Probing the aurone scaffold against Plasmodium falciparum: Design, synthesis and antimalarial activity

Article abstract of DOI:10.1016/j.ejmech.2014.04.076

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC ₅₀ values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds.

Full text of DOI:10.1016/j.ejmech.2014.04.076

European Journal of Medicinal Chemistry 80 (2014) 523e534
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Probing the aurone scaffold against Plasmodium falciparum: Design,
synthesis and antimalarial activity
Marta P. Carrasco a, Ana S. Newton a, Lídia Gonçalves a, Ana Góis b, Marta Machado c,
Jiri Gut d, Fátima Nogueira c, Thomas Hänscheid b, Rita C. Guedes a,
,
,
*
Daniel J.V.A. dos Santos a 1, Philip J. Rosenthal d, Rui Moreira a
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto 1649-003 Lisboa, Portugal
b Unidade de Microbiologia Molecular e Infecção, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, 1649-028 Lisboa, Portugal
c Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 1349-008 Lisboa, Portugal
d Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward
aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Mi-
crowave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy
for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives,
containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening
against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50
values in the low micromolar range. The most potent compounds contained a basic moiety, with the
ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones
revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed
during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite.
Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using
palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the
potential of novel aurone derivatives as promising antimalarial compounds.
Received 9 November 2013
Received in revised form
24 April 2014
Accepted 25 April 2014
Available online 28 April 2014
Keywords:
Malaria
Plasmodium falciparum
Aurones
Cross-coupling reactions
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
compounds isolated from natural sources represents a valuable
approach for the design and development of new drugs.
Malaria remains a critical global health problem, with terrible
social and economic consequences in countries where this disease
is endemic. The problem is exacerbated by the emergence and
spread of parasites that are resistant to well-established antima-
larial drugs [1]. For that reason, development of new, effective,
nontoxic, and affordable antimalarial drugs is a high priority in
medicinal chemistry [2,3]. To achieve this goal, the most important
strategies include not only rational drug design but also exploita-
tion of natural products either as a source of antiparasitic agents or
as an inspiration to design novel molecular scaffolds [4,5]. There-
fore, the evaluation of the antiparasitic activity of plant extracts or
Aurones, 2-benzylidenebenzofuran-3-(2H)-ones (Fig. 1), are
structural isomers of flavones that contain an exocyclic carbone
carbon double bond bridging the benzofuranone and phenyl rings
[6,7]. The therapeutic potential of aurones has been highlighted
with recent studies that revealed their anticancer [8e13], antimi-
crobial [14], antiparasitic [15e18], antiviral [19], and anti-
inflammatory [14,20] activities. In addition, aurones can act as
modulators of ABC drug transporters [21e25] and present inhibi-
tory activity against acetylcholinesterase [26] and MAO-B [27].
The antimalarial potential of aurones was first reported by
Kayser et al. [16]. Moreover, several naturally occurring aurones
were synthesized and tested for their ability to inhibit erythrocytic
stages of Plasmodium falciparum. Some of these compounds
exhibited antiplasmodial activity in the micromolar range, and they
showed no cytotoxicity against mammalian tumor cell lines. More
recently, Souard et al. [18] showed that a series of synthetic aurones
* Corresponding author.
E-mail address: rmoreira@ff.ul.pt (R. Moreira).
Present address: REQUIMTE, Department of Chemistry & Biochemistry, Faculty
1
of Sciences, University of Porto, R. do Campo Alegre, 4169-007 Porto, Portugal.
http://dx.doi.org/10.1016/j.ejmech.2014.04.076
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

524
M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
Table 2
Structure, clogP values and yields for the synthesis (final step) of aurones 4.
Fig. 1. General structure of aurones.
Compound
R3
cLogPa
Yield %
exhibited IC50 values under 5
toxicity against human cells.
mM against P. falciparum without
4a
4b
4c
4d
H
2,4-F
2,4-Cl
3,4,5-OMe
3.09
3.62
4.55
2.93
62
95
59
99
Palladium-catalyzed reactions provide an excellent platform for
compound library extension due to the wide range of trans-
formations mediated by palladium [28]. Most of the aurone-based
libraries already screened against P. falciparum were inspired by
naturally occurring aurones, typically containing hydroxy, methoxy
or acetoxy groups at different positions of this scaffold. Herein we
present the synthesis and screening of a library of novel aurones,
1e7 (Tables 1e4), designed to probe the chemical space around this
scaffold. Many of the target compounds were obtained through
SuzukieMiyaura and BuchwaldeHartwig cross-coupling reactions.
Moreover, taking into account recent data suggesting antimalarial
activity of aurone-Mannich base derivatives [29], some compounds
were also obtained via the introduction of different aliphatic
amines in the benzofuranone ring. All compounds were evaluated
a
clogP values calculated using the AlogPS 2.1 software (http://www.vcclab.org/
lab/alogps/); reference [56].
ability of selected aurones to inhibit hemozoin formation and
falcipain-2, a cysteine protease from P. falciparum, and to address
the possibility of synergism when combined with chloroquine.
2. Results and discussion
2.1. Chemistry
for their activity against
a chloroquine-resistant strain of
A total of 44 aurone derivatives with a wide range of sub-
stituents in ring A and B were synthesized to explore the potential
of this scaffold as a platform to design new antimalarial agents
(Tables 1e4).
P. falciparum and for their cytotoxicity against human cancer cell
lines. To gain insights into the mechanism of action of this class of
compounds, additional studies were performed to evaluate the
Compounds 1aeh, 2jek and 4aed were synthesized from
commercially available benzofuran-3(2H)-one, 8, or 7-methoxy-
3(2H)-benzofuranone, 9, which were reacted with appropriately
substituted benzaldehydes in the presence of neutral alumina [30],
as shown in Scheme 1a.
Table 1
Structure, clogP values and yields for the synthesis (final step) of aurones 1e3.
However, this method failed to provide the 6-hydroxy aurones
5aeg in reasonable yields. In contrast, base-catalyzed aldol
condensation of 6-hydroxy-3(2H)-benzofuranone, 10, with benz-
aldehydes afforded aurones 5aed in good yield (Scheme 1A), while
5eeg were obtained under acidic conditions, in the presence of
glacial acetic acid and hydrochloric acid. The aldehydes required for
aurones 5feg were synthesized from 4-fluorobenzonitrile and the
appropriate phenol, and via the Weinreb amides 13a,b, as described
in Scheme 1B.
Compound
R3
cLogPa
Yield %
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
H
2-Br
3-Br
4-Br
2,4-F
2,4-Cl
3,4,5-OMe
4-NMe2
4-Ph
4-(C6H4-40-F)
4-(C6H4-40-Cl)
4-(C6H4-40-CHO)
4-Bn
4-(30-Quinoline)
4-[50-(Pyridin-20-NH2)]
4-(NHPh)
3.39
4.12
4.13
4.12
3.72
4.60
3.01
3.51
5.06
5.21
5.65
4.70
5.24
5.16
3.69
4.83
4.62
5.40
5.51
5.05
5.21
5.65
4.70
5.24
5.16
3.69
41
55
54
56
30
81
64
49
77
66
64
83
70
87
98
71
49
33
51
71
65
51
63
49
68
76
Compounds 2aeg, 3aeg and 6aec were obtained through a
standard SuzukieMiyaura cross-coupling reaction in the presence
Table 3
Structure, clogP values and yields for the synthesis (final step) of aurones 5.
2g
2h
2i
4-(NHBn)
2j
4-(OC6H4-40-Me)
4-(OC6H4-40-Cl)
3-Ph
Compound
R3
cLogPa
Yield %
2k
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
H
2,4-F
2,4-Cl
3,4,5-OMe
NMe2
4-(OC6H4-40-Me)
4-(OC6H4-40-Cl)
3.02
3.35
4.25
2.60
3.17
5.27
5.13
99
98
99
98
71
51
84
3-(C6H4-40-F)
3-(C6H4-40-Cl)
3-(C6H4-40-CHO)
3-Bn
3-(30-Quinoline)
3-[50-(Pyridin-20-NH2)]
3g
5g
a
a
clogP values calculated using the AlogPS 2.1 software (http://www.vcclab.org/
clogP values calculated using the AlogPS 2.1 software (http://www.vcclab.org/
lab/alogps/); reference [56].
lab/alogps/); reference [56].

M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
525
Table 4
accomplished through the SuzukieMiyaura cross-coupling reac-
tion of triflate 15, obtained from the starting material 10. Aurones
2h and 2i were prepared via the BuchwaldeHartwig cross-coupling
reaction following the Fitzmaurice et al. procedure [28]. Starting
material 1d was reacted with the appropriate amine in the pres-
ence of Pd2(dba)3, (R)-BINAP, and NaOtBu in microwave conditions
to afford 2h and 2i were in moderate yields (Scheme 2).
Finally, the Mannich-base derivatives 7aed were synthesized in
moderate yields by reacting the starting material 5a with the
appropriate aliphatic secondary amine in aqueous formaldehyde
(Scheme 3).
Structure, clogP values and yields for the synthesis (final step) of aurones 6e7.
Compound
R1
R2
cLogPa
Yield %
6a
6b
6c
Ph
H
H
H
5.24
5.05
5.20
78
66
99
C6H4-40-F
The stereochemistry of the carbonecarbon double bond in
aurones 1e7 was assigned as having the (Z)-configuration based
50-(Pyridin-20-NH2)
mainly on 13C chemical shifts for the exocyclic (
b) carbon atom. The
13C NMR data obtained for aurones reveal that the chemical shift for
the exocyclic carbon range from 108 to 112 ppm, in line with the
values reported for (Z)-aurones [32,33], the thermodynamically
more stable isomer [34]. In contrast, the 13C chemical shifts for the
exocyclic carbon in (E)-aurones are usually observed at significantly
higher frequency (ca. 120e130 ppm) [17]. Information from the 1H
NMR spectra was less informative, as the 1H chemical shifts for the
7a
7b
7c
OH
OH
OH
2.55
3.73
2.47
61
63
54
b-hydrogen atoms in most of aurones 1e7 ranged from 6.7 to
7.1 ppm, consistent with reported values for (Z)-aurones (ca.
6.7 ppm) and (E)-aurones (ca. 7.2 ppm) [32e33]. This ambiguity in
1H chemical shifts values might be ascribed to the electronic
properties of substituents at ring B [18].
7d
OH
3.55
29
2.2. Biological evaluation
a
2.2.1. Activity against P. falciparum
clogP values calculated using the AlogPS 2.1 software (http://www.vcclab.org/
lab/alogps/); reference [56].
All compounds were assayed for their antiplasmodial activity
against the chloroquine-resistant P. falciparum W2 strain and for
their toxicity against Human Embryonic Kidney 293T cells. The data
presented in Table 5 reveals that 21 aurones displayed relevant
of Pd(PPh3)2Cl2 as catalyst, as described by Liu et al. [31] (Scheme
2). Performing the cross-coupling reactions on starting materials
1d and 1c afforded aurones 2aeg and 3aeg, respectively, in mod-
erate to very good yields. The synthesis of compounds 6aec was
antiplasmodial activity, with IC50 values ranging from 1.2 to 9.9
mM.
In addition, aurones 1e7 presented negligible cytotoxicity, with
Scheme 1. Reagents and conditions: (a) 8 or 9, Al2O3, MeOH, reflux under N2, 24 h; (b) 10, NaOH (1 M), H2O, RT, 4 h (for 5a to 5d); (c) 10, glacial AcOH, HCl (cat), RT, 4 h (for 5e to
5g), (d) substituted phenol, Na2CO3, dry DMF, reflux, 24 h; (e) H2O2 (30%), KOH, MeOH, EtOH, reflux, 5 h; (f) N,O-dimethylhydroxylamine, TEA, TBTU, dry DMF, RT, overnight; (g)
LiAlH4, dry THF, ꢀ5 ꢁC. R3 is according to Tables 1e3.

526
M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
Scheme 2. Reactions and conditions: (a) 1c, 1d, or 15, Pd(PPh3)2Cl2, Na2CO3 (1 M), 1,4-dioxane, 100 ꢁC, 3 h; (b) 1d, Pd2(dba)3, (R)-BINAP, NaOtBu, dry toluene, 100 ꢁC, 15 min, MW. R3
is according to Tables 1e3.
Scheme 3. Reactions and conditions: (a) aliphatic amine, formaldehyde solution, EtOH, reflux, 5 h.
EC50 values against cultured human cells ranging from 68 to
ꢂ100 M. In general, most of the compounds presented selectivity
Overall, these results indicate that introduction of a basic moiety
to the aurone scaffold can improve antiplasmodial activity. Aurone
7b emerged as the most promising compound, with an IC50 value of
m
indices (SI ¼ EC50(HEK293T)/IC50(W2)) higher than 10, indicating
that aurones are selective and nontoxic antiplasmodial agents.
Inspection of the data in Table 5 shows that aurones containing
basic moieties, specifically 3-(20-amino)pyridine (2g, 3g, 6c), 3-
quinoline (2f, 3f), and Mannich-bases, (7aed) generally present
improved antiplasmodial activity when compared to their non-
basic counterparts. For example, the Mannich-base derivatives,
7aed, are significantly more active than their parent compound 5a,
an observation in line to that reported for similar Mannich-bases in
clinical use such as amodiaquine and pyronaridine [35]. The basic
moiety can exert its beneficial effect on activity independently of its
localization in the aurone scaffold. For example, compound 6c,
which contains a 2-aminopyridine moiety in ring A, is equipotent to
its counterparts 2g and 3g, which contain the basic moiety at C-4
and C-3 of ring B, respectively. A similar effect was reported for
chalcones, where substitution with a quinoline moiety at ring A or
ring B provided compounds with antiplasmodial activity in the low
micromolar range [36].
1.2 mM and an excellent selectivity index SI > 85. Since insight into
the mechanism of action is helpful for drug development, we
screened aurones against two validated targets expressed during
the blood stage of P. falciparum infection: hemozoin formation and
falcipain-2.
2.2.2. Screening against targets localized in the parasite digestive
vacuole
Plasmodium parasites dispose the free, toxic heme that results
from digestion of host erythrocyte hemoglobin by crystallizing it
into hemozoin crystals. Heme detoxification takes place in the
acidic digestive vacuole (DV) of the parasite (pH 5.2), and is
believed to be the target of several antimalarial drugs [37]. It was
recently reported that Mannich-base derivatives of aurones might
interfere with heme polymerization inside the DV of erythrocytic
parasites [29]. Since there is strong evidence that pH trapping plays
a role in the activity of basic antimalarials such as chloroquine, we
assessed the possibility of the quinoline (2f, 3f), 2-aminopyridine
(2g, 3g, 6c) and Mannich-base aurone derivatives (7aed) being
trapped in the acidic DV, by calculating the vacuolar accumulation
ratio (VAR) based on the pH-dependent distribution of these
compounds between water and lipid (log D) (Table 6) [37]. We also
determined the lipid accumulation ratio (LAR), which measures the
expected ratio of compound that would concentrate within the
lipid component within the DV [37].
Aurones containing a second aromatic or benzyl moiety at C-3 of
ring B were shown to be more potent than their C-4 substituted
counterparts (compounds 3a, 3b and 3e versus 2a, 2b and 2e,
respectively). Replacement of the benzyl group at ring B by its
anilino or phenoxy isosters had a detrimental effect on anti-
plasmodial activity (2e versus 2h and 2j,k). The same trend in ac-
tivity was observed when the benzylamine moiety at C-4 of ring B
was replaced by its aniline counterpart (2i versus 2h). In general,
derivatives containing an hydroxyl or methoxy group at ring A were
poorly active, independently of substituents at ring B, with excep-
tion of the 7-methoxy derivative 4c, which presented an IC50 value
As shown by the VAR values presented in Table 6, basic aurones
have the potential to accumulate within the DV, with compound 7a
exhibiting a VAR value comparable to that of chloroquine, reflecting
the predominance of its doubly positively charged form at pH 5.2.
of 4.8 mM.

M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
527
Table 5
Antiplasmodial activity (IC50) against the chloroquine-resistant Plasmodium falciparum W2 strain, cytotoxicity (EC50) against Human Embryonic Kidney 293T cells and
selectivity index (SI ¼ EC50/IC50) for aurones 1e7.
Compd
IC50
/m
M
EC50
/m
M
SI
Compd
IC50
/
mM
EC50
/
mM
SI
Compd
IC50/m
M
EC50/m
M
SI
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
>10
>10
4.70
3.21
>10
>10
>10
>100
>100
>100
>100
>100
68
>100
>100
>100
>100
>100
>100
87
>10
>10
>21
>31
>10
2h
2i
2j
8.59
5.00
>100
>100
>100
>100
>100
>100
>100
>100
71
>100
79
>100
>100
>100
>100
>12
>20
>10
>10
>19
>23
>10
>10
>31
>21
>31
>10
>10
>21
>10
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
7a
7b
7c
7d
CQ
>10
>10
>10
>10
>10
5.84
9.88
>10
>10
3.03
4.01
1.18
3.34
3.47
0.14
>100
>100
>100
>100
>100
95
>10
>10
>10
>10
>10
>16
>1
>10
>10
>33
>25
>85
>30
>29
N.D.
>10
>10
5.21
4.41
>10
>10
2.31
4.78
2.56
>10
>10
4.80
>10
2k
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
<6.8
>10
>13
>10
>10
>10
>10
>12
>15
>27
12
7.99
>100
>100
>100
>100
>100
>100
>100
N.D.
>10
>10
>10
>10
7.34
6.59
3.70
>100
>100
2g
With exception of the piperazine Mannich-base 7a, most of the
basic aurones exhibit high LAR values, suggesting the preference of
these compounds for a lipophilic environment. Consequently, upon
entrance into the digestive food vacuole, the compounds could
accumulate within the neutral lipid particles at the sight of
hemozoin formation [38,39]. However, no clear correlation
emerged between the pIC50 values for antiplasmodial activity of
compounds 2feg, 3feg, 6c, 7aed, and their LAR or VAR values.
Furthermore, none of these basic-aurones inhibited hemozoin
formation at concentrations up to 1 mM, when assayed using a
hemozoin-like crystal inhibition assay [40]. Overall, the VAR value
obtained for these compounds show their ability to accumulate in
the DV, and, as a result, this class of compounds may probably
interact with other targets within this compartment (Table 6).
Aurones 1e6 were also screened for inhibition of cysteine
protease from P. falciparum, falcipain-2, which is of particular
interest as therapeutic target due to its role in parasite devel-
opment [41]. Falcipain-2 localizes in the parasite’s DV and plays a
key role in the hydrolysis of host hemoglobin into amino acids
essential to parasite growth [42]. Additionally, falcipain in-
hibitors that contain a Michael acceptor warhead [43] have been
reported to block the development of cultured erythrocytic par-
asites and to cure mice infected with lethal malaria infections
[44]. Although aurones are well known Michael acceptors [6],
none of the aurones 1e6 inhibited falcipain-2 in concentrations
2.2.3. In vitro drug combination assay
Previous studies showed that aurone derivatives inhibit ABC
transporters, a family of proteins involved in multidrug resistance
[21e25]. Moreover, the importance of this type of transporters was
already demonstrated for resistance of malaria parasites to chlo-
roquine [45,46]. Therefore, we evaluated the antimalarial effects of
compound 7c combined with chloroquine by a modified fixed ratio
isobologram method [47e50]. Isobologram analysis, based on
calculation of the sum of fractional inhibitory concentration values
(FICs ¼ IC50 of drug in the combination/IC50 of drug when tested
alone) or combination index (CI) [50] gives an indication of
whether the interaction is antagonistic, additive or synergistic.
Although there are no defined breakpoints for antimalarial com-
binations, a recent study defined synergism as CI < 0.9, additive
effect as 0.90 ꢃ CI < 1.10 and antagonism as CI ꢂ 1.10 [51]. The CI
value of 1.06 obtained for compound 7c demonstrated an additive
interaction between this compound and chloroquine (Fig. 2).
