Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: Critical role of methoxylation in both inhibition potency and cytotoxicity

Article abstract of DOI:10.1021/jm2016528

ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC₅₀ values below 0.5 μM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6′-hydroxyl-2′,4′-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.

Full text of DOI:10.1021/jm2016528

Article
pubs.acs.org/jmc
Investigation of Chalcones as Selective Inhibitors of the Breast
Cancer Resistance Protein: Critical Role of Methoxylation in both
Inhibition Potency and Cytotoxicity
Glaucio Valdameri,†,‡,∥ Charlotte Gauthier,†,∥ Raphael Terreux,§ Remy Kachadourian,# Brian J. Day,#
́
̈
Sheila M. B. Winnischofer,‡ Maria E. M. Rocha,‡ Veronique Frachet,⊥ Xavier Ronot,⊥
́
Attilio Di Pietro,*,†,∥ and Ahcene Boumendjel*,⊗,∥
̀
†Equipe Labellisee
France
‡Department of Biochemistry and Molecular Biology, Federal University of Parana,
§Equipe Bioinformatique: structures et interactions, BMSSI UMR 5086 CNRS/Universite
Proteines, Lyon, France
#Department of Medicine, National Jewish Health, Denver Colorado 80206, United States
́ ́ ́
Ligue 2012, BMSSI UMR 5086 CNRS/Universite Lyon 1, Institut de Biologie et Chimie des Proteines, Lyon,
́
Curitiba, PR, Brazil
Lyon 1, Institut de Biologie et Chimie des
́
́
⊥AGing Imaging Modeling, FRE 3405, Universite
́
Joseph Fourier, CNRS, EPHE, Faculte
́
de Med
́
ecine, La Tronche, France
ulaire, Grenoble, France
⊗Universite
́
Joseph FourierGrenoble/CNRS, UMR 5063, Departement de Pharmacochimie Molec
́
́
S
* Supporting Information
ABSTRACT: ABCG2 plays a major role in anticancer-drug efflux and
related tumor multidrug resistance. Potent and selective ABCG2 inhibitors
with low cytotoxicity were investigated among a series of 44 chalcones and
analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on
the transport of mitoxantrone, a known ABCG2 substrate. Six compounds
producing complete inhibition with IC50 values below 0.5 μM and high
selectivity for ABCG2 were identified. The number and position of methoxy
substituents appeared to be critical for both inhibition and cytotoxicity. The
best compounds, with potent inhibition and low toxicity, contained an N-
methyl-1-indolyl (compound 38) or a 6′-hydroxyl-2′,4′-dimethoxy-1-phenyl
(compound 27) moiety (A-ring) and two methoxy groups at positions 2
and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to
inhibition at positions 3 and 5, but had a negative effect at position 4.
Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly
increased cytotoxicity and, therefore, should be avoided.
combined with anticancer drugs for increasing their intracellular
concentration. Several types of ABCG2 inhibitors have been
reported, but very few representatives have shown promising
results in preclinical trials.5,6
INTRODUCTION
■
Anticancer agents constitute a large therapeutic arsenal used in
clinics. Although their efficacy is dependent on the cancer
evolution stage and location, most anticancer drugs suffer from
cell resistance to the treatment. This resistance can be related to
several mechanisms leading to subsequent loss of drug efficacy.
One of the well-established mechanisms is associated with
overexpression of ATP-binding cassette (ABC) transporters,
such as P-glycoprotein (P-gp/ABCB1), multidrug resistance
protein 1 (MRP1/ABCC1), and breast cancer resistance
protein (BCRP/ABCG2),1 which bind and extrude the
anticancer drugs out of the cells, creating drug resistance.
ABCG2 was simultaneously discovered in three laboratories
and is therefore known as ABCP for its abundance in placenta,2
BCRP for its discovery in breast cancer,3 and MXR for its
resistance to mitoxantrone.4 ABCG2 is considered to be a
privileged target for drug discovery, with the aim of conceiving
and developing inhibitors of its efflux activity that can be
Pursuing our efforts toward the identification of potent and
selective ABCG2 inhibitors, derived from the naturally
occurring flavonoids,7−9 we investigated chalcones (1,3-diary-
lpropenones) and analogues such as 1-indolyl-3-phenylprope-
nones and 1-phenyl-3-indolylpropenones (Figure 1).
