6-Halogenochromones Bearing Tryptamine: One-Step Access to Potent and Highly Selective Inhibitors of Breast Cancer Resistance Protein

Full text of DOI:10.1002/cmdc.201200154

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DOI: 10.1002/cmdc.201200154
6-Halogenochromones Bearing Tryptamine: One-Step Access to Potent and
Highly Selective Inhibitors of Breast Cancer Resistance Protein
Glaucio Valdameri,[a, b] Estelle Genoux-Bastide,[c] Charlotte Gauthier,[a] Basile Peres,[c] Raphaꢀl Terreux,[d]
Sheila M. B. Winnischofer,[b] Maria E. M. Rocha,[b] Attilio Di Pietro,[a] and Ahcꢁne Boumendjel*[c]
Most anticancer drugs are rendered less efficacious due to cell
resistance to chemotherapy related to various mechanisms. A
major mechanism is associated with the overexpression of ATP
binding cassette (ABC) transporters, especially P-glycoprotein
(Pgp/ABCB1), multidrug resistance-associated protein 1 (MRP1/
ABCC1) and breast cancer resistance protein (BCRP/ABCG2),[1]
which traffic chemotherapeutic agents out of cancer cells.
ABCG2 was simultaneously discovered by three research
groups and named ABCP for its abundance in placenta,[2] BCRP
for its identification in breast cancer cell lines,[3] and MXR for
its resistance to mitoxantrone.[4] ABCG2 constitutes an impor-
tant target for the design of efflux inhibitors that would, when
co-administered with an anticancer agent, give increased intra-
Scheme 1. Retrosynthetic rationale for the synthesis of targeted BCRP inhibi-
tors, and the structures of the commercially available starting materials
1 and 2.
cellular drug concentrations and hence greater cytotoxicity.
While several types of ABCG2 inhibitors have been evaluated
in vitro, very few have entered preclinical trials.[5–9]
We recently discovered that some substituted chromones
are selective and potent ABCG2 inhibitors.[10] These com-
pounds were synthesized in five steps, and the overall yields
were quite low. In pursuing our efforts toward structurally
simple and easily accessible specific inhibitors of BCRP, we in-
vestigated 6-halogenochromones linked to a tryptamine unit,
obtained in only one step, as new potent inhibitors
(Scheme 1).
cule;[10] 2) halogens, especially bromine and iodine, have a posi-
tive contribution to inhibitory activity;[11,12] 3) halogens could
open interesting opportunities for the generation of further
potential inhibitors, as they can be easily replaced by
a number of chemical entities.
Access to target compounds 3–7 was achieved in one step
by coupling 6-substituted-4-oxo-4H-chromene-2-carboxylic
acid (1) with tryptamine (2) in the presence of bis(2-oxo-3-oxa-
zolidinyl)phosphonic chloride (BOP-Cl) as the coupling agent
(Scheme 2; full details are given in the Supporting Informa-
tion). 6-Iodo-4-oxo-4H-chromene-2-carboxylic acid (R=I) was
not commercially available, but was easily obtained by hydrol-
ysis of the commercially available corresponding ethyl ester
with sodium hydrogen carbonate (20% in water) at 808C.
The test compounds were first screened by flow cytometry
for their effects on the inhibition of mitoxantrone efflux in
The choice of C-6 as the site of halogenation was motivated
by a number of considerations: 1) the presence of a hydropho-
bic halogen at the C-6 position fulfills the previously identified
need for a hydrophobic substituent in this part of the mole-
[a] G. Valdameri,+ C. Gauthier, Dr. A. Di Pietro++
Mechanisms and Modulation of Drug Resistance Team
Institute of Biology and Chemistry of Proteins
BMSSI UMR 5086 CNRS/University of Lyon 1
7 Passage du Vercors, 69367 Lyon (France)
[b] G. Valdameri,+ Prof. S. M. B. Winnischofer, Prof. M. E. M. Rocha
Department of Biochemistry and Molecular Biology
Federal University of Paranꢀ
Centro Politꢁcnico, Jardim da Amꢁricas, 81531-980 Curitiba, PR (Brazil)
[c] Dr. E. Genoux-Bastide,+ B. Peres, Prof. A. Boumendjel++
Department of Molecular Medicinal Chemistry
Universitꢁ Joseph Fourier-Grenoble 1/CNRS UMR 5063
Bꢂtiment E Andrꢁ Rassat, Pꢃle Chimie, BP 53, 38041 Grenoble (France)
E-mail: Ahcene.Boumendjel@ujf-grenoble.fr
[d] Dr. R. Terreux
Bioinformatics: Structure and Interactions Team
Institute of Biology and Chemistry of Proteins
BMSSI UMR 5086 CNRS/University of Lyon 1
7 Passage du Vercors, 69367 Lyon (France)
[+] These junior authors contributed equally to this work.
++] These senior authors contributed equally to this work.
[
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200154.
Scheme 2. Synthesis of targeted inhibitors 3–7. Reagents and conditions:
a) BOP-Cl, Et3N, DMF, RT, 24 h, 41–55%.
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Table 1. Inhibition of mitoxantrone efflux in ABCG2-transfected HEK293
Table 2. Inhibition of mitoxantrone efflux (ABCB1) and calcein efflux
cells.
(MRP1) in transfected HEK293 cells.
Compd
Inhibition [%]
ABCB1[a]
ABCC1[b]
1 mm
5 mm
1 mm
5 mm
3
6
7
À23.58Æ13.53
À26.76Æ14.94
À21.49Æ13.44
À31.92Æ20.18
3.97Æ3.05
4.84Æ3.62
0.96Æ0.08
3.07Æ3.02
5.92Æ4.28
5.94Æ4.80
À33.32Æ21.28
À34.63Æ20.93
Compd
R
Inhibition[a] [%]
IC0.5[b] [mm]
1 mm
5 mm
[a] For ABCB1-transfected cells, inhibition (%) was determined using the
control agent GF120918 (5 mm), set as 100% inhibition. [b] For MRP1-
transfected cells, inhibition (%) was normalized using pcDNA3.1-transfect-
ed cells as the control. Data are the mean ÆSD of at least three inde-
pendent experiments.
3
4
5
6
7
H
Me
F
Br
I
44Æ11
28Æ3
58 Æ9
46Æ4
59Æ3
75Æ12
77Æ13
0.55
–
0.60
0.25
0.30
41Æ3
57Æ20
56Æ10
[a] Inhibition (%) was calculated relatively to the control agent GF120918
(5 mm), set as 100% inhibition. Inhibition (%) values were determined by
flow cytometry as described in the Experimental Section. [b] Since the
maximal inhibition was not complete, the affinity was estimated as the
IC0.5 value—the concentration required to cause half maximal inhibition.
Data are the mean ÆSD of at least three independent experiments.
transfected HEK293 cells overexpressing ABCG2, using
GF120918, a known ABCG2 modulator, as a reference (Table 1).
Lead compound 3 that lacks a substituent at C-6 (R=H) was
tested for comparison. The presence of a fluoro substituent at
C-6 (compound 5) leads to an equally active inhibitor. Substitu-
tion of the fluoro with a methyl group (compound 4), which is
approximately the same size but has different electronic ef-
fects, provides a slightly less active inhibitor. As shown in
Table 1, the presence of either a bromo (compound 6) or iodo
(compound 7) atom at C-6 of the chromone moiety leads to
the most active compounds in the series, bromo analogue 6
being the more potent of the two, with a slightly lower IC0.5
value. The improved activity of compounds 6 and 7 might be
due to the size and/or hydrophobicity of the halogen atom.
