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Recently, P218, a new flexible antifolate targeting Plasmodium falciparum dihydrofolate reductase (PfDHFR), has entered its clinical trial with good safety profile and effective Pf infection prevention. However, it carries a free carboxyl terminal, which is hydrophilic and prone to metabolic glucuronidation. Here, a new series of P218 analogues carrying butyrolactone has been synthesized with the purpose of enhancing lipophilicity and minimizing metabolic instability. The inhibition constants against the mutant PfDHFR enzymes are in sub-nanomolar level and the antimalarial activity against antifolate-resistant parasites are in the low micromolar range. The crystal structure of the most potent analogue LA1 bound enzyme complex indicates interaction with multiple residues, including Arg122 and Phe116 in the active site. In vitro log D 7.4 and kinetic solubility confirmed a higher lipophilicity of this butyrolactone series as compared to P218. These outcomes suggest the possibility to further develop butyrolactone derivatives as non-carboxyl antiplasmodial antifolates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sasithorn Decharuangsilp has patent #TH2301005512 pending to National Science and Technology Development Agency (NSTDA). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Malarial dihydrofolate reductase (DHFR) is the target of antifolate antimalarial drugs such as pyrimethamine and cycloguanil, the clinical efficacy of which have been compromised by resistance arising through mutations at various sites on the enzyme. Here, we describe the use of cocrystal structures with inhibitors and substrates, along with efficacy and pharmacokinetic profiling for the design, characterization, and preclinical development of a selective, highly efficacious, and orally available antimalarial drug candidate that potently inhibits both wild-type and clinically relevant mutated forms of Plasmodium falciparum (Pf) DHFR. Important structural characteristics of P218 include pyrimidine side-chain flexibility and a carboxylate group that makes charge-mediated hydrogen bonds with conserved Arg122 (PfDHFR-TS amino acid numbering). An analogous interaction of P218 with human DHFR is disfavored because of three species-dependent amino acid substitutions in the vicinity of the conserved Arg. Thus, P218 binds to the active site of PfDHFR in a substantially different fashion from the human enzyme, which is the basis for its high selectivity. Unlike pyrimethamine, P218 binds both wild-type and mutant PfDHFR in a slow-on/slow-off tight-binding mode, which prolongs the target residence time. P218, when bound to PfDHFR-TS, resides almost entirely within the envelope mapped out by the dihydrofolate substrate, which may make it less susceptible to resistance mutations. The high in vivo efficacy in a SCID mouse model of P. falciparum malaria, good oral bioavailability, favorable enzyme selectivity, and good safety characteristics of P218 make it a potential candidate for further development.