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4.1.2.5. 5-Methoxy-2-phenyl-indol-3-one (5) [22]. Violet solid,
yield: 61%, mp: 208e210 ꢂC. 1H NMR (300 MHz, CDCl3):
: 3.85 (s,
4.3. EPR spectroelectrochemical analysis
d
3H, CH3), 6.96e7.00 (m, 1H), 7.11 (d, J ¼ 2.7 Hz, 1H), 7.31 (d,
J ¼ 8.1 Hz, 1H), 7.47e7.52 (m, 3H), 8.31 (d, J ¼ 8.1 Hz, 2H). IR (KBr,
cmꢁ1): 1762, 1720 (C]O), 1536 (C]N), 768, 690. MS-(þ)APCI, m/z:
238 [MþH]þ. HRMS (DCI, CH4) m/z calcd for C15H12NO2 [MþH]þ
238.0868. Found 238.0879.
The EPR spectrometer was coupled to a Voltalab 80 PGZ 402
(Radiometer). A flat quartz cell adapted to electrochemical mea-
surements (Bruker, Wissembourg, France) was used for analysis.
The electrochemical reduction was performed using a three-
electrode set-up: the working and counter-electrode were plat-
inum and the reference electrode was a silver wire. The applied
potential was chosen to be on the diffusion plateau of the first
reduction wave obtained under stationary conditions:
Eapplied ¼ E1/2 e 0.2 V. The electrolysis potential was applied to the
solution containing the test compound in acetonitrile/TPAB and the
EPR spectra were immediately recorded as a function of time. EPR
spectra were obtained at X-band at room temperature on a Bruker
EMX-8/2.7 (9.86 GHz) equipped with a high-sensitivity cavity
(4119/HS 0205) and a gaussmeter (Bruker). WINEPR and SIMFONIA
software (Bruker) were used for processing the EPR data and
computer simulations spectra. Typical scanning parameters were:
scan rate, 1.2 G/s; scan number, 1; modulation amplitude, 1 G;
modulation frequency, 100 kHz; microwave power, 20 mW; sweep
width, 100 G; sweep time, 83.88 s; time constant, 40.96 ms; centre
field, 3480 G; receiver gain, 1 ꢄ 105.
4.1.2.6. 5-Methoxy-2-(4-methoxy-phenyl)-indol-3-one
(6) [22].
Violet solid, yield: 66%, mp: 215e217 ꢂC. 1H NMR (300 MHz, CDCl3):
d
: 3.85 (s, 3H, CH3), 3.89 (s, 3H, CH3), 6.96e7.02 (m, 3H), 7.11 (d,
J ¼ 3 Hz, 1H), 7.27 (d, J ¼ 9 Hz, 1H), 8.35 (d, J ¼ 9 Hz, 2H). 13C NMR
(75 MHz, DMSO-d6)
d
: 55.4, 55.9, 111.2, 114.3 (ꢄ2), 120.2, 122.1,
122.9, 124,3, 130.8 (ꢄ2), 153.2, 159.4, 159.7, 162.7, 194.4 (C]O). IR
(KBr, cmꢁ1): 1759, 1720 (C]O), 1535 (C]N), 831, 756. MS-(þ)APCI,
m/z: 268 [MþH]þ. HRMS (DCI, CH4) m/z calcd for C16H14NO3
[MþH]þ 268.0974. Found 268.0973.
4.1.2.7. 2-(4-Dimethylamino-phenyl)-5-methoxy-indol-3-one
(7).
Violet solid, yield: 52%, mp: 189e191 ꢂC. 1H NMR (300 MHz, DMSO-
d6):
d
: 3.04 (s, 6H, 2 CH3), 3.80 (s, 3H, CH3), 6.82 (d, J ¼ 9 Hz, 2H),
7.6e7.08 (m, 2H), 7.24 (d, J ¼ 9 Hz, 1H), 8.15 (d, J ¼ 9 Hz, 2H). 13
C
NMR (75 MHz, DMSO-d6)
d: 40.1 (2 CH3), 55.4, 11.5 (ꢄ2), 116.8,
4.4. Biology
122.0, 123.3, 125.4, 132.6 (ꢄ2), 150.8, 154.3, 159.7, 160.0, 162.6, 193.8
(C]O). IR (KBr, cmꢁ1): 1758, 1725 (C]O), 1538 (C]N), 779, 754,
698. MS-(þ)APCI, m/z: 281 [MþH]þ. HRMS (DCI, CH4) m/z calcd for
4.4.1. In vitro antiplasmodial activity
The FcB1 strain of P. falciparum (chloroquine-resistant strain)
was maintained in RPMI 1640 medium (BioWhittaker, Cambrex,
C
17H17N2O2 [MþH]þ 281.1290. Found 281.1292.
Belgium) containing L-glutamine (BioWhittaker), 25 mM HEPES
(BioWhittaker), and 10% human serum (EFS, Toulouse, France) as
previously described [23]. Parasitized RBCs were maintained in
25 cm2 culture flasks (TPP, Switzerland) in a controlled atmosphere
(5% CO2, 100% relative humidity) and synchronized by a combina-
4.1.2.8. 2-(4-Isopropoxy-phenyl)-5-methoxy-indol-3-one
(8).
