New Tripeptide-Based Macrocyclic Calpain Inhibitors Formed by N-Alkylation of Histidine by Hongyuan Chen a ) 1 ), Wanting Jiao a ), Matthew A. Jones a ) b ), James M. Coxon* a ), James D. Morton c ), Roy Bickerstaffe c ), Ashok D. Pehere d ), Ondrej Zvarec d ) 2 ), and Andrew D. Abell* a ) 3 ) a ) Chemistry Department, University of Canterbury, Christchurch, New Zealand (e-mail: jim.coxon@canterbury.ac.nz; andrew.abell@adelaide.edu.au) b ) University of Roehampton, London, UK c ) Lincoln University, Lincoln, New Zealand d )School of Chemistry and Physics, University of Adelaide, South Australia, Australia Dedicated to Professor Dieter Seebach in celebration of his 75th birthday Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The structure with an aldehyde at the C-terminus and the imidazole at P3, i.e. , 4c, shows significant inhibitory activity against calpain 2, with an IC 50 value of 238 nm. The macrocyclic aldehyde with the imidazole at the alternative P1 position, i.e. , 5c, is significantly less active. The relative activities are linked to the ability of the component macrocycles to mimic a b-strand geometry that is known to favor active-site binding. This ability is defined by conformational searches and docking studies with calpain. 1. Introduction. – Much work has been done on constraining the backbone of peptide-based protease inhibitors into a biologically active b-strand conformation by chemically linking the P1 and P3 residue [1 – 3]; for example, see the C-terminal aldehyde macrocycles CAT811 (1) [1], 2, and 3 [2] in Fig. 1. This geometry is almost universally recognized by proteases for active-site binding, with a preorganisation of conformation facilitating active-site binding [1] [3]. Such macrocyclic inhibitors also offer enhanced resistance to proteolytic degradation and thus an improved pharma- cokinetic profile [4]. The introduction of a conformational constraint of this type into peptidomimetics is also of wider interest as a basis of important biological probes and scaffolds [2]. A number of approaches have been reported for introducing such a macrocycle into peptidomimetics, including ring-closing metathesis [1][5], Huisgen cycloaddition [2] [6], and intramolecular nucleophilic cyclization [3] [7]. Here, we extend these studies with methodology for the preparation of histidine-containing macrocycles 4 and CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2473  2012 Verlag Helvetica Chimica Acta AG, Zürich 1 ) Present address: Department of Biological Chemistry, School of Medicine, University of California, Irvine, USA. 2 ) Present address: School of Materials, Science and Engineering, Nanyang Technological University, Singapore. 3 ) Present address: School of Chemistry and Physics, University of Adelaide, South Australia, Australia.