Probing the Azaaurone Scaffold against the Hepatic and Erythrocytic Stages of Malaria Parasites

Article abstract of DOI:10.1002/cmdc.201600327

The potential of azaaurones as dual-stage antimalarial agents was investigated by assessing the effect of a small library of azaaurones on the inhibition of liver and intraerythrocytic lifecycle stages of the malaria parasite. The whole series was screened against the blood stage of a chloroquine-resistant Plasmodium falciparum strain and the liver stage of P. berghei, yielding compounds with dual-stage activity and sub-micromolar potency against erythrocytic parasites. Studies with genetically modified parasites, using a phenotypic assay based on the P. falciparum Dd2-ScDHODH line, which expresses yeast dihydroorotate dehydrogenase (DHODH), showed that one of the azaaurone derivatives has the potential to inhibit the parasite mitochondrial electron-transport chain. The global urgency in finding new therapies for malaria, especially against the underexplored liver stage, associated with chemical tractability of azaaurones, warrants further development of this chemotype. Overall, these results emphasize the azaaurone chemotype as a promising scaffold for dual-stage antimalarials.

Full text of DOI:10.1002/cmdc.201600327

DOI: 10.1002/cmdc.201600327
Full Papers
Probing the Azaaurone Scaffold against the Hepatic and
Erythrocytic Stages of Malaria Parasites
Marta P. Carrasco,*[a, h] Marta Machado,[b] Lꢀdia GonÅalves,[a] Moni Sharma,[a] Jiri Gut,[c]
Amanda K. Lukens,[d, e] Dyann F. Wirth,[d, e] Vꢁnia Andrꢂ,[f] Maria Teresa Duarte,[f]
Rita C. Guedes,[a] Daniel J. V. A. dos Santos,[a, i] Philip J. Rosenthal,[c] Ralph Mazitschek,[d, e, g]
Miguel PrudÞncio,*[b] and Rui Moreira[a]
The potential of azaaurones as dual-stage antimalarial agents
was investigated by assessing the effect of a small library of
azaaurones on the inhibition of liver and intraerythrocytic life-
cycle stages of the malaria parasite. The whole series was
screened against the blood stage of a chloroquine-resistant
Plasmodium falciparum strain and the liver stage of P. berghei,
yielding compounds with dual-stage activity and sub-micromo-
lar potency against erythrocytic parasites. Studies with geneti-
cally modified parasites, using a phenotypic assay based on
the P. falciparum Dd2-ScDHODH line, which expresses yeast di-
hydroorotate dehydrogenase (DHODH), showed that one of
the azaaurone derivatives has the potential to inhibit the para-
site mitochondrial electron-transport chain. The global urgency
in finding new therapies for malaria, especially against the un-
derexplored liver stage, associated with chemical tractability of
azaaurones, warrants further development of this chemotype.
Overall, these results emphasize the azaaurone chemotype as
a promising scaffold for dual-stage antimalarials.
Introduction
Malaria remains a major global public health threat, and is re-
sponsible for high mortality and morbidity burdens in endemic
countries. The most severe form of the disease is caused by
Plasmodium falciparum, which accounts for most fatal cases,
particularly in young children and pregnant women,[1] but
P. vivax also contributes significantly to overall morbidity.[2] Re-
cently reported resistance to artemisinin derivatives, which are
critical components of artemisinin-based combination thera-
pies (ACTs), the current mainstay of malaria therapy, highlights
the urgent need for novel drugs acting on new or underex-
ploited parasite targets.[3]
tal stages of the parasite, including the asymptomatic liver
stage that precedes the invasion of host erythrocytes. Target-
ing liver stage parasites offers advantages, as it can lead to
causal prophylaxis and consequently arrest of parasitic trans-
mission. P. vivax infections can generate dormant hypnozoites
that persist in the liver for long periods of time and, when re-
activated, lead to relapsing malaria, a threat to the goal of
eliminating malaria. Accordingly, targeting the liver stage of
the Plasmodium life-cycle is a high priority in antimalarial drug
discovery.[4,5] However, despite recent advances in liver stage
phenotypic screening methods, the number of scaffolds active
against exoerythrocytic forms (EEFs) is still very limited.
Discovery of novel antimalarials has been focused on target-
ing asexual parasites that invade host erythrocytes and cause
clinical symptoms.[4] However, progress toward malaria elimina-
tion will be facilitated by intervention at multiple developmen-
Aurones (2-benzylidenebenzofuran-3(2H)-ones, 1, Scheme 1)
are secondary metabolites structurally related to flavones.[6]
We[7] and others[8] have reported that aurones are active
[a] Dr. M. P. Carrasco, Dr. L. GonÅalves, Dr. M. Sharma, Dr. R. C. Guedes,
Dr. D. J. V. A. d. . Santos, Prof. R. Moreira
[f] Dr. V. Andrꢀ, Prof. M. T. Duarte
Centro de Quꢁmica Estrutural, Instituto Superior Tꢀcnico, Universidade de
Lisboa, 1049-001 Lisboa (Portugal)
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Uni-
versidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa (Portugal)
[g] Prof. R. Mazitschek
[b] M. Machado, Dr. M. PrudÞncio
Center for System Biology, Massachusetts General Hospital and Harvard
Medical School, Richard B. Simches Research Center, 185 Cambridge Street,
Boston, MA 02114 (USA)
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de
Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
E-mail: mprudencio@medicina.ulisboa.pt
[h] Dr. M. P. Carrasco
[c] Dr. J. Gut, Prof. P. J. Rosenthal
Current address: Department of Chemistry and Molecular Biology, Universi-
ty of Gothenburg, 412 96 Gçteborg (Sweden)
E-mail: marta.carrasco@chem.gu.se
Department of Medicine, San Francisco General Hospital, University of Cali-
fornia San Francisco, 1001 Potrero Avenue, San Francisco CA 94110 (USA)
[d] Dr. A. K. Lukens, Prof. D. F. Wirth, Prof. R. Mazitschek
The Broad Institute, Infectious Diseases Program, Cambridge, MA 02142
(USA)
[i] Dr. D. J. V. A. d. . Santos
Current address: LAQV@REQUIMTE, Department of Chemistry and Biochem-
istry, Faculty of Sciences, University of Porto (Portugal)
[e] Dr. A. K. Lukens, Prof. D. F. Wirth, Prof. R. Mazitschek
Department of Immunology and Infectious Disease, Harvard T.H. Chan
School of Public Health, Boston, MA 02115 (USA)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600327.
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Scheme 1. Synthesis of azaaurones. Reagents and conditions: a) BCl3 (1m in CH2Cl2), chloroacetonitrile, ZnCl2, dry 1,2-DCE, reflux; b) HCl (1m), reflux; c) AcOH,
Ac2O; d) NaH, dry DMF; e) PhR2CHO, piperidine (cat.), toluene, reflux; f) KOH (50% in H2O), MeOH, RT. See Table 1 for R1 and R2 groups.
against intraerythrocytic P. falciparum in the low micromolar
range and non-cytotoxic against mammalian cells. In contrast,
bioisosteric azaaurones (2-benzylideneindolin-3-ones, for exam-
ple, 2, Figure 1) remain largely underexploited as antimalarial
agents. Souard et al. reported that azaaurones inhibit P. falcipa-
rum growth in the low micromolar range.[8b] These azaaurones
were inspired by naturally occurring aurones, with all ana-
logues containing methoxy groups at the C4 and C6 positions
of the A-ring. To better understand the structure-activity rela-
tionships, we designed a small library of azaaurone derivatives
3 and 4 (Table 1) with a diverse substitution pattern in rings A
and B and physicochemical properties. Herein we report
azaaurones as dual-stage antimalarials—that is, capable of in-
hibiting both erythrocytic and liver stage parasites—and de-
scribe the studies to gain insight into their mechanism of
action.
the presence of BCl3 and ZnCl2, to form the 2-chloroacetyl de-
rivatives 6, which were then acetylated at the aniline nitrogen
atom to give intermediates 7.[10] Base-promoted cyclization of
7 afforded the corresponding 1-acetylindolin-3-one (3-oxin-
dole) 8. Piperidine-catalyzed condensation of 3-oxindoles 8[11]
with the appropriate benzaldehyde followed by the alkaline
hydrolysis of the resulting N-acetylazaaurones 9 and 10 afford-
ed the target azaaurones 3 and 4 in good yield. The aldehydes
required to synthesize azaaurones 3d–g were obtained from
4-fluorobenzonitrile and the appropriate phenol,[7] while those
required to prepare compounds 3i–3ab were synthesized
through a standard Suzuki–Miyaura cross-coupling reaction
starting from 4- or 3-bromobenzaldehyde and the appropriate
boronic acids.[12]
Precursors 9 and 10 were obtained as inseparable mixtures
of Z and E isomers, as revealed by the presence of two signals
1
for the acetyl as well as for the olefinic protons in the H NMR
spectra (Supporting Information). Typically, the Z/E ratio varied
from 1:0.1 to 1:0.4. In contrast, the final azaaurones were isolat-
ed as single isomers. The stereochemistry of the carbon–
carbon double bond in derivatives 3 and 4 was assigned as Z-
1
configuration based on both H and 13C chemical shifts for the
exocyclic (b) carbon atom. These values are consistent with
those previously reported by Souard et al.[8b] for (Z)-azaaurones.
Similar to aurones, it is expected that the Z isomer is predomi-
nant, as this is regarded as the thermodynamically more stable
form.[13] Furthermore, the crystallographic structure of com-
pound 3j allowed us to confirm that azaaurones are obtained
as Z isomers (Figure 2).
Azaaurones 3 and 4 and their N-acetyl precursors 9 and 10
were first screened for antiplasmodial activity against the
chloroquine-resistant P. falciparum W2 strain. The most promis-
ing compounds were also assayed against human embryonic
kidney (HEK) 293T cells for potential cytotoxicity. In general,
compounds 3 and 4 revealed moderate to high potency
against intraerythrocytic P. falciparum, with EC50 values within
the sub-micromolar range (Table 1). With selectivity indices rel-
ative to HEK293T cells typically >200, azaaurones can thus be
considered potent and selective antiplasmodial agents. These
Figure 1. Structures of aurone 1, azaaurone 2, and azaaurones from this
study, 3 (R1 =H) and 4 (R2 =H).
Results and Discussion
The library comprising azaaurone derivatives (Table 1) was pre-
pared using the general synthetic approach depicted in
Scheme 1, adapted from Wager and Miller.[9] First, appropriately
substituted anilines 5 were reacted with chloroacetonitrile in
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Table 1. Structures, activity against the chloroquine (CQ)-resistant P. falciparum W2 strain, and cytotoxicity toward HEK293T cells for azaaurones 3 and 4,
and the corresponding N-acetylazaaurone precursors 9 and 10.
Compd
R1
R2
EC50 [mm][a]
Compd
R1
R2
EC50 [mm][a]
Pf(W2) HEK293T
Pf(W2)
HEK293T
3a
3b
3c
3d
3e
3 f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Br
1.76Æ0.11
0.56Æ0.02
2.90Æ0.28
0.36Æ0.01
0.32Æ0.04
0.51Æ0.01
0.59Æ0.02
1.62Æ0.07
0.31Æ0.04
0.27Æ0.01
0.53Æ0.01
0.83Æ0.08
0.73Æ0.06
1.08Æ0.06
0.52Æ0.02
0.44Æ0.01
0.48Æ0.01
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
22
>100
>100
>100
>100
>100
20
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9a
9b
9c
9d
9e
9 f
9h
9i
9j
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Br
>10
5.99Æ0.02
>10
ND
ND
ND
ND
ND
ND
ND
ND
7
4-NMe2
4-OPh
4-(OC6H4-4’-Me)
4-(OC6H4-4’-Cl)
4-(OC6H4-4’-CF3)
4-OBn
4-NMe2
4-OPh
4-(OC6H4-4’-Me)
4-(OC6H4-4’-Cl)
4-OBn
5.02Æ0.23
5.13Æ0.34
>10
>10
4-Ph
8.42Æ0.02
4-Ph
4-(C6H4-4’-F)
4-(C6H4-4’-Me)
4-(C6H4-4’-Cl)
4-(C6H4-4’-CF3)
4-(C6H4-4’-OCF3)
4-Bn
4-(3’-quinolyl)
4-(3’-thiophenyl)
4-(1’-methyl-1H-pyrazolyl)
3-Ph
3-(C6H4-4’-F)
3-(C6H4-4’-Me)
3-(C6H4-4’-Cl)
3-(C6H4-4’-CF3)
3-(C6H4-4’-OCF3)
3-Bn
3-(3’-quinolyl)
3-(3’-thiophenyl)
3-(1’-methyl-1H-pyrazolyl)
H
4.59Æ0.35
3.78Æ0.31
5.64Æ0.17
5.06Æ0.06
3.21Æ0.01
4-(C6H4-4’-F)
4-(C6H4-4’-Me)
4-(C6H4-4’-Cl)
4-(C6H4-4’-CF3)
4-(C6H4-4’-OCF3)
4-Bn
9k
9l
9
ND
ND
8
ND
19
ND
ND
ND
ND
4
ND
ND
ND
ND
14
ND
ND
12
ND
ND
ND
> 100
9m
9n
9o
9p
9q
9r
>10
2.03Æ0.08
9.83Æ0.24
9.43Æ0.81
9.78Æ0.07
9.38Æ0.88
4-(3’-quinolyl)
4-(3’-thiophenyl)
4-(1’-methyl-1H-pyrazolyl) 1.35Æ0.16
9s
9t
3s
3t
3-Ph
0.45Æ0.02
0.26Æ0.01
0.20Æ0.02
0.20Æ0.01
0.11Æ0.01
0.99Æ0.15
0.76Æ0.04
0.26Æ0.01
0.47Æ0.05
3-(C6H4-4’-F)
3-(C6H4-4’-Me)
3-(C6H4-4’-Cl)
3-(C6H4-4’-CF3)
3-(C6H4-4’-OCF3)
3-Bn
9u
9v
9w
9x
9y
9z
9aa
9ab
10a
10b
10c
10d
CQ
>10
3u
3v
3w
3x
3y
3z
3aa
3ab
4a
4b
4c
4d
3.65Æ0.36
6.12Æ0.32
5.08Æ0.01
>10
3.92Æ0.51
8.74Æ1.60
3-(3’-quinolyl)
3-(3’-thiophenyl)
>10
3-(1’-methyl-1H-pyrazolyl) 0.34Æ0.03
5-Ph
5-OPh
5-Bn
3.75Æ0.10
>10
6.77Æ0.78
>10
0.14
5-Ph
5-OPh
5-Bn
H
H
H
H
0.35Æ0.07
0.25Æ0.06
0.17Æ0.01
0.13Æ0.01
H
H
H
6-OPh
6-OPh
[a] Values are the meanÆSD of three independent experiments; ND: not determined.
is also consistent with the poor-to-moderate activity also dis-
played by their isosteric aurone counterparts.[8b]
Inspection of data presented in Table 1 reveals that append-
ing an aromatic moiety to ring B leads to improvement in anti-
plasmodial activity when compared with the parent compound
3a. For example, the 4- (3i) and 3-phenyl (3s) derivatives were
about six- and four-fold more active than the unsubstituted
compound 3a, respectively. The most potent compounds in
the 3 series, 3u–3w, contain the second aromatic moiety ap-
pended to the meta position of ring B. The azaaurone deriva-
tive containing the quinolin-3-yl substituent at the meta posi-
tion, 3z, was also more potent than its para-substituted coun-
terpart, 3p. Incorporating an oxygen or methylene linker be-
tween the two aromatic moieties did not significantly affect ac-
tivity, as compound 3d, containing a 4-phenoxy group, and its
isosteric 4-benzyl analogue, 3o, were equipotent to their 4-
phenyl counterpart, 3i. In contrast, the 4-benzyloxy derivative,
Figure 2. Crystal structure of compound 3j determined by single-crystal X-
ray diffraction data; ellipsoids are set at 50% probability.
results contrast sharply with those obtained for the N-acetyl
counterparts 9 and 10, which were shown to be poorly active,
with IC50 values typically >5 mm, and cytotoxic (Table 1). This
result suggests that the azaaurone NH group is essential to
attain good levels of antiplasmodial activity. Interestingly, this
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3h, was nearly five times less potent than its 4-phenoxy coun-
terpart, 3d, suggesting that additional flexibility is detrimental.
Compounds 4a–d, containing an additional aromatic sub-
stituent in ring A, displayed EC50 values ranging from 0.13 to
0.35 mm, indicating that derivatization at this side of the
azaaurone scaffold also leads to improved antiplasmodial activ-
ity. The 5-phenoxy (4b) and 5-benzyl (4c) derivatives were
more potent than the 5-phenyl analogue (4a), suggesting that
an oxygen linker is beneficial to activity. Azaaurone 4d, con-
taining a 6-phenoxy substituent, was the most potent com-
pound in this series, with a selectivity index of >770 and with
no detectable aggregation up to 10 mm (Supporting Informa-
tion), which could have led to measuring artifacts and false
positive results. In contrast, the N-acetyl counterparts were at
least 10 times less potent in inhibiting P. falciparum growth
(e.g., 4c,d vs. 10c,d).
and 4 using a phenotypic assay based on the P. falciparum
Dd2-ScDHODH line, which expresses yeast dihydroorotate de-
hydrogenase (DHODH).[15,16] This parasite, which uses fumarate
instead of ubiquinone as the final electron acceptor, has been
shown previously to be resistant to mtETC inhibitors.[17] The re-
sults presented in Table 3 indicate that most of the tested
compounds display similar antiplasmodial activity against both
Dd2-ScDHODH and parental Dd2 strains, suggesting that inhib-
ition of the bc1 complex is not the primary antimalarial mode
of action for these azaaurones. The exception was compound
3j, which showed an inhibitory profile similar to that of atova-
quone (Figure 3).
Table 3. Growth inhibition of parental (Dd2) and transgenic (Dd2-
ScDHODH) P. falciparum strains with azaaurones.
Next, azaaurones 3 and 4 were evaluated for their ability to
inhibit the development of liver stage parasites. Compounds
were first assayed at three different concentrations (1, 5, and
10 mm) using an in vitro infection model that employs
a human hepatoma cell line (Huh7) infected with firefly-lucifer-
ase-expressing P. berghei. Results were compared with those
for primaquine (Supporting Information). From the 32 azaaur-
ones studied, 15 led to at least 50% inhibition of the infection
at 1 mm relative to controls, while almost all suppressed infec-
tion at 10 mm without significantly affecting Huh7 cell prolifera-
tion. With these promising results in hand, we then deter-
mined the EC50 values against the liver stage of P. berghei infec-
tion of Huh7 cells for the most potent compounds. Inspection
of Table 2 shows that azaaurones 3n and 3x, both containing
a 4-OCF3 group at the aromatic group appended to ring B,
present sub-micromolar EC50 values for the liver stage inhibito-
ry activity.
Compd
EC50 [mm][a]
Dd2
Dd2-ScDHODH
3e
3j
3t
3v
3w
3z
4a
4b
4c
1.46Æ0.25
1.60Æ0.22
0.414Æ0.08
1.24Æ0.14
0.800Æ0.09
0.573Æ0.05
1.66Æ0.40
2.62Æ0.22
1.33Æ0.11
1.04Æ0.10
0.000298
1.62Æ0.36
>1000
0.343Æ0.02
1.25Æ0.13
0.786Æ0.07
0.493Æ0.07
2.70Æ0.28
4.34Æ1.22
0.906Æ0.08
0.940Æ0.06
>1000
4d
atovaquone
artesunate
0.00287
0.00299
[a] Values are the meanÆSD of three independent experiments.
Table 2. EC50 values of selected azaaurones, primaquine (PQ), and atova-
quone (ATV) for inhibiting the infection of human hepatoma (Huh7) cells
by P. berghei.
Compd
EC50 [mm][a]
Compd
EC50 [mm][a]
3h
3i
3j
3k
3n
3o
3p
2.62Æ1.22
3.05Æ0.14
2.07Æ0.15
1.63Æ0.52
0.75Æ0.19
1.03Æ0.13
3.00Æ0.65
3s
3t
2.49Æ0.21
1.54Æ0.29
0.81Æ0.20
0.53Æ0.26
1.80Æ0.53
9.50Æ2.30
0.0011Æ0.003
3w
3x
3y
PQ
ATV
[a] Values are the meanÆSD of three independent experiments.
Azaaurones are structurally related to quinolin-4(1H)-ones,
and several compounds of this later class have shown antiplas-
modial activity, via inhibition of cytochrome bc1, a key compo-
nent of the mitochondrial electron-transport chain (mtETC).[14]
Atovaquone, the only cytochrome bc1 inhibitor in clinical use,
is active against both erythrocytic and hepatic forms of malaria
parasites.[15] Hence, we investigated whether the activity of
azaaurones was a consequence of bc1 complex inhibition by
screening several of the most potent compounds in series 3
Figure 3. Dose–effect curves for A) azaaurone 3j and B) atovaquone against
&
Dd2 and Dd2-ScDHODH parasite strains. Wild-type Dd2 ( ) and Dd2-
*
ScDHODH ( ) parasite strains were grown in the presence of various drug
concentrations for 48 h. Growth was assessed by [3H]hypoxanthine incorpo-
ration. Growth in the absence of drug was set at 100%. Data are the
meanÆSD of triplicate experiments performed at each condition.
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Azaaurones contain a Michael acceptor moiety, a structural
motif commonly found in cysteine protease inhibitors[18] that
have been reported to block the development of cultured er-
ythrocytic parasites and to cure murine malaria.[19] Falcipains
are the best characterized cysteine proteases of P. falciparum,
with falcipain-2 playing a crucial role in hydrolyzing host he-
moglobin into amino acids essential to parasite growth during
erythrocytic stages of development.[20] We therefore screened
azaaurones 3 and 4 for falcipain-2 inhibition; however none of
the compounds displayed noticeable activity up to 50 mm
(data not shown), ruling out this enzyme as drug target.
To evaluate the potential of azaaurones to undergo Michael
addition with thiols we monitored the reaction of compound
3q with the model thiol, b-mercaptoethanol by 1H NMR. Analy-
sis of the NMR spectra revealed that consumption of 3q was
triggered by addition of triethylamine (1 mol equiv) to the re-
action mixture, resulting in the formation of an adduct in
a 85:15 ratio to the starting azaaurone. This adduct slowly re-
verted to the starting compound 3q, reaching a 60:40 ratio in
three days of incubation, suggesting that azaaurones react in
reversible manner with cysteine residues, which may explain,
at least partially, the lack of inhibition of falcipain-2. These re-
sults are in line with those observed for other scaffolds con-
taining an exocyclic Michael acceptor system such as alkyli-
dene rhodanines, which have been reported to react reversibly
with cysteine residues in catalytic and allosteric binding
sites.[21]
Experimental Section
Chemistry
All reagents and solvents were obtained from commercial suppliers
and were used without further purification. Melting points were
determined using a Kofler camera Bock monoscope M and are un-
corrected. Reactions were monitored by analytical thin-layer chro-
matography (TLC), performed on pre-coated Merck silica gel 60
F254 plates. Flash column chromatography was performed on
Merck silica gel (200–400 mesh). H and 13C NMR spectra were re-
1
corded on a Bruker 400 Ultra-Shield (400 MHz). 1H and 13C NMR
chemical shifts (d) are expressed in parts per million (ppm) refer-
enced to the solvent used, and proton coupling constants (J) are
reported in hertz (Hz). The spin multiplicities are reported as sin-
glet (s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet of
doublets (dd), and broad (br). The purity of final compounds was
determined by elemental analyses conducted on a CE Instruments
EA 1110 apparatus and confirmed to be ꢀ95%.
General procedure for the synthesis of azaaurone derivatives
9a–ab and 10a–d: To a solution of the appropriate 1-acetylindolin-
3-one derivative (see Supporting Information for the synthesis of
8a–e) (0.5 mmol) in toluene (5 mL) at room temperature was
added the proper aldehyde (1.2 mmol) and piperidine (1 drop).
The mixture was held at reflux for 24 h. After reaction completion,
the solvent was removed to provide the crude product.
