Chromones bearing amino acid residues: Easily accessible and potent inhibitors of the breast cancer resistance protein ABCG2

Article abstract of DOI:10.1016/j.ejmech.2020.112503

The Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the G class of ABC (ATP-Binding Cassette) proteins, which is known as one of the main transporters involved in the multidrug resistance (MDR) phenotype that confer resistance to anticancer drugs. The aim of this study was to design, synthesize and develop new potent and selective inhibitors of BCRP that can be used to abolish MDR and potentialize clinically used anticancer agents. In previous reports, we showed the importance of chromone scaffold and hydrophobicity for the inhibition of ABC transporters. In the present study we report the design and development of chromones linked to one or two amino acids residues that are either hydrophobic or found in the structure of FTC, one of most potent (but highly toxic) inhibitors of BCRP. Herewith, we report the synthesis and evaluation of 13 compounds. The studied molecules were found to be not toxic and showed strong inhibition activity as well as high selectivity toward BCRP. The highest activity was obtained with the chromone bearing a valine residue (9c) which showed an inhibition activity against BCRP of 50 nM. The rationalization of the inhibition potential of the most active derivatives was performed through docking studies. Taken together, the ease of synthesis and the biological profile of these compounds render them as promising candidates for further development in the field of anticancer therapy.

Full text of DOI:10.1016/j.ejmech.2020.112503

European Journal of Medicinal Chemistry 202 (2020) 112503
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Chromones bearing amino acid residues: Easily accessible and potent
inhibitors of the breast cancer resistance protein ABCG2
,
b
,
c
b
c
c
Emile Roussel a , Alexis Moreno , Nicolas Altounian , Christian Philouze ,
ꢀ
a
a
c
b
a, *
ꢀ ꢁ
ꢁ
Basile Peres , Aline Thomas , Olivier Renaudet , Pierre Falson , Ahcene Boumendjel
a Univ. Grenoble Alpes, CNRS, DPM UMR 5063, F-38041, Grenoble, France
b Univ. de Lyon 1, CNRS, MMSB UMR 5086, Drug Resistance & Membrane Proteins Lab, 69367, Lyon, France
c Univ. Grenoble Alpes, CNRS, DCM UMR 5250, F-38041, Grenoble, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the G class of ABC (ATP-Binding Cassette)
proteins, which is known as one of the main transporters involved in the multidrug resistance (MDR)
phenotype that confer resistance to anticancer drugs. The aim of this study was to design, synthesize and
develop new potent and selective inhibitors of BCRP that can be used to abolish MDR and potentialize
clinically used anticancer agents. In previous reports, we showed the importance of chromone scaffold
and hydrophobicity for the inhibition of ABC transporters. In the present study we report the design and
development of chromones linked to one or two amino acids residues that are either hydrophobic or
found in the structure of FTC, one of most potent (but highly toxic) inhibitors of BCRP. Herewith, we
report the synthesis and evaluation of 13 compounds. The studied molecules were found to be not toxic
and showed strong inhibition activity as well as high selectivity toward BCRP. The highest activity was
obtained with the chromone bearing a valine residue (9c) which showed an inhibition activity against
BCRP of 50 nM. The rationalization of the inhibition potential of the most active derivatives was per-
formed through docking studies. Taken together, the ease of synthesis and the biological profile of these
compounds render them as promising candidates for further development in the field of anticancer
therapy.
Received 26 February 2020
Received in revised form
20 May 2020
Accepted 23 May 2020
Available online 5 July 2020
Keywords:
Chromones
BCRP
ABCG2
Inhibitors
MDR
Drug efflux
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
(ABCG2), which are overexpressed in tumours and therefore are
actively studied [6].
Multidrug resistance (MDR) against anticancer drugs is one of
the major causes of chemotherapy failure [1e3]. Multidrug resis-
tance (MDR) results in the loss of activity of anticancer agents
against cells possessing the MDR phenotype. Multiple mechanisms
could be involved in the emergence of MDR, including drug inac-
tivation, target alteration, blockade of apoptosis, stimulation of
DNA repair mechanisms and drug efflux [2,4,5]. The latter is caused
by the overexpression of membrane proteins from the ABC super-
family that export anticancer drugs outside the cell, leading to the
reduction of the drug concentration and ultimately decrease the
therapeutic effect on cell proliferation.
BCRP is the most recent discovered ABC human protein [7e9]. It
is widely expressed in many organs like lungs, gut, liver, placenta
and blood-brain barrier [10,11]. Moreover, it is naturally overex-
pressed in stem cells and present in organs responsible for ab-
sorption (intestine), elimination (liver and kidney) and distribution
of drugs (blood-brain barrier and blood-placental barriers) [12,13].
BCRP is a monomer composed of 655 amino acids making it the
smallest ABC protein so far reported [7e9]. BCRP is one of the ABC
proteins that is functional once it is dimerized [7e9]. Recently, the
3D structure of human full-length BCRP by electron crystallography
or cryo-electron microscopy was reported [14e16].
Three ABC exporters play an important role in the MDR
phenotype, P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP
Over the past three decades, the ABC transporters have been
extensively investigated as targets for the development of in-
hibitors aiming to enhance and restore anticancer drugs potential
[4,17,18]. In this regard, BCRP was the subject of extensive studies to
conceive and develop in vivo effective inhibitors to be used as
* Corresponding author.
E-mail address: ahcene.boumendjel@univ-grenoble-alpes.fr (A. Boumendjel).
https://doi.org/10.1016/j.ejmech.2020.112503
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
chemosensitizers that could work additively or synergistically with
an anticancer drug [19e21]. An important number of in cellulo
effective inhibitors has been reported but only a few of them were
subjected to the preclinical studies [22]. Fumitremorgin (FTC) was
the first selective inhibitor of ABCG2, which was considered clini-
cally useless because of its high neurotoxicity [23]. Nevertheless, it
was used an inspiration tool for the development of analogues such
as Ko143, less toxic and potent inhibitor [24,25]. Both FTC and
Ko143 are strong inhibitors of ABCG2 and composed of fragments
found in some amino acids, such as tryptophane, leucine and pro-
line. Otherwise, our previous work on the BCRP/ABCG2 protein led
to the development of chromone derivatives as active, selective,
nontoxic inhibitors. Among them, the MBLeIIe141 (Fig. 1) has
shown promising in vivo activity [19,20,26e28].
the corresponding carboxylic acid with 46e83% yields. The second
peptide coupling reaction was performed in the same conditions as
before to afford compounds 12a-b and 13a-b with 31e41% yields.
Notably, during this second peptide-coupling reaction we observed
a partial racemization on the asymmetric carbon of the primarily
introduced amino acid [32].
Structures of the new compounds were fully elucidated through
NMR characterization. In addition, X-ray analysis of 9b and 9c
confirmed that no racemization had occurred during the first
coupling reactions (Fig. 2). Supplementary data is available on
request from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK,
quoting the deposition number CCDC-1874945 and -1874944 for 9b
and 9c respectively.
Based on the structure of MBLeIIe141, the importance of hy-
drophobicity to the inhibition potential and inspired from FTC and
Ko143, we focused our present study on chromones bearing one or
two hydrophobic amino acids with the aim to develop new class of
potent inhibitors that are biological active, non-cytotoxic and
specific toward BCRP. The introduction of three amino acids was
avoided due anticipated poor solubility that potentially hinder the
biological evaluation. Hence, our study targeted 2 series of
chromones-embedded amino acids analogues (Fig. 1).
2.2. ABCG2 inhibition activity and cytotoxicity
The new compounds of the two series were evaluated for their
ability to inhibit the efflux of mitoxantrone (MX), an anticancer
drug substrate of BCRP that, being fluorescent, can be easily
quantified by flow cytometry. The inhibition potency of the com-
pounds was investigated in human embryotic kidney ABCG2-
transfected cells, HEK293-ABCG2, and their negative control,
HEK293 cells. Moreover, the intrinsic cytotoxicity of the com-
pounds was evaluated using the MTT test on the same cell lines.
Results are displayed in Table 1.
2. Results and discussion
For series I (compounds bearing one amino acid), the maximal
2.1. Chemistry
percentage of inhibition at 1 mM of tested compound is close to the
references, Ko143 and MBLeIIe141. The inhibition activity is
stronger with a bromine atom in position 2 (9a-e) than in position 4
(8a-d) of the benzyloxy group. Such positional effect could be
observed by comparing 8a and 9a, displaying EC50 of ~0.6 vs
The 2 series of analogues shown in Fig. 1 were synthesized ac-
cording to the general synthetic pathway described in Scheme 1.
The chromone moiety used as the building block for the syn-
thesis of acids 6 and 7 was prepared by adapting our previous
described method, using 2,6-dihydroxyacetophenone (1), bromo-
benzyl bromides and ethyl oxalate [19,29,30]. An update of the
general method is available in the experimental section allowing
the synthesis of acids 6 and 7 in several gram scale.
