Synthesis and anticancer cytotoxicity of azaaurones overcoming multidrug resistance

Article abstract of DOI:10.3390/molecules25030764

The resistance of tumors against anticancer drugs is a major impediment for chemotherapy. Tumors often develop multidrug resistance as a result of the cellular efflux of chemotherapeutic agents by ABC transporters such as P-glycoprotein (ABCB1/P-gp), Multidrug Resistance Protein 1 (ABCC1/MRP1), or Breast Cancer Resistance Protein (ABCG2/BCRP). By screening a chemolibrary comprising 140 compounds, we identified a set of naturally occurring aurones inducing higher cytotoxicity against P-gp-overexpressing multidrug-resistant (MDR) cells versus sensitive (parental, non-P-gp-overexpressing) cells. Follow-up studies conducted with the P-gp inhibitor tariquidar indicated that the MDR-selective toxicity of azaaurones is not mediated by P-gp. Azaaurone analogs possessing pronounced effects were then designed and synthesized. The knowledge gained from structure–activity relationships will pave the way for the design of a new class of anticancer drugs selectively targeting multidrug-resistant cancer cells.

Full text of DOI:10.3390/molecules25030764

molecules
Article
Synthesis and Anticancer Cytotoxicity of Azaaurones
Overcoming Multidrug Resistance
Szilárd Tóth 1 , Áron Szepesi 1, Viet-Khoa Tran-Nguyen 2 , Balázs Sarkadi 1, Katalin Német 1,3
,
Pierre Falson 4 , Attilio Di Pietro 4,†, Gergely Szakács 1,5,† and Ahcène Boumendjel 2, ,†
*
1
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences,
1117 Budapest, Hungary; toth.szilard.enzim@ttk.mta.hu (S.T.); aron.szepesi@outlook.hu (Á.S.);
balazs.sarkadi@ttk.mta.hu (B.S.); knemet@gmail.com (K.N.); szakacs.gergely@ttk.mta.hu (G.S.)
Département de Pharmacochimie Moléculaire (DPM), UMR 5063, Univ. Grenoble Alpes, 38041 Grenoble,
France; khoatnv1993@gmail.com or vktrannguyen@unistra.fr
2
3
4
Creative Cell Ltd., 1119 Budapest, Hungary
Drug Resistance & Membrane Proteins group, Molecular Microbiology and Structural Biochemistry
Laboratory (CNRS UMR 5086), University of Lyon, IBCP, 7 Passage du Vercors, 69367 Lyon, France;
pierre.falson@ibcp.fr (P.F.); a.dipietro@ibcp.fr (A.D.P.)
Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria
Correspondence: ahcene.boumendjel@univ-grenoble-alpes.fr; Tel.: +33-4-76-63-53-11
These senior investigators contributed equally.
5
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 December 2019; Accepted: 8 February 2020; Published: 10 February 2020
Abstract: The resistance of tumors against anticancer drugs is a major impediment for chemotherapy.
Tumors often develop multidrug resistance as a result of the cellular efflux of chemotherapeutic
agents by ABC transporters such as P-glycoprotein (ABCB1/P-gp), Multidrug Resistance Protein 1
(ABCC1/MRP1), or Breast Cancer Resistance Protein (ABCG2/BCRP). By screening a chemolibrary
comprising 140 compounds, we identified a set of naturally occurring aurones inducing higher
cytotoxicity against P-gp-overexpressing multidrug-resistant (MDR) cells versus sensitive (parental,
non-P-gp-overexpressing) cells. Follow-up studies conducted with the P-gp inhibitor tariquidar
indicated that the MDR-selective toxicity of azaaurones is not mediated by P-gp. Azaaurone analogs
possessing pronounced effects were then designed and synthesized. The knowledge gained from
structure–activity relationships will pave the way for the design of a new class of anticancer drugs
selectively targeting multidrug-resistant cancer cells.
Keywords: aurone; azaaurone; multidrug resistance; P-gp; overexpression; anticancer; cytotoxicity
1. Introduction
Multidrug resistance constitutes one of the major hurdles to anticancer chemotherapy. One of
the well-described mechanisms via which cancer cells become resistant to structurally different
chemotherapeutic agents is the overexpression of ATPase efflux pumps belonging to the ABC family
of transporters that extrude anticancer drugs from the cells [1–3]. Therefore, to ensure efficiency
in chemotherapy, higher doses of these medications have to be administered, which, on the other
hand, would inevitably lead to severe side effects. In order to counteract multidrug resistance (MDR),
one of the established strategies is to develop inhibitors of ABC proteins involved in drug efflux,
thus optimizing therapeutic effects of anticancer agents [4]. However, the clinical relevance of such
inhibitors remains questionable despite the existence of compounds with potent in vitro activity [
2].
Alternatively, recent reports revealed several compounds selectively killing cells overexpressing ATPase
efflux pumps, such as P-glycoprotein (P-gp/MDR1) and Multidrug resistance associated protein 1
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(MRP1/ABC1) [5–10]. These molecules were shown to target MDR cancer cells, offering an emerging
strategy for anticancer agent development [11,12].
In this study, we report the result of a cytotoxicity screen performed on a pair of drug-sensitive and
multidrug-resistant cancer cell lines. We screened a library of 140 compounds consisting of flavonoidic
derivatives and thiosemicarbazones (Figure 1, full structures are provided in Supplementary Materials,
Table S1). Flavonoidic derivatives have been widely studied as candidates for cancer treatment and
prevention, with various naturally occurring substances reported to be effective against resistant
tumors [13
belonging to this class have been reported to possess increased toxicity against otherwise drug-resistant
cells [15 18]. Primary screening revealed that a subclass of aurones was more toxic to the MDR cell line.
,14]. On the other hand, thiosemicarbazones were included since several compounds
–
Among potential structural analogs of aurones, we found that azaaurones were particularly interesting.
Herein, we focused our efforts on the investigation of azaaurones that target the collateral sensitivity of
MDR cells. The identified structure–activity relationships will pave the way for the design of more
effective compounds with this therapeutic profile.
Figure 1. Scaffolds of the derivatives (library of 140 compounds) tested in the primary screen (at 10
and 100 µM).
2. Results and Discussion
Primary screening using 140 compounds derived from the scaffolds shown in Figure 1
(Supplementary Materials, Table S1) was conducted using two uterine sarcoma cell lines. Multidrug-
resistant MES-SA/Dx5 cells were established from parental MES-SA cells by continuous selection in
doxorubicin [19]. The MDR phenotype of MES-SA/Dx5 cells is conveyed by the overexpression of
the efflux pump P-glycoprotein. MES-SA/Dx5 cells were cultured in doxorubicin (500 nM), and P-gp
expression was regularly checked with the calcein assay (Supplementary Materials, Figure S1) [20].
Compounds were assigned into 3 main categories according to their toxicity against the two cell
lines. Of the 140 tested compounds, 8% were toxic in both cell lines (higher than 50% growth inhibition
at 10
µM), 71% were intermediately toxic (higher than 50% growth inhibition at 100
µM against at
least one of the cell lines), and 21% were not toxic at all (at 100
µM). Among the tested compounds,
xanthones were the least toxic, while chalcones were the most toxic ones. Taken together, the primary
toxicity studies revealed that scaffolds derived from aurones are the most promising candidates as they
induced higher cytotoxicity among resistant cells versus sensitive cell lines (Supplementary Materials,
Table S2).

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Aurones [2-benzylidenebenzofuran-3(2H)-ones], which are structural isomers of flavones,
contribute to the coloration of numerous flowers and vegetables [21]. Despite the limited number
of naturally occurring aurones (compared to flavones), they are emerging as promising scaffolds in
different therapeutic areas [22]. In plants, aurones are mainly hydroxylated and/or methoxylated at
positions 4 and/or 6 (e.g., aureusidin, bracteatin, sulfuretin, hispidol, rengasin, and derivatives) [21].
This substitution pattern guided us to include in our primary screening the aurones bearing methoxyl
groups at these positions. For the sake of optimizing aurones as cytotoxic agents, we decided to
focus on azaaurones where the intracyclic oxygen of aurones was replaced with an N–H group. This
modification proved to be crucial in the design of leishmanicidal and antibacterial agents [23–25].
Moreover, the presence of an indolinone in the core structure of azaaurones makes them closely related
to natural alkaloids and pharmaceuticals exhibiting significant biological activities [26].
2.1. Synthesis of Azaaurones
The synthesis of targeted azaaurones was carried out according to a previously reported procedure
(Scheme 1). The key step was the condensation of a conveniently substituted N-acetylindolin-3-one
A
with a substituted benzaldehyde. It should be highlighted that N-deacetylation occurs during the
condensation step. The indolinone moiety was prepared starting from 3,5-dimethoxyaniline and
chloroacetonitrile as previously reported [23].
Scheme 1. Synthesis of targeted azaaurones.
2.2. Structure–Activity Relationship Study
We determined the cytotoxicity of the novel derivatives against the two human uterine sarcoma
cell lines. Based on the IC50 values (in µM) determined from the effects on both cell lines, a selectivity
ratio (SR) was calculated (Table 1). A value of SR > 1 implies that the compound is more cytotoxic
towards P-gp-overexpressing MES-SA/Dx5 cells than the parental MES-SA cells, displaying a so-called
MDR-selective activity. A value of SR < 1, on the contrary, suggests that the P-gp-expressing cells
are resistant to the compound, as normally observed with P-gp substrates. In order to confirm MDR
selectivity, we tested the most interesting compound (compound 4) on further cell line pairs with or
without P-gp expression and observed only a marginal selectivity (Supplementary Materials, Table S3).
4,6-dimethoxy-40-bromoaurone, bearing a bromine atom at the C-40 position (on the B-ring),
was one of the most active compounds identified in the primary screen, prompting the synthesis of a
similar brominated azaaurone analog. Unfortunately, for unexpected reasons, this azaaurone was not
stable during the purification step, whereas the chlorinated analog (compound
in its pure form. As shown in Table 1, azaaurone displayed a high selectivity toward resistant cells.
Next, we checked the impact of the presence of an alkyl group on the B-ring (compounds ) and
found that butyl chains, either linear (compound ) or ramified (compound ), led to SR values higher
than those obtained with ethyl and propyl groups and comparable to that of compound (the SR of
1, Table 1) was obtained
1
2–6
4
6
1
4
was even slightly better), while a significant improvement in IC50s in both cell lines was also observed,
highlighting the impact of the hydrophobicity and/or the size of the alkyl chain. On the basis of the
importance of alkyl groups linked to the B-ring, we decided to investigate the presence of multiple
methyl groups on this aryl moiety (azaaurones
as it provided the most selective compound of the series (compound
than two methyl groups led to less selective azaaurones ( and 10 versus
of methoxy groups on the B-ring produced much less selective compounds as it can clearly be seen by
8
–
10 and 13). This strategy turned out to be productive
). However, the presence of more
and 13). The introduction
8
9
8

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comparing the azaaurones
8 and 11. Taken together, the above data highlight that compounds 4 and 6,
having the lowest IC50s and giving SR values that were among the highest (Table 1), are the primary
candidates for further development.
Table 1. Structures and activities of azaaurone derivatives determined from the effect on the sensitive
MES-SA cell line and the resistant MES-SA/Dx5 line. IC50 values are in µM.
Compound
1
Aryl
IC50 (MES-SA)
IC50 (Dx5)
SR 1
147.2 ± 32.8
36.6 ± 14.5
4.0
2
3
22.7 ± 1.4
43.9 ± 3.6
10.8 ± 1.7
13.7 ± 3.0
2.1
3.2
4
5
6
7
17.8 ± 4.1
17.1 ± 1.7
11.9 ± 1.6
61.9 ± 10.0
3.7 ± 0.6
5.8 ± 1.3
3.4 ± 0.2
13.1 ± 5.6
4.8
2.9
3.8
2.7
8
9
158.6 ± 43.7
170.8 ± 4.3
32.1 ± 13.3
4.9
1.3
131.7 ± 13.8
10
9.2 ± 2.3
19.5 ± 3.5
0.5
11
12
44.5 ± 12.8
30.4 ± 2.0
27.4 ± 7.8
15.6 ± 1.4
1.6
1.9
13
169.9 ± 30.2
44.2 ± 16.0
3.8
Doxorubicin
NSC57969
0.36 ± 0.11
4.63 ± 1.31
4.15 ± 0.38
0.59 ± 0.10
0.087
7.9
1
SR (selectivity ratio) is a compound’s IC50 against MES-SA cells (µM) divided by its IC50 against Dx5 cells (µM).

