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While in the process of designing more effective synthetic opioid rescue agents, we serendipitously identified a new chemotype of potent synthetic opioid. Here, we report that conformational constraint of a piperazine ring converts a mu opioid receptor (MOR) antagonist into a potent MOR agonist. The prototype of the series, which we have termed atoxifent ( 2 ), possesses potent in vitro agonist activity. In mice, atoxifent displayed long-lasting antinociception that was reversible with naltrexone. Repeated dosing of atoxifent produced antinociceptive tolerance and a level of withdrawal like that of fentanyl. In rats, while atoxifent produced complete loss of locomotor activity like fentanyl, it failed to produce deep respiratory depression associated with fentanyl-induced lethality. Assessment of brain biodistribution demonstrated ample distribution of atoxifent into the brain with a T max of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.

The enantioselective de novo synthesis of pharmacologically important 14-hydroxy-6-oxomorphinans is described. 4,5-Desoxynaltrexone and 4,5-desoxynaloxone were prepared using this route and their biological activities against the opioid receptors were measured.

Background: One of the most prominent opioid analgesics in the United States is the high potency agonist fentanyl. It is used in the treatment of acute and chronic pain and as an anesthetic adjuvant. When used inappropriately, however, ingestion of just a few milligrams of fentanyl or other synthetic opioid can cause opioid-induced respiratory depression (OIRD), often leading to death. Currently, the treatment of choice for OIRD is the opioid receptor antagonist naloxone. Recent reports, however, suggest that higher doses or repeated dosing of naloxone (due to recurrence of respiratory depression) may be required to reverse fully fentanyl-induced respiratory depression, rendering this treatment inadequate. To combat this synthetic opioid overdose crisis, this research aims at identifying a novel opioid reversal agent with enhanced efficacy towards fentanyl and other synthetic opioids., Methods: A series of naltrexone analogues were characterized for their ability to antagonize the effects of fentanyl in vitro utilizing a modified forskolin-induced cAMP accumulation assay. Lead analogue 29 was chosen to undergo further PK studies, followed by in vivo pharmacological analysis to determine its ability to antagonize opioid-induced antinociception in the hot plate assay., Results: A series of potent MOR antagonists were identified, including the highly potent analogue 29 (IC 50  = 2.06 nM). Follow-up PK studies revealed 29 to possess near 100% bioavailability following IP administration. Brain concentrations of 29 surpassed plasma concentrations, with an apparent terminal half-life of ~ 80 min in mice. In the hot plate assay, 29 dose-dependently (0.01-0.1 mg/kg; IP) and fully antagonized the antinociception induced by oxycodone (5.6 mg/kg; IP). Furthermore, the dose of 29 that is fully effective in preventing oxycodone-induced antinociception (0.1 mg/kg) was ineffective against locomotor deficits caused by the KOR agonist U50,488., Conclusions: Methods have been developed that have utility to identify enhanced rescue agents for the treatment of OIRD. Analogue 29, possessing potent MOR antagonist activity in vitro and in vivo, provides a promising lead in our search for an enhanced synthetic opioid rescue agent., (© 2021. The Author(s).)

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.

In the present work, we reported the application of a nitrogen-walk approach on developing a series of novel opioid ligands containing an azaindole moiety at the C6-position of the epoxymorphinan skeleton. In vitro study results showed that introducing a nitrogen atom around the indole moiety not only retained excellent binding affinity, but also led to significant functional switch at the mu opioid receptor (MOR). Further computational investigations provided corroborative evidence and plausible explanations of the results of the in vitro studies. Overall, our current work implemented a series of novel MOR ligands with high binding affinity and considerably low efficacy, which may shed light on rational design of low efficacy MOR ligands for opioid use disorder therapeutics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We have recently reported that N -alkyl and N -acyl naltrindole (NTI) derivatives showed activities for the δ opioid receptor (DOR) ranging widely from full inverse agonists to full agonists. We newly designed sulfonamide-type NTI derivatives in order to investigate the effects of the N -substituent on the functional activities because the side chain and S=O part in the sulfonamide moiety located in spatially different positions compared with those in the alkylamine and amide moieties. Among the tested compounds, cyclopropylsulfonamide 9f (SYK-839) was the most potent full inverse agonist for the DOR, whereas phenethylsulfonamide 9e (SYK-901) showed full DOR agonist activity with moderate potency. These NTI derivatives are expected to be useful compounds for investigation of the molecular mechanism inducing these functional activities.

