Artermisinin and its derivatives are now the mainstays of antimalarial treatment; however, their mechanism of action is only poorly understood. We report on the synthesis of a novel series of epoxy-endoperoxides that can be prepared in high yields from simple starting materials. Endoperoxides that are disubstituted with alkyl or benzyl side chains show efficient inhibition of the growth of both chloroquine-sensitive and -resistant strains of Plasmodium falciparum. A trans-epoxide with respect to the peroxide linkage increases the activity compared to that of its cis-epoxy counterpart or the parent endoperoxide. The novel endoperoxides do not show a strong interaction with artemisinin. We have compared the mechanism of action of the novel endoperoxides with that of artemisinin. Electron microscopy reveals that the novel endoperoxides cause the early accumulation of endocytic vesicles, while artemisinin causes the disruption of the digestive vacuole membrane. At longer incubation times artemisinin causes extensive loss of organellar structures, while the novel endoperoxides cause myelin body formation as well as the accumulation of endocytic vesicles. An early event following endoperoxide treatment is the redistribution of the pH-sensitive probe LysoSensor Blue from the digestive vacuole to punctate structures. By contrast, neither artemisinin nor the novel endoperoxides caused alterations in the morphology of the endoplasmic reticulum nor showed antagonistic antimalarial activity when they were used with thapsigargin. Analysis of rhodamine 123 uptake by P. falciparum suggests that disruption of the mitochondrial membrane potential occurs as a downstream effect rather than as an initiator of parasite killing. The data suggest that the digestive vacuole is an important initial site of endoperoxide antimalarial activity.Artemisinin is a sesquiterpene lactone antimalarial with a 1,2,4-trioxane heterocyclic core that incorporates a peroxide linkage that is essential for its activity (44). Artemisinin is of major importance as a frontline treatment for malaria, particularly as it is active against chloroquine (CQ)-resistant strains of Plasmodium falciparum (20,23,24,45,66,74). Several groups have reported on pathways for the synthesis of artemisinin, but all require numerous steps and have low yields (5, 35). As a result, artemisinin-based antimalarials are produced semisynthetically from extracts of Artemisia annua. A period of over 1 year is needed for the horticultural, harvesting, extraction, and manufacturing processes (34,41,45), which limits the ease of scale-up of production and makes artemisinin derivatives much more expensive than traditional antimalarials, such as CQ and sulfadoxine-pyrimethamine. Indeed, artemisinin combinations are too costly for many patients, and the supply of counterfeit or inferior drugs is a major problem (4, 45, 71). Another issue is the fact that artemisinin and its derivatives are not suitable for prophylaxis or for use as a monotherapy due to their very short half-lives in vivo. T...

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We report the synthesis of a series of novel epoxy endoperoxide compounds that can be prepared in high yields in one to three steps from simple starting materials. Some of these compounds inhibit the growth of Plasmodium falciparum in vitro. Structure-activity studies indicate that an endoperoxide ring bisubstituted with saturated cyclic moieties is the pharmacophore. To study the molecular basis of the action of these novel antimalarial compounds, we examined their ability to interact with oxidized and reduced forms of heme. Some of the compounds interact with oxidized heme in a fashion similar to chloroquine and other 4-aminoquinolines, while some of the compounds interact with reduced heme. However, the level of antimalarial potency is not well correlated with these activities, suggesting that some of the endoperoxides may exert their antimalarial activities by a novel mechanism of action.