Design and Synthesis of FAJANU: a de Novo C 2 Symmetric Cyclopeptide Family Fayna Garcia-Martin, †,‡ Luis J. Cruz,* ,†,| Ricard A. Rodriguez-Mias, †,⊥ Ernest Giralt, †,§ and Fernando Albericio* ,†,‡,§ Institute for Research in Biomedicine, Barcelona Science Park, UniVersity of Barcelona, 08028 Barcelona, Spain, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain, Department of Organic Chemistry, UniVersity of Barcelona, 08028 Barcelona, Spain ReceiVed January 17, 2008 A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C 2 symmetry, cyclic structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first six-member family was previously synthesized and evaluated biologically. Several analogues presented greater activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more exhaustive synthetic and structural analysis: (i) structure-activity relationship provided clues about the key elements in the framework, (ii) NMR assignment confirmed C 2 symmetry, and (iii) confocal images revealed its penetration and cellular localization. Introduction During recent years, several families of antitumoral cyclic peptides with C 2 symmetry have been isolated from marine sources. All of these compounds show head-to-side chain heterodetic (ester for Ser or thioester for Cys) bonds linking the two backbones. Examples include the onchidin B, 1,2 thiocoraline, 3 triostin, 4 IB-01212, 5 sandramycin, 6 and luzopeptin A 7 families. Furthermore, these peptides usually contain N- methyl and D-amino acids in addition to other nonproteogenic amino acids and, in some cases, aromatic heterocycles, a few of which present a disulfide bridge, thereby converting them into bicyclic peptides. 8 Symmetric peptides with these charac- teristics have also been isolated from terrestrial organisms such as valinomycin, the related montanastatin, and korkormicin, all of which are from the actinobacteria phylum. 9 This collection of symmetric natural peptides presents a wide range of biological activity. In some cases, symmetry has proved to be suitable for interaction with highly symmetric complex structures, as is the case of thiocoraline and triostin, which are DNA intercalators. In other cases, symmetry has been considered to be an instrument for the microorganisms themselves to become more complex bioactive structures. Furthermore, in the case of valinomycin, polar groups are oriented toward the center, thereby allowing the delivery of selected ions through the cell membrane. 10 Syntheses of these peptides present an enormous synthetic challenge, which can jeopardize the development of drug discovery programs or even their industrial production. 11–19 Here we explored a de novo C 2 -symmetric cyclopeptide family, taking as the starting point the simplest cytotoxic IB-01212 (1) and its more active analogue 2 (Figure 1), 16,20 where both ester bonds were replaced by amide ones. Results and Discussion Design, Synthesis, and Biological Screening of the New FAJANU Peptide Family. Previous work by our group has described the preparation of 1 analogues, where the substitution of several amino acids using the same cyclic template, and the presence of the amide bond instead of the ester bond to close the cycle were examined. 29 The result of that study was the homodetic analogue 2, which was more stable than 1 and showed a similar activity. In the present study, we designed a new structure by increasing the macrocycle size and by adding aromatic heterocycles with the purpose of improving antitumor activity. The new structure shares the chemical feasibility of IB-01212 analogues and the structural moieties relevant in highly cytotoxic compounds such as triostin or thiocoraline. NMe-Gly (Sar) a , a common amino acid in this kind of peptide, * To whom correspondence should be addressed. Fax: 34934037126 (F.A.). E-mail: albericio@pcb.ub.us (F.A.). † Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona. ‡ CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine. § Department of Organic Chemistry, University of Barcelona. | Present address: Department of Tumor Immunology, NCMLS 278, Radboud University Nijmegen Medical Centre, Geert Grooteplein 26/28, 6525 GA Nijmegen, The Netherlands. ⊥ Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115 a Abbreviations: Abbreviations used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1982, 247, 977-983. The following additional abbreviations are used: aa, amino acid; Alloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl; BPH, biphenyl-4-carboxylic acid; CHR, chromone carboxylic acid; CTC, chlorotrityl chloride (Barlos) resin; Dab, diaminobu- tiric acid; Dap, diaminopropyl carboxylic acid; DBU, 1,8-diazabicyclo- [5.4.0]undec-7-ene; DIPEA, N,N-diisopropylethylamine; DIPCDI, N′,N′′-diisopropylcarbodiimide; DKP, diketopiperazine; DMF, N,N-dim- Figure 1. Structure of IB-01212 (1) and its aza analogue (2). J. Med. Chem. 2008, 51, 3194–3202 3194 10.1021/jm800047b CCC: $40.75  2008 American Chemical Society Published on Web 05/08/2008