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Table 1. GPI and MVD assay of TIPP analoguesCompoundGPIMVDIC 50 (nM) K e (nM) IC 50 (nM) K e (nM)[Cpa 1 ]TIPP 6030 18.3[Hcp 1 ]TIPP 875 (IC 30 ) a 0.392 (IC 30 ) a[Dcap 1 ]TIPP > 1000 8.26[Dgcp 1 ]TIPP 4870 411[Mgcp 1 ]TIPP inactive inactivea Partial agonist (maximal inhibition of contractions = 60 %)from rat or guinea pig brain membrane binding sites, and agonist or antagonist activitieswere determined in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays.Results and DiscussionThe parent peptide [Cpa 1 ]TIPP is a potent δ opioid receptor antagonist with subnanomolarδ receptor binding affinity (Tables 1 and 2). It displays high δ vs. μ receptor selectivity, asdetermined in both the binding assays and in the functional MVD and GPI assays.Interestingly, [Hcp 1 ]TIPP showed subnanomolar δ partial agonist activity and δ receptorbinding affinity, and retained high δ receptor selectivity. The analogue containing a furtherextended alkyl chain, [Dcap 1 ]TIPP, displayed 4-fold lower δ receptor binding affinity than[Hcp 1 ]TIPP and turned out to be a full δ agonist. Surprisingly, the ethyleneglycolcontaininganalogue [Dgcp 1 ]TIPP was a weak δ opioid antagonist. Finally, the analoguecarrying the methoxypolyethyleneglycol moiety on the side chain at the 1-position,[Mgcp 1 ]TIPP, was found to be inactive at concentrations up to 10 μM. These resultsindicate that both the δ opioid receptor binding affinity and the intrinsic efficacy at the δreceptor are highly dependent on the length and the polarity of the carboxamide substituentat the 1-position residue in these TIPP analogues.Table 2. Opioid receptor binding affinities of TIPP analoguesCompound K i μ [nM] K i δ [nM] K i μ / K iδ[Cpa 1 ]TIPP 2190 0.978 2240[Hcp 1 ]TIPP 507 0.369 1370[Dcap 1 ]TIPP 174 1.54 113[Dgcp 1 ]TIPP > 5000 380 -[Mgcp 1 ]TIPP > 10000 > 10000 -AcknowledgmentsThis work was supported by grants from the CIHR (MOP-89716) and NIH (DA-004443).References1. Weltrowska, G., Nguyen, T.M.-D., Lemieux, C., Chung, N.N., Schiller, P.W. Chem. Biol. DrugDesign 72, 337-340 (2008).2. Schiller, P.W., Nguyen, T.M.-D., Weltrowska, G., Wilkes, B.C., Marsden, B.J., Lemieux, C.,Chung, N.N. Proc. Natl. Acad. Sci. U.S.A. 89, 11871-11875 (1992).3. Berezowska, I., Chung, N.N., Lemieux, C., Wilkes, B.C., Schiller, P.W. J. Med. Chem. 52, 6941-6945 (2009).4. Wang, W., Obeyesekere, N.U., McMurray, J.S. Tetrahedron Lett. 37, 6661-6664 (1996).427