High Affinity Conformationally Constrained Nociceptin/Orphanin FQ(1-13) Amide Analogues Laksana Charoenchai, †,§ Hongyan Wang, † Jia Bei Wang, † and Jane V. Aldrich* ,‡ Department of Pharmaceutical Sciences, School of Pharmacy, UniVersity of Maryland Baltimore, Baltimore, Maryland 21201, Department of Medicinal Chemistry, School of Pharmacy, UniVersity of Kansas, Lawrence, Kansas 66045 ReceiVed April 6, 2008 Abstract: A series of cyclic analogues with a lactam linkage were prepared by solid phase peptide synthesis to explore possible biologi- cally active conformation(s) of nociceptin/orphanin FQ (N/OFQ). cyclo[D-Asp 7 ,Lys 10 ]- and cyclo[Asp 6 ,Lys 10 ]N/OFQ(1-13)NH 2 exhibit high affinity (K i ) 0.27 and 0.34 nM, respectively) and high potency in the GTPγS assay (EC 50 ) 1.6 and 4.1 nM, respectively) at human nociceptin/orphanin FQ peptide (NOP) receptors. These analogues exhibit 2- to 3-fold higher affinity and 2- to 5-fold higher potency than the parent peptide. A novel receptor which possesses sequence similarity to the opioid receptors was identified and cloned from human, rat, and mouse brains in the 1990s. 1–3 The endogenous ligand, nociceptin 4,5 or orphanin FQ 5 (N/OFQ), a was subsequently identified by two research groups. Both ligand and receptor are widely distributed in the central nervous system and spinal cord. 6,7 Pharmacological studies of the nociceptin/orphanin FQ system reveal important roles in several physiological and pharmacological effects. 8,9 Despite the sequence similarity to opioid peptides, N/OFQ exhibits distinct physiological and pharmacological profiles different from opioids. 8–10 While early studies clearly demonstrated that N/OFQ modulates nociception, the effects of N/OFQ in nociception are complex and appear to be dependent on a number of factors, including the route of administration (see ref 8). Initially, N/OFQ was reported to induce hyperalgesia in the hot plate and tail flick assays when administered intracerebroventricularly (icv) 4,5 that were subse- quently reclassified as antianalgesic effects. 10,11 N/OFQ admin- istered icv can also functionally antagonize the analgesic effects of morphine and other opiates. 8,10,11 After intrathecal admin- istration, however, N/OFQ has been reported to produce analgesia and/or to potentiate morphine analgesia. 8–10,12 N/OFQ also produces anxiolytic-like effects in several behavioral paradigms of different types of anxiety states in animals, 9,13,14 and thus agonists for this receptor could have potential therapeutic applications as novel anxiolytic agents. Like opioid receptors, the opioid receptor-like 1 (ORL1) receptor or nociceptin/orphanin FQ peptide (NOP) receptor belongs to the G-protein coupled receptor family, and when N/OFQ interacts with its receptor, it triggers effector systems in a similar manner to the opioid receptors. Agonist binding to the NOP receptor inhibit adenylyl cyclase 4,5 and Ca 2+ channels, and activate protein kinases and K + channels. 9 However, N/OFQ has low affinity and negligible activity at opioid receptors, and similarly opioid ligands have low affinity at the NOP receptor. 15 To understand the structural requirements and the physiologi- cal roles of the nociceptin/orphanin FQ system, attempts have been made to develop high affinity and potent ligands for the NOP receptor. A variety of structure-activity relationship studies of N/OFQ have been performed (see refs 9, 16, and 17 for reviews). An alanine scan examined the effects of functional groups at each position and found that the residues in positions 1, 4, and 8 are important for receptor binding and activation. 18,19 D-Amino acid substitution indicated that an amino acid with the D-configuration was well tolerated at positions 2 and 7. 19 In addition, a C-terminal truncation study indicated that OFQ/ N(1-13)NH 2 (1, Figure 1) was the smallest fragment which was as potent as the endogenous ligand, 18,20 and thus 1 was the parent peptide in this study. Because the endogenous ligand is a linear peptide, it exhibits considerable conformational flexibility. Cyclization is one approach to reduce the flexibility of linear peptides and may also increase receptor affinity, potency, and/or metabolic stabil- ity. 21 Also potent cyclic analogues may provide information on possible biologically active conformations of the peptide. Only a limited number of cyclic analogues of N/OFQ have been reported to date, 22–25 and these have all involved cyclization via a disulfide bond; cyclo[Cys 10 ,Cys 14 ]N/OFQ(1-14)NH 2 23,24 and cyclo[Cys 7 ,Cys 10 ]N/OFQ(1-13)NH 2 25 exhibit the highest NOP receptor affinity and agonist potency of the reported cyclic analogues. We prepared conformationally constrained analogues of 1, along with their corresponding linear analogues. The * To whom correspondence should be addressed. Phone: (785) 864-2287. Fax: (785) 864-5326. E-mail: jaldrich@ku.edu. † Department of Pharmaceutical Sciences, School of Pharmacy, Univer- sity of Maryland Baltimore. ‡ Department of Medicinal Chemistry, School of Pharmacy, University of Kansas. § Current address: Faculty of Pharmaceutical Sciences, Ubonrajathanee University, Ubon ratchathani, Thailand 34190. a Abbreviations: Fmoc, fluorenylmethoxycarbonyl; CHO, Chinese ham- ster ovary; GTPγS, guanosine 5′-[γ-S]thiotriphosphate; HOAt, 1-hydroxy- 7-azabenzotriazole; Mtt, 4-methyltrityl; N/OFQ, nociceptin/orphanin FQ; NOP, nociceptin/orphanin FQ peptide; ORL1, opioid receptor-like 1; PAL- PEG-PS, peptide amide linker-polyethylene glycol-polystyrene; Pip, 2-phe- nylisopropyl; PyAOP, 7-azabenzotriazol-1-yloxytris(pyrrolidino)phospho- nium hexafluorophosphate. Figure 1. Structures of N/OFQ(1-13)NH 2 , cyclo[Asp 6 ,Lys 10 ]-, and cyclo[D-Asp 7 ,Lys 10 ]N/OFQ(1-13)NH 2 (1, 2, and 3). J. Med. Chem. 2008, 51, 4385–4387 4385 10.1021/jm800394v CCC: $40.75  2008 American Chemical Society Published on Web 07/15/2008