Macrocyclic inhibitors have emerged as a privileged scaffold in medicinal chemistry, offering enhanced selectivity, stability, and pharmacokinetic profiles compared to their linear counterparts. Here, we describe a novel, on-resin macrocyclization strategy for the synthesis of potent inhibitors targeting the secreted protease Major Aspartyl Peptidase 1 in Cryptococcus neoformans, a pathogen responsible for life-threatening fungal infections. By employing diverse aliphatic linkers and statine-based transition-state mimics, we constructed a focused library of 624 macrocyclic compounds. Screening identified several subnanomolar inhibitors with desirable pharmacokinetic and antifungal properties. Lead compound 25 exhibited a Ki of 180 pM, significant selectivity against host proteases, and potent antifungal activity in culture. The streamlined synthetic approach not only yielded drug-like macrocycles with potential in antifungal therapy but also provided insights into structure-activity relationships that can inform broader applications of macrocyclization in drug discovery.

Department of Biochemistry and Biophysics University of California San Francisco UCSF Genentech Hall 600 16th St Rm N374 San Francisco California 94158 United States

Department of Biochemistry Faculty of Science Charles University Hlavova 8 Prague 2 12843 Czech Republic

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 Prague 2 12843 Czech Republic

Department of Pharmaceutical Chemistry University of California San Francisco UCSF Genentech Hall 600 16th St Rm S512 San Francisco California 94158 United States

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Hlavova 8 Prague 2 12843 Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 Prague 4 14220 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 Prague 6 16610 Czech Republic

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