Tolerance is not detected by current assays and represents a major threat to antimalarial drug policy.

Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1-4 and their Nphenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics. Morphine (1, Fig. 1), the prototypical opioid, has been used for decades for pain relief, and its addictive properties are long and well recognized. Over the years, numerous semisynthetic and synthetic investigations were reported aiming at optimizing morphine's biological actions, especially its safety profile 1-3. These studies have resulted in clinically useful drugs for the treatment of pain and other human disorders (drug abuse, alcohol abuse, and gastrointestinal motility dysfunction), as well as in research tools 1-5. Morphine and structurally-derived compounds (e.g. oxycodone, oxymorphone, hydromorphone) are agonists at the µ opioid receptor (µOR), a G protein-coupled receptor (GPCR), and the opioid receptor subtype that primarily mediates desirable (analgesia) but also undesirable effects (i.e. constipation, respiratory depression, sedation, analgesic tolerance and dependence) of opioids 4-6. Moreover, the number of people misusing opioids, as well as of opioid-related deaths have increased dramatically during the past years 7 .

Morphine and structurally related derivatives are highly effective analgesics, and the mainstay in the medical management of moderate to severe pain. Pharmacological actions of opioid analgesics are primarily mediated through agonism at the µ opioid peptide (MOP) receptor, a G protein-coupled receptor. Position 17 in morphine has been one of the most manipulated sites on the scaffold and intensive research has focused on replacements of the 17-methyl group with other substituents. Structural variations at the N-17 of the morphinan skeleton led to a diversity of molecules appraised as valuable and potential therapeutics and important research probes. Discovery of therapeutically useful morphine-like drugs has also targeted the C-6 hydroxyl group, with oxymorphone as one of the clinically relevant opioid analgesics, where a carbonyl instead of a hydroxyl group is present at position 6. Herein, we describe the effect of N-substituent variation in morphine and oxymorphone on in vitro and in vivo biological properties and the emerging structure-activity relationships. We show that the presence of a N-phenethyl group in position 17 is highly favorable in terms of improved affinity and selectivity at the MOP receptor, potent agonism and antinociceptive efficacy. The N-phenethyl derivatives of morphine and oxymorphone were very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, they were highly effective against acute thermal nociception in mice with marked increased antinociceptive potency compared to the lead molecules. It was also demonstrated that a carbonyl group at position 6 is preferable to a hydroxyl function in these N-phenethyl derivatives, enhancing MOP receptor affinity and agonist potency in vitro and in vivo. These results expand the understanding of the impact of different moieties at the morphinan nitrogen on ligand-receptor interaction, molecular mode of action and signaling, and may be instrumental to the development of new opioid therapeutics.

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Herein, a family of hydrogel-forming peptides was designed starting from the short, tunable and amphipathic hexapeptide hydrogelator H-Phe-Glu-Phe-Gln-Phe-Lys-OH (1). The hydrophobic side chains as well as the nature of both N- and C-termini were modified in order to obtain suitable gelation conditions and drug release profiles for in vivo application. To potentially increase the enzymatic stability, an all-D analogue was prepared as well. After their macroscopic and microscopic characterization by rheology and transmission electron microscopy (TEM) analysis, opioid drugs were encapsulated into the hydrogels and sustained release experiments were carried out. Hydrogel toxicity was assessed in cell viability assays. Based on the physicochemical, mechanical, and noncytotoxic properties, H-Phe-Glu-Phe-Gln-Phe-Lys-NH2 (2) was further investigated for in vivo release of morphine. The antinociceptive effects following subcutaneous injection of the morphine-containing hydrogel 2 was evaluated in a model of thermal nociception using the mouse tail-flick test. Sustained antinociceptive effects over extended periods of time (up to 24 h) for morphine co-formulated with hydrogel 2, compared to morphine injection in solution (effects up to 2 h), were observed