1-acetyl-2-benzylideneindolin-3-one (9a): Purified by flash chro-
matography (hexane/EtOAc=95:5). Obtained as a light-yellow oil,
yield: 72%. Mixture of isomers Z/E=1:0.60. 1H NMR (400 MHz,
CDCl3, major isomer): d=8.30 (d, J=8.3 Hz, 1H), 7.86 (d, J=7.6 Hz,
2H), 7.70–7.61 (m, 1H), 7.55 (d, J=7.6 Hz, 2H), 7.45–7.38 (m, 2H),
7.35 (s, 1H), 7.31 (t, J=7.6 Hz, 1H), 1.96 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=186.0, 170.5, 150.2, 136.4, 135.0, 133.9, 130.2,
129.9, 129.2, 125.0, 124.2, 123.9, 122.4, 117.9, 25.1 ppm.
Conclusions
We have identified, for the first time, azaaurones as a new
class of antimalarials active against the erythrocytic and liver
stages of the malaria parasite. Several compounds displayed
EC50 values against the liver and asexual blood stages within
the same order of magnitude. Although one azaaurone has
shown to inhibit the parasite’s mtETC, the primary mechanism
of action of azaaurones does not appear to involve inhibition
of the bc1 complex. These results suggest that azaaurones may
modulate other targets such as kinases. There is a striking re-
semblance of azaaurones to oxindole inhibitors (e.g., sunitinib)
that are active against Plasmodium[22] and used clinically for
other purposes. The same substitution pattern as in the
azaaurones is also found in indirubin kinase inhibitors, which
are also active against P. falciparum.[23]
1-acetyl-2-(4-bromobenzylidene)indolin-3-one (9b): Purified by
flash chromatography (hexane/EtOAc=80:20). Obtained as yellow
solid, yield: 64%, mp: 138–1418C. Mixture of isomers Z/E=1:0.70.
1H NMR (400 MHz, CDCl3, major isomer): d=8.25 (d, J=8.2 Hz, 1H),
7.87 (d, J=8.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.60–7.54 (m, 2H), 7.41
(d, J=8.3 Hz, 2H), 7.34–7.29 (m, 1H), 7.27 (s, 1H), 2.03 ppm (s, 3H);
13C NMR (101 MHz, CDCl3, major isomer): d=185.8, 170.2, 150.5,
136.6, 135.3, 133.1, 132.6, 131.7, 125.3, 124.4, 124.3, 124.1, 121.0,
118.0, 26.7 ppm.
1-acetyl-2-(4-(dimethylamino)benzylidene)indolin-3-one (9c): Pu-
rified by flash chromatography (CH2Cl2/MeOH=99:1). Obtained as
yellow solid, yield: 70%, mp: 147–1508C. Mixture of isomers Z/E=
1:0.20. 1H NMR (400 MHz, CDCl3, major isomer): d=8.30 (d, J=
8.3 Hz, 1H), 7.84 (d, J=8.2 Hz, 1H), 7.64 (t, J=8.3 Hz, 1H), 7.48 (d,
J=8.8 Hz, 2H), 7.33–7.28 (m, 2H), 6.70 (d, J=8.8 Hz, 2H), 3.06 (s,
6H), 2.14 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=185.9, 171.5, 151.4, 149.7, 135.7, 133.1, 131.812, 125.1, 124.9,
124.7, 123.9, 120.5, 117.9, 112.0, 40.2, 25.5 ppm.
The global urgency in finding new therapies for malaria, es-
pecially against the underexplored liver stage, associated with
the dual activity and chemical tractability of azaaurones, war-
rants further development of this chemotype. In spite of the
concern of potential nonspecific target modulation resulting
from the reactivity of the exocyclic Michael acceptors,[24,25] sev-
eral examples of successful progression of the analogous alkyli-
dene rhodamine-like scaffolds into lead compounds have been
reported.[26,27] Overall, our study has revealed azaaurone start-
ing points for lead optimization of dual-stage antimalarials.
1-acetyl-2-(4-phenoxybenzylidene)indolin-3-one (9d): Purified by
flash chromatography (hexane/CH2Cl2 =50:50). Obtained as yellow
1
oil, yield: 57%. Mixture of isomers Z/E=1:0.30. H NMR (400 MHz,
CDCl3, major isomer): d=8.33 (d, J=8.1 Hz, 1H), 8.01 (d, J=8.0 Hz,
1H), 7.71 (t, J=8.1 Hz, 1H), 7.56 (d, J=8.7 Hz, 2H), 7.39–7.35 (m,
3H), 7.29–7.24 (m, 2H), 7.10–7.07 (m, 4H), 2.15 ppm (s, 3H);
13C NMR (101 MHz, CDCl3, major isomer): d=184.1, 168.9, 159.1,
154.1, 150.0, 135.8, 134.2, 133.8, 131.5, 129.3, 129.0, 128.1, 126.7,
124.1, 122.3, 121.2, 119.2, 117.6, 26.6 ppm.
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1-acetyl-2-(4-(p-tolyloxy)benzylidene)indolin-3-one (9e): Purified
by flash chromatography (hexane/CH2Cl2 =50:50). Obtained as
yellow oil, yield: 53%. Mixture of isomers Z/E=1:0.25. 1H NMR
(400 MHz, CDCl3, major isomer): d=8.27 (d, J=8.2 Hz, 1H), 7.89 (d,
J=8.1 Hz, 1H), 7.71–7.66 (m, 3H), 7.29–7.34 (m, 4H), 7.01–6.98 (m,
4H), 2.18 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=183.9, 167.0, 158.7, 153.8, 148.7, 134.6, 134.3, 132.3, 131.6,
129.9, 128.9, 127.6, 126.1, 123.9, 122.6, 121.6, 120.0, 118.3, 25.9,
21.2 ppm.
1H), 7.69–7.60 (m, 3H), 7.57–7.52 (m, 3H), 7.34–7.27 (m, 3H), 2.41
(s, 3H), 2.06 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.0, 169.3, 148.3, 143.0, 138.2, 137.9, 137.6, 136.0, 134.9,
132.0, 130.9, 130.2, 129.7, 127.1, 126.5, 124.8, 124.3, 117.5, 26.6,
25.4 ppm.
1-acetyl-2-((4’-chlorobiphenyl-4-yl)methylene)indolin-3-one (9l):
Purified by flash chromatography (hexane/EtOAc=80:20). Ob-
tained as yellow solid, yield: 75%, mp: 107–1108C. Mixture of iso-
1
mers Z/E=1:0.35. H NMR (400 MHz, CDCl3, major isomer): d=8.29
1-acetyl-2-(4-(4-chlorophenoxy)benzylidene)indolin-3-one (9 f):
Purified by flash chromatography (hexane/CH2Cl2 =50:50). Ob-
tained as yellow solid, yield: 61%, mp: 108–1118C. Mixture of iso-
(d, J=8.2 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.71–7.55 (m, 7H), 7.45–
7.42 (m, 2H), 7.37 (s, 1H), 7.32 (t, J=8.0 Hz, 1H), 2.06 ppm (s, 3H);
13C NMR (101 MHz, CDCl3, major isomer): d=186.0, 170.6, 150.3,
141.4, 138.3, 136.6, 135.1, 134.4, 133.2, 131.1, 129.3, 128.4, 127.7,
125.2, 124.4, 124.1, 122.0, 118.0, 25.4 ppm.
1
mers Z/E=1:0.70. H NMR (400 MHz, CDCl3, major isomer): d=8.26
(d, J=8.1 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.69–7.62 (m, 1H), 7.55–
7.52 (m, 2H), 7.36–7.31 (m, 3H), 7.03–7.00 (m, 5H), 2.07 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.0, 170.5,
158.9, 154.4, 150.3, 136.4, 134.5, 133.5, 132.5, 130.2, 128.7, 127.3,
125.1, 124.3, 124.3, 121.3, 118.6, 117.6, 25.3 ppm.
1-acetyl-2-((4’-(trifluoromethyl)biphenyl-4-yl)methylene)indolin-
3-one (9m): Purified by flash chromatography (hexane/EtOAc=
95:5). Obtained as yellow solid, yield: 78%, mp: 124–1278C. Mix-
ture of isomers Z/E=1:0.80. 1H NMR (400 MHz, CDCl3, major
isomer): d=8.09 (d, J=8.2 Hz, 1H), 7.98 (d, J=8.2 Hz, 2H), 7.80 (d,
J=8.1 Hz, 1H), 7.76–7.64 (m, 8H), 7.34–7.27 (m, 1H), 2.06 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.1, 170.5,
162.751, 148.4, 141.2, 136.2, 135.4, 134.0 (q, J=32.1 Hz), 132.3,
131.9, 129.5, 127.5, 126.9, 126.0, 124.8 (q, J=271.2 Hz), 124.7,
124.1, 121.7, 117.4, 25.4 ppm.
1-acetyl-2-(4-(4-(trifluoromethyl)phenoxy)benzylidene)indolin-3-
one (9g): Purified by flash chromatography (hexane/CH2Cl2 =
50:50). Obtained as yellow oil, yield: 53%. Mixture of isomers Z/E=
1:0.20. 1H NMR (400 MHz, CDCl3, major isomer): d=8.28 (d, J=
8.1 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.71–7.63 (m, 3H), 7.41–7.32
(m, 3H), 7.02–6.98 (m, 5H), 2.18 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3, major isomer): d=186.1, 169.9, 163.6, 158.8, 153.7, 149.7,
135.6, 134.0, 133.9, 132.0, 130.4, 128.6, 127.7, 124.9, 123.7, 123.5,
120.4, 118.3, 117.8, 24.8 ppm.
1-acetyl-2-((4’-(trifluoromethoxy)biphenyl-4-yl)methylene)indo-
lin-3-one (9n): Purified by flash chromatography (hexane/EtOAc=
95:5). Obtained as yellow solid, yield: 71%, mp: 121–1248C. Mix-
ture of isomers Z/E=1:0.60. 1H NMR (400 MHz, CDCl3, major
isomer): d=8.29 (d, J=8.2 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.72–
7.62 (m, 7H), 7.38 (s, 1H), 7.34–7.31 (m, 3H), 2.06 ppm (s, 3H);
13C NMR (101 MHz, CDCl3, major isomer): d=186.1, 170.4, 166.5,
150.1, 141.2, 138.6, 136.6, 133.1, 131.6, 131.1 (q, J=253.4 Hz),
129.7, 128.6, 127.8, 126.7, 124.4, 123.9, 121.9, 121.5, 118.0,
25.6 ppm.
1-acetyl-2-(4-(benzyloxy)benzylidene)indolin-3-one (9h): Purified
by flash chromatography (hexane/EtOAc=95:5). Obtained as
yellow solid, yield: 57%, mp: 151–1548C. Mixture of isomers Z/E=
1:0.10. 1H NMR (400 MHz, CDCl3, major isomer): d=8.29 (d, J=
8.3 Hz, 1H), 7.85 (d, J=8.2 Hz, 1H), 7.67 (t, J=8.2 Hz, 1H), 7.52 (d,
J=8.5 Hz, 2H), 7.45–7.28 (m, 7H), 7.04 (d, J=8.5 Hz, 2H), 5.11 (s,
2H), 2.06 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.1, 170.9, 160.2, 150.2, 136.3, 133.8, 132.5, 128.8, 128.4,
127.7, 126.4, 125.0, 124.4, 124.2, 123.0, 118.0, 115.7, 70.3, 25.3 ppm.
1-acetyl-2-(4-benzylbenzylidene)indolin-3-one (9o): Purified by
flash chromatography (hexane/EtOAc=92:8). Obtained as yellow
1
1-acetyl-2-(biphenyl-4-ylmethylene)indolin-3-one (9i): Purified by
flash chromatography (hexane/EtOAc=80:20). Obtained as yellow
solid, yield: 94%, mp: 172–1758C. Mixture of isomers Z/E=1:0.10.
1H NMR (400 MHz, CDCl3, major isomer): d=8.31 (d, J=8.3 Hz, 1H),
7.88 (d, J=8.2 Hz, 1H), 7.71–7.60 (m, 6H), 7.48 (t, J=6.9 Hz, 2H),
7.41–7.39 (m, 2H), 7.32 (t, J=8.2 Hz, 1H), 7.27 (s, 1H), 2.06 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.1, 170.7,
150.4, 142.7, 139.8, 136.5, 135.0, 132.8, 131.0, 129.1, 128.2, 127.9,
127.2, 125.1, 124.3, 124.1, 122.3, 118.0, 25.4 ppm.
oil, yield: 73%. Mixture of isomers Z/E=1:0.40. H NMR (400 MHz,
CDCl3, major isomer): d=8.30 (d, J=8.2 Hz, 1H), 7.87–7.84 (m, 1H),
7.69–7.64 (m, 1H), 7.50–7.47 (m, 2H), 7.33–7.18 (m, 9H), 4.02 (s,
2H), 2.00 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.1, 170.7, 150.4, 143.7, 140.1, 136.4, 134.8, 131.8, 130.6,
129.9, 129.1, 128.8, 126.5, 125.0, 124.2, 124.1, 122.6, 118.0, 41.9,
25.2 ppm.
