63.43 (d, J ¼ 6.1 Hz), 16.31 (d,
acid solution and antibiotics (100 mg/mL streptomycin and 100 U/
mL penicillin), all from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO,
USA). HUVEC and MCF-10 A were purchased from the American
Type Culture Collection (ATCC). HUVEC cells were cultured using
EGM-2 Endothelial Medium BulletKit, both purchased from Lonza
(Lonza, Walkersville, MD, USA). For MCF-10 A cells MEGM Mam-
mary Epithelial BulletKit was used. Cells were maintained at 37 ꢀC
in a 5% CO2 atmosphere and grown until 80% confluent.
For biological experiments the compounds were dissolved in
DMSO (Sigma-Aldrich, Louis, MO, USA) and further diluted in cul-
ture medium to obtain less than 0.1% DMSO concentration. In each
experiment controls without and with DMSO were performed.
DMSO in 0.1% concentration had no effects on the observed
parameters.
J ¼ 6.5 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 430.0856, observed 430.0860.
d 6.97. HRMS m/z, calcd
Diethyl (2-(4-chlorophenyl)-4,9-dioxo-4,9-dihydrofuro[3,2-g]qui-
nolin-3-yl)phosphonate (9h): yellow solid, mp 94e96 ꢀC; 1H NMR
(700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.55 (dd, J ¼ 7.9,
1.7 Hz, 1H), 7.90e7.85 (m, 2H), 7.72 (dd, J ¼ 7.9, 4.6 Hz, 1H),
7.49e7.45 (m, 2H), 4.28e4.20 (m, 2H), 4.16 (ddq, J ¼ 10.1, 8.8, 7.1 Hz,
2H), 1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 177.63,
171.60, 164.82 (d, J ¼ 23.6 Hz), 154.57, 152.77 (d, J ¼ 12.3 Hz), 147.86,
138.08, 135.53, 131.74 (d, J ¼ 10.1 Hz), 131.70, 130.62, 128.67, 127.82,
126.23, 108.64 (d, J ¼ 212.9 Hz), 63.28 (d, J ¼ 5.8 Hz), 16.30 (d,
J ¼ 6.5 Hz). 31P NMR (283 MHz, CDCl3)
[MþH]þ 446.0560, observed 446.0567.
d
7.12. HRMS m/z, calcd
4.3.2. Metabolic activity by MTT assay
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium
bromide) assay was performed according to Mossman [39]. In brief,
exponentially growing cells (HL-60, MCF-7, HUVEC, MCF-10 A)
were seeded in 24-well plates at a density of 8 ꢁ 104/mL and left to
grow for 24 h. The cells treated with various concentrations of the
tested analogs were incubated for 24 h or 48 h. Then, MTT solution
Diethyl (2-(4-chlorophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]qui-
nolin-3-yl)phosphonate (10h): yellow solid, mp 144e146 ꢀC; 1H
NMR (700 MHz, CDCl3)
d
9.07 (dd, J ¼ 4.6, 1.7 Hz, 1H), 8.55 (dd,
J ¼ 7.8, 1.7 Hz, 1H), 7.92e7.88 (m, 2H), 7.71 (dd, J ¼ 7.8, 4.6 Hz, 1H),
7.49e7.45 (m, 2H), 4.28 (ddq, J ¼ 10.1, 8.0, 7.1 Hz, 2H), 4.22 (ddq,
J ¼ 10.1, 8.8, 7.1 Hz, 2H), 1.26 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz,
(100 mL; 5 mg/mL in PBS) was added to each well. Following 1.5 h of
CDCl3)
d
176.83, 172.49, 164.90 (d, J ¼ 24.2 Hz), 154.66, 151.79 (d,
incubation, the plates were centrifuged and the supernatant was
discarded. DMSO (1 mL) was added to each well to dissolve the
formazan crystals. The absorbance was measured at 560 nm using
FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices,
LLC,CA, USA) and compared with control (untreated cells). Assays
were performed twice in triplicate for each compound. The IC50
values were calculated from concentrationeresponse curves.
J ¼ 12.2 Hz), 149.07, 138.04, 134.85, 132.30 (d, J ¼ 9.5 Hz), 131.68,
128.79, 128.68, 127.56, 126.31, 109.22 (d, J ¼ 213.6 Hz), 63.50 (d,
J ¼ 6.0 Hz), 16.35 (d, J ¼ 6.4 Hz). 31P NMR (283 MHz, CDCl3)
HRMS m/z, calcd [MþH]þ 446.0560, observed 446.0566.
d 6.79.
