Improved Bicyclic Pyrrolidine Analogues Inhibit <i>Toxoplasma gondii</i> Growth In Vitro and Cure Infection In Vivo

Inhibition of phenylalanine tRNA synthetase (PheRS) by bicyclic pyrrolidines provides a potent and specific inhibition of parasite growth. Herein, we describe novel bicyclic pyrrolidines designed to explore structure-activity relationships with <i>Toxoplasma gondii</i> vs human PheRS. Modification of the biaryl alkyne extension, which fits into the phenylalanine-binding site, showed a strong preference for <i>ortho</i> hydroxyl addition over <i>meta</i> and <i>para</i>. Further addition of N to both the proximal and distal phenyl rings of the biaryl alkyne and to the methoxyphenyl urea moiety, which fits into a unique auxiliary site present in the parasite enzyme, identified compounds with reduced plasma protein binding and lower hERG activity. Finally, we identified a potent lead with improved pharmacokinetics, extended plasma exposure, central nervous system penetration, and low-dose cure of acute infection in mouse. Collectively, these findings advance new candidates for the treatment of toxoplasmosis based on selective and potent inhibitors of parasite PheRS.