Therefore, synergy of this class of compounds with chloroquine was
not demonstrated.
3. Conclusions
Novel aurone derivatives with additional structural complexity
and diversity were synthesized and screened against
a
chloroquine-resistant P. falciparum strain. We demonstrated that
aurones provide a useful scaffold to generate novel bioactive
compounds and that appropriate functionalization of the aurone
scaffold yielded compounds with antiplasmodial activity in the low
micromolar range and with low cytotoxicity. In particular, aurones
containing basic moieties with capacity to protonate under weakly
up to 50
4c, which showed to be a weak falcipain-2 inhibitor with an IC50
value of 18.6 M. The observation that falcipain-2 is inhibited
only in the high M range, suggests that aurone scaffold is not
mM (data not shown). The only exception was compound
m
m
suitable for enzyme binding.
Table 6
Physicochemical parameters for compounds selected compounds. cLogP values were calculated using AlogPS 2.1 software from Virtual Computational Chemistry Laboratory
[56]. pKa values were obtained from SPARC software [57], except when indicated. Values of logD, VAR and LAR were calculated using the equations available in Ref. [37].
Compd
clogP
pKa1
4.49a
pKa2
logD7.4
logD5.2
VAR
LAR
MIC/
m
M
IC50/mM
2f
2g
3f
3g
6c
7a
7b
7c
7d
CQ
5.16
3.69
5.16
3.69
5.20
2.55
3.73
2.47
3.55
7.72
e
5.16
3.67
5.16
3.67
5.18
1.02
2.04
2.42
1.79
0.92
5.08
2.70
5.08
2.70
4.21
ꢀ3.33
ꢀ0.15
1.19
1.19
9.20
1.19
9.20
9.20
144366
4643
144,366
4643
ND
NI
ND
NI
NI
NI
NI
NI
NI
125
6.59
3.70
4.78
2.56
3.03
4.01
1.18
3.34
3.47
0.14
6.14b
4.49a
6.14b
6.14b
8.50
e
e
e
e
150,233
7.78
e
22,589
155
10.6
110
265
62.0
8.25
9.08
6.46
9.15
10.18
e
17.2
e
ꢀ0.40
156
22749
8.38
ꢀ3.44
NI: no inhibition of hemozoin-like crystal formation; ND: not determined.
a
Calculated from Ref. [58].
Calculated from Ref. [59].
b

528
M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
washed with water, dried with anhydrous Na2SO4 and concen-
trated under reduced pressure to give the crude product.
4.1.1.1. (Z)-2-Benzylidenebenzofuran-3(2H)-one (1a). Purified by
flash chromatography (Hexane/CH2Cl2 ¼ 80:20) Obtained as yellow
solid, yield 41%, mp 113e114 ꢁC. 1H NMR (400 MHz, CDCl3)
d
¼ 7.97e7.90 (m, 2H), 7.82 (dd, J ¼ 7.6, 0.8 Hz,1H), 7.66 (ddd, J ¼ 8.5,
7.4, 1.4 Hz, 1H), 7.51e7.38 (m, 3H), 7.34 (d, J ¼ 8.3 Hz, 1H), 7.23 (t,
J ¼ 7.5 Hz, 1H), 6.91 (s, 1H). 13C NMR (101 MHz, MeOD)
d
¼ 186.22,
167.63, 138.77, 132.71, 132.09, 131.23, 130.04, 129.42, 125.38, 124.98,
122.52, 114.24, 114.15. Anal. Calcd. (C15H10O2$0.15H2O): C, 80.09; H,
4.62%. Found: C, 80.35; H, 4.97%.
4.1.1.2. (Z)-2-(2-Bromobenzylidene)benzofuran-3(2H)-one
(1b).
Purified by flash chromatography (Hexane/EtOAc ¼ 95:5). Obtained
as yellow solid, yield 55%, mp 169e171 ꢁC. 1H NMR (400 MHz,
Fig. 2. Isobologram showing the relationship between the FIC50 of chloroquine and
compound 7c against Plasmodium falciparum Dd2 strain. Numbers on each plotted
point correspond to the calculated combination index (CI) value for the utilized
combination ratio.
DMSO-d6):
7.59e7.56 (m, 2H), 7.40e7.33 (m, 2H), 7.07 (s, 1H). 13C NMR
(101 MHz, DMSO-d6):
132.47, 132.09, 131.51, 128.88, 126.06, 125.03, 124.78, 121.02, 113.78,
109.23. Anal. Calcd. (C15H9BrO2): C, 59.82; H, 3.02%. Found: C,
59.67; H, 3.13%.
d
¼ 8.32 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.85e7.79 (m, 3H),
d
¼ 184.15, 166.14, 147.66, 138.55, 133.87,
acidic conditions (2-aminopyridine, Mannich-bases) emerged as
the most active in this series. Our preliminary results indicate that
the primary mechanism of action of these basic aurones does not
involve inhibition of hemozoin formation. Furthermore, aurones
1e7 are only poorly inhibitors of falcipain-2, which is believed to
operate in acidic DV of the parasite. However, the high VAR values
obtained for the basic aurones indicate their ability to accumulate
in the DV which may be related with their mode of action. Further
studies are underway to unravel the antimalarial mechanism of this
class of compounds. In conclusion, our results highlight the po-
tential of the aurone scaffold for future antiplasmodial lead
optimization.
4.1.1.3. (Z)-2-(3-Bromobenzylidene)benzofuran-3(2H)-one
(1c).
Purified by flash chromatography (Hexane/EtOAc ¼ 95:5). Obtained
as yellow solid, yield 54%, mp 123e124 ꢁC. 1H NMR (400 MHz,
DMSO-d6):
d
¼ 8.18 (s, 1H), 8.03 (d, J ¼ 7.9 Hz, 1H), 7.83e7.81 (m,
2H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.61 (d, J ¼ 8.2 Hz, 1H), 7.48 (t, J ¼ 7.9 Hz,
1H), 7.34 (t, J ¼ 8.2 Hz, 1H), 6.96 (s, 1H). 13C NMR (101 MHz, DMSO-
d6):
d
¼ 184.15, 166.02, 147.31, 138.42, 134.81, 133.88, 133.03, 131.56,
130.59, 124.90, 124.67, 122.68, 121.14, 113.82, 110.80. Anal. Calcd.
(C15H9BrO2): C, 59.82; H, 3.02%. Found: C, 59.81; H, 3.05%.
4.1.1.4. (Z)-2-(4-Bromobenzylidene)benzofuran-3(2H)-one
(1d).
Purified by flash chromatography (Hexane/EtOAc ¼ 95:5). Obtained
4. Experimental
as yellow solid, yield 56%, mp 180e182 ꢁC. 1H NMR (400 MHz,
DMSO-d6):
J ¼ 8.3 Hz, 2H), 7.56 (d, J ¼ 8.5 Hz, 1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 6.95 (s,
1H). 13C NMR (101 MHz, DMSO-d6):
137.88, 133.14, 132.10, 131.21, 124.42, 124.16, 123.65, 120.78, 113.28,
110.92. Anal. Calcd. (C15H9BrO2): C, 59.82; H, 3.02%. Found: C, 59.88;
H, 3.04%.
d
¼ 7.91 (d, J ¼ 8.3 Hz, 2H), 7.84e7.80 (m, 2H), 7.72 (d,
4.1. Chemistry
d
¼ 183.66, 165.47, 146.59,
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Melting points
were determined using a Kofler camera Bock monoscope M and are
uncorrected. Merck Silica Gel 60 F254 plates were used as analytical
TLC and flash column chromatography was performed on Merck
Silica Gel (200e400 mesh). 1H and 13C NMR spectra were recorded
on a Bruker 400 Ultra-Shield (400 MHz). 1H and 13C chemical shifts
4.1.1.5. (Z)-2-(2,4-Difluorobenzylidene)benzofuran-3(2H)-one (1e).
Purified by flash chromatography (Hexane/CH2Cl2 ¼ 80:20). Ob-
tained as yellow solid, 30% yield, mp 150e152 ꢁC. 1H NMR
are expressed in parts per million (ppm, d) referenced to the solvent
used and the proton coupling constants (J) in hertz. Proton coupling
patterns were described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), doublet of doublets (dd), and broad (br).
Low-resolution mass spectra were recorded using a VG Quattro
LCMS instruments. HR-ESI-MS were recorded on an ESI-TOF spec-
trometer (Biotof II Model, Bruker). Elemental analyses were per-
formed using an EA 1110 CE Instruments automatic analyser. The
microwave-assisted synthesis was performed in a CEM Corpora-
tion DiscoverÒ LabmateÔ.
(400 MHz, CDCl3)
d
¼ 8.36 (td, J ¼ 8.6, 6.5 Hz, 1H), 7.83 (dd, J ¼ 7.7,
1.3 Hz, 1H), 7.73e7.63 (m, 1H), 7.33 (d, J ¼ 8.3 Hz, 1H), 7.29e7.22 (m,
1H), 7.13 (s, 1H), 7.02 (dd, J ¼ 11.9, 5.6 Hz,1H), 6.95e6.85 (m,1H). 13
C
NMR (101 MHz, CDCl3)
d
¼ 184.55, 166.20, 147.39, 137.25, 133.16,
125.00, 123.91, 121.63, 113.05, 112.33, 104.40, 103.27. Anal. Calcd.
(C15H8F2O2$0.25H2O): C, 68.61; H, 3.27%. Found: C, 68.60; H, 3.27%.
4.1.1.6. (Z)-2-(2,4-Dichlorobenzylidene)benzofuran-3(2H)-one (1f).
Purified by flash chromatography (Hexane/CH2Cl2 ¼ 70:30). Ob-
tained as a yellow solid, 81% yield, mp 213e214 ꢁC. 1H NMR
4.1.1. General procedure for the synthesis of aurones 1aeh, 2jek
and 4aed
(400 MHz, CDCl3)
d
¼ 8.31 (d, J ¼ 8.6 Hz, 1H), 7.84e7.82 (m, 1H),
7.70e7.66 (m, 1H), 7.49 (d, J ¼ 2.1 Hz, 1H), 7.37 (dd, J ¼ 8.6, 2.1 Hz,
To a solution of benzofuran-3(2H)-one (134 mg, 1 mmol) in dry
methanol (20 mL) at room temperature was added the appropriate
aldehyde (1.2 mmol) and Al2O3 (1 mmol). The mixture was
refluxed, under N2, for 48 h. After, the solvent was removed and the
solid residue was dissolved in CH2Cl2. The organic layer was
1H), 7.33 (d, J ¼ 8.3 Hz, 1H), 7.28 (s, 1H), 7.26e7.24 (m, 1H). 13C NMR
(101 MHz, CDCl3)
d
¼ 184.57, 166.23, 147.82, 137.38, 136.61, 135.98,
132.98, 130.03, 129.15, 127.69, 125.07, 124.03, 121.51, 113.08, 106.94.
Anal. Calcd. (C15H8Cl2O2$0.1Hex): C, 62.50; H, 3.17%. Found: C,
62.38; H, 3.32%.

M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
529
4.1.1.7. (Z)-2-(3,4,5-Trimethoxybenzylidene)benzofuran-3(2H)-one
(1g). Purified by flash chromatography (Hexane/CH2Cl2 ¼ 70:30).
Obtained as a yellow solid, 64% yield, mp 200e201 ꢁC. 1H NMR
CH2Cl2. Obtained as yellow solid, 59% yield, mp 289e290 ꢁC. 1H
NMR (400 MHz, CDCl3)
¼ 8.35 (d, J ¼ 8.6 Hz, 1H), 7.48 (d,
J ¼ 2.0 Hz, 1H), 7.42e7.37 (m, 2H), 7.30 (s, 1H), 7.21e7.14 (m, 2H),
4.02 (s, 3H). 13C NMR (101 MHz, CDCl3)
146.04, 136.62, 136.03, 133.17, 129.96, 129.10, 127.88, 124.50, 122.87,
119.02, 116.13, 107.42, 56.47. Anal. Calcd. (C16H10Cl2O3$H2O): C,
56.66; H, 3.57%. Found: C, 56.97; H, 3.45%.
d
(400 MHz, CDCl3)
(d, J ¼ 8.3 Hz, 1H), 7.24 (dd, J ¼ 12.5, 4.9 Hz, 1H), 7.19 (s, 2H), 6.83 (s,
1H), 3.95 (s, 6H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
¼ 7.82 (d, J ¼ 7.6 Hz, 1H), 7.70e7.63 (m, 1H), 7.31
d
¼ 184.73, 155.86, 147.78,
d
¼ 184.70,
166.03, 153.44, 146.53, 140.14, 136.92, 127.82, 124.83, 123.68, 121.83,
113.52, 113.02, 109.01, 61.18, 56.33. Anal. Calcd. (C18H16O5): C, 69.22;
H, 5.17%. Found: C, 69.30; H, 5.36%.
4.1.1.14. (Z)-7-Methoxy-2-(3,4,5-trimethoxybenzylidene)benzofuran-
3(2H)-one (4d). Purified by flash chromatography (Hexane/
CH2Cl2 ¼ 80:20). Obtained as yellow solid, 99% yield, mp 167e
4.1.1.8. (Z)-2-(4-(Dimethylamino)benzylidene)benzofuran-3(2H)-one
(1h). Purified by flash chromatography (Hexane/CH2Cl2 ¼ 50:50).
Obtained as orange solid, 49% yield, mp 189e191 ꢁC. 1H NMR
168 ꢁC. 1H NMR (400 MHz, CDCl3)
d
¼ 7.39 (dd, J ¼ 6.8, 1.9 Hz, 1H),
7.24 (s, 2H), 7.18e7.12 (m, 2H), 6.85 (s, 1H), 4.01 (s, 3H), 3.95 (s, 6H),
(400 MHz, CDCl3):
d
¼ 7.85 (d, 2H, J ¼ 8.8 Hz), 7.81 (d,1H, J ¼ 7.5 Hz),
3.92 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
¼ 184.81, 155.81, 153.41,
7.61 (t, 1H, J ¼ 8.3 Hz), 7.32 (d, 1H, J ¼ 8.3 Hz), 7.19 (t, 1H, J ¼ 7.5 Hz),
146.59, 146.09, 140.08, 127.83, 124.09, 123.22, 118.93, 116.06, 113.95,
109.04, 61.17, 56.69, 56.23. Anal. Calcd. (C19H18O6$H2O): C, 63.33; H,
5.59%. Found: C, 63.07; H, 5.89%.
6.93 (s, 1H), 6.75 (d, 2H, J ¼ 8.8 Hz), 3.07 (s, 6H). 13C NMR (101 MHz,
CDCl3):
d
¼ 184.14, 165.42, 151.47, 145.16, 135.96, 133.78, 124.46,
123.02, 122.59, 120.15, 115.45, 112.90, 112.08, 40.23. Anal. Calcd.
(C17H15NO2ꢄ0.15H2O): C, 76.18; H, 5.77, N, 5.28%. Found: C, 76.20; H,
5.60, N, 5.33%.
4.1.2. General procedure for the synthesis of aurones 5a to 5d
To a solution of 6-hydroxybenzofuran-3(2H)-one (134 mg,
1 mmol) in H2O (15 mL) at room temperature was added the
appropriate aldehyde (1.1 mmol) and KOH 1 M (2 mmol). The
mixture was stirred, at room temperature, for 4 h. After, some drops
of HCl 37% were added to the mixture until the solution reach pH 2.
The mixture was extracted with ethyl acetate and combined
organic layers were washed with brine, dried with anhydrous
Na2SO4 and concentrated under reduced pressure to give the crude
product.
4.1.1.9. (Z)-2-(4-(p-Tolyloxy)benzylidene)benzofuran-3(2H)-one (2j).
Purified by flash chromatography (Hexane/EtOAc ¼ 80:20). Ob-
tained as yellow solid, 33% yield, mp 116e118 ꢁC. 1H NMR
(400 MHz, DMSO-d6):
d
¼ 8.02 (d, 2H, J ¼ 8.8 Hz), 7.82e7.79 (m,
2H), 7.55 (d, 1H, J ¼ 8.6 Hz), 7.32 (t, 1H, J ¼ 7.4 Hz), 7.25 (d, 2H,
J ¼ 8.4 Hz), 7.07 (d, 2H, J ¼ 8.7 Hz), 7.02 (d, 2H, J ¼ 8.4 Hz), 6.97 (s,
1H), 2.32 (s, 3H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 183.39, 165.72,
159.49, 153.41, 146.06, 138.00, 134.10, 133.99, 131.07, 126.92, 124.72,
124.39, 121.47, 120.11, 118.32, 113.66, 112.51, 20.80. Anal. Calcd.
(C22H16O3$0.15H2O): C, 79.81; H, 5.28%. Found: C, 79.59; H, 5.01%.
4.1.2.1. (Z)-2-Benzylidene-6-hydroxybenzofuran-3(2H)-one
(5a).
Purified through recrystallization from CH2Cl2. Obtained as yellow
solid, 99% yield, mp 228e230 ꢁC. 1H NMR (400 MHz, DMSO)
4.1.1.10. (Z)-2-(4-(4-Chlorophenoxy)benzylidene)benzofuran-3(2H)-
d
¼ 11.26 (s, 1H), 7.95 (d, 2H, J ¼ 7.4 Hz), 7.64 (d, 1H, J ¼ 8.4 Hz),
one
CH2Cl2 ¼ 50:50). Obtained as yellow solid, 51% yield, mp 112e
113 ꢁC. 1H NMR (400 MHz, DMSO-d6):
(2k). Purified
by
flash
chromatography
(Hexane/
7.52e7.42 (m, 3H), 6.81e6.80 (m, 2H), 6.73 (d, 1H, J ¼ 8.4 Hz). 13
C
NMR (101 MHz, DMSO)
d
¼ 181.51, 168.02, 166.67, 147.41, 132.11,
d
¼ 8.05 (d, 2H, J ¼ 8.7 Hz),
131.10, 129.71, 129.03, 126.07, 113.15, 112.75, 110.38, 98.69. Anal.
Calcd. (C15H10O3$0.15H2O): C, 74.77; H, 4.32%. Found: C, 74.50; H,
4.19%.
7.83e7.80 (m, 2H), 7.55 (d, 1H, J ¼ 8.6 Hz), 7.48 (d, 2H, J ¼ 8.8 Hz),
7.33 (t, 1H, J ¼ 7.4 Hz), 7.16e7.13 (m, 4H), 6.98 (s, 1H). 13C NMR
(100 MHz, DMSO-d6):
d
¼ 183.95, 165.80, 158.44, 155.01, 146.26,
138.08, 134.06, 130.55, 128.54, 127.76, 124.76, 124.44, 121.57, 121.44,
119.14, 113.68, 112.26. Anal. Calcd. (C21H13ClO3ꢄ0.15H2O): C, 71.76;
H, 3.82%. Found: C, 71.37; H, 3.84%.
4.1.2.2. (Z)-2-(2,4-Difluorobenzylidene)-6-hydroxybenzofuran-
3(2H)-one (5b). Purified through recrystallization from CH2Cl2.
Obtained as yellow solid, 98% yield, mp 277e279 ꢁC. 1H NMR
(400 MHz, DMSO)
d
¼ 8.32e8.22 (m, 1H), 7.65 (d, J ¼ 8.5 Hz, 1H),
4.1.1.11. (Z)-2-Benzylidene-7-methoxybenzofuran-3(2H)-one
(4a).
