On the basis of a previous structure-activity relationship study, we identified some essential parameters, e.g. electronegativity and lipophilicity, required for polar head analogues to inhibit Plasmodium falciparum phospholipid metabolism, leading to parasite death. To improve the in vitro antimalarial activity, 36 cationic choline analogues consisting of mono-, bis-, and triquaternary ammonium salts with distinct substituents of increasing lipophilicity were synthesized. For monoquaternary ammonium salts, an increase in the lipophilicity around nitrogen was beneficial for antimalarial activity: IC(50) decreased by 1 order of magnitude from trimethyl to tripropyl substituents. Irrespective of the polar head substitution (methyl, ethyl, hydroxyethyl, pyrrolidinium), increasing the alkyl chain length from 6 to 12 methylene groups always led to increased activity. The highest activity was obtained for the N,N,N-tripropyl-N-dodecyl substitution of nitrogen (IC(50) 33 nM). Beyond 12 methylene groups, the antimalarial activities of the compounds decreased slightly. The structural requirements for bisquaternary ammonium salts in antimalarial activity were very similar to those of monoquaternary ammonium salts, i.e. polar head steric hindrance and lipophilicity around nitrogen (methyl, hydroxyethyl, ethyl, pyrrolidinium, etc.). In contrast, with bisquaternary ammonium salts, increasing the lipophilicity of the alkyl chain between the two nitrogen atoms (from 5 to 21 methylene groups) constantly and dramatically increased the activity. Most of these duplicated molecules had activity around 1 nM, and the most lipophilic compound synthesized exhibited an IC(50) as low as 3 pM (21 methylene groups). Globally, this oriented synthesis produced 28 compounds out of 36 with an IC(50) lower than 1 microM, and 9 of them had an IC(50) in the nanomolar range, with 1 compound in the picomolar range. This indicates that developing a pharmacological model for antimalarial compounds through choline analogues is a promising strategy.

We characterized the potent in vitro antimalarial activity and biologic assessment of 13 phospholipid polar head analogs on a comparative basis. There was a positive relationship between the abilities of the drugs to inhibit parasite growth in culture and their abilities to specifically inhibit phosphatidylcholine biosynthesis of Plasmodium falciparum-infected erythrocytes. Maximal activity of G25 was observed for the trophozoite stage of the 48-h erythrocytic cycle (50% inhibitory concentration, 0.75 nM), whereas the schizont and ring stages were 12-and 213-fold less susceptible. The compounds exerted a rapid nonreversible cytotoxic effect, with complete clearance of parasitemia after 5 h of contact with the mature stages. The compounds were highly specific against P. falciparum, with much lower toxicity against three other mammalian cell lines, and the in vitro therapeutic indices ranged from 300 to 2,500,000. Finally, the monoquaternary ammonium E10 and two bis-ammonium salts, G5 and G25, were similarly active against multiresistant strains and fresh isolates of P. falciparum. This impressive selective in vitro toxicity against P. falciparum strongly highlights the clinical potential of these quaternary ammonium salts for malarial chemotherapy.The increasing polypharmacoresistance of Plasmodium falciparum to conventional antimalarials, combined with the resistance of mosquitoes to pesticides, partially accounts for the current dramatic resurgence of malaria (7, 16). Malaria has an enormous impact on world health, thus justifying the intensive research under way to design new therapeutic approaches to this disease and to develop a new range of drugs with original structures and modes of action (22,37).In the human host, the parasite undergoes cyclical development within red blood cells (RBC), which accounts for the clinical manifestations of this disease. The rate of multiplication within RBC, with 10 to 20 merozoites produced every 48 h, requires high metabolic activity, including a high membrane formation level. Phospholipids (PL) are essential lipids of Plasmodium membranes, and the erythrocyte PL content increases by as much as 500% after malarial infection (17, 27). Plasmodial PL biosynthesis, which is absent from normal mature human erythrocytes (29), was previously characterized as an ideal potential target for antimalarial chemotherapy due to its vital importance to the parasite (30, 32). Phosphatidylcholine (PC) is the major PL of infected erythrocytes, representing about 45% of total PL, much of which is provided by parasitedriven de novo biosynthesis from choline (4, 31). In the pathway, parasitic cholinephosphate cytidylyltransferase (38) and choline transport, which regulates the precursor supply, are a limiting and likely regulatory step (4, 33).More than 100 analogs of PL polar heads have been synthesized and tested in vitro for their activity against the intraerythrocytic stage of P. falciparum (2,8,9). More particularly, 36 compounds which possess one (or two) quaternary ammonium ions, such as...