1-Azaaurones derived from the naturally occurring aurones as potential antimalarial drugs

Article abstract of DOI:10.1016/j.bmc.2010.06.008

We report the synthesis and in vitro antiplasmodial activity of 35 compounds, designed as analogues of the naturally occurring aurones. Several of these analogues showed submicromolar antimalarial activity against a chloroquine-resistant strain of Plasmodium falciparum (FcB1-Columbia strain) cultured on human erythrocytes. Substitution of the intracyclic oxygen in aurones by a nitrogen atom and systematic variation of the substituent at the B-ring revealed promising leads showing good activity on the CQ-resistant strain. In particular, 4,6-dimethoxy-4′-ethylazaaurone 22 showed antiplasmodial potency without noticeable toxicity. The easy synthesis of this family of compounds and the relevant antiplasmodial activity are in favor of promising candidates for further development.

Full text of DOI:10.1016/j.bmc.2010.06.008

Bioorganic & Medicinal Chemistry 18 (2010) 5724–5731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-Azaaurones derived from the naturally occurring aurones as potential
antimalarial drugs
a
a
b
b,
Florence Souard a, , Sabrina Okombi , Chantal Beney , Séverine Chevalley , Alexis Valentin
,
*
Ahcène Boumendjel b,
a Université de Grenoble I/CNRS, UMR 5063, Département de Pharmacochimie Moléculaire, 470, rue de la Chimie, BP 53, Grenoble Cedex 9, France
b Université de Toulouse/IRD, UMR 152 LPSNPR, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and in vitro antiplasmodial activity of 35 compounds, designed as analogues of
the naturally occurring aurones. Several of these analogues showed submicromolar antimalarial activity
against a chloroquine-resistant strain of Plasmodium falciparum (FcB1-Columbia strain) cultured on
human erythrocytes. Substitution of the intracyclic oxygen in aurones by a nitrogen atom and systematic
variation of the substituent at the B-ring revealed promising leads showing good activity on the CQ-resis-
tant strain. In particular, 4,6-dimethoxy-40-ethylazaaurone 22 showed antiplasmodial potency without
noticeable toxicity. The easy synthesis of this family of compounds and the relevant antiplasmodial activ-
ity are in favor of promising candidates for further development.
Received 25 January 2010
Revised 17 May 2010
Accepted 4 June 2010
Available online 9 June 2010
Keywords:
Aurones
Azaaurones
Plasmodium falciparum
Malaria
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Today’s meta-analysis provides compelling evidence that drug-
resistant malarial parasites are continuing their inexorable global
Malaria kills over 1.5 million people a year and it is responsible
for human misery in tropical countries. Malaria is caused by a pro-
tozoal parasite of the genus Plasmodium and remains world wide
problem and there is an urgent need to identifying new class of
antimalarials.1
Traditional medicine and natural compounds have been and
still a valuable source of drugs for treatment of malaria.2–4 In this
regard, the quinoleic alkaloid, quinine (Q), the active principle of
Cinchona bark is the most known naturally occurring compound
used to treat malaria. Optimization of quinine has led to the
discovery of chloroquine (CQ) which was used as a major antima-
larial drug. More recently, artemisinin has been identified as the
active ingredient in decoctions or beverages of Artemisia annua.5
Artemisinin and its hemi-synthetic derivatives, artemether, and
artesunate share an endoperoxide moiety that is essential for anti-
malarial activity.6,7
march.6 Due to the wide spread of malaria and owing to the resis-
tance of the parasite to major drugs, it is urgent to pursue identifi-
cation for more active and inexpensive drugs to fight malaria.11–14
Aurones, (2-benzylidenebenzofuran-3(2H)-ones) are secondary
metabolite belonging to the flavonoids family ( Fig. 1). Aurones
are structural isomers of flavones,15 and compared to other flavo-
noids subclasses, somehow, aurones still by large less studied.
The natural origin of aurones, theirs inhibitory effects on (1) eryth-
rocytic stages of Plasmodium falciparum strains in vitro15–18 and (2)
efflux pumps involved in the resistance of the parasites19–21 has
prompted us to investigate them as antimalarial agents targeting
resistant strains.
The aim of the present study is to investigate aurones analogs in
order to gather more structural elements required for the antima-
larial activity. The structure of targeted molecules were chosen
according to: (a) from our previous studies, we showed that the
The major hurdle in malaria treatment is the spread of resis-
tance to natural or hemi-synthetic antiplasmodial drugs.8 Among
the various mechanisms identified, those based on drug transport
proteins of the ATP-binding cassette (ABC) family appear to play
an important role by pumping drugs out of their target sites.9,10
4'
4'
B
1'
B
1'
1
1
6
CH3O
X
R
CH3O
6
R
O
C
A
A
C
2
2
3 O
4
3 O
4
OCH3
OCH3
* Corresponding author.
E-mail address: florence.souard@ujf-grenoble.fr (F. Souard).
Equal senior investigators.
Figure 1. Structures of aurones derivatives investigated.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.008

F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
5725
Table 2
presence of two methoxy groups at positions 4 and 6 of the A-ring
was essential for the activity of aurones against the efflux pumps;17
(b) isosteric replacement of the intracyclic oxygen by N–H; (c) intro-
ducing diverse substituents at the B-ring. The general structures of
investigated compounds are shown in Figure 1.
Structures and activity of azaaurones
4'
H
MeO
N1
6
R
1'
4
2. Chemistry
O
OMe
17 - 37
We have previously reported a general and convenient synthe-
sis of intermediates 1 and 2 (Scheme 1).21–23 The condensation of
benzofuranone 1 and dihydroindolyl-3-one 2 with benzaldehyde
derivatives in the presence of KOH in a mixture of MeOH/H2O
led to aurones and azaaurones. It should be highlighted that both
series of compounds were obtained exclusively as Z-isomers. The
configuration of the exocyclic double bond is clearly evidenced
by the chemical shift in the 1H NMR spectrum of the ethylenic
proton. The 4,6-dihydroxyaurones were obtained by demethyla-
tion of 4,6-dimethoxyaurones with BBr3 in CH2Cl2 as previously
reported.21 During the condensation step of compound 2 with
benzaldehydes, the acetyl group was removed. Aurones and azaau-
rones obtained according to Scheme 1 are summarized in Tables 1
and 2.
Compd
R
Yield
IC50 (lM)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
CQ
40-Br
40-Cl
81
80
97
58
44
73
74
25
45
10
18
53
51
59
82
46
60
42
60
11
10
49.8
17
9.9
8.4
9
20-Cl
20,50-Cl
20-Cl, 60-F
40-Et
1
20-Et
12.8
9.1
3.6
5.6
8.9
4.4
7.2
4.1
13.4
5
1.9
1.9
6.7
8.9
3.7
0.19
20,60-Me
20,40-Me
20,40,50-Me
20,30,50,60-Me
40-i-Pr
40-t-Bu
40-Bu
40-CCH
20,40-OMe
20,40,60-OMe
30,40,50-OMe
40-SMe
40-Morpholino
40-N(Me)2
CH3O
R
O
CH3O
O
i
O
OCH3
3 - 16
O
OCH3
1
Ac
N
H
CH3O
N
R
i
CH3O
3. Results
O
Overall, 14 aurones and 21 azaaurones were obtained and
tested in vitro for antiplasmodial activity (Tables 1 and 2).
Antiplasmodial activity was first evaluated in vitro, using P. falci-
parum, the causative agent of fatal malaria. The IC50 (concentration
giving 50% of parasite growth inhibition) was determined and
compared to chloroquine, used as control drug. For all compounds,
cytotoxicity was assessed by MTT assay on human cells and
showed no noticeable toxicity (data not shown).
OCH3
O
OCH3
2
17 - 37
Scheme 1. Synthesis of aurones and azaaurones. Reagents and conditions: (i) KOH
(50% in H2O), MeOH, 70 °C.
Table 1
As shown in Tables 1 and 2, half of the investigated com-
Structures and activity of aurones
pounds were active with an IC50 below 10
them with an IC50 below 5 M. The best candidate was the
4,6-dimethoxy-40-ethylazaaurone 22 with an IC50 of 1
M. Owing
lM, and seven of
4'
l
l
1
R1
6
R
1'
to their known interactions with efflux pumps, the more active
compounds (22, 28, 33, and 34) were evaluated for their poten-
tialization effect on CQ efficiency (Fig. 2). When tested on FcM29
strain (high resistance to CQ), a synergistic effect was induced by
azaaurones 22, 28, and 33. When tested on F32 strain (CQ-sensi-
tive, Pfcrt wild type and PfMDR with no known mutations corre-
lated with resistance), the combination showed only an additive
effect (Fig. 2).
The most active derivative (22) was evaluated for in vivo effi-
cacy. First, toxicity studies were performed on mice before evalua-
tion of the antiplasmodial activity. Compound 22 was apparently
well tolerated by animals, and after 4 days of treatment at
20 mg/kg/day, there were no signs of acute toxicity. In vivo anti-
malarial activity evaluation was then performed following a 4-
day-suppressive test on CD female mice infected by Plasmodium
vinckei petteri.24
O
4
O
R2
3 - 16
Compd
R
R1
R2
Yield
IC50 (lM)
3
4
5
6
7
H
H
OH
OH
OMe
OH
OMe
OH
H
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
40
62
98
85
82
60
78
70
80
77
92
85
97
43
94.5
60.3
63.4
21
113.5
28
13.3
11.8
49.8
86.7
130
11
OMe
OH
OMe
OH
40-Me
20-Et
20-Et
40-Et
40-tBu
40-Bu
40-Br
40-F
8
9
OH
OMe
OMe
OMe
OMe
OH
OMe
OMe
OMe
10
11
12
13
14
15
16
CQ
40-OH
40-OMe
40-Ph
40-Py
Choloroquine CQ was tested at two concentrations whereas 22
was tested at three concentrations (1, 10, and 20 mg/Kg/day). The
DE50 for the title compound was estimated to be higher than
20 mg/ml. The survival data are shown in Table 3 and Figure 3.
234
85
0.19

5726
F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
100
80
60
40
20
0
100
80
60
40
20
0
0
20
40
60
80
100
0
20
40
60
80
100
Figure 2. Combination assays with drugs and chloroquine. Left, FcM29 (CQ-resistant strain), right, F32 strain (CQ-sensitive). Except for 34, the synergistic effect was only
found back for FcM29. Data represent the combination of chloroquine (X-axis: percentage of the IC50 of CQ), Y-axis: percentage of the IC50 of the molecule tested (} molecule
22, h molecule 28, s molecule 33, 4 molecule 34).
In the aurones series, the structure–activity relationship(Table 1)
Table 3
confirmed that dimethoxylation at 4,6-carbons was highly benefi-
cial (compound 3 vs 4 and 7 vs 6). This substitution pattern was pre-
viously found determinant for inhibiting efflux pumps implied in
drug resistance. At the B-ring, the presence of a hydrophobic group
was highly favorable (compound 4 vs 8, 9, 10). The chain homologa-
tion from an ethyl to a butyl group led to a substantial increase in
activity. Further chain elongation led to less active derivatives, prob-
ably due to their poor solubility and/or to the size of the chain. The
presence of a hydrophobic halogen atom at the 40-position was quite
beneficial as illustrated by comparing derivatives 4 and 11. These
preliminary SAR results permitted us to investigate aza analogs of
aurones. The substitution of the intracyclic oxygen atom with
its isosteric equivalent, N–H group, globally led to more active ana-
logs. In azaaurones, the positive effect of a hydrophobic group linked
to the B-ring as well as its position was confirmed. An ethyl group at
the C-40 position was found to be the optimal substituent. When the
ethyl group was placed at the C-20 position, the compound becomes
much less active (22 vs 23). When the ethyl group was replaced by
its oxidized derivative, an acetylenyl group, the activity was sub-
stantiallydecreased (22 vs 31), pointingagain to the size and/or lipo-
philic effects. The methoxylation pattern at the B-ring was highly
favorable as shown by the low IC50 of derivatives 32, 33, and 34.
The presence of a dimethylamino moiety at the 40-position led to po-
tent compounds 37. The choice of the later group was motivated by
its presence in the chloroquine skeleton. Taken together, the biolog-
ical results found among aurones and azaaurones point to the
importance of the hydrophobicity. Concerning the study of synergy
In vivo antiplasmodial activity of (22)
C
CQ
22
10
20.8
6.5
17
Dose (mg/kg/day)
Parasitaemia (D5, %)
Parasitaemia (SD, %)
Inhibition (%)
0
1
10
0
0
1
20
25
16.8
0
25
8.5
0
38.9
18.4
0
24.2
7.1
3
100
DE50 (mg/ml)
5
>20
C: control, CQ: chloroquine, SD: standard deviation.
No differences were observed between the three doses of 22
and CQ at 1 mg/kg/day. Compared to CQ at 10 mg/kg/day, it ap-
peared that compound 22 had only a little effect on the survival
rate (Fig. 3). This low activity was seriously hampered by the low
solubility of 22. Its formulation in more soluble systems would
be helpful for better bio-availability.
4. Structure–activity relationship and discussion
Our previous investigations have allowed the assessment of
aurones potential in inhibiting ABC proteins, a family of proteins
involved in the MDR (multidrug resistance) phenotype.25–28 The
role of protozoal ATP-binding cassette (ABC) transporters in the
resistance of P. falciparum to antimalarial drugs has been
highlighted.29
12
11
10
9
8
7
DMSO
CQ 1 mg/kg/D
CQ 10 mg/kg/D
22- 1 mg/kg/D
22- 10 mg/kg/D
22- 20mg/kg/D
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Days
Figure 3. Representative curves of in vivo assay.

F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
5727
between selected azaaurones and CQ, it seemed that, more the sub-
5.1.5. (Z)-4,6-Dihydroxy-2-(2-ethylbenzylidene)benzofuran-
stituent group was hydrophobic at the 40-position better was the
synergy (28 vs 22).
