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Adequate pain management, particularly chronic pain, remains a major challenge associated with modern-day medicine. Current pharmacotherapy offers unsatisfactory long-term solutions due to serious side effects related to the chronic administration of analgesic drugs. Morphine and structurally related derivatives (e.g., oxycodone, oxymorphone, buprenorphine) are highly effective opioid analgesics, mediating their effects via the activation of opioid receptors, with the mu-opioid receptor subtype as the primary molecular target. However, they also cause addiction and overdose deaths, which has led to a global opioid crisis in the last decades. Therefore, research efforts are needed to overcome the limitations of present pain therapies with the aim to improve treatment efficacy and to reduce complications. This review presents recent chemical and pharmacological advances on 14-oxygenated- N -methylmorphinan-6-ones, in the search of safer pain therapeutics. We focus on drug design strategies and structure-activity relationships on specific modifications in positions 5, 6, 14 and 17 on the morphinan skeleton, with the goal of aiding the discovery of opioid analgesics with more favorable pharmacological properties, potent analgesia and fewer undesirable effects. Targeted molecular modifications on the morphinan scaffold can afford novel opioids as bi- or multifunctional ligands targeting multiple opioid receptors, as attractive alternatives to mu-opioid receptor selective analgesics.

Opioids are the drugs of choice in severe pain management. Unfortunately, their use involves serious, potentially lethal side effects. Therefore, efforts in opioid drug design turn toward safer and more effective mechanisms, including allosteric modulation. In this study, molecular dynamics simulations in silico and 'writhing' tests in vivo were used to characterize potential allosteric mechanism of two previously reported compounds. The results suggest that investigated compounds bind to μ opioid receptor in an allosteric site, augmenting action of morphine at subeffective doses, and exerting antinociceptive effect alone at higher doses. Detailed analysis of in silico calculations suggests that first of the compounds behaves more like allosteric agonist, while the second compound acts mainly as a positive allosteric modulator.

Among the opioid receptors, the kappa opioid receptor (KOR) has been gaining substantial attention as a promising molecular target for the treatment of numerous human disorders, including pain, pruritus, affective disorders (i.e., depression and anxiety), drug addiction, and neurological diseases (i.e., epilepsy). Particularly, the knowledge that activation of the KOR, opposite to the mu opioid receptor (MOR), does not produce euphoria or leads to respiratory depression or overdose, has stimulated the interest in discovering ligands targeting the KOR as novel pharmacotherapeutics. However, the KOR mediates the negative side effects of dysphoria/aversion, sedation, and psychotomimesis, with the therapeutic promise of biased agonism (i.e., selective activation of beneficial over deleterious signaling pathways) for designing safer KOR therapeutics without the liabilities of conventional KOR agonists. In this review, the development of new KOR ligands from the class of diphenethylamines is presented. Specifically, we describe the design strategies, synthesis, and pharmacological activities of differently substituted diphenethylamines, where structure-activity relationships have been extensively studied. Ligands with distinct profiles as potent and selective agonists, G protein-biased agonists, and selective antagonists, and their potential use as therapeutic agents (i.e., pain treatment) and research tools are described.

There is growing evidence on the role of peripheral µ-opioid receptors (MORs) in analgesia and analgesic tolerance. Opioid analgesics are the mainstay in the management of moderate to severe pain, and their efficacy in the alleviation of pain is well recognized. Unfortunately, chronic treatment with opioid analgesics induces central analgesic tolerance, thus limiting their clinical usefulness. Numerous molecular mechanisms, including receptor desensitization, G-protein decoupling, β-arrestin recruitment, and alterations in the expression of peripheral MORs and microbiota have been postulated to contribute to the development of opioid analgesic tolerance. However, these studies are largely focused on central opioid analgesia and tolerance. Accumulated literature supports that peripheral MORs mediate analgesia, but controversial results on the development of peripheral opioid receptors-mediated analgesic tolerance are reported. In this review, we offer evidence on the consequence of the activation of peripheral MORs in analgesia and analgesic tolerance, as well as approaches that enhance analgesic efficacy and decrease the development of tolerance to opioids at the peripheral sites. We have also addressed the advantages and drawbacks of the activation of peripheral MORs on the sensory neurons and gut (leading to dysbiosis) on the development of central and peripheral analgesic tolerance.

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 N-phenethyl 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.

Herein, the synthesis and pharmacological characterization of an extended library of differently substituted N-methyl-14- O-methylmorphinans with natural and unnatural amino acids and three dipeptides at position 6 that emerged as potent μ/δ opioid receptor (MOR/DOR) agonists with peripheral antinociceptive efficacy is reported. The current study adds significant value to our initial structure-activity relationships on a series of zwitterionic analogues of 1 (14- O-methyloxymorphone) by targeting additional amino acid residues. The new derivatives showed high binding and potent agonism at MOR and DOR in vitro. In vivo, the new 6-amino acid- and 6-dipeptide-substituted derivatives of 1 were highly effective in inducing antinociception in the writhing test in mice after subcutaneous administration, which was antagonized by naloxone methiodide demonstrating activation of peripheral opioid receptors. Such peripheral opioid analgesics may represent alternatives to presently available drugs for a safer pain therapy.