The next step in the evaluation process was to check the se-
lectivity of the most active inhibitors. Indeed, ABCG2 inhibitors
often lack selectivity and also target the two other well-known
transporters P-gp/ABCB1 and MRP1/ABCC1. For this purpose,
we evaluated compounds 3, 6 and 7 in P-gp- and MRP1-trans-
fected cells by measuring accumulation of mitoxantrone (P-gp
substrate) in P-gp cells and calcein (MRP1 substrate) in MRP1
cells. As shown in Table 2, the three selected ABCG2 inhibitors
did not induce any inhibitory effect against P-gp and MRP1, in-
dicating good selectivity for ABCG2.
Figure 1. Cytotoxicity of a) compound 3 and b) compound 6. Cell survival
was determined by MTT assays as described in the Experimental Section.
Cell viability of HEK293-ABCG2 cells (&) and HEK293-pcDNA3.1 cells (*),
upon 72 h treatment with the compounds in increasing concentrations, as
indicated.
As mentioned above, our ultimate goal was to obtain suffi-
cient biological data to allow the subsequent preclinical evalu-
ation of the most promising inhibitor. Therefore, evaluation of
the intrinsic cytotoxicity and the ability of these compounds to
chemosensitize cancer cells toward anticancer drugs are essen-
tial. First, we evaluated the cytotoxicity of derivatives 3 and 6
in both resistant (ABCG2 overexpressing) and sensitive
(HEK293-pcDNA3) cell lines. As shown in (Figure 1), the most
potent ABCG2 inhibitor (compound 6) was only toxic at high
concentrations, with an IG50 (concentration producing 50% in-
hibition of cell growth) value of 60 mm, giving a therapeutic
index of 240. A similar IG50 value was obtained with compound
3 in control cells (HEK293-pcDNA3.1).
Finally, we evaluated the ability of the inhibitors to potenti-
ate the effect of anticancer drugs on cancer cells. Compounds
3 or 6 were co-administrated in combination with either mitox-
antrone or SN-38, the active metabolite of irinotecan. The in-
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hibitory activity was evaluated at two concentrations (0.2 and
1 mm) and similar profiles were obtained with mitoxantrone or
SN-38, as illustrated for compound 3 in Figure 2, indicating
that inhibition of BCRP-mediated drug efflux was indeed corre-
lated to increased cytotoxicity. Similar effects were produced
by compound 6 (data not shown). The addition of the chro-
mone-derived inhibitor therefore potentiated the antiprolifera-
tive effect of the anticancer drug.
analogues with potentially improved potency against ABCG2.
For example, compounds 6 and 7 are good reagents for
Suzuki and Heck cross-coupling reactions with diverse boronic
acid derivatives.
Experimental Section
Chemistry
Experimental protocols and characterization data for compounds
3–7 are given in the Supporting Information.
Biology
General: Mitoxantrone, calcein-AM and 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) were purchased from
Sigma–Aldrich (France). All other reagents were commercial prod-
ucts of the highest available purity grade. All compounds were dis-
solved in DMSO and then diluted in Dulbecco’s modified Eagle’s
medium (DMEM). The stock solution was stored at À208C and
warmed to 258C just before use.
Cell cultures: Human fibroblast HEK293 cell lines transfected with
either ABCG2 (HEK293-ABCG2) or the empty vector pcDNA3.1
(HEK293-pcDNA3.1) were obtained as previously described.[14]
Human fibroblast HEK293 cell lines transfected with either MDR1
or MRP1 (HEK293-MDR1 or HEK293-MRP1, respectively) were kindly
provided by Dr. S. E. Bates (US National Cancer Institute–National
Institutes of Health, Bethesda, MD, USA). All cells were maintained
in high-glucose DMEM, supplemented with 10% fetal bovine
serum (FBS), 1% penicillin/streptomycin, and with the selection
drug (0.75 mgmLÀ1 G418 for HEK293-pcDNA3.1 and HEK293-
ABCG2, 2 mgmLÀ1 G418 for HEK293-MDR1, or 5 mm etoposide for
HEK293-MRP1. Selection drug was finally omitted, in flow cytome-
try experiments.
ABCG2- and ABCB1-mediated drug transport: HEK293 cells were
seeded at a density of 1ꢃ105 cells/well into 24-well culture plates.