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In order to compare the activity of azaaurones to those of well-known flavonoids highly
investigated in cancer prevention and treatment, we included the naturally occurring genistein,
kaempferide, and apigenin in the screens [27]. These compounds were toxic against both MES-SA
(IC50 = 53.6
µM for genistein, 57.0
µM for kaempferide, and 36.6 µM for apigenin) and MES-SA/Dx5
cells (IC50 = 40.3, 39.3, and 20.3
µM for genistein, kaempferide and apigenin, respectively), as seen in
Table 2. Based on these results, it can be concluded that azaaurones were more cytotoxic than these
aforementioned flavonoids in most cases.
Table 2. The effect of naturally occurring flavonoids on the sensitive MES-SA cell line and the resistant
MES-SA/Dx5 line. IC50 values are in µM.
Compound
IC50 (MES-SA)
IC50 (Dx5)
SR
Genistein
Kaempferide
Apigenin
53.6 ± 4.63
57.0 ± 2.52
36.6 ± 1.72
40.3 ± 1.8
39.3 ± 3.93
20.3 ± 1.68
1.3
1.5
1.8
As MES-SA/Dx5 cells overexpress the efflux transporter P-gp, we checked if this transporter
contributes to the selective toxicity of the most promising compounds ( , and 10). The P-gp
4, 5, 6, 7
inhibitor tariquidar did not influence the selective toxicity of azaaurones, suggesting that the function
of P-gp is not involved in the collateral sensitivity of MES-SA/Dx5 to these compounds (data shown in
Supplementary Materials, Table S4). While multidrug-resistant MES-SA/Dx5 cells exhibit collateral
sensitivity to azaaurones, in contrast to “MDR-selective” compounds (e.g., isatin thiosemicarbazones
and 8-hydroxyquinoline derivatives) reported by Füredi et al. (2017) [9], our azaaurones’ selective
toxicity was not influenced by the P-gp inhibitor tariquidar (TQ). These results indicate that the
observed hypersensitivity of MES-SA/Dx5 cells is not linked to the activity of P-gp, and should be
rather explained by off-target effects linked to other resistance mechanisms [5].
3. Materials and Methods
3.1. Chemistry
1H NMR and 13C NMR spectra were recorded at 200 MHz and 50 MHz, respectively, using a
Bruker Avance-400 instrument. Chemical shifts (δ) are reported in ppm relative to Me4Si (internal
standard). Electrospray ionization (ESI) mass spectra were performed by the Analytical Department
of Grenoble University on an Esquire 300 Plus Bruker Daltonis instrument with a nanospray inlet.
Elemental analyses were performed at the Analytical Department of Grenoble University. Thin-layer
chromatography (TLC) used Merck silica gel F-254 plates (thickness 0.25 mm). Flash chromatography
used Merck silica gel 60, 200–400 mesh. High-quality solvents used in chromatography were purchased
from commercial sources and were used without further distillation.
3.2. Synthesis of Azaaurones 1–13
Azaaurones were obtained by the condensation of indolinone
A (Scheme 1) and substituted
benzaldehydes [23]. Indolinone was dissolved in MeOH (10 mL/mmol) and treated with KOH
A
(50% in H2O, 1 mL/mmol). The solution was stirred at room temperature and allowed to reach 65
◦C progressively. The reaction was quenched when TLC indicated no reaction progression (3 to 24
h). The solution was acidified by adding HCl (30 mL, 2 N), evaporated, and the crude was dissolved
in H2O and extracted with ethyl acetate. The organic solution was separated, dried over Na2SO4,