Red solid, yield: 78%, mp: 114e116 ꢂC. 1H NMR (300 MHz, DMSO-
d6):
d
: 1.31 (d, J ¼ 6 Hz, 6H, 2 CH3), 3.82 (s, 3H, CH3), 4.74 (m,1H, CH),
7.05e7.13 (m, 4H), 7.32 (d, J ¼ 8.1 Hz, 1H), 8.19 (d, J ¼ 9 Hz, 2H). 13
C
tion of magnetic enrichment followed by D-sorbitol lysis [24,25].
NMR (75 MHz, DMSO-d6) d: 22.3 (2 CH3), 55.3, 69.1, 111.0, 114.8
Chloroquine was dissolved in culture medium and sodium artesu-
nate in ethanol (stock solutions: 10 mg/mL). The stock solutions of
the tested drugs were prepared by mixing 1 mg of drug in 1 mL
DMSO and adding it to a solution of 1 mg BSA in 1 mL of RPMI
medium, giving a stock solution at 0.5 mg/mL. For the drug assays,
serial drug dilutions were made in P. falciparum culture media and
added to 96-well (TPP) culture plates. For the evaluation of Plas-
modium growth inhibition, the culture was plated in 96-well plates
as described elsewhere [4]. [3H]-Hypoxanthine (PerkineElmer)
was added 24 h after the beginning of incubation. At the end of the
incubation (48 h), the microtiter plates were frozen and thawed,
and each well was harvested onto glass-fibre filter paper. The [3H]-
(ꢄ2), 119.8, 121.6, 122.9, 124.3, 130.8 (ꢄ2), 154.4, 160.5, 160.7, 162.8,
193.7 (C]O). IR (KBr, cmꢁ1): 1756, 1722 (C]O), 1538 (C]N), 836,
762. MS-(þ)APCI, m/z: 296 [MþH]þ. HRMS (DCI, CH4) m/z calcd for
C
18H18NO3 [MþH]þ 296.1287. Found 296.1289.
4.1.2.9. 2-(4-Hydroxy-phenyl)-5-methoxy-indol-3-one
(9).
Violet solid, yield: 55%, mp: >300 ꢂC. 1H NMR (300 MHz, DMSO-
d6):
d
: 3.82 (s, 3H, CH3), 6.91 (d, J ¼ 9 Hz, 2H), 7.12e7.13 (m, 2H), 7.31
(d, J ¼ 8.1 Hz, 1H), 8.14 (d, J ¼ 9 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6)
d: 55.4, 110.9, 114.7 (ꢄ2), 120.1, 121.9, 122.7, 124.4, 131.1 (ꢄ2),
153.3, 159.5, 160.0, 162.5. 194.3 (C]O). IR (KBr, cmꢁ1): 1762, 1721
(C]O),1546 (C]N), 782, 761. MS-(þ)APCI, m/z: 254 [MþH]þ. HRMS
(DCI, CH4) m/z calcd for C15H12NO3 [MþH]þ 254.0817. Found
254.0825.
hypoxanthine incorporation was determined with a
b-counter
(1450-Microbeta Trilux, Wallac-PerkinElmer). Growth inhibition
percentages were plotted as a semilogarithmic function of drug
concentration. The IC50 values were determined by linear regres-
sion analysis on the linear segments of the curves. In each assay,
drugs were tested in triplicate and assays were repeated three
times. Controls were carried out to assess the background (negative
control) and parasite growth (positive control).
4.2. Electrochemistry
Experiments were carried out at 25 ꢂC in acetonitrile containing
tetrabutyl ammonium perchlorate, TBAP 0.1 mol Lꢁ1, using an
Autolab (Methrom) and a conventional cell with three electrodes:
reference electrode: a double junction electrode with the AgCl/Ag
couple in KCl 3 M; counter electrode: a platinum electrode sheet
(5 ꢄ 5 mm); working electrode: a platinum disk (0.5 mm diameter)
for cyclic voltammetry and a rotating disk electrode (EDI, Tacussel)
for stationary conditions. All solutions were deoxygenated (argon
bubbling, 15 min) and a blanket of the inert gas was maintained
during the experiment. Voltamperograms were recorded for a
compound concentration around 10ꢁ3 mol Lꢁ1 (ferrocene or
indolone) and a potential scan rate ranging from 0.02 V/s up to
100 V/s depending on experiments.
4.4.2. In vitro cytotoxicity assay
Cytotoxicity was determined on human breast cancer cells
(MCF7). The cells were cultured in the same conditions as those
used for P. falciparum, except that 10% human serum was replaced
by 10% foetal calf serum (Cambrex). After trypsinization, cells were
distributed in 96-well plates at 2 ꢄ 104 cells/well in 100
mL of cul-
ture medium. After an overnight incubation 100 mL culture medium
containing the tested compounds at various concentrations (the
final concentrations in the wells were 1, 10, and 100
g mLꢁ1) was
added. Cell growth inhibition was estimated by a colorimetric assay
m