Can Amphipathic Helices Influence the CNS Antinociceptive Activity of Glycopeptides Related to β‑Endorphin?

Glycosylated β-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to μ-, δ-, or κ-opioid receptors in the low nanomolar range (2.2–35 nM <i>K</i>_i’s). The glycoside moiety was required for intravenous (i.v.) but not for intracerebroventricular (i.c.v.) activity. Circular dichroism and NMR indicated the degree of helicity in H₂O, aqueous trifluoroethanol, or micelles. Glycosylation was essential for activity after i.v. administration. It was possible to manipulate the degree of helicity by the alteration of only two amino acid residues in the helical <i>address</i> region of the β-endorphin analogues without destroying μ-, δ-, or κ-agonism, but the antinociceptive activity after i.v. administration could not be directly correlated to the degree of helicity in micelles

Structural Requirements for CNS Active Opioid Glycopeptides

Glycopeptides related to β-endorphin penetrate the blood–brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [d-Ala²,<i>N</i>-MePhe⁴,Gly-ol⁵]­enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H₂O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (<b>11s</b> → <b>11sG</b> → <b>11</b>) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H₂O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects

Probes for Narcotic Receptor Mediated Phenomena. 34. Synthesis and Structure−Activity Relationships of a Potent μ-Agonist δ-Antagonist and an Exceedingly Potent Antinociceptive in the Enantiomeric C9-Substituted 5-(3-Hydroxyphenyl)-<i>N</i>-phenylethylmorphan Series

Both of the enantiomers of 5-(3-hydroxyphenyl)-N-phenylethylmorphan with C9α-methyl, C9-methylene, C9-keto, and C9α- and C9β-hydroxy substituents were synthesized and pharmacologically evaluated. Three of the 10 compounds, (1R,5R,9S)-(−)-9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl-2-azabicyclo[3.3.1]nonane ((1R,5R,9S)-(−)-10), (1R,5S)-(+)-5-(3-hydroxyphenyl)-9-methylene-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S)-(+)-14), and (1R,5S,9R)-(−)-5-(3-hydroxyphenyl)-9-methyl-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S,9R)-(+)-15) had subnanomolar affinity at μ-opioid receptors (Ki = 0.19, 0.19, and 0.63 nM, respectively). The (1R,5S)-(+)-14 was found to be a μ-opioid agonist and a μ-, δ-, and κ-antagonist in [35S]GTP-γ-S assays and was approximately 50 times more potent than morphine in a number of acute and subchronic pain assays, including thermal and visceral models of nociception. The (1R,5R,9S)-(−)-10 compound with a C9-hydroxy substituent axially oriented to the piperidine ring (C9β-hydroxy) was a μ-agonist about 500 times more potent than morphine. In the single-dose suppression assay, it was greater than 1000 times more potent than morphine. It is the most potent known phenylmorphan antinociceptive. The molecular structures of these compounds were energy minimized with density functional theory at the B3LYP/6-31G* level and then overlaid onto (1R,5R,9S)-(−)-10 using the heavy atoms in the morphan moiety as a common docking point. Based on modeling, the spatial arrangement of the protonated nitrogen atom and the 9β-OH substituent in (1R,5R,9S)-(−)-10 may facilitate the alignment of a putative water chain enabling proton transfer to a nearby proton acceptor group in the μ-opioid receptor