0.10
mM, respectively. The importance of stereochemistry of the
amino acid is marginal as can be observed with inhibitors 8c and
8d. Overall, for this series, the highest activity was reached with
compounds 9a-e for which the hydrophobicity of the amino acid
side chain and the position of the bromine atom are beneficial.
For series II, comparison of compounds 12a-b and 13a-b high-
lighted again the slight contribution of the bromine atom in posi-
tion 2 of the bromobenzyloxy moiety. Besides, the results obtained
with that series also underlined the positive contribution of the
second amino acid. The highest inhibitory activity of ABCG2 was
As shown in Scheme 1, peptide coupling of chromone de-
rivatives 6 or 7 with protected amino acids was realized in DMF
with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetra-
fluoroborate (TBTU) and N,N-diisopropylethylamine (DIEA) to
afford compounds 8a-d and 9a-c in yields ranging from 28 to 90%.
Peptide coupling reactions could also be performed by using N-
obtained with compounds 13a-b (EC50 ¼ 0.07 0.01
mM), regard-
Ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride
less the nature of the side chain as long as it is hydrophobic. For
comparison purposes, the concentration dependence of mitoxan-
trone efflux inhibition by Ko143, MBLeIIe141 and the two most
active compounds (9c and 13a) in HEK293-tranfected cells are
provided in the supporting information section.
(EDCI,HCl) and 1-hydroxybenzotriazole (HOBt) in the presence of
triethylamine or DIEA at room temperature. Overall, the best yields
were obtained with TBTU/DIEA [31e33].
The saponification of esters 8a-d and 9a-e using lithium hy-
droxide in a mixture of THF/CH3OH/H2O allowed the formation of
Taken together, these results highlight the input of the amino
Fig. 1. Structure of MBLeIIe141 and the general structures of the new series.

E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
3
Scheme 1. (a) HCl∙H2NeCH(R1)-COOCH3, TBTU, DIEA, DMF, RT, 12e24 h; (b) i- LiOH, THF/CH3OH/H2O, RT, 2 h; ii- HCl∙H2NeCH(R2)-COOCH, TBTU, DIEA, DMF, RT, 24 h.
Fig. 2. ORTEP schemes for compounds 9b (left) and 9c (right) with ellipsoids drawn at the 50% probability level. Atoms were colored as bromide in dark red, oxygen in red, nitrogen
in blue, carbon in grey and hydrogen in white. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Table 1
Inhibitory activity and cytotoxicity of chromones bearing amino acids residues.
Entry
R1
R2
% Maximal inhibition at 1
m
Ma
EC50
(m
M)
% of Cell viability, at 10e20 m
Mb
8a
8b
8c
8d
9a
9b
9c
9d
9e
12a
12b
13a
13b
(S)eCH(CH3)eCH2CH3
(S)eCH2-Ph
/
/
/
/
/
/
/
/
115 14
92 15
96 14
85 12
0.59 0.08
0.27 0.11
0.48 0.07
0.29 0.05
0.10 0.01
0.14 0.04
0.05 0.03
0.10 0.07
0.6e0.7
0.25 0.1
0.11 0.03
0.07 0.01
0.07 0.01
0.13 0.09
0.09
/
84
77
3
0
1
(S)eCH2-(3-indolyl)
(R)eCH2e(3-indolyl)
(S)eCH(CH3)eCH2CH3
(S)eCH2eCH(CH3)2
(S)eCH(CH3)2
102
78
115 17
87
9
/
87
76
82
88
1
1
6
1
7
(S)eCH2-Ph
100 14
94 17
(S)eCH2-(3-indolyl)
(S,R)eCH(CH3)eCH2CH3
(S,R)eCH(CH3)eCH2CH3
(S,R)eCH(CH3)eCH2CH3
(S,R)eCH(CH3)eCH2CH3
/
/
(S)eCH(CH3)2
(S)eCH2eCH(CH3)2
(S)eCH(CH3)2
(S)eCH2eCH(CH3)2
/
72
52
85
83
98
7
5
7
9
5
89 22b
101 12b
102 11b
104 11b
/
MBL-II-141
Ko143
DMSO 0.5%
/
/
106
1
/
104
8
The values of inhibition %, EC50 and cell viability are the mean of three independent experiments.
a
The percent inhibition of ABCG2 transport activity was studied by comparison with the reference inhibitor Ko143, which fully inhibited; the maximal inhibition for each
derivative was measured at 1 mM.
b
Cytotoxicity was evaluated at 20 mM.
acid hydrophobicity on the inhibition of BCRP. It should be high-
lighted that the introduction of less hydrophobic amino acid was
deleterious for the inhibition activity (independent work, results
not shown). The position of the bromine atom on the benzyloxy
group influences moderately the inhibition activity. Finally, the
introduction of a second hydrophobic amino acid is beneficial to
reach a high level of ABCG2 inhibition at the expense of low
solubility.
The cytotoxicity of chromone derivatives on ABCG2-transfected
HEK293 cells showed a reliable cell viability for all compounds
(Table 1). The absence of cytotoxicity prevails an excellent thera-
peutic index.
2.3. Selectivity toward ABCG2 versus ABCB1 and ABCC1
As introduced above, three exporters are mainly involved in the

4
E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
MDR phenotype, ABCB1, ABCC1 and ABCG2. Hence, we evaluated
the selectivity of our compounds by measuring their ability to
inhibit the drug efflux mediated by ABCB1 and ABCC1. As shown in
Table 2, compounds tested at up to 10 mM did not prevent the efflux
activity of rhodamine-123 for ABCB1 or calcein for ABCC1, indi-
cating that are selective for ABCG2. More specifically, the five more
potent compounds 9b-d and 13a-b displayed the highest selectivity
at 1 mM and 10 mM.
2.4. Interactions of chromone derivatives with the active site of
ABCG2
A docking study was successfully achieved using the cryo-EM
structure of ABCG2-MZ29-Fab complex (PDB ID: 6HIJ) which con-
tains two MZ29 molecules in the binding site [16] (see Supporting
Info). For our study, our most active inhibitors (9c, 13a and 13b),
MBLeIIe141 and ko143 were docked into the active site of ABCG2
defined as comprising both MZ29 ligands.
Except for ko143, all the ligands best poses adopted a bent
configuration that filled both MZ29 binding sites, thanks to the
ligand internal flexibilities. The best pose of ligand ko143, a poly-
cyclic and less flexible compound, binds between the two MZ29
binding sites (Fig. 3).
Fig. 3. Superposition of the best ranked poses of each ligand on the binding poses of
MZ29 colored in cyan, 13a in purple (ꢂ12.5 kcal/mol); 13b in orange; MBL-II-141 in
grey; 9c in dark blue (ꢂ10.94 kcal/mol) and ko143 in green (ꢂ10.1 kcal/mol). (For
interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
3. Conclusions
We observed a large variety of favorable interactions as
p - p
Years ago, we found that conveniently functionalized chro-
mones offer interesting scaffolds for the design of ABCG2 inhibitors.
As, the chemistry needed to access chromones scaffolds is now well
mastered, it allows the building and refinement of new inhibitors
with higher affinity and selectivity. The presence of a carboxylic
acid function at the chromone scaffold allows the introduction of
amino acid residues through peptidic coupling conditions. The
amino acid residues chosen were those found in the FTC and its
derivative Ko143, inhibitors.
As shown here, chromones bearing one bromine atom in posi-
tion 2 of the bromobenzyloxy group and a hydrophobic mono- or
dipeptide constitute promising leads as inhibitors of drug efflux
mediated by ABCG2, being more active than the reference in-
hibitors Ko143 and MBLeIIe141. Their easy chemical synthesis is a
key advantage allowing the synthesis of multigram scale quantities
of the three more potent compounds 9c, 13a and 13b. The three
latter inhibitors are entering in preclinical trials (a patent was filed
under the number FR1908485 on September 2019) dealing with the
synthesis and inhibition activity of ABCG2 with chromones
described in this manuscript. Note that attempts to introduce tri-
peptides on the chromone scaffold led to compounds with extreme
low solubility that excluded any biological evaluation.
and hydrophobic interactions which are in agreement with the
high docking scores and the inhibition activity determined in vitro
(Fig. 4).
The two dipeptides 13a et 13b have small structural difference
where only the second amino acid is different (13a: (R,S)-Ile e (S)-
Val; 13b: (R,S)-Ile e (S)-Leu). Experimentally as well as by docking,
no significant difference of inhibition activity and scores were
observed. Note that the racemization that occurs on the first amino
acid during the second peptide coupling reaction had no effect on
the docking scores.
Table 2
Selectivity of the compounds towards the ABC exporters mainly involved in the MDR
phenotype.
Entry
[Compound] (
m
M)
ABCB1
ABCC1
ABCG2
% Maximal inhibition at 1
mM
8b
8c
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
6
7
7
6
7
6
2
1
3
1
1
1
23.0
19.0
10.3
17.4
11.9
12.6
92 15
/
96 14
/
8d
85 13
/
78
9a
/
/
5
9
1
5
9
4. Experimental section
/
9b
7
5
8
7
5
6
7
5
2
11
4
6
0
0
2
2
1
1
1
2
1
1
0
1
2
20
19
13
18
26
29
23
16
12
15
11
15
11
11
10
13
3
115 17
/
87
4.1. Chemistry
2
1
1
1
2
3
3
0
2
1
1
1
3
1
1
9c
7
/
NMR spectra were recorded on a 400 MHz Bruker Avance-400
instrument (400 MHz) or on a 500 MHz Bruker Avance-500 in-
strument (500 MHz). Chemical shifts (d) are reported in ppm
9d
100 14
/
9e
94 17
/
relatively to Me4Si used as an internal standard. Electrospray
ionization (ESI) mass spectra were acquired by the Analytical
Department of Grenoble University on an Waters Xevo G2-S Q TOF
instrument with a nanospray inlet. Exact mass was given in (m/z).
HPLC analyses were performed with an Agilent 1100 series using a
diode array detector and a C18 reversed-phase column (Nucleosil
C18, Macherey-Nagel, 5 mm particle size,125 mm ꢀ 3 mm) at 45 ꢁC,
with a mobile phase composed of A: water and TFA 0.1% and B:
CH3OH and TFA 0.1% with a gradient 85:15 to 0:100 A:B over
12a
12b
13a
13b
72
52
85
7
5
7
0
/
/
/
0
0
0
0
2
0
83 10
/
The values of inhibition % are the mean of three independent experiments.

E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
5
Fig. 4. Illustrations of the poses in the binding pocket with respectively in green and cyan, hydrophobic and polar residues. (a) Docking of 9c in the chosen binding pocket of BCRP.
(b) Docking of 13a in the chosen binding pocket of BCRP. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
14 min,1 mL/min, 10
m
L injection, detection at 254 nm. Melting
was concentrated under vacuum and then poured into basified
water (K2CO3 20%) and washed with ethyl acetate before to be
acidified with HCl 37%. The aqueous layer was extracted with ethyl
acetate or a mixture of dichloromethane with a small amount of
THF and methanol Then, the combined organic layers were washed
(1 time) with water and brine before dried over magnesium sulfate,
filtrated and evaporated under vacuum.
point (m.p.) expressed in degrees celsius (ꢁC) were obtained on a
Büchi melting point B540. Thin-layer chromatography (TLC) used
Merck silica gel F-254 plates (thickness 0.25 mm). Unless otherwise
stated, reagents were obtained from commercial sources (Alpha
Aesar, Sigma-Aldrich and TCI) and were used without further
purification.
Note: The purity of all products is above 95% and was deter-
mined by HPLC or NMR.
General procedure D: To a solution of 6 or 7 (1.0 equiv.) in dry
DMF (20 mL/mmol) was added TBTU (2 equiv.) in the presence of
DIEA (4 equiv.) under inert atmosphere. The solution was stirred
during 30 min at r.t. Then, a solution of the amino acid methyl ester
hydrochloride (2.0 equiv.) in DMF (10 mL/mmol) under inert at-
mosphere was added to the previous one. The resulting solution
was stirred during 12 he24 h and monitored by TLC (Cyclohexane/
Ethyl acetate 1:1). Then, the solution was concentrated under
vacuum and poured into acidified water (HCl 1 M) and extracted
with ethyl acetate. The combined organic layers were washed (3
times) with a solution of sodium bicarbonate (20%). Then, the
combined organic layers were washed (1 time) with water and
brine before dried over magnesium sulfate, filtrated and evapo-
rated under vacuum.
General procedure D1: To a solution of 10 or 11 (1.0 equiv.) in
dry DMF (20 mL/mmol) was added TBTU (2 equiv.) in the presence
of DIEA (5 equiv.) under inert atmosphere. The solution was stirred
during 30 min at r.t. Then, a solution of the amino acid methyl ester
hydrochloride (2.0 equiv.) in DMF (10 mL/mmol) under inert at-
mosphere was added to the previous one. The resulting solution
was stirred during 12 he24 h and monitored by TLC (Cyclohexane/
Ethyl acetate 3:2). Then, the solution was concentrated under
vacuum and poured into acidified water (HCl 1 M) and extracted
with ethyl acetate. The combined organic layers were washed (3
times) with a solution of sodium bicarbonate (20%). Then, the
combined organic layers were washed (1 time) with water and
brine before dried over magnesium sulfate, filtrated and evapo-
rated under vacuum.
4.1.1. General procedures
General
procedure
A:
To
a
solution
of
2,6-
dihydroxyacetophenone 1 (1,0 equiv.) in acetone (6 mL/mmol)
were added simultaneously K3CO3 (3,0 equiv.) and TBAB (0,4 equiv.)
previously pounded together. The resulting solution was refluxed
during 0.5 or 1 h and then the corresponding bromobenzyl bromide
derivative (1,0 equiv.) in acetone (15 mL/mmol) was added drop by
drop under inter atmosphere. The solution was refluxed during 4 or
5 h and monitored by TLC (Cyclohexane/Ethyl acetate 3:7). The
solution was concentrated under vacuum and then poured into
water before to be extracted with ethyl acetate. The combined
organic layers were washed (1 time) with water and brine before
dried over magnesium sulfate, filtrated and evaporated under
vacuum.
General procedure B: To a solution of 2 or 3 (1.0 equiv.) in dry
THF (10 mL/mmol) wad added a solution of sodium ethoxide
generated in-situ from sodium metal (6.0 equiv.) in dry ethanol
(15 mL/mmol) at 0 ꢁC and under inert atmosphere. Then, the
resulting solution was stirred during 30 min at r.t. before to add the
diethyl oxalate (4.0 equiv.) drop by drop. The solution was warmed
up at 50 ꢁC until precipitation and then refluxed during 2 h and
monitored by TLC (Cyclohexane/Ethyl acetate 1:1). Then, few drops
of concentrated hydrochloric acid (37%) were added to the sus-
pension until a white colour change of the precipitate. The resulting
suspension was refluxed during 1 h before to be cooled down at r.t.
Then, the suspension was concentrated under vacuum and poured
into water before to be extracted with ethyl acetate. The combined
organic layers were washed (1 time) with water and brine before
dried over magnesium sulfate, filtrated and evaporated under
vacuum.
General procedure E: To a solution of 8a or 9a (1.0 equiv.) in
combination of THF (25 mL/mmol) and methanol (10 mL/mmol)
was added a solution of LiOH (1.5 equiv.) in H2O (10 mL/mmol). The
resulting solution was stirred 2 h at r.t. and monitored by TLC
(Cyclohexane/Ethyl acetate 1:1). Then, the solution was concen-
trated under vacuum and poured into basified water (NaHCO3 20%)
and washed (3 times) with ethyl acetate or a mixture of dichloro-
methane with a small amount of THF and methanol. The basic
aqueous layer was acidified with a solution of hydrochloric acid
1 M, 6 M even concentrated hydrochloridric acid 37% if needs and
General procedure C: To a solution of 4 or 5 (1.0 equiv.) in a
combination of THF (25 mL/mmol) and ethanol (8 mL/mmol) was
added a solution of K2CO3 (1.3 equiv.) in water (12 mL/mmol). Then
the resulting solution was warmed up at 50 ꢁC during 4 h and
monitored by TLC (Cyclohexane/ethyl acetate 1:1). The solution

6
E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
extracted with ethyl acetate. Then, the combined organic layers
were washed (1 time) with water and brine before dried over
magnesium sulfate, filtrated and evaporated under vacuum.
product in methanol to afford white crystal. C17H11BrO5.
1H NMR (400 MHz, DMSO‑d6)
7.76 (m, 1H), 7.66e7.59 (m, 4H),
7.24 (dd, J ¼ 8.4, 0.7 Hz, 1H), 7.14e7.10 (m, 1H), 6.76 (s, 1H), 5.27 (s,
2H). 13C NMR (101 MHz, DMSO‑d6)
176.69, 161.44, 157.69, 157.35,
d
d
4.1.2. 1-(2-((4-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one
(2)
151.15, 136.27, 135.14, 131.20, 128.89, 120.58, 115.21, 114.69, 110.58,
108.93, 69.23. m.p. 204.6e205.3 ꢁC. MS (ESI) m/z 374 (79Br), 376
The crude was synthetized according to the General procedure A
starting from 1 (1.500 g, 9.86 mmol). It was purified by a recrys-
tallisation into ethyl acetate to afford 2, pale yellow crystals
(2.321 g, 73%). C15H13BrO3.
(
81Br) [M]þ.
4.1.7. 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic
acid (7)
1H NMR (400 MHz, DMSO‑d6)
d
11.64 (s, 1H), 7.62e7.58 (m, 2H),
The crude was synthetized according to the General procedure C
starting from 5 (1.000 g, 2.48 mmol). It was purified by trituration
into methanol and then washed with diethyl ether to afford 7, a pale
yellow solid (0.777 g, 84%). C17H11BrO5.
7.45e7.41 (m, 2H), 7.30 (t, J ¼ 8.3 Hz, 1H), 6.62 (dd, J ¼ 8.4, 0.5 Hz,
1H), 6.52 (dd, J ¼ 8.3, 0.7 Hz, 1H), 5.14 (s, 2H), 2.48 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6) d 203.43, 159.52, 157.94, 135.96, 133.77, 131.41,
129.98, 121.15, 114.75, 109.63, 103.41, 69.30, 33.04. m.p.
1H NMR (400 MHz, DMSO‑d6)
d
8.14 (dd, J ¼ 7.7, 1.1 Hz, 1H), 7.77
114.8e116.8 ꢁC. MS (ESI) m/z 320 (79Br), 322 (81Br) [M]þ.
(m, 1H), 7.68 (dd, J ¼ 8.0, 0.9 Hz, 1H), 7.51 (m, 1H), 7.32 (m, 1H), 7.26
(d, J ¼ 8.1 Hz,1H), 7.13 (d, J ¼ 8.3 Hz,1H), 6.74 (s,1H), 5.23 (s, 2H). 13
C
4.1.3. 1-(2-((2-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one
(3)
NMR (101 MHz, DMSO‑d6) d 176.70, 161.44, 157.42, 157.36, 151.31,
135.75, 135.24, 132.13, 129.52, 129.13, 127.82, 121.01, 115.17, 114.63,
The crude was synthetized according to the General procedure A
starting from 1 (1.500 g, 9.86 mmol). It was purified by a recrys-
tallisation into methanol or ethyl acetate to afford 3, pale yellow
crystals (2.583 g, 82%). C15H13BrO3.
110.80, 108.71, 69.71. m.p. 244.3 ꢁC. MS (ESI) m/z 373 (79Br), 375
(
81Br) [M ꢂ H]-.
4.1.8. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucinate (8a)
The crude was synthetized according to the General procedure
D starting from 6 (0,300 g, 0.80 mmol) and L-isoleucine methyl
ester hydrochloride (0,290 g, 1.60 mmol). It was purified by
recrystallisation into methanol to afford 8a, crystals (0.318 g, 79%).
1H NMR (400 MHz, DMSO‑d6)
d
11.69 (s, 1H), 7.71 (dd, J ¼ 8.0,
1.1 Hz, 1H), 7.61 (dd, J ¼ 7.6, 1.6 Hz, 1H), 7.46 (m, 1H), 7.39e7.29 (m,
2H), 6.67 (dd, J ¼ 8.4, 0.5 Hz, 1H), 6.56 (dd, J ¼ 8.3, 0.7 Hz, 1H), 5.19
(s, 2H), 2.47 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
203.38, 159.60,
157.95, 135.31, 133.90, 132.72, 130.64, 130.41, 128.00, 123.04, 114.62,
109.83, 103.19, 70.02, 32.86. m.p. 75.5e77.4 ꢁC. MS (ESI) m/z 321
C
24H24BrNO6.
(
79Br), 323 (81Br) [MþH]þ, 319 (79Br), 321 (81Br) [M ꢂ H]þ.
1H NMR (400 MHz, DMSO‑d6)
d
9.17 (d, J ¼ 7.9 Hz, 1H), 7.77 (t,
J ¼ 8.4 Hz, 1H), 7.64e7.56 (m, 4H), 7.34 (dd, J ¼ 8.5, 0.7 Hz, 1H), 7.12
(d, J ¼ 8.1 Hz, 1H), 6.70 (s, 1H), 5.25 (s, 2H), 4.37 (t, J ¼ 7.7 Hz, 1H),
3.68 (s, 3H), 2.07e1.96 (m,1H),1.58e1.45 (m,1H),1.32e1.19 (m,1H),
4.1.4. Ethyl 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carboxylate (4)
The crude was synthetized according to the General procedure B
starting from 2 (1.718 g, 5.35 mmol). It was purified by a recrys-
tallisation into methanol or ethyl acetate to afford 4, yellow crystals
(1.516 g, 70%). C19H15BrO5.
0.95e0.84 (m, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 176.41, 171.40,
159.62, 157.63, 157.04, 152.91, 136.29, 134.95, 131.21, 128.92, 120.59,
114.54, 112.73, 110.86, 108.99, 69.19, 57.24, 51.88, 35.48, 25.09, 15.37,
10.77. m.p. 117 ꢁC. HRMS (ESI/QTOF) calcd for C24H25BrNO6
(M þ Hþ) 502.0865, found 502.0853.
1H NMR (400 MHz, DMSO‑d6)
d 7.74 (m, 1H), 7.64e7.56 (m, 4H),
7.25e7.21 (m, 1H), 7.13e7.08 (m, 1H), 6.77 (s, 1H), 5.24 (s, 2H), 4.38
(q, J ¼ 7.1 Hz, 2H), 1.34 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
4.1.9. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-phenylalaninate (8b)
DMSO‑d6)
d 176.39, 160.01, 157.70, 157.23, 150.19, 136.23, 135.29,
131.21, 128.90, 120.61, 115.43, 114.68, 110.54, 109.02, 69.22, 62.59,
The crude was synthetized according to the General procedure
D starting from 6 (0.472 g, 1.26 mmol) and L-phenylalanine methyl
13.87. m.p. 148.0e149.4 ꢁC. MS (ESI) m/z 402 (79Br), 404 (81Br) [M]þ.
ester hydrochloride (0.453 g, 2.52 mmol). It was purified by
recrystallisation into methanol to afford 8b, crystals (0.551 g, 90%).
4.1.5. Ethyl 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carboxylate (5)
C
27H22BrNO6.
The crude was synthetized according to the General procedure B
starting from 3 (2.583 g, 8,04 mmol). It was purified by a recrys-
tallisation into methanol or ethyl acetate to afford 5, yellow crystals
(2.478 g, 76%). C19H15BrO5.
1H NMR (400 MHz, DMSO‑d6)
d
9.43 (d, J ¼ 7.9 Hz, 1H), 7.79 (t,
J ¼ 8.4 Hz, 1H), 7.68e7.54 (m, 4H), 7.30 (d, J ¼ 12.6 Hz, 5H), 7.23 (d,
J ¼ 6.1 Hz, 1H), 7.13 (d, J ¼ 8.3 Hz, 1H), 6.63 (s, 1H), 5.26 (s, 2H), 4.73
(dd, J ¼ 13.5, 9.1 Hz, 1H), 3.68 (s, 3H), 3.26 (dd, J ¼ 13.8, 5.2 Hz, 1H),
3.16 (dd, J ¼ 13.6, 10.3 Hz, 1H). 13C NMR (101 MHz, DMSO‑d6)
1H NMR (400 MHz, DMSO‑d6)
d
8.11 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.79
(m, 1H), 7.68 (dd, J ¼ 8.0, 1.0 Hz, 1H), 7.51 (m, 1H), 7.33 (m, 1H),
7.30e7.25 (m, 1H), 7.18e7.13 (m, 1H), 6.79 (s, 1H), 5.23 (s, 2H), 4.40
(q, J ¼ 7.1 Hz, 2H), 1.36 (m, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 176.31, 171.22, 159.22, 157.68, 156.94, 152.71, 137.20, 136.26,
135.08, 131.20, 129.05, 128.91, 128.31, 126.62, 120.59, 114.50, 112.56,
110.60, 109.06, 69.21, 54.10, 52.20, 35.93. m.p. (Decomposition)
153.1 ꢁC. HRMS (ESI/QTOF) calcd C27H23BrNO6 (M þ Hþ) for
536.0709, found 536.0704.
d
176.40, 160.01, 158.25, 157.43, 150.24, 135.70, 135.44, 132.17,
129.57, 129.12, 127.85, 121.05, 115.43, 114.62, 110.78, 108.85, 69.72,
62.61, 13.87. m.p. 155.3e157.1 ꢁC. MS (ESI) m/z 403 (79Br), 405 (81Br)
[M ꢂ H]þ.
