Journal of Medicinal Chemistry compounds were tested by 4-point dose−response curves, and IC50 values (between 0.2 and 30 μM) were measured.14 In Vivo PK. In Vivo Pharmacokinetics Studies in Mice. In-life studies were carried out under protocols approved by the Animal Care and Use Committee (IACUC) of GNF. All compounds were formulated at a concentration of 2.5 mg/mL in 75% PEG300 and 25% D5W (5% dextrose in distilled water) solution and were ﬁltered using a 0.45 μm syringe ﬁlter. The ﬁltered solutions were dosed intravenously via the lateral tail vein at 5 mg/kg with a dosing volume of 2 mL/kg to male Balb/c mice (n = 3 per group). The ﬁltered solutions were also administered orally at 20 mg/kg (with a dosing volume of 8 mL/kg) to another group of mice (n = 3 per group). Six blood samples of 50 μL each were collected serially from each animal via retro orbital sinus up to 24 h after dosing. Blood samples were collected into heparin microtainer and centrifuged and plasma separated and frozen until analysis. Plasma samples were analyzed by LC-MS/MS. In Vivo Pharmacokinetics Studies in Rats (Snapshot and Full PK). Compounds were formulated at a concentration of 2.5 mg/mL in 75% PEG300 and 25% D5W solution. In rat snapshot PK, two rats (Wistar rats) were administrated orally at 10 mg/kg dose and four blood samples (100 μL) were taken at 0.5, 1, 3, and 5 h post dosing. Before the sample analysis, the plasma samples were pooled based on the sampling time. In the rat full PK studies, the formulations were ﬁltered before the administration to rats. Three rats were dosed intravenously (3 mg/kg), and three rats were administrated orally (10 mg/kg). Six blood samples (100 μL per sample) were collected serially from each rat within 24 h post dosing. Extraction and LCMS Analysis. Plasma samples (20 μL) were extracted with solution of acetonitrile:methanol (3:1) containing internal standard. The samples were vortexed and then centrifuged with an Eppendorf centrifuge 5810R (Eppendorf, Hamburg, Germany) at a setting of 4000 rpm for 5 min at 4 °C. The supernatant was transferred to a clean 96-well analysis plate for LC/ MS/MS analysis. An aliquot (10 μL) was injected onto a Phenomenex C18 guard column (4 mm × 2 mm) followed by a Zorbax SB-C8 analytical column (2.1 mm × 30 mm, 3.5 μm, Agilent Technologies Inc., Palo Alto, CA, USA). Separation was carried out using a gradient elution method with a mobile phase consisting of 0.05% formic acid in water (solvent A) and 0.05% formic acid in acetonitrile (solvent B). The ﬂow rate was 600 μL/min. The HPLC system, consisting of Agilent 1100 series binary pump (Agilent Technologies Inc.), Agilent 1100 series micro vacuum degasser (Agilent Technologies Inc.), HTC PAL CTC analytics autosampler (LEAP Technologies, Carborro, NC), and VICI two-position actuator (Valco Int., Houston, TX, USA) was interfaced to a MDS SCIEX API-4000 triple quadruple mass spectrometer (MDS Inc., Toronto, Canada). Mass spectral analyses were carried out using electrospray source in the positive ion mode and using multiple reaction monitoring (MRM) for quantiﬁcation. MRM transition of compound 15 383.9/101.2, compound 22 412.2/ 312.1, compound 24 430.1/330.1, compound 27 412.2/327.2, compound 29 436.1/308.1, compound 34 430.0/346.1, and compound 36 355.0/272.3 was used with its optimized MS parameters, respectively. Pharmacokinetic Analysis. The mean value from the three animals at each time point was plotted against time to give plasma concentration time proﬁle. Pharmacokinetic parameters were determined using Watson LIMS, version 7.2 (Thermo Electron Corporation, PA, USA), by noncompartmental analysis. Pharmacoki- netic parameters for compounds were calculated by noncompartmen- tal regression analysis using an in-house ﬁtting program developed at GNF. The oral bioavailability (F) was calculated as the ratio between the area under the curve (AUC) following oral administration and the AUC following intravenous administration corrected for dose (F = AUCpo × doseiv/AUCiv × dosepo). In Vivo Antimalarial Activity. All in vivo eﬃcacy studies were approved by the veterinary authorities of the Canton Basel-Stadt. In vivo antimalarial activity was usually assessed for groups of three female NMRI mice (20−22 g) intravenously infected on day zero with Article 2 × 107 erythrocytes parasitized with P. berghei GFP ANKA malaria strain (PbGFPCON donation from A. P. Waters and C. J. Janse, Leiden University).8a,15 Untreated control mice die typically between day six and day seven post infection. Experimental compounds were formulated in 75% PEG300/25% D5W as indicated. Chloroquine and artesunate were formulated in 10% ethanol, 30% PEG400, and 6% vitamin E TPGS. Compounds were administered orally in a volume of 10 mL/kg either as a single dose (24 h post infection) or as three consecutive daily doses (24, 48, and 72 h post infection). With the single dose regimen, parasitemia was determined (72 h post infection) and for the triple dose regimen (96 h post infection) using standard ﬂow cytometry techniques.15 Activity was calculated as the diﬀerence between the mean percent parasitemia for the control and treated groups expressed as a percentage of the control group. The survival time in days was also recorded up to 30 days after infection. A compound was considered curative if the animal survived to day 30 after infection with no detectable parasites. Experimental Protocols (Chemistry). Materials and Meth- ods. Unless otherwise noted, materials were obtained from commercial suppliers and were used without puriﬁcation. Removal of solvent under reduced pressure refers to distilla- tion using Büchi rotary evaporator attached to a vacuum pump (∼3 mmHg). Products obtained as solids or high boiling oils were dried under vacuum (∼1 mmHg). Puriﬁcation of com- pounds by high pressure liquid chromatography was achieved using a Waters 2487 series with Ultra 120 5 μm C18Q column with a linear gradient from 10% solvent A (acetonitrile with 0.035% triﬂuoroacetic acid) in solvent B (water with 0.05% triﬂuoroacetic acid) to 90% A in 7.5 min, followed by 2.5 min elution with 90% A. 1H NMR spectra were recorded on Bruker XWIN-NMR (400 or 600 MHz). Proton resonances are reported in parts per million (ppm) downﬁeld from tetramethylsilane (TMS). 1H NMR data are reported as multiplicity (s singlet, d doublet, t triplet, q quartet, quint quinted, sept septed, dd doublet of doublets, dt doublet of triplet, bs broad singlet), number of protons, and coupling constant in hertz. For spectra obtained in CDCl3, DMSO-d6, and CD3OD, the residual protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the reference. Analytical thin-layer chromatography (TLC) was performed on commercial silica plates (Merck 60-F 254, 0.25 mm thickness); compounds were visualized by UV light (254 nm). Flash chromatography was performed either by CombiFlash (separation system Sg. 100c, ISCO) or using silica gel (Merck Kieselgel 60, 230− 400 mesh). The purity and quantitative analysis were determined with a Waters ZQ 2000 LC/MS system, which employed an Acquity UPLC system (binary pump, column compartment, autosampler, and diode array UV detector). The eluent was split between the mass spectrometer and an Antek chemiluminescent nitrogen detector (CLND). The mobile phases used were (A) 95% H2O/5% MeOH/ IPA (75/25, v/v) + 0.05% formic acid and (B) MeOH/IPA (75/25, v/v) + 0.035% formic acid. A gradient HPLC method with ﬂow of 0.4 mL/min started at 2% B, with a hold of 1.0 min before a linear increase to 95% B at 3.50 min, with a 30 s hold. From 4.0−4.25 min, the mobile phases were returned to starting conditions. Total run time was 5.0 min. The column used was a Thermo Syncronis C18 2.1 mm × 30 mm, 3 μm. The mass spectrometer was operated in positive mode, with a spray voltage of 3.2 kV and cone voltage of 30 V. The source and desolvation temperatures were 130 and 400 °C, respectively, with 600 L/h of nitrogen desolvation ﬂow. All the ﬁnal compounds reported in this communication had the purity of at least 95%. Elemental analyses were carried out by Midwest microlabs LLC, Indianapolis, IN, USA. Synthesis of 1a. Compound 1a was prepared from 1 by the following method: to a stirred solution of 1 (12 mg, 0.03 mmol) was added MnO2 (52 mg, 0.60 mmol). The reaction mixture was stirred at room temperature (rt) for 2 h. LC/MS test showed that 1 was almost consumed and the desired product ([M + 1] = 428) was detected as one of the major peaks. Solid was ﬁltered oﬀ, and solvent was 4251 dx.doi.org/10.1021/jm300041e | J. Med. Chem. 2012, 55, 4244−4273