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.

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.

We report the synthesis of macrocycles 1-6 via ring closing metathesis of dienes 7-12. Addition of chlorodicyclohexylborane to thermal and microwave assisted RCM of dienes 8 and 9 markedly improves the yield. The geometric isomers of macrocycles 1-3 and 5 have been assigned using X-ray crystallography and NMR.

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

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.

Ring closing metathesis and cross metathesis approaches to a new macrocyclic peptidomimetic aldehyde 2 have been developed, with the former route being the most convenient. Aldehyde 2 is a potent inhibitor of calpain II (IC50 of 45 nM) with comparable activity to the benchmark acyclic inhibitor SJA6017 4. Both compounds contain an N-terminal 4-fluorophenylsulfonyl group. The P2 Ile analogue of 2 (16) is significantly less active (IC50 of 2000 nM) which reflects an unusually subtle importance of the P2 residue for active site binding.

The introduction of a macrocycle into a biologically active peptide can increase potency [1,2] and selectivity [3] by reducing the entropic penalty of inhibitor-enzyme binding. The incorporation of macrocycles in peptides has been used to mimic secondary structure, such as extended b-strand-like and bent b-turn-like conformations. [3,4] The challenge is to devise protocols for the design, evaluation, and synthesis of macrocyclic templates that provide access to families of inhibitors. [5] Such templates should 1) have a well-defined conformation that can be readily assessed by computational screening, 2) be readily synthesized from natural building blocks, and 3) be easily modified to target an enzyme for a specific biological application.Herein we present studies on the 16-19-membered macrocycles 1-4 (Scheme 1) designed to be constrained into a b-strand-like geometry, a conformation universally adopted by inhibitors and substrates on binding to a protease. [6] We report a versatile approach based on ring-closing metathesis (RCM) to these orthogonally protected templates as well as computational analysis of their potential to form a b strand and of their binding to a target protease. The templates were converted into aldehydes 1 d-4 d as potential inhibitors of the calcium-activated cysteine protease calpain. The alcohols 1 c-4 c were evaluated to establish whether the macrocycles negate the need for a reactive warhead. Calpain was chosen as a challenging target for the design of selective inhibitors, [7] and because its link to cortical cataracts [8] provides an opportunity to assess the in vivo efficacy of our approach. The acyclic tripeptide analogues 5 a-d were also investigated to establish the importance (or unimportance) of the macrocyclic constraint for potency and selectivity of inhibition.Conformational searches [9] were carried out on 1 a-d to 5 a-d to assess their ability to adopt a b-strand conformation. The resulting ensembles of low-energy conformers were examined by XCluster (see the Supporting Information for detailed results). For the 16-membered macrocycles 1 a-d, all conformers (> 99 %) within 12 kJ mol À1 of their global minima adopt a b-strand conformation (see Figure 1 for Scheme 1. Macrocyclic templates and acyclic peptides. Dihedral angles F and Y that describe a b strand are shown. Boc = tert-butoxycarbonyl, Cbz = carbobenzyloxy. Figure 1. Overlaid conformers of the two clusters of 1 d.

Olefin metathesis, a process by which alkylidene groups on alkenes are exchanged, is reviewed with reference to the historical development of functional catalysts and microwave catalysis. The advent of new catalysts and improved reaction conditions has paved the way for the development of new and versatile domino processes that combine either different metathesis events or a key metathesis reaction with another reaction in a particularly atom economic and ‘green’ sense. We review this area as a convenient route to some otherwise difficult to prepare systems.