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1.
Scheme 1

Scheme 1. From: Thiazole, Oxadiazole, and Carboxamide Derivatives of Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma gondii.

Scheme 1 outlines the preparation of the 23 DART derivatives starting from the natural trioxane artemisinin. The specific C-10 functional groups in this study were chosen based largely on their chemical accessibility. The syntheses of the DART-1,3-thiazole derivatives were accomplished in a three-step, one-pot procedure while the DART-1,2,4-oxadiazoles and DART-carboxamides were prepared in four or five chemical steps. The DART-1,3-thiazole series were prepared by the addition to artemisinin of the lithium thiazole species, from the respective 2-bromo-1,3-thiazole that was synthesized from the corresponding aryl or alkyl ketone using published procedures.32 The resulting thiazolyl alcohol was acetylated and then acetic acid was eliminated, in situ. The DART-1,2,4-oxadiazole series was prepared from the DART-acid33 via the amidoxime34 followed by cyclization with tetrabutylammonium fluoride (TBAF).35 The DART-carboxamide series was prepared from this same DART-acid and the requisite amine via N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)/hydroxybenzotriazole (HOBt) amide coupling.

Christopher P. Hencken, et al. J Med Chem. ;53(9):3594-3601.
2.
Figure 2

Figure 2. From: Thiazole, Oxadiazole, and Carboxamide Derivatives of Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma gondii.

Quantification of replication inhibition using replication assay. Compounds were tested for replication inhibition using established procedures.24 HFF monolayers were inoculated with tachyzoites and then incubated (37 °C, 5% CO2) for 2 h. Compounds (final 10 μM) or DMSO (vehicle) were then added to the medium. Parasite replication proceeded for 24-26 h after which time the monolayers were fixed, permeablilized, and immunolabeled with Rb anti-p30 followed by Gt antirabbit Alexa Fluoro 594 (red). DAPI was added to secondary antibody. Cells were examined by reflected fluorescence as in Figure 1. Data were compiled from three independent experiments, each from 10 random fields per sample. The number of vacuoles containing 1, 2, 4, or 8+ parasites/vacuole were enumerated by eye. Data are expressed as the modal number of parasites per vacuole.

Christopher P. Hencken, et al. J Med Chem. ;53(9):3594-3601.
3.
Figure 1

Figure 1. From: Thiazole, Oxadiazole, and Carboxamide Derivatives of Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma gondii.

Quantification of invasion/attachment inhibition using red/green assay. The red/green invasion assay was performed using published methods.24 Tachyzoites (5 × 106) were mixed with DMSO [vehicle (VHL)] or compound (final 10 μM) and allowed to sit at room temperature for 20 min. before being added to HFF monolayers growing in 8-well chamber slides. After 1 hour at 37 °C, 5% CO2, the cells were rinsed and fixed. Attached/extracellular parasites were detected using Rb anti-p30 (SAG1) (AbD Serotec, UK) followed by Alexa Fluoro 594 (red) (Invitrogen, CA). After permeabilization, penetrated/intracellular parasites were stained with MAb 9e11 anti-SAG1 (Argene Inc., NY) followed by Gt antimouse Alexa Fluoro 488 (green). DAPI (Invitrogen) for staining nuclei was added to secondary antibody. BAPTA-AM (20 μM) and cytochalasin D (2 μM) are included as controls for defects in attachment and penetration, respectively.24 Stained cells were examined by phase contrast and reflected fluorescence using an Olympus BX41 microscope. Numbers of green and red tachyzoites per host cell were enumerated by visual counting. Data are mean values ±SEM of three independent experiments, counting 10 random fields per well at 600× magnification. A single asterisk indicates tachyzoite penetration significantly lower (P ≤ 0.05, two-tailed Students' t-test) than vehicle. A double asterisk indicates a significant effect (P ≤ 0.05, one-tailed Students' t-test) on parasite attachment relative to vehicle.

Christopher P. Hencken, et al. J Med Chem. ;53(9):3594-3601.

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