1-acetyl-2-(4-(quinolin-3-yl)benzylidene)indolin-3-one (9p): Puri-
fied by flash chromatography (hexane/EtOAc=70:30). Obtained as
yellow solid, yield: 69%, mp: 168–1718C. Mixture of isomers Z/E=
1:0.70. 1H NMR (400 MHz, CDCl3, major isomer): d=9.25 (s, 1H),
8.39 (s, 1H), 8.29 (d, J=8.3 Hz, 1H), 8.17 (d, J=8.0 Hz, 1H), 7.93–
7.88 (m, 2H), 7.84–7.60 (m, 7H), 7.40 (s, 1H), 7.33 (t, J=7.5 Hz, 1H),
2.07 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer): d=
186.2, 170.5, 150.4, 149.5, 147.7, 139.3, 137.8, 136.6, 136.2, 135.4,
133.9, 133.7, 131.3, 129.4, 128.1, 128.0, 126.9, 125.2, 124.8, 124.4,
123.6, 122.7, 118.0, 25.4 ppm.
1-acetyl-2-((4’-fluorobiphenyl-4-yl)methylene)indolin-3-one (9j):
Purified by flash chromatography (hexane/EtOAc=80:20). Ob-
tained as yellow solid, yield: 80%, mp: 70–738C. Mixture of isomers
Z/E=1:0.40. 1H NMR (400 MHz, CDCl3, major isomer): d=8.30 (d,
J=8.3 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.69 (t, J=8.3 Hz, 1H), 7.65–
7.58 (m, 6H), 7.37 (s, 1H), 7.32 (d, J=8.0 Hz, 1H), 7.18–7.14 (m,
2H), 2.06 ppm (s, 1H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.1, 170.6, 163.0 (d, J=268.7 Hz), 150.3, 141.7, 136.5, 135.9,
135.1, 132.9, 131.0, 128.8 (d, J=8.1 Hz), 127.7, 125.2, 124.3, 124.1,
122.1, 118.0, 116.1 (d, J=22.2 Hz), 25.4 ppm.
1-acetyl-2-(4-(thiophen-3-yl)benzylidene)indolin-3-one (9q): Puri-
fied by flash chromatography (hexane/EtOAc=70:30). Obtained as
yellow solid, yield: 72%, mp: 126–1298C. Mixture of isomers Z/E=
1:0.25. 1H NMR (400 MHz, CDCl3, major isomer): d=8.30 (d, J=
8.2 Hz, 1H), 7.87 (d, J=8.1 Hz, 1H), 7.70–7.56 (m, 6H), 7.45 (s, 2H),
7.36 (s, 1H), 7.31 (t, J=8.1 Hz), 2.05 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3, major isomer): d=186.2, 170.8, 150.4, 137.3,
1-acetyl-2-((4’-methylbiphenyl-4-yl)methylene)indolin-3-one
(9k): Purified by flash chromatography (hexane/EtOAc=90:10).
Obtained as yellow solid, yield: 65%, mp: 135–1388C. Mixture of
isomers Z/E=1:0.50. 1H NMR (400 MHz, CDCl3, major isomer): d=
8.15 (d, J=8.2 Hz, 1H), 7.98 (d, J=8.2 Hz, 2H), 7.80 (d, J=8.1 Hz,
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136.5, 135.0, 134.4, 132.6, 131.1, 127.1, 126.9, 126.1, 125.1, 124.3,
124.2, 122.3, 118.0, 25.3 ppm.
1-acetyl-2-((4’-(trifluoromethoxy)biphenyl-3-yl)methylene)indo-
lin-3-one (9x): Purified by flash chromatography (hexane/EtOAc=
90:10). Obtained as yellow solid, yield: 72%, mp: 115–1178C. Mix-
ture of isomers Z/E=1:0.45. 1H NMR (400 MHz, CDCl3, major
isomer): d=8.18 (s, 1H), 8.10 (d, J=8.3 Hz, 1H), 7.82–7.49 (m, 7H),
7.32–7.27 (m, 4H), 2.06 ppm (s, 3H); 13C NMR (101 MHz, CDCl3,
major isomer): d=186.1, 169.3, 151.4, 150.4, 148.4, 138.6, 138.8,
136.2, 132.8, 130.4 (q, J=254.1 Hz), 130.0, 129.8, 129.6, 128.8,
128.7, 124.9, 124.7, 124.5, 121.6, 121.4, 117.4, 26.9 ppm.
1-acetyl-2-(4-(1-methyl-1H-pyrazol-4-yl)benzylidene)indolin-3-
one (9r): Purified by flash chromatography (CH2Cl2/MeOH=99:1).
Obtained as yellow solid, yield: 77%, mp: 161–1648C. Mixture of
isomers Z/E=1:0.20. 1H NMR (400 MHz, CDCl3, major isomer): d=
8.29 (d, J=8.2 Hz, 1H), 7.86 (d, J=8.1 Hz, 1H), 7.81 (s, 1H), 7.69–
7.65 (m, 2H), 7.54–7.52 (m, 4H), 7.33–7.29 (m, 2H), 3.96 (s, 3H),
2.05 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer): d=
186.1, 170.8, 150.3, 137.1, 136.4, 134.7, 134.5, 131.7, 131.2, 127.5,
126.0, 125.1, 124.3, 124.2, 122.5, 122.3, 118.0, 39.4, 25.3 ppm.
1-acetyl-2-(3-benzylbenzylidene)indolin-3-one (9y): Purified by
flash chromatography (hexane/EtOAc=92:8). Obtained as yellow
1
oil, yield: 72%. Mixture of isomers Z/E=1:0.40. H NMR (400 MHz,
1-acetyl-2-(biphenyl-3-ylmethylene)indolin-3-one (9s): Purified
by flash chromatography (hexane/EtOAc=80:20). Obtained as
yellow solid, yield: 65%, mp: 100–1038C. Mixture of isomers Z/E=
1:0.30. 1H NMR (400 MHz, CDCl3, major isomer): d=8.31 (d, J=
8.3 Hz, 1H), 7.87 (d, J=8.2 Hz, 1H), 7.76 (s, 1H), 7.71–7.67 (m, 1H),
7.63–7.52 (m, 5H), 7.48 (t, J=7.6 Hz, 2H), 7.41–7.37 (m, 2H), 7.32 (t,
J=8.2 Hz, 1H), 2.03 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major
isomer): d=186.1, 170.6, 150.4, 142.4, 140.0, 136.6, 135.3, 134.5,
129.8, 129.1, 129.0, 128.9, 128.8, 128.0, 127.2, 125.1, 124.4, 124.0,
122.3, 117.9, 25.4 ppm.
CDCl3, major isomer): d=8.29 (d, J=8.2 Hz, 1H), 7.86 (d, J=8.0 Hz,
1H), 7.70–7.62 (m, 2H), 7.41–7.19 (m, 10H), 4.01 (s, 2H), 1.94 ppm
(s, 3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.1, 170.5,
150.4, 142.5, 140.3, 136.5, 135.1, 134.2, 130.8, 130.7, 129.5, 129.0,
128.8, 129.1, 126.5, 125.0, 124.3, 124.0, 122.5, 118.0, 41.9, 25.2 ppm.
1-acetyl-2-(3-(quinolin-3-yl)benzylidene)indolin-3-one (9z): Puri-
fied by flash chromatography (hexane/EtOAc=70:30). Obtained as
yellow solid, yield: 62%, mp: 145–1488C. Mixture of isomers Z/E=
1:0.70. 1H NMR (400 MHz, CDCl3, major isomer): d=9.27 (s, 1H),
8.43 (s, 1H), 8.28 (d, J=8.2 Hz, 1H), 8.80 (d, J=8.0 Hz, 1H), 7.94–
7.57 (m, 10H), 7.32 (t, J=7.5 Hz, 1H), 2.07 ppm (s, 1H); 13C NMR
(101 MHz, CDCl3, major isomer): d=186.0, 169.4, 150.5, 149.9,
148.3, 139.1, 137.6, 136.2, 135.2, 133.8, 133.2, 130.7, 130.2, 129.7,
129.5, 129.2, 128.9, 128.3, 127.2, 124.9, 124.7, 124.5, 124.0, 122.0,
117.4, 25.4 ppm.
1-acetyl-2-((4’-fluorobiphenyl-3-yl)methylene)indolin-3-one (9t):
Purified by flash chromatography (hexane/EtOAc=80:20). Ob-
tained as yellow solid, yield: 69%, mp: 165–1688C. Mixture of iso-
1
mers Z/E=1:0.15. H NMR (400 MHz, CDCl3, major isomer): d=8.30
(d, J=8.0 Hz, 1H), 7.87 (d, J=7.9 Hz, 1H), 7.71–7.49 (m, 7H), 7.39
(s, 1H), 7.32 (t, J=7.9 Hz, 1H), 7.16 (t, J=8.4 Hz, 2H), 2.02 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.0, 170.6,
164.1 (d, J=269.1 Hz), 161.7, 150.4, 141.4, 136.6, 135.4, 134.7,
129.9, 128.9, 128.8, 128.6 (d, J=7.9 Hz), 125.1, 124.4, 123.9, 122.1,
117.9, 116.2, 116.0 (d, J=22.3 Hz), 25.4 ppm.
1-acetyl-2-(3-(thiophen-3-yl)benzylidene)indolin-3-one (9aa): Pu-
rified by flash chromatography (hexane/EtOAc=70:30). Obtained
as yellow solid, yield: 69%, mp: 150–1538C. Mixture of isomers Z/
1
E=1:0.75. H NMR (400 MHz, CDCl3, major isomer): d=8.31 (d, J=
8.2 Hz, 1H), 7.87 (d, J=8.1 Hz, 1H), 7.80–7.60 (m, 4H), 7.50–7.38
(m, 5H), 7.31 (t, J=8.1 Hz, 1H), 2.01 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3, major isomer): d=186.1, 170.7, 150.4, 141.2,
137.0, 136.6, 135.4, 134.6, 129.9, 128.8, 128.1, 126.9, 126.2, 125.1,
124.4, 124.0, 122.1, 121.4, 117.8, 25.4 ppm.
1-acetyl-2-((4’-methylbiphenyl-3-yl)methylene)indolin-3-one
(9u): Purified by flash chromatography (hexane/EtOAc=90:10).
Obtained as yellow oil, yield: 62%. Mixture of isomers Z/E=1:0.40.
1H NMR (400 MHz, CDCl3, major isomer): d=8.16–8.12 (m, 2H),
7.81–7.79 (m, 2H), 7.69–7.57 (m, 4H), 7.51–7.47 (m, 2H), 7.29–7.26
(m, 3H), 2.41 (s, 3H), 2.02 ppm (s, 3H); 13C NMR (101 MHz, CDCl3,
major isomer): d=186.1, 169.3, 148.3, 141.0, 137.9, 137.4, 136.1,
135.2, 132.5, 139.2, 129.8, 129.7, 128.8, 128.5, 127.2, 124.8, 124.7,
124.3, 122.4, 117.5, 26.5, 25.3 ppm.
1-acetyl-2-(3-(1-methyl-1H-pyrazol-4-yl)benzylidene)indolin-3-
one (9ab): Purified by flash chromatography (CH2Cl2/MeOH=99:1).
Obtained as yellow solid, yield: 68%, mp: 198–2018C. Mixture of
isomers Z/E=1:0.60. 1H NMR (400 MHz, CDCl3, major isomer): d=
8.30 (d, J=8.2 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.77 (s, 1H), 7.70–
7.63 (m, 3H), 7.53–7.38 (m, 3H), 7.35 (s, 1H), 7.33 (t, J=8.0 Hz, 1H),
3.96 (s, 3H), 2.00 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major
isomer): d=186.1, 170.7, 150.4, 136.9, 136.6, 135.3, 134.6, 133.9,
129.9, 128.0, 127.3, 127.1, 127.0, 125.1, 124.4, 123.9, 122.3, 122.2,
117.8, 39.3, 25.3 ppm.
