European Journal of Medicinal Chemistry 120 (2016) 51e63

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Anticancer properties of new synthetic hybrid molecules combining

naphtho[2,3-b]furan-4,9-dione or benzo[f]indole-4,9-dione motif
with phosphonate subunit

ski c, Dorota Pomorska a,
Katarzyna Gach a, 1, Jakub Modranka b, 1, Jacek Szyman
Urszula Krajewska , Marek Mirowski , Tomasz Janecki b, **, Anna Janecka a, *
d d

a
Department of Biomolecular Chemistry, Faculty of Medicine, Medicinal University of Lodz, Mazowiecka 6/8, 92-215, Lodz, Poland
b
Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Lodz, Poland
c
Central Scientific Laboratory, Division of Public Health, Faculty of Health Sciences, Medical University of Lodz, Mazowiecka 6/8, 92-215, Lodz, Poland
d
Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz, Muszynskiego 1, 90-151, Lodz, Poland

a r t i c l e i n f o a b s t r a c t

Article history: In this paper we report an efficient and general synthesis of substituted 3-diethoxyphosphorylnaphtho
Received 13 February 2016 [2,3-b]furan-4,9-diones and 3-diethoxyphosphorylbenzo [f]indole-4,9-diones which integrate the nat-
Received in revised form ural 1,4-naphtalenedione scaffold, present in several anticancer agents with the phosphonate moiety.
26 April 2016
The cytotoxicity of such hybrid molecules was tested against two leukemia cell lines, HL-60 and NALM-6
Accepted 1 May 2016
Available online 3 May 2016
and against a breast adenocarcinoma MCF-7 cell line. Selected compounds were also tested on normal
human cells: HUVEC and MCF-10A. In general, naphthofuran-4,9-diones showed much higher cytotoxic
activity (IC50 values below 10 mM) than benzoindole-4,9-diones. The most promising 2-(2-chlorophenyl)-
Keywords:
Naphthofurandiones
3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-dione, with the highest cytotoxic activity in the MTT
Phosphonate moiety test, was chosen for further evaluation of its anticancer potential. This compound, tested on HL-60 and
Hybrid molecules MCF-7 cells inhibited cell proliferation, generated DNA damage and induced apoptosis. The suggested
Cytotoxicity mechanism of its cytotoxic activity was the generation of intracellular reactive oxygen species and the
Apoptosis induction of mitochondrial membrane potential dissipation.
© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction isolated from Streptomyces peucetius and its 2-hydroxyacetyl

The 1,4-naphtalenedione scaffold, usually ortho-condensed with
one or two planar rings is a well-known pharmacophoric unit
present in many natural and synthetic compounds displaying
diverse biological activities. Natural 1,4-naphthoquinones such as
plumbagin 1 or lapachol 2 (Fig. 1) had been investigated during the
past years, mainly due to their antibacterial, antifungal, trypano-
cidal and anticancer activities [1e3]. Many synthetic derivatives of
such compounds were obtained and several of them turned out to
be highly cytotoxic against various cancer cell lines [4e8]. Well
known examples of 1,4-naphtalenediones are daunorubicin [9] 3
* Corresponding author.
** Corresponding author.
E-mail addresses: tomasz.janecki@p.lodz.pl (T. Janecki), anna.janecka@umed.
lodz.pl (A. Janecka).
1
K.G. and J.M. contributed equally.
analog, doxorubicin [10] 4 (Fig. 1), both containing a tetracene-
dione framework and both used as drugs in cancer chemotherapy
for the treatment of various human neoplastic diseases including
leukaemias, lymphomas, sarcomas, carcinomas and breast cancers
[11].
The mechanism of antitumor activity of quinones has been
extensively investigated. Due to their structural properties, qui-
nones participate in multiple biological oxidative processes. The
fundamental feature of quinones is their ability to act as oxidizing
or dehydrogenating agents, the driving force of this being the for-
mation of a fully aromatic system [12]. The two major mechanisms
of quinone cytotoxicity are the stimulation of oxidative stress and/
or alkylation of cellular nucleophiles, which encompasses a large
range of biomolecules [7,12e14]. Quinones undergo either one- or
two-electron reduction, leading to the formation of reactive oxygen
species (ROS), including superoxide, hydrogen peroxide, and ulti-
mately the hydroxyl radicals. The production of ROS can cause se-
vere oxidative stress within cells through the formation of oxidized

http://dx.doi.org/10.1016/j.ejmech.2016.05.002
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
52 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
Fig. 1. Structures of Plumbagin, Lapachol, Daunorubicin, Doxorubicin and compounds containing naphtho [2,3-b]furan-4,9-dione or benzoindole-4,9-dione framework.
cellular macromolecules, including lipids, proteins and DNA, lead-
ing to cell damage and apoptosis. Alternatively, quinones are
Michael-type acceptors, and cellular damage can occur through
alkylation of crucial cellular proteins and/or DNA [14]. Generating
oxidative stress and cellular damage results in the inhibition of cell
proliferation, cell cycle arrest and apoptosis [2,4,14,15]. ROS are
important participants in the regulation of normal cellular pro-
cesses, such as gene expression, metabolism, cell differentiation,
proliferation, and apoptosis. However, at higher concentrations,
ROS become toxic and induce cell death by various signaling
pathways [15]. Compounds that are able to modulate the redox
balance in cancer cells are potential candidates for the develop-
ment of anticancer drugs. Therefore, various chemical modifica-
tions of compounds containing the 1,4-naphtalenedione scaffold
are sought in order to obtain analogs with enhanced bioactivity and
reduced toxicity, thereby maximizing their therapeutic potential.
Interesting groups of such analogs are compounds with naphtho
[2,3-b]furan-4,9-dione or benzo [f]indole-4,9-dione framework
which often display cytotoxic and other biological activities. For
example, maturone 5 (Fig. 1) was isolated from a Mexican plant,
Cacalia decomposita, the root extract of which has been used for the
treatment of diabetes and other diseases [16e19]. Other natural
compounds with the naphthofuranodione framework are avice-
quinones 6 (Fig. 1). These compounds were isolated from the stem
bark of Avicennia alba or root and stem extracts of Mendonica
cowanii and display cytotoxic activity [20,21]. Also several synthetic
naphtho [2,3-b]furan-4,9-diones containing alkyl substituents in
position 2 and/or EWG groups, such as carboxyl, alkoxycarbonyl or
acyl, in position 3 were prepared and some of these analogs
possessed significant selectivity in antitumor activity compared to
normal cells [22e24].
Additionally, 2-substituted naphtho [2,3-b]furan-4,9-diones
suppressed hyperproliferation of human keratinocytes and could
be potentially useful in the treatment of psoriasis, a common,
immune-mediated inflammatory and scaling skin disease [25].
Likewise, compounds with the benzoindole-4,9-dione frame-
work were found in nature and show promising biological activity.
Thus, 3-methyl-1H-benzo [f]indole-4,9-dione 7a [26] and Utha-
mycin B 7b [27] (Fig. 1) were isolated from Goniothalamus tapis Miq.
and Streptomyces albus, respectively. Furthermore, their synthetic
analogs of general structure 8 (Fig. 1), containing ester functionality
in position 3 were extensively tested for their cytotoxicity against
numerous human cancer cell lines and it turned out that several of
these analogs are very potent anticancer agents [28e32]. For
example, 3-ethoxycarbonylbenzoindole-4,9-dione 8a (R1 ¼ Et,
R2 ¼ cyclohexyl) exhibited greater cytotoxic activity against a wide
variety of human tumor cell lines than etoposite [29] and ben-
zoindole-4,9-dione 8b (R1 ¼ Et, R2 ¼ 3,4-dimethylphenyl) displayed
selective cytotoxicity against lung cancer cells which was compa-
rable to doxorubicin [30].
Continuing our research on biologically important phosphory-
lated oxa- and aza-heterocycles we decided to synthesize com-
pounds with naphtho [2,3-b]furan-4,9-dione and benzo [f]indole-
4,9-dione skeletons containing phosphoryl functionality instead of
a carboxyl group in position 3. It is well known, that the phosphoryl
group can mimic the tetrahedral intermediates formed in enzy-
matic reactions involved in the carboxyl group metabolism
[33e35]. This rationale has resulted in the design of some
 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
important drugs, such as N-phosphonoacetyl-L-aspartic acid (PALA)
with anticancer activity or fosinopril which is a clinically useful
antihypertensive agent [36]. We reasoned that such replacement
can produce biologically active analogs which might inhibit key
enzymes or interact with receptors which normally bind the cor-
responding original compounds. Also, it is well known that intro-
ducing organophosphorus functionalities into organic molecules
can increase their biological activity [33,34].
Accordingly, in this paper we report on efficient and general
synthesis of substituted 3-diethoxyphosphorylnaphtho [2,3-b]
furan-4,9-diones 13 and 3-diethoxyphosphorylbenzo [f]indole-4,9-
diones 18 which integrate the natural 1,4-naphtalenedione scaffold
and the phosphonate moiety. Such hybrid molecules could poten-
tially improve their precursors’ cytotoxic properties and/or help to
overcome their limitations [37,38].
2. Results and discussion
2.1. Chemistry
The target compounds were synthesized applying modified
methodology originally reported for the synthesis of 3-
alkoxycarbonylnaphtho [2,3-b]furan-4,9-diones [39] or 3-
alkoxycarbonylbenzo [f]indole-4,9-diones [29], i.e. performing the
reaction of 2,3-dichloro-1,4-naphthoquinone with b-ketophosph-
onates or diethoxyphosphorylacetate which were used instead of
b-ketoesters or malonate, respectively.
A series of 2-substituted 3-diethoxyphosphorylnaphtho [2,3-b]
furan-4,9-diones 13a-f were synthesized as shown in Scheme 1.
Starting diethyl acylmethylphosphonates 9a,b are commercially
available and phosphonates 9c-f were synthesized applying
modified literature procedure [40], from diethyl methyl-
phosphonate and corresponding ester in the presence of LDA.
Phosphonates 9aef were next used in the reaction with commer-
cially available 2,3-dichloro-1,4-naphthoquinone 10 and after
standard work-up and purification by column chromatography
expected 3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-diones
13aef were obtained. Reactions with aryloylmethylphosphonates
9bef proceeded effectively and final 13bef were obtained in
satisfactory yields (Table 1). Only the reaction with acetylmethyl-
phosphonate 9a was much less efficient and a complex mixture of
products was formed. Fortunately, it was possible to isolate the
desired naphthofurandione 13a in low, 10% yield. Structures of the
obtained naphthofurandiones 13aef were unambiguously
Scheme 1. Synthesis of 3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-diones 13a-f.
53
Table 1
Yields of the obtained 13a-f.
Compound 13 R Yielda [%]
a Me 10
b Ph 61
c 55
d 63
e 76
f 88
a
Yield of pure, isolated products based on 9.
confirmed by MS and spectroscopic methods. Formation of 13 can
be easily rationalized by assuming the Michael addition of phos-
phonate 9 to naphthoquinone 10, followed by elimination of the
chlorine anion and formation of the substitution product 11 which
is in equilibrium with the enol form 12. Intramolecular substitution
of the second chlorine ion in 12, via addition/elimination mecha-
nism, gave the target furandiones 13.
We also decided to prepare the monoester derivative 14 which
better mimics tetrahedral intermediates involved in carboxyl group
metabolism in comparison to the diester [36]. In this respect,
naphthofurandione 13c was chemoselectively hydrolized to 3-
hydroxyethoxyphosphoryl-2-(2-chlorophenyl)naphtho [2,3-b]
furan-4,9-dione 14 in 93% yield by the treatment with lithium
bromide followed by hydrolysis with 1 M hydrochloric acid
(Scheme 2).
In turn, 3-diethoxyphosphorylbenzo [f]indole-4,9-diones 18aee
were synthesized in the reaction sequence shown in Scheme 3.
Reaction of 2,3-dichloro-1,4-naphthoquinone 10 with ethyl
54 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63

2.2. Biological evaluation

2.2.1. In vitro cytotoxicity of new compounds against cancer cell

lines
The obtained 3-diethoxyphosphorylnaphtho [2,3-b]furan-4,9-
diones 13aef and 3-hydroxyethoxyphosphoryl-2-(2-
chlorophenyl)naphtho [2,3-b]furan-4,9-dione 14 as well as 3-
diethoxyphosphorylbenzo [f]indole-4,9-diones 18aee were tested
in vitro against three human cancer cell lines: leukemia NALM-6
and HL-60, as well as breast cancer MCF-7, and the results are
Scheme 2. Synthesis of 3-hydroxyethoxyphosphoryl-2-(2-chlorophenyl)naphtho [2,3-
shown in Table 3. Doxorubicin was used as the positive control. In
b]furan-4,9-dione 14.

Scheme 3. Synthesis of 3-diethoxyphosphorylbenzo [f]indole-4,9-diones 18a-e.

diethoxyphosphorylacetate (15) in the presence of NaH furnished
2-chloro-3-(2-ethoxy-1-diethoxyphosphoryl-2-oxoethyl)naphtha-
lene-1,4-dione 16 in 96% yield, via an addition/elimination mech-
anism. This compound treated with various aliphatic amines gave,
after standard work up and purification by column chromatog-
raphy, target diethoxyphosphorylbenzoindole-4,9-diones 18aee in
the yields given in Table 2. Structures of the obtained benzoindo-
lediones 18aee were unambiguously confirmed by MS and spec-
troscopic methods. Clearly, in the first step of the reaction, amine
displaces the chlorine ion to form 17 which undergoes intra-
molecular nucleophilic substitution on acyl group.
Table 2
Yields of the obtained 18a-e.
general, naphthofuran-4,9-diones, except for the monoester de-
rivative 14, showed high cytotoxic activity with the IC50 values
below 10 mM (after 48 h incubation), in all three cancer cell lines. On
the other hand benzoindole-4,9-diones were moderately active,
with IC50 values of 20e60 mM. It is worth noticing that cytotoxicity
of naphthofurandiones was about 2e3-fold higher in MCF-7 cells
than in the tested leukemia cell lines. Selected naphthofurandiones
were also tested on human mammary gland/breast cell line
(MCF10A) and human umbilical vein endothelial cells (HUVEC) to
evaluate their influence on normal cells. The toxicity of the tested
compounds against leukemia and HUVEC cells was similar. How-
ever, these compounds were 4-fold less toxic for MCF-10A as
compared with MCF-7 cells. Compound 13c was selected for more
detailed evaluation of its anticancer potential against HL-60 and
MCF-7 cells. The MTT assay was performed for 13c after 24 h in-
cubation. The IC50 values were 7.27 ± 0.08 and 3.40 ± 0.15 for HL-60
and MCF-7 cells, respectively, and these concentrations were used
in all further experiments.

Compound 18 R Yielda [%]

a Me
b n-Pr
c c-Pr
d n-Hex
e 52
a
Yield of pure, isolated products based on 16.
2.2.2. Inhibition of cell proliferation, induction of DNA damage and
94 apoptosis
88
81
The ability of 13c to inhibit cell proliferation, generate DNA
98 damage and induce apoptotic cell death was examined by flow
cytometry using the ‘Apoptosis, DNA Damage, and Cell Proliferation
Kit’ (BD Bioscience). After 24 h treatment with compound 13c at
IC50 concentration, HL-60 and MCF-7 were incubated with bro-
modeoxyuridine (BrdU) for additional 8 h. Cells were simulta-
neously stained with fluorochrome-labeled anti-BrdU, H2AX
 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63 55

Table 3
In vitro cytotoxic activity of naphthofurandiones 13a-f, monoacid 14 and benzo [f]indolediones 18a-e tested on three cancer cell lines and two normal cell lines (HUVEC and
MCF-10A).

Compound IC50a (mM)

HL-60 NALM-6 MCF-7 HUVEC MCF-10A

13a 8.13 ± 0.16 5.85 ± 0.55 5.70 ± 0.21

13b 6.01 ± 0.18 6.06 ± 0.40 2.40 ± 0.30
13c 6.35 ± 0.46 5.07 ± 0.58 2.34 ± 0.18 3.73 ± 0.18 10.03 ± 0.43
13d 5.44 ± 0.25 4.82 ± 0.26 2.90 ± 0.12 4.06 ± 0.25 11.31 ± 0.71
13e 8.66 ± 0.88 6.65 ± 0.35 2.55 ± 0.05
13f 5.94 ± 0.26 5.32 ± 0.53 2.85 ± 0.85 4.85 ± 0.49 9.79 ± 0.48
14 486.72 ± 27.7 478.64 ± 45.68 150 ± 10.08
18a 59.66 ± 4.23 61.74 ± 4.7 50 ± 6.01
18b 56.20 ± 3.84 60.03 ± 2.02 43.93 ± 5.18
18c 65.05 ± 2.21 51.30 ± 3.99 102.0 ± 6.98
18d 30.74 ± 3.55 44.3 ± 3.2 19.5 ± 4.4
18e 42.35 ± 4.47 60.07 ± 2.16 65.0 ± 7.2
Doxorubicin 0.11 ± 0.01 e 0.89 ± 0.12 1.4 ± 0.02 1.2 ± 0.05
a
Compound concentration required to inhibit metabolic activity by 50%. Data are expressed as the mean ± SD from the concentrationeresponse curves of at least three
experiments.

(pS139), and cleaved PARP (Asp214) antibodies. BrdU incorporation
into DNA was used as an index of cell proliferation. The induction of
apoptosis was assessed using PE Anti-Cleaved PARP (Asp214) con-
jugated antibodies recognizing 89 kDa-cleaved fragment of PARP
that can serve as a marker of cellular apoptosis. The DNA damage
was evaluated using Alexa Fluor ® 647 Anti-H2AX (pS139) anti-
bodies, directed against the phosphorylated form of human H2AX
protein at the pS139 residue that is considered a biomarker for DNA
double-strand breaks (DSB).
The obtained results demonstrated that 13c significantly
inhibited cell proliferation in both tested cell lines. In MCF-7 cells,
inhibition of cell proliferation was observed in 34%, whereas in HL-
60 in about 91% of cell population (Fig. 2A). Similar tendency was
observed on the generation of DNA damage. As shown in Fig. 2B,
13c induced DNA damage in about 40.7 ± 1.65% and 11.03 ± 0.5% of
HL-60 and MCF-7 cell populations, respectively. The tested com-
pound increased also the number of cleaved PARP positive
(apoptotic) cells in both tested cell lines. In MCF-7 cells only very
slight effect on apoptosis induction was observed. On the contrary,
in HL-60 cells 13c enhanced the number of cleaved PARP positive
cells up to 82.8 ± 5.47%, compared with control 1.2 ± 0.2% (Fig. 2C).
The representative multiparameter flow cytometry analysis of the
effects of 13c on cell proliferation, induction of DNA damage and
apoptosis is shown in Fig. 3.
2.2.3. Induction of apoptosis
Pro-apoptotic activity of 13c was also tested using APC-Annexin
V and PI staining. The results of this experiment are shown in Fig. 4.
Compound 13c did not significantly influence the number of early
apoptotic cells in the tested cell lines but increased the number of
late apoptotic cells (27.8 ± 1.3% and 19 ± 1.5% compared with
control 2.7 ± 0.2% and 6.1 ± 1.0%, in HL-60 and MCF-7 cells,
respectively).
2.2.4. ROS generation
One of the possible mechanisms of the cytotoxic activity of
compounds with 1,4-naphtalenedione scaffold can be associated
with generation of ROS in cancer cells. To test the ability of 13c to
induce ROS generation, HL-60 and MCF-7 cells treated for 24 h with
the tested compound at IC50 concentration, were incubated with
CellROX® Green Oxidative Stress Reagent (Molecular Probes, Life
Technologies). As shown in Fig. 5 13c increased intracellular ROS
levels about 10 times in both tested cell lines, as compared with
control. Interestingly, the effect induced by this compound was
much stronger than that of redox-cycling agent e menadione, used
as a positive control.
2.2.5. Dissipation of mitochondrial membrane potential
Mitochondrial dysfunction has been shown to participate in the

Fig. 2. Inhibition of cell proliferation (BrdU test) (A), induction of DNA damage (H2AX test) (B) and induction of apoptosis (PAPR test) (C) by 13c in MCF-7 cells. After 24 h incubation
with tested compounds, BrdU was added and cells were incubated for additional 8 h. The cells were stained with PerCP-Cy™5.5 Mouse Anti-BrdU, Alexa Fluor® 647 Mouse Anti-
H2AX (pS139), PE Mouse Anti-Cleaved PARP (Asp214) antibodies for 20 min at room temperature and analyzed by flow cytometry. Data represent mean ± SEM of three independent
experiments in triplicate. Statistical significance was assessed by the Student t-test. **p < 0.01, ***p < 0.001 was considered as significantly different from control.
56 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
induction of apoptosis. Opening of the mitochondrial permeability
transition pore induces depolarization of the transmembrane po-
tential (DJm). The DJm disruption is a critical step for the intrinsic
pathway of apoptosis. To investigate the changes in DJm, HL-60
and MCF-7 cells treated for 24 h with 13c at IC50 concentrations,
were stained with JC-10. The majority of untreated cells (control)
were within the population with high DJm. A mitochondrial un-
coupler e carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
(FCCP) was used as a positive control and caused dissipation of
DJm in 67.1 ± 3.5% and 92.5 ± 2.0% of HL-60 and MCF-7 cells,
respectively. Compound 13c caused similar effects in both cell lines.
In HL-60, 13c decreased DJm in about 44%, while in MCF-7 in 50%
of cell population (Fig. 6).
2.2.6. Cell cycle arrest
Cell cycle distribution in HL-60 and MCF-7 cells was studied
after 24 h exposure to 13c at IC50 concentration. DNA staining with
DAPI (1mg/mL) was performed. In HL-60, 13c cased cell cycle arrest
in S phase, whereas in MCF-7 in G0/G1 phase (Fig. 7). A decrease in
G2/M was observed in both tested cell lines.
3. Conclusions
We developed novel and efficient synthesis of substituted 3-
diethoxyphosphorylnaphtho [2,3-b]furan-4,9-diones 13 and 3-
diethoxyphosphorylbenzo [f]indole-4,9-diones 18 which integrate
the natural 1,4-naphtalenedione scaffold and the phosphonate
moiety. The rational to obtain such hybrid molecules was to search
for compounds with potentially improved cytotoxic properties.
Screening of the synthesized 3-diethoxyphosphorylnaphtho
[2,3-b]furan-4,9-diones 13aef and 3-hydroxyethoxyphosphoryl-
2-(2-chlorophenyl)naphtho [2,3-b]furan-4,9-dione 14, as well as 3-
diethoxyphosphorylbenzo [f]indole-4,9-diones 18aee was per-
formed in vitro against three human cancer cell lines: leukemia HL-
60 and NALM-6 and breast cancer MCF-7. Only naphtho [2,3-b]
furan-4,9-diones 13aef showed high cytotoxic activity with the
IC50 values below 10 mM. In the naphthodione moiety, the
replacement of the ortho-fused 3-diethoxyphosphoryl-2-
hydroxypirol by 3-diethoxyphosphoryl-2-arylfuran ring increased
the IC50 value about 10 fold. The activity of the most potent analog
13c was completely abolished by the hydrolysis of one ester group,
indicating that both ester groups were important for anticancer
activity of the hybrids. The toxicity of the tested compounds against
leukemia and HUVEC cells was similar. However, these compounds
were 4-fold less toxic to MCF-10A as compared with MCF-7 cells.
On the example of compound 13c we have shown that naphtho
[2,3-b]furan-4,9-diones display a broad range of anticancer activ-
ities including inhibition of cell proliferation, generation of DNA
damage and induction of apoptosis in two cancer cell lines. We
have shown that the underling mechanism of the anticancer ac-
tivity of this compound was the generation of intracellular ROS and
imbalance between ROS production and a cell's ability to readily
detoxify the reactive intermediates or to repair the resulting
damage. Excess of ROS may induce lipid and protein oxidative
modifications, DNA damage, and disruption of mitochondrial
membrane potential, resulting in cell damage or apoptosis [42].
It is well documented that most cancer cells contain higher
levels of endogenous ROS comparing with their non-cancerous
counterparts, caused largely by the products of their highly
Fig. 3. Multiparameter analysis of cell proliferation, DNA damage and apoptosis in HL-60 and MCF-7 cells treated for 24 h with 13c, at IC50 concentration. Panel A: BrdU vs DAPI
staining profile. BrdU positive cells (proliferating cells) are in the inside frame. Panel B: H2AX (pS139) vs cleaved PARP (Asp214) staining profile. Quadrants Q1þQ2 represent H2AX
(pS139) positive cells (cells with DNA damage), quadrants Q2þQ4 represent cleaved PARP (Asp214) positive cells (apoptotic cells), whereas quadrant Q3 represents H2AX (pS139)
and cleaved PARP (Asp214) negative cells.
57
metabolic nature. This biochemical difference increases cancer cell
susceptibility to oxidative stress-induced cell death and may be
exploited in the design of selective drugs [15,43]. Naphtho [2,3-b]
furan-4,9-diones are highly redox active molecules that are able to
modulate the redox balance in cancer cells. Therefore, naph-
thofurandiones increasing intracellular ROS levels over a critical
threshold may induce different signaling pathways leading to
apoptosis in cancer cells.
4. Experimental
4.1. Synthesis
4.1.1. General methods
NMR spectra were recorded on a Bruker DPX 250 at 250.13 MHz
for 1H, 62.9 MHz for 13C, and 101.3 MHz for 31P NMR using tetra-
methylsilane as internal and 85% H3PO4 as external standard. 31P
NMR spectra were recorded using broadband proton decoupling. IR
spectra were recorded on a Bruker Alpha ATR spectrophotometer.
Melting points were determined in open capillaries and are un-
corrected. Column chromatography was performed on Aldrich®
silica gel 60 (230e400 mesh). Thin-layer chromatography was
performed with precoated TLC sheets of silica gel 60 F254
(Aldrich®). HRMS spectra were performed on Waters Acquity UPLC/
Q-TOF LC-HRMS apparatus. Reagents and starting materials were
purchased from commercial vendors and used without further
purification. All organic solvents were dried over appropriate dry-
ing agents and distilled prior to use. Standard syringe techniques
were used for transferring dry solvents.
Diethyl (2-oxopropyl)phosphonate (9a), diethyl (2-oxo-2-
phenylethyl)phosphonate (9b) and 2,3-dichloro-1,4-
naphthoquinone (10) were purchased from SigmaeAldrich®.
4.1.2. General procedure for preparing compounds 13aef
A mixture of 2,3-dichloronaphthalene-1,4-dione (10) (1.14 g,
5.0 mmol) K2CO3 (1.73 g, 12.5 mmol) and corresponding diethyl
acylmethylphosphonate (9aef) (5.0 mmol) in MeCN (50 mL) was
stirred at 60  C for 24 h. The reaction mixture was poured into sat.
aq. NaCl solution (50 mL). Extraction with CHCl3 (3  50 mL), drying
(MgSO4) and evaporation of the solvent gave the crude product,
which was purified by column chromatography [petroleum ether-
eEtOAc (2:1)].
4.1.2.1. Diethyl (2-methyl-4,9-dioxo-4,9-dihydronaphtho [2,3-b]
furan-3-yl)phosphonate (13a). yellow solid, mp 148e150  C; 1H
NMR (250 MHz, CDCl3) d 8.25e8.12 (m, 2H, HeAr), 7.81e7.69 (m,
2H, HeAr), 4.39e4.13 (m, 4H, (CH3CH2O)2P(O)), 2.80 (d,
3
JHeP ¼ 2.1 Hz, 3H, CH3), 1.36 (t, 3JHeH ¼ 7.1 Hz, 6H,
(CH3CH2O)2P(O)); 13C NMR (63 MHz, CDCl3) d 178.79 (s, C(O)),
173.18(s, C(O)), 167.90 (d, 2JCeP ¼ 26.5 Hz, C-2), 151.79 (d,
2
JCeP ¼ 11.3 Hz, C-9a), 133.98 (s, CHeAr), 133.73 (s, CHeAr), 133.06
(s, CeAr), 131.54 (s, CeAr), 130.41 (d, 2JCeP ¼ 10.1 Hz, C-3a), 127.13
(s, CHeAr), 126.46 (s, CHeAr), 107.42 (d, 1JCeP ¼ 214.7 Hz, C-3),
62.75 (d, 2JCeP ¼ 5.8 Hz, (CH3CH2O)2P(O)), 16.27 (d, 3JCeP ¼ 6.6 Hz,
(CH3CH2O)2P(O)), 14.36 (s, CH3); 31P NMR (101 MHz, CDCl3) d 9.69;
IR n(cm1): 2984, 1685, 1360, 1287, 1235, 1020, 976; HRMS m/z,
calcd [MþH]þ 349.08355, observed 349.08377.
58 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63

Fig. 4. Induction of apoptosis in HL-60 (A) and MCF-7 (B) cells treated for 24 h with 13c, at IC50 concentration. The cells were stained with APC-conjugated Annexin V in a buffer
containing propidium iodide and analyzed by flow cytometry. For each group of cells, the percentage of surviving cells is shown in quadrant Q3. Quadrant Q4 indicates the
percentage of early apoptotic cells (Annexin V-positive cells). The percentage of late apoptotic cells is shown in Q2 (Annexin V- and PI-positive cells). Data represent mean ± SEM of
three independent experiments in triplicate. Statistical significance was assessed by the Student t-test. **p < 0.01, ***p < 0.001 was considered as significantly different from control.

13
C NMR (63 MHz, CDCl3) d 178.79 (s, C(O)), 173.36 (d,
3
JCeP ¼ 0.7 Hz, C(O)), 165.19 (d, 2JCeP ¼ 23.6 Hz, C-2), 152.19 (d,
2
JCeP ¼ 12.5 Hz, C-9a), 134.11 (s, CHeAr), 133.75 (s, CHeAr), 133.32
(s, CeAr), 131.80 (d, 2JCeP ¼ 10.3 Hz, C-3a), 131.63 (s, CeAr), 131.13 (s,
CeAr), 130.15 (s, CHeAr), 128.04 (s, CHeAr), 127.96 (s, CHeAr),
127.28 (s, CHeAr), 126.50 (s, CHeAr), 107.91 (d, 1JCeP ¼ 214.2 Hz, C-
3), 62.95 (d, 2JCeP ¼ 6.0 Hz, (CH3CH2O)2P(O)), 16.05 (d,
3
JCeP ¼ 6.8 Hz, (CH3CH2O)2P(O)). 31P NMR (101 MHz, CDCl3) d 7.18.
IR n(cm1): 2982, 1674, 1360, 1260, 1209, 1010, 936. HRMS m/z,
calcd [MþH]þ 411.09920, observed 411.09915.

4.1.2.3. Diethyl (2-(2-chlorophenyl)-4,9-dioxo-4,9-dihydronaphtho

Fig. 5. Effect of 13c on intracellular ROS generation in HL-60 and MCF-7 cells. Cells
were treated for 24 h with 13c at IC50 concentration and stained with 5 mM CellROX®
Green Reagent in a 37  C, 5% CO2 incubator for 30 min. Menadione (200 mM, 1 h in-
cubation) was used as a positive control. Data represent mean ± SEM of 3e4 inde-
pendent experiments in triplicate. Statistical significance was assessed using one-way
ANOVA and a post-hoc multiple comparison StudenteNewmaneKeuls test. **p < 0.01,
***p < 0.001 was considered as significantly different from control.
4.1.2.2. Diethyl (4,9-dioxo-2-phenyl-4,9-dihydronaphtho [2,3-b]
furan-3-yl)phosphonate (13b). orange oil; 1H NMR (250 MHz,
CDCl3) d 8.29e8.10 (m, 2H, HeAr), 7.89e7.82 (m, 2H, HeAr),
7.78e7.73 (m, 2H, HeAr), 7.56e7.43 (m, 3H, HeAr), 4.28e4.07 (m,
4H, (CH3CH2O)2P(O)), 1.19 (t, 3JHeH ¼ 7.0 Hz, 6H, (CH3CH2O)2P(O)).
[2,3-b]furan-3-yl)phosphonate (13c). yellow solid, mp 145e148  C;
1
H NMR (250 MHz, CDCl3) d 8.27e8.19 (m, 2H, HeAr), 7.81e7.74 (m,
2H, HeAr), 7.55e7.49 (m, 2H, HeAr), 7.49e7.43 (m, 1H, HeAr),
7.42e7.33 (m, 1H, HeAr), 4.30e4.01 (m, 4H, (CH3CH2O)2P(O)), 1.16
(t, 3JHeH ¼ 7.1 Hz, 6H, (CH3CH2O)2P(O)); 13C NMR (63 MHz, CDCl3)
d 178.65 (s, C(O)), 173.32 (d, 3JCeP ¼ 0.9 Hz, C(O)), 161.92 (d,
2
JCeP ¼ 22.5 Hz, C-2), 152.54 (d, 2JCeP ¼ 12.7 Hz, C-9a), 134.43 (d,
2
JCeP ¼ 0.9 Hz, C-3a), 134.09 (s, CCl), 133.74 (s, CHeAr), 133.00 (s,
CHeAr), 132.38 (s, CHeAr), 131.85 (s, CeAr), 131.53 (s, CeAr), 130.54
(s, CHeAr), 130.38 (s, CHeAr), 129.32 (s, CeAr), 128.20 (s, CHeAr),
127.16 (s, CHeAr), 126.49 (s, CHeAr), 126.16 (s, CHeAr), 111.40 (d,
1
JCeP ¼ 215.5 Hz, C-3), 62.70 (d, 2JCeP ¼ 6.1 Hz, (CH3CH2O)2P(O)),
15.88 (d, 3JCeP ¼ 7.3 Hz, (CH3CH2O)2P(O)); 31P NMR (101 MHz,
CDCl3) d 7.50; IR n(cm1): 2984, 1671, 1363, 1256, 1210, 1011, 940;
HRMS m/z, calcd [MþH]þ 445.06023, observed 445.06033.
 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63 59

Fig. 6. Effect of 13c on dissipation of mitochondrial membrane potential (DJm) in HL-60 (A) and MCF-7 (B) cells. Cells were treated for 24 h with 13c at IC50 concentration and
stained with JC-10 (5 mL/mL) for 45 min in 37  C, 5% CO2. Changes in the fluoresence were analyzed by flow cytometry. FCCP (30 mM) was used as a positive control. Data represent
mean ± SEM of three independent experiments in triplicate. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Stu-
denteNewmaneKeuls test. **p < 0.01, ***p < 0.001 was considered as significantly different from control.