Pain, particularly chronic pain, is still an unsolved medical condition. Central goals in pain control are to provide analgesia of adequate efficacy and to reduce complications associated with the currently available drugs. Opioids are the mainstay for the treatment of moderate to severe pain. However, opioid pain medications also cause detrimental side effects, thus highlighting the need of innovative and safer analgesics. Opioids mediate their actions via the activation of opioid receptors, with the mu-opioid receptor as the primary target for analgesia, but also for side effects. One long-standing focus of drug discovery is the pursuit for new opioids exhibiting a favorable dissociation between analgesia and adverse effects. In this study, we describe the in vitro and in vivo pharmacological profiles of the 14- O -phenylpropyl substituted analog of the mu-opioid agonist 14- O -methyloxymorphone (14-OMO). The consequence of the substitution of the 14- O -methyl in 14-OMO with a 14- O -phenylpropyl group on in vitro binding and functional activity, and in vivo behavioral properties (nociception and gastrointestinal motility) was investigated. In binding studies, 14- O -phenylpropyloxymorphone (POMO) displayed very high affinity at mu-, delta-, and kappa-opioid receptors ( K i values in nM, mu:delta:kappa = 0.073:0.13:0.30) in rodent brain membranes, with complete loss of mu-receptor selectivity compared to 14-OMO. In guinea-pig ileum and mouse vas deferens bioassays, POMO was a highly efficacious and full agonist, being more potent than 14-OMO. In the [ 35 S]GTPγS binding assays with membranes from CHO cells expressing human opioid receptors, POMO was a potent mu/delta-receptor full agonist and a kappa-receptor partial agonist. In vivo , POMO was highly effective in acute thermal nociception (hot-plate test, AD 50 = 0.7 nmol/kg) in mice after subcutaneous administration, with over 70- and 9000-fold increased potency than 14-OMO and morphine, respectively. POMO-induced antinociception is mediated through the activation of the mu-opioid receptor, and it does not involve delta- and kappa-opioid receptors. In the charcoal test, POMO produced fourfold less inhibition of the gastrointestinal transit than 14-OMO and morphine. In summary, POMO emerges as a new potent mixed mu/delta/kappa-opioid receptor agonist with reduced liability to cause constipation at antinociceptive doses.

Position 6 of the morphinan skeleton plays a key role in the μ-opioid receptor (MOR) activity in vitro and in vivo. We describe the consequence of the 6-carbonyl group deletion in N-methylmorphinan-6-ones 1-4 on ligand-MOR interaction, signaling, and antinociception. While 6-desoxo compounds 1a, 2a, and 4a show similar profiles to their 6-keto counterparts, the 6-desoxo-14-benzyloxy substituted 3a displays significantly increased MOR binding and agonist potency and a distinct binding mode compared with its analogue 3.

We previously reported on a series of small molecules targeting the κ-opioid (KOP) receptor featuring a diphenethylamine scaffold and showed the promise of these ligands as effective analgesics with reduced liability for adverse effects. This study expands the structure-activity relationships on our original series by presenting several modifications in the lead compounds 1 (HS665) and 2 (HS666). A library of new diphenethylamines was designed, synthesized, and pharmacologically evaluated. In comparison with 1 and 2, the KOP receptor affinity, selectivity, and agonist activity were modulated by introducing bulkier N-substituents, a 2-fluoro substitution, and additional hydroxyl groups at positions 3' and 4'. Several analogues showed subnanomolar affinity and excellent KOP receptor selectivity acting as full or partial agonists, and one as an antagonist. The new diphenethylamines displayed antinociceptive efficacies with increased potencies than U50,488, 1 and 2 in the writhing assay and without inducing motor dysfunction after sc administration in mice.

Background and Purpose: The κ receptor has a central role in modulating neurotransmission in central and peripheral neuronal circuits that subserve pain and other behavioural responses. Although κ receptor agonists do not produce euphoria or lead to respiratory suppression, they induce dysphoria and sedation. We hypothesized that brain-penetrant κ receptor ligands possessing biased agonism towards G protein signalling over β-arrestin2 recruitment would produce robust antinociception with fewer associated liabilities., Experimental Approach: Two new diphenethylamines with high κ receptor selectivity, HS665 and HS666, were assessed following i.c.v. administration in mouse assays of antinociception with the 55°C warm-water tail withdrawal test, locomotor activity in the rotorod and conditioned place preference. The [ 35 S]-GTPγS binding and β-arrestin2 recruitment in vitro assays were used to characterize biased agonism., Key Results: HS665 (κ receptor agonist) and HS666 (κ receptor partial agonist) demonstrated dose-dependent antinociception after i.c.v. administration mediated by the κ receptor. These highly selective κ receptor ligands displayed varying biased signalling towards G protein coupling in vitro, consistent with a reduced liability profile, reflected by reduced sedation and absence of conditioned place aversion for HS666., Conclusions and Implications: HS665 and HS666 activate central κ receptors to produce potent antinociception, with HS666 displaying pharmacological characteristics of a κ receptor analgesic with reduced liability for aversive effects correlating with its low efficacy in the β-arrestin2 signalling pathway. Our data provide further understanding of the contribution of central κ receptors in pain suppression, and the prospect of dissociating the antinociceptive effects of HS665 and HS666 from κ receptor-mediated adverse effects., (© 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Among opioids, morphinans are of major importance as the most effective analgesic drugs acting primarily via μ-opioid receptor (μ-OR) activation. Our long-standing efforts in the field of opioid analgesics from the class of morphinans led to N-methylmorphinan-6-ones differently substituted at positions 5 and 14 as μ-OR agonists inducing potent analgesia and fewer undesirable effects. Herein we present the first thorough molecular modeling study and structure-activity relationship (SAR) explorations aided by docking and molecular dynamics (MD) simulations of 14-oxygenated N-methylmorphinan-6-ones to gain insights into their mode of binding to the μ-OR and interaction mechanisms. The structure of activated μ-OR provides an essential model for how ligand/μ-OR binding is encoded within small chemical differences in otherwise structurally similar morphinans. We reveal important molecular interactions that these μ-agonists share and distinguish them. The molecular docking outcomes indicate the crucial role of the relative orientation of the ligand in the μ-OR binding site, influencing the propensity of critical non-covalent interactions that are required to facilitate ligand/μ-OR interactions and receptor activation. The MD simulations point out minor differences in the tendency to form hydrogen bonds by the 4,5α-epoxy group, along with the tendency to affect the 3-7 lock switch. The emerged SARs reveal the subtle interplay between the substituents at positions 5 and 14 in the morphinan scaffold by enabling the identification of key structural elements that determine the distinct pharmacological profiles. This study provides a significant structural basis for understanding ligand binding and μ-OR activation by the 14-oxygenated N-methylmorphinan-6-ones, which should be useful for guiding drug design.

