Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquine's low therapeutic ratio and toxicity.

Ro 42-1611 (arteflene), a new effective antimalarial: chemical structure and biological activity.

The discovery of the natural peroxides qinghaosu (arteannuin A, artemisinin) (1) and yingzhaosu A (3) from traditional Chinese herbal medicines was a major advance in the search for new antimalarials (Fig. 1). Whereas qinghaosu can be produced from natural sources and has been well studied, yingzhaosu A has never been available for full evaluation as anti-malarial. We have designed a synthesis of the novel ring system present in yingzhaosu A, the 2,3-dioxabicyclo[3.3.1]nonane and prepared a series of yingzhaosu A analogues which were tested against Plasmodium berghei in mice. Structure-activity rules could be established and used for lead optimization. The best anti-malarial activity was observed for analogues having a keto group within the ring system and an aliphatic or aromatic lipophilic tail as ring substituent. The optimized analogues possessed activity comparable to qinghaosu. In spite of the presence of a peroxide ring, the new compounds were chemically stable against common reagents. In contrast to qinghaosu and its derivatives, they were also stable against hydrolytic decomposition and could therefore be expected to show improved pharmacokinetic properties. As one of the best compounds, Ro 42-1611 (arteflene) (26n, Fig. 2) was selected for detailed preclinical evaluation. Ro 42-1611 (arteflene) was found negative in a battery of mutagenicity tests. It had low acute toxicity after oral or subcutaneous administration. In a 4-week oral tolerance study in rats, doses of up to 400 mg/kg/day were well tolerated.(ABSTRACT TRUNCATED AT 250 WORDS)