Synthesis and antimalarial evaluation of amide and urea derivatives based on the thiaplakortone A natural product sca ﬀ old †

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Introduction
Malaria is an infectious disease caused by parasites belonging to the genus Plasmodium and is a major health and economic problem globally.An estimated 3.2 billion people living in tropical and subtropical regions of the world, such as Sub-Saharan Africa, Central and South America, the Middle East, India and South East Asia are at risk from this disease. 1The World Health Organisation estimated that in 2010 there were 207 million clinical cases and 627 000 deaths due to malaria, with more than 90% of malaria cases and the majority of malaria deaths occurring in Sub-Saharan Africa. 2 Despite recent data on a partially effective pre-erythrocytic stage vaccine (RTS,S), 3,4 malaria prevention and treatment currently relies on small molecule drugs and vector control.It is predicted that even if the RTS,S vaccine is approved for clinical use, it will be implemented as part of a multi-pronged approach that will also include antimalarial drugs.6][7][8] Consequently, new antimalarial drugs with novel targets are urgently needed in order to combat the global problem of parasite drug resistance.
In recent efforts to discover new antimalarial leads from nature, a high-throughput antimalarial screening campaign using a pre-fractionated natural product library 9,10 identified four novel antimalarial thiazine-derived alkaloids, thiaplakortones A-D (1-4; Fig. 1), from the Australian marine sponge Plakortis lita. 11Thiaplakortone A (1) was the most active natural product with IC 50 values of 51 and 6.6 nM against chloro-Fig. 1 Chemical structures of the natural products thiaplakortones A-D (1-4) and previously synthesised methyl analogues (5-7).† Electronic supplementary information (ESI) available: 1 H and representative quine-sensitive (3D7) and multi-drug resistant (Dd2) P. falciparum malaria parasite lines, respectively. 11he first total synthesis of 1, along with a series of monoand di-methyl analogues (5-7), has been reported and some preliminary structure-activity relationships ascertained. 12hile in vivo toxicity effects for several of the synthetic compounds indicated potential liabilities associated with this structure class, the limited number of analogues investigated made it difficult to assess their true potential as antimalarial leads.To more thoroughly explore this compound class we generated a series of amide and urea analogues based on the thiaplakortone A scaffold.Herein we report the synthesis of these compounds (8-46) along with their in vitro antimalarial and cytotoxicity activities, in vivo tolerability, ADME profiles and antimalarial efficacy in a murine malaria model.

Chemistry
With the synthetic route for the lead molecule thiaplakortone A previously optimised, 12 suitable quantities of the natural product were available to commence more extensive structureactivity investigations.To address the possible toxicity associated with the primary amino group of thiaplakortone A, initial analogues focused on replacement of this motif with a series of amides and ureas.
The amide series (8-32) was accessed through established procedures, by either exposure of thiaplakortone A to acid anhydrides, acyl chlorides or amide formation under standard HBTU protocols (Scheme 1). 13On scale-up, methods using HBTU were avoided, since yields were poor and purification was not trivial.These shortcomings were addressed by reaction of 1 with carbonyl imidazoles, 14 that allowed for enhanced yields and undemanding purifications.During the course of these studies, single crystal X-ray structures were obtained on the hydrochloride salt of the natural product 1 and its propyl amide analogue 9.These data represent the first X-ray struc-tures of this class and confirmed the previously reported NMRbased assignment 11 of the thiazine ring regiochemistry (Fig. 2).
Synthesis of analogues (33-34, 36-39) in the urea series was accomplished by treatment of thiaplakortone A with commercially available isocyanates.In the case of compounds 37 and 39 these molecules were synthesised using their respective carbamoyl imidazoles (Scheme 1). 15,16he indole-N-alkylated acetamides 40-45 were accessed from either indole-N-alkylation of Boc-protected thiaplakortone A (35) 12 followed by subsequent deprotection and acetamide formation with N-acetyl imidazole or from direct alkylation of the acetamide 8 (Scheme 2).Compound 40 was accessed from acetylation of the previously reported N-methyl analogue 5 12 with N-acetylimidazole.

In vitro antimalarial activity and mammalian cell cytotoxicity
Synthetic analogues 8-45 were all evaluated for in vitro antimalarial activity against the chloroquine-sensitive 3D7 and the multidrug-resistant Dd2 P. falciparum lines.To compare the selectivity of the compounds for malaria parasites versus normal mammalian cells, cytotoxicity tests were carried out using the human neonatal foreskin fibroblast (NFF) cell line.All biological data, including selectivity indices (SI) are detailed in Tables 1-3.

Structure-activity relationships
In regards to the amide analogues (8-32), the series of aliphatic amides showed minimal differences in potency and selectivity as the aliphatic chain increased in length or branch-ing.For instance, replacing the acetamide side chain in 8 with an n-pentylamide unit in 13 resulted in a 1.3-fold and 2.8-fold decrease in potency against the 3D7 and Dd2 lines, respectively.The SI for the straight chain amides (8-10, 13) clearly showed a decreasing trend as the chain was extended from   In vitro profiling of several analogues towards an atovaquoneresistant Plasmodium falciparum line (C2B) Atovaquone is a synthetic quinone-based antimalarial drug, which is typically co-administered with proguanil for the treatment of P. falciparum infection.1][22] In vitro screening results using the C2B line showed that 1, 2, 8, 16, 33 and 38 had IC 50 values of 52 ± 9.8, 377 ± 49, 324 ± 70, 286 ± 26, 383 ± 108, and 331 ± 124 nM, respectively.These C2B data when compared to the 3D7 strain (Tables 1 and 2) showed only minimal differences, with most compounds displaying slightly more activity towards the C2B line (cf.to 3D7).Compound 39 showed the largest IC 50 difference, however this equated to only a 2.0-fold change in IC 50 value.These data indicate that the thiaplakortone structure class does not affect Plasmodium parasite growth via inhibition of the cytochrome bc1 complex.

ADME and pharmacokinetic profiling
A series of eight compounds was assessed for physicochemical (Table 4) and metabolic stability characteristics (Table 5).All compounds exhibited high total polar surface area values in excess of 120 Å 2 and a high number of hydrogen bond accepting groups, suggesting that absorption following oral administration would likely be compromised.With the exception of 32 and 39, all compounds had relatively low log D 7.4 values (0.3-1.3) and the majority had good aqueous solubility (50-100 µg mL −1 or higher) at pH values representative of the gastric (pH 2.0) and intestinal (pH 6.5) environments.For 32 and 39, log D 7.4 values   This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
The metabolic stability of the same eight compounds was then assessed in vitro using NADPH supplemented mouse liver microsomes. 23All of the compounds exhibited good metabolic stability (in vitro intrinsic clearance (CL int ) of ≤10 µL min −1 mg −1 protein) with the exception of 32 and 39, which both exhibited a high degree of metabolic lability (CL int > 100 µL min −1 mg −1 protein).Two compounds, the amide analogue 8 and the urea analogue 33, were progressed to an in vivo study where they were administered to mice at a dose of 32 mg kg −1 via either the oral or subcutaneous route (Fig. 3).The apparent half-lives were approximately 2 h, and as expected given the physicochemical properties, the exposure [area under the curve (AUC) over 8 h and maximum plasma concentration (C max )] following oral administration was more than 10-fold lower than that after subcutaneous dosing.Following subcutaneous administration, plasma concentrations for each compound remained above 0.2 µM for at least 8 h.

In vivo tolerability studies in mice
The amide (8, 16, 32) and urea (33, 34, 37, 38) analogues were evaluated for tolerability in mice at 8 mg kg −1 , 16 mg kg −1 and 32 mg kg −1 given twice daily for 4 days by subcutaneous administration.At these doses the analogues were found to be well tolerated in healthy mice with no observable adverse events such as tremors, panting, loss of weight (>20%) and lack of natural movement.These findings are in contrast to the poor tolerance seen with the methyl derivatives of thiaplakortone A reported elsewhere. 12 vivo efficacy studies in the P. berghei malaria mouse model Based on the favorable tolerability findings of the thiaplakortone A amide derivatives (8, 16, 32) and urea (33, 34, 37, 38)  analogues, the efficacy of these compounds was assessed in the rodent-P.berghei model using the highest tested dose of 32 mg kg −1 .Compounds were administered subcutaneously twice daily for 4 days.Of the amide analogues tested, 8 was the most active with a mean suppression of blood stage parasitemia of 52% on D + 4 post inoculation of P. berghei to mice.The next most active amide analogue was 32 with 11% suppression on D + 4. Fig. 4 compares the mean parasitemia profile of P. berghei infected mice treated with either 8, chloroquine ( positive control) or drug-free vehicle (control).Chloroquine suppression at D + 4 was 65% at 1 mg kg −1 day −1 given subcutaneously, which is comparable to the 50% suppression at 1.5 mg kg −1 day −1 described by Ridley et al. 24 Of the urea analogues tested, 33 was the most active with 26% suppression of parasitemia on D + 4 following subcutaneous administration.Analogues 37 and 38 had similar suppression values of 16% and 14%, respectively, on D + 4. When orally administered at 32 mg kg −1 twice daily for 4 days both compounds 8 and 33 did not significantly inhibit parasitemia (<3%) on D + 4.

Conclusions
In summary, 31 amide and 7 urea derivatives based on the thiaplakortone A tricyclic scaffold were synthesised and evaluated for their in vitro antimalarial activity and mammalian cell  toxicity.Of these compounds, eight were chosen for ADME profiling after which several analogues were prioritised for in vivo pharmacokinetic analysis, followed by tolerability and antimalarial efficacy in a murine malaria model.Compounds 8 and 33 were both well tolerated in mice when administered subcutaneously at 32 mg kg −1 twice daily for 4 days.Furthermore, using this regimen blood stage P. berghei was suppressed by 52% for 8 and 26% for 33, relative to the vehicle control.Unfortunately, oral administration of these compounds at 32 mg kg −1 twice daily for 4 days did not suppress parasitemia, most likely due to poor oral bioavailability and limited exposure.While these data highlight the potential of the natural product derived thiazino-quinone scaffold as an anti-malarial starting point, the lack of oral bioavailability currently impinges on the further development of this series.

General procedures
Melting points were recorded on a capillary melting point apparatus and are uncorrected.Unless otherwise specified, General procedure A -HBTU mediated amide formation.A solution of carboxylic acid (46 µmol) in DMF (1 mL) and N,Ndiisopropylethylamine (DIPEA) (25 µL, 182 µmol), was treated with HBTU (21 mg, 54 µmol) in one portion and stirred for 1 h at rt. Thiaplakortone A hydrochloride (15 mg, 46 µmol) was added and stirred for a further 2 h.The mixture was concentrated in vacuo and then the residue was triturated with a solution of DCM-ether (1 : 3, 3 × 1 mL).The remaining solid was purified by flash chromatography (silica, 1 : 5 v/v MeOH-DCM elution) to afford the amide.
General procedure Cisocyanate mediated urea formation.A suspension of thiaplakortone A hydrochloride (10 mg 30 µmol) in DMF (1 mL), was treated with DIPEA (10 µL) and stirred vigorously for 5 min.The mixture was then cooled to 0 °C and treated with isocyanate (33 µmol) in one portion.After 1 h the solution was concentrated in vacuo and the residue purified by flash chromatography (amino-bonded silica, 1 : 10 v/v MeOH-DCM elution) to afford the urea.
General procedure Dcarbonyl imidazole mediated amide formation.A solution of carboxylic acid (0.66 mmol) and 1,1′carbonyldiimidazole (98 mg, 0.60 mmol) in anhydrous THF (2 mL) was refluxed for 1 h and then cooled to rt.This solution was then transferred via syringe to a mixture of thiaplakortone A hydrochloride (200 mg, 0.60 mmol) and DIPEA (93 µL, 0.66 mmol) in DMF (6 ml) and the reaction was magnetically stirred for 18 h.The mixture was concentrated to dryness in vacuo and the residue purified by flash chromatography (silica, 1 : 10 v/v MeOH-DCM elution) to afford the amide.
General procedure Efreebasing protocol.A solution of amine hydrochloride (0.4 mmol) in H 2 O (2 mL) was treated with aqueous NH 3 (1 mL, 30% w/w) and stirred for 0.25 h.The solution was then concentrated in vacuo to afford the amine that was used without further purification.
General procedure F -Boc deprotection protocol.Boc-protected amine (45 µmol) was treated with HCl (1 mL, 4 M solution in 1,4-dioxane) at 0 °C and stirred for 0.5 h.The mixture was warmed to 40 °C and then concentrated with a gentle stream of nitrogen (CAUTION!Highly corrosive vapours).The resulting residue was then triturated with ether (3 × 1 mL) and dried in vacuo to afford the amine that was used without further purification.