The κ opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calculations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the KOP receptor attained by molecular dynamics simulations. The structure-activity relationships derived from computational studies was in accordance with pharmacological activities of targeted diphenethylamines at the KOP receptor established by competition binding and G protein activation in vitro assays. Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond, which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed. Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists. Thus, combination of experimental and molecular modeling strategies provides an initial framework for understanding the structural features of diphenethylamines that are essential to promote binding affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding event into molecular changes, ultimately leading to receptor activation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

While μ-opioid receptor (MOR) agonists remain the most powerful analgesics for the treatment of severe pain, serious adverse side effects that are secondary to their central nervous system actions pose substantial barriers to therapeutic use. Preclinical and clinical evidence suggest that peripheral MORs play an important role in opioid analgesia, particularly under inflammatory conditions. However, the mechanisms of peripheral MOR signaling in primary afferent pain fibres remain to be established. We have recently introduced a novel ex vivo optical imaging approach that, for the first time, allows the study of physiological functioning within individual peripheral nociceptive fibre free nerve endings in mice. In the present study, we found that MOR activation in selectively identified, primary afferent CGRP nociceptive terminals caused inhibition of N-type Ca(2+) channel signaling and suppression of action potential-evoked Ca(2+) fluorescent transients mediated by 'big conductance' Ca(2+)-activated K(+) channels (BKCa). In the live animal, we showed that the peripherally acting MOR agonist HS-731 produced analgesia and that BKCa channels were the major effectors of the peripheral MOR signaling. We have identified two key molecular transducers of MOR activation that mediate significant inhibition of nociceptive signaling in primary afferent terminals. Understanding the mechanisms of peripheral MOR signaling may promote the development of pathway selective μ-opioid drugs that offer improved therapeutic profiles for achieving potent analgesia while avoiding serious adverse central side effects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.

Tubulin is a major molecular target for anticancer drugs. The dynamic process of microtubule assembly and disassembly can be blocked by various agents that bind to distinct sites on tubulin, usually its β-subunit. Among the antimitotic agents that perturb microtubule dynamics, noscapinoids represent an emerging class of agents. In particular, 9'-bromonoscapine (EM011) has been identified as a potent noscapine analog. Here we present high yielding, efficient synthetic methods based on Suzuki coupling of 9'-alkyl and 9'-arylnoscapines and an evaluation of their antiproliferative properties. Our results showed that 9'-alkyl and 9'-aryl derivatives inhibit proliferation of human cancer cells. The most active compounds were the 9'-methyl and the 9'-phenyl derivatives, which showed similar cytotoxic potency in comparison to the 9'-brominated derivative. Interestingly these newly synthesized derivatives did not induce cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. All of these derivatives, except 9'-(2-methoxyphenyl)-noscapine, efficiently induced a cell cycle arrest in the G2/M phase of the cell cycle in HeLa and Jurkat cells. Furthermore, we showed that the most active compounds in HeLa cells induced apoptosis following the mitochondrial pathway with the activation of both caspase-9 and caspase-3. In addition, these compounds significantly reduced the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures., Results: This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone., Conclusion: Development of such novel chemotypes by targeting position 6 provides valuable insights on ligand-receptor interaction and molecular mode of action, and may aid in identification of opioid therapeutics with enhanced analgesic properties and fewer undesirable effects.

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.

Although the µ opioid receptor (MOR) was pharmacologically and biochemically identified in binding studies forty years ago, its structure, function, and true complexity only have emerged after its cloning in 1993. Continuous efforts from many laboratories have greatly advanced our understanding of MORs, ranging from their anatomic distribution to cellular and molecular mechanisms, and from cell lines to in vivo systems. The MOR is recognized as the main target for effective pain relief, but its involvement in many other physiological functions has also been recognized. This review provides a synopsis on the history of research on MORs and ligands acting at the MOR with the focus on their clinical and potential use as therapeutic drugs, or as valuable research tools. Since the elucidation of the chemical structure of morphine and the characterization of endogenous opioid peptides, research has stimulated the development of new generations of MOR ligands with distinct pharmacological profiles (agonist, antagonist, mixed agonist/antagonist and partial agonist) or site of action (central/peripheral). Discovery of therapeutically useful morphine-like drugs and innovative drugs with new scaffolds, with several outstanding representatives, is discussed. Extensive efforts on modifications of endogenous peptides to attain stable and MOR selective analogs are overviewed with stimulating results for the development of peptide-based pharmaceuticals. With pharmacophore modeling as an important tool in drug discovery, application of modern computational methodologies for the development of morphinans as new MOR ligands is described. Moreover, the crystal structure of the MOR available today will enable the application of structure-based approaches to design better drugs for the management of pain, addiction and other human diseases, where MORs play a key role.

Here we have studied regulatory changes of µ-opioid receptors accompanying in vivo 14-methoxymetopon treatments of rats. Previously, this ligand has been shown to be an extremely potent, centrally acting µ-opioid specific analgesic with low physical dependence, tolerance, respiratory depression, constipation and other side effects. Our work shows that it is a highly potent full agonist of µ-opioid receptor coupled G-protein signaling in vitro, alike the well-known opioid agonist, etorphine. However, unlike etorphine, which desensitized and down-regulated the endogenous µ-opioid receptors, 14-methoxymetopon, given to rats intraperitoneally (i.p.) either acutely or chronically, did not change the binding or G-protein signaling of µ-opioid receptors in rat brain subcellular membranes. Thereby, these data provide further evidence that there is no direct relationship between the efficacy of the ligand in signaling and its ability to internalize or desensitize the receptor. Viewed collectively with published work, it is discussed that µ-opioid receptors display functional selectivity, also called 'biased agonism'. This concept implies that each ligand may induce unique, ligand-specific receptor conformation that can result in distinct agonist- directed trafficking and/or signal transduction pathways associated with the receptor. Ligand-specific signaling may open up new directions for designing potent analgesics that do not interact with unwanted signaling pathways, which mediate undesired side-effects, such as tolerance and dependence.

The well-known opioid agonists, oxycodone and oxymorphone, and the opioid antagonists, naloxone and naltrexone, are commonly used clinical agents and research tools in the opioid field. They belong to the class of morphinan-6-ones, and produce their pharmacological effects by interacting with opioid receptors, i.e. mu (MOR), delta (DOR) and kappa (KOR). The search for potent agonists and antagonists has continuously engaged the interest of pharmaceutical research, aiming for the identification of safer therapeutic agents or discovery of opioids with novel therapeutic properties and with lesser unwanted side effects. The chemically highly versatile carbonyl group in position 6 of mophinan-6-ones permits functionalization and modification leading to numerous opioid ligands. We have focused on representative examples of various derivatives and interesting approaches for the development of structurally distinct molecules with substitution at C6 (e.g. 6-methylene, 6-hydroxy, 6-amido, bifunctional ligands), as preclinically and clinically valuable opioids. In this work, the development of 6-amino and 6-guanidino substituted 14-alkoxymorphinans, including the synthesis and pharmacological investigations is presented. The new approach represented by the introduction of amino and guanidino groups into position 6 of the morphinan skeleton of 14-O-methyloxymorphone, led to compounds with high efficacy, MOR affinity and selectivity, which act as potent antinociceptive agents. Altogether, as a consequence of target drug design and synthetic efforts in the field of morphinan-6-ones, we achieve a better understanding of the function of the opioid system, and such efforts may open new avenues for further investigations.

The κ opioid receptor (KOR) plays a significant role in many physiological functions, including pain relief, stress, depression, drug abuse, anxiety and psychotic behaviors. KORs are widely distributed in the central and peripheral nervous systems, and are specifically activated by endogenous opioids derived from prodynorphin. They are members of the G protein-coupled receptor superfamily, and the crystal structure of the human KOR was recently elucidated. KORs were initially studied for their involvement in mediation of pain as stimulation of KOR produces analgesia and minimizes abuse liability and other side effects. Nowadays, the KOR is rapidly emerging as an important target for the treatment of a variety of other human disorders. Specifically, the KOR system has become increasingly implicated as a modulator of stress-related and addictive behaviors. Several selective KOR partial agonists and antagonists have been developed as potential antidepressants, anxiolytic and anti-addiction medications. Although many KOR ligands have not demonstrated desirable pharmacological properties, some have been shown to be viable drug candidates. Herein, we describe chemical and pharmacological developments on KOR ligands, advantages and challenges, and potential therapeutic applications of ligands for KORs. In the second part, recent advances in the KOR drug design utilizing computational approaches are presented, with focus on the discovery of a new naturally derived scaffold, sewarine, as a novel class of selective KOR ligands with antagonist properties, using a pharmacophore-based virtual screening strategy.

Here we report on the design, synthesis, and biological characterization of novel κ opioid (KOP) receptor ligands of diphenethylamines. In opioid receptor binding and functional assays, the N-cyclobutylmethyl substituted derivative 4 (HS665) showed the highest affinity and selectivity for the KOP receptor and KOP agonist potency. Compound 4 inhibited acetic acid induced writhing after subcutaneous administration in mice via KOP receptor-mediated mechanisms, being equipotent as an analgesic to the KOP agonist U50,488.

A new procedure was elaborated for the synthesis of potentially opioid-active thiazolo- and thiazinomorphinans. These derivatives and some intermediates related with the synthesis were tested in opioid receptor binding studies. Two compounds showed remarkable µ opioid activities and specificities. The ligand-stimulated [(35) S]GTPγS assays confirmed for both compounds the potent full agonist profile at the µ receptor and for the benzothiazinomorphinan derivative also the δ receptor full agonist character. The structures of these remarkably effective compounds were analyzed with the aid of computational chemistry calculations., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the most important functions of the opioid system is the control of pain. Among the three main opioid receptor classes (μ, δ, κ), the μ (MOR) is the main type targeted for pharmacotherapy of pain. Opioid analgesics such as morphine, oxycodone and fentanyl are agonists at the MOR and are the mainstay for the treatment of moderate-to-severe pain. However, adverse effects related to opioid use are severe and often lead to early discontinuation and inadequate analgesia. The development of more effective and safer medications for the management of pain still remains a major direction in pharmaceutical research. Chemical approaches towards the identification of novel MOR analgesics with reduced side effects include structural modifications of 14-alkoxy-N-methylmorphinan-6-ones in key positions that are important for binding, selectivity, potency, and efficacy at opioid receptors. This paper describes a representative strategy to improve the therapeutic usefulness of opioid analgesics from the morphinan class of drugs by targeting position 5. The focus is on chemical and biological studies and structure-activity relationships of this series of ligands. We report on 14-alkoxymorphinan-6-ones having a methyl and benzyl group at position 5 as strong opioid antinociceptive agents with reduced propensity to cause undesired effects compared to morphine although interacting selectively with MORs.

The synthesis and the effect of a combination of 6-glycine and 14-phenylpropoxy substitutions in N-methyl- and N-cycloproplymethylmorphinans on biological activities are described. Binding studies revealed that all new 14-phenylpropoxymorphinans (11-18) displayed high affinity to opioid receptors. Replacement of the 14-methoxy group with a phenylpropoxy group led to an enhancement in affinity to all three opioid receptor types, with most pronounced increases in δ and κ activities, hence resulting in a loss of μ receptor selectivity. All compounds (11-18) showed potent and long-lasting antinociceptive effects in the tail-flick test in rats after subcutaneous administration. For the N-methyl derivatives 13 and 14, analgesic potencies were in the range of their 14-methoxy analogues 9 and 10, respectively. Even derivatives 15-18 with an N-cyclopropylmethyl substituent acted as potent antinociceptive agents, being several fold more potent than morphine. Subcutaneous administration of compounds 13 and 14 produced significant and prolonged antinociceptive effects mediated through peripheral opioid mechanisms in carrageenan-induced inflammatory hyperalgesia in rats.

Among opioids, morphinans play an important role as therapeutically valuable drugs. They include pain relieving agents such as naturally occurring alkaloids (e.g. morphine, codeine), semisynthetic derivatives (e.g. oxycodone, oxymorphone, buprenorphine), and synthetic analogs (e.g. levorphanol). Currently used opioid analgesics also share a number of severe side effects, limiting their clinical usefulness. The antagonist morphinans, naloxone and naltrexone are used to treat opioid overdose, opioid dependence, and alcoholism. All these opioid drugs produce their biological actions through three receptor types, mu, delta, and kappa, belonging to the G-protein-coupled receptor family. Considerable effort has been put forward to understand the appropriate use of opioid analgesics, while medicinal chemistry and opioid pharmacology have been continuously engaged in the search for safer, more efficacious and nonaddicting opioid compounds, with the final goal to reduce complications and to improve patient compliance. Toward this goal, recent advances in chemistry, ligand-based structure activity relationships and pharmacology of 14-alkoxymorphinans are reviewed in this chapter. Current developments of different structural patterns of 14-alkoxymorphinans as research tools and their potential therapeutic opportunities are also summarized.

Opioids are the most effective analgesics for pain management, and efficient pain control is a therapeutic priority. Herein, we describe the synthesis and pharmacological activities of the 5-benzyl analogue of the mu opioid analgesic 14-methoxymetopon (14-MM). The result of the replacement of the 5-methyl in 14-MM with a benzyl group on in vitro opioid receptor binding and functional profiles, and in vivo behavioural properties, i.e. nociception and motor activity, was investigated. In rodent brain membranes, the 5-benzyl derivative showed high affinity at the micro opioid receptor and decreased interaction with delta and kappa receptors, hence displaying a similar binding profile as 14-MM. It displayed potent agonist activity in vitro and in vivo. In in vitro guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding assay, it activated G-proteins in rat brain membranes through a micro opioid receptor-mediated mechanism having significantly enhanced potency compared to DAMGO (D-Ala(2),Me-Phe(4),Gly-ol(5)]enkephalin), and to the micro opioid agonist morphinans 14-MM, 14-O-methyloxymorphone (14-OMO) and morphine. In vivo, the 5-benzyl analogue of 14-MM elicited dose-dependent and naloxone-sensitive antinociceptive effects in hot-plate and tail-flick tests in mice after subcutaneous (s.c.) administration. Its analgesic potency was comparable to 14-MM, and was 50-fold higher than that of morphine. Contrary to morphine, 14-MM and 14-OMO, no motor dysfunction was produced by the new opioid in the mouse rotarod test at any of the tested doses. In summary, the 5-benzyl analogue of 14-MM emerged as a novel potent mu opioid antinociceptive agent with reduced propensity to cause unwanted motor impairment., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

14-O-Methyloxymorphone and 14-methoxymetopon were reported as highly selective and potent micro opioid receptor agonists. The aim of this study was to demonstrate the opioid activity of these compounds in vitro and in vivo in comparison to oxymorphone, morphine and DAMGO. The micro opioid receptor efficacy, full or partial agonist nature of opioids was analyzed in the rat vas deferens (RVD) bioassay. Compared to oxymorphone, 14-O-methyloxymorphone and 14-methoxymetopon showed greater affinities to the rodent brain micro opioid receptors in receptor binding assays. In isolated organs 14-O-methyloxymorphone and 14-methoxymetopon were 3-10-fold more potent than the micro agonist opioid peptide, DAMGO. All tested compounds reached at least 70% maximum inhibition in mouse vas deferens (MVD) except morphine and oxymorphone. In the RVD, morphine could not exceed 50% inhibition of the twitches while 14-O-methyloxymorphone and 14-methoxymetopon showed inhibitory effects more than 70%. Oxymorphone reached only 4% maximal agonist effect and antagonized the inhibitory effect of DAMGO. The investigated morphinans produced dose-dependent antinociceptive activities in mice and rats. Both, 14-O-methyloxymorphone and 14-methoxymetopon are highly efficacious micro opioid receptor agonists in the RVD exhibiting full micro agonist properties. The RVD tissue contains mu receptors indicated by the comparable K(e) values of the micro antagonist naltrexone against DAMGO in the MVD. RVD may be a good alternative to assess the mu receptor efficacy of opioid agonists providing a more physiological environment for the ligand-receptor interaction than other efficacy measuring methods such as the [(35)S]GTPgammaS binding assay.

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Opioid receptors are expressed not only on neuroendocrine cells but also on immunocompetent cells such as lymphocytes, monocytes and macrophages. micro-Opioid receptor agonists were found to exert immunosuppressive effects, whereas delta-opioid receptor agonists have been shown to act as immunostimulants. delta-Opioid receptor agonists stimulate T and B cells and activate granulocytes and monocytes, conversely, immunostimulation can be blocked by the non-peptidic delta-opioid receptor antagonist (NTI). We investigated the impact of NTI and of the two structurally related compounds HS-378 and HS-459 on degradation of tryptophan and formation of neopterin in mitogen-stimulated human peripheral blood mononuclear cells (PBMC). Both these biochemical pathways were found to be suppressed by all three opioid receptor antagonists, HS-378 and HS-459 exhibiting slightly greater potency than NTI. The suppression of tryptophan degradation suggests that the tested delta-opioid antagonists are able to influence the serotonergic system via a non-opioid action.

Peripheral micro-opioid receptors (MOR) have emerged as important components of inhibitory nociceptive pathways. Here, the antinociceptive effects of MOR agonists, the 6beta-glycine derivative of 14-O-methyloxymorphone (HS-731), DAMGO and morphine were evaluated in a mouse model of visceral pain. The abdominal acetic acid-induced writhing test was used to examine the peripheral, preemptive antinociceptive opioid action on visceral nociception. HS-731 administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) dose-dependently and completely inhibited writhing, being 24-598-fold more potent, depending on the administration route, than two selective MOR agonists, the enkephalin analogue [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO) and morphine. A longer duration of action (2-3 h) was induced by HS-731 given before acetic acid, while shorter effect was produced by morphine (30-60 min) and DAMGO (30-45 min). The antinociceptive effects of systemic opioids were reversed by the s.c. opioid antagonist, naloxone. Blocking of central MOR by the selective MOR antagonist D-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, i.c.v.) resulted in a significant reduction of antinociception of s.c. morphine, whereas it completely failed to antagonize the effects of systemic HS-731 or DAMGO. In in vitro studies, HS-731 and DAMGO, but not morphine showed high intrinsic efficacy, naltrexone-sensitive agonist effect at MOR of the rat vas deferens. These data demonstrate that selective activation of peripheral MOR by systemic s.c. HS-731 or DAMGO produces potent peripheral, preemptive visceral antinociception, while morphine's effects are mediated primarily through central mechanisms. Our findings support the role of peripheral MOR in the pathology of pain states involving sensitization of peripheral nociceptors.

Rationale: Increased opioidergic activity is thought to increase the propensity to consume ethanol. However, the dose monotonicity and receptor subtype for this effect remain uncertain. 14-methoxymetopon is a centrally acting, selective micro opioid receptor agonist with greater systemic antinociceptive potency than morphine and a putatively improved therapeutic index., Objective: To determine whether 14-methoxymetopon influenced voluntary ethanol intake in Sardinian alcohol-preferring (sP) rats., Methods: Male sP rats with continuous 2-bottle choice access to ethanol (10% v/v) or water were subjects. The effects of systemic 14-methoxymetopon administration (2, 5, 12.25, 30 micro/kg, s.c.) on 4-h ethanol intake were determined. The ability of naltrexone (50 micro/kg, s.c.), an opioid antagonist, to block actions of 14-methoxymetopon (12.25, 30 micro/kg, s.c.) was examined as were the effects of 14-methoxymetopon (12.25 micro/kg, s.c.) on self-administered blood alcohol levels (BALs) and clearance of a passive ethanol bolus (1 g/kg). Finally, the effects of central 14-methoxymetopon administration (0.0003-100 ng, i.c.v.) on 4-h ethanol intake were evaluated., Results: Systemic 14-methoxymetopon very potently and dose-dependently suppressed ethanol and food intake for 30 min, followed by a greater, longer-lasting, and behaviorally specific increase in ethanol intake. The increased ethanol intake led to threefold higher BALs, was naltrexone-reversible, and not due to altered ethanol clearance. Intracerebroventricular 14-methoxymetopon administration rapidly altered ethanol intake per an inverted U-shaped dose-response function, increasing it at a 10 pg dose, while suppressing it at a 10,000-fold higher dose., Conclusions: The novel mu analgesic increases ethanol intake, a potential therapeutic liability, and results suggest a non-monotonic influence of brain mu opioid receptor stimulation on ethanol intake.

Antinociception achieved after peripheral administration of opioids has opened a new approach to the treatment of severe and chronic pain. Additionally, opioid analgesics with restricted access to the central nervous system could improve safety of opioid drugs used in clinical practice. In the present study, peripheral components of antinociceptive actions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone were investigated after local intraplantar (i.pl.) administration in rat models of inflammatory and neuropathic pain. Their antinociceptive activities were compared with those of morphine, the classical mu-opioid receptor agonist. Intraplantar administration of morphine and the 6-amino acid derivatives produced dose-dependent reduction of formalin-induced flinching of the inflamed paw, without significant effect on the paw edema. Local i.pl. administration of the new derivatives in rats with neuropathic pain induced by sciatic nerve ligation produced antiallodynic and antihyperalgesic effects; however, the antinociceptive activity was lower than that observed in inflammatory pain. In both models, the 6-amino acid derivatives and morphine at doses that produced analgesia after i.pl. administration were systemically (s.c.) much less active indicating that the antinociceptive action is due to a local effect. Moreover, the local opioid antinociceptive effects were significantly attenuated by naloxone methiodide, a peripherally acting opioid receptor antagonist, demonstrating that the effect was mediated by peripheral opioid receptors. The present data indicate that the peripherally restricted 6-amino acid conjugates of 14-O-methyloxymorphone elicit antinociception after local administration, being more potent in inflammatory than in neuropathic pain. Opioid drugs with peripheral site of action can be an important target for the treatment of long lasting pain.

Receptor binding studies of 5,14-O-dimethyloxymorphone (14-methoxymetopon) in brain membranes have established its high affinity for mu-binding sites, but its analgesic potency far exceeds the modest increase in binding affinity relative to other opioids. The current study has established the selectivity of [(3)H]14-methoxymetopon for mu sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine mu-opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [(3)H]14-methoxymetopon for the variants expressed in Chinese hamster ovary cells was quite high, with K(D) values around 0.2 nM for all of the variants with the exception of mMOR-1F (K(D) of 1.2 nM). The affinity for most of the expressed variants was greater than that seen in the brain membranes (K(D) of 0.99 nM). Functionally, in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays with the MOR-1 variants, 14-methoxymetopon and the mu-opioid peptide [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) showed similar efficacies, as determined by maximal stimulation, but 14-methoxymetopon was up to 65-fold more potent than DAMGO. The greatest difference was seen with mMOR-1E and the least with mMOR-1C, which displayed only a 10-fold difference. These potency differences in the stimulation of [(35)S]GTPgammaS binding far exceeded the differences in binding affinity. The differences between 14-methoxymetopon and DAMGO remained after normalizing the potency shifts based upon receptor binding affinities and varied from 1.2-fold with mMOR-1C to 21-fold for mMOR-1 and 42-fold with mMOR-1F. Thus, 14-methoxymetopon is a potent agonist against all of the mMOR-1 splice variants, but its potency ranged widely despite similar binding affinities for most of the variants and may give insight into its unusual pharmacological profile.

Opioids induce analgesia by activating opioid receptors not only within the central nervous system but also on peripheral sensory neurons. This study investigated peripherally mediated antinociception produced by the mu-opioid receptor agonist 2-[(4,5alpha-epoxy-3-hydroxy-14beta-methoxy-17-methylmorphinan-6beta-yl)amino]acetic acid (HS-731) after s.c. and oral administration in rats with carrageenan-induced hindpaw inflammation. Antinociceptive effects after s.c. administration were assessed 3 h after intraplantar carrageenan injection and compared with those of centrally acting mu-opioid agonists 14-methoxymetopon and morphine. Opioid agonists caused dose-dependent increases in inflamed paw withdrawal latencies to mechanical and thermal stimulation. The time course of action was different, in that HS-731 (20 microg/kg s.c.) produced significant long-lasting effects up to 4 h after administration, whereas 14-methoxymetopon (20 microg/kg) and morphine (2 mg/kg) reached their peak of action at 10 to 30 min, and their effect declined rapidly thereafter. Subcutaneous administration of the peripherally selective opioid antagonist naloxone methiodide inhibited antinociception elicited by HS-731 (20 microg/kg s.c.), whereas it was ineffective against 14-methoxymetopon (20 microg/kg s.c.). Moreover, the antinociception produced by 100 microg/kg s.c. HS-731 was dose-dependently reversed by s.c. naloxone methiodide. This indicates that HS-731 preferentially activates peripheral opioid receptors, whereas 14-methoxymetopon mediates analgesia via central mechanisms. Orally administered HS-731 significantly reduced hyperalgesia in the inflamed paw induced by carrageenan, which was reversible by s.c. administered naloxone methiodide. These results show that systemic (s.c. and oral) treatment with the mu-opioid agonist HS-731 produces potent and long-lasting antinociception through peripheral mechanisms in rats with carrageenan-induced hindpaw inflammation.

Tosylmethyl isocyanide was used to convert 7,8-didehydro-6-ketomorphinans to 6,7-didehydromorphinan-6-carbonitriles with retainment of the 4,5-epoxy ring. However, ring opening occurred in the presence of NaH giving 5,6,7,8-tetradehydromorphinan-6-carbonitriles. Addition of nucleophiles such as Li diisopropylamide or Grignard reagents to the acrylonitrile substructure yielded ring-opened 5,6-didehydro products. Seven products were characterized by X-ray crystal structure analysis and revealed insight into the mechanistic diversity of the van Leusen reaction.

Opioid analgesics with restricted access to the central nervous system represent a new approach to the treatment of severe pain with an improved safety profile. The objective of this study was to investigate the peripheral and central components of the antinociceptive actions of the 6-amino acid conjugates (glycine, alanine, and phenylalanine) of 14-O-methyloxymorphone. Their antinociceptive activities were compared with those of the centrally penetrating mu-opioid agonists morphine, fentanyl, and 14-O-methyloxymorphone. In the tail-flick test in rats, the 6-amino acid conjugates were 45- to 1170-fold more potent than morphine after i.c.v. administration and 19- to 209-fold after s.c. administration. They showed potencies similar to fentanyl after s.c. administration and were more potent after i.c.v. application. The time course of action was different between s.c. and i.c.v. administration, with significant long-lasting effects after i.c.v. administration. Systemic administration of the peripherally selective opioid antagonist naloxone methiodide antagonized the effects after s.c. but not after i.c.v. administration in the tail-flick test. Subcutaneous 6-amino acid derivatives also elicited antihyper-analgesic effects in the formalin test in rats, which were reversed by systemically administered naloxone methiodide. Although morphine exerts its analgesic effects by central and peripheral mechanisms, the investigated new opioids interact primarily with peripheral opioid receptors after s.c. administration. The present data indicate that the 6-amino acid conjugates of 14-O-methyloxymorphone have limited access to the central nervous system and can mediate antinociception at peripheral sites. Also, they might find clinical application when the central actions of opioids are unwanted.

The synthesis, biological, and pharmacological evaluation of novel derivatives of cyprodime are described. Their binding affinities at mu, delta, and kappa opioid receptors were evaluated using receptor binding assay. It was observed that the affinity of these compounds was sensitive to the character and length of the substituent in position 4. Further prolongation of the 4-alkoxy group of cyprodime (1) and its 4-butoxy analogue 2 is detrimental for the mu opioid receptor affinity. Introduction of an arylalkoxy group at C-4 does not increase mu affinity in the case of benzyloxy, while a phenylpropoxy group reduces mu affinity. The delta and kappa affinities were also reduced compared to the reference compounds. A significant increase in the affinity at the mu opioid receptors was achieved by introducing a 14-phenylpropoxy group. Increases in the affinity at delta and kappa receptors were also observed. These findings provide further evidence that the nature of the substituent at position 14 has a major impact on the abilities of morphinans to interact with opioid receptors. In the [(35)S]GTPgammaS binding assay, all tested compounds were partial agonists at mu and delta receptors. Compounds 8 and 17 showed antagonism at kappa receptors, while compound 7 exhibited some partial agonist activity at this receptor. The novel derivatives of cyprodime containing a 14-phenylpropoxy group acted as potent antinociceptives. When tested in vivo, compounds 7, 8, and 17 were considerably more potent than morphine, with phenol 7 showing the highest antinociceptive potency (21-fold in the hot plate test, 38-fold in the tail flick test, and 300-fold in the paraphenylquinone writhing test) in mice. Introduction of a 14-phenylpropoxy substituent leads to a profound alteration in the pharmacological profile of this class of compounds.

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.

