Opioid Activity of 4-Imidazolidinone Positional Analogues of Leu-Enkephalin Marke´ta Rinnova´, Adel Nefzi and Richard A. Houghten* Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA Received 22 May 2002; accepted 23 July 2002 Abstract—Modulation of opioid activity was accomplished for analogues of Leu-enkephalin through incorporation of a 4-imi- dazolidinone moiety. The peptide backbone was constrained via a methylene bridge between two neighboring amides within its regular peptide sequence, which was expected to disrupt the secondary structure of the original molecule. Five positional analogues of Leu-enkephalin based on the same sequence and different location of the imidazolidinone-constrict were designed, synthesized, and examined for their affinity to m-, d- and k-opioid receptors. # 2002 Elsevier Science Ltd. All rights reserved. Peptidomimetics 1 are generally designed to mimic the topology of peptides using a range of synthetic approa- ches to change typical amide backbone characteristics. Alteration of amide bonds using a variety of isosteres or insertion of unnatural amino acids into a peptide sequence has widely proven to enhance enzymatic sta- bility of peptides. 2 Various approaches have also been used to rigidify an active structure by cyclization or by introduction of constraints into regular peptide mol- ecules in order to amplify conformational and func- tional features beneficial to enzymatic stability and biological activity. Diverse peptidomimetics have found applications in immunology, 3 receptor binding interac- tions, 4 and in the design of enzyme inhibitors. 5 Our interest focused on the introduction of a simple hetero- cyclic constraint based upon the generation of an 4- imidazolidinone moiety at individual amide bonds while preserving the remaining amide backbone and side chain functionalities. 4-Imidazolidinone is a five-mem- bered ring that can be formed directly on an N-terminus of a peptide backbone via a cyclic condensation with an aldehyde or ketone. 6,7 Traditionally, this cyclization mechanism has been used for the temporary protection of primary amines 7 or as a synthon for the preparation of unnatural amino acids. 8 Since the hydrolytic stability of N-terminal imidazolidinones is reported to be rela- tively low and C-2 substituent-dependent, its incor- poration to ‘temporarily’ modify the peptide, has been attempted in order to create bioreversible prodrugs. 9 This concept was investigated by several groups on Leu-enkephalin analogues with diverse N-terminal tyrosine-based imidazolidinones. 10 13 The goal of those studies was to obtain an analogue having a slow rate of hydrolysis while providing complete recovery of the original active substance. Investigation of imidazolidi- none derivatives formed from monosaccharide-modified enkephalins by intramolecular rearrangement 14,15 has also been carried out. To our knowledge, the incor- poration of the 4-imidazolidinone group into specific sequences, in order to reduce the memory loss asso- ciated with aging, 16 is the only example of biological activity of imidazolidinone-modified peptides. 4-Imizo- lidinones, derived from single aromatic amino acids and acetone, have also been found to be moderate inhibitors of tyrosine and histidine decarboxylase. 17 Recently, we developed a method for a solid-phase synthesis of 1,2,5-trisubstituted 4-imidazolidinones 18 based on Katritzky’s benzotriazole-mediated reaction. 19 We have adopted this synthetic protocol in the solid- phase preparation of various imidazolidinone-based peptidomimetics. 20 This enables the incorporation of individual imidazolidinone constraints into each posi- tion of the Leu-enkephalin sequence and thus prepara- tion of a set of peptidomimetic analogues (Scheme 1). This paper presents the relationship between the posi- tion of the imidazolidinone insert within a sequence and the opioid activity of such analogues. We have prepared 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00678-9 Bioorganic & Medicinal Chemistry Letters 12 (2002) 3175–3178 *Corresponding author. Tel.: +1-858-455-3803; fax: +1-858-455- 3804; e-mail: houghten@tpims.org