Abstact

Malaria remains one of the world's most devastating parasitic diseases, with increasing resistance to current therapies highlighting the need for new antimalarial agents with novel mechanisms of action. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in pyrimidine biosynthesis, represents a validated molecular target for antimalarial drug discovery. In this study, a structure-based optimization strategy was applied to a previously identified hydroxypyrazole hit compound to design, synthesize, and evaluate new derivatives as selective PfDHODH inhibitors. Through three iterative rounds of rational design, we identified several analogs with notable PfDHODH activity and, in some cases, selectivity over the human isoform (SI > 30). X-ray crystallographic studies of PfDHODH in complex with the most active inhibitors confirmed their predicted binding modes and demonstrated the enzyme's ability to accommodate bulky hydrophobic substituents. The best compounds, notably derivatives 6, 7, 12, 13 and 22, exhibited low micromolar enzymatic inhibition (IC(50) values from 1.3 to 5.9 muM), favourable physicochemical properties (PBS solubility 0.03 - 2.8 mM, logD(7.4) 0.25 - 1.7), and antiparasitic activity in P. falciparum 3D7 cultures (EC(50) values from 15 to 22 muM), with negligible cytotoxicity in mammalian cells (CC(50) > 150 muM except for compound 22). While these hydroxypyrazole-based scaffolds have poor antiplasmodial activity, this study provides structure activity relationship data that may be useful for the development of next-generation PfDHODH inhibitors for malaria chemoprevention and treatment.