All chemicals were purchased from commercial sources and used without further purification. All glassware was pre-dried in an oven. All stirring was performed with an internal magnetic stirrer. All solvents were distilled or purified where necessary in accordance with D. D. Perrin and W. L. F. Armarego in “Purification of Laboratory Chemicals”, Pergamon Press, Fourth Edition, 1996. All chemicals 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 disc. Absorption maxima (νmax) are recorded in wave numbers (cm−1) 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 assigned, where possible, with use of DEPT, 1H-1H (COSY) and 1H-13C (HMQC) correlation spectra. Melting points were recorded using a Büchi-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 pre-coated silica gel 60 F254 plates and determined using u.v fluorescence. Flash column chromatography was performed with Merck Kieselgel 60 (239–400) 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 Büchi Rotavapor or in the Genevac HT series. Microwave Irradiation was carried out in a Biotage® Initiator 60
General Procedure for chalcone formation
A mixture of the acetophenone (1 eq.) and the corresponding aldehyde (1 eq.) in anhydrous ethanol (70 mL/23 mmol of acetophenone) was stirred at room temperature for 5 min. NaOH (3 eq.) 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 re-crystallized from ethanol.
General procedure for selective MW enone reduction
Chalcone (1 eq.), 1,4-cyclohexene (10 eq.) and 10% Pd/C (0.1eq.) were placed in a microwave vial. Ethanol (18mL/500mg of propafenone) was added and the vial was sealed. The vial was placed in the microwave, pre-stirred for 30 s and irradiated for 10 min. at 100–300 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 re-crystallized from ethanol.
General procedure for epoxide formation
o-Hydroxyphenone (1 eq.) was dissolved in epichlorohydrin (30 eq.), and powdered NaOH (1.2 eq.) was added. The reaction mixture was refluxed for 18 h then allowed to cool to room temperature and concentrated in vacuuo. The yellow oil was dissolved in Et2O, washed with water, dried with MgSO4 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 eq.) were weighed into microwave vials. The epoxide (1eq.) was dissolved in ethanol (0.24 M solution) and added to the vials. Each vial was irradiated for 15 min. at 100–300 W and 160 °C. Un-reacted amine was extracted with PS-Isocyanate. Alternatively the product was purified by flash chromatography. On a large scale the products can generally be re-crystallized from ethanol.
General procedure for selective nitro reduction
The 4-nitro-phenyl piperidine compounds (1 eq.), cyclohexene (10 eq.), and 10% Pd/C (0.1 eq.) 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, pre-stirred 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 and the resulting mixture filtered through a 2 µM syringe filter to remove the Pd/C. The reaction was concentrated in vacuuo then dissolved in ethyl acetate and washed with 10% K2CO3 solution (aq.). The product was re-crystallized 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 compounds 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.
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 Trager30
. Parasites were grown in presence of fresh group O-positive erythrocytes (Lifeblood Memphis, TN) in Petri dishes at a hematocrit of 4–6% 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° C in a gas mixture of 90% N2
, 5% O2
, 5% CO2
. For IC50
determinations, 20 µL of RPMI 1640 with 5 µg/mL gentamycin were dispensed per well in an assay plate (Corning 8807BC 384-well microtiter plate). 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 (10X 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, then read with the Envision spectrophotometer at Ex/Em 485nm/535nm. EC50
s were calculated with the RISE (Robust Investigation of Screening Experiments) in-house protocol.
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 mycoplasma 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 ten testing concentrations. Cytotoxicity was determined following a 72 h incubation using Promega Cell Titer Glo Reagent according to the manufacturer’s recommendation. Luminescence was measured on an Envision plate reader (Perkin Elmer)
Ion Channel Activity Panel Screen by Chantest
The in vitro effects of one test article 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. Propafenone was evaluated at 10 µM in two cells (n ≥ 2) for each channel. The duration of exposure was 5 minutes. The effects were evaluated using IonWorks Quattro system (MDS-AT). In case of maximal blocking effect less then 50% the IC50 value was not calculated.
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 for IKr cloned hNav1.5 sodium channel (encoded by the human SCN5A gene and expressed in CHO cells), responsible for INa, fast sodium current. Cloned Kir6.2/SUR2A potassium channels (expressed by the human KCNJ11 and SUR2A genes and co-expressed in HEK293 cells), responsible for the ATP-sensitive current, IKATP.
The in vitro effects of four test articles were evaluated using these cells at room temperature using the PatchXpress 7000A (Molecular Devices), an automatic parallel patch clamp system. Each test article was evaluated at 0.2, 0.6, 1.6, and 5 µM with each concentration tested in two cells (n ≥ 2). The duration of exposure to each test article concentration was 5 minutes.
Solubility assays were carried out on a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using µSOL Evolution software (pION Inc., Woburn, MA). The detailed method is described as follows: 10 µL of compound stock was added to 190 µL 1-propanol to make a reference stock plate. 5 µL from this reference stock plate was mixed with 70 µL 1-propanol and 75 µL phosphate buffered saline (PBS, pH 7.4 and 4) to make the reference plate, and the UV spectrum (250 nm – 500 nm) of the reference plate was read. 6 µL of 10 mM test compound stock was added to 600 µL PBS in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate (pION Inc., Woburn, MA). 75 µL of filtrate was mixed with 75 µL 1-propanol to make the sample plate, and the UV spectrum of the sample plate was read. Calculation was carried out by µSOL Evolution software based on the AUC (area under curve) of UV spectrum of the sample plate and the reference plate. All compounds were tested in triplicate.
The Parallel Artificial membrane Permeability Assay (PAMPA) was conducted on a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using the PAMPA Evolution software (pION Inc., Woburn, MA). The detailed method is described as follows: 3 µL 10 µM test compound stock was mixed with 600 µL of SSB (system solution buffer, pH 7.4 or 4, pION Inc., Woburn, MA) to make diluted test compound. 150 µL of diluted test compound was transferred to a UV plate (pION Inc., Woburn, MA) and the UV spectrum was read as the reference plate. The membrane on pre-loaded PAMPA sandwich (pION Inc., Woburn, MA) was painted with 4 µL GIT lipid (pION Inc., Woburn, MA). The acceptor chamber was then filled with 200 µL ASB (acceptor solution buffer, pION Inc., Woburn, MA), and the donor chamber was filled with 180 µL diluted test compound. The PAMPA sandwich was assembled, placed on the Gut-box and stirred for 30 minutes. The aqueous Boundary Layer was set to 40 µm for stirring. The UV spectrum (250–500 nm) of the donor and the acceptor were read. The permeability coefficient was calculated using PAMPA Evolution software (pION Inc., Woburn, MA) based on the AUC of the reference plate, the donor plate, and the acceptor plate. All compounds were tested in triplicate.
Liver microsomal stability
0.633 mL of mouse liver microsomes (20 mg/mL, female CD9 mice, Fisher Scientific, #NC9567486) or human liver microsomes (20 mg/mL, 200 pooled mixed gender, Fisher Scientific #50-722-552) was mixed with 0.051 mL of 0.5 M EDTA solution and 19.316 mL potassium phosphate buffer (0.1M, pH 7.4, 37°C) to make 20 mL of liver microsome solution. 1 part of 10 mM DMSO compound stock was mixed with 4 part of acetonitrile to make 2 mM diluted compound stock in DMSO and acetonitrile. 29.1 µL of the diluted compound stock was added to 2.3 mL of liver microsomal solution and vortexed to make a microsomal solution with compound. 180 µL of the microsomal solutions with different compounds were dispensed into respective rows of a 96-well storage plate (pION Inc., MA, #110323). For 0 hour time point, 450 µL pre-cooled (4 °C) internal standard (10 µM warfarin in methanol) was added to the first three columns before the reaction starts. 1.25 mL of microsome assay solution A (Fisher Scientific, #NC9255727) was combined with 0.25 mL of solution B (Fisher Scientific, #NC9016235) in 3.5 mL of potassium phosphate buffer (0.1 M, pH 7.4). 45 µL of this A+B solution was added to each well of the 96-well storage plate (reaction plate). Liquid in the first 3 columns was moved to another storage plate (quenched plate). The reaction plate was then sealed and incubated at 37 °C, shaken at a speed of 60 rpm. The solutions were sampled at 0.5 hr, 1 hr, and 2 hr time points. At each time point, 450 µL of pre-cooled internal standard was added to 3 rows in the reaction plate, and the liquid was then transferred to the quenched plate. The quenched plate was then centrifuged (model 5810R, Eppendorf, Westbury, NY) at 4000 rpm for 20 minutes. 200 µL supernatant was then transferred to a 96-well plate and analyzed by UPLC-MS (Waters Inc., Milford, MA). The compounds and internal standard were detected by SIR. The log peak area ratio (compound peak area / internal standard peak area) was plotted against time and the slope was determined to calculate the elimination rate constant [k = (−2.303) * slope]. The half life (hour) was calculated as t (1/2) = 0.693 / k.