4.1.10. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-tryptophanate (8c)
4.1.6. 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic
acid (6)
The crude was synthetized according to the General procedure C
starting from 4 (1.586 g, 3.93 mmol). It was purified by trituration
into methanol and then washed with diethyl ether to afford 6, a
white solid (1.259 g, 85%). It’s possible to recrystallize the desired
The crude was synthetized according to the General procedure
D starting from 6 (0.100 g, 0.27 mmol) and L-tryptophan methyl
ester hydrochloride (0.136 g, 0.533 mmol). It was purified by
recrystallisation into methanol to afford 8c, crystals (0.777 g, 82%).
C
29H23BrN2O6.
1H NMR (400 MHz, DMSO‑d6)
d
10.90 (s, 1H), 9.34 (d, J ¼ 7.8 Hz,

E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
7
1H), 7.79 (t, J ¼ 8.4 Hz, 1H), 7.69e7.52 (m, 5H), 7.35 (d, J ¼ 8.1 Hz,
4.1.14. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-valinate (9c)
1H), 7.29 (d, J ¼ 8.1 Hz,1H), 7.25 (d, J ¼ 2.2 Hz,1H), 7.12 (d, J ¼ 8.3 Hz,
1H), 7.10e7.03 (m, 1H), 7.02e6.94 (m, 1H), 6.63 (s, 1H), 5.25 (s, 2H),
The crude was synthetized according to the General procedure
D starting from 7 (0,200 g, 0,53 mmol) and valine methyl ester
hydrochloride (0.179 g, 1.07 mmol). It was purified recrystallisation
in methanol and then washed with diethyl ether to afford 9c, a pale
yellow solid (0.126 g, 48%). C23H22BrNO6.
4.80e4.69 (m, 1H), 3.68 (s, 3H). Two signals under water peak. 13
C
NMR (101 MHz, DMSO‑d6)
d 176.34, 171.50, 159.19, 157.67, 156.92,
152.76, 136.25, 136.10, 135.04, 131.20, 128.92, 127.03, 123.78, 121.02,
120.59, 118.44, 118.04, 114.48, 112.54, 111.48, 110.61, 109.47, 109.06,
69.23, 53.79, 52.18, 26.39. m.p. 235.3e236.2 ꢁC. HRMS (ESI/QTOF)
calcd for C29H24BrN2O6 (M þ Hþ) 575.0818, found 575.0807.
1H NMR (400 MHz, DMSO‑d6)
d
9.20 (d, J ¼ 7.8 Hz, 1H), 8.12 (d,
J ¼ 7.4 Hz,1H), 7.82 (m,1H), 7.69 (d, J ¼ 7.8 Hz,1H), 7.51 (m,1H), 7.39
(d, J ¼ 8.4 Hz, 1H), 7.33 (m, 1H), 7.16 (d, J ¼ 8.3 Hz, 1H), 6.74 (s, 1H),
5.24 (s, 2H), 4.33 (m, 1H), 3.70 (s, 3H), 2.32e2.19 (m, 1H), 1.06e0.92
4.1.11. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-D-tryptophanate (8d)
(m, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 176.42, 171.34, 159.69,
157.41, 157.07, 153.03, 135.76, 135.09, 132.17, 129.57, 129.18, 127.84,
121.07, 114.53, 112.74, 111.10, 108.88, 69.76, 58.56, 51.90, 29.46,
19.04, 18.95. m.p. 127.0e128.2 ꢁC. HRMS (ESI/QTOF) calcd for
The crude was synthetized according to the General procedure
D starting from 6 (0,300 g, 0,80 mmol) and D-tryptophan methyl
ester hydrochloride (0.408 g, 1.60 mmol). It was purified by pre-
cipitation into ethyl acetate/cyclohexane (2:1) and then washed
with diethyl ether to afford 8d, a pale yellow solid (0.375 g, 81%).
C
23H23BrNO6 (M þ Hþ) 488.0709, found 488.0719.
4.1.15. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-phenylalaninate (9d)
The crude was synthetized according to the General procedure
D starting from 7 (0,194 g, 0,52 mmol) and L-phenylalanine methyl
ester hydrochloride (0.223 g, 1.03 mmol). It was purified by pre-
cipitation into ethyl acetate/cyclohexane (2:1) and then washed
with diethyl ether to afford 9d, a pale yellow solid (0.187 g, 70%).
C
29H23BrN2O6.
1H NMR (400 MHz, DMSO‑d6)
d
10.90 (s, 1H), 9.34 (d, J ¼ 7.8 Hz,
1H), 7.79 (t, J ¼ 8.4 Hz, 1H), 7.66e7.57 (m, 5H), 7.35 (d, J ¼ 8.1 Hz,
1H), 7.29 (d, J ¼ 8.3 Hz,1H), 7.25 (d, J ¼ 2.1 Hz,1H), 7.12 (d, J ¼ 8.3 Hz,
1H), 7.07 (t, J ¼ 7.2 Hz, 1H), 6.98 (t, J ¼ 7.4 Hz, 1H), 6.63 (s, 1H), 5.25
(s, 2H), 4.79e4.70 (m, 1H), 3.69 (s, 3H), 3.43e3.38 (m, 1H),
3.33e3.27 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 176.33, 171.51,
C
27H22BrNO6.
159.18, 157.67, 156.93, 152.75, 136.26, 136.10, 135.04, 131.20, 128.91,
127.03, 123.78, 121.01, 120.59, 118.43, 118.04, 114.48, 112.54, 111.48,
110.60, 109.47, 109.04, 69.21, 53.79, 52.18, 26.38. m.p.
236.2e237.9 ꢁC. HRMS (ESI/QTOF) calcd for C29H24BrN2O6 (M þ Hþ)
575.0818, found 575.0822.
1H NMR (400 MHz, DMSO‑d6)
d
9.47 (s, 1H), 8.13 (dd, J ¼ 7.7,
1.5 Hz, 1H), 7.82 (m, 1H), 7.68 (dd, J ¼ 8.0, 1.1 Hz, 1H), 7.51 (m, 1H),
7.35e7.27 (m, 6H), 7.25e7.19 (m, 1H), 7.16 (d, J ¼ 8.0 Hz, 1H), 6.64 (s,
1H), 5.24 (s, 2H), 4.77e4.71 (m, 1H), 3.69 (s, 3H), 3.26 (dd, J ¼ 13.8,
5.4 Hz, 1H), 3.21e3.13 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d
176.33,171.26,159.21, 157.42,156.95, 152.77,137.22,135.74,135.24,
4.1.12. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucinate (9a)
The crude was synthetized according to the General procedure
D starting from 7 (0,511 g, 1.36 mmol) and L-isoleucine methyl ester
132.15, 129.54, 129.11, 129.06, 128.31, 127.84, 126.62, 121.01, 114.43,
112.57, 110.82, 108.85, 69.69, 54.13, 52.20, 30.67. m.p.
145.5e147.5 ꢁC. HRMS (ESI/QTOF) calcd for C27H23BrNO6 (M þ Hþ)
536.0709, found 536.0711.
hydrochloride (0.495 g, 2.72 mmol). It was purified by recrystalli-
sation in methanol and then washed with diethyl ether to afford 9a,
crystal, (0.352 g, 51%). C24H24BrNO6.
4.1.16. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-tryptophanate (9e)
1H NMR (400 MHz, DMSO‑d6)
d
9.23 (d, J ¼ 7.7 Hz, 1H), 8.14 (d,
The crude was synthetized according to the General procedure
D starting from 7 (0,100 g, 0,27 mmol) and L-tryptophan methyl
ester hydrochloride (0.137 g, 0.54 mmol). It was purified by pre-
cipitation into methanol and then washed with diethyl ether to
afford 9e, a white solid (0.043 g, 28%). C29H23BrN2O6.
J ¼ 7.4 Hz,1H), 7.82 (m,1H), 7.69 (d, J ¼ 7.9 Hz,1H), 7.52 (m,1H), 7.40
(d, J ¼ 8.4 Hz, 1H), 7.33 (m, 1H), 7.17 (d, J ¼ 8.3 Hz, 1H), 6.74 (s, 1H),
5.25 (s, 2H), 4.39 (t, J ¼ 7.7 Hz, 1H), 3.70 (s, 3H), 2.10e1.99 (m, 1H),
1.60e1.48 (m, 1H), 1.33e1.23 (m, 1H), 0.97e0.86 (m, 6H). 13C NMR
1H NMR (400 MHz, DMSO‑d6)
d
10.89 (s, 1H), 9.36 (d, J ¼ 7.8 Hz,
(101 MHz, DMSO‑d6)
d 176.44, 171.41, 159.61, 157.38, 157.05, 152.97,
135.76, 135.11, 132.17, 129.56, 129.14, 127.85, 121.04, 114.49, 112.75,
111.09, 108.81, 69.70, 57.26, 51.90, 35.48, 25.09, 15.37, 10.77. m.p.
123.1e125.7 ꢁC. HRMS (ESI/QTOF) calcd for C24H25BrNO6 (M þ Hþ)
502.0865, found 502.0860.
1H), 8.12 (d, J ¼ 7.7 Hz, 1H), 7.83 (m, 1H), 7.69 (dd, J ¼ 8.0, 0.9 Hz,
1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.51 (m, 1H), 7.39e7.28 (m, 3H), 7.25 (d,
J ¼ 2.2 Hz, 1H), 7.17 (d, J ¼ 8.3 Hz, 1H), 7.07 (m, 1H), 6.98 (m, 1H),
6.65 (s, 1H), 5.24 (s, 2H), 4.80e4.70 (m, 1H), 3.69 (s, 3H). Two proton
signals under water peak 13C NMR (101 MHz, DMSO‑d6)
d 176.36,
4.1.13. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-leucinate (9b)
The crude was synthetized according to the General procedure
D starting from 7 (0,500 g, 1,33 mmol) and L-leucine methyl ester
171.52, 159.18, 157.43, 156.95, 152.81, 136.09, 135.74, 135.21, 132.17,
129.57, 129.15, 127.85, 127.03, 123.79, 121.05, 121.02, 118.44, 118.04,
114.43, 112.55, 111.48, 110.83, 109.47, 108.89, 69.73, 53.80, 52.19,
26.38. m.p. 250e252.8 ꢁC. HRMS (ESI/QTOF) calcd for C29H24BrN2O6
(M þ Hþ) 575.0818, found 575.0821.
hydrochloride (0.483 g, 2.66 mmol). It was purified recrystallisation
in methanol and then washed with diethyl ether to afford 9b, a pale
yellow solid (0.234 g, 35%). C24H24BrNO6.
4.1.17. (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-
1H NMR (400 MHz, CDCl3)
7.48 (d, J ¼ 7.9 Hz, 1H), 7.38 (m, 1H), 7.18e7.05 (m, 3H), 6.98 (s, 1H),
6.90 (d, J ¼ 8.3 Hz, 1H), 5.18 (s, 2H), 4.82e4.73 (m, 1H), 3.74 (s, 3H),
1.80e1.60 (m, 3H), 0.99e0.87 (m, 6H). 13C NMR (101 MHz, CDCl3)
d
8.13 (d, J ¼ 7.4 Hz, 1H), 7.57 (m, 1H),
L
-alloisoleucine (10)
The crude was synthetized according to the General procedure E
starting from 8a (0,671 g, 0.51 mmol). It was purified by recrys-
tallisation in methanol and then washed with diethyl ether to
afford 10, white crystals, (0.419 g, 51%). C23H22BrNO6.
d
177.52, 172.93, 159.05, 158.46, 157.33, 152.34, 135.55, 134.74,
132.10, 129.07, 128.79, 128.12, 120.82, 115.42, 114.21, 110.63, 108.66,
70.38, 52.73, 51.14, 41.81, 25.02, 22.82, 22.05. m.p. 115.1e116.7 ꢁC.
HRMS (ESI/QTOF) calcd for C24H25BrNO6 (M þ Hþ) 502.0865, found
502.0865.
1H NMR (400 MHz, DMSO)
7.78 (m, 1H), 7.67e7.57 (m, 4H), 7.37 (dd, J ¼ 8.5, 0.6 Hz, 1H), 7.13 (d,
J ¼ 8.2 Hz, 1H), 6.72 (s, 1H), 5.26 (s, 2H), 4.40e4.29 (m, 1H),
2.07e1.96 (m, 1H), 1.61e1.45 (m, 1H), 1.38e1.20 (m, 1H), 0.96 (d,
d
12.91 (s, 1H), 8.98 (d, J ¼ 8.1 Hz,1H),

8
E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
J ¼ 6.8 Hz, 3H), 0.90 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO)
4.1.21. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucyl-L-valinate (13a)
d
176.44, 172.23, 159.44, 157.63, 157.04, 153.12, 136.28, 134.91,
131.20, 128.93, 120.59, 114.54, 112.63, 110.89, 108.99, 69.21, 57.19,
35.63, 25.04, 15.52, 10.96. m.p. 230.1e230.4 ꢁC. HRMS (ESI/QTOF)
calcd for C23H23BrNO6 (M þ Hþ) 488.0709, found 488.0715.
The crude was synthetized according to the General procedure
D1 starting from 11 (0,091 g, 0.19 mmol) and L-valine methyl ester
hydrochloride (0.064 g, 0.38 mmol). It was purified by precipitation
into ethyl acetate and a minimum amount of cyclohexane and then
washed with diethyl ether to afford 13a, pale yellow solid, (0.042 g,
37%). C29H33BrN2O7.
4.1.18. (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-
L
-alloisoleucine (11)
1H NMR (400 MHz, DMSO‑d6)
d
8.79 (d, J ¼ 8.4 Hz, 0.5H),
The crude was synthetized according to the General procedure E
8.60e8.50 (m, 1H), 8.45 (d, J ¼ 7.2 Hz, 0.5H), 8.13 (d, J ¼ 7.5 Hz, 1H),
7.81 (m, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H), 7.52 (m, 1H), 7.40 (d, J ¼ 8.1 Hz,
1H), 7.33 (m, 1H), 7.16 (d, J ¼ 8.4 Hz,1H), 6.72 (d, J ¼ 5.0 Hz,1H), 5.25
(s, 2H), 4.73e4.65 (m, 0.5H), 4.54e4.45 (m, 0.5H), 4.27e4.21 (m,
0.5H), 4.21e4.14 (m, 0.5H), 3.65 (d, J ¼ 4.7 Hz, 3H), 2.13e1.92 (m,
2H), 1.64e1.47 (m, 1H), 1.28e1.14 (m, 1H), 1.04e0.70 (m, 12H)$13C
starting from 9a (0,256 g, 0.51 mmol). It was purified by recrys-
tallisation into methanol and then washed with diethyl ether to
afford 11, pale yellow crystals, (0.114 g, 46%). C23H22BrNO6.
1H NMR (400 MHz, DMSO‑d6)
d
12.91 (s, 1H), 8.98 (d, J ¼ 8.1 Hz,
1H), 7.78 (t, J ¼ 8.4 Hz, 1H), 7.67e7.57 (m, 4H), 7.37 (dd, J ¼ 8.5,
0.6 Hz, 1H), 7.13 (d, J ¼ 8.2 Hz, 1H), 6.72 (s, 1H), 5.26 (s, 2H),
4.40e4.29 (m, 1H), 2.07e1.96 (m, 1H), 1.61e1.45 (m, 1H), 1.38e1.20
(m, 1H), 0.96 (d, J ¼ 6.8 Hz, 3H), 0.90 (t, J ¼ 7.4 Hz, 3H). 13C NMR
NMR (101 MHz, DMSO‑d6)
d 172.31, 171.91, 171.88, 157.84, 157.83,
157.82, 157.51, 157.49, 157.46, 157.43, 157.42, 153.69, 153.67, 153.65,
135.92, 132.79, 130.34, 129.78, 128.33, 121.94, 114.74, 114.72, 114.70,
114.66, 112.89, 112.87, 112.69, 111.52, 111.46, 109.48, 109.46, 109.37,
109.35, 70.33, 70.31, 58.31, 58.30, 58.21, 58.05, 58.02, 57.96, 52.33,
52.26, 52.23, 52.21, 36.32, 30.00, 25.02, 19.31, 19.17, 19.13, 18.66,
18.59, 15.29, 15.28, 10.85. m.p. (Decomposition) 277 ꢁC. HRMS (ESI/
QTOF) calcd for C29H34BrN2O7 (M þ Hþ) 601.1549, found 601.1533.
(101 MHz, DMSO‑d6) d 176.46, 172.23, 159.43, 157.39, 157.06, 153.18,
135.76, 135.07, 132.16, 129.57, 129.16, 127.84, 121.06, 114.49, 112.64,
111.12, 108.84, 69.73, 57.21, 35.62, 25.04, 15.52, 10.96. m.p.
230.1e230.4 ꢁC. HRMS (ESI/QTOF) calcd for C23H23BrNO6 (M þ Hþ)
488.0709, found 488.0715.
4.1.19. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucyl-L-valinate (12a)
4.1.22. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucyl-L-leucinate (13b)
The crude was synthetized according to the General procedure
D1 starting from 10 (0,123 g, 0.25 mmol) and
L
-valine methyl ester
The crude was synthetized according to the General procedure
hydrochloride (0.084 g, 0.50 mmol). It was purified by trituration
into diethyl ether to afford 12a, pale yellow solid, (0.061 g, 41%).
D1 starting from 11 (0,082 g, 0.16 mmol) and L-leucine methyl ester
hydrochloride (0.061 g, 0.32 mmol). It was purified by trituration
into diethyl ether to afford 13b, pale yellow solid, (0.030 g, 31%).
C
29H33BrN2O7.
1H NMR (400 MHz, DMSO‑d6)
d
8.76 (d, J ¼ 8.6 Hz, 0.5H), 8.54 (d,
C
30H35BrN2O7.
J ¼ 9.4 Hz, 1H), 8.43 (d, J ¼ 7.5 Hz, 0.5H), 7.76 (t, J ¼ 8.3 Hz, 1H), 7.60
(m, 4H), 7.35 (d, J ¼ 8.2 Hz, 1H), 7.11 (d, J ¼ 8.3 Hz, 1H), 6.69 (d,
J ¼ 5.4 Hz, 1H), 5.25 (s, 2H), 4.67 (dd, J ¼ 8.8, 6.9 Hz, 0.5H), 4.48 (t,
J ¼ 8.8 Hz, 0.5H), 4.22 (dd, J ¼ 7.9, 6.9 Hz, 0.5H), 4.15 (m, 0.5H), 3.63
(d, J ¼ 4.9 Hz, 3H), 2.12e1.90 (m, 2H), 1.59e1.36 (m, 1H), 1.25e1.11
1H NMR (400 MHz, DMSO‑d6)
d
8.79 (d, J ¼ 8.8 Hz, 0.5H), 8.64 (d,
J ¼ 7.6 Hz, 1H), 8.56 (d, J ¼ 7.7 Hz, 0.5H), 8.13 (d, J ¼ 7.6 Hz, 1H), 7.81
(m, 1H), 7.69 (d, J ¼ 8.0 Hz,1H), 7.52 (m, 1H), 7.40 (dd, J ¼ 8.3, 4.4 Hz,
1H), 7.33 (m, 1H), 7.16 (d, J ¼ 8.1 Hz, 1H), 6.73 (d, J ¼ 7.3 Hz, 1H), 5.25
(s, 2H), 4.64e4.56 (m, 0.5H), 4.41 (m, 1H), 4.37e4.27 (m, 0.5H), 3.63
(s, 3H), 2.05e1.92 (m, 1H), 1.74e1.43 (m, 4H), 1.27e1.13 (m, 1H),
(m,1H), 0.99e0.81 (m,12H). 13C NMR (101 MHz, DMSO‑d6)
d 176.43,
171.93, 171.64, 170.88, 170.67, 159.10, 158.94, 157.64, 157.01, 153.21,
136.29, 134.90, 131.19, 128.91, 120.58, 114.52, 112.61, 112.54, 110.83,
109.00, 69.21, 57.66, 57.42, 57.24, 56.72, 51.72, 51.53, 37.09, 35.99,
29.85, 29.58, 25.65, 24.62, 19.02, 18.84, 18.35, 18.20, 14.95, 14.58,
11.37, 10.55. m.p. (Decomposition) 268 ꢁC. HRMS (ESI/QTOF) calcd
for C29H34BrN2O7 (M þ Hþ) 601.1549, found 601.1545.
0.98e0.81 (m, 12H)). 13C NMR (126 MHz, DMSO‑d6)
d 177.64, 174.72,
173.46, 173.29, 171.55, 171.48, 159.81, 159.64, 157.82, 157.41, 153.65,
153.63, 135.89, 135.84, 132.77, 130.31, 129.75, 128.32, 121.89, 114.67,
112.93, 112.88, 111.50, 109.43, 70.31, 58.05, 57.49, 52.53, 52.42,
52.32, 51.52, 51.02, 50.90, 41.57, 40.89, 37.32, 36.39, 25.92, 24.96,
24.70, 24.64, 23.10, 23.01, 22.99, 21.82, 21.57, 21.44, 21.23, 15.31,
14.95, 11.64, 10.85. m.p. (Decomposition) 280 ꢁC. HRMS (ESI/QTOF)
calcd for C30H36BrN2O7 (M þ Hþ) 615.1690, found 615.1684.
4.1.20. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-
carbonyl)-L-alloisoleucyl-L-leucinate (12b)
The crude was synthetized according to the General procedure
D1 starting from 10 (0,123 g, 0.25 mmol) and L-leucine methyl ester
4.2. Biology
hydrochloride (0.091 g, 0.50 mmol). It was purified by trituration
into diethyl ether to afford 12b, pale yellow solid, (0.052 g, 34%).
4.2.1. Materials
DMEM (Dulbecco/Vogt modified Eagle’s minimal essential me-
dium) High glucose with GlutaMAX™ (Gibco) and fetal bovine
serum (FBS, GE Healthcare Hyclone) were purchased from Fisher
Scientific. The penicillin/streptomycin (10 000 unit/10 mg per mL),
G418, trypsine and Dulbecco’s Phosphate Buffered Saline (DPBS)
were purchased from Sigma Aldrich (France) as well as mitoxan-
trone (MX), rhodamine 123 (R123), calcein AM (cAM) and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT).
All commercial products were of the highest available purity grade.
C
30H35BrN2O7.
1H NMR (400 MHz, DMSO‑d6)
d
8.76 (d, J ¼ 8.7 Hz, 0.5H), 8.62 (d,
J ¼ 7.8 Hz, 0.5H), 8.54 (dd, J ¼ 8.0, 4.5 Hz, 1H), 7.76 (m, 1H),
7.66e7.56 (m, 4H), 7.35 (dd, J ¼ 8.4, 4.2 Hz, 1H), 7.11 (d, J ¼ 8.2 Hz,
1H), 6.70 (d, J ¼ 7.5 Hz, 1H), 5.25 (s, 2H), 4.58 (m, 0.5H), 4.39 (m,
0.5H), 4.36e4.24 (m, 1H), 3.62 (d, J ¼ 2.3 Hz, 3H), 2.03e1.89 (m, 1H),
1.70e1.55 (m, 2H), 1.55e1.45 (m, 2H), 1.24e1.10 (m, 1H), 0.96e0.79
(m, 12H). 13C NMR (101 MHz, DMSO‑d6) 176.43, 172.81, 172.60,
170.56, 170.42, 159.14, 158.95, 157.63, 157.01, 153.19, 136.28, 134.90,
131.19, 128.91, 120.57, 114.51, 112.62, 112.54, 110.84, 109.00, 69.20,
57.29, 56.64, 51.86, 51.71, 50.39, 50.23, 36.98, 36.00, 25.59, 24.56,
24.23, 24.18, 22.78, 22.66, 21.28, 20.90, 14.99, 14.62, 10.54. m.p.
(Decomposition) 273 ꢁC. HRMS (ESI/QTOF) calcd for C30H36BrN2O7
(M þ Hþ) 615.1690, found 615.1690.
4.2.2. Compounds
All chromone derivatives were dissolved in DMSO, and then
diluted in DMEM high glucose medium. Stock solutions were stored
at ꢂ20 ꢁC and warmed to 25 ꢁC just before to use.

E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
9
4.2.3. Cell lines and cultures
(a.u.) of accumulated fluorophore in the cells expressing the efflux
pump incubated with the substrate only. HEKFA corresponds to the
fluorescence emission (a.u.) of accumulated fluorophore in the
control cells incubated with the substrate and the tested com-
pound. HEKFBG corresponds to the resulting background fluores-
cence emission (a.u.) in the control cells (no substrate and no tested
compound). All values are given as the geometric mean of fluo-
rescence emission (a.u.) in a 655e730 nm filter (excitation 635 nm)
measured in 5000 events. Assays were performed in triplicate.
ABCC1-transfected, ABCB1-transfected and ABCG2-transfected,
Flp-In-MDR, NIH/3T3-MDR [34] HEK293-MDR [35] were obtained
as previously published elsewhere [36].
Flp-In293, NIH/3T3 and HEK293 cell lines were cultured and
obtained in DMEM-High glucose with GlutaMAX™ supplemented
with 10% heat inactivated FBS and 1% of penicillin/streptomycin in
humidified atmosphere at 37 ꢁC with 5% CO2. In addition, 200
m
g/
mL hygromycin B, 90 ng/mL colchicine or 750
mg/mL of G418 was
added to the growth medium as selection agent for NIH/3T3, Flp-
In293 and HEK293 transfected cells respectively. Specifically, for
HEK293-MDR the monoclonal cell line was obtained by
4.2.6. Selectivity assays
NIH/3T3 and Flp-In were seeded into a 96-well plates cells at a
Fluorescence-Activated
Cell
Sorting
(FACS)
using
the
density of 5 ꢀ 104 cells/well in 200
mL of medium and incubated
phycoerythrin-coupled 5D3 antibody (Santa Cruz Biotech) as a
native expression reporter.
overnight. Then, growth medium was changed with fresh medium
containing the compounds 3a-h and 4a-c and in presence of 0.5
R123 (NIH/3T3) or 0.2 M cAM (Flp-In) as fluorescence probe to a
final concentration of DMSO 0,5% (v/v). Finally, medium was
mM
m
4.2.4. Cytotoxicity assays
removed, washed with 100
m
m
L of PBS and cells were trypsinized
L of trypsin. Then, each medium well
mL of ice-cold PBS with 2% BSA.