In this study, microemulsions capable of transforming into nanostructured hexagonal phase gels in vivo upon uptake of biological fluids for naltrexone prolonged release were investigated as a strategy for management of alcohol use disorder (AUD). Microemulsions were prepared using monoolein, tricaprylin, water and propylene glycol; after preliminary characterization, one formulation was selected, which contained 55% of monoolein-tricaprylin (M-55). This microemulsion displayed size below 200 nm and Newtonian rheological behavior. Liquid crystalline gels formed in vitro upon 8 h of contact with water following a second order kinetics. After 120 h, <50% of naltrexone was released in vitro independently on drug loading (5 or 10%). In vivo, gels formed within 24 h of M-55 subcutaneous administration, and persisted locally for over 30 days providing slow release of the fluorescent marker Alexa fluor compared to a solution. Using the conditioned place preference paradigm, a test used to measure drug's rewarding effects, a single dose of M-55 containing 5% naltrexone reduced the time spent in the ethanol-paired compartment by 1.8-fold compared to saline; this effect was similar to that obtained with daily naltrexone injections, demonstrating the formulation efficacy and its ability to reduce dosing frequency. A more robust effect was observed following administration of M-55 containing 10% of naltrexone, which was compatible with aversion. These results support M-55 as a platform for sustained release of drugs that can be further explored for management of AUD to reduce dosing frequency and aid treatment adherence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the κ opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in the literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single-molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization, and trafficking. Our findings indicate that most KORs labeled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the μ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [ 35 S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile 5,6 deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The 7-benzylidenenaltrexone (BNTX) derivatives 2a-v, 3a-c, 13a-c, and 14a were synthesized from naltrexone (1) and evaluated for their antitrichomonal activity. The structure-activity-relationship studies found that 4-iodo-BNTX (2g) showed the highest activity (IC 50 =10.5µM) and the affinity for the opioid receptor was less important for antitrichomonal activity against Trichomonas vaginalis. The morphinan skeleton bearing both the double bond for a Michael acceptor and the phenolic hydroxy group would be a specific template for development of antitrichomonal agents. In addition, the mechanism of the antitrichomonal activity of the BNTX derivatives may differ from that of the standard drug, metronidazole., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation., (Published by Elsevier Ltd.)

The 6β-N-heterocyclic naltrexamine derivative, NAP, has been demonstrated to be a peripherally selective mu opioid receptor modulator. To further improve peripheral selectivity of this highly potent ligand, its pyridal ring was quaterinized with benzyl bromide to produce BNAP. In radioligand binding assay, the Ki of BNAP for MOR was 0.76 ± 0.09 nM and was >900-fold more selective for MOR than DOR. The Ki for KOR was 3.46 ± 0.05 nM. In [(35)S]GTPγS ligand stimulated assay, BNAP showed low agonist efficacy with 14.6% of the maximum response of DAMGO with an EC50 of 4.84 ± 0.6 nM. However, unlike its parent compound NAP, BNAP displayed partial agonist activity at KOR with % maximum response at 45.9 ± 1.7% of U50,488H. BNAP did not reverse morphine-induced antinociception when administered subcutaneously but did antagonize when administered intracerebroventricularly. BNAP antagonized morphine-induced contractions of the circular muscle in mice colon. BNAP inhibition of field-stimulated contractions in longitudinal muscle strips for the guinea-pig ileum were also blocked by nor-BNI, a kappa opioid receptor antagonist. BNAP induced inhibition of acetic acid induced abdominal stretching in chronic morphine treated mice. These findings suggest that BNAP is a dual MOR antagonist/KOR agonist and may have functional use in irritable bowel patients.

As the reports about C-homomorphinans with the seven-membered C-ring are much fewer than those of morphinan derivatives with a six-membered C-ring, we attempted to synthesize C-homomorphinan derivatives and to evaluate their opioid activities. C-Homomorphinan 5 showed sufficient binding affinities to the opioid receptors. C-Homomorphinan derivatives possessing the δ address moiety such as indole (NTI-type), quinoline, or benzylidene (BNTX-type) functionalities showed the strongest binding affinities for the δ receptor among the three types of opioid receptors, which indicated that the C-homomorphinan skeleton sufficiently functions as a message-part in the ligand. Although NTI-type compound 8 and quinoline compound 9 with C-homomorphinan scaffold exhibited lower affinities and selectivities for the δ receptor than the corresponding morphinan derivatives did, both the binding affinity and selectivity for the δ receptor of BNTX-type compound 12 with a seven-membered C-ring were improved compared with the corresponding compounds with a six-membered C-ring including BNTX itself. BNTX-Type compound 12 was the most selective δ receptor antagonist among the tested compounds., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We evaluated antitrichomonal effects of δ opioid receptor (DOR) agonists and antagonists. Although all the agonists were inactive, the DOR antagonists BNTX (2a) and its derivatives 2b-d showed antitrichomonal activity with MIC of 20-40 μM. In addition, the development of a more effective synthetic method for the BNTX derivatives was achieved by using the Knoevenagel condensation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a-f). 6b, 6d, and 6 e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6 e) exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1h)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

10-Nornaltrexones (3-(cyclopropylmethyl)-4a,9-dihydroxy-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one, 1) have been underexploited in the search for better opioid ligands, and their enantiomers have been unexplored. The synthesis of trans-isoquinolinone 2 (4-aH, 9-O-trans-9-methoxy-3-methyl-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one) was achieved through a nonchromatographic optimized synthesis of the intermediate pyridinyl compound 12. Optical resolution was carried out on 2, and each of the enantiomers were used in efficient syntheses of the "unnatural" 4aR,7aS,12bR-(+)-1) and its "natural" enantiomer (-)-1. Addition of a 14-hydroxy (the 4a-hydroxy) group in the enantiomeric isoquinolinones, (+)- and (-)-2), gave (+)- and (-)-10-nornaltrexones. A structurally unique tetracyclic enamine, (12bR)-7,9-dimethoxy-3-methyl-1,2,3,7-tetrahydro-7,12b-methanobenzo[2,3]oxocino[5,4-c]pyridine, was found as a byproduct in the syntheses and offers a different opioid-like skeleton for future study.

On the basis of a mu opioid receptor (MOR) homology model and the isosterism concept, three generations of 14-heteroaromatically substituted naltrexone derivatives were designed, synthesized, and evaluated as potential MOR-selective ligands. The first-generation ligands appeared to be MOR-selective, whereas the second and the third generation ones showed MOR/kappa opioid receptor (KOR) dual selectivity. Docking of ligands 2 (MOR selective) and 10 (MOR/KOR dual selective) to the three opioid receptor crystal structures revealed a nonconserved-residue-facilitated hydrogen-bonding network that could be responsible for their distinctive selectivity profiles. The MOR/KOR dual-selective ligand 10 showed no agonism and acted as a potent antagonist in the tail-flick assay. It also produced less severe opioid withdrawal symptoms than naloxone in morphine-dependent mice. In conclusion, ligand 10 may serve as a novel lead compound to develop MOR/KOR dual-selective ligands, which might possess unique therapeutic value for opioid addiction treatment.

Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique 'address' domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the 'message-address' concept, also facilitated our next generation ligand design and development., (Published by Elsevier Ltd.)

Isosterism is commonly used in drug discovery and development to address stability, selectivity, toxicity, pharmacokinetics, and efficacy issues. A series of 14-O-substituted naltrexone derivatives were identified as potent mu opioid receptor (MOR) antagonists with improved selectivity over the kappa opioid receptor (KOR) and the delta opioid receptor (DOR), compared to naltrexone. Since esters are not metabolically very stable under typical physiological conditions, their corresponding amide analogs were thus synthesized and biologically evaluated. Unlike their isosteres, most of these novel ligands seem to be dually selective for the MOR and the KOR over the DOR. The restricted flexibility of the amide bond linkage might be responsible for their altered selectivity profile. However, the majority of the 14-N-substituted naltrexone derivatives produced marginal or no MOR stimulation in the (35)S-GTP[γS] assay, which resembled their ester analogs. The current study thus indicated that the 14-substituted naltrexone isosteres are not bioisosteres since they have distinctive pharmacological profile with the regard to their opioid receptor binding affinity and selectivity., (Published by Elsevier Ltd.)

Beta-funaltrexamine is a unique irreversible antagonist for the mu-opiate receptor and would be useful as a tritiated radioligand. Starting from high specific activity [15, 16-³H] naltrexone, [15, 16-³H] beta-funaltrexamine was synthesized and characterized by means of a two-step reductive amination-acylation process., (Copyright © 2013 John Wiley & Sons, Ltd.)

A contracted ring degradation product, WYE-120318 (compound 2), was discovered during the development phase for methylnaltrexone bromide (compound 1) drug substance. The compound was isolated by high-performance liquid chromatography fractionation, and its structure was determined by spectroscopic data analyses. WYE-120318 is formed from methylnaltrexone through a benzyl-benzilic acid type rearrangement reaction to yield an α-hydroxy-cyclopentanecarboxylic acid substructure. The proposed structure and the formation mechanism are confirmed by the synthesis of WYE-120318 from methylnaltrexone (compound 1). A similar benzyl-benzilic acid type rearrangement reaction can be envisioned as the biological origin of remisporine A (compound 3), a naturally occurring cyclopentadienyl compound that autocatalytically dimerizes to remisporine B (compound 4). The structure of remisporine A was deduced from its dimer 4. Coniothyione (compound 5) can be considered as the first example of a stable natural product bearing the remisporine A skeleton. However, the regiochemistry of the chlorosubstitution in the coniothyrione structure needs to be revised to compound 6 on the basis of the nuclear magnetic resonance data and biogenesis analysis., (Copyright © 2012 John Wiley & Sons, Ltd.)

In an effort to better understand the conformational preferences that inform the biological activity of naltrexone and related naltrexol derivatives, a new synthesis of the restricted analog 3-OBn-6β,14-epoxymorphinan 4 is described. 4 was synthesized starting from naltrexone in 50% overall yield, proceeding through the OBn-6α-triflate intermediate 8. Key steps to the synthesis include benzylation (96% yield), reduction (90% yield, α:β:3:2), followed by a one-pot triflation/displacement sequence (96% yield) to yield the desired bridged epoxy derivative 4. X-ray crystallographic analysis of intermediate 3-OBn-6α-naltrexol 7a supports population of the key boat conformation required for the epoxy ring closure. We also report that the 6β-mesylate 10-a high affinity opioid receptor ligand, the epimeric derivative of 11, and an analog of 12-functions as an inverse agonist at the mu opioid receptor using herkinorin pre-conditioned cells and an agonist at the kappa opioid receptor when evaluated in independent in vitro [(35)S]-GTP-γ-S assays., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloroquine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activities. The synthesized derivatives showed CQ-resistance reversing effects. They also reacted with glutathione (GSH) both enzymatically and chemically, and inhibited glutathione reductase activity. 7-Benzyl derivative, which was obtained by reduction of the olefin group in α,β-unsaturated ketone structure of BNTX, also exhibited CQ-resistance reversing effect, but its potency was significantly lower than that of BNTX. These outcomes suggested that the decrease in GSH level could be one of the mechanisms of CQ-resistance reversing effects induced by BNTX derivatives., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The synthesis and biological evaluation of hybrid opioids consisting of Naloxone or Naltrexone and a partial opioid peptide are described. These compounds were synthesized in a homogeneous solution as well as in solid phase. A hydrazone linkage was employed to connect the alkaloids to the tetrapeptides. In synthesizing the peptides some non-traditional methods, which provided excellent results, were explored. The solid phase synthesis was achieved by anchoring the Fmoc-Phe to the 2-chlorotrityl resin, followed by stepwise addition of two Fmoc-Gly units. Each addition step preceded by standard piperidine removal of the Fmoc from the prior amino acid (AA) residue. The final AA, Tyr, was added as its Boc derivative. The Boc-tetrapeptide was then separated from the resin with a TFE/AcOH/CH(2)Cl(2) mixture. In the solution synthesis, each peptide elongation step was accomplished by one-pot removal of the Fmoc from the prior AA residue and addition of the next Fmoc-AA. TBAF-thiol was used to cleanly remove the Fmoc, before adding the next Fmoc-AA in the presence of DIPEA and TBTU. All prepared hybrid ligands exhibited high affinities toward all three opioid receptors; moderate preferences for κ and μ receptors over δ receptor were observed. [(35)S]GTPγS binding assays indicated that these hybrid opioids are δ and μ antagonists but partial κ agonist.

Using the selective mu-kappa agonist, N-naphthoyl-β-naltrexamine 1, as the prototype ligand, a series of closely related naphthalene analogues were synthesized to study the chemical space around the naphthalene moiety in an effort to evaluate how receptor selectivity is affected by chemical modification. Nine analogues (2-10) of compound 1 were synthesized and tested on HEK-293 cells expressing homomeric and heteromeric opioid receptors, and in the mouse tail-flick assay. It was found that a small change in structure produces profound changes in selectivity in this series. This is exemplified by the discovery that introduction of a 6-fluoro group transforms 1 from a selective mu-kappa heteromeric receptor agonist to a delta-preferring agonist 7. The in vivo studies reveal that many of the ligands are more potent spinally than supraspinally and devoid of tolerance.

As important pharmacological probes, highly selective opioid receptor antagonists are essential in opioid receptor structural characterization and opioid agonist functional studies. At present, a nonpeptidyl, highly selective, and reversible mu opioid receptor antagonist is still not available. Among a series of novel naltrexamine derivatives that have been designed and synthesized following molecular modeling studies, two compounds, NAP and NAQ, were identified as leads based on the results of in vitro and in vivo pharmacological assays. Both of them displayed high binding affinity and selectivity to the mu opioid receptor. Further pharmacokinetic and functional characterization revealed that NAP seems to be a peripheral nervous system agent while NAQ seems to be a central one. Such characteristics provide two distinguished potential application routes for these two agents and their derivatives. These results also supported our hypothesis that they may serve as leads to develop more potent and selective antagonists for the mu opioid receptor.

We synthesized symmetrical and nonsymmetrical triplet drugs with 1,3,5-trioxazatriquinane skeletons. The isolation of key intermediates, oxazoline dimers, made it possible to effectively produce nonsymmetrical triplets. Among the synthesized triplets, KNT-93, composed of three identical opioid μ receptor agonists, showed dose-dependent antinociception via the μ receptor. The effect was 56-fold more potent than that of morphine, a representative μ agonist. The profound analgesic effect induced by KNT-93 might result from simultaneous occupation of three μ opioid receptors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Acetylcholinesterase inhibitor (-)-homogalanthamine 3 was synthesized from μ opioid antagonist naltrexone (2) in 16% total yield. The synthesis features Grob fragmentation as a key reaction, which was especially accelerated in the presence of 15-crown-5.

We synthesized novel 15-16 nornaltrexone derivatives 9, 11 and 22 to examine the importance of the cavity in the Beckett-Casy model, which was proposed to interact with the 15-16 ethylene moiety in the morphine structure. All the synthesized compounds showed lower affinities for the opioid receptor than did the naltrexone (10). The binding affinities of 14-OH derivatives 11, in which the rotation of the 9-17 bond would be restricted by an intramolecular hydrogen bond, was improved compared to the corresponding 14-H derivatives 9. Compound 22 whose 9-17 bond was strictly fixed by the ethylene bridge hardly bound to the opioid receptor. Compound 26 also showed very weak binding affinity in spite of the existence of the 15-16 ethylene unit. We proposed an important role for the orientation of the lone electron pair on the 17-nitrogen rather than the significance of the cavity in the Beckett-Casy model., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Novel 16,17-seco-naltrexone derivatives 3 were synthesized using a 16-17 bond cleavage reaction of naltrexone as the key reaction to examine the Beckett-Casy model. All the prepared 16,17-seco-naltrexone derivatives 3 showed lower affinities for opioid receptors than naltrexone. Although the results of binding assay seem to support the existence of a cavity in the model, further investigation using 15,16-nornaltrexone derivatives 26 will be needed to confirm the model., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

In an effort to develop antagonists for kappa-mu opioid receptor heterodimers, a series of bivalent ligands 3-6 containing kappa- and mu-antagonist pharmacophores were designed and synthesized. Evaluation of the series in HEK-293 cells revealed 4 (KMN-21) to selectively antagonize the activation of kappa-mu heterodimers, suggesting possible bridging of receptors when the bivalent ligand spacer contains 21 atoms.

We re-examined the accessory site of the 4,5-epoxymorphinan skeleton by camdas conformational analysis in an effort to deign novel delta opioid receptor antagonists. We synthesized three novel compounds (SN-11, 23 and 28) with a 10-methylene bridge and without a 4,5-epoxy ring. Among them, compounds SN-23 (17-isobutyl derivative) and SN-28 (17-methyl derivative) showed very strong agonist activity (over 10 times more than TAN-67). SN-28 also showed high delta selectivity. The delta agonist activity of SN-23 was weaker than that of SN-28, but in terms of the delta selectivity, SN-23 was higher than that of SN-28. These unexpected results indicated that the 4,5-epoxy ring, but not the 10-methylene bridge, was an accessory site in delta opioid receptor agonists.

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring 'open' derivatives display very high affinity for mu and kappa receptors and much less affinity for delta. The observation that these target compounds have much higher receptor affinity than the corresponding ring 'closed' carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.

Opioid receptor selective antagonists are important pharmacological probes in opioid receptor structural characterization and opioid agonist functional study. Thus far, a nonpeptidyl, highly selective and reversible mu opioid receptor (MOR) antagonist is unavailable. On the basis of our modeling studies, a series of novel naltrexamine derivatives have been designed and synthesized. Among them, two compounds were identified as leads based on the results of in vitro and in vivo assays. Both of them displayed high binding affinity for the MOR (K(i) = 0.37 and 0.55 nM). Compound 6 (NAP) showed over 700-fold selectivity for the MOR over the delta receptor (DOR) and more than 150-fold selectivity over the kappa receptor (KOR). Compound 9 (NAQ) showed over 200-fold selectivity for the MOR over the DOR and approximately 50-fold selectivity over the KOR. Thus these two novel ligands will serve as leads to further develop more potent and selective antagonists for the MOR.

A twin drug consisting of 8-oxaendoethanotetrahydromorphides with a 1,4-dioxane spacer, NS29, was synthesized from a naltrexone derivative. The structure of compound 8, the precursor of NS29, was determined by X-ray crystallography. Monomeric NS28 showed mu opioid receptor antagonist activity, whereas dimeric NS29, consisting of two NS28 units, showed antagonist activities for mu, kappa, and the putative epsilon opioid receptor agonists. Twin drug NS29 and its derivatives are expected to be unique pharmacological tools for investigation of opioid receptor types.

Selective ring opening reaction of the N-cyclopropylmethyl group in naltrexone (1d) was effected in the presence of platinum (IV) oxide and hydrobromic acid under a hydrogen atmosphere at rt to selectively afford N-isobutyl derivative 10. The binding affinity of N-i-Bu derivative 10 for opioid receptors was 11-17 times less than that of the corresponding N-CPM compound, naltrexone (1d). However, compound 10 showed dose-dependent analgesic effects. Contrary to expectations based on previous structure-activity relationship studies for a series of N-substituted naltrexone derivatives that compound 10 would be an opioid antagonist, 10 showed dose-dependent analgesia in the mouse acetic acid writhing test (ED(50): 5.05 mg/kg, sc), indicating it was an opioid agonist. This finding may have a great influence on the drug design of opioid agonists.

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

We have previously reported on antitussive effect of (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol(1b) methanesulfonate (TRK-850), a selective delta opioid receptor antagonist which markedly reduced the number of coughs in a rat cough model. We designed TRK-850 based on naltrindole (NTI), a typical delta opioid receptor antagonist, to improve its permeability through the blood-brain barrier by introducing hydrophobic moieties to NTI. The ED(50) values of NTI and compound 1b by intraperitoneal injections were 104 microg/kg and 2.07 microg/kg, respectively. This increased antitussive potency probably resulted from the improved brain exposure of compound 1b. However, 1b was extremely unstable toward metabolism by cytochrome P450. In this study, we designed and synthesized compound 1b derivatives to improve the metabolic instability, which resulted in affording highly potent and metabolically stable oral antitussive agent (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-8'-fluoro-5',6'-dihydro-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-3,14-diol (1c) methanesulfonate (TRK-851).

