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The synthesis of conformationally restricted dipeptidic moieties 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba)-Gly ([(4 S)-amino-3-oxo-1,2,4,5-tetrahydro-1 H-2-benzazepin-2-yl]-acetic acid) and 8-hydroxy-4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Hba)- d-Ala ([(4S)-amino-8-hydroxy-3-oxo-1,2,4,5-tetrahydro-benzo[c]azepin-2-yl]-propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N-acyliminium precursor. Introducing these Aba scaffolds into the N-terminal tetrapeptide of dermorphin (H-Tyr- d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)-induced remarkable shifts in affinity and selectivity towards the opioid μ- and δ-receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N-terminal tetrapeptide H-Tyr- d-Ala-Phe-Gly-NH2, which is the minimal subunit of dermorphin needed for dermorphin-like opiate activity. [ABSTRACT FROM AUTHOR]

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in l- or d-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [ d-1-Nal4]- and [ d-2-Nal4]endomorphin-2, with IC50 values 14 ± 1.25 and 19 ± 2.1 n m, respectively, compared with 1.9 ± 0.21 n m for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [ d-1-Nal4]- and [ d-2-Nal4]-endomorphin-2, indicating that these analogs were μ-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [ d-1-Nal4]endomorphin-2 in about 46%. [ABSTRACT FROM AUTHOR]

The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH2 (Dmt = 2′,6′-dimethyltyrosine) ([Dmt1]DALDA) is a highly potent and selectiveμ-opioid agonist capable of crossing the blood–brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt1]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2′-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and inμ-,δ- andκ-opioid receptor-binding assays. The analogues showed various degrees ofμ receptor-binding selectivity, but all of them were lessμ-selective than the [Dmt1]DALDA parent peptide. Most analogues retained potent, fullμ-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partialμ-agonist) and one containingβ-(6′-dimethylamino-2′-naphthoyl)alanine (aladan) in place of Phe3 (4) (μ- andκ-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy. [ABSTRACT FROM AUTHOR]