3(2H)-one (6)
Yield: 85%; yellow powder; 1H NMR (400 MHz, CDCl3) d: 8.10
(m, 1H, H60), 7.25 (m, 3H, H30-H50), 6.79 (s, 1H, @CH–), 6.26 (d,
1H, J = 2 Hz, H7), 6.09 (d, 1H, J = 2 Hz, H5), 3.90 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 2.80 (q, 2H, J = 7.6 Hz, CH2), 1.18 (t, 3H,
In conclusion, throughout the present study, we report the preli-
minary results regarding the structural requirements for the
antiplasmodial activity of aurones. We have found that the replace-
ment of the intracyclic oxygen by a N–H was revealed to be highly
advantageous for the activity. We also optimized (at least in part)
the nature of substituents to be present at the A and B-rings. The
present results bring essential elements which will be used to go
more straightforward for the synthesis of more active azaaurones.
This investigation will be facilitated by the easy synthesis of azaau-
rones through the optimized method reported in this study.
J = 7.6 Hz, CH3); IR (NaCl, CH2Cl2): 1695 (CO), 1614 (C@C) cmꢀ1
HRMS calcd for C19H18O4: 310.353; found: 311.24 [MꢀH]+.
;
5.1.6. (Z)-4,6-Dihydroxy-2-(4-ethylbenzylidene)benzofuran-
3(2H)-one (7)
Yield: 82%; yellow powder; mp 208 °C; 1H NMR (400 MHz,
CD3COCD3) d: 7.80 (d, 2H, J = 8.4 Hz, H20, H60), 7.27 (d, 2H,
J = 8.4 Hz, H30, H50), 6.56 (s, 1H, @CH–), 6.29 (d, 1H, J = 1.6 Hz,
H7), 6.09 (d, 1H, J = 1.6 Hz, H5), 2.62 (q, 2H, J = 7.6 Hz, CH2), 1.20
(t, 3H, J = 7.2 Hz, CH3); 13C NMR (CD3COCD3, 100 MHz) d: 180.67,
167.89, 167.64, 158.12, 148.27, 144.53, 130.85, 130.11, 129.44,
129.15, 126.39, 105.57, 103.26, 97.84, 91.29, 59.64, 15.38); IR
(NaCl, CH2Cl2): 3400 (OH), 1634 (CO), 1456 (C@C) cmꢀ1; MS
(DCI) m/z 283 [MAH]+.
5. Experimental
5.1. Chemistry
5.1.1. Materials and methods
1H and 13C NMR spectra were recorded on a Bruker AC-200
(200 MHz for 1H, 50 MHz for 13C) and Bruker AC-400 instrument
(400 MHz for 1H, 100 MHz for 13C). Chemical shifts (d) are reported
in ppm relative to Me4Si as an internal standard. EI and DCI mass
spectra were recorded on a Fisons Trio 1000 instrument. Elemental
analyses were performed by the Analytical Department of CNRS,
Vernaison, France. IR spectra were recorded on Perkin–Elmer
(spectrum one) FT-IR spectrometer. Thin-layer chromatography
(TLC) was carried out using Merck Silica Gel F-254 plates (thick-
ness 0.25 mm). Flash chromatography was carried out using Merck
Silica Gel 60, 200–400 mesh. All solvents were distilled prior to
use. Chemicals and reagents were obtained either from Aldrich or
Acros companies and used as obtained.
5.1.7. (Z)-2-(4-Ethylbenzylidene)-6-hydroxybenzofuran-3(2H)-
one (8)
Yield: 60%; yellow powder; mp 228 °C; 1H NMR (400 MHz,
CD3COCD3) d: 7.83 (d, 2H, J = 8 Hz, H20, H60), 7.56 (d, 1H,
J = 8.4 Hz, H4), 7.28 (d, 2H, J = 8 Hz, H30, H50), 6.78 (d, 1H, J = 2 Hz,
H7), 6.72 (dd, 1H, J1 = 2 Hz, J2 = 8.4 Hz, H5), 6.65 (s, 1H, @CH–),
2.63 (q, 2H, J = 7.8 Hz, CH2), 1.19 (t, J = 7.8 Hz, CH3); 13C NMR
(CD3COCD3, 100 MHz) d: 181.77, 168.36, 165.94, 147.49, 146.24,
131.37, 130.11, 128.42, 125.73, 113.97, 112.74, 110.45, 95.47,
29.06, 14.97; IR (NaCl, CH2Cl2): 3400 (OH), 1694 (CO), 1615
(C@C) cmꢀ1
; HRMS calcd for C17H14O3: 266.09429; found:
266.0919 [MꢀH]+. Anal. (C17H14O3) C 76.51, H 5.30.
5.1.2. (Z)-2-Benzylidene-4,6-dihydroxybenzofuran-3(2H)-
one (3)
5.1.8. (Z)-2-(4-tert-Butylbenzylidene)-4,6-dimethoxybenzofu-
ran-3(2H)-one (9)
Yield: 40%; yellow powder; mp 252 °C; 1H NMR (400 MHz,
CD3COCD3) d: 7.87 (m, 2H, H20, H60); 7.42 (m, 2H, H30, H50); 7.35
(m, 1H, H40); 6.57 (s, 1H, @CH–); 6.30 (d, 1H, J = 1.6 Hz, H5); 6.09
(d, 1H, J = 1.6 Hz, H7); 13C NMR (CD3COCD3, 100 MHz) d: 181.36,
168.74, 148.50, 132.59, 130.96, 129.31, 128.91, 109.66, 109.04,
91.87, 89.86; IR (NaCl, CH2Cl2): 3400 (OH), 1687 (CO), 1546
Yield: 78%; yellow powder; mp 210–211 °C; 1H NMR (400 MHz,
CDCl3) d: 7.79 (d, 2H, J = 8.4 Hz, H20, H60), 7.39 (d, 2H, J = 8.4 Hz,
H30, H50), 6.72 (s, 1H, @CH–), 6.32 (d, 1H, J = 1.6 Hz, H5), 6.07 (d,
1H, J = 1.6 Hz, H7), 3.89 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 1.27 (s,
9H, tBu); 13C NMR (CDCl3, 100 MHz) d: 180.91, 169.18, 169.06,
159.59, 153.03, 147.80, 131.15, 129.96, 125.98, 111.10, 105.56,
94.15, 89.41, 56.39, 56.28, 35.08, 31.36; IR (NaCl, CH2Cl2): 1694
(CO), 1615 (C@C) cmꢀ1; HRMS calcd for C21H22O4: 338.407; found:
339.19 [MꢀH]+.
(C@C) cmꢀ1
; HRMS calcd C15H10O4: 252.05; found 253.05
[MꢀH]+, Anal. (C15H10O4) C, 70.85; H, 4.74.
5.1.3. (Z)-2-Benzylidene-4,6-dimethoxybenzofuran-3(2H)-
one (4)
Yield: 62%; yellow powder; mp 154–156 °C; 1H NMR (400 MHz,
CDCl3) d: 7.87–7.35 (m, 5H, H20- H60), 6.76 (s, 1H, @CH–), 6.39 (d,
1H, J = 1.5 Hz, H7), 6.14 (d, 1H, J = 1.5 Hz, H5), 3.95 (s, 3H, OCH3),
3.91 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz) d: 180.73, 169.10,
169.01, 159.45, 147.88, 132.60, 131.12, 129.33, 129.75, 110.79,
105.24, 94.07, 89.26, 56.24, 56.14; IR (NaCl, CH2Cl2): 1694 (CO),
1590 (C@C) cmꢀ1; HRMS calcd C17H14O4: 282.09; found 283.08
[MꢀH]+, Anal. (C17H14O4) C, 71.27; H, 5.06.
5.1.9. (Z)-2-(4-Butylbenzylidene)-4,6-dimethoxybenzofuran-
3(2H)-one (10)
Yield: 70%; white powder, mp 150 °C, 1H NMR (400 MHz,
CDCl3): d 7.89 (d, 2H, J = 8.4 Hz, H20, H60), 7.26 (d, 2H, J = 8.4 Hz,
H30, H50), 6.56 (s, 1H, @CH–), 6.31 (d, 1H, J = 2 Hz, H7), 6.11 (d,
1H, J = 2 Hz, H5), 3.89 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 2.59 (t,
2H, J = 7.2 Hz, CH2), 1.58 (m, 2H, CH2), 1.29 (m, 2H, CH2), 0.88 (t,
3H, J = 7.6 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d: 180.94, 169.21,
169.07, 159.62, 147.70, 144.99, 131.36, 130.20, 129.15, 111.32,
105.60, 94.20, 89.43, 56.43, 56.32, 35.86, 33.59, 22.54, 14.14; IR
(NaCl, CH2Cl2): 1695 (CO), 1615 (C@C) cmꢀ1; HRMS calcd for
5.1.4. (Z)-4,6-Dihydroxy-2-(4-methylbenzylidene)benzofuran-
3(2H)-one (5)
Yield: 98%; yellow powder; mp 250–252 °C; 1H NMR (400 MHz,
CDCl3) d: 7.71 (d, 2H, J = 8 Hz, H20, H60), 7.18 (d, 2H, J = 8 Hz, H30,
H50), 6.71 (s, 1H, @CH–), 6.33 (d, 1H, J = 1.6 Hz, H7), 6.08 (d, 1H,
J = 2 Hz, H5), 1.50 (s, 3H, CH3); 13C NMR (CD3COCD3, 100 MHz) d:
180.69, 167.61, 167.31, 157.94, 147.46, 139.65, 130.97, 129.86,
129.56, 109.31, 103.26, 97.75, 91.19, 20.59; IR (NaCl, CH2Cl2):
3400 (OH), 1615 (CO), 1452 (C@C) cmꢀ1; HRMS calcd C16H12O4:
268.0735; found 268.0721 [MꢀH]+, Anal. (C16H12O4) C, 71.59; H,
4.72.
C
21H22O4: 338.407; found: 329.25 [MꢀH]+.
5.1.10. (Z)-2-(4-Bromobenzylidene)-4,6-dimethoxybenzofuran-
3(2H)-one (11)
Yield: 80%; yellow powder; mp 171–172 °C; 1H NMR (200 MHz,
CDCl3) d: 7.72 (d, 2H, J = 1.5 Hz, H20, H60), 7.54 (d, 2H, J = 1.5 Hz,
H30, H50), 6.68 (s, 1H, @CH–), 6.38 (d, 1H, J = 1.7 Hz, H7), 6.14 (d,
1H, J = 1.7 Hz, H5), 3.96 (s, 3H, OCH3), 3.92 (s, 3H, OCH3); 13C
NMR (CDCl3, 50 MHz) d: 180.49, 169.14, 169.06, 159.58, 148.11,

5728
F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
132.35, 131.94, 131.56, 123.59, 109.27, 105.11, 94.12, 89.34, 56.26,
H7), 6.14 (d, 1H, J = 1.7 Hz, H5), 3.96 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3); 13C NMR (CDCl3, 50 MHz) d: 180.44, 169.16, 169.01,
159.50, 148.15, 132.35, 131.90, 131.52, 123.54, 109.28, 105.15,
94.15, 89.34, 56.24, 56.19; IR (NaCl, CH2Cl2): 3315 (NH), 1680
(CO), 1615 (C@C) cmꢀ1; HRMS calcd C17H13O4Br: 360.7935; found
361.7930 [MꢀH]+.
56.13 IR (NaCl, CH2Cl2): 1697 (CO), 1594 (C@C) cmꢀ1; HRMS calcd
C
17H13BrO4: 359.9997; found 361.7930 [MꢀH]+.
5.1.11. (Z)-2-(4-Fluorobenzylidene)-4,6-dimethoxybenzofuran-
3(2H)-one (12)
Yield: 77%; yellow powder; mp 173–174 °C; 1H NMR (200 MHz,
CDCl3): d 7.85 (dd, 2H, J1 = 8.7 Hz, J2 = 5.5 Hz, H arom), 7.16 (t, 2H,
J = 8.7 Hz, H arom), 6.73 (s, 1H, @CH–), 6.38 (d, 1H, J = 1.7 Hz, H7),
6.14 (d, 1H, J = 1.7 Hz, H5), 3.96 (s, 3H, OCH3), 3.92 (s, 3H, OCH3);
13C NMR (CDCl3, 50 MHz): d: 180.7 (CO), 169.1 (C-8), 169.0 (d,
J = 250 Hz, C-4), 159.69, 147.66, 133.14, 129.09, 116.13, 109.79,
105.09, 94.27, 89.43, 56.31, 56.26; IR (NaCl, CH2Cl2): 1644 (CO)
cmꢀ1; HRMS calcd C17H13FO4: 300.0798; found 300.8880 [MꢀH]+.
5.1.17. (Z)-2-(4-Chlorobenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (18)
Yield: 80%; yellow powder, mp 146–148 °C, 1H NMR (200 MHz,
CDCl3): d: 7.4 (dd, 2H, J1 = 8.5 Hz, J2 = 0.4 Hz, H30, H50), 7.3 (dd, 2H,
J1 = 8.5 Hz, J2 = 0.4 Hz, H20, H60), 6.6 (s, 1H, @CH–), 6.1 (d, 1H,
J = 1.6 Hz, H7), 5.85 (d, 1H, J = 1.6 Hz, H5), 3.85 (s, 3H, OCH3), 3.8
(s, 3H, OCH3); 13C NMR (CDCl3, 50 MHz) d: 182.33, 168.89,
160.61, 156.64, 136.64, 133.87, 133.68, 130.54, 129.44, 108.36,
105.07, 91.56, 88.69, 56.06, 56.03, 27.09; IR (NaCl, CH2Cl2): 3400
5.1.12. (Z)-2-(4-Hydroxybenzylidene)-6-hydroxybenzofuran-
3(2H)-one (13)
(NH), 1624 (CO); HRMS calcd
C17H14O3NCl: 315.751; found
Yield: 92%; yellow powder; mp 286–288 °C; 1H NMR (400 MHz,
DMSO-d6): d: 10.1 (br s, 1H, OH), 7.73 (d, 2H, J = 8.8 Hz, H20, H60),
7.52 (d, 1H, J = 8.8 Hz, H4), 6.81 (d, 2H, J = 8.8 Hz, H30, H50), 6.71
(d, 1H, J = 1.6 Hz, H7), 6.65 (s, 1H, @CH–), 6.63 (dd, 1H,
J1 = 1.6 Hz, J2 = 8.8 Hz, H5); 13C NMR (DMSO-d6, 100 MHz) d:
181.70, 167.97, 166.64, 159.70, 146.1, 133.72, 126.18, 123.49,
116.53, 113.61, 113.34, 111.84, 98.98, 56.2; MS (DCI: NH3 + isobu-
tane) m/z 255 [MꢀH]+; Anal. (C15H10O4) C, H.
316.12 [MꢀH]+.
5.1.18. (Z)-2-(2-Chlorobenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (19)
Yield: 97%, brown powder, mp 194–195, 1H NMR (200 MHz,
CD3COCD3): d: 7.75 (m, 1H, H30), 7.33 (m, 3H, H40, H50, H60),
6.75 (s, 1H, @CH–), 6.21 (d, J = 1.6 Hz, 1H, H7), 6.03 (d,
J = 1.7 Hz, 1H, H5), 3.67 (s, 3H, OCH3), 3.65 (s, 3H, OCH3); 13C
NMR (CD3COCD3, 50 MHz) d: 182.00, 169.62, 161.30, 158.19,
138.42, 134.84, 134.11, 130.79, 130.65, 129.71, 128.23, 104.72,
102.76, 91.74, 89.70, 56.22, 56.05; IR (NaCl, CH2Cl2): 3400
5.1.13. (Z)-2-(4-Methoxybenzylidene)-6-hydroxybenzofuran-
3(2H)-one (14)
Yield: 85%, yellow powder, mp 167–168 °C, 1H NMR (200 MHz,
DMSO-d6): d: 7.73 (d, 2H, J = 8.6 Hz, H30, H30, H50), 6.87 (d, 2H,
J = 8.6 Hz, H20, H60), 6.62 (s, 1H, @CH–), 6.29 (d, 1H, J = 1.7 Hz,
H7), 6.05 (d, 1H, J = 1.7 Hz, H5), 3.85 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3), 3.76 (s, 3H, OCH3); 13C NMR (DMSO-d6, 100 MHz) d:
178.94, 168.74, 168.04, 160.36, 158.85, 146.13, 132.80, 124.67,
114.58, 109.86, 104.22, 94.33, 89.84, 56.52, 56.14, 55.38; IR (NaCl,
CH2Cl2): 1638 (CO) cmꢀ1; HRMS calcd for C18H16O5: 312.325;
found: 313.22 [MꢀH]+.
(NH), 1630 (CO) cmꢀ1
; HRMS calcd C17H14ClNO3: 316.0502;
found 316 [MꢀH]+.
5.1.19. (Z)-2-(2,4-Dichlorobenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (20)
Yield: 58%, yellow powder, mp 235–236 °C, 1H NMR (200 MHz,
CD3COCD3): d: 7.45 (m, 3H, H30,50,60), 6.38 (s, 1H, @CH–), 6.08 (d,
J = 1.8 Hz, 1H, H7), 6.0 (d, J = 1.9 Hz, 1H, H5), 3.88 (s, 3H, OCH3),
3.84 (s, 3H, OCH3); 13C NMR (CD3COCD3, 50 MHz) d: 181.54,
170.05, 161.44, 157.77, 139.84, 135.75, 133.56, 130.62, 129.25,
128.12, 104.33, 101.27, 91.33, 89.04, 56.20, 56.01; IR (NaCl,
CH2Cl2): 3400 (NH), 1682 (CO), 1615 (C@C) cmꢀ1; HRMS calcd
5.1.14. (Z)-2-(4-Phenylbenzylidene)-6-hydroxybenzofuran-
3(2H)-one (15)
Yield: 97%, yellow powder, mp 193–195 °C, 1H NMR (400 MHz,
CDCl3): d: 7.92 (d, 2H, J = 8.7 Hz, H30, H50), 7.59 (m, 4H, H30, H50,
H200, H600), 7.44 (t, 2H, J = 7.5 Hz, H300, H500), 7.35 (t, 1H, J = 7.5 Hz,
H400), 6.77 (s, 1H, @CH–), 6.39 (s, 1H, H7), 6.13 (s, 1H, H5), 3.94
(s, 3H, OCH3), 3.90 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz) d:
180.73, 169.11, 159.56, 148.10, 142.01, 140.40, 131.75, 131.71,
129.04, 127.92, 127.51, 127.18, 110.56, 105.40, 94.21, 89.43,
56.35, 56.27; IR (NaCl, CH2Cl2): 1616 (CO) cmꢀ1; HRMS calcd
C
17H13Cl2NO3: 350.20; found 350 [MꢀH]+.
5.1.20. (Z)-2-(2-Chloro-6-fluorobenzylidene)-4,6-dimethoxy-
2,3-dihydro-1H-indol-3-one (21)
Yield: 44%, yellow powder, mp 200–201 °C, 1H NMR (200 MHz,
CD3COCD3): d: 7.4 (m, 2H, H30, H40), 7.2 (m, 1H, H40), 6.6 (s, 1H,
@CH–), 6.1 (d, J = 1.9 Hz, 1H, H7), 5.85 (d, J = 1.9 Hz, 1H, H5), 3.68
(s, 3H, OCH3), 3.65 (s, 3H, OCH3); 13C NMR (CD3COCD3, 50 MHz)
d: 181.43, 169.94, 161.44, 130.77, 130.67, 126.59, 126.56, 115.74,
115.52, 96.82, 91.55, 89.40, 56.27, 56.11; IR (NaCl, CH2Cl2): 3400
(NH), 1630 (CO) cmꢀ1; HRMS calcd C17H13ClFNO3: 333.74; found
334 [MꢀH]+.
C
23H18O4: 358.29; found 359.32 [MꢀH]+.
5.1.15. (Z)-4,6-Dimethoxy-2-(pyridin-4-ylmethylene)benzofu-
ran-3(2H)-one (16)
Yield: 43%, white powder, mp 186–188 °C, 1H NMR (400 MHz,
CDCl3): d: 8.42 (d, 2H, J = 5.2 Hz, H30, H50), 7.03 (d, 2H, J = 5.5 Hz,
H20, H60), 6.63 (s, 1H, @CH–), 6.14 (d, 1H, J = 1.46 Hz, H7), 6.01 (d,
1H, J = 1.46 Hz, H5), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13C
NMR (CDCl3, 100 MHz) d: 182.57, 168.63, 160.43, 156.71, 149.30,
135.92, 132.58, 130.59, 129.61, 127.30, 110.23, 105.09, 91.25,
88.50, 56.01, 55.94; IR (NaCl, CH2Cl2): 1634 (CO) cmꢀ1; HRMS calcd
C16H13NO4: 283.28; found 284.29 [MꢀH]+.
5.1.21. (Z)-2-(4-Ethylbenzylidene)-4,6-dimethoxy-2,3-dihydro-
1H-indol-3-one (22)
Yield: 70%; yellow powder; mp 215–217 °C 1H NMR (200 MHz,
CDCl3) d: 7.45 (dd, 2H, J1 = 8.2 Hz, J2 = 0.4 Hz, H20, H60), 7.25 (dd,
2H, J1 = 8.2 Hz, J2 = 0.4 Hz, H30, H50), 7.1 (br s, 1H), 6.7 (s, 1H), 6.1
(d, 1H, J = 1.8 Hz, H7), 5.9 (d, J = 1.8 Hz, 1H, H5), 3.9 (s, 3H), 3.8
(s, 3H), 2.7 (q, 2H, J = 6 Hz, –CH2), 1.3 (t, J = 6 Hz, 3H, CH3); 13C
NMR (CD3COCD3, 50 MHz) d: 182.89, 169.88, 161.74, 158.71,
145.59, 137.21, 134.12, 130.91, 129.89, 108.70, 105.62, 92.23,
90.13, 56.77, 56.60, 16.56; IR (NaCl, CH2Cl2): 3247 (NH), 1672
(CO), 1615 (C@C) cmꢀ1; HRMS calcd C19H19NO3: 309.359; found
3310.43 [MꢀH]+.
5.1.16. (Z)-2-(4-Bromobenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (17)
Yield: 80%; yellow powder; mp 171–172 °C; 1H NMR (CDCl3,
200 MHz) d: 7.72 (d, 2H, J = 1.5 Hz,
H arom), 7.54 (d, 2H,
J = 8.6 Hz, H arom), 6.68 (s, 1H, @CH–), 6.38 (d, 1H, J = 1.7 Hz,

F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
5729
5.1.22. (Z)-2-(2-Ethylbenzylidene)-4,6-dimethoxy-2,3-dihydro-
1H-indol-3-one (23)
(s, 3H, OCH3), 3.01 (m, 1H, CH(CH3)2), 1.28 (d, 6H, J = 6,8 Hz,
(CH3)2); 13C NMR (CDCl3, 100 MHz) d: 179.94, 166.27, 158.10,
154.23, 147.01, 133.50, 130.23, 127.22, 124.97, 107.99, 102.78,
88.95, 86.07, 53.88, 53.62, 31.69, 21.58, 21.48; IR (NaCl, CH2Cl2):
3294 (NH), 1677 (CO), 1615 (C@C) cmꢀ1; HRMS calcd C20H21O3N:
323.5857; found 323.6541 [MꢀH]+.
Yield: 74%; yellow powder; mp 197–198 °C 1H NMR (400 MHz,
CDCl3) d: 7.51–7.49 (m, H6), 7.25 (br s, 3H, H30, H40, H50), 6.90
(s,1H, @CH–), 6.78 (br s, 1H, NH), 6.00 (s, 1H, H7), 5.92 (s, 1H,
H5), 3.94 (s, 3H, OCH3), 2.79–2.73 (q, J = 7.28 Hz, 2H, CH2), 1.22–
1.18 (t, J = 7.52 Hz, 3H, CH3); 13C NMR (CDCl3, 100 MHz) d:
183.58, 170.14, 162.06, 158.04, 145.81, 138.58, 134.67, 130.76,
129.95, 129.83, 127.77, 109.11, 106.66, 92.53, 89.58, 57.38, 57.34,
28.22, 16.04; IR (NaCl, CH2Cl2): 3269 (NH), 1675 (CO), 1615
5.1.28. (Z)-2-(4-tert-Buthylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (29)
Yield: 51%; brown powder; mp 196–197 °C; 1H NMR (CDCl3,
400 MHz) d: 7.46 (br s, 2H, H20, H60), 7.25 (br s, 2H, H30, H50),
6.84 (br s,1H, NH), 6.77 (s, 1H, @CH–), 6.06 (d, J = 1.69 Hz, 1H,
H7), 5.94 (d, J = 1.67 Hz, 1H, H5), 3.94 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 1.37 (br s, 9H, -C(CH3)3); 13C NMR (CDCl3, 400 MHz) d:
184.07, 170.04, 161.92, 158.12, 152.94, 137.50, 133.69, 130.70,
127.57, 111.34, 106.59, 92.67, 89.88, 57.32, 36.30, 32.73; IR (NaCl,
CH2Cl2): 3416 (NH), 1678 (CO), 1613 (C@C) cmꢀ1; HRMS calcd
(C@C) cmꢀ1
[MꢀH]+.
; HRMS calcd C19H19O3N: 309.37; found 310.21
5.1.23. (Z)-2-(2,6-Dimethylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (24)
Yield: 25%; yellow powder; mp 217–218 °C; 1H NMR (400 MHz,
CDCl3) d: 7.31–7.03 (m, J = 7.62 Hz, 4H, H30, H40, H50, NH), 6.84 (s,
1H, @CH–), 6.03 (s, 1H, H7), 5.92 (s, 1H, H5), 3.93 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 2.37 (s, 3H, CH3), 2.35 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz) d: 183.49, 170.05, 162.09, 157.90, 138.21,
137.24, 136.81, 135.27, 132.35, 130.49, 130.05, 109.52, 106.79,
92.53, 89.56, 57.47, 57.35, 22.69, 21.19; IR (NaCl, CH2Cl2): 3400
(NH), 1592 (CO) cmꢀ1; HRMS calcd C19H19O3N: 309.37; found
310.23 [MꢀH]+.
C
21H23O3N: 337.42; found 336.22 [MꢀH]+.
5.1.29. (Z)-2-(4-Buthylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (30)
Yield: 59%; yellow powder; mp 90–92 °C; 1H NMR (400 MHz,
CDCl3) d: 7.44 (d, J = 8.2 Hz, 2H, H20, H60), 7.27 (d, J = 8.2 Hz, 2H,
H30, H50), 6.8 (br s, 1H, NH), 6.75 (s, 1H, @CH–), 6.07 (d,
J = 1.69 Hz, 1H, H7), 5.93 (d, J = 1.72 Hz, 1H, H5), 3.92 (s, 3H,
OCH3), 3.87 (s, 3H, OCH3), 2.63 (t, J = 7.5 Hz, 2H, CH2-Ph), 1.63 (br
s, 4H, CH2–CH2), 0.94 (t, J = 7.5 Hz, 3H, CH3); 13C NMR (CDCl3,
100 MHz) d: 170.02, 161.91, 158.5, 144.86, 137.37, 133.89,
130.85, 111.56, 106.62, 92.69, 89.85, 57.33, 37.06, 35.01, 90.3,
23.91, 15.51; IR (NaCl, CH2Cl2): 3402 (NH), 1677 (CO), 1614
5.1.24. (Z)-2-(2,4-Dimethylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (25)
Yield: 45%, brown powder; mp 235 °C; 1H NMR (200 MHz,
CD3COCD3): d: 7.5 (m, 1H, H20), 7.06 (m, 2H, H40, H60), 6.6 (s, 1H,
@CH–), 6.2 (d, J = 1.8 Hz, 1H, H7), 5.99 (d, J = 1.78 Hz, 1H, H5),
3.67 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 2.34 (s, 3H, CH3), 2.28 (s,
3H, CH3); 13C NMR (CD3COCD3, 100 MHz) d: 182.07, 169.26,
161.19, 158.18, 138.42, 138.36, 137.22, 132.19, 131.81, 129.26,
127.79, 105.56, 105.11, 91.47, 89.39, 56.18, 56.02, 21.28, 20.13.;
IR (NaCl, CH2Cl2): 3201 (NH), 1668 (CO), 1615 (C@C) cmꢀ1; HRMS
calcd C19H19O3N: 309.37; found 310.23 [MꢀH]+.
(C@C) cmꢀ1
[MꢀH]+.
; HRMS calcd C21H23O3N: 337.42; found 338.22
5.1.30. (Z)-2-(4-Ethynylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (31)
Yield: 82%; yellow powder; mp 135 °C; 1H NMR (400 MHz,
CDCl3) d: 7.46 (d, 2H, J = 8.4 Hz, H30, H50), 7.39 (d, 2H, J = 8.4 Hz,
H20, H60), 6.80 (br s, 1H, NH), 6.62 (s, 1H, @CH–), 6.01 (d, 1H,
J = 2 Hz, H7), 5.88 (d, 1H, J = 1,6 Hz, H5), 3.85 (s, 3H, OCH3), 3.81
(s, 3H, OCH3), 3.11 (s, 1H, CH); 13C NMR (CDCl3, 100 MHz) d:
182.19, 168.75, 140.47, 156.50, 136.50, 133.73, 133.54, 130.40,
129.31, 108.21, 104.93, 91.52, 88.55, 77.27, 55.92, 55.88, 26.95;
IR (NaCl, CH2Cl2): 3284 (NH), 1677 (CO), 1612 (C@C) cmꢀ1; HRMS
calcd C19H15O3N: 305.327; found 306.241 [MꢀH]+.
5.1.25. (Z)-2-(2,4,5-Trimethylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (26)
Yield: 10%; yellow powder; mp 229 °C; 1H NMR (400 MHz,
CDCl3) d: 7.27 (s, 1H, H60), 7.03 (s, 1H, H30) 6.84 (br s, 2H, NH,
@CH–), 6.04–6.03 (d, J = 1.61 Hz, 1H, H7), 5.91 (d, J = 1.58 Hz, 1H,
H5), 3.92 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 2.33 (s, 3H, CH3), 2.28
(s, 3H, CH3), 2.25 (s, 3H, CH3); 13C NMR (CDCl3, 100 MHz) d:
183.56, 169.95, 162.03, 157.89, 138.46, 137.83, 132.29, 135.87,
13.86, 132.73, 130.62, 109.71, 106.84, 92.56, 89.55, 57.41–57.34,
21.13; IR (NaCl, CH2Cl2): 3400 (NH), 1671 (CO), 1592 (C@C)
cmꢀ1; HRMS calcd C20H21O3N: 323.40; found 324.19 [MꢀH]+.
5.1.31. (Z)-2-(2,4-Dimethoxybenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (32)
Yield: 46%, orange powder, 214–216 °C, 1H NMR (400 MHz,
CDCl3) d: 7.42–7.39 (d, 1H, J = 8.42, H60), 6.82 (s, 1H, H30), 6.56
(d, 1H, J = 8.37 Hz, H50), 6.51 (s, 1H, @CH–), 6.01 (s, 1H, H7), 5.89
(s, H, H5), 3.92 (s, 6H, 2 OCH3), 3.86 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3); 13C NMR (CDCl3, 100 MHz) d: 183.56, 169.95, 162.03,
157.89, 138.46, 137.83, 132.29, 135.87, 13.86, 132.73, 130.62,
109.71, 106.84, 92.56, 89.55, 57.41, 57.34, 21.13; IR (NaCl, CH2Cl2):
3400 (NH), 1650 (CO), 1591 (C@C) cmꢀ1; HRMS calcd C19H19O5N:
341,369; found 342.42 [MꢀH]+.
5.1.26. (Z)-2-(2,3,5,6-Tetramethylbenzylidene)-4,6-dimethoxy-
2,3-dihydro-1H-indol-3-one (27)
Yield: 18%; yellow powder; mp 260 °C; 1H NMR (400 MHz,
CDCl3) d: 6.97 (s, 1H, H40), 6.85 (s, 1H, @CH–), 5.89 (d, J = 1.66 Hz,
1H, H7), 5.87 (d, J = 1.69 Hz, 1H, H5), 3.93 (s, 3H, OCH3), 3.82 (s,
3H, OCH3), 2.25 (s, 6H, 2 CH3), 2.15 (s, 6H, 2 CH3); 13C NMR (CDCl3,
100 MHz) d: 182.79, 170.18, 162.10, 157.57, 139.32, 135.63,
134.55, 134.02, 132.65, 111.82, 92.24, 89.08, 57.43, 57.29, 21.64,
18.33; IR (NaCl, CH2Cl2): 3338 (NH), 1682 (CO), 1615 (C@C)
cmꢀ1; HRMS calcd C21H23O3N: 337.41; found 338.23 [MꢀH]+.
5.1.32. (Z)-2-(2,4,6-Trimethoxybenzylidene)-4,6-dimethoxy-
2,3-dihydro-1H-indol-3-one (33)
Yield: 60%; yellow powder; mp 109–111 °C; 1H NMR (400 MHz,
CDCl3) d: 7.29 (br s, 1H, NH), 6.98 (s, 1H, @CH–), 6.19 (br s, 2H, H30,
H50), 5.97 (d, 1H, J = 1.51 Hz, H7), 5.86 (d, 1H, J = 1.47 Hz, 1H, H5),
3.91 (s, 3H, OCH3), 3.89 (s, 6H, 2 OCH3), 3.85 (s, 6H, 2 OCH3); 13C
NMR (CDCl3, 100 MHz) d: 182.34, 168.20, 162.09, 160.48, 159.32,
155.44, 135.60, 106.88, 105.31, 102.19, 92.04, 90.52, 87.86, 56.61,
56.00, 55.83, 55.64; IR (NaCl, CH2Cl2): 3390 (NH), 1671 (CO),
5.1.27. (Z)-2-(4-Isopropylbenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (28)
Yield: 53%; yellow powder; mp 252 °C; 1H NMR (400 MHz,
CDCl3) d: 7.47 (d, 2H, J = 4 Hz, H20, H60), 7.31 (d, 1H, J = 4 Hz, H30,
H50), 6,86 (br s, 1H, NH), 6.77 (s, 1H, @CH–), 6.07 (d, 1H,
J = 1,6 Hz, H5), 5.94 (d, 1H, J = 2 Hz, H7), 3.94 (s, 3H, OCH3), 3.88

5730
F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
1621 (C@C) cmꢀ1; HRMS calcd C20H21O6N: 371.396; found 372.06
[MꢀH]+.
tration curves as described elsewhere.33 Combination effect of the
drugs was analyzed by combining drugs at sub-inhibitory concen-
tration (Fractional IC50 FIC) on either FcM29, (CQ-resistant strain)
or F32-Tanzania (CQ-sensitive strain) as described by Garavito
et al.34 Isobolograms representing the combination activities were
plotted with an X-axis representing the fractional IC50 of CQ and
the Y-axis representing the fractional IC50 of the tested drug.