We have previously reported antitussive effects of naltrindole (NTI), a typical delta opioid receptor antagonist, in a rat model. The ED50 values of NTI by intraperitoneal and peroral injections were 104 microg/kg and 1840 microg/kg, respectively, comparable to those of codeine. Codeine, one of the most reliable centrally acting antitussive drugs, has micro agonist activity and thus the same side effects as morphine, e.g., constipation, dependency, and respiratory depression. Because NTI is a delta opioid antagonist, its derivatives have potential as highly potent antitussives, free from the mu opioid agonist side effects. We attempted to optimize the NTI derivatives to develop novel antitussive agents. On the basis of the studies of structure-antitussive activity relationships of alkyl substituted NTI derivatives, we designed NTI derivatives with extra ring fused structures. As a clinical candidate, we identified a highly potent new compound, (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol (5b) methanesulfonate (TRK-850) which was effective even by oral administration (ED50 6.40 microg/kg).

Substituted aryl and aliphatic amide analogues of 6-naltrexamine were synthesized and used to characterize the binding to and functional activity of human mu-, delta-, and kappa-opioid receptors. Competition binding assays showed 11-25 and 27-31 bound to the mu (K(i) = 0.05-1.2 nM) and kappa (K(i) = 0.06-2.4 nM) opioid receptors. Compounds 11-18 possessed significant binding affinity for the delta receptor (K(i) = 0.8-12.4 nM). Functional assays showed several compounds acted as partial or full agonists of delta or kappa receptors while retaining an antagonist profile at the mu receptor. Structure-activity relationship for aryl amides showed that potent compounds possessed lipophilic groups or substituents capable of hydrogen bonding. Metabolic stability studies showed that 11, 12, and 14 possessed considerable stability in the presence of rat, mouse, or human liver preparations. The ED 50 of inhibition of 10% ethanol self-administration in trained rats, using operant techniques for 11, was 0.5 mg/kg.

Our investigation of bivalent ligands at mu, delta, and kappa opioid receptors is focused on the preparation of ligands containing kappa agonist and mu agonist/antagonist pharmacophores at one end joined by a chain containing the mu antagonist pharmacophores (naltrexone, naloxone, or nalbuphine) at the other end. These ligands were evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors and their relative efficacy in the [35S]GTPgammaS assay.

We have identified a series of hydrophilic indium-labeled DOTA and DO3A conjugates of naltrindole (NTI) that are suited to in vivo studies of peripheral delta opioid receptors. Indium(III) complexes, linked to the indole nitrogen of NTI by six- to nine-atom spacers, display high affinities (0.1-0.2 nM) and excellent selectivities for binding to delta sites in vitro. The [111In]-labeled complexes can be prepared in good isolated yields ( approximately 65%) with high specific radioactivities (>3300 mCi/mumol). The spacers serve as pharmacokinetic modifiers, and log D7.4 values range from -2.74 to -1.79. These radioligands exhibit a high level of specific binding (75-94%) to delta opioid receptors in mouse gut, heart, spleen, and pancreas in vivo. Uptakes of radioactivity are saturable by the non-radioactive complexes, inhibited by naltrexone, and blocked by NTI. Thus, these radiometal-labeled NTI analogues warrant further study by single-photon emission computed tomography.

A codrug approach for simultaneous treatment of alcohol abuse and tobacco dependence is considered as very desirable because of substantial evidence that smoking is increased significantly during drinking, and that smoking is regarded as a behavioral 'cue' for the urge to consume alcohol. The purpose of this study was to design and synthesize codrugs for simultaneous treatment of alcohol abuse and tobacco dependence. Two novel tripartate codrugs of naltrexone (NTX) and naltrexol (NTXOL) covalently linked to hydroxybupropion (BUPOH) were synthesized (25 and 26, respectively), and their hydrolytic cleavage to the parent drugs was determined. These codrugs were generally less crystalline when compared to NTX, or NTXOL, as indicated by their lower melting points, and were expected to be more lipid-soluble. Also, the calculated clogP values were found to be higher for the codrugs compared to those for NTX and NTXOL. The studies on the hydrolysis of the codrugs provided good evidence that they could be efficiently converted to the parent drugs in buffer at physiological pH. Thus, these codrugs are likely to be cleaved enzymatically in vivo to generate the parent drugs, and are considered to be potential candidates for simultaneous treatment of alcohol abuse and tobacco dependence.