1-acetyl-2-((4’-chlorobiphenyl-3-yl)methylene)indolin-3-one (9v):
Purified by flash chromatography (hexane/EtOAc=80:20). Ob-
tained as yellow solid, yield: 66%, mp: 180–1838C. Mixture of iso-
1
mers Z/E=1:0.20. H NMR (400 MHz, CDCl3, major isomer): d=8.29
(d, J=8.3 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.70–7.50 (m, 7H), 7.44
(d, J=8.4 Hz, 2H), 7.39 (s, 1H), 7.32 (t, J=8.0 Hz, 1H), 2.02 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=186.0, 170.5,
150.4, 141.2, 138.5, 136.7, 135.4, 134.7, 134.2, 129.9, 129.3, 129.2,
128.8, 128.6, 128.5, 125.2, 124.4, 123.9, 122.0, 117.9, 25.4 ppm.
1-acetyl-2-benzylidene-5-phenylindolin-3-one (10a): Purified by
flash chromatography (hexane/EtOAc=90:10). Obtained as yellow
solid, yield: 67%, mp: 131–1348C. Mixture of isomers Z/E=1:0.25.
1H NMR (400 MHz, CDCl3, major isomer): d=8.36 (d, J=8.1 Hz, 1H),
8.08 (s, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.64–7.56 (m, 4H), 7.49–7.38
(m, 7H), 1.99 ppm (s, 1H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.1, 170.6, 149.4, 140.2, 135.1, 134.1, 133.8, 131.8, 129.4,
128.9, 128.6, 128.4, 128.0, 127.7, 127.1, 127.0, 121.1, 117.7,
26.0 ppm.
1-acetyl-2-((4’-(trifluoromethyl)biphenyl-3-yl)methylene)indolin-
3-one (9w): Purified by flash chromatography (hexane/EtOAc=
95:5). Obtained as yellow solid, yield: 75%, mp: 144–1458C. Mix-
ture of isomers Z/E=1:0.80. 1H NMR (400 MHz, CDCl3, major
isomer): d=8.28 (d, J=8.2 Hz, 1H), 8.21 (s, 1H), 7.88 (d, J=8.1 Hz,
1H), 7.81–7.51 (m, 8H), 7.40 (s, 1H), 7.32 (t, J=8.1 Hz, 1H),
2.04 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer): d=
186.0, 170.5, 150.4, 148.3, 140.9, 139.5, 136.3, 135.4, 134.9 (q, J=
31.9 Hz), 132.9, 130.8, 130.1, 129.7, 128.7, 127.7, 125.9, 124.9 (q, J=
272.1 Hz), 124.5, 123.9, 121.7, 117.4, 26.6 ppm.
1-acetyl-2-benzylidene-5-phenoxyindolin-3-one (10b): Purified
by flash chromatography (hexane/EtOAc=90:10). Obtained as
yellow oil, yield: 62%. Mixture of isomers Z/E=1:0.20. 1H NMR
(400 MHz, CDCl3, major isomer): d=8.30 (d, J=8.1 Hz, 1H), 7.56 (d,
J=7.5 Hz, 2H), 7.47–7.33 (m, 8H), 7.16 (t, J=7.4 Hz, 1H), 7.04–6.99
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(m, 2H), 1.98 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer):
d=186.0, 170.5, 155.1, 146.2, 135.5, 134.0, 131.2, 130.4, 130.2,
129.4, 127.4, 125.2, 124.1, 123.0, 120.3, 119.6, 119.3, 112.7,
25.1 ppm.
mp: 175–1768C. 1H NMR (400 MHz, [D6]DMSO): d=9.74 (s, 1H),
7.75 (d, J=8.4 Hz, 2H), 7.58 (d, J=7.5 Hz, 1H), 7.52 (t, J=7.5 Hz,
1H), 7.24 (d, J=8.0 Hz, 2H), 7.13 (d, J=7.5 Hz, 1H), 7.05–6.99 (m,
4H), 6.92 (t, J=7.5 Hz, 1H), 6.65 (s, 1H), 2.32 ppm (s, 3H); 13C NMR
(101 MHz, [D6]DMSO): d=186.1, 157.5, 154.1, 153.5, 136.2, 133.8,
133.3, 131.8, 130.5, 128.9, 124.1, 120.2, 119.6, 119.3, 118.1, 112.6,
109.7, 20.3 ppm.
1-acetyl-5-benzyl-2-benzylideneindolin-3-one (10c): Purified by
flash chromatography (hexane/EtOAc=90:10). Obtained as yellow
1
oil, yield: 65%. Mixture of isomers Z/E=1:0.45. H NMR (400 MHz,
CDCl3, major isomer): d=8.20 (d, J=8.1 Hz, 1H), 7.67 (s, 1H), 7.54–
7.51 (m, 2H), 7.47–7.36 (m, 3H), 7.32–7.15 (m, 7H), 4.04 (s, 2H),
1.98 ppm (s, 3H); 13C NMR (101 MHz, CDCl3, major isomer): d=
186.1, 170.5, 149.0, 140.4, 138.6, 137.3, 135.4, 134.0, 131.2, 130.3,
129.3, 129.0, 128.1, 126.6, 124.3, 124.1, 122.5, 118.1, 41.4, 25.2 ppm.
(Z)-2-(4-(4-chlorophenoxy)benzylidene)indolin-3-one (3 f): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
yield: 89%, mp: 192–1948C. H NMR (400 MHz, CDCl3): d=7.83 (d,
J=8.2 Hz, 2H), 7.62 (d, J=7.5 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.26
(d, J=8.1 Hz, 2H), 7.17 (d, J=7.5 Hz, 1H), 7.15–7.00 (m, 5H),
6.71 ppm (br, 2H); 13C NMR (101 MHz, CDCl3): d=186.0, 156.9,
154.0, 152.8, 136.1, 133.5, 133.2, 131.1, 130.6, 129.0, 124.0, 120.3,
119.3, 119.1, 117.9, 111.9, 110.6 ppm.
1
1-acetyl-2-benzylidene-6-phenoxyindolin-3-one (10d): Purified
by flash chromatography (hexane/EtOAc=90:10). Obtained as
yellow oil, yield: 61%. Mixture of isomers Z/E=1:0.30. 1H NMR
(400 MHz, CDCl3, major isomer): d=8.22 (d, J=8.3 Hz, 1H), 7.67–
7.61 (m, 5H), 7.53 (t, J=7.6 Hz, 2H), 7.31–7.12 (m, 6H), 2.01 ppm (s,
3H); 13C NMR (101 MHz, CDCl3, major isomer): d=185.9, 170.1,
149.1, 145.6, 136.7, 134.0, 130.9, 129.8, 129.7, 128.7, 127.7, 125.5,
123.9, 123.1, 121.2, 119.2, 118.3, 115.7, 26.0 ppm.
(Z)-2-(4-(4-(trifluoromethyl)phenoxy)benzylidene)indolin-3-one
(3g): Purified by TLC (toluene/EtOH=80:20). Obtained as orange
solid, yield: 91%, mp: 204–2068C. 1H NMR (400 MHz, CDCl3): d=
7.76 (d, J=8.3 Hz, 1H), 7.63–7.57 (m, 3H), 7.54–7.47 (m, 2H), 7.13–
7.10 (m, 3H), 7.02–6.97 (m, 2H), 6.98 (s, 1H), 6.79 ppm (br, 1H);
13C NMR (101 MHz, CDCl3): d=186.4, 156.1, 153.1, 153.3, 136.2,
135.1 (q, J=32.2 Hz), 130.8, 128.9, 127.3, 125.1, 124.1 (q, J=
270.9 Hz), 121.9, 120.9, 120.1, 118.6, 112.0, 110.7 ppm.
General procedure for the synthesis of azaaurone derivatives
3a–ab and 4a–d: To a solution of the proper acetylated azaaurone
derivative (0.25 mmol) in MeOH (2.5 mL) at room temperature was
added a solution of KOH 50% in water (375 mL). The mixture was
stirred for 45 min. After reaction completion, the reaction mixture
was neutralized with extracted with EtOAc. The organic layer was
dried with anhydrous Na2SO4 and concentrated under reduced
pressure to give the crude product.
(Z)-2-(4-(benzyloxy)benzylidene)indolin-3-one (3h): Purified by
TLC (toluene/EtOH=80:20). Obtained as orange solid, yield: 89%,
1
mp: 231–2338C. H NMR (400 MHz, CDCl3): d=7.76 (d, J=7.42 Hz,
1H), 7.52–7.35 (m, 8H), 7.05 (d, J=8.72 Hz, 2H), 7.01–6.95 (m, 2H),
6.87 (s, 1H), 6.74 (br, 1H), 5.12 ppm (br, 2H); 13C NMR (101 MHz,
CDCl3): d=186.0, 159.2, 153.1, 136.5, 136.0, 134.4, 131.3, 128.8,
128.3, 127.7, 127.6, 125.1, 122.2, 120.7, 115.8, 112.3, 112.2,
70.3 ppm.
(Z)-2-benzylideneindolin-3-one (3a): Purified by TLC (toluene/
EtOH=80:20). Obtained as orange solid, yield: 90%, mp: 189–
1918C. 1H NMR (400 MHz, CDCl3): d=7.75 (d, J=7.7 Hz, 1H), 7.56
(d, J=7.5 Hz, 2H), 7.50–7.43 (m, 3H), 7.34 (t, J=7.4 Hz, 1H), 7.01–
6.98 (m, 2H), 6.87 ppm (br, 2H); 13C NMR (101 MHz, CDCl3): d=
186.7, 153.3, 136.3, 135.5, 134.9, 129.6, 129.4, 128.7, 125.2, 121.9,
120.8, 112.1, 111.7 ppm.
(Z)-2-(biphenyl-4-ylmethylene)indolin-3-one (3i): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 94%, mp:
233–2358C. 1H NMR (400 MHz, CDCl3): d=7.77 (d, J=7.6 Hz, 1H),
7.70–7.62 (m, 6H), 7.51–7.45 (m, 3H), 7.38 (t, J=7.3 Hz, 1H), 7.04–
6.97 (m, 2H), 6.91 ppm (br, 2H); 13C NMR (101 MHz, CDCl3): d=
186.6, 153.2, 141.3, 140.2, 136.3, 135.5, 133.9, 130.1, 129.1, 128.0,
127.5, 127.1, 125.2, 121.9, 120.9, 112.2, 111.4 ppm.
(Z)-2-(4-bromobenzylidene)indolin-3-one (3b): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 91%, mp:
241–2438C. 1H NMR (400 MHz, [D6]DMSO): d=9.84 (s, 1H), 7.69–
764 (m, 4H), 7.69 (d, J=7.7 Hz, 1H), 7.53 (t, J=7.7 Hz, 1H), 7.13 (d,
J=7.7 Hz), 6.93 (t, J=7.7 Hz, 1H), 6.59 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=186.3, 154.2, 136.5, 134.8, 133.3, 131.8,
131.6, 124.2, 121.4, 119.8, 112.6, 108.3 ppm.
(Z)-2-((4’-fluorobiphenyl-4-yl)methylene)indolin-3-one (3j): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
1
yield: 91%, mp: 265–2678C. H NMR (400 MHz, [D6]DMSO): d=9.90
(s, 1H), 7.80 (d, J=7.6 Hz, 1H), 7.66–7.60 (m, 6H), 7.52 (t, J=7.6 Hz,
1H), 7.19 (d, J=7.8 Hz, 2H), 7.06–7.02 (m, 2H), 6.93 ppm (s, 1H);
13C NMR (101 MHz, [D6]DMSO): d=186.4, 162.6 (d, J=267.5 Hz),
154.1, 138.8, 136.4, 135.8, 134.5, 133.3, 130.6, 128.7 (d, J=8.2 Hz),
127.1, 124.2, 120.0, 118.9, 115.9 (d, J=22.2 Hz), 112.7, 109.3 ppm.
(Z)-2-(4-(dimethylamino)benzylidene)indolin-3-one (3c): Purified
by TLC (toluene/EtOH=80:20). Obtained as red solid, yield: 87%,
mp: 250–2528C. 1H NMR (400 MHz, CDCl3): d=7.76 (d, J=7.7 Hz,
1H), 7.49–7.43 (m, 3H), 7.02 (d, J=7.7 Hz, 1H), 6.95 (t, J=7.7 Hz,
1H), 6.90 (s, 1H), 6.77–6.75 ppm (m, 3H); 13C NMR (101 MHz,
CDCl3): d=186.2, 153.0, 135.4, 133.1, 131.6, 124.9, 122.5, 120.4,
118.4, 117.1, 114.3, 122.3, 112.2, 40.3 ppm.
(Z)-2-((4’-methylbiphenyl-4-yl)methylene)indolin-3-one (3k): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
1
yield: 92%, mp: 169–1718C. H NMR (400 MHz, CDCl3): d=7.77 (d,
1H, J=7.7 Hz, 1H), 7.68 (d, J=8.1 Hz, 2H), 7.62 (d, J=8.1 Hz, 2H),
7.56–7.47 (m, 3H), 7.28 (d, J=8.0 Hz, 2H), 7.03–6.97 (m, 2H), 6.91
(s, 1H), 6.88 (br, 1H), 2.41 ppm (s, 3H); 13C NMR (101 MHz, CDCl3):
d=186.1, 153.2, 147.9, 137.9, 137.3, 136.2, 135.4, 133.6, 130.1,
129.8, 127.7, 127.0, 125.2, 120.9, 112.1, 11.6, 21.1 ppm.
(Z)-2-(4-phenoxybenzylidene)indolin-3-one (3d): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 93%, mp:
209–2118C. 1H NMR (400 MHz, CDCl3): d=7.78 (d, J=7.6 Hz, 1H),
7.55 (d, J=8.7 Hz, 2H), 7.50 (t, J=7.5 Hz, 1H), 7.43–7.39 (m, 2H),
7.20 (t, J=7.6 Hz, 1H), 7.10–7.08 (m, 4H), 7.03–7.01 (m, 2H), 6.89 (s,
1H), 6.78 ppm (br, 1H); 13C NMR (101 MHz, CDCl3): d=186.1, 153.2,
136.2, 131.3, 130.1, 125.2, 124.2, 120.9, 119.7, 119.1, 112.2,
111.5 ppm.
(Z)-2-((4’-chlorobiphenyl-4-yl)methylene)indolin-3-one (3l): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
1
yield: 87%, mp: 259–2618C. H NMR (400 MHz, [D6]DMSO): d=9.92
(s, 1H), 7.85–7.78 (m, 5H), 7.71–7.52 (m, 5H), 7.16 (d, J=8.1 Hz,
1H), 6.94 (t, J=8.1 Hz, 1H), 6.68 ppm (s, 1H); 13C NMR (101 MHz,
(Z)-2-(4-(p-tolyloxy)benzylidene)indolin-3-one (3e): Purified by
TLC (toluene/EtOH=80:20). Obtained as orange solid, yield: 92%,
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[D6]DMSO): d=186.3, 154.1, 138.4, 138.1, 136.4, 134.5, 133.7, 132.7,
130.6, 129.0, 128.4, 127.0, 124.2, 120.0, 119.9, 112.7, 109.2 ppm.
(Z)-2-((4’-fluorobiphenyl-3-yl)methylene)indolin-3-one (3t): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
yield: 95%, mp: 297–2998C. H NMR (400 MHz, [D6]DMSO): d=8.98
1
(Z)-2-((4’-(trifluoromethyl)biphenyl-4-yl)methylene)indolin-3-one
(3m): Purified by TLC (toluene/EtOH=80:20). Obtained as orange
solid, yield: 90%, mp: 289–2918C. 1H NMR (400 MHz, [D6]DMSO):
d=9.95 (s, 1H), 7.99 (d, J=8.1 Hz, 2H), 7.87–7.84 (m, 6H), 7.60 (d,
J=7.6 Hz, 1H), 7.55 (d, J=7.6 Hz, 1H), 7.17 (d, J=7.6 Hz, 1H), 6.94
(t, J=7.6 Hz, 1H), 6.70 ppm (s, 1H); 13C NMR (101 MHz, [D6]DMSO):
d=186.4, 154.2, 143.3, 138.0, 136.5, 134.7, 134.4 (q, J=32.3 Hz),
130.6, 127.5, 127.5, 125.9, 124.2 (q, J=270.4 Hz), 120.0, 119.9, 112.7,
108.9 ppm.
(s, 1H), 7.95 (s, 1H), 7.84–7.80 (m, 2H), 7.74 (d, J=7.6 Hz, 1H),
7.64–7.52 (m, 4H), 7.36–7.32 (m, 2H), 7.16 (d, J=7.6 Hz, 1H), 6.95
(t, 1H, J=7.6 Hz), 6.73 ppm (s, 1H); 13C NMR (101 MHz, [D6]DMSO):
d=186.4, 162.1 (d, J=268.1 Hz), 154.3, 139.93, 136.5, 136.2, 123.8
(d, J=22.1 Hz), 134.7, 129.6, 129.1, 128.7, 128.2, 126.8, 124.2, 120.1,
119.9, 115.6 (d, J=22.1 Hz), 112.7, 109.7 ppm.
(Z)-2-((4’-methylbiphenyl-3-yl)methylene)indolin-3-one (3u): Pu-
rified by TLC (toluene/EtOH=80:20). Obtained as orange solid,
1
yield: 94%, mp: 201–2028C. H NMR (400 MHz, [D6]DMSO): d=9.88
(Z)-2-((4’-(trifluoromethoxy)biphenyl-4-yl)methylene)indolin-3-
one (3n): Purified by TLC (toluene/EtOH=80:20). Obtained as
orange solid, yield: 92%, mp: 280–2828C. 1H NMR (400 MHz,
[D6]DMSO): d=9.92 (s, 1H), 7.89–7.79 (m, 7H), 7.61–7.46 (m, 3H),
7.16 (d, J=7.8 Hz, 1H), 6.94 (t, J=7.8 Hz, 1H), 6.69 ppm (s, 1H);
13C NMR (101 MHz, [D6]DMSO): d=186.3, 154.1, 152.2, 149.1, 148.1,
138.6, 136.4, 134.6, 133.8, 130.6 (q, J=253.7 Hz), 128.6, 127.3,
126.5, 124.2, 121.6, 120.0, 112.6, 109.1 ppm.
(s, 1H), 7.91 (s, 1H), 7.71 (d, J=7.6 Hz, 1H), 7.65–7.52 (m, 6H), 7.30
(d, J=7.8 Hz, 2H), 7.12 (d, J=7.6 Hz, 1H), 6.94 (t, J=7.6 Hz, 1H),
6.72 (s, 1H), 2.35 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=
186.6, 154.4, 141.0, 137.2, 136.9, 136.6, 134.8, 134.8, 129.7, 128.5,
128.2, 127.0, 126.8, 124.3, 120.2, 120.0, 112.8, 110.1, 20.8 ppm.
(Z)-2-((4’-chlorobiphenyl-3-yl)methylene)indolin-3-one (3v): Puri-
fied by TLC (toluene/EtOH=80:20). Obtained as orange solid,
1
yield: 90%, mp: 189–1918C. H NMR (400 MHz, [D6]DMSO): d=9.89
(Z)-2-(4-benzylbenzylidene)indolin-3-one (3o): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 91%, mp:
194–1968C. 1H NMR (400 MHz, CDCl3): d=7.75 (d, J=7.6 Hz, 1H),
7.47–7.45 (m, 3H), 7.13–7.19 (m, 7H), 6.99–6.95 (m, 2H), 6.86 (s,
1H), 6.82 (br, 1H), 4.01 ppm (s, 2H); 13C NMR (101 MHz, CDCl3): d=
186.6, 153.2, 142.1, 140.6, 136.2, 135.3, 132.7, 129.9, 129.8, 129.1,
128.7, 126.5, 125.2, 121.9, 120.9, 112.1, 111.8, 41.9 ppm.
(s, 1H), 7.97 (s, 1H), 7.82–7.73 (m, 4H), 7.67–7.52 (m, 5H), 7.16 (d,
J=7.6 Hz, 1H), 6.94 (t, J=7.6 Hz, 1H), 6.73 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=186.1, 154.3, 139.6, 138.6, 136.5, 134.9,
134.7, 132.6, 129.6, 129.0, 128.9, 128.8, 128.3, 126.8, 124.2, 119.9,
112.7, 109.6 ppm.
(Z)-2-((4’-(trifluoromethyl)biphenyl-3-yl)methylene)indolin-3-one
(3w): Purified by TLC (toluene/EtOH=80:20). Obtained as orange
solid, yield: 91%, mp: 228–2308C. 1H NMR (400 MHz, CDCl3): d=
7.76 (d, J=7.7 Hz, 1H), 7.69 (s, 1H), 7.62 (d, J=8.5 Hz, 2H), 7.55–
7.47 (m, 4H), 7.33 (d, J=8.5 Hz, 2H), 7.02–6.97 (m, 2H), 6.91 (s,
1H), 6.88 ppm (br, 1H); 13C NMR (101 MHz, CDCl3): d=186.5, 164.9,
153.1, 149.0, 140.9, 139.2, 136.3, 135.7, 135.4 (q, J=32.1 Hz), 129.8,
128.6, 128.4, 128.3, 127.3, 125.1 (q, J=272.1 Hz), 121.7, 121.4,
120.9, 112.0, 111.0 ppm.
(Z)-2-(4-(quinolin-3-yl)benzylidene)indolin-3-one (3p): Purified by
TLC (toluene/EtOH=80:20). Obtained as orange solid, yield: 97%,
1
mp: 271–2738C. H NMR (400 MHz, CDCl3): d=9.22 (s, 1H), 8.37 (s,
1H), 8.17 (d, J=7.9 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 7.83–7.71 (m,
6H), 7.62 (t, J=7.6 Hz, 1H), 7.51 (t, J=7.6 Hz, 1H), 7.05–6.98 (m,
3H), 6.92 ppm (s, 1H); 13C NMR (101 MHz, CDCl3): d=186.3, 153.3,
149.5, 138.8, 136.4, 134.8, 133.6, 130.4, 130.0, 129.2, 128.2, 127.5,
125.3, 121.9, 121.1, 112.2, 110.7 ppm.
(Z)-2-((4’-(trifluoromethoxy)biphenyl-3-yl)methylene)indolin-3-
one (3x): Purified by TLC (toluene/EtOH=80:20). Obtained as
orange solid, yield: 92%, mp: 223–2258C. 1H NMR (400 MHz,
[D6]DMSO): d=9.92 (s, 1H), 7.93 (s, 1H), 7.84–7.70 (m, 5H), 7.65–
7.43 (m, 5H), 6.98 (t, J=7.6 Hz, 1H), 6.78 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=186.4, 153.0, 144.8, 141.8, 141.5, 139.3,
135.3, 129.6 (q, J=252.9 Hz), 128.2, 127.3, 126.9, 126.1, 125.1,
123.1, 121.8, 121.2, 121.0, 120.2, 114.2, 109.6 ppm.
(Z)-2-(4-(thiophen-3-yl)benzylidene)indolin-3-one (3q): Purified
by TLC (toluene/EtOH=80:20). Obtained as orange solid, yield:
93%, mp: 255–2578C. 1H NMR (400 MHz, [D6]DMSO): d=9.88 (s,
1H), 8.03 (s, 1H), 7.84 (d, J=8.3 Hz, 2H), 7.78 (d, J=8.3 Hz, 2H),
7.67 (s, 2H), 7.59 (d, J=7.6 Hz, 1H), 7.53 (t, J=7.6 Hz, 1H), 7.16 (d,
J=7.6 Hz, 1H), 6.93 (t, J=7.6 Hz, 1H), 6.67 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=186.2, 154.0, 140.8, 136.3, 134.9, 134.2,
132.9, 130.6, 127.4, 126.5, 126.2, 124.1, 121.9, 120.0, 119.8, 112.6,
109.6 ppm.
