Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.The antimalarial artemisinin contains a pharmacophoric peroxide bond within its 1,2,4-trioxane heterocycle. A longstanding hypothesis (17,24,29) to account for the antimalarial specificity of artemisinin is that the peroxide bond undergoes reductive activation by heme released by parasite hemoglobin (Hb) digestion. Although most (95%) of the heme released from parasite digestion is incorporated into the relatively inert hemozoin (12), it is estimated (8) that there is some 100 M of "free" heme/hematin in malaria-infected red blood cells, a more than adequate quantity to react with artemisinin. The irreversible redox reaction between artemisinin and heme produces carbon-centered free radicals or carbocations that alkylate heme (25,32,33) and membrane-associated parasite proteins (2), one of which is the translationally controlled tumor protein (PfTCTP) (4) and another is likely to be PfATP6, a SERCA-type Ca 2ϩ -ATPase (11). Eckstein-Ludwig et al. (11) studied the latter protein in some detail and provided evidence that it may be an important target of the artemisinins.The structural diversity of semisynthetic artemisinins (41) and synthetic peroxides (37) is quite remarkable and, we suggest, has implications for the mechanism of action for this class of antimalarial drugs. In this respect, we thought it might be useful to compare the activities of artemisinin and synthetic peroxide (1,2,4-trioxolane) OZ277 (RBx11160) (40) and their nonperoxidic counterparts deoxyartemisinin (6) and carbaOZ277 (Fig. 1) against a range of protozoan parasites. CarbaOZ277 (1,3-dioxolane) was prepared as a mixture of cis-trans isomers by an acidcatalyzed reaction (9) between the requisite adamantane diol and amino amide cyclohexanone. In vitro antiprotozoal assays for Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, Giardia duodenalis, Babesia divergens, and Neospora caninum were performed as previously described (1,5,16,28,35,38,40) with standard drug controls ( Table 1).The data in Table 1 shows a clear demarcation in the antiplasmodial activities of artemisinin and OZ277 versus their nonperoxidic analogs deoxyartemisinin (6) and carbaOZ277, confirming the peroxide bond-dependent activity of the former. For artemisinin and OZ277, there were losses of activity greater than 3 orders of magnitude against all of the other protozoa compared to activities against P. falciparum. In contrast, for deoxyartemisinin and carbaOZ277, there were only small differences in activity between P. falciparum and the other protozoa. Only for T. b. rhodesiense was there a significant difference between artemisinin and deoxyartemisinin ve...