A series of 6-amino acid conjugates (glycine, alanine and phenylalanine) of the highly potent opioid analgesic 14-O-methyloxymorphone was developed in an effort to obtain agonists that would have potentially limited ability to cross the blood-brain barrier. Binding studies revealed that all derivatives displayed high affinities (0.77-2.58 nM) at the mu-opioid receptor in rat brain membranes. They were potent agonists in mouse vas deferens preparation (IC(50)=5.52-26.8 nM). While the alpha-amino acid epimers are favoured by mu-opioid receptors, the beta-epimers proved to have increased interaction with delta-sites. Only the beta-phenylalanine conjugate showed some preference for delta- over mu-opioid receptors and delta-opioid receptor agonist activity. The relatively high delta-opioid receptor affinity of this analogue was also predicted by molecular modelling studies. The newly developed ionizable derivatives could find clinical applications as potent analgesics without the adverse actions of centrally acting opioids.

The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level.

The synthesis, biological, and pharmacological evaluations of 14beta-O-phenylpropyl-substituted morphinan-6-ones are described. The most striking finding of this study was that all of the compounds from the novel series of differently N-substituted 14beta-O-phenylpropylmorphinans acted as powerful opioid agonists. Even with N-substituents such as cyclopropylmethyl and allyl, which are usually associated with distinct antagonist properties, only agonists were obtained. Compared to morphine, the N-cyclopropylmethyl derivative 15 showed considerably increased potency in the in vivo assays in mice (600-fold in the tail-flick assay, 60-fold in the paraphenylquinone writhing test, and 400-fold in the hot-plate assay). Remarkably, most of the new ligands were nonselective and exhibited binding affinities in the subnanomolar range at opioid receptors (mu, kappa, delta), with the N-propyl derivative 19 displaying the highest affinity for the mu-receptor (K(i) = 0.09 nM).

We have studied the possible interaction between three selective opioid-receptor antagonists, nor-binaltorphimine (NB: kappa) (5 mg/kg), cyprodime (CY: mu) (10 mg/kg) and naltrindole (NTI: delta) (1 mg/kg), and the cannabinoid receptor agonist CP 55,940, in the modulation of anxiety (plus-maze) and adrenocortical activity (serum corticosterone levels by radioimmunoassay) in male rats. The holeboard was used to evaluate motor activity and directed exploration. CP 55,940 (75 microg/kg, but not 10 microg/kg) induced an anxiogenic-like effect, which was antagonised by NB. The other effects of CP 55,940 (75 microg/kg), a decreased holeboard activity and stimulation of adrenocortical activity, were not antagonised by any of the three opioid receptor antagonists. CY and NTI, when administered alone, induced marked reductions in motor activity, anxiogenic-like effects and stimulation of adrenocortical activity. The selective kappa-opioid receptor antagonist NB, on its own, did not modify the level of anxiety but stimulated adrenocortical activity. We provide the first pharmacological evidence about the involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940.

14-Methoxymetopon is a potent opioid analgesic. When given systemically, it is approximately 500-fold more active than morphine. However, this enhanced potency is markedly increased with either spinal or supraspinal administration, where its analgesic activity is more than a million-fold greater than morphine. It was mu-opioid receptor selective in binding assays and its analgesia was blocked only by mu-opioid receptor-selective antagonists. Yet, it had a different selectivity profile than either morphine or morphine-6beta-glucuronide. Unlike morphine, 14-methoxymetopon was antagonized by 3-O-methylnaltrexone, it was sensitive to antisense probes targeting exons 1, 2 and 8 of the opioid receptor gene and was inactive both spinally and supraspinally in CXBK mice. Although it retarded gastrointestinal transit, it displayed a ceiling effect with no dose lowering transit by more than 65%, in contrast to the complete inhibition of transit by morphine. These finding demonstrate that 14-methoxymetopon is a highly potent mu-opioid with a pharmacological profile distinct from that of the traditional mu-opioid morphine.

14-Alkoxy analogues of naltrindole and naltriben differently substituted in positions 5 and 17 and at the indole nitrogen (compounds 28-44) have been synthesized in an effort to enhance the delta potency and/or delta selectivity and in order to further elaborate on structure-activity relationships of this class of compounds. Introduction of a 14-alkoxy instead of the 14-hydroxy group present in naltrindole resulted in somewhat lower delta binding affinity, while in many cases (compounds 31, 34, 37, 40, 41, 44, HS 378) the delta receptor selectivity was considerably increased. An ethoxy group in position 14 is superior to other alkoxy groups concerning delta affinity and selectivity (34, 41, 42, 44, HS 378). In [35S]GTP gamma S binding, compounds 34, 41, and HS 378 exhibited about one-tenth the antagonist potency of naltrindole at delta opioid receptors while their delta antagonist selectivity was considerably higher. 17-Methyl-substituted compounds 35 and 44 were found to be pure delta receptor agonists in this test.

We have studied the effects of 14-methoxymetopon (HS 198), a potent opioid agonist, on the responses to the tail electric stimulation test and plus-maze activity of adult male rats. The prototype mu agonist morphine was used as the drug of reference. Besides we addressed the effects of HS 198 on the serum corticosterone levels and on serotonergic systems of discrete brain regions. Both drugs were administered subcutaneously. Morphine (5 mg/kg) and HS 198 (30 microg/kg) induced a similar effect on the nociceptive test, with both drugs significantly increasing the threshold for the vocalization afterdischarge, which is related to the emotional component of pain. In the plus-maze, morphine (5 mg/kg) and HS 198 (20 and 30 microg/kg) induced similar increases in the percentages of entries and time in the open arms, two parameters related to the anxiety state of the animals. The results indicate that HS 198 is far more potent than morphine in reducing the emotive/affective component of pain and in inducing an anxiolytic effect. HS 198 (30 microg/kg) also induced parallel increases in the serum corticosterone levels and the hypothalamic serotonin content. A possible correlation between the anxiolytic action of the drug and its effect on the hypothalamic serotonergic system is suggested.