Journal of Medicinal Chemistry
ARTICLE
were handled in accordance with the safety instructions in “Good
Laboratory Practice”. Infrared spectra were recorded on a Nicolet-IR-
100 with the sample in thin film (solution in CHCl3) between NaCl
plates, as a Nujol Mull or as a KBr disk. Absorption maxima (νmax) are
recorded in wave numbers (cmꢀ1) and the following abbreviations are
used: w, weak; m, medium; s, strong; br, broad. Proton magnetic
resonance spectra were recorded on a Bruker 400 NMR spectrometer.
Chemical shifts (δH) are quoted in parts per million (ppm) and are
referenced to CDCl3 (δ 7.26 ppm). NMR peaks were assigned by
MestReNova (5.2.2) Carbon magnetic resonance spectra were recorded
on a Bruker 400 NMR spectrometer. Chemical shifts (δC) are quoted in
parts per million (ppm) and are referenced to CDCl3 (δ = 77 ppm).
NMR peaks were assigned by MestReNova (5.2.2). Spectra have been
and the resulting mixture filtered through a 2 μM syringe filter to remove
the Pd/C. The reaction was concentrated in vacuo and then dissolved in
ethyl acetate and washed with 10% K2CO3 solution (aq). The product
was recrystallized from ethanol.
General Procedure for HCl Salt Formation. The propafenone
analogues were dissolved in 1.25 M solution of HCl in methanol then
evaporated to dryness to form HCl salts.
All compounds were purified using normal phase chromatography to
a minimum standard of 95% purity. To improve solubility, all com-
pounds were converted to the hydrochloride salts by dissolving in a 1.25
M solution of HCl in methanol, followed by evaporation of the solvent in
vacuo. Purity was confirmed both by NMR and by UPLC/UV/ELSD/
MS (Waters Affinity).24 Testing was carried out using the purified
hydrochloride salts.
1
assigned, where possible, with use of DEPT, 1Hꢀ H (COSY) and
13
1Hꢀ C (HMQC) correlation spectra. Melting points were recorded
Growth of Parasites and IC50 Determinations. Two P. falciparum
strains were used in this study and were provided by the MR4 Unit of the
American Type Culture Collection (ATCC, Manassas, VA). Those two
strains were the chloroquine sensitive strain 3D7 and the chloroquine resistant
strain K1. Asynchronous parasites were maintained in culture based on the
method of Trager.30 Parasites were grown in presence of fresh group
O-positive erythrocytes (Lifeblood Memphis, TN) in Petri dishes at a
hematocrit of 4ꢀ6% in media consisted of RPMI 1640 supplemented with
0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO3 (pH 7.3), 100 μg/mL
hypoxanthine, and 5 μg/mL gentamycin. Cultures were incubated at 37 °Cin
a gas mixture of 90% N2, 5% O2, and 5% CO2. For IC50 determinations,
20 μLofRPMI1640with5μg/mL of gentamycin were dispensed per well in
an assay plate (Corning 8807BC 384-well microtiter plate). Then 40 nL of
each compound, previously serial diluted in a separate assay plate (Corning
3657 384-well white polypropylene plate), were dispensed in the assay plate,
followed by 20 μL of a synchronized culture suspension (1% rings, 10%
hematocrit), were added per well thus giving a final hematocrit and
parasitemia of 5% and 1%, respectively. Assay plates were incubated for 72
h, and the parasitemia were determined by a method previously described.31
Briefly, 10 μL of the development solution (10ꢁ Sybr Green I, 0.5% v/v
triton, 0.5 mg/mL saponin, in RPMI) was added per well, assay plates were
shaken for 30 s, incubated in the dark for 4 h, and then read with the Envision
spectrophotometer at Ex/Em 485 nm/535 nm. EC50s were calculated with
the robust investigation of screening experiments (RISE) in-house protocol.