Diethyl (2-(2-chlorophenyl)-4,9-dioxo-4,9-dihydrofuro[3,2-g]qui-
nolin-3-yl)phosphonate (9i): brown solid, mp 140e142 ꢀC; 1H NMR
(700 MHz, CDCl3)
d
9.05 (dd, J ¼ 4.7, 1.7 Hz, 1H), 8.56 (dd, J ¼ 7.9,
1.7 Hz,1H), 7.72 (dd, J ¼ 7.9, 4.7 Hz,1H), 7.52e7.45 (m, 3H), 7.40e7.35
(m, 1H), 4.19 (ddq, J ¼ 10.1, 8.0, 7.1 Hz, 2H), 4.08 (ddq, J ¼ 10.1, 8.8,
4.3.3. Apoptosis, DNA damage and cell proliferation assay
The cell-cycle distribution, cell proliferation, apoptosis and DNA
damage were assessed using Apoptosis, DNA Damage and Cell
Proliferation Kit (BD Biosciences, San Jose, CA, USA). In brief,
2.0 ꢁ 105/mL HL-60 cells were seeded in 6-well plates and cultured
for 24 h. Then, the cells were treated with various concentrations of
9i and 9c. Cells incubated without tested compound were used as a
control. Following 24 h incubation, cells were treated with BrdU
7.1 Hz, 2H),1.14 (t, J ¼ 7.1 Hz, 6H). 13C NMR (176 MHz, CDCl3)
d 177.77,
171.71, 162.92 (d, J ¼ 21.8 Hz), 154.59, 153.18 (d, J ¼ 12.0 Hz), 147.92,
135.48, 134.81, 132.65, 132.33, 130.66 (d, J ¼ 10.2 Hz), 130.48, 129.71,
128.22, 127.85, 126.50, 111.91 (d, J ¼ 215.3 Hz), 63.11 (d, J ¼ 6.1 Hz),
16.17 (d, J ¼ 6.9 Hz). 31P NMR (283 MHz, CDCl3)
calcd [MþH]þ 446.0560, observed 446.0567.
d 6.16. HRMS m/z,
Diethyl
(2-(2-chlorophenyl)-4,9-dioxo-4,9-dihydrofuro[2,3-g]
solution (10 mM final concentration)for 8 h and collected by
9

J. Modranka, J. Drogosz-Stachowicz, A. Pietrzak et al.
European Journal of Medicinal Chemistry 219 (2021) 113429
centrifugation. Then, the cells were fixed, permeabilized and
measured at 440 nm for 5 min with 20 s interval and shaking before
and between readings using FlexStation 3 Multi-Mode Microplate
Reader (Molecular Devices, LLC,CA, USA). NQO1 activity is
expressed as the change in absorbance/min/amount of sample
loaded into the well.
incubated with DNase (300 m
g/mL in PBS) for 1 h at 37 ꢀC. After-
wards, the cells were stained with Anti-BrdU, Anti-H2AX (pS139)
and Anti-Cleaved PARP (Asp214) fluorescent antibodies for 20 min,
in the dark at room temperature. For cell cycle analysis, total DNA
was stained with DAPI solution (1 mg/mL in staining buffer). Cells
were analyzed by flow cytometry using CytoFLEX (Beckman
Coulter, Inc). The data was analyzed using Kaluza Analysis Software
v2 (Beckman Coulter, Inc).
4.3.8. Statistical analysis
Data are expressed as mean SEM. Assays were performed in
triplicate. Statistical analyses were performed using Prism 6.0
(GraphPad Software Inc., San Diego, CA, USA). Statistical signifi-
cance was assessed using Student’s t-test (for comparisons of two
groups) or one-way ANOVA followed by a post-hoc multiple com-
parison Student-Newman-Keuls test (for comparisons of three or
more groups).*p < 0.05, **p < 0.01, and ***p < 0.001 were
considered significant.
4.3.4. Apoptosis detection by Annexin V and Propidium iodide
staining
Induction of apoptosis was determined using FITC Annexin V
Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA).
Briefly, 2.0 ꢁ 105/mL HL-60 cells were plated in 6-well plates,
cultured overnight and treated with various concentrations of 9i
and 9c for 24 h or untreated for control. The cells were then
collected by centrifugation, washed with PBS, resuspended in 1x
binding buffer and stained with FITC Annexin V and Propidium
Iodide at room temperature for 15 min in the dark. The percentage
of apoptotic cells was assessed by flow cytometry using CytoFLEX
(Beckman Coulter, Inc., Brea, CA, USA). Data analysis was performed
using Kaluza Analysis Software v2 (Beckman Coulter, Inc., Indian-
apolis, IN, USA).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This study was supported financially by the grant from the
Medical University of Lodz No. 503/1-156-02/503-11-001-19-00
and from Lodz University of Technology No. 501-3-32-1-2.
4.3.5. ROS detection
The CellROX Oxidative Stress Green Reagent (LifeTechnologies,
Carlsbad, CA, USA) was used to detect changes of ROS. HL-60 cells
were plated in a 24-well plate, cultured overnight and then treated
with IC50 concentrations of 9i and 9c for 24 h. Afterwards, the
CellROX Green Reagent was added to each well and incubated for
30 min at 37 ꢀC. The cells were then washed with PBS and analyzed
by flow cytometry using CytoFLEX (Beckman Coulter, Inc). Data
analysis was performed using Kaluza Analysis Software v2 (Beck-
man Coulter, Inc).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113429.
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