Despite recent progress in the fight against malaria,the emergenceand spread of drug-resistant parasites remains a serious obstacleto the treatment of infections. We recently reported the developmentof a novel antimalarial drug that combines the 4-aminoquinoline pharmacophoreof chloroquine with that of clotrimazole-based antimalarials. Herewe describe the optimization of this class of hybrid drug throughin-depth structure–activity relationship studies. Antiplasmodialproperties and mode of action were characterized in vitro and in vivo, andinteractions with the parasite’s 'chloroquine resistancetransporter' were investigated in a Xenopus laevisoocyte expression system. These tests indicated that piperazinederivatives 4band 4dmay be suitable forcoadministration with chloroquine against chloroquine-resistant parasites.The potential for metabolism of the drugs by cytochrome P450 was determinedin silico, and the lead compounds were tested for toxicity and mutagenicity.A preliminary pharmacokinetic analysis undertaken in mice indicatedthat compound 4bhas an optimal half-life. [ABSTRACT FROM AUTHOR]