(Z)-2-(3-benzylbenzylidene)indolin-3-one (3y): Purified by TLC
1
(toluene/EtOH=80:20). Obtained as orange oil, yield: 96%. H NMR
(Z)-2-(4-(1-methyl-1H-pyrazol-4-yl)benzylidene)indolin-3-one
(3r): Purified by TLC (toluene/EtOH=80:20). Obtained as orange
solid, yield: 92%, mp: 247–2498C. 1H NMR (400 MHz, [D6]DMSO):
d=9.82 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.73 (d, J=8.1 Hz, 2H),
7.67 (d, J=8.1 Hz, 2H), 7.59 (d, J=7.5 Hz, 1H), 7.52 (t, J=7.5 Hz,
1H), 7.15 (d, J=7.5 Hz, 1H), 6.92 (t, J=7.4 Hz, 1H), 6.65 (s, 1H),
3.88 ppm (s, 3H); 13C NMR (101 MHz, [D6]DMSO): d=186.7, 153.4,
136.7, 136.1, 135.9, 135.4, 133.5, 130.4, 128.0, 125.8, 125.0, 123.8,
121.8, 119.4, 112.4, 109.8, 38.5 ppm.
(400 MHz, CDCl3): d=7.73 (d, J=7.7 Hz, 1H, H4), 7.47 (t, J=7.7 Hz,
1H), 7.38–7.31 (m, 4H), 7.27–7.17 (m, 5H), 6.98–6.93 (m, 2H), 6.82
(s, 1H), 6.71 (br, 1H), 4.04 ppm (s, 2H); 13C NMR (101 MHz, CDCl3):
d=186.6, 153.2, 142.4, 140.7, 136.2, 135.5, 135.1, 130.2, 129.5,
129.3, 129.2, 128.8, 127.3, 126.5, 125.2, 121.9, 120.8, 112.0, 111.7,
41.8 ppm.
(Z)-2-(3-(quinolin-3-yl)benzylidene)indolin-3-one (3z): Purified by
TLC (toluene/EtOH=80:20). Obtained as orange solid, yield: 94%,
mp: 266–2688C. 1H NMR (400 MHz, [D6]DMSO): d=9.95 (s, 1H),
9.37 (s, 1H), 8.76 (s, 1H), 8.20 (s, 1H), 8.09 (d, J=8.1 Hz, 2H), 7.89–
7.79 (m, 3H), 7.70–7.61 (m, 3H), 7.55 (t, J=7.7 Hz, 1H), 7.17 (d, J=
7.7 Hz, 1H), 6.95 (d, J=7.7 Hz, 1H), 6.78 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=186.4, 154.3, 149.7, 146.9, 137.9, 136.5,
135.1, 134.8, 133.3, 132.4, 129.8, 129.7, 129.4, 128.7, 128.5, 127.6,
127.2, 127.1, 124.2, 120.0, 119.9, 112.7, 109.5 ppm.
(Z)-2-(biphenyl-3-ylmethylene)indolin-3-one (3s): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 93%, mp:
1
173–1758C. H NMR (400 MHz, [D6]DMSO): d=9.90 (s, 1H), 8.00 (s,
1H), 7.77–7.74 (m, 3H), 7.76–7.49 (m, 6H), 7.40 (t, J=7.5 Hz, 1H),
7.16 (d, J=7.6 Hz, 1H), 6.93 (t, J=7.6 Hz, 1H), 6.74 ppm (s, 1H);
13C NMR (101 MHz, [D6]DMSO): d=186.5, 153.2, 141.4, 140.2, 136.2,
135.5, 133.5, 130.1, 129.3, 128.7, 128.48, 128.1, 126.8, 126.6, 123.9,
121.8, 119.6, 112.4, 109.6 ppm.
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Full Papers
(Z)-2-(3-(thiophen-3-yl)benzylidene)indolin-3-one (3aa): Purified
by TLC (toluene/EtOH=80:20). Obtained as orange solid, yield:
93%, mp: 202–2048C. 1H NMR (400 MHz, [D6]DMSO): d=9.88 (s,
1H), 8.02–8.01 (m, 2H), 7.72–7.68 (m, 4H), 7.60 (d, J=7.8 Hz, 1H),
7.56–7.50 (m, 2H), 7.16 (d, J=7.8 Hz, 1H), 6.94 (t, J=7.8 Hz, 1H),
6.71 ppm (s, 1H); 13C NMR (101 MHz, [D6]DMSO): d=186.2, 154.0,
140.8, 136.3, 134.9, 134.2, 132.9, 129.3, 128.1, 127.6, 126.9, 126.3,
126.0, 124.0, 121.5, 120.0, 119.7, 112.5, 109.6 ppm.
anisotropic thermal parameters. All the hydrogen atoms were in-
serted in idealized positions and allowed to refine in the parent
carbon or oxygen atom. OLEX2.1.2.6[32] was used for packing dia-
grams. CCDC 1419162 contains the supplementary crystallographic
data for this paper. These data are provided free of charge by The
Cambridge Crystallographic Data Centre. Further details are provid-
ed in the Supporting Information, Table S2.
(Z)-2-(3-(1-methyl-1H-pyrazol-4-yl)benzylidene)indolin-3-one
(5ab): Purified by TLC (toluene/EtOH=80:20). Obtained as orange
solid, yield: 94%, mp: 212–2138C. 1H NMR (400 MHz, CDCl3): d=
7.80 (s, 1H), 7.75 (d, J=7.8 Hz, 1H), 7.66 (s, 1H), 7.62 (s, 1H), 7.47
(t, J=7.8 Hz, 1H), 7.43–7.39 (m, 3H), 7.02 (d, J=7.8 Hz, 1H), 6.97 (t,
J=7.8 Hz, 1H), 6.86 (br, 2H), 3.94 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=186.8, 153.4, 136.6, 136.3, 135.7, 135.5, 133.5, 129.8,
127.5, 127.4, 126.8, 125.8, 125.2, 122.7, 121.8, 120.8, 112.2, 111.4,
39.3 ppm.
Biological assays
Activity against erythrocyte-stage P. falciparum: Drug assays
were performed as previously described,[33] with modifications for
384-well format. Briefly, synchronized ring-stage parasites were cul-
tured in the presence of 12-point two-fold serial dilutions of test
compounds in 40 mL RPMI supplemented with 0.5% AlbuMAXꢅ II
at 1.0% hematocrit and an initial parasitemia of 1.0% in black
clear-bottom plates (Greiner Bio-one 781090). Following 72 h incu-
bation under standard culture conditions, SYBR Green I dye (Invi-
trogen S7563) was added to a dilution of 1:5000, and plates were
stored at room temperature until fluorescence signal was read on
(Z)-2-benzylidene-5-phenylindolin-3-one (4a): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 91%, mp:
1
234–2368C. H NMR (400 MHz, CDCl3): d=8.00 (s, 1H), 7.75 (d, J=
a Spectramax M5 plate reader (Molecular Devices, lex: 480 nm, lem
:
8.1 Hz, 1H), 7.59–7.57 (m, 4H), 7.49–7.42 (m, 4H), 7.38–7.32 (m,
2H), 7.08 (d, J=8.1 Hz), 6.91 ppm (br, 2H); 13C NMR (101 MHz,
CDCl3): d=186.7, 152.5, 140.2, 135.8, 135.4, 134.82 134.3, 129.7,
129.4, 129.0, 128.8, 127.3, 126.8, 123.3, 122.3, 112.4, 112.1 ppm.
530 nm). After background subtraction and normalization, EC50
values were calculated using a nonlinear regression curve fit imple-
mented in the GraphPad Prism software package v. 5.00 (GraphPad
Software, CA, USA).
(Z)-2-benzylidene-5-phenoxyindolin-3-one (4b): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 91%, mp:
212–2148C. 1H NMR (400 MHz, CDCl3): d=7.82 (s, 1H), 7.77–7.68
(m, 4H), 7.48–7.27 (m, 7H), 7.03 (d, J=7.6 Hz, 1H), 6.85 ppm (br,
2H); 13C NMR (101 MHz, CDCl3): d=186.5, 152.0, 141.0, 136.0,
135.3, 134.1, 133.0, 129.0, 128.9, 128.9, 128.3, 128.0, 126.2, 124.1,
122.4, 112.0, 111.0 ppm.
Activity against liver-stage P. berghei: Huh7 cells, a human hepa-
toma cell line, were cultured in RPMI 1640 medium supplemented
with 10% v/v fetal calf serum (FCS), 1% v/v non-essential amino
acids, 1% v/v penicillin/streptomycin, 1% v/v glutamine, and
10 mm HEPES, pH 7, and maintained at 378C with 5% CO2. Inhibi-
tion of P. berghei liver stage infection was determined by measur-
ing the luminescence of Huh7 cell lysates 48 h after infection with
a firefly-luciferase-expressing P. berghei line, PbGFP-Luccon, as pre-
viously described.[34] Briefly, cells (1.2ꢆ104 per well) were seeded in
96-well plates the day before drug treatment and infection. Tested
compounds were prepared in the following way: 10 mm stock sol-
utions were obtained by dissolving accurately weighed com-
pounds in MeOH and dilutions subsequently made with medium
to the desired concentration. Medium was replaced by fresh
medium containing the appropriate concentration of each com-
pound 1 h prior to infection. Sporozoites (10000 per well), freshly
obtained through disruption of salivary glands of infected female
Anopheles stephensi mosquitoes, were added to the wells 1 h after
compound addition. Sporozoite addition was followed by centrifu-
gation at 1700 g for 5 min. At 24 h post-infection, medium was
again replaced by fresh medium containing the appropriate con-
centration of each compound. Parasite load was determined 48 h
after infection by luminescence measurement using a Firefly Luci-
ferase Assay Kit (Biotium). The effect of the compounds on the via-
bility of Huh7 cells was assessed by the Alamar Blue assay (Invitro-
gen, UK) using the manufacturer’s protocol. Nonlinear regression
analysis was employed to fit the normalized results of the dose–re-
sponse curves, and EC50 values, determined using SigmaPlot soft-
ware v. 12.0 (Systat Software, San Jose, CA, USA), are the mean
value averaged from three independent experiments, each per-
formed in triplicate wells.
(Z)-5-benzyl-2-benzylideneindolin-3-one (4c): Purified by TLC (tol-
uene/EtOH=80:20). Obtained as orange solid, yield: 92%, mp:
1
203–2058C. H NMR (400 MHz, CDCl3): d=7.61 (s, 1H), 7.57 (d, J=
7.9 Hz, 2H), 7.47 (t, J=7.9 Hz, 2H), 7.38–7.20 (m, 7H), 6.94 (d, J=
7.6 Hz, 1H), 6.87 (s, 1H), 6.79 (br, 1H), 3.98 ppm (s, 2H); 13C NMR
(101 MHz, CDCl3): d=186.7, 152.0, 140.7, 137.3, 135.9, 134.9, 134.0,
129.6, 129.3, 129.0, 128.7, 128.6, 126.4, 125.0, 122.1, 112.2, 111.7,
41.3 ppm.
(Z)-2-benzylidene-6-phenoxyindolin-3-one (4d): Purified by TLC
(toluene/EtOH=80:20). Obtained as orange solid, yield: 89%, mp:
216–218. 1H NMR (400 MHz, CDCl3): d=8.02 (d, J=8.3 Hz, 1H),
7.57–7.43 (m, 5H), 7.53 (t, J=7.6 Hz, 2H), 7.36–7.24 (m, 5H), 6.91 (s,
1H), 6.85 ppm (br, 1H); 13C NMR (101 MHz, CDCl3): d=186.5, 150.4,
144.3, 137.0, 134.9, 131.0, 130.0, 129.2, 128.2, 127.3, 125.1, 124.0,
122.9, 121.0, 118.8, 112.0, 110.8 ppm.
