Cyclic Enkephalins with a Diversely Substituted Guanidine Bridge or a Thiourea Bridge: Synthesis, Biological and Structural Evaluations

Two series of 22 and 15 atom cyclic enkephalins incorporating a diversely substituted guanidine bridge have been prepared to assess the potential effect of the bridge substitutions on their opioid activity profile. The most notable results were obtained with the shortest cyclic analogues, which showed a significant variation of their binding affinity toward μ and δ opioid receptors in relation to bridge substitution. NMR studies were performed to rationalize these data. Some small analogues were found to exist as at least one major and one minor stable forms, which could be separated by chromatography. In particular, the compounds <b>13</b> and <b>14</b> with a cyclic substituent were separated in three isomers and the basis of this multiplicity was explored by 2D NMR spectroscopy. All compounds were agonists with slight selectivity for the μ opioid receptor. Compounds <b>7a</b> (thiourea bridge) and <b>10a</b> (<i>N</i>-Me-guanidine bridge) showed nanomolar affinity toward μ receptor, the latter being the more selective for this receptor (40-fold)

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt¹]­DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂ (<b>9</b>; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c­[-d-Arg-Phe-Lys-Dmt-] (<b>1</b>) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp¹²⁷ and Glu²²⁹ receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c­[-Arg-d-Phe-d-Lys-d-Dmt-] (<b>2</b>), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c­[-d-Arg-Phe-LysΨ­[CH₂NH]­Dmt-] (<b>8</b>), with MOR agonist activity

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt¹]­DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂ (<b>9</b>; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c­[-d-Arg-Phe-Lys-Dmt-] (<b>1</b>) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp¹²⁷ and Glu²²⁹ receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c­[-Arg-d-Phe-d-Lys-d-Dmt-] (<b>2</b>), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c­[-d-Arg-Phe-LysΨ­[CH₂NH]­Dmt-] (<b>8</b>), with MOR agonist activity

χ‑Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the <i>N</i>-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH₂) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH₂ was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the <i>C</i>-terminus of the peptide sequences, (partial) (ant)­agonism at MOP and weak (ant)­agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism

In Vitro Membrane Permeation Studies and in Vivo Antinociception of Glycosylated Dmt¹‑DALDA Analogues

In this study the μ opioid receptor (MOR) ligands DALDA (Tyr-d-Arg-Phe-Lys-NH₂) and Dmt¹-DALDA (Dmt-d-Arg-Phe-Lys-NH₂, Dmt = 2′,6′-dimethyltyrosine) were glycosylated at the N- or C-terminus. Subsequently, the modified peptides were subjected to in vitro and in vivo evaluation. In contrast to the N-terminally modified peptide (<b>3</b>), all peptide analogues derivatized at the C-terminus (<b>4</b>–<b>7</b>) proved to possess high affinity and agonist potency at both MOR and DOR (δ opioid receptor). Results of the Caco-2 monolayer permeation, as well as in vitro blood–brain barrier model experiments, showed that, in the case of compound <b>4</b>, the glycosylation only slightly diminished the lumen-to-blood and blood-to-lumen transport. Altogether, these experiments were indicative of transcellular transport but not active transport. In vivo assays demonstrated that the peptides were capable of (i) crossing the blood–brain barrier (BBB) and (ii) activating both the spinal ascending as well as the descending opioid pathways, as determined by the tail-flick and hot-plate assays, respectively. In contrast to the highly selective MOR agonist Dmt¹-DALDA <b>1</b>, compounds <b>4</b>–<b>7</b> are mixed MOR/DOR agonists, expected to produce reduced opioid-related side effects

Variation of the Net Charge, Lipophilicity, and Side Chain Flexibility in Dmt¹‑DALDA: Effect on Opioid Activity and Biodistribution

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH₂ ([Dmt¹]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt¹]-DALDA and to investigate the Phe³ side chain flexibility, the final amide bond was <i>N</i>-methylated and Phe³ was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt¹]-DALDA and its <i>N</i>-methyl analogue, Dmt-d-Arg-Phe-<i>N</i>MeLys-NH₂, showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit² generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt¹]-DALDA and [Dmt¹,<i>N</i>MeLys⁴]-DALDA

Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice

The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure–activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions