New, highly potent and non-toxic, chromone inhibitors of the human breast cancer resistance protein ABCG2

Article abstract of DOI:10.1016/j.ejmech.2016.05.053

Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of anticancer compounds, contributing to multidrug resistance (MDR). Inhibition of ABCG2-mediated transport is then considered a promising strategy for o

Full text of DOI:10.1016/j.ejmech.2016.05.053

European Journal of Medicinal Chemistry 122 (2016) 291e301
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
New, highly potent and non-toxic, chromone inhibitors of the human
breast cancer resistance protein ABCG2
,
,
Amanda do Rocio Andrade Pires a 1, Florine Lecerf-Schmidt b 1, Nathalie Guragossian a,
Jaqueline Pazinato a, Gustavo Jabor Gozzi a, Evelyn Winter a, Glaucio Valdameri a,
Alexander Veale , Ahcene Boumendjel , Attilio Di Pietro , Basile Peres
b
b
a,
2
b, *, 2
ꢀ
ꢁ ꢀ
a
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Equipe Labellisee Ligue 2014 “Mecanisme et Modulation de la Resistance aux Medicaments”, Universite Lyon 1, Univ. Lyon, CNRS UMR 5086 Bases
ꢁ
ꢀ
Moleculaires et Structurales des Systemes Infectieux, IBCP, 7 Passage du Vercors, 69367 Lyon Cedex 07, France
b
ꢁ
ꢁ
ꢁ
Universite Grenoble-Alpes/CNRS, Departement de Pharmacochimie Moleculaire UMR 5063, 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:
Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of
anticancer compounds, contributing to multidrug resistance (MDR). Inhibition of ABCG2-mediated
transport is then considered a promising strategy for overcoming MDR in tumors. We recently identi-
fied a chromone derivative, namely MBL-II-141 as a selective ABCG2 inhibitor, with relevant in vivo
activity. Here, we report the pharmacomodulation of MBL-II-141, with the aim of identifying key phar-
macophoric elements to design more potent selective and non-toxic inhibitors. Through rational
structural modifications of MBL-II-141, using simple and affordable chemistry, we obtained highly active
and easily-made inhibitors of ABCG2. Among the investigated compounds, derivative 4a, was found to be
3-fold more potent than MBL-II-141. It was similarly efficient as the reference inhibitor Ko143 but with
the advantage of a lower intrinsic cytotoxicity, and therefore constitutes the best ABCG2 inhibitor ever
reported displaying a very high therapeutic ratio.
Received 21 January 2016
Received in revised form
28 April 2016
Accepted 22 May 2016
Available online 27 June 2016
Keywords:
Chromones
ABCG2 modulators
Chemoresistance
Efflux
© 2016 Published by Elsevier Masson SAS.
1. Introduction
cytotoxic drugs. ABCG2 is the most recently ABC transporter
identified to be involved in cross-resistance to a wide panel of
ATP-Binding Cassette (ABC) transporters belong to one of the
largest membrane protein superfamily expressed in both pro-
karyotic and eukaryotic cells. Acting as ATP-powered pumps, ABC
transporters are able to extrude a wide variety of structurally-
unrelated compounds from the cells. As expressed in major phys-
iological barriers, ABC transporters are crucial for cell detoxification
and survival, by effluxing exogenous toxic substances outside the
cell. Their overexpression in tumor cells contributes to chemo-
resistance through the efflux of anticancer drugs. P-glycoprotein
(Pgp/ABCB1) [1], multidrug resistance protein 1 (MRP1/ABCC1) [2]
and breast cancer resistance protein (BCRP/ABCG2) [3e5] are so far
the three major ABC proteins recognized to strongly contribute to
the multidrug resistance developed by cancer cells against
structurally-unrelated anticancer drugs and other compounds. As
opposed to Pgp and MRP1, ABCG2 is a half-transporter which re-
quires at least dimerization [6], or even tetramerization [7,8], to be
functional. Since its discovery, ABCG2 has attracted intense interest
in the MDR context, particularly as a target for the development of
new inhibitors to be used in combination with conventional anti-
cancer drugs for restoring their efficacy. In the absence of high-
resolution structural information regarding ABCG2, the design of
new inhibitors is exclusively based on ligand-based drug design
[9,10].
Fumitremorgin C (FTC) [11] was the first selective inhibitor re-
ported for ABCG2. Unfortunately, it was found to be clinically un-
usable due to its high neurotoxicity. Targeting powerful, selective
and less toxic inhibitors has led to the design and development of
FTC synthetic analogues. In this regard, Ko143 was considered as a
reference ABCG2 inhibitor on the basis of its high potency [12,13];
however, its selectivity for ABCG2 versus ABCB1 (P-gp) was recently
challenged [14]. Despite the number of known ABCG2 inhibitors
[15e22], only very few of them were evaluated in vivo, in animal-
* Corresponding author.
ꢁ ꢀ
E-mail address: Basile.Peres@univ-grenoble-alpes.fr (B. Peres).
First co-authors.
1
2
Last co-authors.
http://dx.doi.org/10.1016/j.ejmech.2016.05.053
0223-5234/© 2016 Published by Elsevier Masson SAS.

292
A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
based models to assess the restoration of antitumor activity of
conventional anticancer drugs. Pursuing our efforts to develop
effective modulators of ABC proteins, we recently reported that a
chromone-derived compound, namely MBL-II-141 (Fig. 1) was an
excellent in vivo ABCG2 inhibitor, based on its potency, high
selectivity, lack of transport and very low toxicity [19,22].
temperature in a mixture of water/ethanol/tetrahydrofuran to
afford carboxylic acids 7aec which were engaged in a classic
peptide coupling reaction (TBTU, DIEA) with 5-methoxytryptamine
to give the desired final compounds 8aec with a 7e29% yield.
3. Results and discussion
With the aim to establish structure-activity relationships gov-
erning MBL-II-141 inhibition and obtain more potent ABCG2 in-
hibitors, we targeted its pharmacomodulation (Fig. 1). We
especially investigated: a) the nature and position of the halogen
present within the benzyloxy group (series I), b) the influence of
the methoxy group on the indole ring (series II), and c) the variation
of the linker between the chromone and indole moieties through
the insertion of an amino acid residue (series III).
The newly synthesized compounds were evaluated for their
ability to inhibit the efflux of mitoxantrone (an anticancer drug and
substrate of ABCG2) in ABCG2-transfected HEK293 cells and for
their cytotoxicity (Table 1). The inhibition results underline that
compound 4a, bearing a 2-bromine atom on the benzyloxy moiety,
was 3-fold more potent than MBL-II-141 where the bromine atom
is at C-4 position (EC50 ¼ 0.086
same potency as Ko143 (0.074 M). In contrast, bromine shift to the
C-3 position in 4b was detrimental to inhibition (0.54 M). Inter-
mM versus 0.26 mM), reaching the
m
2. Chemistry
m
estingly, substitution of the bromine by fluorine induced different
types of effects (4c-f compounds). There was no effect at C-4 po-
sition as compared to MBL-II-141, whereas negative effects were
observed at all other positions including C-2 as compared to 4a.
Such an influence of halogen nature and position may indicate
potential Hammet contribution at the benzyl group. This is
consistent with previous observations that replacement of the
MBL-II-141 bromine with a methoxy group or its omission led to
less active derivatives [19].
The modulation performed with 5aee derivatives (Series II)
showed the beneficial presence of a hydrophobic 5-alkoxy group on
the indole ring of chromones 5bed. Indeed, the dealkylation or the
suppression of the alkyloxy group led to a sharp decrease of activity
The synthesis of series I compounds is shown in Scheme 1. The
chromone moiety was prepared according to our previously
described process, starting from 2,6-dihydroxyacetophenone and
the corresponding benzylbromide derivatives [19,23]. 3aeg acids
were stepped into
a peptide coupling reaction with 5-
methoxytryptamine, using 1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide (EDCI) and hydroxybenzotriazole (HOBt) as coupling
agent, in the presence of triethylamine to give target analogues
4aeg with 5e50% yields [24].
The synthesis of series II compounds (5aee) is shown in Scheme
2. Starting from previously described derivative 3g, a peptide
coupling reaction was performed with either serotonin or trypt-
amine to provide 5a and 5e, respectively. Finally, compound 5a was
alkylated with different alkyl iodide in refluxing acetone to afford
compounds 5bed with a 17e31% yield.
in 5a (4.35 mM) and 5e (1.05 mM) as compared to MBL-II-141. This is
consistent with previous observation that a methoxy group was
important at either position C-5 or C-6 [22].
The synthetic pathway for compounds 8aec (series III) is shown
in Scheme 3. Starting from 3g, a classical peptide-coupling reaction
was performed with the corresponding protected amino acid to
give derivatives 6aed. This time, the peptide-coupling reaction was
performed in DMF with O-(Benzotriazol-1-yl)-N,N,N0,N’-tetrame-
thyluronium tetrafluoroborate (TBTU) in the presence of N,N-dii-
sopropylethylamine (DIEA). The mild hydrolysis of the ester
function was performed with lithium hydroxide at room
Series III was targeted to explore if the presence of an amino acid
residue might impact the inhibitory activity. This pharmacomo-
dulation was inspired from the chemical structures of FTC and
Ko143, considered as reference inhibitors of ABCG2, in which the
presence of an isobutyl moiety was found to be important for in-
hibition. A shown in Table 1, we noticed a slight decrease in potency
for the 8b derivative bearing a leucine residue, while a valine res-
idue in 8a had no effect. Finally, mono methylation at one of the
O
O
O
X
H
N
NH
O
Series I: halogen variation
O
O
O
Br
H
O
N
O
O
O
NH
O
Br
MBLII-141
H
N
NH
O
O
Series II: methoxy modulation
R
O
O
O
O
NH
O
Br
H
N
N
H
O
R
O
Series III: amino acid introduction
Fig. 1. Structure of MBL-II-141 and different types of modulations.

A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
293
NH
O
O
O
CO2H
NH
O
O
N
X
H
a
X
+
H2N
OMe
O
O
OMe
4a-g
3a-g
Scheme 1. Reagents and conditions: (a) K2CO3, Bu4NþBrꢀ, acetone, reflux; (b) (i) EtOꢀNaþ, Ethyl oxalate, THF-EtOH, reflux, (ii) HCl 37%, THF-EtOH, reflux; (c) NaHCO3, EtOH-THF-
H2O, reflux; (d) EDCI, HOBt, Et3N, DMF, rt, overnight.
O
O
O
O
O
O
Br
b
Br
H
N
OH
NH
3g
O
O
5e
a
O
O
O
O
O
Br
Br
c
H
N
H
N
O
NH
NH
O
O
5a
5b R = Et
5c R = Pr
5d R= iPr
O
HO
R
Scheme 2. Reagents and conditions: (a) 5-hydroxytryptamine, EDCI, HOBt, ET3N, DMF, rt, overnight; (b) tryptamine, EDCI, HOBt, ET3N, DMF, rt, overnight; (c) corresponding alkyl
iodide, K2CO3, Bu4NþBrꢀ, acetone, reflux (4e5 h).
O
O
O
O
O
O
Br
O
a
Br
H
N
R2
OH
O
3h
R1
6a R1 = CH(CH3)2, R2 = CH3
6b R1 = CH2CH(CH3)2, R2 = CH3
O
O
6c R1 = CH(CH3)CH2CH3, R2 = CH3
6d R1 = CH(CH3)2, R2 = C(CH3)3
b
for R2 = CH3
O
O
O
O
O
O
c
NH
Br
O
H
N
Br
O
N
H
H
N
R1
O
OH
O
R1
O
8a R1 = CH(CH3)2
8b R1 = CH2CH(CH3)2
8c R1 = CH(CH3)CH2CH3
7a R1 = CH(CH3)2
7b R1 = CH2CH(CH3)2
7c R1 = CH(CH3)CH2CH3
Scheme 3. Reagents and conditions: (a) corresponding protect racemic amino acid, TBTU, DIEA, DMF, rt, overnight; (b) LiOH, THF-MeOH-H2O, rt, 1 h; (c) 5-methoxytryptamine,
TBTU, DIEA, DMF, rt, overnight.
two nitrogen atoms or dimethylation of MBL-II-141 altered the
inhibitory activity (results not shown here, in agreement with
previous observations on other derivatives [22]).
Cell cytotoxicity assays were performed on control, untrans-
fected, HEK293 cells. As shown in Table 1, 4a displayed a very low
(MBL-II-141) did not induce cytotoxicity, the presence of two
fluorines in 4f was quite detrimental (IG50 ¼ 10.2 M and TR ¼ 16).
Similarly, the introduction of a valine residue (8a) within the linker
chain markedly increased cytotoxicity (IG50 ¼ 7.6 M) giving low TR
m
m
value (TR ¼ 22). Such increased cytotoxicity might be related to
additional interaction of the chromone derivative with unknown
cellular target(s) important for cell survival. When ABCG2-
transfected HEK293 cells were used (not shown here), the same
cytotoxicity values as in control cells were observed for all com-
pounds, suggesting that none of these chromone derivatives was
transported by ABCG2. This confirms the lack of transport
cytotoxicity (IG50 > 100
very high TR value (>1163). It is worth mentioning that Ko143 was
M), then resulting in a lower
mM), similarly to MBL-II-141, leading to a
significantly more toxic (IG50 ¼ 60.2
m
TR value. This makes the new chromone derivative 4a the most
potent and promising ABCG2 inhibitor reported to date. Whereas a
bromine substituent at either position C-2 (4a), C-3 (4b) or C-4

294
A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
Table 1
Inhibition of mitoxantrone efflux, and cytotoxicity.
O
O
O
O
O
O
O
O
O
X
NH
O
Br
H
N
Br
NH
H
N
H
N
N
H
NH
O
R
O
O
O
Series III
Series I
Series II
R
O
Compound
EC50
(m
M)b
(%) Maximal inhibition at 10
m
Ma
IG50 for cytotoxicity (
m
M)c
Therapeutic ratio (TR)d
Series I
4a X ¼ 2-Br
4b X ¼ 3-Br
4c X ¼ 2-F
4d X ¼ 3-F
4e X ¼ 4-F
0.086 0.02
0.54 0.05
0.55 0.01
0.38 0.06
0.26 0.06
0.63 0.02
102
98
131
107
106
140
8
>100
>100
94
>100
>100
10.2 0.4
>1163
>185
171
>263
>385
16
8
3
3
4
3
1
4f X ¼ 3,4-difluoro
Series II
5a R ¼ OH
4.35 0.47
0.36 0.02
0.28 0.04
0.36 0.2
1.05 0.2
104
ND
ND
ND
7
61.2 7.6
>80
>60
>100
>100
14
5b R ¼ OC2H5
5c R ¼ OC3H7
5d R ¼ OCH(CH3)2
5e R ¼ H
>222
>214
>278
>95
97 10
Series III
8a R ¼ CH(CH3)2
8b R ¼ CH2CH(CH3)2
Reference inhibitors
Ko143
0.34 0.02
0.91 0.06
101
105
7
9
7.6 0.2
>100
22
>110
0.074 0.01
0.26 0.02
99 5e
60.2
1
814
>385
MBL-II-141 (4g)
133 11f
>100
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
M.
The affinity of inhibitor interaction was expressed as EC50 values (compound concentrations giving a half-maximal inhibition).
The cytotoxicity of inhibitors was evaluated by IG50 (concentration producing 50% inhibition of cell growth) values on control HEK293 cells using the MTT test.
The therapeutic ratio (TR) was calculated as the ratio between IG50 and EC50 values.
derivative was measured at 10
m
b
c
d
e
f
The maximal inhibition with Ko143 was measured at 1
mM.
The maximal inhibition with MBL-II-141 was measured at 5
mM.
previously reported for MBL-II-141 [19] and other derivatives [22].
chromatography (TLC) used Merck silica gel F-254 plates (thickness
0.25 mm), and flash chromatography used Merck silica gel 60,
0.04e0.063 mesh. Unless otherwise stated, reagents were obtained
from commercial sources (Alpha Aesar and Sigma-Aldrich) and
were used without further purification.
4. Conclusion
The new chromone derivative 4a, with a 2-bromine atom on the
benzyloxy group appears a quite promising compound since it is: i)
3-fold more potent that MBL-II-141, ii) better than the reference
Ko143 inhibitor based on its a remarkably low cytotoxicity, and iii)
available through an easy synthesis process [25]. It therefore con-
stitutes the best, non-transported, ABCG2 inhibitor ever reported.
Although introduction of amino acids residues into the structure
did not provide more active modulators, it indicated that further
efforts could be spent, for example by investigating other amino
5.1.1. Synthesis of alkylated compounds 1aeg
5.1.1.1. General procedure A. A solution of dihydroxyacetophenone
(1 equiv.), potassium carbonate (3 equiv.) and tetrabutylammonium
bromide (0.35 equiv.) in acetone (7 mL/mmol) was refluxed for
20 min. A solution of halogenated derivative (1.2 equiv.) in acetone
(3 mL/mmol) was added dropwise and the resulting mixture was
refluxed for 2e6 h (the end of the reaction was controlled by TLC
using acetone/cyclohexane 25:75). The resulting solution was
cooled to room temperature and concentrated under reduced
pressure. The residue was poured into water and extracted with
ethyl acetate. The combined organic layers were washed with
water, dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure.
acids residues, and introducing
in the structure.
D-amino acids, or even dipeptides,
5. Experimental section
5.1. Chemistry
NMR spectra were recorded on a 400 MHz Bruker Avance-400
instrument (400 MHz). Chemical shifts ( ) are reported in ppm
5.1.1.2. 6-(20-Bromobenzyloxy)-2-hydroxyacetophenone
(1a). The
d
crude was prepared according to general procedure A starting
from 2,6-dihydroxyacetophenone (2 g, 13 mmol) and
2-bromobenzylbromide (4 g, 16 mmol) and was purified by flash
silica column chromatography, using cyclohexane/ethyl acetate
(100:0 to 98:2) as eluent to afford 1a as a yellow solid (1.5 g, 36%).
relatively to Me4Si used as an 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 in the same Analytical Department, and all the
C
15H13BrO3. m.p. 68e70 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.45
tested compounds had
a purity of at least 95%. Thin-layer
(s, 3H, COCH3), 5.17 (s, 2H, OCH2), 6.54 (dd, 1H, J ¼ 0.7 Hz,

A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
295
J ¼ 8.3 Hz, ArH), 6.64 (m, 1H, ArH), 7.28e7.37 (m, 2H, ArH), 7.44 (m,
1H, ArH), 7.59 (dd, 1H, J ¼ 1.7 Hz, J ¼ 7.6 Hz, ArH), 7.69 (dd, 1H,
J ¼ 1.1 Hz, J ¼ 7.9 Hz, ArH), 11.67 (s, 1H, OH). 13C NMR (100 MHz,
m.p. 127e128 ꢁC. 1H NMR (400 MHz, DMSO-d6)
COCH3), 5.14 (s, 2H, OCH2), 6.52 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.3 Hz, ArH),
6.62 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.3 Hz, ArH), 7.30 (m, 1H, ArH), 7.34 (m,
1H, ArH), 7.50 (m, 1H, ArH), 7.56 (m, 1H, ArH), 11.62 (s, 1H, OH). 13C
d
2.48 (s, 3H,
DMSO-d6)
d 32.9, 70.0, 103.2, 109.8, 114.7, 123.1, 128.0, 130.4, 130.7,
132.7, 133.9, 135.3, 157.9, 159.6, 203.3. MS (ESI) m/z 321 (79Br), 323
NMR (100 MHz, DMSO-d6) d 33.0, 68.8, 103.4, 109.7, 114.9, 116.9 (d, JC-
(
81Br) [MþH]þ.
¼ 17.5 Hz), 117.5 (d, JC-F ¼ 17.1 Hz), 124.7 (m) 133.7, 134.3 (m), 147.9
F
(m), 150.4 (m), 157.8, 159.4, 203.4. MS (ESI) m/z 277 [M ꢀ H]ꢀ.
5.1.1.3. 6-(30-Bromobenzyloxy)-2-hydroxyacetophenone
crude was prepared according to general procedure A starting from
2,6-dihydroxyacetophenone (2 g, 13 mmol) and 3-
(1b). The
5.1.1.8. 6-(40-Bromobenzyloxy)-2-hydroxyacetophenone
(1g). The
crude was prepared according to general procedure A starting from
commercially available 2,6-dihydroxyacetophenone (2 g, 13 mmol)
and 4-bromobenzylbromide (4 g, 16 mmol) and was triturated in
diethyl ether to afford 1h as a white solid (2.8 g, 68%). C15H13BrO3.
bromobenzylbromide (4 g, 16 mmol) and was washed with diethyl
ether to afford 1b as a beige solid (3 g, 73%). C15H13BrO3. m.p.