Toxicity. BJ, HEK293, Hep G2, and Raji cell lines were purchased
from the American Type Culture Collection (ATCC, Manassas, VA)
and were cultured according to recommendations. Cell culture media
were purchased from ATCC. Cells were routinely tested for mycoplas-
ma contamination using the MycoAlert Mycoplasma Detection Kit
(Lonza). Exponentially growing cells were plated in Corning 384-well
white custom assay plates and incubated overnight at 37 °C in a
humidified incubator with atmosphere controlled at 5% CO2 and
100% humidity. DMSO inhibitor stock solutions were added the
following day to a final concentration of 25 μM, 0.25% DMSO, and
then diluted 1/3 for a total of 10 testing concentrations. Cytotoxicity was
determined following a 72 h incubation using Promega Cell Titer Glo
Reagent according to the manufacturer’s recommendation. Lumines-
cence was measured on an Envision plate reader (Perkin-Elmer)
Ion Channel Activity Panel Screen by Chantest. The in vitro
effects of one test article of propafenone on 12 cardiac ion channels that
are responsible for major components of the cardiac action potential
were evaluated at room temperature using the PatchXpress 7000A
(Molecular Devices), an automatic parallel patch clamp system. Propa-
fenone was evaluated at 10 μM in two cells (n g 2) for each channel. The
duration of exposure was 5 min. The effects were evaluated using
IonWorks Quattro system (MDS-AT). In case of maximal blocking
effect, less than 50% the IC50 value was not calculated.
using a B€uchi-545. Optical rotations were recorded using a Jasco P-1010
at the D line of sodium (λ = 589 nm) to the nearest tenth of a degree.
Thin layer chromatography was performed on precoated silica gel 60
F254 plates and determined using UV fluorescence. Flash column
chromatography was performed with Merck Kieselgel 60 (239ꢀ400)
mesh silica or the Biotage SP1 flash column system. Rf values are quoted
for the eluent given unless otherwise stated. Evaporation took place on a
B€uchi Rotavapor or in the Genevac HT series. Microwave iIrradiation
was carried out in a Biotage Initiator 60
General Procedure for Chalcone Formation. A mixture of the
acetophenone (1 equiv) and the corresponding aldehyde (1 equiv) in
anhydrous ethanol (70 mL/23 mmol of acetophenone) was stirred at
room temperature for 5 min. NaOH (3 equiv) was added, and the
reaction mixture was stirred at room temperature until completion. HCl
(10%) was added to dissolve the sodium salt, and the product was
extracted with EtOAc and washed with brine to give the products as
bright-yellow solids. The product was recrystallized from ethanol.
General Procedure for Selective MW Enone Reduction.
Chalcone (1 equiv), 1,4-cyclohexene (10 equiv), and 10% Pd/C (0.1
equiv) were placed in a microwave vial. Ethanol (18 mL/500 mg of
propafenone) was added, and the vial was sealed. The vial was placed in
the microwave, prestirred for 30 s, and irradiated for 10 min at 100ꢀ
300 W and 140 °C. The vial was cooled, the cap removed, and an equal
volume of acetonitrile was added to the reaction mixture and the
resulting mixture filtered through a 2 μM syringe filter to remove the
Pd/C. The reaction was concentrated in vacuo and dissolved in ethyl
acetate and washed with 10% K2CO3 solution (aq). The product was
recrystallized from ethanol.
General Procedure for Epoxide Formation. o-Hydroxyphe-
none (1 equiv) was dissolved in epichlorohydrin (30 equiv), and
powdered NaOH (1.2 equiv) was added. The reaction mixture was
refluxed for 18 h and then allowed to cool to room temperature and
concentrated in vacuo. The yellow oil was dissolved in Et2O, washed with
water, dried with MgSO4, and then concentrated in vacuo to give a colorless
solid. The product was purified by flash chromatography with 100% DCM.
General Procedure for Epoxide Opening. Amines (1 equiv)
were weighed into microwave vials. The epoxide (1equiv) was dissolved
in ethanol (0.24 M solution) and added to the vials. Each vial was
irradiated for 15 min at 100ꢀ300 W and 160 °C. Unreacted amine was
extracted with PS-isocyanate. Alternatively the product was purified by
flash chromatography. On a large scale, the products can generally be
recrystallized from ethanol.
General Procedure for Selective Nitro Reduction. The
4-nitro-phenyl piperidine compounds (1 equiv), cyclohexene (10
equiv), and 10% Pd/C (0.1 equiv) were placed in a microwave vial.
Ethanol (18 mL/500 mg of starting material) was added, and the vial was
sealed. The vial was placed in the microwave, prestirred for 30 s, and
irradiated for 10 min at 300 W and 140 °C. The vial was cooled, the cap
removed, and an equal volume of acetonitrile was added to the reaction
Ion Channel Activity of hERG, hNav1.5, and Kir6.2/SUR2A.
The channels tested were as follows: Cloned hERG potassium channels
(encoded by the KCNH2 gene and expressed in CHO cells), responsible
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dx.doi.org/10.1021/jm2005546 |J. Med. Chem. 2011, 54, 7477–7485