Purpose: Physicochemical characterization and in vitro human skin diffusion studies of branched-chain ester and carbonate prodrugs of naltrexone (NTX) were compared and contrasted with straight-chain ester and carbonate NTX prodrugs., Methods: Human skin permeation rates, thermal parameters, solubilities in mineral oil and buffer, and stabilities in buffer and plasma were determined. Partition coefficients between stratum corneum and vehicle were determined for straight- and branched-chain esters with the same number of carbon atoms., Results: Branched prodrugs had lower melting points, lower buffer solubilities, and higher mineral oil solubilities than NTX. The transdermal flux values from all of these branched prodrugs were significantly lower than flux values from the straight-chain ester and the methyl carbonate prodrugs. Straight-chain prodrugs had higher partition coefficient values and higher calculated thermodynamic activities than their branched-chain counterparts. The prodrug hydrolysis to NTX in buffer and plasma was slower for prodrugs with increased branching., Conclusions: Branched-chain prodrugs with bulky moieties had smaller stratum corneum-vehicle partition coefficients and lower thermodynamic activities that resulted in smaller transdermal flux values than straight-chain prodrugs.

Very high affinity for opioid receptors (e.g., K(i)=0.052nM for mu) has been observed in the rationally designed naltrexone analogue 5. SAR and physical data supports the hypothesis that the 4-OH group of 5 stabilizes the 3-carboxamido group in the putative bioactive conformation.

To characterize delta- and kappa-opioid receptor phenotypes, bivalent ligands (KDAN series) containing delta-antagonist (naltrindole) and kappa(1)-agonist (ICI-199,441) pharmacophores were synthesized and evaluated by the intrathecal route using the mouse tail-flick assay and binding studies. The data have suggested that KDAN-18 (2) bridges phenotypic delta(2)- and kappa(1)-receptors. A conceptual model is presented to explain the organizational differences between the opioid receptors that give rise to the phenotypes (delta(1), delta(2), kappa(1), kappa(2)).

Indolic N-benzylation of naltrindole reportedly extends the duration of delta-opioid receptor (DOR) antagonism. Similar modification of the kappa-opioid receptor (KOR) antagonist norBNI (1a) and its 17,17'-diNMe analogue (1d), a low potency mu-opioid receptor (MOR) partial agonist, was found to affect predominantly their MOR activity. When administered systemically in mouse antinociceptive assays, N-benzyl-norBNI (1b) had only MOR agonist activity of relatively short duration whereas on central administration it had only a KOR-antagonist action of extremely long duration.

The well-established Nenitzescu reaction of imines of beta-dicarbonyl systems, as their enamine tautomers, with benzoquinone has been applied to a wide range of such imines to give 5-hydroxyindoles, some of which are of significant biological importance. This reaction has now been extended to the benzylimines of simple ketones, including those of the potent mu-opioid receptor antagonists naltrexone and naloxone. The products of the latter reactions, 1'-benzyl-5'-hydroxyindolomorphinans (7), are potent delta-opioid receptor (DOR) antagonists, confirming the enhancement of DOR antagonist potency and selectivity resulting from the introduction of the 1'-benzyl group.

The effect of substitution of the pyrrolo- and indolo-N atoms in tetrahydronaltrindole (TNTI), tetrahydrooxymorphindole (TOMI), and 17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-4'-phenyl-6,7:2',3'-pyrrolomorphinan (4) is reported. In opioid functional assays 4 were potent deltaopioid receptor (DOR) antagonists while the TNTI derivatives (7) were potent DOR antagonists or low-efficacy DOR partial agonists without substantial selectivity. The TOMI derivatives (8) were DOR agonists with significant selectivity. In vivo the DOR antagonist activity of 7d was confirmed, but the predominant agonist effect of 8d was shown to be mu opioid receptor mediated.