After 48 h incubation, cells were exposed to 5 mm mitoxantrone
(HEK293-ABCG2 and HEK293-MDR1) for 30 min at 378C, in the pres-
ence or absence of test compounds at various concentrations.
After cell washing with phosphate buffer saline (PBS), the cells
were trypsinized. The intracellular drug fluorescence was moni-
tored with a FACS Calibur cytometer (Becton–Dickinson). At least
10000 events were collected, for which the maximal fluorescence
(100%) was the difference between geometric mean fluorescence
of cells incubated with 5 mm GF120918 and without inhibitor (con-
trol). Mitoxantrone was used as an ABCB1 substrate for direct com-
parison with ABCG2. It is transported by ABCB1 at a lower rate
than rhodamine 123 and anthracyclines but indeed constitutes
a true substrate,[15] the transport of which is fully inhibited by the
characteristic inhibitor GF120918. In addition, many experiments
showed the same efficiency of an established ABCB1 inhibitor on
various substrates.
Figure 2. Sensitization to mitoxantrone and SN-38. Cell viability of HEK293-
ABCG2 cells upon co-treatment with compound 3 and a) mitoxantrone
(MXR) or b) SN-38 (0–0.5 mm) for 72 h. Parallel experiments with only mitox-
antrone or SN-38 were performed with HEK293-pcDNA3.1 and ABCG2-trans-
fected cells. The values represent the mean ÆSD of percent cell viability
with respect to the untreated control. Data correspond to at least three
independent experiments performed in triplicate. ABCG2: *; ABCG2+3
(0.2 mm): &; ABCG2+3 (0.1 mm): &; empty vector: *.
In conclusion, the present work describes the very short and
easy access to potent, selective and nontoxic inhibitors of
ABCG2. The most active inhibitor (compound 6) can be pre-
pared in multigram scale without limitation. Owing to its po-
tency, selectivity and very low toxicity, compound 6 is being
considered for in vivo evaluation in ABCG2-xenograft models
with the aim of confirming its effectiveness on the sensitiza-
tion of tumors to anticancer agents.[13] Furthermore, com-
pound 6, as well as its analogues 5 and 7, constitute ideal
starting blocks for the generation of further chemical diverse
MRP1-mediated transport: HEK293 cells transfected with either
MRP1 or the empty vector (pcDNA3.1) were seeded at a density of
1ꢃ105 cells/well into 24-well culture plates. After 48 h incubation,
cells were exposed to 0.2 mm calcein-AM and analyzed by flow cy-
tometry as described above. The maximal fluorescence (100%) was
the difference between geometric mean fluorescence of control
cells (HEK293-pcDNA3.1) and MRP1-transfected cells, incubated
with substrate but without inhibitor.
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Acknowledgements
The authors acknowledge Drs. R. W. Robey and S. E. Bates (US
National Cancer Institute–National Institutes of Health, Bethesda,
MD, USA) for providing transfected HEK293 cells, and Glaxo-
SmithKline for providing GF120918. C.G. received a doctoral fel-
lowship from the Ligue Nationale Contre le Cancer (France). G.V.
received a mobility fellowship from the Brazilian government
through the Coordenażo de AperfeiÅoamento de Pessoal de
Nꢄvel Superior (CAPES) initiative (2303/10-8). Financial support
was provided by the French National Centre for Scientific Re-
search (CNRS)/Universitꢁ Lyon 1 (France) (UMR 5086), the Ligue
Nationale Contre le Cancer (Equipe labellisꢁe Ligue 2012), the
Rꢁgion Rhꢃne-Alpes Council (France) (CIBLE 2010), and an inter-
national grant from the French National Research Agency (ANR)
and the Hungarian National Institutes of Health (2010-INT-1101-
01).
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Keywords: ABCG2
· breast cancer resistance protein ·
Received: March 20, 2012
chromones · inhibitors · multidrug resistance
Published online on May 21, 2012
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