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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis and in vitro Characterization of New, Potent andSelective Oxytocin Receptor AgonistsKazimierz Wiśniewski, Robert Galyean, Claudio D. Schteingart,Hiroe Tariga, Glenn Croston, Sudarkodi Alagarsamy, andPierre J.-M. RivièreFerring Research Institute Inc., 4245 Sorrento Valley Boulevard,San Diego, CA, 92121, U.S.A.IntroductionOxytocin (OT, 1) exerts its numerous biological functions by interacting with oxytocinreceptors (OTR) located in the periphery (uterine contractions, milk ejection) and in theCNS (social and maternal behavior) [1]. OT is widely used as a labor-inducing agent [2]and, in a few markets, for lactation support in term mothers. Substantial research has beenfocused on identifying OTR antagonists suitable for treatment of premature labor [3], whileefforts to design new OTR agonists have been relativelysparse. Side effects associated with the intravenous orintranasal administration of OT are well described in theliterature and are attributed to lack of selectivity versusrelated receptors such as the V 2 R, which may result inhyponatremia [4]. Therefore, the identification of oxytocinagonists that selectively activate the OTR could betherapeutically useful and free of these dose-limiting sideeffects.The biological activity of the OT molecule has beenshown to be very sensitive to changes in position 7. OTanalogs with Gly 7 or Sar 7 have shown an improvedselectivity profile versus the related vasopressin receptors [5]whereas other modifications in position 7 [6] resulted inFig. 1. Structure of OTanalogues synthesized inthis study.compounds with different degrees of antagonistic activity.Here we report the synthesis and in vitro evaluation of aseries of OT agonists with N-alkylglycine residues(-NR 2 -CH 2 -CO-) in position 7 as the key modification(Figure 1). All new compounds were prepared as desamino analogs (R 0 = H) containingeither an unaltered disulfide bridge or its monocarba modifications, and some compoundswere also modified in position 2 with Phe.Results and DiscussionThe linear peptides were synthesized by standard SPPS methods using the Fmoc strategy.The N-alkylglycine residues in position 7 were introduced by a two step procedurecomprising the acylation of the resin-bound C-terminal dipeptide with bromoacetic acidfollowed by a treatment with an appropriate primary amine. Several Fmoc-N-alkylglycineswere also prepared separately by a modified literature procedure [7] and used in SPPS. Thelinear peptides were cleaved from the resin and cyclized in solution and purified bypreparative HPLC.The compounds were tested in in vitro functional assays for their agonistic potencyand efficacy at the OTR and for the selectivity versus the related vasopressin receptors(Table 1). While the natural ligand OT (1) lacks OTR specificity, carbetocin (carba-1-[Tyr(Me) 2 ]dOT) 2, clinically used for prevention of postpartum bleeding [8], is fairlyselective versus both the hV 2 R (selectivity ratio of 240) and the V 1a R. To improve theselectivity profile of 2, a substituent in position 4 of the pyrrolidine ring of the Pro 7 residuewas introduced. The modification led to compound 3 with selectivity versus hV 2 R of 700.Analogue 4 in which the Pro residue was replaced with Gly (R 2 = R 3 = H) showed furtherimprovement in V 2 R selectivity as compared to 2. That result prompted us to investigatethe position 7 open ring analogues in which the Pro 7 residue was replaced with a variety ofN-alkylglycines. Compounds 5-22 were prepared with the disulfide bridge or its monocarbamodification. The Tyr 2 analogues 8 (R 2 = 3-MeBzl), 12 (R 2 = 4-FBzl) and 22 (R 2 = 3-306