Developmentof Macrocyclic Peptide-Based ProteasomeInhibitors with Enhanced Blood-Brain Barrier Penetration for TreatingBrain Neoplasms

Abstract Proteasome inhibitors are effective in treating hematologic cancers but have limited utility in brain tumors due to poor blood–brain barrier (BBB) penetration and metabolic instability. In this study, we developed novel macrocyclic peptide epoxyketone inhibitors with improved drug-like properties. Compounds were screened for cytotoxicity against brain cancer cell lines, permeability (PAMPA-BBB and Caco-2), and metabolic stability. Lead compound 10 demonstrated potent in vitro activity (IC50 < 100 nM), low P-gp efflux, and favorable microsomal and plasma stability. In vivo pharmacokinetic studies in mice showed that compound 10 maintained therapeutic plasma levels and achieved measurable brain concentrations without toxicity. Co-administration of a P-gp inhibitor significantly enhanced brain exposure of compound 35, confirming efflux as a key parameter. The incorporation of fluorinated phenyl and α,α-dimethylglycine moieties contributed to improved BBB permeability and metabolic stability. These findings support further development of macrocyclic epoxyketone inhibitors as promising candidates for brain cancer therapy.