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and concentrated. The crude mixture was purified by flash chromatography over silica gel, eluted with
a mixture of ethyl acetate:cyclohexane (1:2) to afford pure azaaurones. The synthesized compounds
were isolated in 96–98% pure form as evidenced by elemental analysis. For each azaaurone, carbon,
hydrogen, and nitrogen atoms were analyzed and were found in nearly full (96–98%) agreement
1
with the expected values. The compounds were fully characterized by H NMR, 13C NMR, and
high-resolution mass spectrometry (HRMS) (data shown in Supplementary Materials, Table S5).
3.3. Biological Evaluation
In the primary screen, we investigated the toxicity of 140 compounds (dissolved in DMSO).
Pipetting was performed by an automated liquid handling robot (Hamilton StarLet). Both MES-SA
and MES-SA/Dx5 cells were purchased from ATCC (ATCC codes: ATCC-CRL-1976, ATCC-CRL-1977,
respectively), where they were characterized by DNA fingerprinting. Upon arrival, the cells were
cultured, and stocks (with passage number under 5) were frozen and stored. Cells were kept in culture
for maximum 2–3 months, with passage numbers below 30. These cells were seeded for 24 h prior
to drug treatment on 96-well plates (5000 cells/100
(10 M and 100 M) for 96 h in a final volume of 200
we used a toxic concentration (25 M) of NSC-57969 [28]; and for negative control, we used 0.4%
µL). Toxicity was screened in 2 concentrations
µ
µ
µL. For positive control (representing toxicity),
µ
DMSO-containing medium (referring to 100% growth). Fluorescent intensity of mCherry was detected
by Perkin Elmer EnSpire Multimode Plate Reader (fluorescent cells were characterized in our earlier
publications) [15,29]. Excitation wavelength was set to 585 nm and emission was set to 607 nm as a
result of a wavelength optimization with the instrument.
Secondary assays were performed by PrestoBlue viability reagent (Thermo Fisher Scientific).
Non-fluorescent MES-SA and MES-SA/Dx5 cells were seeded on 96-well plates (5000 cells/100
Dulbecco’s Modified Eagle Medium—DMEM). The next day, serially diluted compounds were added
manually in an additional 100 L volume, and plates were further incubated for 72 h. For negative
µL of
µ
control, we used 0.4% DMSO-containing DMEM; and for positive control, we prepared cell-free wells.
After the 72 h incubation, the drug-containing medium was replaced by 5% PrestoBlue diluted in PBS
and incubated for 1 h. Fluorescent detection of the PrestoBlue was carried out by exciting on 555 nm
and reading emission on 585 nm. Tariquidar (TQ) was applied at 1
least 3 independent experiments.
µM. The results were based on at
For other cell lines on which the compound
4 was tested (Supplementary Materials, Table S3):
A431 and MDCK II were also purchased from ATCC; KB-3-1 and KB-V1 were provided by Dr. Michael
M. Gottesman (NIH, NCI/CCR); MDCK II-B1 (which stably expresses the human wild-type ABCB1)
was created by the Sleeping Beauty transposon-based gene delivery system, using the 100
SB transposase [30]. All cells were regularly tested for Mycoplasma contamination with the MycoAlert
Mycoplasma Detection Kit from Lonza, giving negative results.
×
hyperactive
4. Conclusions
Targeting the collateral sensitivity of drug-resistant cancer cells is an emerging strategy developed
to overcome intrinsic or acquired multidrug resistance (MDR). In this paper, we characterize novel
aurone derivatives that show selective toxicity against MDR cells. Our structure–activity relationship
(SAR) analysis suggests that the substitution of the intracyclic oxygen in aurones by a nitrogen atom
contributes to selective toxicity. These novel azaaurone derivatives reveal a new chemotype that
can serve as a starting point for further development and systematic SAR studies of a new class of
MDR-selective anticancer agents.
Supplementary Materials: The following are available online. Table S1: Structures of the screened compounds,
Table S2: IC50 of aurone representatives (in µM), Table S3: Effects of azaaurone 4 in various P-gp-expressing
cell lines, Table S4: Contribution of P-gp to the selective toxicity, Table S5: Physicochemical characterization of
azaaurones (compounds 13) synthesized according to Scheme 1, Figure S1: Characterization of P-glycoprotein
expression and activity in MES-SA/Dx5 cells.
1–

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Author Contributions: S.T. contributed to the biological work and manuscript writing.
Á
.S. contributed to the
biological experiments. V.-K.T.-N. contributed to the chemical work and manuscript writing. B.S., K.N., P.F.,
A.D.P., G.S., and A.B. designed and supervised the study. G.S. and A.B. wrote the manuscript. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was supported by CBH-EUR-GS (ANR-17-EURE-0003). GS was supported by a Momentum
grant of the Hungarian Academy of Sciences and the National Research, Development and Innovation Fund
of Hungary (FIEK_16-1-2016-0005). Project no. 2018-1.2.1-NKP-2018-00005 has been implemented with the
support provided by the National Research, Development and Innovation Fund of Hungary, financed under the
2018-1.2.1-NKP funding scheme.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of these compounds are not available from the authors.
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