4 .1. 2 . 4 . D i e t h y l ( 2 - ( 2 , 4 - d i c h l o r o p h e n y l ) - 4 , 9 - d i o x o - 4 , 9 -
dihydronaphtho [2,3-b]furan-3-yl)phosphonate (13d). yellow solid,
mp 122e124  C; 1H NMR (250 MHz, CDCl3) d 8.28e8.17 (m, 2H,
HeAr), 7.82e7.75 (m, 2H, HeAr), 7.54 (d, 4JHeH ¼ 1.9 Hz, 1H, HeAr),
7.46 (d, 3JHeH ¼ 8.3 Hz, 1H, HeAr), 7.37 (dd, 3JHeH ¼ 8.3 Hz,
4
JHeH ¼ 1.9 Hz, 1H, HeAr), 4.33e4.04 (m, 4H, (CH3CH2O)2P(O)), 1.21
(t, 3JHeH ¼ 7.1 Hz, 6H, (CH3CH2O)2P(O)). 13C NMR (63 MHz, CDCl3)
d 178.51 (s, C(O)), 173.25 (d, 3JCeP ¼ 0.7 Hz, C(O)), 160.98 (d,
2
JCeP ¼ 22.3 Hz, C-2), 152.69 (d, 2JCeP ¼ 12.2 Hz, C-9a), 137.44 (s,
CCl), 135.35 (d, 2JCeP ¼ 0.9 Hz, C-3a), 134.15 (s, CHeAr), 133.79 (s,
CHeAr), 133.19 (s, CCl), 132.96 (s, CeAr), 131.48 (s, CeAr), 130.36 (s,
CHeAr), 130.20 (s, CHeAr), 129.30 (s, CHeAr), 127.18 (s, CeAr),
126.74 (s, CHeAr), 126.63 (s, CHeAr), 126.52 (s, CHeAr), 111.83 (d,
1
JCeP ¼ 214.4 Hz, C-3), 62.79 (d, 2JCeP ¼ 6.1 Hz, (CH3CH2O)2P(O)),
15.97 (d, 3JCeP ¼ 6.7 Hz, (CH3CH2O)2P(O)). 31P NMR (101 MHz,
CDCl3) d 7.18. IR n(cm1): 2986, 1674, 1357, 1238, 1209, 939. HRMS
m/z, calcd [MþH]þ 479.02126, observed 479.02118.
4 .1. 2 . 5 . D i e t hyl ( 2 - ( 2 - ch l o r o p y r i d i n - 3 - yl ) - 4 , 9 - d i o x o - 4 , 9 -
dihydronaphtho [2,3-b]furan-3-yl)phosphonate (13e). yellow solid,
mp 204e206  C; 1H NMR (250 MHz, CDCl3) d 8.56 (dd,
3
JHeH ¼ 4.9 Hz, 4JHeH ¼ 1.9 Hz, 1H, HeAr), 8.28e8.19 (m, 2H, HeAr),
60 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
Fig. 7. Effect of 13c on cell cycle distribution in HL-60 and MCF-7 cells. Cells were
treated for 24 h with 13c at IC50 concentration. The percentage of cells at each stage of
the cycle was analyzed by flow cytometry, following DNA staining with DAPI and
compared with untreated cells regarded as control, C. The results are representative of
three independent experiments. Statistical significance was assessed by the Student t-
test. *p < 0.05, **p < 0.01 was considered as significantly different from control.
7.87 (dd, 3JHeH ¼ 7.6 Hz, 4JHeH ¼ 1.9 Hz, 1H, HeAr), 7.82e7.77 (m,
2H, HeAr), 7.39 (dd, 3JHeH ¼ 7.6 Hz, 3JHeH ¼ 4.9 Hz, 1H, HeAr),
4.30e4.09 (m, 4H, (CH3CH2O)2P(O)), 1.19 (t, 3JHeH ¼ 7.1 Hz, 6H,
(CH3CH2O)2P(O)). 13C NMR (63 MHz, CDCl3) d 178.69 (s, C(O)),
173.51 (d, 3JCeP ¼ 1.8 Hz, C(O)), 160.25 (d, 2JCeP ¼ 22.1 Hz, C-2),
153.07 (d, 2JCeP ¼ 12.1 Hz, C-9a), 151.44 (s, CHeAr), 150.97 (d,
3 4.37e4.09 (m, 6H, (CH3CH2O)2P(O), (CH3CH2O)C(O)), 1.29 (t, 3JH-
JCeP ¼ 1.1 Hz, CeAr), 141.33 (s, CCl), 134.45 (s, CHeAr), 134.08 (s,
CHeAr), 133.18 (s, CeAr), 131.73 (s, CeAr), 130.46 (d, 2JCeP ¼ 9.9 Hz,
C-3a), 127.46 (s, CHeAr), 126.82 (s, CHeAr), 125.44 (s, CeAr), 121.74
(s, CHeAr), 112.52 (d, 1JCeP ¼ 213.9 Hz, C-3), 63.10 (d, 2JCeP ¼ 6.0 Hz,
(CH3CH2O)2P(O)), 16.17 (d, 3JCeP ¼ 6.7 Hz, (CH3CH2O)2P(O)). 31P
NMR (101 MHz, CDCl3) d 6.72. IR n(cm1): 2983, 1676, 1383, 1261,
1214, 1025, 938. HRMS m/z, calcd [MþH]þ 446.05548, observed
446.05560.
4.1.2.6. Diethyl (2-(2,6-dichloro-5-fluoropyridin-3-yl)-4,9-dioxo-4,9-
dihydronaphtho [2,3-b]furan-3-yl)phosphonate (13f). yellow solid,
mp 103e105  C; 1H NMR (250 MHz, CDCl3) d 8.28e8.21 (m, 2H,
HeAr), 7.85e7.78 (m, 2H, HeAr), 7.70 (d, 3JHeF ¼ 7.0 Hz, 1H, HeAr),
4.35e4.13 (m, 4H, (CH3CH2O)2P(O)), 1.27 (t, 3JH-H ¼ 7.1 Hz, 6H,
(CH3CH2O)2P(O)). 13C NMR (63 MHz, CDCl3) d 178.32 (s, C(O)),
173.24 (s, C(O)), 157.81 (d, 2JC-P ¼ 22.3 Hz, C-2), 153.22 (d, 2JC-
1 4
P ¼ 11.9 Hz, C-9a), 153.07 (d, JC-F ¼ 263.1 Hz, CF), 144.17 (d, JC-
3 hexylamine, 2-chlorobenzylamine) was added, and the resulting
P ¼ 3.2 Hz, CCl), 140.00 (d, JC-P ¼ 20.9 Hz, CeAr), 134.46 (s, CH-Ar),
134.10 (s, CH-Ar), 132.96 (s, CeAr), 131.51 (s, CeAr), 129.99 (d, 2JC-
2 was evaporated, and DCM (15 mL), 1 M HClaq (20 mL) were added.
P ¼ 9.3 Hz, C-3a), 129.42 (d, JC-P ¼ 21.5 Hz, CCl)), 127.38 (s, CH-Ar),
126.75 (s, CH-Ar), 125.24 (d, 2JC-P ¼ 3.6 Hz, CeAr), 113.21 (d, 1JC-
2 extracted with DCM (2  10 mL). The combined organic extracts
P ¼ 212.5 Hz, C-3), 63.24 (d, JC-P ¼ 6.0 Hz, (CH3CH2O)2P(O)), 16.13
(d, JC-P ¼ 6.2 Hz, (CH3CH2O)2P(O)). 31P NMR (101 MHz, CDCl3)
3 were washed with brine, dried over MgSO4, filtered and concen-
d 5.71. IR n(cm1): 2989, 1679, 1394, 1283, 1207, 1018, 983. HRMS m/
z, calcd [MþH]þ 498.00708, observed 498.00714.
4.1.3. Ethyl hydrogen (2-(2-chlorophenyl)-4,9-dioxo-4,9-
dihydronaphtho [2,3-b]furan-3-yl)phosphonate (14)
To a solution of diethyl phosphonate 13c (0.44 g, 1 mmol) in
butanone (10 mL) lithium bromide (0.11 g, 1.25 mmol) was added
and the mixture was stirred at 80  C for 5 h. The solvent was
evaporated and 1 M HClaq (10 mL) was added to the solid residue.
Extraction with CHCl3 (3  30 mL), drying (MgSO4) and evaporation
of the solvent gave the crude product. The solid was suspended in
Et2O (10 mL), stirred, filtered and dried to give 0.39 g (93%) of 14 as
yellow solid, mp 208e209  C; 1H NMR (250 MHz, DMSO-d6)
d 8.19e8.11 (m, 2H, HeAr), 7.99e7.92 (m, 2H, HeAr), 7.74e7.59 (m,
3H, HeAr), 7.60e7.49 (m, 1H, HeAr), 3.86 (p, 3JH-H ¼ 2JH-P ¼ 7.2 Hz,
2H, (CH3CH2O)P(O)), 1.01 (t, 3JH-H ¼ 7.0 Hz, 3H, (CH3CH2O)2P(O)). 13C
NMR (63 MHz, DMSO-d6) d 178.43 (s, C(O)), 172.99 (s, C(O)), 161.01
(d, 2JC-P ¼ 21.6 Hz, C-2), 152.71 (d, 2JC-P ¼ 12.1 Hz, C-9a), 134.43 (s,
CHeAr), 134.20 (s, CHeAr), 133.37 (s, CCl), 132.76 (s, CHeAr), 132.76
(s, CHeAr), 132.55 (s, CeAr), 131.44 (s, CeAr), 129.56 (d, 2JC-
P ¼ 10.7 Hz, C-3a), 129.34 (s, CeAr), 128.13 (s, CHeAr), 126.96 (s,
CHeAr), 126.70 (s, CHeAr), 126.13 (s, CHeAr), 111,02 (d,
1
JCeP ¼ 212.6 Hz, C-3), 62.15 (d, 2JC-P ¼ 5.4 Hz, (CH3CH2O)P(O)),
15.78 (d, 3JC-P ¼ 6.8 Hz, (CH3CH2O)P(O)).31P NMR (101 MHz, DMSO-
d6) d 2.43; IR n(cm1): 2923, 1676, 1365, 1209, 1038, 998, 963, 937;
HRMS m/z, calcd [MþH]þ 417.02893, observed 417.02948.
4.1.4. Ethyl 2-(3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-
(diethoxyphosphoryl)acetate (14)
To a stirred solution of 2,3-dichloronaphthalene-1,4-dione (10)
(4.5 g, 20.0 mmol) and ethyl 2-(diethoxyphosphoryl)acetate (15)
(4.0 mL, 20.0 mmol) in THF (50 mL) NaH (1.0 g, 42 mmol) was added
at 0  C, and the resulting mixture was stirred at room temperature
for 1 week. Cold (0  C) water (100 mL), 1 M HClaq (20 mL) and DCM
(150 mL) were added. The organic layer was separated and the
aqueous layer was extracted with DCM (2  50 mL). The combined
organic extracts were washed with brine, dried over MgSO4,
filtered and concentrated. The crude product was purified by flash
chromatography (chloroform) to give pure product as yellow solid,
mp 70e71  C; 1H NMR (250 MHz, CDCl3) d 8.24e8.08 (m, 2H,
HeAr), 7.90e7.70 (m, 2H, HeAr), 5.02 (d, J ¼ 27.3 Hz, 1H, CHP),
3
H ¼ 7.1 Hz, 6H, (CH3CH2O)2P(O)), 1.25 (t, JH-H ¼ 7.1 Hz, 3H,
(CH3CH2O)C(O)). 13C NMR (63 MHz, CDCl3) d 180.15 (d, 2JC-
3
P ¼ 2.4 Hz, C(O)), 176.81 (d, JC-P ¼ 1.8 Hz, C(O)), 165.15 (s, C(O)),
145.13 (d, 2JC-P ¼ 9.7 Hz, CCl), 140.06 (d, 2JC-P ¼ 8.4 Hz, C-2), 134.23
(s, CH-Ar), 133.99 (s, CH-Ar), 130.87 (s, CeAr), 130.73 (s, CeAr),
127.00 (s, CH-Ar), 126.94 (s, CH-Ar), 63.33 (d, 3JC-P ¼ 6.6 Hz,
(CH3CH2O)C(O)), 63.00 (d, 2JC-P ¼ 6.7 Hz, (CH3CH2O)2P(O)), 46.23 (d,
1
JC-P ¼ 143.4 Hz, CHP), 15.99 (d, 3JC-P ¼ 4.3 Hz, (CH3CH2O)2P(O)),
13.64. 31P NMR (101 MHz, CDCl3) d 16.09. IR n(cm1): 2986, 2898,
1736, 1667, 1262, 1248, 962, 877, 720, 570; HRMS m/z, calcd
[MþH]þ 415.07079, observed 415.07031.
4.1.5. General procedure for preparing compounds 18aee
To a stirred solution of phosphonate (16) (0.83 g, 2 mmol) in
MeOH (10 mL) amine (30 mmol, 21.4 mL, C ¼ 1.4 M methylamine
solution in MeOH or 8 mmol for n-propylamine, c-propylamine, n-
mixture was stirred at room temperature for 1 day. Then, solvent
The organic layer was separated and the aqueous layer was
trated. The crude product was purified by flash chromatography
(chloroform).
4.1.5.1. Diethyl (2-hydroxy-1-methyl-4,9-dioxo-4,9-dihydro-1H-
benzo[f]indol-3-yl)phosphonate (18a). red solid, mp 74e75  C; 1H
NMR (250 MHz, CDCl3) d 8.20e7.97 (m, 2H, HeAr), 7.76e7.57 (m,
2H, HeAr), 4.34e3.98 (m, 4H, (CH3CH2O)2P(O)), 3.91 (s, 3H, CH3),
1.33 (t, 3JH-H ¼ 7.0 Hz, 6H, (CH3CH2O)2P(O)). 13C NMR (63 MHz,
CDCl3) d 179.73 (s, C(O)), 175.22 (s, C(O)), 160.58 (d, 2JC-P ¼ 18.3 Hz,
C-2), 133.29 (s, CeAr), 133.19 (s, CH-Ar), 132.84 (s, CH-Ar), 132.79 (s,
CeAr), 127.18 (d, 2JC-P ¼ 8.2 Hz, C-9a), 126.54 (d, 2JC-P ¼ 11.9 Hz, C-
3a), 126.30 (s, CH-Ar), 126.00 (s, CH-Ar), 81.76 (d, 1JC-P ¼ 215.7 Hz, C-
3), 63.44 (d, 2JC-P ¼ 5.4 Hz, (CH3CH2O)2P(O)), 30.83 (s, CH3), 16.27 (d,
3
JC-P ¼ 7.0 Hz, (CH3CH2O)2P(O)). 31P NMR (101 MHz, CDCl3) d 18.24.
IR n(cm1): 2983, 1652, 1552, 1289, 1021, 966, 854, 552. HRMS m/z,
calcd [MþH]þ 364.09445, observed 364.09448.
 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
4.1.5.2. Diethyl (2-hydroxy-4,9-dioxo-1-propyl-4,9-dihydro-1H-
benzo[f]indol-3-yl)phosphonate (18b). red solid, mp 81e82  C; 1H
NMR (250 MHz, CDCl3) d 8.11e8.06 (m, 2H, HeAr), 7.71e7.58 (m,
2H, HeAr), 4.34 (t, 3JH-H ¼ 7.4 Hz, 2H, CH2CH2N), 4.29e4.04 (m, 4H,
(CH3CH2O)2P(O)), 1.83 (h, 3JH-H ¼ 7.4 Hz, 2H, CH2CH2N), 1.33 (t, 3JH-
3
H ¼ 7.1 Hz, 6H, (CH3CH2O)2P(O)), 0.97 (t, JH-H ¼ 7.4 Hz, 3H,
CH3CH2).13C NMR (63 MHz, CDCl3) d 179.78 (s, C(O)), 174.84 (s,
C(O)), 160.52 (d, 2JC-P ¼ 19.7 Hz, C-2), 133.42 (s, CeAr), 133.16 (s,
CeAr), 132.80 (s, CH-Ar), 132.75 (s, CH-Ar), 127.47 (d, 2JC-P ¼ 8.4 Hz,
C-9a), 126.21 (d, 2JC-P ¼ 12.1 Hz, C-3a), 126.24 (s, CH-Ar), 126.03 (s,
CH-Ar), 81.73 (d, 1JC-P ¼ 216.1 Hz, C-3), 63.42 (d, 2JC-P ¼ 5.5 Hz,
(CH3CH2O)2P(O)), 45.48 (s, CH2N), 23.05 (s, CH3CH2), 16.24 (d, 3JC-
31
P ¼ 7.0 Hz, (CH3CH2O)2P(O)), 11.02 (s, CH3CH2). P NMR (101 MHz,
1
CDCl3) d 18.45. IR n(cm ): 2974, 2932, 2874, 1660, 1589, 1387, 981,
962, 751, 703, 575, 506. HRMS m/z, calcd [MþH]þ 392.12575,
observed 392.12552.
4.1.5.3. Diethyl (1-cyclopropyl-2-hydroxy-4,9-dioxo-4,9-dihydro-1H-
benzo[f]indol-3-yl)phosphonate (18c). red solid, mp 90e91  C; 1H
NMR (250 MHz, CDCl3) d 11.95 (bs, 1H, OH), 8.31e8.10 (m, 2H,
HeAr), 7.88e7.66 (m, 2H, HeAr), 4.47e4.10 (m, 4H,
(CH3CH2O)2P(O)), 3.47 (p, 3JH-H ¼ 3.5 Hz, 1H, CHN), 1.44 (t, 3JH-
H ¼ 7.1 Hz, 6H, (CH3CH2O)2P(O)), 1.38e1.31 (m, 2H, CH2), 1.25e1.19
(m, 2H, CH2). 13C NMR (63 MHz, CDCl3) d 179.59 (s, C(O)), 173.88 (s,
C(O)), 161.63 (d, 2JC-P ¼ 20.3 Hz, C-2), 133.32 (s, CeAr), 133.07 (s,
CeAr), 132.47 (s, CH-Ar), 132.41 (s, CH-Ar), 127.59 (d, 2JC-P ¼ 11.6 Hz,
C-9a), 127.29 (d, 2JC-P ¼ 8.2 Hz, C-3a), 125.99 (s, CH-Ar), 125.94 (s,
CH-Ar), 81.32 (d, 1JC-P ¼ 215.3 Hz, C-3), 63.26 (d, 2JC-P ¼ 5.6 Hz,
(CH3CH2O)2P(O)), 26.95 (s, CHN), 16.07 (d, 3JC-P ¼ 6.8 Hz,
(CH3CH2O)2P(O)), 8.37 (s, CH2). 31P NMR (101 MHz, CDCl3) d 18.41.
IR n(cm1): 2982, 2929, 2906, 1719, 1507, 1248, 1017, 965, 789, 680,
578, 560. HRMS m/z, calcd [MþH]þ 390.11010, observed 390.11017.
4.1.5.4. Diethyl (1-hexyl-2-hydroxy-4,9-dioxo-4,9-dihydro-1H-benzo
[f]indol-3-yl)phosphonate (18d). red solid, mp 67e68  C; 1H NMR
(250 MHz, CDCl3) d 11.95 (bs, 1H, OH), 8.14e8.07 (m, 2H, HeAr),
7.69e7.63 (m, 2H, HeAr), 4.38 (t, 3JH-H ¼ 7.4 Hz, 2H, CH2CH2N),
4.30e4.08 (m, 4H, (CH3CH2O)2P(O)), 1.88e1.74 (m, 2H, CH2),
1.46e1.22 (m, 12H, (CH3CH2O)2P(O), 3  CH2), 0.88 (t, 3JH-H ¼ 6.7 Hz,
3H, CH3). 13C NMR (63 MHz, CDCl3) d 179.76 (s, C(O)), 174.94 (s,
C(O)), 160.15 (d, 2JC-P ¼ 20.1 Hz, C-2), 133.37 (s, CeAr), 133.21 (s, CH-
Ar), 132.82 (s, CH-Ar), 132.73 (s, CeAr), 127.38 (d, 2JC-P ¼ 11.6 Hz, C-
9a), 126.28 (s, CH-Ar), 126.20 (d, 2JC-P ¼ 7.2 Hz, C-3a), 126.09 (s, CH-
Ar), 81.89 (d, 1JC-P ¼ 215.4 Hz, C-3), 63.47 (d, 2JC-P ¼ 5.5 Hz,
(CH3CH2O)2P(O)), 44.27 (s, CH2N), 31.35 (s, CH2), 29.73 (s, CH2),
26.29 (s, CH2), 22.55 (s, CH2), 16.27 (d, 3JC-P ¼ 6.7 Hz,
(CH3CH2O)2P(O)), 13.99 (s, CH3). 31P NMR (101 MHz, CDCl3) d 18.45.
IR n(cm1): 3060, 2976, 2925, 1643, 1591, 1541, 1235, 1019, 955, 787,
724, 622, 576. HRMS m/z, calcd [MþH]þ 434.17270, observed
434.17249.
4.1.5.5. Diethyl (1-(2-chlorobenzyl)-2-hydroxy-4,9-dioxo-4,9-
dihydro-1H-benzo[f]indol-3-yl)phosphonate (18e). red solid, mp
86e88  C; 1H NMR (250 MHz, CDCl3) d 8.16e7.82 (m, 2H, HeAr),
7.63e7.49 (m, 2H, HeAr), 7.32e7.15 (m, 1H, HeAr), 7.07e6.80 (m,
2H, HeAr), 6.73e6.51 (m, 1H, HeAr), 5.50 (s, 2H, CH2), 4.21e3.91
(m, 4H, (CH3CH2O)2P(O)), 1.16 (t, 3JH-H ¼ 6.6 Hz, 6H,
(CH3CH2O)2P(O)). 13C NMR (63 MHz, CDCl3) d 179.83 (s, C(O)),
174.97 (s, C(O)), 160.72 (d, 2JC-P ¼ 20.0 Hz, C-2),133.78 (s, CCl), 133.39
(s, CeAr), 133.24 (s, CH-Ar), 133.05 (s, CH-Ar), 132.80 (s, CeAr),
132.49 (s, CeAr), 129.76 (s, CH-Ar), 128.76 (s, CH-Ar), 127.72 (d, 2JC-
P ¼ 8.0 Hz, C-3a), 127.14 (s, CH-Ar), 126.46 (s, CH-Ar), 126.29 (s, CH-
Ar), 126.26 (s, CH-Ar), 126.25 (d, 2JC-P ¼ 11.8 Hz, C-9a), 82.34 (d, 1JC-
2
P ¼ 215.7 Hz, C-3), 63.70 (d, JC-P ¼ 5.6 Hz, (CH3CH2O)2P(O)), 45.25
(s, CH2), 16.35 (d, 3JC-P ¼ 6.6 Hz, (CH3CH2O)2P(O)). 31P NMR
61
(101 MHz, CDCl3) d 19.66. IR n(cm1): 2959, 2928, 2859, 1732, 1649,
1509, 1392, 1232, 1166, 1022, 963, 793, 622. HRMS m/z, calcd
[MþH]þ 474.08678, observed 474.08612.
4.2. Biological activity
4.2.1. Cell culture and treatment
Two human leukemia cell lines, promyelocytic leukemia (HL-
60) and lymphoblastic leukemia (NALM-6), and a solid tumor-
derived human breast adenocarcinoma cell line (MCF-7) were
purchased from the European Collection of Cell Cultures (ECACC).
Leukemia cells were cultured in RPMI 1640 plus GlutaMax I me-
dium (Invitrogen, Grand Island, NY, USA), supplemented with 10%
heat-inactivated FBS (Biological Industries, Beit-Haemek, Israel)
and antibiotics (100 mg/mL streptomycin and 100 U/mL penicillin)
(SigmaeAldrich, St. Louis, MO, USA). MCF-7 cells were cultured in
Minimum Essential Medium Eagle (MEME, SigmaeAldrich, 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 acid solution and an-
tibiotics (100 mg/mL streptomycin and 100 U/mL penicillin), all from
SigmaeAldrich (SigmaeAldrich, St. Louis, MO, USA). Normal hu-
man mammary gland/breast cell line (MCF10A) and normal human
umbilical vein endothelial cells (HUVECs) were purchased from the
American Type Culture Collection (ATCC). MCF-10A cells were
cultured using MEGM Mammary Epithelial BulletKit, while HUVECs
using EGM-2 Endothelial Medium BulletKit, both purchased from
Lonza (Lonza, Walkersville, MD, USA). Cells were maintained at
37  C in a 5% CO2 atmosphere and were grown until 80% confluent.
For all experiments tested compounds were dissolved in DMSO
(SigmaeAldrich, Louis, MO, USA) and further diluted in culture
medium to obtain less than 0.1% DMSO concentration. In each
experiment controls without and with 0.1% DMSO were performed.
DMSO in this concentration had no effects on the observed
parameters.
4.2.2. Metabolic activity assay (MTT)
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium
bromide) assay, which measures activity of cellular de-
hydrogenases, was performed according to a known procedure
[41]. Exponentially growing cells were seeded on 96-well plates
(Nunc, Roskilde, Denmark) in 100 mL and left to growth for 24 h.
Subsequently, various concentrations of the tested compounds
(freshly prepared in DMSO and diluted with complete culture
medium) were added in 100 mL of culture medium to obtain the
concentration range from 107 to 103 M and the plates were
incubated for 24 or 48 h. Afterwards, MTT (5 mg/mL in PBS) was
added and incubation was continued for 2 h. The metabolically
active cells reduced MTT to blue formazan crystals. Then, the MTT-
containing medium was carefully aspirated and 100 mL DMSO was
added to dissolve the crystals. The absorbance of the blue formazan
product was measured at 560 nm using an automated plate reader
(iMark Bio-Rad, Hercules, CA, USA) and compared with control
(untreated cells). The IC50 values were calculated from concen-
trationeresponse curves.
4.2.3. Analysis of cell proliferation, apoptosis and DNA damage by
flow cytometry
Cell proliferation, DNA damage and apoptotic cell death were
determined using the “Apoptosis, DNA Damage, and Cell Prolifer-
ation Kit” (BD Bioscience, San Jose, CA, USA), according to the
manufacturer guidelines. The test kit includes all the necessary
components, such as: BD Cytofix/Cytoperm™ Fixation/Per-
meabilization Solution; BD Perm/Wash™ Buffer; BD Cytofix/Cyto-
perm™ Plus Permeabilization Buffer, DAPI, (Bromodeoxyuridine)
62 K. Gach et al. / European Journal of Medicinal Chemistry 120 (2016) 51e63
BrdU, DNase and antibodies (PerCP-Cy™5.5 Mouse Anti-BrdU;
Alexa Fluor® 647 Mouse Anti-H2AX (pS139); PE Mouse Anti-
Cleaved PARP (Asp214)). Briefly, cells were seeded on 25 cm2 cell
culture flask at a density 8.0  104/mL in 10 mL of culture medium
(MCF-7 cells) or on 6-well plates at a density 2.0  105/mL in 2 mL
of growth medium (HL-60 cells) and left to grow for 18e24 h. Af-
terwards, compound 13c diluted in medium was added to the cells
at IC50 concentration. The cells incubated without tested com-
pounds were used as control. After 24 h of incubation, cells were
treated with BrdU (final concentration of 10 mM) for additional 8 h.
Then, MCF-7 cells were harvested by trypsinization and
centrifuged (200  g, 5 min). HL-60 cells were collected by
centrifugation (200  g, 5 min). The total number of cells was
determined. Cells were fixed, permeabilized according to the
manufacturer's protocol and treated with DNase (300 mg/mL in
DPBS) for 1 h at 37  C in order to expose BrdU epitopes. Following
this treatment, cells were simultaneously stained with
fluorochrome-labeled anti-BrdU, H2AX (pS139), and cleaved PARP
(Asp214) antibodies for 20 min at room temperature. After
washing, DNA staining for cell cycle analysis using DAPI solution
(1mg/mL of staining buffer) was performed. Cells were re-
suspended in a staining buffer and analyzed by flow cytomtery
(Becton Dickinson Canto II). The data was visualized and quantified
by constructing a dot-plot using the BD FACSDiva software.
4.2.4. Analysis of apoptosis by flow cytometry using APC-Annexin
Vand propidium iodide (PI) double staining
Apoptotic cell death was determined using APC-Annexin V
Apoptosis Detection Kit (BD Bioscience, San Jose, CA, USA). Briefly,
the cells were seeded on 6-well plates (4.5  105 cells/well for MCF-
7 cells and 4.0  105 cells/well for HL-60 cells) and left to growth for
18e24 h. Subsequently, tested compound 13c diluted in medium
was added to the cells at IC50 concentration. The cells incubated
without tested compounds were used as a control. After 24 h of
incubation MCF-7 cells were harvested by trypsinization and
centrifuged (200g, 5 min). HL-60 cells were collected by centri-
fugation (200  g, 5 min). Cells were stained with annexin V and
propidium iodide (PI) according to the manufacturer guidelines
(15 min, at room temperature). Apoptosis of cells was analyzed by
flow cytometry (Becton Dickinson Canto II). Early and late apoptosis
was visualized and quantified by constructing a dot-plot using the
BD FACSDiva software.
4.2.5. Analysis of ROS induction by flow cytometry
The change of intracellular ROS levels was determined by flow
cytometry, based on the staining with cell-permeable CellROX®
Green Oxidative Stress Reagent (Molecular Probes, Life Technolo-
gies) which is a fluorogenic probe designed to reliably measure ROS
in the live cells. CellROX® Green Reagent is non-fluorescent or very
weakly fluorescent while in a reduced state and upon oxidation it
exhibits a strong fluorogenic signal localized in the mainly in nu-
cleus and mitochondria. Briefly, cells were seeded on 6-well plates
(4.5  105 cells/well for MCF-7 cells and 4.0  105 cells/well for HL-
60 cells) and left to growth for 18e24 h. Cells were treated with
tested compound 13c as described above. After 24 h of treatment,
cells were incubated with 5 mM CellROX® Green Reagent at 37  C for
30 min in the dark. Then, MCF-7 cells were harvested by trypsini-
zation and centrifuged (200  g, 5 min). HL-60 cells were collected
by centrifugation (200  g, 5 min). Cells were washed twice with
PBS, re-suspended in PBS and analyzed by flow cytometry (Becton
Dickinson Canto II). A redox-cycling agent - menadione (Sigma-
eAldrich, Louis, MO, USA) was used as a positive control.
4.2.6. Assessment of mitochondrial membrane potential
Changes in mitochondrial membrane potential (DJm) were
determined using the Cell Meter™ JC-10 Mitochondrial Membrane
Potential Assay Kit (AAT, Bioquest Inc., Sunnyvale, CA, USA), ac-
cording to the manufacturer guidelines. JC-10 dye enters selectively
into mitochondria and reversibly changes color from green to or-
ange as membrane potential increases. Briefly, cells were seeded on
6-well plates (4.5  105 cells/well for MCF-7 cells and
4.0  105 cells/well for HL-60 cells) and left to growth for 18e24 h.
Then, cells were treated with tested compound 13c, as previously
described. After 24 h of treatment MCF-7 cells were harvested by
trypsinization and centrifuged (200  g, 5 min). HL-60 cells were
collected by centrifugation (200  g, 5 min). Afterwards, cells were
washed once with PBS, stained with JC-10 at 37  C for 45 min in
dark and analyzed by flow cytometry (Becton Dickinson Canto II). A
mitochondrial uncoupler - carbonyl cyanide-4-(trifluoromethoxy)
phenylhydrazone (FCCP, SigmaeAldrich, Louis, MO, USA) was
used as a positive control. Changes in DJm were visualized and
quantified by constructing a dot-plot using the BD FACSDiva
software.
4.2.7. Statistical analysis
Statistical analysis was performed using Prism 4.0 (GraphPad
Software Inc., San Diego, CA, USA). Results from at least three in-
dependent experiments in triplicate are expressed as mean ± SD or
SEM. Statistical significance was analyzed using either Student's t
test (for two-group comparison) or one-way ANOVA followed by a
post-hoc multiple comparison StudenteNewmaneKeuls test (for
multiple-group comparison). p < 0.05 was considered statistically
significant.
Notes
The authors declare no competing financial interest.
Acknowledgments
Financial support from Lodz University of Technology Statutory
founds I-18/13/DzS and from Medical University of Lodz (No 502-
03/1-156-02/502-14-191 to KG and No 503/1-156-02/503-11-002).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.05.002.
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