Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors Ronan Gealageas a , Séverine Schneider a , Jean-Paul Humbert b , Isabelle Bertin b , Martine Schmitt a , Emilie Laboureyras c , Christophe Dugave d , Catherine Mollereau e , Guy Simonnet c , Jean-Jacques Bourguignon a , Frédéric Simonin b,⇑,  , Frédéric Bihel a,⇑,  a University of Strasbourg, CNRS, UMR7200, Faculty of Pharmacy, F-67400 Illkirch Graffenstaden, France b University of Strasbourg, CNRS, UMR7242, Inst Rech ESBS, F-67412 Illkirch Graffenstaden, France c University of Bordeaux 2, INCIA, CNRS UMR 5287, F-33076 Bordeaux, France d CEA, IBiTecS, F-91191 Gif Sur Yvette, France e University of Toulouse, CNRS, Inst Pharmacol & Biol Struct, UMR 5089, F-31077 Toulouse, France article info Article history: Received 31 August 2012 Revised 8 October 2012 Accepted 9 October 2012 Available online 17 October 2012 Keywords: Opioid-induced hyperalgesia NPFF receptors Pain Dipeptidic ligand GPCR abstract Based on our earlier reported neuropeptide FF receptors antagonist (RF9), we carried out an extensive structural exploration of the N-terminus part of the amidated dipeptide Arg-Phe-NH 2 in order to establish a structure-activity relationships (SAR) study towards both NPFF receptor subtypes. This SAR led to the discovery of dipeptides (12, 35) with subnanomolar affinities towards NPFF1 receptor subtype, similar to endogenous ligand NPVF. More particularly, compound 12 exhibited a potent in vivo preventive effect on opioid-induced hyperalgesia at low dose. The significant selectivity of 12 toward NPFF1-R indicates that this receptor subtype may play a critical role in the anti-opioid activity of NPFF-like peptides. Ó 2012 Elsevier Ltd. All rights reserved. The mammalian neuropeptide FF (FLFQPQRF-NH 2 ) belongs to the family of RFamide neuropeptides, characterized by an Arg-Phe-NH 2 sequence at their C-termini. NPFF and NPFF-related peptides are recognized by two G protein-coupled receptors named NPFF1-R (GPR147) and NPFF2-R (GPR74), and have been linked to various physiological activities. 1 At first, NPFF was iden- tified as an inducer of hyperalgesia and a modulator of opiate anal- gesia. 2 Moreover, several in vitro and in vivo studies involved NPFF in the regulation of cardiovascular system, 3 regulation of body temperature, 4 and water balance. 5 Several structure–activity rela- tionships studies were reported on NPFF-related peptides. 6 Among the various modifications, the N-terminus part of NPFF-related octapeptides were shortened, but by never more than 4 amino acids. In 2006, based on the observation that the C-terminus se- quence Arg-Phe-NH 2 is common to each NPFF-related peptide, we reported the N-acylated dipeptides RF2 and RF9 as non-selec- tive antagonists of both NPFF1-R and NPFF2-R (Fig. 1). 7 Stable en- ough to be subcutaneously co-administrated with opiates, RF9 completely blocked the delayed and long lasting paradoxical opi- oid-induced hyperalgesia (OIH) and prevented the development of associated tolerance. Moreover, in agreement with its antagonist function of both NPFF receptor subtypes, RF9 was shown to pre- vent hypothermia and hypertension induced by NPFF or NPVF. 7,8 More recently, on the basis of mice treated with RF9, Simonin and collaborators highlighted NPFF receptors as interesting thera- peutic targets to improve the analgesic efficacy of opioids by lim- iting the development of tolerance, and for the treatment of physical dependence. 9 We describe here an extensive structure–activity relationships study of the N-terminus part of the amidated dipeptide Arg-Phe- NH 2 , along with the configuration effects of both Ca. All dipeptidic compounds were synthesized by standard Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) on Rink-amide resin. For the side-chain protection, 2,2,4,6,7-pentamethyl-dihydrobenzofuran- 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.10.049 ⇑ Corresponding authors. Tel.: +33 0368854875 (F.S.); tel.: +33 0368854130 (F.B.). E-mail addresses: simonin@unistra.fr (F. Simonin), fbihel@unistra.fr (F. Bihel).   These authors contributed equally to this work. R N H O HN HN NH 2 H N O NH 2 O RF9 RF2 R Figure 1. Structure of compounds RF2 and RF9. Bioorganic & Medicinal Chemistry Letters 22 (2012) 7471–7474 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl