Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD- scid IL2Rγ ^null Mouse Model of Malaria

Abstract

A series of 4-amino 2-anilinoquinazolines optimized for activity against the most lethal malaria parasite of humans, Plasmodium falciparum, was evaluated for activity against other human Plasmodium parasites and related apicomplexans that infect humans and animals. Four of the most promising compounds from the 4-amino 2-anilinoquinazoline series were equally as effective against the asexual blood stages of the zoonotic P. knowlesi, suggesting that they could also be effective against the closely related P. vivax, another important human pathogen. The 2-anilinoquinazoline compounds were also potent against an array of P. falciparum parasites resistant to clinically available antimalarial compounds, although slightly less so than against the drug-sensitive 3D7 parasite line. The apicomplexan parasites Toxoplasma gondii, Babesia bovis, and Cryptosporidium parvum were less sensitive to the 2-anilinoquinazoline series with a 50% effective concentration generally in the low micromolar range, suggesting that the yet to be discovered target of these compounds is absent or highly divergent in non-Plasmodium parasites. The 2-anilinoquinazoline compounds act as rapidly as chloroquine in vitro and when tested in rodents displayed a half-life that contributed to the compound’s capacity to clear P. falciparum blood stages in a humanized mouse model. At a dose of 50 mg/kg of body weight, adverse effects to the humanized mice were noted, and evaluation against a panel of experimental high-risk off targets indicated some potential off-target activity. Further optimization of the 2-anilinoquinazoline antimalarial class will concentrate on improving in vivo efficacy and addressing adverse risk.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4

“…The asexual parasite viability was determined using LDH following an assay protocol previously described …”

“…■ EXPERIMENTAL SECTION P. falciparum 3D7 Asexual Stage LDH Assay. The asexual growth assay was adapted 43 from the previously described method. 44 HepG2 Viability Assay.…”

“…The asexual parasite viability was determined using LDH following an assay protocol previously described. 43 Gamete Assay. P. falciparum NF54 Stage V gametocytes for gamete transduction were produced using a protocol by Delves et al 40 Male and female gamete induction and viability were assessed as previously described.…”

Substrate Peptidomimetic Inhibitors of P. falciparum Plasmepsin X with Potent Antimalarial Activity

“…P. falciparum LDH viability assays were performed as previously described. [40] Briefly, a ten‐point 1 : 3‐fold dilution series of the compounds were pre‐dispensed in 384‐well assay plates (Grenier), and RPMI/AlbuMAX growth media inoculated with synchronized ring stage P. falciparum parasites. 10 mM compound stocks were transferred into the assay plates such that the starting concentration was 10 μM.…”

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway

“…No reuse allowed without permission. Parasite killing rate assay: Assay was performed as previously described with some alterations (47). Briefly, sorbitol synchronized ring or trophozoite stage parasites were cultured in the presence of 10x the EC50 of 3 or artesunate for either 24 or 48 h. Cultures that were incubated with either compound for 48 h were fed at 24 h with fresh media containing 10x the EC50 of compound.…”