Synthesis and evaluation of phenoxyoxazaphospholidine, phenoxyoxazaphosphinane, and benzodioxaphosphininamine sulfides and related compounds as potential anti-malarial agents.

A series of phenoxyoxazaphospholidine, phenoxyoxazaphosphinane and benzodioxaphosphininamine sulfides and related cyclic organophosphorus compounds based on the lead anti-tubulin herbicides amiprophos methyl and butamifos were synthesised and evaluated for anti-malarial activity. Of these compounds, while none of the phenoxyoxazaphospholidines, phenoxyoxazaphosphinanes or benzodioxaphosphininamine sulphides were more potent than APM, phosphorothioamidate 30, a dual compound also bearing an aminoquinoline motif, showed promising activity against Plasmodium falciparum (IC50 0.038 µM) and warrants further study.

Antimitotic herbicides bind to an unidentified site on malarial parasite tubulin and block development of liver-stage Plasmodium parasites.

Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin. Here we show for the first time that the antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel antimalarial agents binding to tubulin at the herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on α-tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the herbicides amiprophosmethyl and oryzalin for these proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (oryzalin) higher than wild-type. We conclude that the herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite tubulin remains to be proven.

Synthesis and evaluation of phosphoramidate and phosphorothioamidate analogues of amiprophos methyl as potential antimalarial agents.

A series of phosphoramidate and phosphorothioamidate compounds based on the lead antitubulin herbicidal agents amiprophos methyl (APM) and butamifos were synthesised and evaluated for antimalarial activity. Of these compounds, phosphorothioamidates were more active than their oxo congeners and the nature of both aryl and amido substituents influenced the desired activity. The most active compound was 46, O-ethyl-O-(2-methyl-4-nitrophenyl)-N-cyclopentyl phosphorothioamidate, which was more effective than the lead compound.

Identification of plasmepsin inhibitors as selective anti-malarial agents using ligand based drug design.

We describe the application of ligand based virtual screening technologies towards the discovery of novel plasmepsin (PM) inhibitors, a family of malarial parasitic aspartyl proteases. Pharmacophore queries were used to screen vendor libraries in search of active and selective compounds. The virtual hits were biologically assessed for activity and selectivity using whole cell Plasmodium falciparum parasites and on target in PM II, PM IV and the closely related human homologue, Cathepsin D assays. Here we report the virtual screening highlights, structures of the hits and their demonstrated biological activity.

Cellular and molecular actions of dinitroaniline and phosphorothioamidate herbicides on Plasmodium falciparum: tubulin as a specific antimalarial target.

Microtubules play important roles in cell division, motility and structural integrity of malarial parasites. Some microtubule inhibitors disrupt parasite development at very low concentrations, but most of them also kill mammalian cells. However, the dinitroaniline family of herbicides, which bind specifically to plant tubulin, have inhibitory activity on plant cells but are relatively non-toxic to human cells. Certain dinitroanilines are also inhibitory to various protozoal parasites including Plasmodium. Here we demonstrate that the dinitroanilines trifluralin and oryzalin inhibited progression of erythrocytic Plasmodium falciparum through schizogony, blocked mitotic division, and caused accumulation of abnormal microtubular structures. Moreover, radiolabelled trifluralin interacted with purified, recombinant parasite tubulins but to a much lesser extent with bovine tubulins. The phosphorothioamidate herbicide amiprophos-methyl, which has the same herbicidal mechanism as dinitroanilines, also had antimalarial activity and a similar action on schizogony. These data suggest that P. falciparum tubulin contains a dinitroaniline/phosphorothioamidate-binding site that is not conserved in humans and might be a target for new antimalarial drugs.