5.1.33. (Z)-2-(3,4,5-Trimethoxybenzylidene)-4,6-dimethoxy-
2,3-dihydro-1H-indol-3-one (34)
Yield: 42%, brown powder, mp 200 – 201 °C; 1H NMR (400 MHz,
CDCl3) d: 7.61 (br s, 1H, NH), 6.95 (s, 1H, @CH–), 6.54 (s, 2H, H20,
H60), 6.01 (s, 1H, H7), 5.86 (s, 1H, H5), 3.91 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 3.87 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3); 13C NMR (CDCl3, 100 MHz) d: 183.74, 169.85, 161.83,
157.19, 153.22, 151.96, 145.46, 136.72, 117.82, 116.32, 108.11,
106.51, 100.63, 92.20, 89.50, 59.36, 58.13, 57.67, 57.35, 57.29; IR
5.2.2. In vivo antiplasmodial activity
In vivo effect was evaluated following a 4-day-suppressive as-
say performed on CD female mice, using P. vinckei petteri. Mice
(mean body weight: 20 2 g) were infected with 106 infected red
blood cells in RPMI at day 0. Groups of five mice were treated intra-
peritoneally from day 0–3 with doses (1, 10, 20 mg/kg/day) with
the most efficient drug, compound 22. On day 4, Giemsa stained
smears were made for each mouse after tail blood sampling. Para-
sitaemia was estimated by visual counting of at least 5000 erythro-
cytes. Controls were mice treated either with RPMI alone or with
chloroquine (1 and 10 mg/kg). The inhibition percentage was cal-
culated with the following formula: (control parasitaemia ꢀ para-
sitaemia with drugs)/(control parasitaemia) ꢁ 100.35 Mice were
maintained under institutional animal guidelines. Mice were then
followed for 15 days after inoculation in order to evaluate survival
time of treated animals.
(NaCl, CH2Cl2): 3400 (NH), 1603 (C@C) cmꢀ1
C20H21O6N: 371.396; found 372.10 [MꢀH]+.
; HRMS calcd
5.1.34. (Z)-2-(4-Thiomethylbenzylidene)-4,6-dimethoxybenzofu-
ran-3(2H)-one (35)
Yield: 60%, yellow powder, mp 228–230 °C; 1H NMR (200 MHz,
CDCl3) d: 7.4 (dd, 2H, J1 = 8.5 Hz, J2 = 0.4 Hz, H20, H60), 7.3 (dd, 2H,
J1 = 8.5 Hz, J2 = 0.4 Hz, H30, H50), 6.8 (s, 1H, @CH–), 6.7 (s, 1H,
NH), 6.1 (d, 1H, J = 1.6 Hz, H7), 5.9 (d, 1H, J = 1.6 Hz, H5), 3.9 (s,
3H, OCH3), 3.8 (s, 3H, OCH3), 2.5 (s, 3H, SCH3); 13C NMR (CDCl3,
100 MHz) d: 182.50, 168.73, 160.46, 156.65, 139.30, 135.97,
131.62, 130.59, 129.88, 126.56, 125.05, 109.80, 105.10, 91.40,
88.62, 77.45, 55.98, 15.50; IR (NaCl, CH2Cl2): 3400 (NH), 1625
(CO) cmꢀ1; HRMS calcd C18H17O3NS: 327.399; found 328.23
[MꢀH]+.
References and notes
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5.1.35. (Z)-2-(4-Morpholinobenzylidene)-4,6-dimethoxy-2,3-
dihydro-1H-indol-3-one (36)
Yield: 11%; yellow powder; mp 257 °C; 1H NMR (400 MHz,
CDCl3) d: 7.46–7.44 (d, 2H, J = 8.7 Hz, H20, H60), 6.93 (d, 2H,
J = 8.6 Hz, H30, H50), 6.82 (br s, 1H, NH), 6.74 (s, 1H, @CH–), 6.08
(d, 1H, J = 1.37 Hz, H7), 5.93 (d, 1H, J = 1.37 Hz, H5), 3.93 (s, 4H, 2
OCH2), 3.89 (br s, 6H, 2 OCH3), 3.24 (m, 4H, J = 4.46 Hz, 2 CH2N);
13C NMR (CDCl3, 100 MHz) d: 183.83, 169.71, 161.88, 157.66,
152.27, 136.30, 132.25, 127.63, 116.89, 112.05, 92.70, 89.82,
68.27, 57.32, 49.91; IR (NaCl, CH2Cl2): 3417 (NH), 1662 (CO),
1615 (C@C) cmꢀ1; HRMS calcd C20H20O4N2: 366.16; found 367.18
[MꢀH]+.
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5.1.36. (Z)-2-(4-(Dimethylamino)benzylidene)-4,6-dimethoxy-
2,3-dihydro-1H-indol-3-one (37)
Yield: 10%; red powder; mp 170 °C; 1H NMR (400 MHz, CDCl3)
d: 7.43 (d, 2H, J = 8.62 Hz, H20, H60), 6.33 (br s, 1H, NH), 6.78 (s,
1H, @CH–), 6.75 (d, 2H, J = 1.42 Hz, H30, H50), 6.08 (d, J = 1.51 Hz,
1H, H7), 5.94 (d, J = 1.72 Hz, 1H, H5), 3.93 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 3.03 (s, 6H, 2 CH3); 13C NMR (CDCl3, 100 MHz) d:
169.44, 161.80, 157.42, 151.68, 135.38, 132.50, 113.97, 113.36,
112.57, 107.25, 92.62, 89.70, 57.41, 57.29, 41.77, 41.67; IR (NaCl,
CH2Cl2): 3400 (Harom), 1629 (CO), 1615 (C@C) cmꢀ1; HRMS calcd
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5.2. Biology
5.2.1. Antiplasmodial activity
The latter was evaluated in vitro, using P. falciparum. Blood
stages (FcB1-Columbia, FcM29 and F32) were cultured in human
erythrocytes maintained in RPMI 1640 supplemented with 5%
human serum according to the method described by Trager and
Jensen30 with modifications described by Valentin et al.31 The cul-
tures were synchronized every 48 h by 5% D-sorbitol lysis (Merck,
Darmstadt, Germany) in order to discard old stage parasite. In vitro
antimalarial activity testing was performed by following [3H]-
hypoxanthine (Perkin–Elmer, France) incorporation.32 The IC50
values were graphically determined from inhibition versus concen-

F. Souard et al. / Bioorg. Med. Chem. 18 (2010) 5724–5731
5731
29. Sauvage, V.; Aubert, D.; Bonhomme, A.; Pinon, J. M.; Millot, J. M. Mol. Biochem.
Parasitol. 2004, 134, 89.
33. Munoz, V.; Sauvain, M.; Mollinedo, P.; Callapa, J.; Rojas, I.; Gimenez, A.;
Valentin, A.; Mallie, M. Planta Med. 1999, 65, 448.
30. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
34. Garavito, G.; Bertani, S.; Rincon, J.; Maurel, S.; Monje, M. C.; Landau, I.; Valentin,
A.; Deharo, E. Parasite 2007, 14, 135.
35. Cachet, N.; Hoakwie, F.; Bertani, S.; Bourdy, G.; Deharo, E.; Stien, D.; Houel, E.;
Gornitzka, H.; Fillaux, J.; Chevalley, S.; Valentin, A.; Jullian, V. Antimicrob. Agents
Chemother. 2009, 53, 4393.
31. Valentin, A.; BenoitVical, F.; Moulis, C.; Stanislas, E.; Mallie, M.; Fouraste, I.;
Bastide, J. M. Antimicrob. Agents Chemother. 1997, 41, 2305.
32. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710.