Reversed-phase HPLC method 1. Isocratic conditions of 90% H 2 O (0.1% TFA)/10% MeOH (0.1% TFA) were employed for the first 10 min, then a linear gradient to MeOH (0.1% TFA) was run over 40 min, followed by isocratic conditions of MeOH (0.1% TFA) for a further 10 min, all at a flow rate of 9 mL min −1 .Sixty fractions (60 × 1 min) were collected by time from the start of the HPLC run.All UV active fractions were dried down and analyzed by 1 H NMR spectroscopy, with relevant fractions combined.
Reversed-phase HPLC method 2. Isocratic conditions of 95% H 2 O (0.1% TFA)/5% MeOH (0.1% TFA) were employed for the first 10 min, followed by a 40 min linear gradient to 50% H 2 O (0.1% TFA)/50% MeOH (0.1% TFA) then isocratic conditions of MeOH (0.1% TFA) for a further 10 min, all at a flow rate of 9 mL min −1 .Sixty fractions (60 × 1 min) were collected by time from the start of the HPLC run.All UV active fractions were dried down and analyzed by 1 H NMR spectroscopy, with relevant fractions combined.
Compound 1.The hydrochloride salt of thiaplakortone A was prepared in an analogous method to that previously reported in the literature. 12The NMR and MS data of 1 were identical to the literature values. 12Recrystallised from H 2 O to afford brown crystals; mp decomp.>280 °C.
Compound 8. Prepared using a method analogous to General Procedure B, orange powder (7 mg, 59%); 1   Compound 11.A magnetically stirred mixture of thiaplakortone A hydrochloride (10 mg, 30 µmol), pyridine (0.5 mL) and dioxane (0.5 mL) maintained under nitrogren at 0 °C was treated with cyclopropanecarbonyl chloride (6.3 mg, 61 µmol) and warmed to rt.The mixture was stirred for 18 h then concentrated in vacuo to afford a residue that was purified by flash chromatography (silica, 1 : 5 v/v MeOH-DCM elution) to afford compound 11 (1.5 mg, 14%) as a brown powder. 1  Compound 16.A mixture of thiaplakortone A hydrochloride (100 mg, 0.30 mmol), anhydrous DMF (5 mL) and benzoic anhydride (460 mg, 1.82 mmol, 90% grade) was treated with TEA (500 µL, excess) and stirred overnight under an atmosphere of nitrogen.The mixture was concentrated in vacuo and purified by flash chromatography (silica, 1 : 10 v/v MeOH-DCM elution) to afford the compound 16 (54 mg, 45%) as a yellow powder. 1   Compound 31.Prepared using a method analogous to General Procedure A, brown powder (2.5 mg, 20%); 1  Compound 35.Boc-protected thiaplakortone A was prepared in an analogous method to that previously reported in the literature. 12The NMR and MS data of 35 were identical to the literature values. 12ompound 36.Prepared according to General Procedure C, yellow powder (5 mg, 34%); 1  Compound 37. A mixture of thiaplakortone A hydrochloride (220 mg, 0.67 mmol), N-methoxy-N-methyl-1H-imidazole-1-carboxamide (400 mg, 2.60 mmol) 15 and TEA (220 µL, 1.57 mmol) in DMF (10 mL) was heated at 50 °C under an atmosphere of nitrogen for 18 h.After solvent removal the residue was purified by flash chromatography (silica, 1 : 10 v/v MeOH-DCM elution) to afford urea 37 (67 mg, 26%) as an orange powder; 1  X-ray structure determination of thiaplakortone A (1) and n-propyl amide analogue (9)   Unique data sets for compounds 1 as the dihydrate and 9 with solvated DMSO were measured at 223 and 200 K respectively on an Oxford-Diffraction GEMINI S Ultra CCD diffractometer (Mo-K α radiation, graphite monochromation) utilising CrysAlis software. 27The structures were solved by direct methods and refined by full matrix least squares refinement on F 2 .Anisotropic thermal parameters were refined for non-hydrogen atoms; (x, y, z, U iso ) H were included and constrained at estimated values.Conventional residuals at convergence are quoted; statistical weights were employed.Computation used, SIR-97, 28 SHELX97, 29 TeXsan, 30 ORTEP-3 31 and PLATON 32 programs and software systems.

In vitro P. falciparum growth inhibition assays
The in vitro growth inhibitory activity of compounds was tested against P. falciparum lines 3D7, Dd2, and C2B using the [ 3 H]hypoxanthine incorporation assay.Chloroquine ( positive control) was prepared as a 10 mM stock solution in PBS.All other compounds were prepared as 20 mM stock solutions in DMSO.Serial dilutions of compounds/controls were prepared in hypoxanthine-free culture media, followed by addition of [ 3 H]-hypoxanthine (0.5 µCi per well) and asynchronous cultures at 1% parasitemia and 1% final haematocrit.The antimalarial drugs chloroquine or atovaquone were included in assays as internal controls.Following incubation for 48 h, the amount of [ 3 H]-hypoxanthine incorporated into parasites was determined by harvesting cultures onto glass fiber filter mats and counting using a Perkin Elmer/Wallac Trilux 1450 MicroBeta scintillation counter.Percentage inhibition of growth compared to matched DMSO controls (0.5%) was determined.IC 50 values were determined using log-linear interpolation of inhibition curves and are presented as mean (± SD) of three independent assays, each carried out in triplicate wells.Chloroquine [IC 50 = 0.109 ± 0.060 µM (Dd2); IC 50 = 0.012 ± 0.004 µM (3D7)] and atovaquone [IC 50 = 0.0002 ± 0.0001 µM (3D7) IC 50 = 5.750 ± 0.778 µM (C2B)] served as positive controls.

In vitro cytotoxicity assay
Cytotoxicity against a mammalian cell line (neonatal foreskin fibroblast cells; NFF) was assessed by culturing cells in RPMI media (Life Technologies) supplemented with 10% heat inactivated foetal calf serum (CSL Biosciences) and 1% streptomycin (Life Technologies).Chloroquine was prepared as a 10 mM stock solution in PBS.All other compounds were prepared as 20 mM stock solutions in DMSO.Cells were seeded into wells of 96-well tissue culture plates (3000 cells per well) and cultured for 24 h at 37 °C in 5% CO 2 before being treated with a dilution series of each compound/control.After 72 h, medium was removed and plates washed in phosphate buffered saline pH 7.4 (PBS), before fixing with denatured alcohol.Fixative was removed and washed from cells before the addition of sulforhodamine B (0.4%; Sigma 50 µL).After staining for 1 h, plates were washed three times with 1% acetic acid, then 100 µL of 10 mM Tris base (unbuffered, pH > 9) was added to each well.Plates were read at 564 nm in an ELISA micro-plate reader.Percentage inhibition of growth as compared to matched DMSO controls (0.5%) was determined and IC 50 values calculated using log-linear interpolation of inhibition curves.Data are presented as mean ± SD of three independent assays, each carried out in triplicate wells.The antimalarial drug chloroquine was included as an internal control in each assay, and was shown to display an IC 50 of 46 ± 14 µM.

Physicochemical and in vitro metabolic stability assays
In silico parameters were calculated using JChem for excel (ChemAxon, Budapest).Kinetic solubility in 0.01 M HCl ( pH 2) and phosphate buffer ( pH 6.5) was determined by serial dilution of a concentrated stock solution prepared in DMSO and the solubility range was determined by nephelometry.Log D was estimated at pH 7.4 using a chromatographic method as described previously. 23etabolic stability was assessed in vitro by incubating with mouse liver microsomes (Xenotech, Lenexa, KS) at 37 °C using substrate concentrations of 1 µM and a microsomal protein concentration of 0.4 mg mL −1 .Addition of an NADPH-regenerating buffer system (containing 1 mg mL −1 NADP, 1 mg mL −1 glucose-6-phosphate, 1 U mL −1 glucose-6-phosphate dehydrogenase and 0.67 mg mL −1 MgCl 2 ) was used to initiate the metabolic reactions, and samples were quenched at various time points over 60 min by the addition of chilled MeCN.Control samples (containing no NADPH) were included to monitor for potential degradation in the absence of cofactor.Samples were analysed by LC-MS on a Waters/Micromass Xevo G2 QTOF or a Waters/Micromass Quattro Ultima PT Triple Quadrupole MS, each coupled to a Waters Acquity uPLC (Milford, MA) under positive electrospray ionisation.Chromatography was conducted using a Supelco Ascentis Express reverse phase C 8 or C 18 column (50 × 2.1 mm, 2.7 µm) (Sigma-Aldrich, St Louis, MO), equipped with a Phenomenex Security-Guard column hosting a Luna C 8 cartridge (Torrance, CA) and both were maintained at a temperature of 40 °C.The mobile phase consisted of MeCN and H 2 O (containing 0.05% formic acid) mixed using a binary gradient at a flow rate of 0.4 mL min −1 .The first-order rate constant for substrate depletion was used to calculate the in vitro intrinsic clearance.
In vivo amide and urea analogue exposure in mice All animal studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee.The systemic exposure of the amide 8 and the urea 33 analogues was assessed in non-fasted, noninfected female Swiss Outbred mice following administration of 32 mg kg −1 by oral gavage (0.2 mL per mouse) or subcutaneous injection (0.2 mL per mouse) under the skin in the abdomen.Formulations for both routes comprised 10% v/v EtOH, 10% v/v Tween 80 (Sigma Chemical Co., St Louis, MO), and 80% v/v MilliQ H 2 O. Blood samples were taken over 8 h with two samples per mouse; one via submandibular bleed (∼120 µL, conscious sampling) and the other via terminal cardiac puncture (0.6 mL under isofluorane anesthesia) after which animals were euthanised by cervical dislocation.Blood samples were transferred to tubes containing heparin and a stabilisation cocktail (complete protease inhibitor cocktail (Roche), potassium fluoride, and EDTA) to minimise the potential for ex vivo degradation.
Following collection, blood samples were centrifuged and plasma stored at −20 °C prior to analysis.Plasma samples were assayed by first thawing, spiking with internal standard (diazepam) and precipitating plasma proteins with the addition of MeCN (2-fold volume ratio), centrifuging and injecting the supernatant onto an LCMS system (Micromass Xevo TQ triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC).The column was a Supelco Ascentis Express RP Amide column (2.7 µm, 50 mm × 2.1 mm i.d.) equipped with a Phenomenex Security Guard column with Synergy Polar packing material, and both columns were maintained at 40 °C.The mobile phase consisted of MeCN and H 2 O, both containing 0.05% v/v formic acid, and delivered using a linear gradient over 3.3 min followed by re-equilibration to the starting conditions.The flow rate was 0.4 mL min −1 , and the injection volume was 3 µL.LCMS analysis was conducted in positive mode electrospray ionisation and elution of the analytes monitored in MRM mode.Concentrations were determined by comparison to a set of calibration standards prepared in blank mouse plasma.Accuracy was within ± 10%, and precision (% RSD) was less than 11%.The limit of quantitation was 0.5 ng mL −1 for 8 and 1 ng mL −1 for 33.
Pharmacokinetic parameters were determined using noncompartmental analysis (WinNonlin version 5.2, Pharsight, Mountain View, CA) and included the maximum plasma concentration (C max ), the time to reach the maximum concentration (T max ), the apparent terminal elimination half-life (where it could be determined) and the area under the plasma concentration versus time profile from 0 to 8 h (AUC 0-8h ).

In vivo tolerability assessment in mice
Animal studies were approved by the Army Malaria Institute Animal Ethics Committee (AEC no.05/13) in accord with the Australian Code of Practice for the care and Use of Animals for Scientific Purposes.Tolerability assessment of amide and urea analogues were carried out in groups of three healthy mice administered different doses of compound.The mice were male and female Outbred ARC Swiss mice (Animal Resource Centre, Western Australia) aged between 6 and 7 weeks, with a mean weight of 28 ± 3 g.The animals were observed for physical distress twice daily.Physical adverse events that were monitored included reduced activity and movement, tremour, panting, behaviour changes, reduced appetite, extreme pallor, ruffled coat and weight loss.If any animal exhibited physical adverse events that appeared highly stressful they were euthanised.All compounds were dissolved in 10% EtOH/10% Tween 80/80% distilled H 2 O and administered subcutaneously to mice twice daily at about 6 h apart for 4 consecutive days.
In vivo efficacy assessment in the rodent-P.berghei model The in vivo efficacy of amide and urea analogues were determined using a modified Peters 4 day test. 33This test measures the suppressive activity of blood schizontocides over 4 days at a high-tolerated dose that does not cause physical stress in healthy mice.Briefly, female ARC mice (groups of six mice, age 6-7 weeks with a mean weight of 28 ± 3 g) were inoculated intraperitoneally with 20 × 10 6 P. berghei (ANKA strain) infected erythrocytes.The mice were then treated subcutaneously or orally at about 1 h and 6 h after parasite inoculation (D0) and then twice daily at about 6 h apart for 3 consecutive days with either the analogue and drug-free vehicle (control).Chloroquine
( positive control) was given daily for 4 days.The analogues were dissolved in 10% EtOH/ 10% Tween 80/80% distilled H 2 O and chloroquine was dissolved in H 2 O.The drug-free vehicle was 10% EtOH/10% Tween 80/80% distilled H 2 O. Thin blood smears were made on D + 1, D + 2, D + 3, and D + 4 and stained with Giemsa.The degree of infection ( parasitemia expressed as percentage of infected erythrocytes) was determined microscopically.Blood smears were read independently by two microscopists with results averaged.This journal is © The Royal Society of Chemistry 2015 Org.Biomol.Chem., 2015, 13, 1558-1570 | 1569 Open Access Article.Published on 26 November 2014.Downloaded on 9/16/2023 11:59:33 AM.This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online grateful to Stephen McLeod-Robertson and Thomas Travers for the in vivo tolerability and efficacy mice studies.

Table 2
Biological profiles of urea and carbamate analogues33-39 5; for example the C 2 amide 8 displayed 3.6-fold (Dd2 line) and 1.7-fold (3D7 line) better SI values compared to the C 5 amide 13.While substitution with cyclic sidechains, as in the cyclopropyl and cyclobutyl derivatives 11 and 15, respectively, showed no improvement to the potency or

Table 5
In vitro metabolic stability of selected thiaplakortone analogues in NADPH supplemented mouse liver microsomesIn vitro CL int (µL min −1 mg −1 protein) a CQ = chloroquine.
26ash chromatographic separations were carried out following protocols defined by Still et al.25with silica gel 60 (40-63 mm, supplied by GRACE) or amino bonded silica gel (Davisil®) as the stationary phase and using the AR-or HPLC-grade solvents indicated.Semi-preparative HPLC work was performed using a Waters 600 pump and 966 PDA detector, a Gilson 715 liquid handler and a C 18bonded silica Betasil 5 μm 143 Å column (21.2 × 150 mm).MS studies.All compounds were analysed for purity using LC-MS and shown to be >95% pure, unless otherwise stated.Starting materials and reagents were generally available from the Sigma-Aldrich, Merck, AK Scientific Inc., Matrix Scientific Chemical Companies and were used as supplied.THF, MeOH and DCM were dried using a Glass Contour solvent purification system that is based upon a technology originally described by Grubbs et al.26Triethylamine (TEA) was freshly distilled over calcium hydride before use.Where necessary, reactions were performed under a nitrogen atmosphere and glassware was heated in an oven at 140 °C then dried under vacuum prior to use.Temperatures quoted as 0 °C and −78 °C were obtained by cooling the reaction vessel in baths of ice/H 2 O and CO 2(s) /acetone, respectively.Compounds for biological studies were place under high vacuum (0.05 mmHg) for several hours before testing to remove trace, residual solvents.
1H and13C NMR spectra were recorded at 30 °C in DMSO-d 6 on a Varian INOVA 500 NMR spectrometer.The 1 H and13C NMR chemical shifts were referenced to the solvent peak for DMSOd 6 at δ H 2.50 and δ C 39.5.LRESIMS was obtained from LC-MS data generated using a Waters Alliance 2790 HPLC equipped with a Waters 996 photodiode array detector and an Alltech evaporative light scattering detector that was attached to a Water ZQ mass spectrometer.HRESIMS were recorded on a Bruker MicrOTof-Q spectrometer (Dionex UltiMate 3000 micro LC system, ESI mode).Analytical thin layer chromatography (TLC) was performed on aluminum-backed 0.2 mm thick silica gel 60 F 254 plates as supplied by Merck.Eluted plates were visualised using a 254 nm UV lamp and/or by treatment with a suitable dip followed by heating.These dips included phosphomolybdic acid-Ce(SO 4 ) 2 -H 2 SO 4 (conc.)-H 2 O (37.5 g : 7.5 g : 37.5 g : 720 mL) or KMnO 4 -K 2 CO 3 -5% NaOH aqueous solu-tion-H 2 O (3 g : 20 g : 5 mL : 300 mL).