Novel Antimalarial Aminoquinolines: Heme Binding and Effects on Normal or Plasmodium falciparum-Parasitized Human Erythrocytes

Two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM-1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Their accumulation in parasitized RBC (pRBC) is increased twofold compared to levels in normal RBC. Binding of AM1 or AP4b to both normal and pRBC is higher than that of CQ, in agreement with the lower pK_a and higher lipophilicity of the compounds. AM1 or AP4b is not hemolytic per se and is less hemolytic than CQ when hemolysis is accelerated (induced) by hematin. Moreover, AM-1 and AP4b bind heme with a stoichiometry of interaction similar to that of CQ (about 1:1.7) but with a lower affinity. They both inhibit dose dependently the formation of beta-hematin in vitro with a 50% inhibitory concentration comparable to that of CQ. Taken together, these results suggest that the antimalarial activity of AM1 or AP4b is likely due to inhibition of hemozoin formation and that the efficacy of these compounds against the CQ-resistant strains can be ascribed to their hydrophobicity and capacity to accumulate in the vacuolar lipid (elevated lipid accumulation ratios).Chloroquine (CQ) has been effectively used for decades as an antimalarial drug for its efficacy, safety, and stability but is now largely ineffective because of widespread parasite resistance. This has led to the necessity of utilizing artemisinin-based combination therapy as a first-line treatment for uncomplicated malaria (20). However, the recent reports of artemisinin-based combination therapy treatment failure in southeast Asia and the potential emergence of artemisinin resistance (26) indicate that the search for new drugs or new drug combinations is still highly necessary (40, 41). Quinoline-type antimalarials remain an attractive class of compounds because their mechanism of action and resistance are unrelated (14) and resistance has emerged very slowly over time. CQ and quinoline antimalarial drugs are thought to kill parasites by inhibiting the process of crystallization necessary to detoxify ferriprotoporphyrin IX (FP) into hemozoin (HZ) (5, 30, 34). This process is vital for Plasmodium falciparum and can be reproduced in vitro with hematin under acidic conditions, leading to the formation of beta-hematin (BH), a crystal product showing physicochemical properties identical to those of HZ (29).FP in the food vacuole of the parasite is considered the target of quinoline antimalarials (6, 15). The inhibition of HZ formation leads to the accumulation of free FP, potentially toxic for its prooxidant and lytic activities (7, 27). The resistance mechanism of CQ is based on the reduced drug accumulation at the site of action, the food vacuole, due to mutation of PfCRT (P. falciparum CQ resistance transporter) (5, 16, 39) and does not involve modification of the target.In the last few years, the research of our group has been directed to the study of new quinolizidinyl and quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline (33). The terminal quinolizidine ring (octahydro-2H-quinolizine) confers basicity and lipophilicity to the molecules and prevents the metabolic oxidative dealkylation known to limit the usefulness of quinoline derivatives (28). Lead candidates have been obtained that are very stable and highly effective in vitro against both the D10 (CQ susceptible [CQ-S]) and W2 (CQ resistant [CQ-R]) strains of P. falciparum (33) and in vivo in the murine Plasmodium berghei model (23). Two of these compounds, named AM-1 and AP4b, have been selected for further characterization (Fig. ​(Fig.1):1): AM-1, a pure enantiomer, is semisynthetic and derives from l-lupinine, a natural alkaloid extracted from the seeds of Lupinus luteus; AP4b is synthetic and a racemate.Open in a separate windowFIG. 1.Molecular structures of the compounds used in the study.For the present article, we evaluated the binding of these compounds to human red blood cells (RBC), normal or parasitized by the P. falciparum strain D10 or W2. In addition, an analysis of the heme binding properties of these compounds and of their ability to affect RBC membrane stability and inhibit FP crystallization in vitro has been performed.