Benzomorphan and non-benzomorphan agonists differentially alter sigma-1 receptor quaternary structure, as does types of cellular stress.

Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.

Structure-Affinity relationships of novel σ2R/TMEM97 ligands.

The sigma 2 receptor (σ2R), which was recently identified as the transmembrane protein 97 (TMEM97), is increasingly attracting interest as a possible therapeutic target for indications in neuroscience. Toward identifying novel modulators of σ2R/TMEM97, we prepared a collection of benzoxazocine, benzomorphan, and methanobenzazepine ligands related to the known bioactive norbenzomorphans DKR-1677, FEM-1689, and EES-1686 and determined their Ki values for σ2R/TMEM97 and the sigma 1 receptor (σ1R). The σ2R/TMEM97 binding affinities and selectivities relative to σ1R of these new benzoxazocine, benzomorphan, and methanobenzazepine analogs are lower, often significantly lower, than their respective norbenzomorphan counterparts, suggesting the spatial orientation of pharmacophoric substituents is critical for binding to the two proteins. The benzoxazocine, benzomorphan, and methanobenzazepine congeners of DKR-1677 and FEM-1689 tend to be weakly selective for σ2R/TMEM97 versus σ1R, whereas EES-1686 derivatives exhibit the greatest selectivity, suggesting the size and/or nature of the substituent on the nitrogen atom of the scaffold may be important for selectivity. Computational docking studies were performed for the 1S,5R-and 1R,5S-enantiomers of DKR-1677, FEM-1689, and EES-1686 and their benzoxazocine, benzomorphan, and methanobenzazepine counterparts. These computations predict that the protonated amino group of each ligand forms a highly conserved salt bridge and a H-bonding interaction with Asp29 as well as a cation-π interaction with Tyr150 of σ2R/TMEM97. These electrostatic interactions are major driving forces for binding to σ2R/TMEM97 and are similar, though not identical, for each ligand. Other interactions within the well-defined binding pocket also tend to be comparable, but there are some major differences in how the hydrophobic aryl groups of various ligands interact with the protein surface external to the binding pocket. Overall, these studies show that the orientations of aryl and N-substituents on the norbenzomorphan and related scaffolds are important determinants of binding affinity of σ2R/TMEM97 ligands, and small changes can have significant effects upon binding profiles.

Potent MOR Agonists from 2'-Hydroxy-5,9-dimethyl-N-phenethyl Substituted-6,7-benzomorphans and from C8-Hydroxy, Methylene and Methyl Derivatives of N-Phenethylnormetazocine.

(-)-5,9-Dimethyl-6,7-benzomorphan (normetazocine) derivatives with a para-OH or ortho-F substituent in the aromatic ring of the N-phenethyl moiety were synthesized and found to have subnanomolar potency at MOR, and both were fully efficacious in vitro. These new compounds, (1R,5R,9R)-6,11-dimethyl-3-(2-fluorophenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol and (1R,5R,9R)-6,11-dimethyl-3-(4-hydroxyphenethyl)-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol, were more potent than the unsubstituted compound N-phenethylnormetazocine and about 30 or 40 times more potent than morphine, respectively. A variety of substituents in the ortho, meta, or para position in the aromatic ring of the N-phenethyl moiety were synthesized, 25 of these compounds, and found to have varying effects on potency and efficacy as determined by the forskolin-induced cAMP accumulation assay. The N-phenethyl moiety was also modified by increasing chain length to form a N-phenylpropyl side chain with and without a para-nitro moiety, and by an N-cinnamyl side chain. Also, an indole ethylamine normetazocine was synthesized to replace the N-phenethylamine side chain in normetazocine. The phenylpropylamine, propenylamine (cinnamyl) and the para-nitropropylamine had little or no MOR potency. The indole-ethylamine on the normetazocine nucleus, however, had moderate potency (MOR EC50 = 12 nM), and was fully efficacious (%Emax = 102%) in the cAMP assay. Retention of the N-phenethyl moiety and the addition of alkyl and alkenyl moieties on C8 in (-)-N-phenethylnormetazocine gave a C8-methylene derivative that had subnanomolar potency at MOR and a C8-methyl analog that had nanomolar potency. Five C8-substituted compounds were synthesized.

LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief.

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the "one-molecule-multiple targets" strategy nowadays is highly recognized. Indeed, multitarget ligands displaying a better antinociceptive activity with fewer side effects, combined with favorable pharmacokinetic and pharmacodynamic characteristics, have already been shown. Multitarget ligands possessing non-opioid/opioid and opioid/opioid mechanisms of action are considered as potential drug candidates for the management of various pain conditions. In particular, dual-target MOPr (mu opioid peptide receptor)/DOPr (delta opioid peptide receptor) ligands exhibit an improved antinociceptive profile associated with a reduced tolerance-inducing capability. The benzomorphan-based compounds LP1 and LP2 belong to this class of dual-target MOPr/DOPr ligands. In the present manuscript, the structure-activity relationships and the pharmacological fingerprint of LP1 and LP2 compounds as suitable drug candidates for persistent pain relief is described.

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists.

The opioid pharmacological profile of cis-(-)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a-d and 6a-d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(-)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50 of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(-)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment.

Development of novel LP1-based analogues with enhanced delta opioid receptor profile.

Pain relief achieved by co-administration of drugs acting at different targets is more effective than that obtained with conventional MOR selective agonists usually associated to relevant side effects. It has been demonstrated that simultaneously targeting different opioid receptors is a more effective therapeutic strategy. Giving the promising role for DOR in pain management, novel LP1-based analogues with different N-substituents were designed and synthesized with the aim to improve DOR profile. For this purpose, we maintained the phenyl ring in the N-substituent of 6,7-benzomorphan scaffold linked to an ethyl spacer bearing a hydroxyl/methyl or methoxyl group at carbon 2 or including it in a 1,4-benzodioxane ring. LP1 analogues were tested by competition binding assays. Compounds 6 (KiMOR=2.47nM, KiDOR=9.6nM), 7 (KiMOR=0.5nM and KiDOR=0.8nM) and 9 (KiMOR=1.08nM, KiDOR=6.6nM) retained MOR affinity but displayed an improved DOR binding capacity as compared to LP1 (KiMOR=0.83nM, KiDOR=29.1nM). Moreover, GPI and MVD functional assays indicated that compounds 6 (IC50=49.2 and IC50=10.8nM), 7 (IC50=9.9 and IC50=11.8nM) and 9 (IC50=21.5 and IC50=4.4nM) showed a MOR/DOR agonist profile, unlike LP1 that was a MOR agonist/DOR antagonist (IC50=1.9 and IC50=1240nM). Measurements of their antinociceptive effect was evaluated by mice radiant tail flick test displaying for compounds 6, 7 and 9 ED50 values of 1.3, 1.0 and 0.9mg/kg, i.p., respectively. Moreover, the antinociceptive effect of compound 9 was longer lasting with respect to LP1. In conclusion the N-substituent nature of compounds 6, 7 and 9 shifts the DOR profile of LP1 from antagonism to agonism.

An LP1 analogue, selective MOR agonist with a peculiar pharmacological profile, used to scrutiny the ligand binding domain.

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an 'allosteric' site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5, bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (Kiµ=0.5±0.2nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with 'orthosteric' and 'allosteric' binding sites.

Evaluation of N-substituent structural variations in opioid receptor profile of LP1.

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9-15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (Ki(MOR)=38±4nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA2) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD50=2.0mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function.

Predicted structures for kappa opioid G-protein coupled receptor bound to selective agonists.

Human kappa opioid receptor (κ-OR), a G protein-coupled receptor (GPCR), has been identified as a drug target for treatment of such human disorders as pain perception, neuroendocrine physiology, affective behavior, and cognition. In order to find more selective and active agonists, one would like to do structure based drug design. Indeed, there is an X-ray structure for an antagonist bound to κ-OR, but structures for activated GPCRs are quite different from those for the inactive GPCRs. Here we predict the ensemble of 24 low-energy structures of human kappa opioid receptor (κ-OR), obtained by application of the GEnSeMBLE (GPCR Ensemble of Structures in Membrane Bilayer Environment) complete sampling method, which evaluates 13 trillion combinations of tilt and rotation angles for κ-OR to select the best 24. To validate these structures, we used the DarwinDock complete sampling method to predict the binding sites for five known agonists (ethylketocyclazocine, bremazocine, pentazocine, nalorphine, and morphine) bound to all 24 κ-OR conformations. We find that some agonists bind selectively to receptor conformations that lack the salt bridge between transmembrane domains 3 and 6 as expected for active conformations. These 3D structures for κ-OR provide a structural basis for understanding ligand binding and activation of κ-OR, which should be useful for guiding subtype specific drug design.

Design, synthesis, in vitro evaluation, and molecular modeling studies of N-substituted benzomorphans, analogs of LP2, as novel MOR ligands.

6,7-Benzomorphans have been investigated in medicinal chemistry for developing new drugs. This nucleus could be considered a versatile scaffold. The physicochemical properties of benzomorphan N-substituent are crucial in achieving a definite pharmacological profile at opioid receptors. Thus, the dual-target MOR/DOR ligands LP1 and LP2 were obtained through N-substituent modifications. Specifically, LP2, bearing as N-substituent the (2R/S)-2-methoxy-2- phenylethyl group, is a dual-target MOR/DOR agonist and is successful in animal models of inflammatory and neuropathic pain. To obtain new opioid ligands, we focused on the design and synthesis of LP2 analogs. First, the 2-methoxyl group of LP2 was replaced by an ester or acid functional group. Then, spacers of different lengths were introduced at N-substituent. In-vitro, their affinity profile versus opioid receptors has been performed through competition binding assays. Molecular modeling studies were conducted to deeply analyze the binding mode and the interactions between the new ligands and all opioid receptors.

Determination of separate inhibitor and substrate binding sites in the dehaloperoxidase-hemoglobin from Amphitrite ornata.

Dehaloperoxidase-hemoglobin (DHP A) is a dual function protein found in the terrebellid polychaete Amphitrite ornata. A. ornata is an annelid, which inhabits estuary mudflats with other polychaetes that secrete a range of toxic brominated phenols. DHP A is capable of binding and oxidatively dehalogenating some of these compounds. DHP A possesses the ability to bind halophenols in a distinct, internal distal binding pocket. Since its discovery, the distal binding pocket has been reported as the sole binding location for halophenols; however, data herein suggest a distinction between inhibitor (monohalogenated phenol) and substrate (trihalogenated phenol) binding locations. Backbone (13)Calpha, (13)Cbeta, carbonyl (13)C, amide (1)H, and amide (15)N resonance assignments have been made, and various halophenols were titrated into the protein. (1)H-(15)N HSQC experiments were collected at stoichiometric intervals during each titration, and binding locations specific for mono- and trihalogenated phenols have been identified. Titration of monohalogenated phenol induced primary changes around the distal binding pocket, while introduction of trihalogenated phenols created alterations of the distal histidine and the local area surrounding W120, a structural region that corresponds to a possible dimer interface region recently observed in X-ray crystal structures of DHP A.

Evaluation of N-substitution in 6,7-benzomorphan compounds.

6,7-benzomorphan derivatives, exhibiting different mu, delta, and kappa receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2'-hydroxy-6,7-benzomorphan derivatives (12-22). Data obtained by competition binding assays showed that the mu opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide. Highly stringent features are required for delta receptor interaction, while an N-acetamide spacer and/or bulkier amide could preferentially lead to kappa receptor selectivity. In the propanamide series, compound 12 (named LP1) displayed high mu affinity (Ki=0.83 nM), good delta affinity (Ki=29 nM) and low affinity for the kappa receptor (Ki=110 nM), with a selectivity ratio delta/mu and kappa/mu of 35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a mu/delta agonist profile, with IC50 values of 4.8 and 12 nM at the mu and delta receptors, respectively. The antinociceptive potency of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine (ED50=2.03 and 2.7 mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a mu/delta agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent in vivo analgesic activity and a reduced tendency to induce side effects.

Exploiting the Power of Stereochemistry in Drug Action: 3-[(2S,6S,11S)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide as Potent Sigma-1 Receptor Antagonist.

(+)-(2S,6S,11S)- and (-)-(2R,6R,11R)-Benzomorphan derivatives have a different binding affinity for sigma-1 (σ1R) and opioid receptors, respectively. In this study, we describe the synthesis of the (+)-enantiomer [(+)-LP1] of the benzomorphan MOR agonist/DOR antagonist LP1 [(-)-LP1]. The binding affinity of both (+)-LP1 and (-)-LP1 for σ1R and sigma-2 receptor (σ2R) was tested. Moreover, (+)-LP1 opioid receptor binding affinity was also investigated. Finally, (+)-LP1 was tested in a mouse model of inflammatory pain. Our results showed a nanomolar σ1R and binding affinity for (+)-LP1. Both (+)-LP1 and (-)-LP1 elicited a significant analgesic effect in a formalin test. Differently from (-)-LP1, the analgesic effect of (+)-LP1 was not reversed by naloxone, suggesting a σ1R antagonist profile. Furthermore, σ1R agonist PRE-084 was able to unmask the σ1R antagonistic component of the benzomorphan compound. (+)-LP1 could constitute an useful lead compound to develop new analgesics based on mechanisms of action alternative to opioid receptor activation.

Effects of atypical kappa-opioid receptor agonists on intrathecal morphine-induced itch and analgesia in primates.

Itch/pruritus is the most common side effect associated with spinal administration of morphine given to humans for analgesia. The aim of this study was to investigate the effectiveness of kappa-opioid receptor (KOR) agonists with diverse chemical structures as antipruritics and to elucidate the receptor mechanism underlying the antipruritic effect in monkeys. In particular, previously proposed non-KOR-1 agonists, including nalfurafine [TRK-820, 17-cyclopropylmethyl-3,14 beta-dihydroxy-4,5 alpha-epoxy-6 beta-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan], bremazocine [(+/-)-6-ethyl-1,2,3,4,5,6-hexahydro-3-[(1-hydroxycyclopropy)-methyl]-11,11-dimethyl-2,6-methano-3-benzazocin-8-ol], and GR 89696 [4-[(3,4-dichlorophenyl)acetyl]-3-(1-pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl ester] were studied in various behavioral assays for measuring itch/scratching, analgesia, and respiratory depression. Systemic administration of nalfurafine (0.1-1 microg/kg), bremazocine (0.1-1 microg/kg), or GR 89696 (0.01-0.1 microg/kg) dose-dependently attenuated intrathecal morphine (0.03 mg)-induced scratching responses without affecting morphine antinociception. The combination of intrathecal morphine with these KOR agonists did not cause sedation. In addition, pretreatment with effective antiscratching doses of nalfurafine, bremazocine, or GR 89696 did not antagonize systemic morphine-induced antinociception and respiratory depression. The dose-addition analysis revealed that there is no subadditivity for nalfurafine in combination with morphine in the antinociceptive effect. Furthermore, the KOR antagonist study revealed that antiscratching effects of both nalfurafine and a prototypical KOR-1 agonist, U-50488H [trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide], could be blocked completely by a selective KOR antagonist, nor-binaltorphimine (3 mg/kg). These findings suggest that the agonist action on KOR mainly contributes to the effectiveness of these atypical KOR agonists as antipruritics, and there is no evidence for KOR subtypes or mu-opioid antagonist action underlying the effects of these KOR agonists. This mechanism-based study further supports the clinical potential of KOR agonists as antipruritics under the context of spinal opioid analgesia.

Psychotomimesis mediated by kappa opiate receptors.

The kappa opioid agonists are analgesics that seem to be free of undesired morphine-like effects. Their dysphoric actions observed with the kappa agonist cyclazocine are thought to be mediated by an action at sigma-phencyclidine receptors. The benzomorphan kappa agonist MR 2033 is inactive at sigma-phencyclidine receptors. In male subjects, the opiate-active (-)-isomer, but not the (+)-isomer, elicited dose-dependent dysphoric and psychotomimetic effects that were antagonized by naloxone. Thus, kappa opiate receptors seem to mediate psychotomimetic effects. In view of the euphorigenic properties of mu agonists, our results imply the existence of opposed opioid systems affecting emotional and perceptual experiences.

(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan compound (2S-LP2): Discovery of a biased mu/delta opioid receptor agonist.

The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synthesized and their pharmacological profile was evaluated in in vitro and vivo assays. From our results, 2S-LP2 (5) showed an improved pharmacological profile in comparison to LP2 (1) and 2R-LP2 (4). 2S-LP2 (5) elicited an antinociceptive effect with a 1.5- and 3-times higher potency than LP2 (1) and R-antipode (4), respectively. In vivo effect of 2S-LP2 (5) was consistent with the improved MOR/DOR efficacy profile assessed by radioligand binding assay, to evaluate the opioid receptor affinity, and BRET assay, to evaluate the capability to promote receptor/G-protein and receptor/ß-arrestin 2 interaction. 2S-LP2 (5) was able to activate, with different efficacy, G-protein pathway over ß-arrestin 2, behaving as biased agonist at MOR and mainly at DOR. Considering the therapeutic potential of both multitarget MOR/DOR agonism and functional selectivity over G-protein, the 2S-LP2 (5) biased multitarget MOR/DOR agonist could provide a safer treatment opportunity.

Molecular modeling and biological studies show that some µ-opioid receptor agonists might elicit analgesia acting as MMP-9 inhibitors.

Aim: Despite the serious side effects, analgesics acting on opioid receptors are still considered the best way to get antinociception. Matrix metalloproteinases, a large family of zinc-dependent proteases implicated in many pathological conditions, such as diabetes and osteoarthritis, are also involved in inflammation and pain. Methodology & results: Looking for evidence of possible interactions of opioid pathways and inflammation mediators, molecular modeling studies of a series of recently developed µ-opioid receptor benzomorphanic agonists together with biological data on pain and inflammation molecular targets, allowed us to hypothesize a possible correlation between µ-opioid receptor system and MMP-9. Conclusion: A new compound, (-)-MML1017, emerged as a possible dual-acting agent able to interact selectively and potently with the two molecular targets.

The mu-opioid receptor agonist morphine, but not agonists at delta- or kappa-opioid receptors, induces peripheral antinociception mediated by cannabinoid receptors.

BACKGROUND AND PURPOSE: Although participation of opioids in antinociception induced by cannabinoids has been documented, there is little information regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids could be involved in peripheral antinociception induced by activation of mu-, delta- and kappa-opioid receptors. EXPERIMENTAL APPROACH: Nociceptive thresholds to mechanical stimulation of rat paws treated with intraplantar prostaglandin E2 (PGE2, 2 microg) to induce hyperalgesia were measured 3 h after injection using an algesimetric apparatus. Opioid agonists morphine (200 microg), (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) (80 microg), bremazocine (50 microg); cannabinoid receptor antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (20-80 microg), 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl) methanone (AM630) (12.5-100 microg); and an inhibitor of methyl arachidonyl fluorophosphonate (MAFP) (1-4 microg) were also injected in the paw. KEY RESULTS: The CB1-selective cannabinoid receptor antagonist AM251 completely reversed the peripheral antinociception induced by morphine in a dose-dependent manner. In contrast, the CB2-selective cannabinoid receptor antagonist AM630 elicited partial antagonism of this effect. In addition, the administration of the fatty acid amide hydrolase inhibitor, MAFP, enhanced the antinociception induced by morphine. The cannabinoid receptor antagonists AM251 and AM630 did not modify the antinociceptive effect of SNC80 or bremazocine. The antagonists alone did not cause any hyperalgesic or antinociceptive effect. CONCLUSIONS AND IMPLICATIONS: Our results provide evidence for the involvement of endocannabinoids, in the peripheral antinociception induced by the mu-opioid receptor agonist morphine. The release of cannabinoids appears not to be involved in the peripheral antinociceptive effect induced by kappa- and delta-opioid receptor agonists.

The influence of esters and carboxylic acids as the N-substituent of opioids. Part 1: Benzomorphans.

To investigate the effects of carboxylic ester and acid moieties as the N-substituent of opioids, a short series of racemic N-substituted normetazocines was prepared. The introduction of both groups as the normetazocine N-substituent produced compounds which displayed low potency in vitro and in vivo, with the esters displaying the greater activity. The pharmacology of the compounds is discussed with implications resulting from potential in vivo metabolic hydrolysis.

Benzomorphan scaffold for opioid analgesics and pharmacological tools development: A comprehensive review.

Benzomorphan, derived by morphine skeleton simplification, has been the subject of exploration in medicinal chemistry for the development of new drugs and pharmacological tools to explore opioid pharmacology in vitro and in vivo. Building upon these evidences, the design and synthesis of benzomorphan-based compounds, appropriately modified at the basic nitrogen and/or the phenolic hydroxyl (8-OH) group, represent a valid and versatile strategy to obtain analgesics. In this review, to improve the body of information in this field, we report structure activity-relationships (SARs) of benzomorphan-based compounds analysing data literature of last 25 years. Collectively, SARs data highlighted that the benzomorphan nucleus represents a template in the achievement of a specific functional profile, by modifying N-substituent or 8-OH group.