Drug Design DOI: 10.1002/anie.200805014 Molecular Modeling, Synthesis, and Biological Evaluation of Macrocyclic Calpain Inhibitors** Andrew D. Abell,* Matthew A. Jones, James M. Coxon, James D. Morton, Steven G. Aitken, Stephen B. McNabb, Hannah Y.-Y. Lee, Janna M. Mehrtens, Nathan A. Alexander, Blair G. Stuart, Axel T. Neffe, and Roy Bickerstaffe 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 1d–4d as potential inhibitors of the calcium-activated cysteine protease calpain. The alcohols 1c– 4c 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 5a–d were also investigated to establish the importance (or unimportance) of the macro- cyclic constraint for potency and selectivity of inhibition. Conformational searches [9] were carried out on 1a–d to 5a–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 1a–d, all conformers (> 99 %) within 12 kJ mol 1 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 1d. [*] Prof. A. D. Abell, [+] Dr. M. A. Jones, Prof. J. M. Coxon, Dr. S. G. Aitken, Dr. S. B. McNabb, Dr. J. M. Mehrtens, Dr. N. A. Alexander, B. G. Stuart, Dr. A. T. Neffe [#] Department of Chemistry, University of Canterbury Private Bag 4800, Christchurch (New Zealand) Dr. J. D. Morton, Dr. H. Y.-Y. Lee, Prof. R. Bickerstaffe Agriculture and Life Sciences Division, Lincoln University Post Office Box 84, Canterbury (New Zealand) Fax: (+ 64) 3-325-3851 [ + ] Present address: School of Chemistry & Physics The University of Adelaide North Terrace, Adelaide, SA 5005 (Australia) Fax: (+ 61) 8-8303-4358 E-mail: andrew.abell@adelaide.edu.au [ # ] Present address: Institute of Polymer Research GKSS Research, Centre Geesthacht GmbH Kantstrasse 55, 14513 Teltow (Germany) [**] We acknowledge the assistance of Dr. D. Q. McDonald (computa- tional chemistry), Dr. A. Muscroft-Taylor (synthesis), and M. Muir (supply of calpain), and financial support from the New Zealand Public Good Science and Technology Fund, Foundation for Research Science and Technology, the Australian Research Council, and Douglas Pharmaceuticals Limited. Supporting information (confirmation of the structure and purity of all compounds, details on modeling studies, and biological assay data) for this article is available on the WWW under http://dx.doi. org/10.1002/anie.200805014. Angewandte Chemie 1455 Angew. Chem. Int. Ed. 2009, 48, 1455 –1458  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim