The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

A new cyclic pentapeptide, chrysosporide (1), was isolated from a New Zealand sample of the mycoparasitic fungus Sepedonium chrysospermum by bioactivity-guided fractionation. The planar structure was deduced by detailed spectroscopic analysis, and the absolute configurations of the amino acid residues were defined by Marfey's method. As both enantiomers of Leu occurred in chrysosporide, molecular mechanics calculations were applied to the analysis to distinguish between the possible structural isomers. Only the lowest energy conformers of the cyclo-(L-Val-D-Ala-L-Leu-L-Leu-D-Leu) isomer were in agreement with the observed NOEs, suggesting that this was the most probable amino acid sequence for chrysosporide (1).

The photoswitchable N-terminal diazo and triazene-dipeptide aldehydes 8a-d, 10a,b, and 17a,b present predominantly as the (E)-isomer, which purportedly binds deep in the S3 pocket of calpain. All compounds are potent inhibitors of m-calpain, with 8b being the most active (IC50 of 35 nM). The diazo-containing inhibitors 8a, 8c, and 10a were irradiated at 340 nm to give a photostationary state enriched in the (Z)-isomer, and in all cases, these were less active. The most water soluble triazene 17a (IC50 of 90 nM) retards calpain-induced cataract formation in lens culture.

Studies of 17 analoges of 3 (SJA6017) in an in silico calpain model are reconciled to measured IC50 values against ovine calpain. The studies validate the potential of the “model” and criteria established for inhibition as a tool to select structures for synthesis to test as calpain inhibitors. Using this screening methodology of virtual libraries led us to synthesize several inhibitors including macrocycle 33, which in vitro sheep eye lens culture experiments showed to substantially slow opacification.