131e133 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 2.49 (s, 3H, COCH3), 5.17
(s, 2H, OCH2), 6.52 (d, 1H, J ¼ 8.2 Hz, ArH), 6.61 (d, 1H, J ¼ 8.3 Hz,
ArH), 7.29 (m, 1H, ArH), 7.37 (m, 1H, ArH), 7.46e7.55 (m, 2H, ArH),
7.68 (m, 1H, ArH), 13.58 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
m.p.116e117 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 2.48 (s, 3H, COCH3),
5.14 (s, 2H, OCH2), 6.54 (d, 1H, J ¼ 8.0 Hz, ArH), 6.63 (d, 1H, J ¼ 8.0 Hz,
ArH), 7.31 (m, 1H, ArH), 7.44 (d, 2H, J ¼ 8.0 Hz, ArH), 7.61 (d, 2H,
J ¼ 8.0 Hz, ArH), 11.69 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
d
33.0, 69.1, 103.5, 109.7, 115.0, 121.7, 126.7, 130.4, 130.7, 130.8, 133.7,
139.4, 157.8, 159.3, 203.4. MS (ESI) m/z 321 (79Br), 323 (81Br) [MþH]þ.
d 33.0, 69.3, 103.4, 109.7, 114.6, 121.2, 130.0, 131.4, 133.8, 135.9, 158.0,
159.7, 203.4. MS (ESI) m/z 321 (79Br), 323 (81Br) [MþH]þ.
5.1.1.4. 6-(20-Fluorobenzyloxy)-2-hydroxyacetophenone
crude was prepared according to general procedure A starting from
2,6-dihydroxyacetophenone (2 g, 13 mmol) and 2-
(1c). The
5.1.2. Synthesis of chromones 2aeg
5.1.2.1. General procedure B. To a solution of sodium (6 equiv.) in a
mixture of anhydrous THF/EtOH (1/1, 10 mL/mmol) under argon
were added alkylated derivative 1aeg (1 equiv.) and diethyloxalate
(4 equiv.) at OꢁC. The resulting mixture was warmed to room
temperature and refluxed for 2e4 h (the end of the reaction was
controlled by TLC using acetone/cyclohexane 30:70). The solution
was cooled to room temperature and concentrated under reduced
pressure. The residue was poured into 1 M hydrochloric acid and
extracted with ethyl acetate. The combined organic layers were
washed with water, dried over anhydrous magnesium sulfate,
filtered and concentrated under reduced pressure. The residue was
dissolved in THF/EtOH (1/1,10 mL/mmol) and 37% hydrochloric acid
(1 mL/mmol) was added dropwise. The resulting mixture was
refluxed for 2e4 h (the end of the reaction was controlled by TLC
using acetone/cyclohexane 30:70). The solution was cooled to room
temperature and concentrated under reduced pressure. The residue
was poured into water and extracted with ethyl acetate. The
combined organic layers were washed with water, dried over
anhydrous magnesium sulfate, filtered and concentrated under
reduced pressure.
fluorobenzylbromide (3 g, 16 mmol) and was washed with diethyl
ether to afford 1c as a white solid (1.3 g, 39%). C15H13FO3. m.p.
106e108 ꢁC.1H NMR (400 MHz, DMSO-d6)
d 2.43 (s, 3H, COCH3), 5.20
(s, 2H, OCH2), 6.52 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.3 Hz, ArH), 6.69 (d, 1H,
J ¼ 7.8 Hz, ArH), 7.23e7.29 (m, 2H, ArH), 7.32 (m, 1H, ArH), 7.43 (m,
1H, ArH), 7.57 (m, 1H, ArH). 13C NMR (100 MHz, DMSO-d6)
d 32.8,
64.5 (d, JC-F ¼ 3.7 Hz),103.3,109.8,114.7,115.5 (d, JC-F ¼ 21.9 Hz), 123.2
(d, JC-F ¼ 14.5 Hz),124.6 (d, JC-F ¼ 3.5 Hz),130.6 (d, JC-F ¼ 8.3 Hz),130.9
(d, JC-F ¼ 3.9 Hz), 133.9, 158.0, 159.6, 160.4 (d, JC-F ¼ 246.4 Hz), 203.4.
MS (ESI) m/z 283 [MþNa]þ.
5.1.1.5. 6-(30-Fluorobenzyloxy)-2-hydroxyacetophenone
crude was prepared according to general procedure A starting from
2,6-dihydroxyacetophenone (2 g, 13 mmol) and 3-
(1d). The
fluorobenzylbromide (2.8 g, 15 mmol) and recrystallized with
cyclohexane/ethyl acetate (3:1) to afford 1d as a white solid (1.7 g,
50%). C15H13FO3. m.p. 123e124 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.5 (s, 3H, COCH3), 5.18 (s, 2H, OCH2), 6.52 (d, 1H, J ¼ 8.3 Hz, ArH),
6.62 (d,1H, J ¼ 7.9 Hz, ArH), 7.17 (m,1H, ArH), 7.27e7.31 (m, 2H, ArH),
7.42e7.48 (m, 2H, ArH), 11.62 (s, 1H, OH). 13C NMR (100 MHz, DMSO-
d6)
d
33.0, 69.3, 103.4, 109.7, 114.4 (d, JC-F ¼ 21.9 Hz), 114.6,114.8 (d, JC-
5.1.2.2. 5-(20-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2a). The crude was prepared according to general
procedure B starting from 1a (273 mg, 0.8 mmol) and was purified
by flash silica column chromatography using cyclohexane/ethyl
acetate (9:1 to 8:2) as eluent to afford 2a as a beige solid (150 mg,
44%). C19H15BrO5. m.p. 155e157 ꢁC. 1H NMR (400 MHz, DMSO-d6)
¼ 5.1 Hz), 123.6 (d, JC-F ¼ 3 Hz), 130.5 (d, JC-F ¼ 8.3 Hz), 133.7, 139.4
F
(d, JC-F ¼ 7.6 Hz),157.8,159.4,162.1 (d, JC-F ¼ 243.7 Hz), 203.4. MS (ESI)
m/z 259 [M ꢀ H]ꢀ.
5.1.1.6. 6-(40-Fluorobenzyloxy)-2-hydroxyacetophenone
crude was prepared according to general procedure A starting from
2,6-dihydroxyacetophenone (2 g, 13 mmol) and 4-
(1e). The
d
1.35 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 4.39 (q, 2H, J ¼ 7.1 Hz,
CO2CH2CH3), 5.23 (s, 2H, OCH2), 6.80 (s, 1H, COCH), 7.15 (d, 1H,
J ¼ 8.0 Hz, ArH), 7.25e7.35 (m, 2H, ArH), 7.50 (m, 1H, ArH), 7.67 (dd,
1H, J ¼ 1.1 Hz, J ¼ 7.1 Hz, ArH), 7.79 (m, 1H, ArH), 8.10 (dd, 1H,
fluorobenzylbromide (3 g, 16 mmol) and was washed with diethyl
ether to afford 1e as a beige solid (1.7 g, 50%). C15H13FO3. m.p.
137e139 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.47 (s, 3H, COCH3),
J ¼ 1.5 Hz, J ¼ 7.7 Hz, ArH). 13C NMR (100 MHz, DMSO-d6)
d 13.9,
5.14 (s, 2H, OCH2), 6.51 (dd, 1H, J ¼ 0.8 Hz, J ¼ 8.3 Hz, ArH), 6.65
(dd, 1H, J ¼ 0.8 Hz, J ¼ 8.4 Hz, ArH), 7.19e27 (m, 2H), 7.31 (m, 1H),
7.49e7.56 (m, 2H), 11.69 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
62.6, 69.8, 108.9, 110.8, 114.7, 115.5, 121.1, 127.9, 129.2, 129.6, 132.2,
135.5, 135.7, 150.3, 157.3, 157.5, 160.0, 176.5. MS (ESI) m/z 403 (79Br),
405 (81Br) [MþH]þ.
d
33.1, 69.4, 103.4, 109.6, 114.7, 115.3 (d, JC-F ¼ 21.4 Hz), 130.1 (d, JC-
¼ 8.4 Hz), 132.7 (d, JC-F ¼ 2.9 Hz), 133.9, 158.1, 159.7, 161.8 (d, JC-
¼ 243.9 Hz), 203.5. MS (ESI) m/z 259 [M ꢀ H]ꢀ.
5.1.2.3. 5-(30-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2b). The crude was prepared according to general
procedure B starting from 1b (3 g, 9.4 mmol) and washed with
diethyl ether to afford 2b as a yellow solid (2 g, 50%). C19H15BrO5.
F
F
5.1.1.7. 6-(30,40-Difluorobenzyloxy)-2-hydroxyacetophenone
(1f).
The crude was prepared according to general procedure A starting
from 2,6-dihydroxyacetophenone (5 g, 33 mmol) and 3,4-
difluorobenzylbromide (8.2 g, 40 mmol) and was washed with
diethyl ether to afford 1f as a white solid (2.5 g, 27%). C15H12F2O3.
m.p. 147e149 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 1.75 (t, 3H,
J ¼ 7.2 Hz, CO2CH2CH3), 4.80 (q, 2H, J ¼ 7.2 Hz, CO2CH2CH3), 5.69 (s,
2H, OCH2), 7.22 (s, 1H, COCH), 7.12 (d, 1H, J ¼ 8.0 Hz, ArH), 7.25 (d,
1H, J ¼ 8.0 Hz, ArH), 7.38 (m, 1H, ArH), 7.50e7.65 (m, 2H, ArH), 7.77

296
A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
(m, 1H, ArH), 7.92 (m, 1H, ArH). 13C NMR (100 MHz, DMSO-d6)
13.9, 62.6, 69.0, 109.0, 110.6, 114.7, 115.5, 121.7, 125.6, 129.4, 130.3,
ether to afford 2g as a yellow solid (1.7 g, 69%). C19H15BrO5. m.p.
151e153 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
d
1.34 (t, 3H, J ¼ 7.1 Hz,
130.5, 135.3, 139.6, 150.2, 157.3, 157.6, 160.0, 176.5. MS (ESI) m/z 425
CO2CH2CH3), 4.38 (q, 2H, J ¼ 7.1 Hz, CO2CH2CH3), 5.25 (s, 2H, OCH2),
6.79 (s, 1H, COCH), 7.13 (m, 1H, ArH), 7.24 (dd, 1H, J ¼ 0.8 Hz,
J ¼ 8.5 Hz, ArH), 7.55e7.65 (m, 4H, ArH), 7.75 (m, 1H, ArH). 13C NMR
(
79Br), 427 (81Br) [MþNa]þ.
5.1.2.4. 5-(20-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2c). The crude was prepared according to general
procedure B starting from 1c (900 mg, 3.5 mmol) and was purified
by flash silica column chromatography using cyclohexane/ethyl
acetate (1:0 to 8:2) as eluent to afford 2c as a white solid (265 mg,
24%). C19H15FO5. m.p 156e157 ꢁC. 1H NMR (400 MHz, DMSO-d6)
(100 MHz, DMSO-d6) d 13.9, 62.6, 69.2, 109.0, 110.5, 114.7, 115.4,
120.6, 128.9, 131.2, 135,2, 136.2, 150.1, 157.2, 157.7, 160.0, 176.4. MS
(ESI) m/z 403 (79Br), 405 (81Br) [MþH]þ.
5.1.3. Synthesis of chromones-2-carboxylic acids 3aeg
5.1.3.1. General procedure C. To a solution of ethyl ester 2aeg
(1 equiv.) in THF/EtOH (1/1, 9 mL/mmol) were added a solution of
sodium bicarbonate (7% in water, 4 mL/mmol) and water (4 mL/
mmol). The resulting mixture was refluxed for 2 h (the end of the
reaction was controlled by TLC using acetone/cyclohexane 50:50).
The solution was cooled to room temperature and poured into 1 M
hydrochloric acid in order to form a solid which was filtered and
washed with diethyl ether.
d
1.34 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 4.38 (q, 2H, J ¼ 7.1 Hz,
CO2CH2CH3), 5.29 (s, 2H, OCH2), 6.76 (s, 1H, COCH), 7.16e7.32 (m,
4H, ArH), 7.42 (m, 1H, ArH), 7.76 (m, 1H, ArH), 7.92 (m, 1H, ArH). 13C
NMR (100 MHz, DMSO-d6)
d
13.9, 62.6, 64.5 (d, JC-F ¼ 4.2 Hz), 109.1,
110.7, 114.7, 115.0 (d, JC-F ¼ 20.5 Hz), 115.4, 123.7 (d, JC-F ¼ 14.1 Hz),
124.5 (d, JC-F ¼ 3.2 Hz),129.6 (d, JC-F ¼ 4.0 Hz),129.8 (d, JC-F ¼ 8.1 Hz),
135.4, 150.2, 157.3, 157.7, 159.6 (d, JC-F ¼ 245.1 Hz), 160.0, 176.4. MS
(ESI) m/z 343 [MþH]þ, 365 [MþNa]þ.
5.1.3.2. 5-(20-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3a). The crude was prepared according to general procedure
C starting from 2a (150 mg, 0.4 mmol) to afford 3a as a white solid
(103 mg, 74%). C17H11BrO5. m.p. 244e245 ꢁC. 1H NMR (400 MHz,
5.1.2.5. 5-(30-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2d). The crude was prepared according to general
procedure B starting from 1d (1.5 g, 5.7 mmol) and was washed
with diethyl ether to afford 2d as white solid (1.7 g, 87%). C19H15FO5.
DMSO-d6)
d 5.22 (s, 2H, OCH2), 6.74 (s, 1H, COCH), 7.13 (d, 1H,
m.p. 108e109 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
1.34 (t, 3H,
J ¼ 8.2 Hz, ArH), 7.25 (d, 1H, J ¼ 8.1 Hz, ArH), 7.30 (m, 1H, ArH), 7.49
(m, 1H, ArH), 7.67 (dd, 1H, J ¼ 0.9 Hz, J ¼ 7.9 Hz, ArH), 7.77 (m, 1H,
ArH), 8.11 (d, 1H, J ¼ 7.6 Hz, ArH). 13C NMR (100 MHz, DMSO-d6)
J ¼ 7.1 Hz, CO2CH2CH3), 4.38 (q, 2H, J ¼ 7.1 Hz, CO2CH2CH3), 5.30 (s,
2H, OCH2), 6.80 (s, 1H, COCH), 7.11e7.26(m, 2H, ArH), 7.26 (d, 1H,
J ¼ 8.4 Hz, ArH), 7.41e7.47 (m, 2H, ArH), 7.56 (m, 1H, ArH), 7.76 (m,
d
69.8,108.7, 110.9,114.7,115.0, 121.1,127.9, 129.2,129.6,132.2,135.3,
1H, ArH). 13C NMR (100 MHz, DMSO-d6)
d
13.9, 62.6, 69.1, 109.0,
135.8, 157.4, 157.5, 161.4, 176.9. MS (ESI) m/z 373 (79Br), 375 (81Br)
[M ꢀ H]ꢀ.
110.6, 113.4 (d, JC-F ¼ 22.5 Hz), 114.2 (d, JC-F ¼ 20.9 Hz), 114.7, 115.5,
122.4 (d, JC-F ¼ 2.6 Hz), 130.2 (d, JC-F ¼ 8.4 Hz), 135.3, 139.8 (d, JC-
¼ 7.8 Hz), 150.2, 157.2, 157.6, 160.0, 162.3 (d, JC-F ¼ 242.9 Hz), 176.5.
5.1.3.3. 5-(30-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3b). The crude was prepared according to general procedure
C starting from 2b (1.8 mg, 4.5 mmol) to afford 3b as a beige solid
(821 mg, 49%). C17H11BrO5. Decomposition at 230 ꢁC. 1H NMR
F
MS (ESI) m/z 343 [MþH]þ.
5.1.2.6. 5-(40-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2e). The crude was prepared according to general
procedure B starting from 1e (1.2 g, 4.6 mmol) and was washed
with diethyl ether to afford 2e as a yellowish solid (590 mg, 39%).
(400 MHz, DMSO-d6)
d 5.26 (s, 2H, OCH2), 6.74 (s, 1H, COCH), 7.07
(d, 1H, J ¼ 8.2 Hz, ArH), 7.20 (d, 1H, J ¼ 8.4 Hz, ArH), 7.37 (m, 1H,
ArH), 7.51 (d, 1H, J ¼ 7.8 Hz, ArH), 7.60 (d, 1H, J ¼ 7.8 Hz, ArH), 7.72
(m, 1H, ArH), 7.92 (m, 1H, ArH). 13C NMR (100 MHz, DMSO-d6)
C
19H15FO5. m.p. 125e126 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 1.34 (t,
3H, J ¼ 7.1 Hz, CO2CH2CH3), 4.38 (q, 2H, J ¼ 7.2 Hz, CO2CH2CH3), 5.25
(s, 2H, OCH2), 6.77 (s, 1H, COCH), 7.13 (d, 1H, J ¼ 7.7 Hz, ArH),
7.21e7.29 (m, 3H, ArH), 7.62e7.69 (m, 2H, ArH), 7.75 (m, 1H, ArH).
d 69.0, 108.7, 110.7, 114.4, 114.7, 121.8, 125.6, 129.4, 130.3, 130.5,
134.9, 139.7, 157.5, 157.6, 161.5, 177.2. MS (ESI) m/z 373 (79Br), 375
(
81Br) [M ꢀ H]ꢀ, 397 (79Br), 399 (81Br) [MþNa]þ.
13C NMR (100 MHz, DMSO-d6)
d 13.9, 62.6, 69.4, 109.1, 110.5, 114.7,
115.1 (d, JC-F ¼ 21.4 Hz), 115.5, 128.9 (d, JC-F ¼ 8.2 Hz), 132.9, 135.3,
150.2, 157.3, 157.8, 160.1, 161.6 (d, JC-F ¼ 243.2 Hz), 176.4. MS (ESI) m/
z 343 [MþH]þ, 365 [MþNa]þ.
5.1.3.4. 5-(20-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3c). The crude was prepared according to general procedure C
starting from 2c (265 mg, 0.8 mmol) to afford 3c as a white solid
(157 mg, 65%). C17H11FO5. m.p. 213e214 ꢁC. 1H NMR (400 MHz,
5.1.2.7. 5-(30,40-Difluorobenzyloxy)-4-oxo-4H-chromene-2-
carboxylic acid ethyl ester (2f). The crude was prepared according to
general procedure B starting from 1f (900 mg, 3.2 mmol) and was
purified by flash silica column chromatography using cyclohexane/
ethyl acetate (100:0 to 50:50) as eluent to afford 2f as a white solid
(200 mg, 18%). C19H14F2O5. m.p. 145e146 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
ArH), 7.40 (m, 1H, ArH), 7.75 (m, 1H, ArH), 7.93 (m, 1H, ArH). 13C
NMR (100 MHz, DMSO-d6)
114.8,115.1 (d, JC-F ¼ 21.9 Hz),115.2,123.7 (d, JC-F ¼ 14.1 Hz),124.5 (d,
JC-F ¼ 3.3 Hz), 129.7 (d, JC-F ¼ 3 Hz), 129.8 (d, JC-F ¼ 8.1 Hz), 135.3,
151.3, 157.4, 157.7, 159.6 (d, JC-F ¼ 245.3 Hz), 161.5, 176.8. MS (ESI) m/
z 313 [M ꢀ H]ꢀ, 337 [MþNa]þ.
d 5.29 (s, 2H, OCH2), 6.73 (s, 1H, COCH), 7.14e7.32 (m, 4H,
d
64.5 (d, JC-F ¼ 3.7 Hz), 108.9, 110.8,
DMSO-d6)
d
1.34 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 4.38 (q, 2H,
J ¼ 7.1 Hz, CO2CH2CH3), 5.26 (s, 2H, OCH2), 6.80 (s, 1H, COCH), 7.12
(d, 1H, J ¼ 8.0 Hz, ArH), 7.25 (d, 1H, J ¼ 7.9 Hz, ArH), 7.46e7.50 (m,
2H, ArH), 7.73e7.82 (m, 2H, ArH). 13C NMR (100 MHz, DMSO-d6)
5.1.3.5. 5-(30-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3d). The crude was prepared according to general procedure
C starting from 2d (1.2 g, 3.5 mmol) to afford 3d as a white solid
(840 mg, 76%). C17H11FO5. m.p. 241e242 ꢁC. 1H NMR (400 MHz,
d
13.9, 62.6, 68.7, 109.0, 110.7, 114.7, 115.4, 115.8 (d, JC-F ¼ 18.2 Hz),
117.4 (d, JC-F ¼ 17.2 Hz), 123.2 (m), 134.6 (m), 135.4, 147.8 (m), 150.1
(m), 150.2, 157.2, 157.5, 160.0, 176.5. MS (ESI) m/z 361 [MþH]þ, 383
[MþNa]þ.
DMSO-d6) d 5.29 (s, 2H, OCH2), 6.76 (s, 1H, COCH), 7.05e7.18 (m, 2H,
ArH), 7.22 (d, 1H, J ¼ 8.5 Hz, ArH), 7.38e7.50 (m, 2H, ArH), 7.56 (d,
1H, J ¼ 10.2 Hz, ArH), 7.77 (m, 1H, ArH). 13C NMR (100 MHz, DMSO-
5.1.2.8. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid ethyl ester (2g). The crude was prepared according to general
procedure B from 1g (2 g, 6.2 mmol) and was washed with diethyl
d6)
d
69.1, 108.8, 110.6, 113.4 (d, JC-F ¼ 22.5 Hz), 114.1 (d, JC-F ¼ 21 Hz),
114.7, 115.2, 122.4, 130.2, 135.2, 138.8 (d, JC-F ¼ 7.8 Hz), 151.2, 157.4,
157.6, 161.5, 162.3 (d, JC-F ¼ 242.7 Hz), 176.8. MS (ESI) m/z 313

A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
297
[M ꢀ H]ꢀ, 337 [MþNa]þ.
H, 4.22; N, 5.12.
5.1.3.6. 5-(40-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3e). The crude was prepared according to general procedure C
starting from 2e (500 mg,1.5 mmol) to afford 3e as a yellowish solid
(220 mg, 47%). m.p. 178e179 ꢁC. C17H11FO5. 1H NMR (400 MHz,
5.1.4.3. 5-(30-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol 3-yl)-ethyl]-amide (4b). The crude was
prepared according to general procedure D starting from 3b (0.6 g,
1.7 mmol) and 5-methoxytryptamine hydrochloride (323 mg,
1.7 mmol) and was recrystallized in ethyl acetate/toluene (9:1) to
afford 4b as a cream solid (456 mg, 49%). C28H23BrN2O5. m.p.
DMSO-d6)
d 5.24 (s, 2H, OCH2), 6.69 (s, 1H, COCH), 7.10 (d, 1H,
J ¼ 8.3 Hz, ArH), 7.20 (d, 1H, J ¼ 8.5 Hz, ArH), 7.18e7.28 (m, 2H, ArH),
7.63e7.77 (m, 2H, ArH), 7.72 (m, 1H, ArH). 13C NMR (100 MHz,
199e200 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H, J ¼ 6.8 Hz,
DMSO-d6)
d
69.3, 108.7, 110.6, 114.3, 114.7, 115.1 (d, JC-F ¼ 21.3 Hz),
CH2CH2NH), 3.57 (m, 2H, CH2CH2NH), 3.74 (s, 3H, OCH3), 5.27 (s, 2H,
OCH2), 6.69 (s, 1H, COCH), 6.73 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.7 Hz, ArH),
7.08 (d, 1H, J ¼ 1.8 Hz, ArH), 7.10 (d, 1H, J ¼ 8.3 Hz, ArH), 7.17 (d, 1H,
J ¼ 1.7 Hz, ArH), 7.23 (d, 1H, J ¼ 8.7 Hz, ArH), 7.26 (d, 1H, J ¼ 8.3 Hz,
ArH), 7.38 (m, 1H, ArH), 7.52 (d, 1H, J ¼ 8.0 Hz, ArH), 7.60 (d, 1H,
J ¼ 7.6 Hz, ArH), 7.77 (m, 1H, ArH), 7.91 (m, 1H), 9.19 (t, 1H,
J ¼ 5.2 Hz, CONH), 10.68 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
128.9 (d, JC-F ¼ 8.1 Hz), 133.0 (d, JC-F ¼ 2.8 Hz), 134.8, 157.5, 157.8,
158.6, 161.3, 161.6 (d, JC-F ¼ 242.9 Hz), 177.1. MS (ESI) m/z 313
[M ꢀ H]ꢀ.
5.1.3.7. 5-(30,40-Difluorobenzyloxy)-4-oxo-4H-chromene-2-
carboxylic acid (3f). The crude was prepared according to general
procedure C starting from 2f (196 mg, 0.5 mmol) to afford 3f as a
white solid (142 mg, 79%). C17H10F2O5. m.p. 198e199 ꢁC. 1H NMR
d
24.9, CH2 under DMSO, 55.2, 69.0, 100.2, 108.9, 110.7, 111.1, 111.2,
112.0, 112.1, 114.5, 121.7, 123.4, 125.6, 127.5, 129.4, 130.3, 130.5, 131.4,
(400 MHz, DMSO-d6)
1H, J ¼ 7.9 Hz, ArH), 7.23 (d, 1H, J ¼ 7.7 Hz, ArH), 7.42e7.54 (m, 2H,
ArH), 7.73e7.84 (m, 2H, ArH). 13C NMR (100 MHz, DMSO-d6)
68.6,
d
5.25 (s, 2H, OCH2), 6.74 (s,1H, COCH), 7.10 (d,
135.0, 139.7, 153.0, 153.6, 157.0, 157.6, 158.9, 176.6. MS (ESI) m/z 569
(
59.48; H, 4.46; N, 4.95; Found: C, 59.73; H, 4.29; N, 4.82.
79Br), 571 (81Br) [MþNa]þ; Anal. Calcd for C28H23BrN2O5, H2O: C,
d
108.9, 110.7, 114.7, 115.1, 115.8 (d, JC-F ¼ 18.1 Hz), 117.4 (d, JC-
¼ 17.1 Hz), 123.2 (m), 134.7, 135.2, 148.0 (m), 150.0, 151.5, 157.4,
5.1.4.4. 5-(20-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4c). The crude
was prepared according to general procedure D starting from 3c
(157 mg, 0.5 mmol) and was recrystallized in acetonitrile to afford
4c as a white solid (18 mg, 7%). C28H23FN2O5. m.p. 216e217 ꢁC. 1H
F
157.5, 161.5, 176.9. MS (ESI) m/z 331 [M ꢀ H]ꢀ.
5.1.3.8. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid (3g). The crude was prepared according to general procedure B
from 2g (1.7 g, 4.3 mmol) to afford 3g as a white solid (1.5 g, 90%).
NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H, J ¼ 7.5 Hz, CH2CH2NH), 3.56
C
17H11BrO5. Decomposition at 200 ꢁC. 1H NMR (400 MHz, DMSO-
(m, 2H, CH2CH2NH), 3.73 (s, 3H, OCH3), 5.29 (s, 2H, OCH2), 6.66 (s,
1H, COCH), 6.71 (m, 1H, ArH), 7.07 (m, 1H, ArH), 7.17e7.29 (m, 6H,
ArH), 7.41 (m, 1H), 7.78 (m, 1H, ArH), 7.95 (m, 1H, ArH), 9.21 (t, 1H,
J ¼ 5.5 Hz, CONH), 10.69 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d6)
ArH), 6.76 (d, 1H, J ¼ 8.0 Hz, ArH), 7.08e7.20 (m, 4H, ArH), 7.28 (m,
1H, ArH). 13C NMR (100 MHz, DMSO-d6)
69.2, 108.9, 110.6, 114.7,
d
4.79 (s, 2H, OCH2), 6.29 (s, 1H, COCH), 6.64 (d, 1H, J ¼ 8.2 Hz,
d
115.2, 120.6, 128.9, 131.2, 135.1, 136.3, 151.2, 157.4, 157.7, 161.4, 176.7.
d
24.9, CH2 under DMSO, 55.3, 64.5 (d, JC-F ¼ 4.0 Hz), 100.1, 108.9,
MS (ESI) m/z 374 (79Br), 476 (81Br) [M]þ.
110.7, 111.1, 111.2, 112.1, 114.5,115.0 (d, JC-F ¼ 14.2 Hz), 123.4, 123.8 (d,
JC-F ¼ 14.2 Hz), 124.5 (d, JC-F ¼ 2.9 Hz), 127.5, 129.6 (d, JC-F ¼ 4.3 Hz),
129.8 (d, JC-F ¼ 8.3 Hz), 131.4, 135.0, 153.0, 153.6, 157.0, 157.7, 158.9,
159.6 (d, JC-F ¼ 245.2 Hz), 176.5. MS (ESI) m/z 485 [M ꢀ H]ꢀ, 487
[MþH]þ, 509 [MþNa]þ. Anal. Calcd for C28H23FN2O5, C, 69.13; H,
4.77; N, 5.76; Found: C, 69.09; H, 4.81; N, 6.13.
5.1.4. Synthesis of targeted compounds 4aeg
5.1.4.1. General procedure D. To a solution of tryptamine derivative
(1 equiv.) in DMF (10 mL/mmol) were added successively a solution
of acid derivative 3aeg (1 equiv.) in DMF (10 mL/mmol), HOBt
(2 equiv.), triethylamine (4 equiv.) and EDCI (2 equiv.). The mixture
was stirred overnight at room temperature. The resulting mixture
was poured into 1 M hydrochloric acid and extracted with ethyl
acetate. The combined organic layers were washed with sodium
bicarbonate and water, dried over anhydrous magnesium sulfate,
filtered and concentrated under reduced pressure.
5.1.4.5. 5-(30-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4d). The crude
was prepared according to general procedure D starting from 3d
(370 mg, 1.2 mmol) and was washed with diethyl ether to afford 4d
as a white solid (300 mg, 51%). C28H23FN2O5. Decomposition at
260 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H, J ¼ 7.6 Hz,
5.1.4.2. 5-(20-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4a). The crude
was prepared according to general procedure D starting from 3a
(103 mg, 0.3 mmol) and 5-methoxytryptamine hydrochloride
(105 mg, 0.55 mmol) and was purified by flash silica column
chromatography, using dichloromethane/methanol (95:5) as
eluent to afford 4a as a white solid (36 mg, 24%). C28H23BrN2O5.
CH2CH2NH), 3.57 (m, 2H, CH2CH2NH), 3.75 (s, 3H, OCH3), 5.29 (s,
2H, OCH2), 6.69 (s, 1H, COCH), 6.71 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.7 Hz,
ArH), 7.08 (d, 1H, J ¼ 2.3 Hz, ArH), 7.11 (d, 1H, J ¼ 8.3 Hz, ArH), 7.15
(m, 1H, ArH), 7.17 (d, 1H, J ¼ 2.2 Hz, ArH), 7.23 (d, 1H, J ¼ 8.8 Hz,
ArH), 7.26 (d, 1H, J ¼ 8.0 Hz, ArH), 7.41e7.49 (m, 2H, ArH), 7.57 (m,
1H, ArH), 7.77 (m, 1H, ArH), 9.19 (t, 1H, J ¼ 5.8 Hz, CONH), 10.68 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6)
d 24.9, CH2 under DMSO,
m.p. 204e205 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H,
55.3, 69.1, 100.1, 108.8, 110.6, 111.0, 111.1, 112.0, 112.1, 113.4 (d, JC-
J ¼ 7.6 Hz, CH2CH2NH), 3.56 (m, 2H, CH2CH2NH), 3.74 (s, 3H, OCH3),
5.23 (s, 2H, OCH2), 6.69 (s, 1H, COCH), 6.71 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 8.8 Hz, ArH), 7.08 (d, 1H, J ¼ 2.3 Hz, ArH), 7.12e7.18 (m, 2H, ArH),
7.23 (d, 1H, J ¼ 8.6 Hz), 7.26e7.35 (m, 2H, ArH), 7.50 (m, 1H, ArH),
7.67 (m, 1H, ArH), 7.80 (m, 1H, ArH), 8.12 (m, 1H, ArH), 9.20 (t, 1H,
J ¼ 5.7 Hz, CONH), 10.7 (s, 1H, NH). 13C NMR (500 MHz, DMSO-d6)
¼ 22.6 Hz), 114.1 (d, JC-F ¼ 21 Hz), 114.5, 122.4 (d, JC-F ¼ 2.6 Hz),
F
123.4, 127.5, 130.2 (d, JC-F ¼ 8.4 Hz), 131.4, 135.0, 139.8 (d, JC-
¼ 7.9 Hz), 153.0, 153.6, 157.0, 157.6, 158.9, 161.3 (d, JC-F ¼ 242.9 Hz),
F
176.6. MS (ESI) m/z 487 [MþH]þ.
5.1.4.6. 5-(40-Fluorobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4e). The crude
was prepared according to general procedure D starting from 3e
(180 mg, 0.6 mmol) and 5-methoxytryptamine hydrochloride
(114 mg, 0.6 mmol) and was precipitated with diethyl ether to
afford 4e as a yellow solid (118 mg, 50%). C28H23FN2O5. m.p.
d
24.9, CH2 under DMSO, 55.3, 69.7, 100.1, 108.8, 110.8, 111.1, 111.2,
112.0, 112.1, 114.5, 121.1, 123.4, 127.6, 127.9, 129.2, 129.6, 131.4, 132.2,
135.2, 135.8, 153.0, 153.7, 157.1, 157.5, 158.9, 176.6. MS (ESI) m/z 547
(
79Br), 549 (81Br) [MþH]þ, 545 (79Br), 547 (81Br) [M ꢀ H]ꢀ. Anal.
Calcd for C28H23BrN2O5, C, 61.43; H, 4.24; N, 5.12; Found: C, 61.74;

298
A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
234e236 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H, J ¼ 7.8 Hz,
under DMSO, 69.2, 102.2, 108.9, 110.5, 110.6, 111.4, 111.7, 112.1, 114.5,
120.6, 123.2, 127.9, 128.9, 130.8, 131.2, 134.9, 136.3, 150.3, 153.6,
157.0,157.7,158.9,176.6. MS (ESI) m/z 533 (79Br), 535 (81Br) [MþH]þ.
Anal. Calcd for C27H21BrN2O4, 1/2 H2O: C, 59.79; H, 4.09; N, 5.16.
Found: C, 59.66; H, 3.76; N, 5.41.
CH2CH2NH), 3.56 (m, 2H, CH2CH2NH), 3.74 (s, 3H, OCH3), 5.24 (s, 2H,
OCH2), 6.67 (s, 1H, COCH), 6.72 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.7 Hz, ArH),
7.07 (d, 1H, J ¼ 2.4 Hz, ArH), 7.12 (d, 1H, J ¼ 8.0 Hz, ArH), 7.17 (d, 1H,
J ¼ 2.3 Hz, ArH), 7.20e7.30 (m, 4H, ArH), 7.60e7.75 (m, 2H, ArH),
7.76 (m, 1H, ArH), 9.18 (t, 1H, J ¼ 5.8 Hz, CONH), 10.7 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6)
d
24.8, CH2 under DMSO, 55.3, 69.3,100.1,
5.1.5.2. General procedure E. A solution of 5a (1 equiv.), potassium
carbonate (6 equiv.) and tetrabutylammonium bromide
(0.35 equiv.) in acetone (10 mL/mmol) was refluxed for 20 min. A
solution of the corresponding alkyl iodide (1.2 equiv.) in acetone
(3 mL/mmol) was added dropwise and the resulting mixture was
refluxed for 2e6 h (the end of the reaction was controlled by TLC
using acetone/cyclohexane 40:60). The resulting solution was
cooled to room temperature and concentrated under reduced
pressure. The residue was poured into water and extracted with
ethyl acetate. The combined organic layers were washed with
water, dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure.
109.0, 110.5, 111.1, 111.2, 112.0, 112.1, 114.5, 115.1 (d, JC-F ¼ 21.5 Hz),
123.4, 127.5, 128.9 (d, JC-F ¼ 8.3 Hz), 131.4, 133.0 (d, JC-F ¼ 2.9 Hz),
134.9, 153.0, 153.6, 157.0, 157.8, 158.9, 161.6 (d, JC-F ¼ 243.0 Hz),
176.5. MS (ESI) m/z 487 [MþH]þ, 509 [MþNa]þ. Anal. Calcd for
C
28H23FN2O5, 1/3 H2O: C, 68.29; H, 4.84; N, 5.69; Found: C, 68.63; H,
4.63; N, 5.66.
5.1.4.7. 5-(30,40-Difluorobenzyloxy)-4-oxo-4H-chromene-2-
carboxylic acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4f).
The crude was prepared according to general procedure D starting
from 3f (142 mg, 0.4 mmol) and was recrystallized in acetonitrile to
afford 4f as a white solid (13.5 mg, 5%). C28H22F2N2O5. m.p.
234e236 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.95 (t, 2H, J ¼ 7.7 Hz,
5.1.5.3. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-ethoxy-1H-indol-3-yl)-ethyl]-amide (5b). The crude was
prepared according to general procedure E starting from 5a
(200 mg, 0.4 mmol) and iodoethane (56 mg, 0.4 mmol) and was
purified by flash silica column chromatography, using dichloro-
methane/methanol (99:1) as eluent to afford 5b as a white solid
(36.5 mg,17%). C29H25BrN2O5. m.p. 234e236 ꢁC. 1H NMR (400 MHz,
CH2CH2NH), 3.57 (m, 2H, CH2CH2NH), 3.74 (s, 3H, OCH3), 5.25 (s, 2H,
OCH2), 6.69 (s, 1H, COCH), 6.71 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.7 Hz, ArH),
7.08 (d, 1H, J ¼ 2.3 Hz, ArH), 7.10 (d, 1H, J ¼ 8.3 Hz, ArH), 7.17 (d, 1H,
J ¼ 2.2 Hz, ArH), 7.20e7.30 (m, 2H, ArH), 7.40e7.55 (m, 2H, ArH),
7.73e7.83 (m, 2H, ArH), 9.19 (t, 1H, J ¼ 5.8 Hz, CONH), 10.7 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6)
d 24.9, CH2 under DMSO, 55.3,
68.6, 100.1, 108.9, 110.7, 111.0, 111.2, 112.0, 112.1, 114.5, 115.8 (d, JC-
DMSO-d6)
d
1.28 (t, 3H, J ¼ 6.8 Hz, OCH2CH3), 2.92 (t, 2H, J ¼ 7.2 Hz,
¼ 18.1 Hz), 117.4 (d, JC-F ¼ 17.1 Hz), 123.3 (m), 123.4, 127.5, 131.4,
CH2CH2NH), 3.53 (m, 2H, CH2CH2NH), 3.94 (q, 2H, J ¼ 6.8 Hz,
OCH2CH3), 5.23 (s, 2H, OCH2), 6.64 (s, 1H, COCH), 6.68 (dd, 1H,
J ¼ 2.0 Hz, J ¼ 8.4 Hz, ArH), 7.06e7.24 (m, 5H, ArH), 7.55e7.66 (m,
4H, ArH), 7.74 (m, 1H, ArH), 9.15 (t, 1H, J ¼ 5.6 Hz, CONH), 10.64 (s,
F
134.7 (m),135.0,147.8 (m),150.3 (m),153.0,153.7,157.0,157.5,158.9,
176.5. MS (ESI) m/z 505 [MþH]þ, 527 [MþNa]þ. Anal. Calcd for
C
28H22F2N2O5: C, 66.65; H, 4.40; N, 5.55. Found: C, 66.57; H, 4.36; N,
5.49.
1H, NH). 13C NMR (100 MHz, DMSO-d6)
d 14.9, 24.8, CH2 under
DMSO, 63.3, 69.2, 101.2, 109.0, 110.6, 111.2, 111.5, 112.0, 112.1, 114.5,
120.6,123.5,127.6,128.9,131.2,131.4,134.9,136.3,152.1,153.6,157.0,
157.7, 158.9, 176.5. MS (ESI) m/z 561 (79Br), 563 (81Br) [MþH]þ; 583
5.1.4.8. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide (4g or MBL-II-141).
The crude was prepared according to general procedure D from 3g
(860 mg, 2.3 mmol) and 5-methoxytryptamine hydrochloride
(430 g, 2.3 mmol) and was purified by flash silica column chro-
matography using dichloromethane/ethyl acetate (9:1) as eluent to
afford MBL-II-141 as a beige powder (566 mg, 45%). C28H23BrN2O5.
(
79Br), 585 (81Br) [MþNa]þ. Anal. Calcd for C29H25BrN2O5: C, 62.04;
H, 4.49; N, 4.99. Found: C, 61.82; H, 4.28; N, 4.75.
5.1.5.4. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-propyloxy-1H-indol-3-yl)-ethyl]-amide (5c). The crude
was prepared according to general procedure E starting from 5a
(200 mg, 0.4 mmol) and 1-iodopropane (60 mg, 0.4 mmol) and was
purified by flash silica column chromatography using dichloro-
methane/methanol (99:1) as eluent to afford 5c as a white solid
(67 mg, 31%). C30H27BrN2O5. m.p. 203e206 ꢁC. 1H NMR (400 MHz,
mp 159e160 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 2.95 (t, 2H,
J ¼ 7.8 Hz, CH2CH2NH), 3.56 (m, 2H, CH2CH2NH), 3.74 (s, 3H, OCH3),
5.25 (s, 2H, OCH2), 6.67 (s, 1H, COCH), 6.71 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 8 Hz, ArH), 7.06e7.29 (m, 5H, ArH), 7.55e7.65 (m, 4H, ArH), 7.76
(m, 1H, ArH), 9.18 (t, 1H, J ¼ 5.8 Hz, CONH), 10.68 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6)
d
24.8, CH2 under DMSO, 55.3, 69.2,100.1,
DMSO-d6)
d
0.94 (t, 3H, J ¼ 8 Hz, CH3), 1.66 (m, 2H, OCH2CH2CH3),
108.9, 110.6, 111.0, 111.2, 112.0, 114.5, 120.6, 123.4, 127.5, 128.9, 131.2,
131.4, 134.9, 136.3, 153.0, 153.6, 157.0, 157.7, 158.9, 176.6. MS (ESI) m/
z 546 (79Br), 548 (81Br) [M]þ. Anal. Calcd for C28H23BrN2O5: C,
61.44; H, 4.24; N, 5.12. Found C, 61.38; H, 4.23; N, 5.10.
2.93 (t, 2H, J ¼ 8 Hz, CH2CH2NH), 3.55 (m, 2H, CH2CH2NH), 3.86 (t,
2H, J ¼ 6.4 Hz, OCH2CH2CH3), 5.23 (s, 2H, OCH2), 6.66 (s, 1H, COCH),
6.70 (dd, 1H, J ¼ 2.0 Hz, J ¼ 8.8 Hz, ArH), 7.02e7.31 (m, 5H, ArH),
7.55e7.68 (m, 4H, ArH), 7.76 (m, 1H, ArH), 9.17 (t, 1H, J ¼ 5.6 Hz,
CONH), 10.66 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d 10.5, 22.3,
5.1.5. Synthesis of compounds 5aee belonging to the second type of
modulation
24.8, CH2 under DMSO, 69.2, 69.4, 101.2, 109.0, 110.6, 111.3, 111.5,
112.0, 112.1, 114.5, 120.6, 123.4, 127.6, 128.9, 131.2, 131.4, 134.9, 136.3,
152.3, 153.6, 157.0, 157.7, 158.9, 176.5. MS (ESI) m/z 575 (79Br), 577
5.1.5.1. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-hydroxy-1H-indol-3-yl)-ethyl]-amide (5a). The crude was
prepared according to general procedure D starting from 3g (1 g,
2.66 mmol) and commercially available serotonine hydrochloride
(567 mg, 2.7 mmol) and was washed with diethylether to afford 5a
as a white solid (794 mg, 56%). C27H21BrN2O5. m.p. 222e225 ꢁC. 1H
(
81Br) [MþH]þ; 597 (79Br), 599 (81Br) [MþNa]þ. Anal. Calcd for
C30H27BrN2O5, 1/2 H2O: C, 61.65; H, 4.83; N, 4.79. Found: C, 61.92;
H, 4.77; N, 4.97.
5.1.5.5. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(5-isopropyloxy-1H-indol-3-yl)-ethyl]-amide (5d). The crude
was prepared according to general procedure E starting from 5a
(280 mg, 0.5 mmol) and 2-iodopropane (89 mg, 0.5 mmol) and was
purified by flash silica column chromatography using dichloro-
methane/methanol (99:1) as eluent to afford 5d as a white solid
(90 mg, 30%). C30H27BrN2O5. m.p. 240e243 ꢁC. 1H NMR (400 MHz,
NMR (400 MHz, DMSO-d6)
d
2.88 (t, 2H, J ¼ 8 Hz, CH2CH2NH), 3.53
(m, 2H, CH2CH2NH), 5.24 (s, 2H, OCH2), 6.59 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 8.8 Hz, ArH), 6.68 (s, 1H, COCH), 6.89 (d, 1H, J ¼ 2.4 Hz, ArH),
7.05e7.17 (m, 3H, ArH), 7.25 (d, 1H, J ¼ 8.4 Hz, ArH), 7.55e7.67 (m,
4H, ArH), 7.76 (m, 1H, ArH), 8.60 (s, 1H, OH), 9.17 (t, 1H, J ¼ 5.7 Hz,
CONH), 10.5 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d 25.0, CH2

A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
299
DMSO-d6)
d
1.21 (d, 6H, J ¼ 6.0 Hz, CH(CH3)2), 2.93 (t, 2H, J ¼ 7.5 Hz,
135e138 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
0.89 (d, 3H, J ¼ 4.3 Hz,
CH2CH2NH), 3.54 (m, 2H, CH2CH2NH), 4.46 (septuplet, 1H,
J ¼ 6.0 Hz, CH(CH3)2), 5.24 (s, 2H, OCH2), 6.66 (s, 1H, COCH), 6.69
(dd, 1H, J ¼ 2.3 Hz, J ¼ 8.7 Hz, ArH), 7.00e7.30 (m, 5H, ArH),
7.53e7.65 (m, 4H, ArH), 7.76 (t, 1H, J ¼ 8.4 Hz, ArH), 9.17 (t, 1H,
J ¼ 5.9 Hz, CONH), 10.65 (s, 1H, NH). 13C NMR (125 MHz, DMSO-d6)
CH3), 0.92 (d, 3H, J ¼ 4.4 Hz, CH3), 1.64 (m, 2H, CHCH2CH(CH3)2),
1.84 (m, 1H, CHCH2CH(CH3)2), 3.67 (s, 3H, OCH3), 4.53 (m, 1H,
CHCH2CH(CH3)2), 5.25 (s, 2H, OCH2), 6.70 (s, 1H, COCH), 7.11 (d, 1H,
J ¼ 8.4 Hz, ArH), 7.30 (d,1H, J ¼ 8.2 Hz, ArH), 7.55e7.68 (m, 4H, ArH),
7.77 (m, 1H, ArH), 9.33 (d, 1H, J ¼ 7.3 Hz, CONH). 13C NMR (100 MHz,
d
22.0, 24.8, CH2 under DMSO, 69.2, 70.1, 104.0, 109.0, 110.6, 111.2,
DMSO-d6) d 21.1, 22.8, 24.3, signal under DMSO, 50.9, 52.1, 69.2,
111.9, 112.0, 112.9, 120.6, 123.5, 127.7, 128.9, 131.2, 131.6, 134.9, 136.3,
109.1, 110.7, 112.7, 114.6, 120.6, 128.9, 131.2, 135.0, 136.3, 152.8, 157.0,
157.7,159.4,172.1,176.4. MS (ESI) m/z 502 (79Br), 504 (81Br) [MþH]þ.
Anal. Calcd for C24H24BrNO6: C, 57.38; H, 4.82; N, 2.79. Found: C,
57.00; H, 5.12; N, 2.87.
150.7, 152.6, 153.6, 157.7, 158.9, 176.7. MS (ESI) m/z 575 (79Br), 576
(
81Br) [MþH]þ. Anal. Calcd for C30H27BrN2O5: C, 62.62; H, 4.73; N,
4.87. Found: C, 64.16; H, 4.75; N, 4.85.
5.1.5.6. 5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-carboxylic
acid [2-(1H-indol-3-yl)-ethyl]-amide (5e). The crude was prepared
according to general procedure D starting from 3g (200 mg,
0.5 mmol) and commercially available tryptamine hydrochloride
(98 mg, 0.5 mmol) and was purified by flash silica column chro-
matography using dichloromethane/methanol (95:5) as eluent to
afford to afford 5e as a white solid (80 mg, 31%). C27H21BrN2O4. m.p.
5.1.7. Synthesis of 7aeb acids
5.1.7.1. General procedure G. To a solution of methyl ester 6aed
(1 equiv.) in tetrahydrofuran/methanol (1/1) (10 mL/mmol) was
added a solution of lithium hydroxide (1 equiv.) in water (5 mL/
mmol). The resulting mixture was stirred at room temperature for
1 h. The solution was poured into 1 M hydrochloric acid and
extracted with dichloromethane. The combined organic layers
were washed with water, dried over anhydrous magnesium sulfate,
filtered and concentrated under reduced pressure.
221 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
2.99 (t, 2H, J ¼ 7.5 Hz,
CH2CH2NH), 3.58 (m, 2H, CH2CH2NH), 5.24 (s, 2H, OCH2), 6.68 (s,1H,
COCH), 6.92e7.40 (m, 6H, ArH), 7.52e7.68 (m, 5H, ArH), 7.77 (m, 1H,
ArH), 9.20 (t, 1H, J ¼ 5.7 Hz, CONH), 10.8 (s, 1H, NH). 13C NMR
5.1.7.2. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-3-methylbutyric acid (7a). The crude was pre-
pared according to general procedure G starting from 6a (400 mg,
0.8 mmol) and recrystallized in acetonitrile to afford 7a as a white
solid (282 mg, 72%). C22H20BrNO6. m.p. 250e251 ꢁC. 1H NMR
(100 MHz, DMSO-d6)
d 24.8, CH2 under DMSO, 69.2, 109.0, 110.6,
111.4, 112.0, 114.5, 118.2, 118.3, 120.6, 121.0, 122.7, 127.2, 128.9, 131.2,
135.0, 136.2,136.3, 153.6, 157.0, 157.7, 158.9, 162.3, 176.6. MS (ESI) m/
z 539 (79Br), 541 (81Br) [MþNa]þ. Anal. Calcd for C27H21BrN2O4: C,
62.67; H, 4.10; N, 5.41. Found: C, 62.45; H, 4.12; N, 5.48.
(400 MHz, DMSO-d6)
d 0.97 (m, 6H, CH(CH3)2), 2.24 (m, 1H,
CHCH(CH3)2), 4.25 (m, 1H, CHCH(CH3)2), 5.25 (s, 2H, OCH2), 6.70 (s,
1H, COCH), 7.11 (d, 1H, J ¼ 8.3 Hz, ArH), 7.34 (d, 1H, J ¼ 8.4 Hz, ArH),
7.54e7.69 (m, 4H, ArH), 7.77 (m, 1H, ArH), 8.89 (d, 1H, J ¼ 7.8 Hz,
5.1.6. Synthesis of esters 6aeb
5.1.6.1. General procedure F. To a solution of acid 3g (1 equiv.) in
DMF (10 mL/mmol) were added successively a solution of corre-
CONH). 13C NMR (100 MHz, DMSO-d6)
d 19.0, 19.5, 29.8, 58.9, 69.5,
sponding
protected
amino-acid
derivative
or
5-
109.3, 111.2, 112.8, 114.7, 120.9, 129.3, 131.5, 135.3, 136.5, 153.5, 157.3,
157.9, 159.7, 172.5, 176.9. MS (ESI) m/z 472 (79Br), 474 (81Br)
[M ꢀ H]ꢀ, 496 (79Br), 498 (81Br) [MþNa]þ. Anal. Calcd for
methoxytryptamine hydrochloride (2 equiv.) in DMF (10 mL/
mmol), TBTU (1.5 equiv.) and DIEA (5 equiv.). The mixture was
stirred overnight at room temperature. The resulting mixture was
poured into sodium bicarbonate and extracted with ethyl acetate.
The combined organic layers were washed with water, dried over
anhydrous magnesium sulfate, filtered and concentrated under
reduced pressure.
C22H20BrNO6: C, 55.70; H, 4.26; N, 2.95. Found: C, 55.54; H, 4.16; N,
3.06.
5.1.7.3. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-3-methylpentanoic acid (7b). The crude was pre-
pared according to general procedure G starting from 6b (450 mg,
0.9 mmol) and recrystallized in acetonitrile to afford 7b as a white
solid (163 mg, 37%). C23H22BrNO6. m.p. 258e259 ꢁC. 1H NMR
5.1.6.2. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-3-methylbutyric acid methyl ester (6a). The crude
was prepared according to general procedure F starting from 3g
(400 MHz, DMSO-d6)
d
0.88 (d, 3H, J ¼ 6.1 Hz, CH3), 0.93 (d, 3H,
(500 mg, 1.3 mmol) and
L-valine methylester hydrochloride
J ¼ 6.2 Hz, CH3), 1.66 (m, 2H, CHCH2CH(CH3)2), 1.81 (m, 1H,
CHCH2CH(CH3)2), 4.45 (m, 1H, CHCH2CH(CH3)2), 5.25 (s, 2H, OCH2),
6.70 (s, 1H, COCH), 7.11 (d, 1H, J ¼ 8.0 Hz, ArH), 7.30 (dd, 1H,
J ¼ 0.8 Hz, J ¼ 8.5 Hz, ArH), 7.55e7.65 (m, 4H, ArH), 7.77 (m, 1H,
ArH), 9.33 (d, 1H, J ¼ 7.6 Hz, CONH). 13C NMR (100 MHz, DMSO-d6)
(446 mg, 2.7 mmol) and was washed with diethyl ether to afford 6a
as a white solid (540 mg, 83%). C23H22BrNO6. m.p. 151e152 ꢁC. 1H
NMR (400 MHz, DMSO-d6)
d
0.95 (d, 3H, J ¼ 6.7 Hz, CH3), 0.98 (d,
3H, J ¼ 6.8 Hz, CH3), 2.23 (m, 1H, CHCH(CH3)2), 3.69 (s, 3H, OCH3),
4.30 (m, 1H, CHCH(CH3)2), 5.25 (s, 2H, OCH2), 6.70 (s, 1H, COCH),
7.11 (d, 1H, J ¼ 8.3 Hz, ArH), 7.34 (d, 1H, J ¼ 8.3 Hz, ArH), 7.55e7.68
(m, 4H, ArH), 7.77 (m, 1H, ArH), 9.16 (d, 1H, J ¼ 7.9 Hz, CONH). 13C
d
21.1, 22.9, 24.5, signal under DMSO, 50.9, 69.3, 109.0, 110.8, 112.6,
114.6, 120.6, 129.0, 131.2, 135.0, 136.3, 153.1, 157.1, 157.7, 159.3, 173.2,
176.5. MS (ESI) m/z 486 (79Br), 488 (81Br) [M ꢀ H]ꢀ, 488 (79Br), 490
NMR (100 MHz, DMSO-d6)
d
19.0, 19.1, 29.5, 51.9, 58.6, 69.2, 109.0,
(
81Br) [MþH]þ. Anal. Calcd for C23H22BrNO6: C, 56.57; H, 4.54; N,
110.9, 112.7, 114.6, 120.6, 129.0, 131.2, 135.0, 136.3, 153.0, 157.0, 157.7,
159.7,171.4,176.4. MS (ESI) m/z 488 (79Br), 490 (81Br) [MþH]þ. Anal.
Calcd for C23H22BrNO6: C, 56.57; H, 4.54; N, 2.87. Found: C, 56.42;
H, 4.49; N, 3.01.
2.87. Found: C, 56.54; H, 4.65; N, 3.06.
5.1.8. Synthesis of targeted compounds 8aeb
5.1.8.1. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-3-methylbutyric acid [2-(5-methoxy-1H-indol-3-
yl)-ethyl]-amide (8a). The crude was prepared according to gen-
eral procedure F starting from 7a (263 mg, 0.55 mmol) and 5-
methoxytryptamine hydrochloride (211 mg, 1.1 mmol) and was
purified by flash silica column chromatography using cyclohexane/
acetone (8:2 to 5:5) as eluent to afford 8a as a white solid (62.5 mg,
18%). C33H32BrN3O6. m.p. 170e171 ꢁC. 1H NMR (400 MHz, DMSO-
5.1.6.3. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-4-methylpentanoic acid methyl ester (6b).
The crude was prepared according to general procedure F starting
from 3g (500 mg, 1.3 mmol) and L-leucine methylester hydrochlo-
ride (484 mg, 2.7 mmol) and was purified by flash silica column
chromatography, using cyclohexane:acetone (8:2 to 5:5) as eluent
to afford 6b as a white solid (531 mg, 79%). C24H24BrNO6. m.p.
d6)
d
0.90 (d, 6H, J ¼ 6.7 Hz, CH(CH3)2), 2.14 (m, 1H, CHCH(CH3)2),

300
A.R.A. Pires et al. / European Journal of Medicinal Chemistry 122 (2016) 291e301
2.81 (t, 2H, J ¼ 7.2 Hz, CH2CH2NH), 3.42 (m, 2H, CH2CH2NH), 3.75 (s,
3H, OCH3), 4.26 (m, 1H, CHCH(CH3)2), 5.25 (s, 2H, OCH2), 6.67e6,73
(m, 2H, ArH), 7.01 (d, 1H, J ¼ 2.3 Hz, ArH), 7.07e7.13 (m, 2H, ArH),
7.20 (d, 1H, J ¼ 8.8 Hz, ArH), 7.35 (d, 1H, J ¼ 7.9 Hz, ArH), 7.55e7.65
(m, 4H, ArH), 7.77 (m, 1H), 8.32 (t,1H, J ¼ 5.6 Hz, CONHCH2), 8.77 (d,
1H, J ¼ 4.8 Hz, CONHCH), 10.64 (s, 1H, NH). 13C NMR (100 MHz,
After 48-h incubation, the cells were exposed to 5 mM mitoxantrone
for 30 min at 37 ꢁC (HEK293-ABCG2 cells), in the presence or
absence of compounds at various concentrations. After cell washing
with phosphate buffer saline (PBS), the cells were trypsinized and
subsequently resuspended in ice-cold PBS for analysis by flow
cytometry. The intracellular drug fluorescence was monitored with
a FACS Calibur cytometer (Becton Dickinson). At least 5000e10,000
events were collected, for which the maximal fluorescence (taken
as 100%) was the difference between the geometric mean fluores-
DMSO-d6)
d 19.2, 19.7, 25.4, 30.7, CH2 under DMSO, 55.9, 59.8, 69.8,
100.6, 109.6, 111.4, 111.6, 111.9, 112.7, 113.0, 114.9, 121.3, 124.0, 128.0,
129.6, 131.8, 131,9, 135.8, 136.7, 153.5, 153.8, 157.6, 158.1, 159.7, 171.0,
177.6. MS (ESI) m/z 644 (79Br), 646 (81Br) [M ꢀ H]ꢀ, 646 (79Br), 648
cence of cells incubated with 1 mM Ko143 and without inhibitor.
(
81Br) [MþH]þ. Anal. Calcd for C33H32BrN2O6: C, 61.31; H, 4.99; N,
6.50. Found: C, 61.27; H, 5.48; N, 5.81.
5.2.5. Cytotoxicity assays
HEK293-ABCG2 and wild-type (control) cells were seeded into
96-well culture plates at a 1 ꢂ 104 cells/well density. After over-
night incubation, the cells were treated with increasing concen-
trations of compounds for 72 h, at 37 ꢁC under 5% CO2. Cell viability
was then evaluated with a MTT colorimetric assay [27]. Control
experiments were performed with DMEM high glucose containing
0.1% DMSO (v/v). The results were expressed as percentage of viable
cells versus initial control cells taken as 100%.
5.1.8.2. 2-[(5-(40-Bromobenzyloxy)-4-oxo-4H-chromene-2-
carbonyl)-amino]-3-methylpentanoic acid [2-(5-methoxy-1H-indol-
3-yl)-ethyl]-amide (8b). The crude was prepared according to
general procedure E starting from 7b (163 mg, 0.3 mmol) and 5-
methoxytryptamine hydrochloride (127 mg, 0.7 mmol) and was
washed with diethyl ether to afford 8b as a white solid (64 mg,
29%). C34H34BrN3O6. m.p. 130e131 ꢁC. 1H NMR (400 MHz, DMSO-
d6)
d
0.86 (d, 3H, J ¼ 5.7 Hz, CH3), 0.90 (d, 3H, J ¼ 5.8 Hz, CH3), 1.58
(m, 2H, CHCH2CH(CH3)2), 1.71 (m, 1H, CHCH2CH(CH3)2), 2.80 (t, 2H,
J ¼ 7.3 Hz, CH2CH2NH), 3.39 (m, 2H, CH2CH2NH), 3.75 (s, 3H, OCH3),
4.48 (m, 1H, CHCH2CH(CH3)2), 5.25 (s, 2H, OCH2), 6.66e6.75 (m, 2H,
ArH), 7.02 (m, 1H, ArH), 7.07e7.15 (m, 2H, ArH), 7.21 (d, 1H,
J ¼ 8.7 Hz, ArH), 7.33 (d,1H, J ¼ 8.5 Hz, ArH), 7.55e7.65 (m, 4H, ArH),
7.77 (m, 1H, ArH), 8.22 (m, 1H, CONHCH2), 9.00 (d, 1H, J ¼ 8.3 Hz,
Acknowledgments
The authors are grateful to ANR Labex Arcane (ANR-11-LABX-
0003-01) and the Ligue Nationale contre le Cancer (Equipe Label-
ꢁ
lisee 2014) for financial support and postdoctoral fellowship A.R.A.P
from the Brazilian CNPq (Science without Borders Program 201403/
2014-5).
CONHCH), 10.63 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d 21.4,
23.0, 24.4, 25.1, 2 signals under DMSO, 51.9, 55.4, 69.2, 100.1, 109.0,
110.9, 111.0, 111.4, 112.0, 112.5, 114.6, 120.6, 123.4, 127.5, 129.0, 131.3,
131.4, 135.0, 136.4, 153.0, 153.3, 157.1, 157.7, 159.1, 171.1, 176.6. MS
(ESI) m/z 658 (79Br), 660 (81Br) [M ꢀ H]ꢀ, 660 (79Br), 662 (81Br)
[MþH]þ. Anal. Calcd for C34H34BrN3O6: C, 61.81; H, 5.20; N, 6.36.
Found: C, 60.49; H, 5.22; N, 6.16.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.05.053.
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