Solid-state characterization of compound 3j: Crystals of 3j suita-
ble for X-ray diffraction study were mounted with Fomblinꢃ in
a cryoloop. Data were collected on a Bruker AXS-KAPPA APEX II dif-
fractometer with graphite-monochromated radiation (MoKa, l=
0.17073 ꢄ) at 150 K. The X-ray generator was operated at 50 kV
and 30 mA, and X-ray data collection was monitored by the
APEX2[28] program v. 2014.11-0. All data were corrected for Lorent-
zian, polarization, and absorption effects using SAINT[28] (v. 8.34A)
and SADABS[28] (v. 2014/5) programs. SIR97[29] and SHELXS-97[30]
were used for structure solution, and SHELXL-97 was used for full
matrix least-squares refinement on F2. These three programs are in-
cluded in the package of programs WINGX-Version 1.80.05.[31] Non-
hydrogen atoms were refined anisotropically. A full-matrix least-
squares refinement was used for the non-hydrogen atoms with
In vitro cytotoxicity: Cytotoxicity was assessed using the 3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
assay.[35] Briefly, HEK293T (human embryonic kidney epithelial cell
line, ATCC CRL-11268) cells were seeded in 96-well tissue culture
plates one day before the experiment, in RPMI 1640 culture
medium supplemented with 10% fetal bovine serum, 2 mm l-glu-
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Full Papers
[13] Atta-Ur-Rahman, M. I. Choudhary, S. Hayat, A. M. Khan, A. Ahmed,
Chem. Pharm. Bull. 2001, 49, 105–107.
tamine, 100 U penicillin G (sodium salt) and 100 mg streptomycin
sulfate, at a concentration allowing exponential growth during the
assay. Compounds were diluted in DMSO, serially diluted in culture
medium, and added to cells incubated at 378C in humidified 5%
CO2 atmosphere. After 48 h, culture media was removed and re-
placed with fresh medium containing MTT dye. After 3 h of incuba-
tion the complete media was removed and intracellular formazan
crystals were solubilized and extracted with DMSO. After 15 min at
room temperature absorbance was measured at 570 nm in a micro-
plate reader. The percentage of cell viability was determined for
each tested compound as described previously.[35] EC50 values were
determined from concentration–response curves using nonlinear
regression with GraphPad Prism software package v. 5.02 (Graph-
Pad Software, CA, USA).
[14] a) F. P. da Cruz, C. Martin, K. Buchholz, M. J. Lafuente-Monasterio, T. Ro-
drigues, B. Sonnichsen, R. Moreira, F. J. Gamo, M. Marti, M. M. Mota, M.
Hannus, M. Prudencio, J. Infect. Dis. 2012, 205, 1278–1286; b) T. G. Nam,
C. W. McNamara, S. Bopp, N. V. Dharia, S. Meister, G. M. C. Bonamy, D. M.
Plouffe, N. Kato, S. McCormack, B. Bursulaya, H. J. Ke, A. B. Vaidya, P. G.
Schultz, E. A. Winzeler, ACS Chem. Biol. 2011, 6, 1214–1222; c) R.
Cowley, S. Leung, N. Fisher, M. Al-Helal, N. G. Berry, A. S. Lawrenson, R.
Sharma, A. E. Shone, S. A. Ward, G. A. Biagini, P. M. O’Neill, MedChem-
Comm 2012, 3, 39–44.
[15] E. R. Derbyshire, M. Prudencio, M. M. Mota, J. Clardy, Proc. Natl. Acad.
Sci. USA 2012, 109, 8511–8516.
[16] C. K. Dong, S. Urgaonkar, J. F. Cortese, F. J. Gamo, J. F. Garcia-Bustos,
M. J. Lafuente, V. Patel, L. Ross, B. I. Coleman, E. R. Derbyshire, C. B.
Clish, A. E. Serrano, M. Cromwell, R. H. Barker, J. D. Dvorin, M. T. Durai-
singh, D. F. Wirth, J. Clardy, R. Mazitschek, Chem. Biol. 2011, 18, 1602–
1610.
Abbreviations: ACTs: artemisinin-based combination therapies,
EEFs: exoerythrocytic forms, mtETC: mitochondrial electron-trans-
port chain, DHODH: dihydroorotate dehydrogenase.
[17] H. J. Painter, J. M. Morrisey, M. W. Mather, A. B. Vaidya, Nature 2007, 446,
88–91.
[18] M. M. M. Santos, R. Moreira, Mini-Rev. Med. Chem. 2007, 7, 1040–1050.
[19] J. E. Olson, G. K. Lee, A. Semenov, P. J. Rosenthal, Bioorg. Med. Chem.
1999, 7, 633–638.
Acknowledgements
[20] a) P. J. Rosenthal, Adv. Exp. Med. Biol. 2011, 712, 30–48; b) E. L. Dahl, P. J.
Rosenthal, Mol. Biochem. Parasitol. 2005, 139, 205–212; c) C. Teixeira,
J. R. B. Gomes, P. Gomes, Curr. Med. Chem. 2011, 18, 1555–1572.
[21] T. Tomasic, L. P. Masic, Expert Opin. Drug Discovery 2012, 7, 549–560.
[22] S. Praveen Kumar, J. Gut, R. C. Guedes, P. J. Rosenthal, M. M. M. Santos,
R. Moreira, Eur. J. Med. Chem. 2011, 46, 927–933.
[23] S. Holton, A. Merckx, D. Burgess, C. Doerig, M. Noble, J. Endicott, Struc-
ture 2003, 11, 1329–1337.
[24] J. B. Baell, G. A. Holloway, J. Med. Chem. 2010, 53, 2719–2740.
[25] J. B. Baell, L. Ferrins, H. Falk, G. Nikolakopoulos, Aust. J. Chem. 2013, 66,
1483–1494.
[26] L. A. Dakin, M. H. Block, H. W. Chen, E. Code, J. E. Dowling, X. M. Feng,
A. D. Ferguson, I. Green, A. W. Hird, T. Howard, E. K. Keeton, M. L. Lamb,
P. D. Lyne, H. Pollard, J. Read, A. J. Wu, T. Zhang, X. L. Zheng, Bioorg.
Med. Chem. Lett. 2012, 22, 4599–4604.
[27] a) V. Pomel, J. Klicic, D. Covini, D. D. Church, J. P. Shaw, K. Roulin, F.
Burgat-Charvillon, D. Valognes, M. Camps, C. Chabert, C. Gillieron, B.
Francon, D. Perrin, D. Leroy, D. Gretener, A. Nichols, P. A. Vitte, S. Car-
boni, C. Rommel, M. K. Schwarz, T. Ruckle, J. Med. Chem. 2006, 49,
3857–3871; b) A. Mital, D. Murugesan, M. Kaiser, C. Yeates, I. H. Gilbert,
Eur. J. Med. Chem. 2015, 103, 530–538.
[28] Bruker Analytical Systems, Madison, WI (USA), 2005: https://www.bru-
ker.com/ (accessed August 10, 2016).
[29] A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giacovazzo, A.
Guagliardi, A. G. G. Moliterni, G. Polidori, R. Spagna, J. Appl. Crystallogr.
1999, 32, 115–119.
This work was supported financially by the Fundażo para a CiÞn-
cia e a Tecnologia (FCT, Portugal) through projects PTDC/SAU-
FCF/098734/2008 and UID/DTP/04138/2013. FCT is also acknowl-
edged for grants SFRH/BD/61611/2009 (to M.P.C.) and SFRH/BPD/
94692/2013 (to M.S.). We thank Dr. Jo¼o Lavrado for technical
assistance and support in preparing the manuscript.
Keywords: antiprotozoal agents · azaaurones · erythrocytic
stage · liver stage · malaria
[1] World Malaria Report 2014, World Health Organization: http://
www.who.int/malaria/publications/world_malaria_report_2014/en/ (ac-
cessed August 10, 2016).
[2] a) R. N. Price, N. M. Douglas, N. M. Anstey, Curr. Opin. Infect. Dis. 2009,
22, 430–435; b) I. Mueller, M. R. Galinski, J. K. Baird, J. M. Carlton, D. K.
Kochar, P. L. Alonso, H. A. del Portillo, Lancet Infect. Dis. 2009, 9, 555–
566.
[3] a) D. A. Fidock, R. T. Eastman, S. A. Ward, S. R. Meshnick, Trends Parasitol.
2008, 24, 537–544; b) R. Arav-Boger, T. A. Shapiro, Annu. Rev. Pharma-
col. 2005, 45, 565–585.
[4] E. R. Derbyshire, M. M. Mota, J. Clardy, PLoS Pathog. 2011, 7, e1002178.
[5] D. Mazier, L. Renia, G. Snounou, Nat. Rev. Drug Discovery 2009, 8, 854–
864.
[6] R. Haudecoeur, A. Boumendjel, Curr. Med. Chem. 2012, 19, 2861–2875.
[7] M. P. Carrasco, A. S. Newton, L. Goncalves, A. Gois, M. Machado, J. Gut,
F. Nogueira, T. Hanscheid, R. C. Guedes, D. J. V. A. dos Santos, P. J. Rosen-
thal, R. Moreira, Eur. J. Med. Chem. 2014, 80, 523–534.
[8] a) O. Kayser, A. F. Kiderlen, R. Brun, Planta Med. 2001, 67, 718–721; b) F.
Souard, S. Okombi, C. Beney, S. Chevalley, A. Valentin, A. Boumendjel,
Bioorg. Med. Chem. 2010, 18, 5724–5731.
[9] C. A. B. Wager, S. A. Miller, J. Labelled Compd. Radiopharm. 2006, 49,
615–622.
[10] M. A. Lawson, A. M. Mariotte, A. N. Boumendjel, Heterocycl. Commun.
2003, 9, 149–152.
[30] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112–122.
[31] L. Farrugia, J. Appl. Crystallogr. 1999, 32, 837–838.
[32] O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, H. Pusch-
mann, J. Appl. Crystallogr. 2009, 42, 339–341.
[33] J. D. Johnson, R. A. Dennull, L. Gerena, M. Lopez-Sanchez, N. E. Roncal,
N. C. Waters, Antimicrob. Agents Chemother. 2007, 51, 1926–1933.
[34] a) I. H. J. Ploemen, M. Prudencio, B. G. Douradinha, J. Ramesar, J. Fonag-
er, G.-J. van Gemert, A. J. F. Luty, C. C. Hermsen, R. W. Sauerwein, F. G.
Baptista, M. M. Mota, A. P. Waters, I. Que, C. W. G. M. Lowik, S. M. Khan,
C. J. Janse, B. M. D. Franke-Fayard, PLoS One 2009, 4, e7881; b) M. Pru-
dÞncio, M. M. Mota, A. M. Mendes, Trends Parasitol. 2011, 27, 565–574.
[35] a) S. P. Kumar, P. M. C. Gloria, L. M. Goncalves, J. Gut, P. J. Rosenthal, R.
Moreira, M. M. M. Santos, MedChemComm 2012, 3, 489–493; b) S. D.
Lucas, L. M. Goncalves, T. A. F. Cardote, H. F. Correia, R. Moreira, R. C.
Guedes, MedChemComm 2012, 3, 1299–1304.
[11] S. C. Leung, P. Gibbons, R. Amewu, G. L. Nixon, C. Pidathala, W. D. Hong,
B. Pacorel, N. G. Berry, R. Sharma, P. A. Stocks, A. Srivastava, A. E. Shone,
S. Charoensutthivarakul, L. Taylor, O. Berger, A. Mbekeani, A. Hill, N. E.
Fisher, A. J. Warman, G. A. Biagini, S. A. Ward, P. M. O’Neill, J. Med. Chem.
2012, 55, 1844–1857.
[12] Q. S. Liu, J. W. Chang, J. H. Wang, S. A. Kang, C. C. Thoreen, A. Markhard,
W. Hur, J. M. Zhang, T. Sim, D. M. Sabatini, N. S. Gray, J. Med. Chem.
2010, 53, 7146–7155.
Received: June 28, 2016
Published online on && &&, 0000
ChemMedChem 2016, 11, 1 – 12
www.chemmedchem.org
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ꢃ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
M. P. Carrasco,* M. Machado,
L. GonÅalves, M. Sharma, J. Gut,
A. K. Lukens, D. F. Wirth, V. Andrꢀ,
M. T. Duarte, R. C. Guedes,
D. J. V. A. d. . Santos, P. J. Rosenthal,
R. Mazitschek, M. PrudÞncio,* R. Moreira
&& – &&
Once bitten, twice potent: Novel syn-
thetic azaaurone derivatives were syn-
thesized and screened against the
blood stage of a chloroquine-resistant P.
falciparum strain and the liver stage of
P. berghei. Our report shows several
compounds with dual-stage activity and
nanomolar potency against erythrocytic
parasite, indicating that the azaaurone
chemotype is a promising scaffold for
dual-stage antimalarial agents.
Probing the Azaaurone Scaffold
against the Hepatic and Erythrocytic
Stages of Malaria Parasites
&
ChemMedChem 2016, 11, 1 – 12
www.chemmedchem.org
12
ꢃ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!