The cytotoxicity of the compounds 8a-d, 9a-e, 12a-b and 13a-b
was determined using a MTT colorimetric assay as reported in
literature [37,38]. Briefly, cells were seeded into 96-well plates at a
density of 1 ꢀ 105 cells/well for a total growth medium volume of
5 min at 37 ꢁC thanks to 25
was suspended with 175
Intracellular fluorescence was measured with a MacsQuant VRB
Analyzer flow cytometer (Miltenyi Biotec) with, at least, 5000
events recorded. While MX was excited at 635 nm and fluorescence
emission recorded in a 655e730 nm window, R123 and cAM were
excited at 488 nm and recorded in a 525/50 nm filter. The com-
pound inhibition efficacy was estimated by using the previous
equation. Assays were performed in triplicate.
100 mL and incubated overnight. Following, 100 mL of fresh medium
containing sequential concentrations of compounds to be tested
(dissolved in DMSO in a 0, 1 and 10 M concentration range) were
added to each well while DMSO control was fixed at 0.5% (v/v).
After 72 h incubation, 22 L of MTT dye in PBS (5 mg/mL) were
m
m
added to each well and plates were incubated for an additional
4 h at 37 ꢁC. After medium removal and drying the formazan dye
4.3. Docking
crystals were solubilized with 200 mL of DMSO/ethanol (1:1, v/v).
The absorbance was measured using spectrophotometry at 570 nm
and 690 nm as reference wavelength. The effect of each compound
on cell viability in all cell lines was calculated as the difference in
absorbance between test wells and medium control wells.
The structure of the ternary complex of human ABCG2
-MZ29eFab (PDB ID: 6HIJ) was taken as our starting template [16].
It was solved by cryo-EM at a resolution of 3.56 Å. The protein exists
as a homodimer, and two MZ29 molecules are bound at the central,
inward-facing cavity of ABCG2.
We used the Protein Preparation Wizard tool to build an all-
atom structure [39]. The missing loops of the protein lie beyond
30 Å of the ligands and were thus not considered in the
calculations.
4.2.5. Inhibition tests of MDR-related drug efflux
Cells were seeded into 96-well plates at a density of 5 ꢀ 104 cells/
well in 200 mL of medium and incubated overnight. Then, growth
medium was switched for fresh medium containing the compounds
8a-d, 9a-e, 12a-b or 13a-b and in the presence of 4 M MX as fluo-
m
The partial atomic charges for the protein and ligand atoms
were derived from the OPLS3 force field [40]. All the water mole-
cules were retrieved. The histidine residues were treated as neutral.
The selection of the histidine enantiomers and the orientation of
the asparagine and glutamine side-chains were chosen so as to
maximize the hydrogen bond network. An all-atom energy mini-
mization with a 0.3 Å heavy-atom RMSD criteria for termination
was performed using the Impref module of Impact and OPLS3 [41].
Low-energy conformers of the chemical compounds, including
MZ29, were generated using the LigPrep module. Relevant ioniza-
tion and tautomeric states at pH comprised between 5 and 9 were
generated using the Hammett and Taft methodology implemented
in Epik. For compounds available as racemic mixtures, all stereo-
isomers were generated. The resulting conformations were energy-
minimized using OPLS3 force field and a maximum of 32 confor-
mations per ligand were generated and subjected to post-docking
minimization [40].
rescent probe for BCRP-mediated efflux to a final concentration of
0.5% DMSO (v/v). After 30 min incubation at 37 ꢁC, the medium was
removed and cells were washed with 100
cells dissociation during 5 min at 37 ꢁC mediated by 25
Finally, trypsinwas neutralized with 175 L of ice-cold DPBS with 2%
mL of DPBS followed by
m
L of trypsin.
m
Bovine Serum Albumin (BSA) and cells were carefully resuspended.
As selectivity assay, the same experiment was performed for P-gp-
and MRP1-mediated efflux with 0.5
mM R123 or 0.2 mM cAM as
respective fluorescent substrates instead of MX.
Intracellular fluorescence was measured with a MacsQuant VRB
Analyzer flow cytometer (Miltenyi Biotec) with, at least, 5000
events recorded. While MX was excited at 635 nm and fluorescence
emission recorded in a 655e730 nm window, R123 and cAM were
excited at 488 nm and recorded in a 525/50 nm filter. The com-
pound inhibition efficacy was estimated by using the following
equation:
½ðG2FA ꢂ G2FBGÞ ꢂ ðG2S ꢂ G2FBGÞꢃ
% inhibition ¼ 100 ꢀ
The obtained ligands were subjected to a rigid-receptor docking
calculation using Glide in Extra Precision mode [42]. The shape and
physico-chemical properties of the binding site centered on one
MZ29 molecule were mapped onto a cubic grid where the innerbox
and outerbox have respective dimensions of 18*20*18 and
39*41*39 Å3, thus encompassing both MZ29 binding sites. The
hydroxyls of Ser, Thr and Tyr residues were allowed to rotate.
During the docking calculations, the parameters for van der
Waals radii were scaled by 0.80 for receptor atoms with partial
½ðHEKFA ꢂ HEKFBGÞ ꢂ ðG2S ꢂ G2FBGÞꢃ
Where G2FA corresponds to the fluorescence emission (a.u.) of
accumulated fluorophore in cells expressing the efflux pump
incubated with a fluorescent substrate and the tested compound.
G2FBG corresponds to the resulting background fluorescence
emission (a.u.) in the ABCG2-transfected cells (no substrate and no
tested compound). G2S corresponds to the fluorescence emission

10
E. Roussel et al. / European Journal of Medicinal Chemistry 202 (2020) 112503
(2016) 723e737, https://doi.org/10.1111/1440-1681.12581.
charges less than 0.15e. A maximum of 100 poses were retained and
ranked according to the Glide XP docking scoring function. This
docking protocol was able to recover the crystallographic binding
pose of MZ29 ligand as the first-ranked pose with a Glide docking
score of ꢂ10.26 kcal/mol (see Figures), and RMSD of thus allowing
the binding of a second MZ29 ligand in cavity 1. The second-ranked
pose of MZ29, with a docking score of ꢂ10.23 kcal/mol, shows a
slightly titled orientation, which avoids the binding of a second
MZ29 ligand in cavity 1. Thus, as debated in the literature (Jackson
2018 and references therein), one cannot state that there is a single
or two inhibitors that bind in the protein.
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annurev.biochem.71.102301.093055.
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Declaration of competing interest
[11] E.M. Leslie, R.G. Deeley, S.P.C. Cole, Multidrug resistance proteins: role of P-
glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense, Toxicol. Appl.
The authors declare no conflicts of interest regarding the pub-
lication of this manuscript.
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(2005)
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https://doi.org/10.1016/
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Acknowledgments
ꢀ
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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Abbreviations
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1.
ABC
BBB
BCRP
BSA
ATP Binding Cassette
BloodeBrain Barrier
Breast Cancer Resistance Protein
bovine Serum Albumin
ꢁ
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E. Winter, G. Valdameri, A. Veale, A. Boumendjel, A. Di Pietro, et al., New,
highly potent and non-toxic, chromone inhibitors of the human Breast cancer
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doi.org/10.1016/j.ejmech.2016.05.053.
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R. Terreux, H. Gervot, C. Rioufol, A. Boumendjel, et al., MBL-II-141, a chromone
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cAM
DIEA
DMEM
DMF
EDCI
FBS
MDR
MRP1
MTT
Calcein Acetoxymethyl
N,N-Diisopropylethylamine
Dulbecco modified Eagle’s minimal essential medium
Dimethylformamide
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Fetal Bovine Serum
Multidrug Resistance
Multidrug Resistance-associated protein 1
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
Mitoxantrone
Parental
Phosphate buffered saline
Rhodamine 123
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
tetrafluoroborate
MX
PAR
PBS
R123
TBTU
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overcoming multidrug resistance in cancer, J. Bio. Sci. 41 (2) (2016) 313e324,
https://doi.org/10.1007/s12038-016-9601-5.
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Opin. Ther. Pat. 25 (11) (2015) 1229e1237, https://doi.org/10.1517/
13543776.2015.1076796.
THF
Tetrahydrofuran
[23] S.K. Rabindran, H. He, M. Singh, E. Brown, K.I. Collins, T. Annable,
L.M. Greenberger, Reversal of a novel multidrug resistance mechanism in
human colon carcinoma cells by fumitremorgin C, Can. Res. 58 (24) (1998)
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