Chalcones were early shown to bind to P-gp10,11 and inhibit
its transport activity.12 Different substituents on the A-ring
appeared to be required for inhibiting ABCG2,13 but the
inhibition was rather low due to inadequate substitutions in
both rings A and B.13 Very recently, the inhibition was found to
be decreased by replacing the A-ring with a naphthyl moiety, or
Received: December 6, 2011
Published: March 26, 2012
© 2012 American Chemical Society
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Figure 1. General structures of targeted chalcones and analogues.
a
Scheme 1
a
(a) KOH (50% in H2O), EtOH, 60 °C.
by B-ring methoxylation, but no effect on cytotoxicity was
reported.14 Interestingly, replacement of one phenyl with an
indolyl moiety was shown to significantly increase the chalcone
activity,15 in agreement with its presence in other types of
potent and specific ABCG2 inhibitors.16−18
The aim of this work was to shed light on structural
requirements responsible not only for selective inhibition of
ABCG2 but also for cytotoxicity. The results led to the
identification of selective chalcones and chalcone-like com-
pounds with both high potency and low cytotoxicity, making
them good candidates for future in vivo preclinical evaluation.
from 3-acetyl-N-methylindole and N-methylindolyl-3-carbox-
aldehyde in the presence of KOH in a mixture of EtOH/
H2O.20
BIOLOGICAL EVALUATION, SAR, AND
DISCUSSION
■
The substituents to be introduced on the chalcone scaffold
were chosen among those frequently found in naturally
occurring compounds, such as methoxy (OMe) and hydroxyl
(OH) groups often present at positions 2′, 4′, and 6′ of the A-
ring and 2, 3, 4, 5, and 6 of the B-ring.21 Furthermore, a primary
screening of diversely substituted chalcones concluded that the
OR substituents are quite promising in providing the most
active inhibitors and relevant structure−activity relationship
(unpublished results). By contrast, substitution of the B-ring by
halogens gave variable results: a single Cl at either position 2 or
3 was found to increase inhibition of mitoxantrone efflux,13 in
contrast to the negative effect produced at position 4, whereas
all substitutions at either position 2, 3, or 4 were negative to
inhibition of ABCG2-mediated efflux.14 Interestingly, substitu-
tion at position 2 by Cl, or by F to a lower extent, was recently
found to promote glutathione biosynthesis.22 Taking into
account such structural elements, we synthesized and evaluated
a series of 34 chalcones (Table 1) and 10 chalcone-like
analogues containing an indolyl moiety (Table 2). The
chalcones and analogues were evaluated by flow cytometry
for their ability to restore, through ABCG2 inhibition, the
intracellular accumulation of mitoxantrone, a well-known
transported anticancer agent, in an ABCG2-transfected human
fibroblast HEK293 cell line, using GF120918 as a control for
complete inhibition.
CHEMISTRY
■
The investigated chalcones and analogues were synthesized
according to the classical method illustrated in Scheme 1. For
chalcones (upper reaction A), the conveniently substituted
acetophenones and benzaldehydes were reacted together in the
presence of KOH in a mixture of water and ethanol. Some
methoxylated or ethoxylated chalcones required the preparation
of methoxylated acetophenones, ethoxylated acetophenones,
and/or ethoxylated benzaldehydes (when not commercially
available). In such a case, they were obtained by methylation or
ethylation of hydroxylated acetophenones and benzaldehydes,
by using methyl iodide or ethyl iodide in the presence of
K2CO3 in acetone.19 For 1-indolyl-3-phenylpropenones (mid-
dle reaction B), the same synthesis conditions as for reaction A
were used, starting from 3-acetyl-N-methylindol and a
derivative of benzaldehyde.20 The 3-indolyl-1-phenylprope-
nones (bottom reaction C) were also synthesized under the
same conditions, starting from an acetophenone derivative and
N-methylindolyl-3-carboxaldehyde.20 Finally, the synthesis of
1,3-diindolylpropenone (44; Table 2) was performed starting
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Table 1. Inhibition of Mitoxantrone Efflux by Substituted Chalcones in ABCG2-Transfected Cells
a
ABCG2 inhibition (%)
b
compd
2′
4′
6′
2
3
4
5
6
at 2 μM at 10 μM
class
1
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
2.0 0.61
−0.9 8.2
−6.8 9.4
2.5 13.6
−0.9 10.5
2.7 13.9
2.7 12.2
13.0 14.4
22.1 0.13
1.3 10.7
21.2 11.7
42.6 18.4
16.7 6.6
11.9 14.3
11.5 15.7
37.0 15.7
0.02 9.81
56.4 13.7
44.7 10.3
71.1 10.3
45.9 14.8
27.8 15.1
47.5 5.1
50.1 19.8
58.2 19.3
59.6 11.1
130.7 15.9
115.0 16.7
67.9 1.0
75.7 8.9
93.0 12.9
72.9 6.9
72.5 6.67
54.9 11.1
2.6 2.4
0.7 7.7
3
3
3
3
3
3
3
3
3
3
3
2
3
3
3
2
3
2
2
2
2
2
2
3
2
2
1
1
2
3
1
3
2
3
2
OMe
3
OMe
1.9 11.3
6.7 13.1
18.3 15.6
9.4 13.3
18.9 18.0
40.4 19.6
55.1 11.7
17.4 12.7
45.4 13.3
55.3 13.4
71.8 11.4
54.3 17.4
15.7 9.4
55.0 11.2
26.7 10.2
63.2 9.6
59.7 9.0
76.5 6.7
68.4 12.4
48.5 14.0
41.6 23.1
101.2 14.0
79.3 13.8
54.7 10.8
130.5 12.6
117.9 22.8
60.4 17.2
93.1 8.9
97.9 2.2
90.0 14.5
84.9 10.7
95.0 12.6
4
OMe
OMe
OMe
OMe
OMe
OMe
5
OMe
OMe
OMe
OMe
OMe
6
OMe
OMe
7
OMe
OMe
8
9
OMe
OMe
OMe
OMe
OMe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
OEt
OEt
OEt
OEt
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
OMe
OMe
OMe
OMe
OEt
OMe
OMe
OMe
OMe
OMe
OEt
OMe
OMe
OMe
OEt
OMe
OEt
OEt
OMe
OMe
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
OMe
OMe
OMe
OMe
OH
OH
OMe
OMe
OMe
OMe
OH
OMe
OMe
OH
OMe
OMe
OH
OMe
OMe
OH
OMe
OMe
OMe
OMe
OMe
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
a
The percent inhibition on ABCG2-mediated mitoxantrone transport was determined by flow cytometry as described under Experimental Section,
b
and GF120918 (5 μM) was used as a control (100 9.7%). Data are the mean SD of at least three independent experiments. The synthesis,
physical characteristics, and structural evidence were previously reported19 and are given here in the Supporting Information.
The inhibitory activity against ABCG2-mediated mitoxan-
trone efflux is reported in Tables 1 and 2, expressed as percent
inhibition observed at 2 and 10 μM concentrations of each
compound, relative to 5 μM GF120918 taken as a reference
inhibitor. For facilitating comparison and interpretation of the
results, we ranked the studied compounds in three different
classes, according to their apparent affinity and maximal extent
of inhibition:
Class 1 included high-affinity inhibitors (IC50 < 1 μM)
producing a complete inhibition at both 2 and 10 μM, similarly
to recently reported chromones.23
Class 2 comprised high-affinity inhibitors producing an
incomplete maximal inhibition (limited to 50−75%), similarly
to hydrophobic flavones7 and methoxy-trans-stilbenes.24
Class 3 was composed of low-affinity inhibitors. The
following compounds displayed especially low, if any,
inhibition: 1−7, 10, 15, 17, 42, and 43.
As shown in Table 1, chalcones in which the A-ring (1-
phenyl moiety) was substituted by OMe at positions 2′ and 6′
displayed either a low inhibition (class 3 chalcone 8) or no
inhibition at all (class 3 chalcones 1−4 and 6), independently
of the number and positions of OMe groups on the B-ring (3-
phenyl moiety). The substitution of 2′,6′-OMe groups with
ethoxyls slightly improved the activity in class 3 chalcone 9 (vs
5), but not in class 3 chalcone 10 (vs 4). Shifting the 6′-OMe
group to the 4′-position produced an increased inhibition in
class 2 chalcone 12 (vs 5 and 7). The presence of three OMe
groups on the A-ring led to the moderately active class 3
chalcones 13 (vs 5), whereas their replacement by ethoxy
groups had limited, if any, effect in chalcone 16 (vs 13).
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comparing 32 to 27 and 33 to 28. This explains why our
compound 31, without OMe at position 4, was 2−3-fold more
potent than the 4-OMe-containing lead recently reported.14
Chalcone 34, also, was ranked in the less active class 3. A
critical role of methoxy groups toward inhibition, depending on
their number and positions, was also recently demonstrated in
our group in the case of trans-stilbenes as specific ABCG2
inhibitors.24
Table 2. Inhibition by Indolylphenylpropenones
The positive contribution of the OH at position 6′ was also
observed for inhibition of ABCB1 (P-gp) by both chalcones
and flavonoids.25 However, it was not investigated, in addition
to OMe at position 2′ in previous ABCG2 studies.12,13 As
shown in the present work, the role of the OH at position 6′
was not due to a hydrogen bond with the adjacent carbonyl
group (by reducing free rotation around the bound linking A-
ring and carbonyl group) because the presence of OMe at
position 2′ (as in 27, 28, and 31) induces steric hindrance,
preventing such a conformation.
The activity of indolyl-containing chalcone analogues
(derivatives 35−44) is shown in Table 2. From this series,
we obtained three highly active derivatives (class 1 compounds
37−39). By comparing the latter series to chalcones, the indolyl
contribution undoubtedly appeared to be crucial: it was as
efficient as 2′,4′-diOMe,6′-OH-1-phenyl (A-ring), which con-
trasts with the negative effect observed for a naphthyl moiety.14
On the contrary, in the 3-indolyl series (derivatives 40−43),
the results were generally disappointing, with compounds
ranked in class 3. The great difference observed between the 1-
indolyl and 3-indolyl series may point to the impact of the
electronic distribution within the molecule on the inhibition
activity. The last investigated compound was 1,3-diindolylpro-
penone (class 3, 44): its moderate activity indicates that the
presence of at least one methoxylated phenyl group (B-ring)
was required. The importance of methoxy substituents for
ABCG2 inhibition was previously observed in other types of
compounds such as tectochrysin versus chrysin7 and methoxy
derivatives of both rotenoids26 and trans-stilbene.24
Displaying the contribution volumes indicates relationships
between compound structure and inhibitory activity, whereas
the central core was kept constant (Figure 2). As expected, the
positive effect of OH at position 6′ (in 27, 28, and 31) was due
to electrostatic contribution (red volume). This was also the
case for OMe at position 5 (in 28). By difference, the positive
contributions of OMe at position 6 (in 27, 37, and 38)
appeared to be due to steric effect (yellow volume), as well as
OMe at position 2′ (in 27, 28, and 31). The negative effect of
a
b
The conditions were the same as in Table 1. The synthesis, physical
characteristics, and structural evidence were previously reported19 and
are given in the Supporting Information.
Introduction of an OH group at the 2′-position, in chalcones
18−26, had an effect similar to that of the insertion of OMe by
providing essentially class 2 compounds similarly to 12. The
highest inhibition was observed in 27, 28, and 31, in the
concomitant presence of 6′-OH and 2′,4′-diOMe groups, which
constituted the optimal substitution pattern of the A-ring. This
was consistent with the efficiency produced by the same
substitution on the efflux of Hoechst 33342.14 The positive role
of 6′-OH in the series 27−34 was evident by comparison to the
lack of activity of the series 1−8; in contrast, it allowed a
complete inhibition to be reached by contrast to the class 2
compounds 18−23, 25, and 26. On the B-ring, both position
and number of OMe groups appeared to be important: pairs of
OMe at either positions 2 and 6 (in 27) or 3 and 5 (in 28) gave
the best inhibitors, whereas a single OMe at either position 3
(in 31) or 2 (in 30) was better than no OMe (in 29). In
contrast, substitution at position 4 was unfavorable when
Figure 2. 3D-QSAR analyses. (Left) The 44 molecules were modeled, aligned on the central core, and inserted in the databank for a computed 3D-
QSAR study. The calibration and validation coefficients were, respectively, 0.924 and 0.651. Two highly active compounds, chalcone 27 and its
indolyl analogue, 38, are displayed in capped stick, and the other molecules are represented in lines. (Right) CoMFA contribution volumes with all
molecules. Volumes were plotted with 20 for positive contribution (yellow for steric and red for electrostatic fields) and 70 for negative contribution
(green for steric and blue for electrostatic fields).
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Figure 3. Inhibition specificity for ABCG2 relative to ABCB1 and ABCC1. (A) The inhibition on ABCB1-mediated mitoxantrone efflux was
determined by flow cytometry as described under Experimental Section and normalized using control cells transfected by the empty vector (taken as
100% inhibition). (B) The percent inhibition on ABCC1-mediated calcein transport was determined using the same approach. Data are the mean
SD of two independent experiments.
OMe at position 4, in 32 versus 27 and in 33 versus 28,
appeared to be electrostatic (blue volume). Surprisingly, the N-
methyl-1-indolyl did not display particularly positive inter-
actions, suggesting that hydrophobic interactions (not dis-
played here because all compounds were essentially hydro-
phobic) were most likely essential for the binding. In contrast,
the negative effect produced by a naphthyl moiety, as recently
reported,14 might be due to steric hindrance (green volume on
the left side), similarly to the N-methyl-3-indolyl of derivatives
40−44 on the right side.
On the basis of above results, we selected all class 1 inhibitors
(27, 28, 31, and 37−39) for further experimental inves-
tigations. They were first evaluated for their selectivity, under
similar inhibitory conditions, on HEK293 cell lines transfected
with the two other multidrug transporters, ABCB1 (P-gp) and
ABCC1 (MRP1), and using HEK293 cells transfected with the
empty vector (HEK293-pcDNA3.1) as a control. As shown in
Figure 3, the six derivatives did not show any significant
inhibition up to 10 μM, indicating a high selectivity for ABCG2
(BCRP).
Second, the IC50 values for ABCG2 were determined from
Figure 4A and are indicated in Table 3: they were all below 0.5
μM, with compound 28 being the most potent inhibitor (IC50
= 0.17 μM). Finally, the cytotoxicity of each inhibitor was
evaluated on both sensitive control cells and resistant ABCG2-
transfected cells. As shown in Figure 4B, the six compounds
induced very similar cytotoxicity profiles in both types of cell
lines.
The absence of any apparent cross-resistance in ABCG2-
transfected cells suggests that the chalcones were not
transported by ABCG2, as this was also the case for other
ABCG2-specific inhibitors such as flavones,7 chromones,23 and
methoxystilbenes.24 Cytotoxicities were produced at higher
concentrations than for inhibition of ABCG2 transport activity.
However, marked differences were observed among the
different compounds: the IG50 value of compound 39 was
50-fold lower than the high value obtained for compounds 27
and 38, whereas the remaining chalcones, 28, 31, and 37,
displayed intermediate effects (Table 3).
diOMe-substituted lead reported by Juvale et al.,14 our present
results indicate that it should be at least 10−50-fold more
cytotoxic than our compounds 27 and 38, which therefore
could prevent its use for in vivo experiments. The OMe
substituents at positions 3 and 5 played a dual role because they
also increased ABCG2 inhibition. In contrast, OMe sub-
stitutions at positions 2 and 6 were not critical for cytotoxicity,
neither was the use of an 1-indolyl, instead of a 1-phenyl,
moiety (in 38 versus 27). Such marked differences in
cytotoxicity were mostly responsible for the differences
observed in calculating the therapeutic ratio because the IC50
values for ABCG2 inhibition were more constant. The two less
cytotoxic compounds with IG50 values around 50 μM, that is,
chalcone 38 and indolylphenylpropenone 27, therefore
displayed the highest values of therapeutic ratio (185 and
143, respectively), which constitutes an important parameter
for considering future in vivo experiments. A much lower
therapeutic ratio was obtained previously with other types of
inhibitor: around 40 for 6-prenylchrysin7 and even less for
acridones,6,27 which nevertheless displayed a significant in vivo
activity.28
Overall, our results emphasize the differential roles of up to
six OMe substituents: (i) the OMe at position 2′ of the 1-
phenyl moiety greatly contributes through steric effects, in
addition to OH at position 6′, to inhibition potency; (ii) the
two OMe at positions 2 and 6 of the 3-phenyl moiety also
greatly contribute to inhibition potency without apparent effect
on cytotoxicity; (iii) the two OMe at vicinal positions 3 and 5
contribute, even more strongly, to inhibition potency but also
induce additive effects on cytotoxicity; (iv) finally, the most
negative role is played by OMe at position 4, which is
unfavorable to inhibition, both sterically and electrostatically,
and greatly increases the cytotoxicity. This is the first work
describing structure−activity relationships of cytotoxicity for
ABCG2 inhibitors.
In conclusion, chalcones and indolyphenylpropenones
constitute promising leads as inhibitors of ABCG2. The easy
chemical access to such compounds and their high activity and
specificity are fundamental prerequisites for preclinical trials.
The two compounds selected here with a high therapeutic ratio,
38 and 27, without OMe at position 4, constitute good
candidates to enter preclinical trials to validate their interest for
further development. Our recent studies on in vivo evaluation
of acridones28 will be helpful to speed in vivo testing of the
present lead chalcones.
These results show the important role played by OMe
groups at position 4 (in 39 and 37) as well as at both positions
3 (in 31 and 28) and 5 (in 28) on cytotoxicity, probably due to
interaction with additional target(s), different from ABCG2,
which are present in both control and ABCG2-transfected cells.
Although no cytotoxicity data were reported for the 3,4-
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Table 3. Inhibitory Activity and Cytotoxicity of the Most
Potent Chalcones and Indolylphenylpropenones
IG50
a
IC50 (drug efflux
inhibition, μM)
(cytotoxicity,
therapeutic ratio
(IG50/IC50)
compd
μM)
27
28
31
37
38
39
0.35 0.14
0.17 0.07
0.26 0.04
0.45 0.09
0.27 0.08
0.35 0.09
50.0 3.9
6.0 0.4
10.0 2.1
4.0 0.2
50.0 6.2
1.0 0.1
143
35
38
9
185
3
a
The therapeutic ratio of class 1 compounds was calculated from the
values of IG50 (concentration producing 50% inhibition of cell growth)
and IC50 (concentration producing half-maximal inhibition of ABCG2-
mediated mitoxantrone efflux).
mesh. Unless otherwise stated, reagents were obtained from
commercial sources and were used without further purification.
Biology. Materials. Mitoxantrone, calcein-AM, and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
were purchased from Sigma-Aldrich (France). All other
reagents were commercial products of the highest available
purity grade.
Compounds. All compounds were dissolved in DMSO and then
diluted in DMEM high-glucose medium. The stock solution was
stored at −20 °C and warmed to 25 °C just before use.
Cell Cultures. The human fibroblast HEK293 cell line transfected
with either ABCG2 (HEK293-ABCG2) or the empty vector
(HEK293-pcDNA3.1 cells) was obtained as previously described.28
The human fibroblast HEK293 cell line transfected with either ABCB1
(P-gp) or ABCC1 (MRP1) was kindly provided by Dr. S. E. Bates
(NCI, NIH, Bethesda, MD, USA). All cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM high glucose),
supplemented with 10% fetal bovine serum (FBS), 1% penicillin/
streptomycin, and drug supplemented in some cases with either 0.75
mg/mL G418 (HEK293-pcDNA3.1 and HEK293-ABCG2) or 2 mg/
mL G418 (HEK293-ABCB1) or 5 μM etoposide (HEK293-ABCC1).
ABCG2- and ABCB1-Mediated Mitoxantrone Transport. The
efflux assays were determined as previously reported29 with minor
modifications. Cells were seeded at a density of 1 × 105 cells/well in
24-well culture plates. After 48 h, HEK293-ABCG2 and HEK293-
ABCB1 cells were exposed to mitoxantrone (5 μM) with or without
compounds at 2 or 10 μM, and incubated at 37 °C in 5% CO2 for 30
min. The cells were then washed with phosphate buffer saline (PBS)
and, after being trypsinized and subsequently resuspended in ice-cold
PBS (0.2 mL), they were kept on ice until analysis by flow cytometry.
The data of intracellular drug fluorescence were acquired using a
FACSCalibur flow cytometer equipped with a 635 nm red diode laser
and a 670 nm bandpass filter (FL4-H) controlled by CellQuest Pro
software. At least 10,000 events were collected, and the geometric
mean fluorescence (GMean) for each histogram was used as the
measure of fluorescence for calculation of efflux values. Cells in PBS
alone yielded the Blank histogram (cell autofluorescence), whereas
cells in the presence of mitoxantrone alone, or GF120918 (5 μM) and
mitoxantrone, constituted the controls. In ABCG2-mediated mitox-
antrone transport, the percentage of inhibition was calculated by using
eq 1
Figure 4. Inhibition of ABCG2 efflux activity and intrinsic cytotoxicity.
(A) The IC50 values (concentrations for half-maximal inhibition) on
ABCG2-mediated mitoxantrone efflux were determined by flow
cytometry, as described under Experimental Section. The curves
were fitted using GraphPad Prism 5 software. (B) Cell viability of
ABCG2-transfected HEK293 cells and control cells upon treatment
with compounds 27, 28, 31, and 37−39, up to 100 μM, for 72 h. The
values represent the mean SD of percent cell viability with respect to
the untreated control. Data correspond to at least two independent
experiments performed in triplicate.
EXPERIMENTAL SECTION
■
1
Chemistry. H and 13C NMR spectra were recorded on a Bruker
̈
% inhibition = (C − M)/(I − M) × 100
(1)
AC-400 instrument (400 MHz). Chemical shifts (δ) are reported in
parts per million relative to Me4Si (internal standard). Electrospray
ionization (ESI) mass spectra were acquired by the Analytical
Department of Grenoble University on an Esquire 300 Plus Bruker
Daltonis instrument with a nanospray inlet. Combustion analyses were
performed at the Analytical Department of Grenoble University, and
all tested compounds have a purity of at least 95%. Thin-layer
chromatography (TLC) used Merck silica gel F-254 plates (thickness
= 0.25 mm). Flash chromatography used Merck silica gel 60, 200−400
where C corresponds to the intracellular fluorescence of cells in the
presence of compounds and mitoxantrone, and M to the intracellular
fluorescence of cells in the presence of only mitoxantrone. Here, I is
the intracellular fluorescence of cells in the presence of the reference
inhibitor GF120918 and mitoxantrone. In ABCB1-mediated mitoxan-
trone transport, the cells transfected with the empty vector (HEK293-
pcDNA3.1) were used as control. The percentage of inhibition was
calculated by using eq 2
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Journal of Medicinal Chemistry
Article
% inhibition = (C − M)/(Cev − M) × 100
(2)
ACKNOWLEDGMENTS
■
Drs. R. W. Robey and S. E. Bates, NCI-NIH, Bethesda, MD,
USA, are acknowledged for providing HEK-293 cells trans-
fected by either ABCB1 (MDR-19) or ABCC1, and
GlaxoSmithKline is acknowledged for providing GF120918.
C.G. is the recipient of a doctoral fellowship from the Ligue
Nationale Contre le Cancer. G.V. is recipient of a mobility
fellowship from the Brazilian CAPES (Process 2303/10-8).
Financial support was provided by the CNRS and Universite
Lyon 1 (UMR 5086), the Ligue Nationale Contre le Cancer
where C and M are defined as above. In contrast to eq 1, Cev
corresponds to the intracellular fluorescence of cells transfected with
the empty vector in the presence of mitoxantrone.
MRP1-Mediated Calcein Transport. HEK293 cells transfected with
either ABCC1 or the empty vector were seeded at a density of 1 × 105
cells/well in 24-well culture plates. After 48 h, cells were exposed to
calcein-AM (0.2 μM) with or without compounds at 2 or 10 μM and
incubated at 37 °C in 5% CO2 for 30 min. The cells were treated as
described above and analyzed with a FACSCalibur flow cytometer
equipped with a 488 nm argon laser and a 530 nm bandpass filter
(FL1-H) controlled by CellQuest Pro software. In ABCC1-mediated
calcein transport, the cells transfected with the empty vector
(HEK293-pcDNA3.1) were used as a control, and the percentage of
inhibition was calculated with eq 2.
Cytotoxicity Assays. The cytotoxicity assays were performed using
the MTT colorimetric assay,30 based on mitochondrial dehydro-
genases of viable cells that cleave the MTT, yielding purple MTT
formazan which is quantified spectrophotometrically. HEK293-ABCG2
and HEK293 cells transfected with the empty vector (HEK293-
pcDNA3.1) were seeded in 96-well culture plates at a 1 × 104 cells/
well density and allowed to attach for 24 h at 37 °C in 5% CO2. The
treatment was done with various concentrations of compounds in a
final volume of 200 μL and allowed to grow for 72 h at 37 °C in 5%
CO2. After the appropriate treatment time, 20 μL of MTT solution (5
mg/mL) was added to each well and incubated for 4 h at 37 °C in 5%
CO2. Then the culture medium was discarded, each well was washed
with PBS, and 100 μL of DMSO was added into each well and mixed
by gentle shaking for 10 min. Absorbance was measured in a
microplate reader at 570 nm. Experimental conditions were set in
triplicate, and control experiments were performed with DMEM high
glucose containing 0.1% DMSO (v/v). Data are plotted as a
percentage of reduced cell viability compared to control (untreated
cells) that was taken as 100%.
Molecular Modeling. The compounds were analyzed by using the
Sybyl X 1.3 molecular modeling suite software31 and minimized by the
MMFF94 force field32 with a dielectric constant of 80 and an
electrostatic cutoff of 16 Å. Minimized molecules were aligned on the
central common core and put in a database; lateral chains of molecules
were manually checked and aligned on a common position, and the
modified conformation was minimized. The differences in internal
energy between the two conformations must be <20 kcal mol−1 to
validate the aligned conformations. Using the 44 aligned molecules, a
3D-QSAR study was initiated with the CoMFA program.33 The grids
of electrostatic and steric potential fields were computed using the C3
atom with a charge of +2 as a probe. The grid was filtered with 6 kcal
mol−1 as the minimal variation to select probes, and validation by the
leave-one-out (LOO) method was chosen.34
́
(Equipe labellisee
(CIBLE 2010).
́ ́ ̂
Ligue 2012), and the Region Rhone-Alpes
ABBREVIATIONS USED
■
ABC, ATP-binding cassette; MDR, multidrug resistance;
MRP1, multidrug resistance protein 1; MTT, 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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ASSOCIATED CONTENT
■
S
* Supporting Information
Characterization of chemical compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(A.B.) Phone: (33) 4 7663 5311. E-mail: ahcene.
boumendjel@ujf-grenoble.fr. (A.D.P.) Phone: (33) 4 7272
2629. E-mail: a.dipietro@ibcp.fr.
Author Contributions
∥Both Ph.D. students (G.V. and C.G.) and senior investigators
(A.D.P. and A.B.) equally contributed to the work.
Notes
The authors declare no competing financial interest.
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