Pitstop 2 binds to the clathrin terminal domain. This work explores the nature of the benzylidene moiety, R. The synthesis of 56 novel analogues reveals enhanced potency of a multiple analogues, with catechol-free 2,3-dihydroxybenzo[b][1,4]dioxone (54) returning a 1.2 μm IC₅₀, ca 10-fold more active than Pitstop 2.

Pitstop 2, (Z)-N-(5-(4-bromenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide (1) inhibits the clathrin terminal domain–amphiphysin interaction (NTD-PPI) and has been widely used to investigate endocytosis. Herein, the synthesis of 56 novel Pitstop 2 analogues via four discrete focused libraries is reported. Specific modification to the 4-bromonenzylidene moiety is explored, and their ability to inhibit the NTD-PPI interaction is examined by ELISA. In cell endocytosis, effects are measured for selective analogues as is the inhibition of dynamin, another protein involved in the endocytosis process. The most NTD-PPI active analogues retain 2- and 4-disposed substituents on the aromatic head, with 2,3,4-trihydroxy (28) the most active (IC₅₀ 0.94 μm). Catechol-free 2,3-dihydroxybenzo[b][1,4]dioxone (54) is a more promising lead with an NTD-PPI IC₅₀ = 1.16 μm. The corresponding benzo[d][1,3]dioxole (53) is threefold less active suggesting ring size preference at this position. Nine analogues show improved or comparable NTD-PPI activity to Pitstop 2 with IC₅₀ values from 0.94 to 2.1 μM. Heterocyclic analogues are well tolerated and potent inhibitors of CME in U2OS cells, in particular, benzofuran 67 (NTD-PPI IC₅₀ 1.5 μm and CME IC₅₀ 6.8 ± 2.7 μm). This positions 67 as one of the most cell active inhibitors of clathrin-mediated endocytosis yet reported.