In Vitro Efficacy of Next Generation Dihydrotriazines and Biguanides Against Babesiosis and Malaria Parasites

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutics strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus are vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates such as the aminopyrimidine, pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here we utilized an in vitro culture condition suitable for continuous propagation of B. duncani, B. divergens, B. MO1 and P. falciparum in human erythrocytes to screen a library of 51 dihydrotriazines and 28 biguanides for their efficacy in vitro and to compare their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3 with IC ₅₀ values below 10 nM and demonstrated excellent therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs.