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.

[reaction: see text] Stabilized primary radicals can be formed from alkyl halides in an atom transfer process with Et(3)B. This process depends on the strength of the carbon-halogen bond and the stability of the resulting primary radical. Radicals formed from benzyl iodide and ethyl iodoacetate add to glyoxylic oxime ethers; however, more electrophilic radicals do not. Glyoxylic oxime ethers are also good radical acceptors for heterocyclic carbon-centered secondary radicals, giving novel alpha-amino acid derivatives.

A range of dehydro amino acid derivatives has been prepared and subjected to halogenation using either molecular bromine or chlorine, or NBS. Allylic halogenation of the unsaturated amino acid side chains occurs through radical bromination with NBS. The procedure is complementary to treatment with chlorine, which also affords allyl halides. This latter and unusual reaction is shown through a deuterium labelling study to proceed via an ionic mechanism. The choice of NBS or chlorine for allyl halide synthesis is shown to depend on the potential to avoid competing reactions, such as halolactonization of leucine derivatives with chlorine, and hydrogen abstraction and bromine incorporation at multiple sites on treatment of isoleucine derivatives with NBS. The synthetic utility of the allyl halides prepared in this study is indicated through the synthesis of a cyclopropyl amino acid derivative and the extension of the carbon skeleton of an amino acid side chain.

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.

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