7.48e7.38 (m, 1H), 7.32e7.25 (m, 1H), 6.80 (d, J ¼ 1.9 Hz, 1H), 6.73
Purified by flash chromatography (Hexane/EtOAc ¼ 90:10). Ob-
(dd, J ¼ 8.5, 2.0 Hz, 1H), 6.71 (s, 1H). 13C NMR (101 MHz, DMSO)
tained as yellow solid, 62% yield, mp 168e169 ꢁC. 1H NMR
d
¼ 181.19, 168.19, 167.07, 148.20, 132.69, 126.36, 116.77, 116.69,
(400 MHz, CDCl3):
7.35 (d, 1H, J ¼ 7.8 Hz), 7.26 (t, 1H, J ¼ 7.8 Hz), 6.99 (s, 1H), 4.01 (s,
3H). 13C NMR (101 MHz, DMSO-d6):
145.62, 131.89, 131.44, 130.29, 129.17, 129.71, 122.16, 119.64, 115.17,
112.75, 56.35. Anal. Calcd. (C16H12O3): C, 76.18; H, 4.80%. Found: C,
76.32; H, 5.03%.
d
¼ 8.00 (d, 2H, J ¼ 7.3 Hz), 7.56e7.47 (m, 4H),
113.47, 112.78, 112.47, 104.66, 99.74, 98.85. Anal. Calcd.
(C15H8F2O3$0.15Hex): C, 66.50; H, 3.55%. Found: C, 66.74; H, 3.86%.
d
¼ 183.88, 154.91, 146.32,
4.1.2.3. (Z)-2-(2,4-dichlorobenzylidene)-6-hydroxybenzofuran-
3(2H)-one (5c). Purified through recrystallization from CH2Cl2.
Obtained as yellow solid, 99% yield, mp > 300 ꢁC. 1H NMR
(400 MHz, DMSO)
d
¼ 8.26 (d, J ¼ 8.6 Hz, 1H), 7.80 (s, 1H), 7.66 (d,
4.1.1.12. (Z)-2-(2,4-Difluorobenzylidene)-7-methoxybenzofuran-
3(2H)-one (4b). Purified by flash chromatography (Hexane/
CH2Cl2 ¼ 50:50). Obtained as yellow solid, 95% yield, mp 167e
J ¼ 8.4 Hz, 1H), 7.60 (d, J ¼ 8.6 Hz, 1H), 6.88 (s, 1H), 6.79 (s, 1H), 6.74
(d, J ¼ 8.5 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
¼ 181.17, 168.19,
167.14, 148.69, 134.98, 134.61, 132.68, 129.58, 128.75, 128.17, 126.42,
113.50, 112.29, 103.15, 98.84. Anal. Calcd. (C15H8Cl2O3$H2O): C,
55.41; H, 3.10%. Found: C, 55.10; H, 3.23%.
168 ꢁC. 1H NMR (400 MHz, CDCl3)
d
¼ 8.38 (td, J ¼ 8.6, 6.6 Hz, 1H),
7.38 (dd, J ¼ 6.7, 2.1 Hz, 1H), 7.20e7.14 (m, 2H), 7.13 (s, 1H), 7.07e
6.99 (m, 1H), 6.92e6.84 (m, 1H), 4.02 (s, 3H). 13C NMR (101 MHz,
CDCl3)
d
¼ 184.66, 155.83, 147.35, 146.05, 133.38, 124.36, 122.96,
4.1.2.4. (Z)-6-Hydroxy-2-(3,4,5-trimethoxybenzylidene)benzofuran-
3(2H)-one (5d). Purified through recrystallization from CH2Cl2.
Obtained as yellow solid, 98% yield, mp 264e266 ꢁC. 1H NMR
118.81, 116.04, 112.48, 104.31, 103.68, 56.44. Anal. Calcd.
(C16H10F2O3): C, 66.66; H, 3.50%. Found: C, 66.65; H, 3.84%.
(400 MHz, DMSO)
d
¼ 7.62 (d, J ¼ 8.4 Hz, 1H), 7.32 (s, 2H), 6.83 (s,
4.1.1.13. (Z)-2-(2,4-Dichlorobenzylidene)-7-methoxybenzofuran-
3(2H)-one (4c). Purified through recrystallization from Hexane/
1H), 6.76 (s, 1H), 6.72 (d, J ¼ 8.5 Hz, 1H), 3.85 (s, 6H), 3.72 (s, 3H). 13
C
NMR (101 MHz, DMSO)
d
¼ 181.48, 167.93, 166.68, 153.10, 146.95,

530
M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
139.18, 127.64, 126.07, 113.25, 112.88, 111.03, 108.84, 98.91, 60.33,
56.10. Anal. Calcd. (C18H16O6): C, 65.84; H, 4.92%. Found: C, 65.62; H,
5.00%.
(400 MHz, DMSO-d6):
d
¼ 8.10 (d, J ¼ 8.3 Hz, 2H), 7.84e7.80 (m, 6H),
7.61 (d, J ¼ 8.5 Hz, 1H), 7.36e7.31 (m, 3H), 7.03 (s, 1H). 13C NMR
(101 MHz, DMSO-d6):
d
¼ 183.61, 165.45, 162.27, 146.43, 140.41,
137.75, 135.60, 132.08, 131.05, 128.90, 127.18, 124.39, 124.09, 120.94,
115.94, 113.31, 111.90. Anal. Calcd. (C21H13FO2$0.1H2O): C, 79.28; H,
4.19%. Found: C, 79.07; H, 4.33%.
4.1.3. General procedure for the synthesis of aurones 5eeg
To a solution of 6-hydroxybenzofuran-3(2H)-one (0.57 mmol) in
glacial acetic acid (5.7 mL) at room temperature was added the
appropriate aldehyde (0.68 mmol) and HCl (cat, 3 drops). The re-
action mixture was stirred for 4 h at room temperature. After, the
mixture was dropped in cold water and the precipitate formed was
filtered and washed with water.
4.1.4.3. (Z)-2-((40-chlorobiphenyl-4-yl)methylene)benzofuran-3(2H)-
one
(2c). Purified
by
flash
chromatography
(Hexane/
EtOAc ¼ 90:10). Obtained as yellow solid, 64% yield, mp 171e173 ꢁC.
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.10 (d, J ¼ 8.3 Hz, 2H), 7.86e7.79
(m, 6H’), 7.61 (d, J ¼ 8.7 Hz, 1H), 7.56 (d, J ¼ 8.5 Hz, 2H), 7.35 (t,
4 .1. 3 .1. ( Z ) - 2 - ( 4 - ( D i m e t h y l a m i n o ) b e n z y l i d e n e ) - 6 -
hydroxybenzofuran-3(2H)-one (5e). Obtained as orange solid, 71%
J ¼ 7.5 Hz, 1H), 7.03 (s, 1H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 184.05, 165.91, 146.95, 140.52, 138.35, 138.20, 133.47, 132.53,
yield, mp 235e237 ꢁC. 1H NMR (400 MHz, CDCl3):
d
¼ 10.00 (s, 1H),
7.73 (d, 2H, J ¼ 8.9 Hz), 7.55 (d, 1H, J ¼ 8.4 Hz), 6.70e6.61 (m, 5H),
2.99 (s, 6H). 13C NMR (101 MHz, CDCl3):
131.86, 129.49, 129.01, 127.63, 124.82, 124.54, 121.36, 113.74, 112.22.
Anal. Calcd. (C21H13ClO2$0.15H2O): C, 75.18; H, 4.00%. Found: C,
75.16; H, 3.97%.
d
¼ 182.10, 167.38, 165.43,
150.17, 145.56, 132.77, 125.15, 119.69, 114.04, 112.79, 112.27, 111.59,
98.40. Anal. Calcd. (C17H15NO3): C, 72.58; H, 5.39, N, 4.98%. Found:
C, 72.19; H, 5.31, N, 5.09%.
4.1.4.4. (Z)-40-((3-Oxobenzofuran-2(3H)-ylidene)methyl)biphenyl-4-
carbaldehyde (2d). Purified by flash chromatography (Hexane/
EtOAc ¼ 90:20). Obtained as yellow solid, 83% yield, mp 189e
4.1.3.2. (Z)-6-Hydroxy-2-(4-(p-tolyloxy)benzylidene)benzofuran-
3(2H)-one (5f). Obtained as yellow solid, 51% yield, mp 248e
190 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
¼ 10.07 (s, 1H), 8.14 (d,
250 ꢁC. 1H NMR (400 MHz, CDCl3):
d
¼ 11.22 (br, 1H), 7.96 (d, 2H,
J ¼ 8.3 Hz, 2H), 8.04e7.99 (m, 4H), 7.94 (d, J ¼ 8.3 Hz, 2H), 7.83e7.81
(m, 2H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.35 (t, J ¼ 7.4 Hz, 1H), 7.04 (s, 1H).
J ¼ 8.8 Hz), 7.62 (d, 1H, J ¼ 8.5 Hz), 7.24 (d, 1H, J ¼ 8.1 Hz), 7.06e6.99
13C NMR (101 MHz, DMSO-d6):
d
¼ 192.81, 183.69, 165.52, 146.70,
(m, 4H), 6.79e6.77 (m, 2H), 6.71 (dd, 1H, J ¼ 8.5, 1.8 Hz). 13C NMR
144.76, 139.95, 137.85, 135.49, 132.22, 132.13, 130.26, 127.75, 127.53,
124.45, 124.17, 120.89, 113.34, 111.61. Anal. Calcd.
(C21H14O3$0.75H2O): C, 77.74; H, 4.61%. Found: C, 77.43; H, 4.47%.
(101 MHz, CDCl3):
d
¼ 181.38, 167.82, 166.53, 158.67, 153.14, 146.71,
133.59, 133.17, 130.64, 126.73, 125.99, 119.64, 117.92, 113.11, 112.92,
110.14, 98.61, 20.38. Anal. Calcd. (C22H14O4$0.2H2O): C, 75.93; H,
5.05%. Found: C, 75.66; H, 5.03%.
4.1.4.5. (Z)-2-(4-Benzylbenzylidene)benzofuran-3(2H)-one
(2e).
4 .1. 3 . 3 . ( Z ) - 2 - ( 4 - ( 4 - C h l o r o p h e n o x y ) b e n z yl i d e n e ) - 6 -
Purified by flash chromatography (Hexane/EtOAc ¼ 92:8). Obtained
as yellow solid, 70% yield, 154e156 ꢁC. 1H NMR (400 MHz, DMSO-
hydroxybenzofuran-3(2H)-one (5g). Obtained as yellow solid, 84%
yield, mp 243e244 ꢁC. 1H NMR (400 MHz, CDCl3):
d
¼ 10.21 (br,1H),
d6):
d
¼ 7.93 (d, J ¼ 7.9 Hz, 2H), 7.81e7.79 (m, 2H), 7.56 (d, J ¼ 8.6 Hz,
7.80 (d, 2H, J ¼ 8.8 Hz), 7.56 (d, 1H, J ¼ 9.1 Hz), 7.27e7.25 (m, 2H),
1H), 7.39 (d, J ¼ 7.9 Hz, 2H), 7.34e7.25 (m, 5H), 7.20 (t, J ¼ 7.2 Hz,
6.97e6.93 (m, 4H), 6.67e6.63 (m, 3H). 13C NMR (101 MHz, CDCl3):
1H), 6.93 (s, 1H), 4.01 (s, 2H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 181.39, 167.86, 166.56, 157.59, 154.71, 146.88, 133.24, 130.11,
d
¼ 183.60, 165.43, 146.11, 143.86, 140.71, 137.69, 131.67, 129.75,
128.00, 127.56, 126.02, 121.06, 118.72, 113.12, 112.87, 109.91, 98.62.
Anal. Calcd. (C21H13ClO4ꢄ0.15H2O): C, 68.63; H, 3.66%. Found: C,
68.57; H, 3.64%.
129.48, 128.77, 128.54, 126.16, 124.33, 124.01, 120.95, 113.26, 112.33,
41.01. Anal. Calcd. (C22H16O2$0.15H2O): C, 83.86; H, 5.23%. Found: C,
83.87; H, 5.12%.
4.1.4. General procedure for the synthesis of aurones derivatives
2aeg, 3aeg, and 6aec via Suzuki coupling
To a solution of the appropriate aurone (0.23 mmol) in dioxane
(2.3 mL) was added Pd(PPh3)2Cl2 (0.023 mmol) and Na2CO3 1 M
4.1.4.6. (Z)-2-(4-(Quinolin-3-yl)benzylidene)benzofuran-3(2H)-one
(2f). Purified by flash chromatography (Hexane/EtOAc ¼ 70:30).
Obtained as yellow solid, 87% yield, mp 205e207 ꢁC. 1H NMR
(400 MHz, DMSO-d6):
d
¼ 9.35 (s, 1H), 8.77 (s, 1H), 8.19 (d,
(690
mL) followed by the proper boronic acid (0.28 mmol). The
resulting mixture was degassed and stirred at 100 ꢁC for 3 h under
N2. After cooling to room temperature, the reaction mixture was
diluted with CH2Cl2, filtered under celite and concentrated under
pressure to give the crude product.
J ¼ 8.4 Hz, 2H), 8.09e8.07 (m, 4H), 7.86e7.78 (m, 3H), 7.69e7.62 (m,
2H), 7.35 (t, J ¼ 7.4 Hz, 1H), 7.07 (s, 1H). 13C NMR (101 MHz, DMSO-
d6):
d
¼ 184.08, 165.93, 149.78, 147.47, 147.07, 138.80, 138.24, 133.68,
132.65, 132.24, 130.38, 129.15, 129.02, 128.12, 128.05, 127.65, 127.62,
124.86, 124.58, 121.35, 113.78, 112.15. Anal. Calcd.
(C24H15NO2$0.4H2O): C, 80.87; H, 4.48; N, 3.93%. Found: C, 80.67; H,
4.27; N, 4.06%.
4.1.4.1. (Z)-2-(biphenyl-4-ylmethylene)benzofuran-3(2H)-one (2a).
Purified by flash chromatography (Hexane/EtOAc ¼ 90:10). Ob-
tained as yellow solid, 77% yield, mp 133e135 ꢁC. 1H NMR
(400 MHz, DMSO-d6):
d
¼ 8.10 (d, J ¼ 8.0 Hz, 2H), 7.85e7.75 (m, 6H),
4.1.4.7. (Z)-2-(4-(6-aminopyridin-3-yl)benzylidene)benzofuran-
3(2H)-one (2g). Purified by flash chromatography (EtOAc/
MeOH ¼ 97:3). Obtained as orange solid, 99% yield, mp 195e197 ꢁC.
7.61 (d, J ¼ 8.4 Hz, 1H), 7.52e7.49 (m, 2H), 7.41 (m, 1H), 7.34 (t,
J ¼ 7.3 Hz, 1H), 7.03 (s, 1H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 183.60, 165.45, 146.42, 141.50, 139.11, 137.73, 132.08, 131.08,
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.38 (s, 1H), 8.04 (d, J ¼ 8.4 Hz,
129.10, 128.15, 127.23, 126.82, 124.38, 124.07, 120.94, 113.30, 111.97.
Anal. Calcd. (C21H14O2$0.15H2O): C, 83.78; H, 4.80%. Found: C,
83.67; H, 4.89%.
2H), 7.84e7.80 (m, 3H), 7.76 (d, J ¼ 8.4 Hz, 2H), 7.61 (d, J ¼ 8.5 Hz,
1H), 7.34 (t, J ¼ 7.4 Hz, 1H), 7.00 (s, 1H), 6.55 (d, J ¼ 8.6 Hz, 1H), 6.24
(s, 2H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 183.91, 165.75, 160.10,
146.75, 146.52, 140.16, 138.03, 135.74, 132.62, 130.16, 125.95, 124.75,
124.44, 123.02, 121.46, 113.72, 112.83, 108.48. Anal. Calcd.
(C20H14N2O2$0.4H2O): C, 74.70; H, 4.65; N, 8.71%. Found: C, 74.57;
H, 4.43; N, 8.79%.
4.1.4.2. (Z)-2-((40-Fluorobiphenyl-4-yl)methylene)benzofuran-3(2H)-
one (2b). Purified by flash chromatography (Hexane/EtOAc ¼ 92:8).
Obtained as yellow solid, 66% yield, mp 174e175 ꢁC. 1H NMR

M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
531
4.1.4.8. (Z)-2-(biphenyl-3-ylmethylene)benzofuran-3(2H)-one (3a).
Purified by flash chromatography (Hexane/EtOAc ¼ 92:8). Obtained
as yellow solid, 71% yield, mp 123e125 ꢁC. 1H NMR (400 MHz,
122.24,113.85,112.55. Anal. Calcd. (C24H15NO2$0.3H2O): C, 81.24; H,
4.44; N, 3.95%. Found: C, 81.59; H, 4.83; N, 3.92%.
DMSO-d6):
d
¼ 8.24 (s, 1H), 8.08 (d, J ¼ 7.8 Hz, 1H), 7.84e7.80 (m,
4.1.4.14. (Z)-2-(3-(6-Aminopyridin-3-yl)benzylidene)benzofuran-
3(2H)-one (3g). Purified by flash chromatography (CH2Cl2/
MeOH ¼ 97:3). Obtained as yellow solid, 76% yield, mp 187e189 ꢁC.
2H), 7.77e7.73 (m, 3H), 7.64e7.60 (m, 2H), 7.54e7.50 (m, 2H), 7.42
(t, J ¼ 7.3 Hz, 1H), 7.34 (t, J ¼ 7.4 Hz, 1H), 7.07 (s, 1H). 13C NMR
(101 MHz, DMSO-d6):
d
¼ 183.79, 165.59, 146.57, 140.96, 139.51,
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.32 (s, 1H), 8.14 (s, 1H), 7.98 (d,
137.84, 132.62, 130.06, 129.99, 129.76, 129.13, 128.47, 127.90, 126.87,
124.43, 124.13, 120.89, 113.41, 112.27. Anal. Calcd.
(C21H14O2$0.1H2O): C, 84.03; H, 4.78%. Found: C, 83.94; H, 4.80%.
J ¼ 7.7 Hz, 1H), 7.83e7.80 (m, 2H), 7.77 (dd, J ¼ 8.6, 2.5 Hz, 1H), 7.67
(d, J ¼ 7.9 Hz, 1H), 7.63e7.61 (m, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.34 (t,
J ¼ 7.4 Hz, 1H), 7.03 (s, 1H), 6.56 (d, J ¼ 8.6 Hz, 1H), 6.15 (s, 2H). 13
C
NMR (101 MHz, DMSO-d6):
d
¼ 184.17, 165.97, 159.89, 145.87,
4.1.4.9. (Z)-2-((40-Fluorobiphenyl-3-yl)methylene)benzofuran-3(2H)-
one (3b). Purified by flash chromatography (Hexane/EtOAc ¼ 92:8).
Obtained as yellow solid, 65% yield, mp 103e105 ꢁC. 1H NMR
(400 MHz, DMSO-d6):
7.78 (m, 5H), 7.64e7.60 (m, 2H), 7.37e7.32 (m, 3H), 7.06 (s, 1H). 13
NMR (101 MHz, DMSO-d6):
139.89, 137.85, 135.95, 132.63, 129.96, 129.78, 128.93, 128.85,
128.38, 124.43, 124.14, 120.88, 116.03e115.82, 113.41, 112.18. Anal.
Calcd. (C21H13FO2$0.1H2O): C, 79.28; H, 4.19%. Found: C, 79.23; H,
4.15%.
146.35, 138.19, 135.87, 132.93, 131.99, 130.09, 129.28, 127.50, 124.80,
123.52, 121.31, 113.82, 112.89, 108.49. Anal. Calcd.
(C20H14N2O2$0.5H2O): C, 74.28; H, 4.69; N, 8.67%. Found: C, 74.43;
H, 4.55; N, 8.97%.
d
¼ 8.22 (s, 1H), 8.08 (d, J ¼ 7.8 Hz, 1H), 7.81e
C
4.1.4.15. (Z)-2-Benzylidene-6-phenylbenzofuran-3(2H)-one
(6a).
d
¼ 183.78, 165.59, 162.15, 146.58,
Purified by flash chromatography (Hexane/CH2Cl2 ¼ 70:30). Ob-
tained as yellow solid, 78% yield, mp 141e143 ꢁC. 1H NMR
(400 MHz, CDCl3):
7.57 (d, 2H, J ¼ 7.1 Hz), 7.56 (s, 1H), 7.53e7.40 (m, 7H), 6.92 (s, 1H).
13C NMR (101 MHz, DMSO-d6):
¼ 183.14, 166.24, 149.52, 146.92,
138.58, 131.99, 131.44, 130.16, 129.23, 129.20, 129.10, 127.44, 124.79,
122.86, 124.48, 119.81, 112.18, 111.06. Anal. Calcd.
d
¼ 7.95 (d, 2H, J ¼ 7.3 Hz), 7.87 (d,1H, J ¼ 8.0 Hz),
d
4.1.4.10. (Z)-2-((40-Chlorobiphenyl-3-yl)methylene)benzofuran-
3(2H)-one (3c). Purified by flash chromatography (Hexane/
EtOAc ¼ 85:15). Obtained as yellow solid, 51% yield, mp 187e
(C21H14O2$0.6H2O): C, 81.58; H, 4.97%. Found: C, 81.41; H, 5.28%.
189 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
¼ 8.24 (s, 1H), 8.09 (d,
4.1.4.16. (Z)-2-Benzylidene-6-(4-fluorophenyl)benzofuran-3(2H)-one
(6b). Purified by flash chromatography (Hexane/EtOAc ¼ 90:10).
Obtained as yellow solid, 66% yield, mp 172e174 ꢁC. 1H NMR
J ¼ 7.8 Hz, 1H), 7.85e7.76 (m, 5H), 7.65e7.56 (m, 4H), 7.34 (t, 1H,
J ¼ 7.5 Hz), 7.06 (s, 1H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 183.82,
165.62, 146.64, 139.63, 138.33, 137.91, 132.83, 132.73, 130.35, 129.94,
129.89, 129.11, 128.67, 128.38, 124.47, 124.19, 120.88, 113.44, 112.11.
Anal. Calcd. (C21H13ClO2$0.15H2O): C, 75.18; H, 4.00%. Found: C,
74.79; H, 4.20%.
(400 MHz, DMSO-d6):
d
¼ 8.04 (d, 2H, J ¼ 7.2 Hz), 7.94e7.90 (m, 3H),
7.87 (d, 1H, J ¼ 8.1 Hz), 7.64 (dd, 1H, J ¼ 8.1, 1.3 Hz), 7.55e7.48 (m,
3H), 7.38 (t, 2H, J ¼ 8.8 Hz), 6.98 (s, 1H). 13C NMR (101 MHz, DMSO-
d6):
d
¼ 183.10, 166.22, 164.08e161.63, 148.35, 146.91, 135.03,
131.98, 131.43, 130.17, 129.73e129.64, 129.10, 124.81, 122.78, 119.76,
116.21e115.99, 112.21, 111.04. Anal. Calcd. (C21H13FO2$0.3H2O): C,
78.39; H, 4.27%. Found: C, 78.14; H, 4.22%.
4.1.4.11. (Z)-30-((3-oxobenzofuran-2(3H)-ylidene)methyl)biphenyl-4-
carbaldehyde (3d). Purified by flash chromatography (Hexane/
EtOAc ¼ 75:25). Obtained as yellow solid, 63% yield, mp 193e
195 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
¼ 10.09 (s, 1H), 8.35 (s, 1H),
4.1.4.17. (Z)-6-(6-Aminopyridin-3-yl)-2-benzylidenebenzofuran-
3(2H)-one (6c). Purified by flash chromatography (CH2Cl2/
MeOH ¼ 98:2). Obtained as orange solid, 98% yield, mp 191e192 ꢁC.
8.15 (d, J ¼ 7.8 Hz,1H), 8.06e7.98 (m, 4H), 7.88e7.82 (m, 3H), 7.68 (t,
1H, J ¼ 7.8 Hz), 7.62 (d, 1H, J ¼ 8.5 Hz), 7.35 (t, 1H, J ¼ 7.4 Hz), 7.09 (s,
1H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 192.83, 183.76, 165.59,
1H NMR (400 MHz, DMSO-d6):
d
¼ 8.50 (s, 1H), 8.02 (d, 2H,
146.66, 145.13, 139.54, 137.86, 135.38, 132.81, 130.97, 130.29, 128.73,
127.95, 127.55, 124.43, 124.15, 120.84, 113.42, 111.89. Anal. Calcd.
(C22H14O3$0.25H2O): C, 79.86; H, 4.43%. Found: C, 79.48; H, 4.57%.
J ¼ 7.3 Hz), 7.92 (dd, 1H, J ¼ 8.7, 2.5 Hz), 7.82e7.78 (m, 1H), 7.58 (d,
1H, J ¼ 8.1 Hz), 7.53 (t, 2H, J ¼ 7.3 Hz), 7.48e7.45 (m,1H), 6.92 (s,1H),
6.56 (d, 1H, J ¼ 8.7 Hz), 6.44 (s, 2H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 182.75, 166.53, 160.35, 147.83, 147.36, 147.12, 135.82, 132.09,
4.1.4.12. (Z)-2-(3-Benzylbenzylidene)benzofuran-3(2H)-one
Purified by flash chromatography (Hexane/EtOAc ¼ 92:8). Obtained
as yellow oil, 60%. 1H NMR (400 MHz, DMSO-d6):
(3e).
131.32, 130.01, 129.08, 124.76, 121.94, 121.00, 118.51, 111.52, 108.53,
108.02. Anal. Calcd. (C20H14N2O2$0.3H2O): C, 75.12; H, 4.61; N,
8.76%. Found: C, 74.91; H, 4.55; N, 8.64%.
d
¼ 7.86e7.79 (m,
4H), 7.52 (t,1H, J ¼ 8.2 Hz), 7.44 (t,1H, J ¼ 7.6 Hz), 7.34e7.27 (m, 6H),
7.23e7.22 (m, 1H), 6.90 (s, 1H), 4.02 (s, 2H). 13C NMR (101 MHz,
4.1.5. General procedure for the synthesis of aurones derivatives
2hei via Buchwald coupling
DMSO-d6):
d
¼ 183.80, 165.54, 146.41, 142.41, 140.94, 137.90, 132.11,
131.77, 130.72, 129.32, 129.26, 128.93, 128.67, 126.26, 124.48, 124.17,
120.94, 113.33, 112.39, 41.00. HRMS calc. (C22H17O2): 313.1223.
Found: 313.1226.
(Z)-2-(4-bromobenzylidene)benzofuran-3(2H)-one
(1d)
(0.23 mmol), Pd2(dba)3 (0.0115 mmol), (R)-BINAP (0.075 mmol) and
NaOtBu (0.322 mmol) were dissolved in dry toluene (2.3 mL). The
resulting mixture was degassed and the appropriate amine
(0.276 mmol) was added. The mixture was stirred at 100 ꢁC for
15 min under MW conditions. After cooling to room temperature,
the reaction mixture was diluted with Et2O, filtered under celite
and concentrated under pressure to give the crude product.
4.1.4.13. (Z)-2-(3-(Quinolin-3-yl)benzylidene)benzofuran-3(2H)-one
(3f). Purified by flash chromatography (Hexane/EtOAc ¼ 80:20).
Obtained as yellow solid, 68% yield, mp 195e196 ꢁC. 1H NMR
(400 MHz, pyridine-d6):
d
¼ 9.53 (s, 1H), 8.53 (s, 1H), 8.42 (d, 1H,
J ¼ 8.4 Hz), 8.37 (s, 1H), 8.19 (d, 1H, J ¼ 7.8 Hz), 8.05 (d, 1H,
J ¼ 8.1 Hz), 7.90 (d, 1H, J ¼ 7.7 Hz), 7.85 (d, 1H, J ¼ 7.4 Hz), 7.78 (t, 1H,
J ¼ 7.6 Hz), 7.67e7.61 (m, 3H), 7.40 (d, 1H, J ¼ 8.1 Hz), 7.26e7.18 (m,
4.1.5.1. (Z)-2-(4-(Phenylamino)benzylidene)benzofuran-3(2H)-one
(2h). Purified by flash chromatography (Hexane/EtOAc ¼ 80:20).
Obtained as orange solid, 71% yield, mp 191e192 ꢁC. 1H NMR
2H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 184.80, 166.74, 148.56,
147.97, 139.23, 137.84, 134.13, 133.94, 133.66, 131.43, 131.21, 130.56,
130.29, 130.13, 129.46, 129.11, 128.83, 127.83, 125.05, 124.45, 124.17,
(400 MHz, DMSO-d6):
7.76 (m, 2H), 7.55 (d, 1H), 7.34e7.29 (m, 3H), 7.20 (d, 2H, J ¼ 7.6 Hz),
d
¼ 8.87 (s, 1H), 7.90 (d, 2H, J ¼ 8.7 Hz), 7.79e

532
M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
7.15 (d, 2H, J ¼ 8.7 Hz), 6.98 (t, 1H, J ¼ 7.3 Hz), 6.91 (s, 1H). 13C NMR
4.1.7.3. 2-Benzylidene-6-hydroxy-7-(morpholinomethyl)benzofuran-
3(2H)-one (7c). Purified by flash chromatography (CH2Cl2/
MeOH ¼ 99:1). Obtained as yellow solid, 54% yield, 179e181 ꢁC.
Mixture of isomers Z/E ¼ 1:0.1. 1H NMR (400 MHz, acetone-d6):
(100 MHz, DMSO-d6):
d
¼ 183.21, 165.18, 146.57, 144.95, 141.80,
137.36, 134.02, 129.80, 124.46, 124.08, 122.61, 122.13, 121.92, 119.41,
115.66, 114.30, 113.58. Anal. Calcd. (C21H15NO2$0.15H2O): C, 79.80;
H, 4.89; N, 4.43%. Found: C, 79.56; H, 5.12; N, 4.68%.
d
¼ 7.99 (d, 2H, J ¼ 7.3 Hz), 7.58e7.44 (m, 4H), 6.74 (s, 1H), 6.67 (d,
1H, J ¼ 8.4 Hz), 4.11 (s, 2H), 3.96e3.75 (m, 8H). 13C NMR (101 MHz,
4.1.5.2. (Z)-2-(4-(Benzylamino)benzylidene)benzofuran-3(2H)-one
(2i). Purified by flash chromatography (Hexane/EtOAc ¼ 80:20)
followed by TLC (Hexane/EtOAc ¼ 70:30). Obtained as orange oil,
acetone-d6):
d
¼ 167.85,133.51,132.06,129.83,125.38,114.11,111.03,
67.21, 53.71. Anal. Calcd. (C20H19NO4$0.25H2O): C, 70.26; H, 5.76; N,
4.10%. Found: C, 70.17; H, 5.72; N, 4.24%.
49% yield. 1H NMR (400 MHz, CDCl3):
d
¼ 7.82e7.80 (m, 3H), 7.62 (t,
1H, J ¼ 7.6 Hz), 7.39e728 (m, 6H’), 7.20 (m, 1H, J ¼ 7.6 Hz), 6.91 (s,
4.1.7.4. (Z)-2-Benzylidene-7-((diethylamino)methyl)-6-
hydroxybenzofuran-3(2H)-one (7d). Purified by flash chromatog-
1H), 6.70 (d, 2H, J ¼ 8.5 Hz), 4.66 (br, H), 4.43 (s, 2H). 13C NMR
(100 MHz, CDCl3):
d
¼ 184.22, 165.41, 149.82, 145.12, 138.47, 136.08,
raphy (CH2Cl2/MeOH
¼
97:3) followed by TLC (CH2Cl2/
133.93, 128.86, 127.59, 127.44, 124.40, 123.03, 122.37, 121.48, 115.21,
112.90, 112.86, 47.71. HRMS calc. (C22H17NO2): 328.1332. Found:
328.1334.
MeOH ¼ 95:5). Obtained as yellow solid, 29% yield, mp 165e167 ꢁC.
1H NMR (400 MHz, CDCl3):
d
¼ 7.83 (d, 2H, J ¼ 7.3 Hz), 7.60 (d, 1H,
J ¼ 8.5 Hz), 7.46 (t, 2H, J ¼ 7.5 Hz), 7.40e7.38 (m, 1H), 6.78 (s, 1H),
6.62 (d, 1H, J ¼ 8.5 Hz), 4.06 (s, 2H), 2.78 (q, 4H, J ¼ 7.2 Hz), 1.21 (t,
4.1.6. Synthesis of (Z)-2-benzylidene-3-oxo-2,3-
dihydrobenzofuran-6-yl trifluoromethanesulfonate (15)
6H, J ¼ 7.2 Hz). 13C NMR (101 MHz, CDCl3):
d
¼ 168.93, 148.28,
132.88, 131.23, 129.48, 129.01, 125.22, 114.05, 112.94, 111.17, 104.51,
49.54, 47.10, 11.34.
Compound 5a (0.25 mmol) was dissolved in dry CH2Cl2 (1 mL)
and dry TEA (0.275 mmol) was added to the solution followed by
Tf2O (0.275 mmol). The mixture was stirred for 45 min at room
temperature under N2. After completion, water was added to the
reaction mixture and the product was extracted with CH2Cl2. The
organic layer was washed with water, dried with anhydrous Na2SO4
and concentrated under reduced pressure permitting to obtain the
pure product. Obtained as yellow solid, 98% yield. 1H NMR
5. Biological assays
5.1. Activity against P. falciparum W2 strain
Compounds were assayed against human red blood cells infec-
ted with 1% ring stage W2-strain P. falciparum synchronized with
5% sorbitol. These cells were incubated with tested compounds in
96-well plates at 37 ꢁC for 48 h in RPMI-1640 medium supple-
mented with 25 mM HEPES pH 7.4, 10% heat inactivated human
(400 MHz, DMSO-d6):
d
¼ 8.04e7.97 (m, 4H), 7.53e7.44 (m, 4H),
7.05 (s, 1H). 13C NMR (101 MHz, DMSO-d6):
d
¼ 182.17, 165.67,
154.11, 146.55, 131.69, 131.59, 130.56, 129.13, 126.56, 121.37, 118.23,
117.57, 113.51, 107.89.
serum (or 0.5% Albumax/2% human serum), and 100
mM hypo-
xanthine under an atmosphere of 3% O2, 5% CO2, 91% N2. After 48 h,
the cells were fixed in 2% formaldehyde in PBS, transferred into PBS
with 100 mM NH4Cl, 0.1% Triton X-100, 1 nM YOYO-1, and then
analyzed in a flow cytometer (FACSort, Beckton Dickinson; EX
488 nm, EM 520 nm). Values of IC50 were calculated using Graph-
Pad PRISM software.
4.1.7. General procedure for the synthesis of Mannich bases 7aed
To a solution of (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-
one (0.29 mmol) in absolute ethanol (1 mL) was added the
appropriate amine (0.32 mmol) followed by formaldehyde solution
(0.32 mmol). The mixture was refluxed for 3 h30. After, the solvent
was removed and the solid residue was dissolved in CH2Cl2 and
extracted with HCl 1 M. The aqueous layer was neutralized with
NaHCO3 saturated solution and extracted with CH2Cl2. The organic
layer was dried with anhydrous Na2SO4 and concentrated under
reduced pressure to give the crude product.
5.2. Hemozoin-like crystals growth inhibition
Inhibition of hemozoin-like crystals formation by tested com-
pounds was assessed using the previously described in vitro
method [39,40]. In short, a hemozoin-like crystals stock suspension
was sonicated for 3 min and diluted in fresh broth medium to the
4.1.7.1. 2-Benzylidene-6-hydroxy-7-((4-methylpiperazin-1-yl)
methyl)benzofuran-3(2H)-one (7a). Purified by flash chromatog-
raphy (CH2Cl2/MeOH ¼ 98:2). Obtained as yellow solid, 61% yield,
mp 178e179 ꢁC. Mixture of isomers Z/E ¼ 1:0.15. 1H NMR (400 MHz,
final concentration of 2 mM (heme equivalents) in the wells of a 96-
wells plate. Stock solutions of tested compounds were prepared at
25 mM in DMSO. Stock solutions of chloroquine (positive control)
and gentamicin (negative control) were prepared at 100 mM in
DMSO-d6):
d
¼ 7.97 (d, 2H, J ¼ 7.4 Hz), 7.54e7.49 (m, 3H), 7.45e7.41
(m, 1H), 6.77 (s, 1H), 6.65 (d, 1H), 3.90 (s, 2H), 2.50 (br, 8H), 2.18 (s,
3H). 13C NMR (101 MHz, DMSO-d6):
¼ 181.24, 167.75, 166.19,
147.67, 132.32, 131.07, 129.57, 129.05, 124.62, 113.64, 111.48, 109.91,
105.77, 54.30, 52.00, 50.83, 45.47. Anal. Calcd.
distilled water; and all were 0.22 mm-filtered previous to being
d
diluted to 0e1000 mM final concentrations in the wells. Plates were
incubated at 37 ꢁC in a 5% CO2 atmosphere for 5 days to observe the
presence or absence of crystal growth. All tests were performed in
triplicate.
(C21H22N2O3$0.3H2O): C, 70.88; H, 6.42; N, 7.87%. Found: C, 70.75;
H, 6.48; N, 7.66%.
5.3. In vitro drug combination assay
4.1.7.2. 2-Benzylidene-6-hydroxy-7-(piperidin-1-ylmethyl)benzo-
furan-3(2H)-one (7b). Purified by flash chromatography (CH2Cl2/
MeOH ¼ 98:2). Obtained as yellow solid, 63% yield, mp 198e200 ꢁC.
Mixture of isomers Z/E ¼ 1:0.4. 1H NMR (400 MHz, DMSO-d6):
5.3.1. Parasite cultivation
Laboratory-adapted P. falciparum Dd2 (chloroquine-resistant)
strain was continuously cultured as previously described by Trager
and Jensen [52], with minor modifications. Briefly, parasites were
cultivated on human erythrocytes suspended in RPMI 1640 me-
dium supplemented with 25 mM HEPES, 6.8 mM hypoxanthine and
10% AlbuMAX II, at pH 7.2. Cultures were maintained at 37 ꢁC under
an atmosphere of 5% O2, 3e5% CO2, and N2 and synchronized by
double sorbitol treatment prior to the assays [53]. Staging and
d
¼ 7.94 (d, 2H, J ¼ 7.4 Hz), 7.51e7.41 (m, 4H), 6.70 (s, 1H), 6.50e6.45
(m, 1H), 4.06 (s, 2H), 2.82e2.76 (m, 4H), 1.63e1.48 (m, 6H). 13C NMR
(101 MHz, DMSO-d6):
d
¼ 180.26, 171.55, 166.39, 148.16, 132.50,
130.91, 129.05, 128.28, 124.90, 115.03, 109.47, 109.07, 103.14, 52.69,
51.91, 24.49, 22.69. HRMS calc. (C21H22NO3): 336.1594. Found:
336.1594.

M.P. Carrasco et al. / European Journal of Medicinal Chemistry 80 (2014) 523e534
533
parasitaemia were determined by light microscopy of Giemsa-
stained thin blood smears.
for grants SFRH/BD/41276/2007 and SFRH/BD/61611/2009, respec-
tively. P.J.R. is a Doris Duke Charitable Foundation Distinguished
Clinical Scientist.
5.3.2. In vitro antimalarial activity
The antimalarial activity of compound 7c and chloroquine were
determined using the SYBR Green I assay as previously described.
Stock solutions of the drugs (10 mM) were prepared in DMSO and
serially diluted in complete media. Parasitized erythrocytes at the
early ring stage were added to a final 1% parasitaemia and 3% he-
matocrit to each triplicate well of a 96-well plate, and incubated for
48 h at 37 ꢁC prior to growth assessment with SYBR Green I nucleic
acid. Each compound was analyzed at a final concentration range of
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.076.
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