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Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55 °C hot plate and the 55 °C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, G α • βγ Classic pharmacological methods, such as [ 35 S]GTP γ S binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous G α proteins in a given cell type. Bioluminescence resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of G α subunit-specific effects on opioid pharmacology. Using a Venus-tagged G βγ and nanoluciferase-tagged truncated G protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific G α protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM 2-(3,4-dichlorophenyl)- N -methyl- N -[(1 R ,2 R )-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50,488) for the kappa OR (KOR). Opioids reached equilibrium with the KOR, and concentration-response curves were generated. Although the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the G α subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through G α z compared with other G α i/o subunits. In contrast, the G α subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classic mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC 50 values of 7.3 and 110 nM and maximal stimulation values of 79% and 35% when the KOR signaled through G α z and G α i1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific G α subunit coupled to the KOR. SIGNIFICANCE STATEMENT: Opioid receptors couple to various heterotrimeric Gαβγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various inhibitory Gα subunits influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared with other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

A combination of buprenorphine (BUP) and samidorphan (SAM) at a 1:1 (mg/mg) fixed-ratio dose is being investigated as an adjunctive treatment of major depressive disorder (BUP/SAM, ALKS 5461). Both [ 3 H]BUP and [ 3 H]SAM bound to the μ -, κ -, and δ -opioid receptors (MOR, KOR, and DOR, respectively) with K d values of 3 nM or less. [ 3 H]BUP dissociated from the MOR more slowly than [ 3 H]SAM did. In the [ 35 S]GTP γ S assay, BUP was a partial agonist at the MOR, KOR, and DOR. SAM was an antagonist at the MOR and a partial agonist at the KOR and DOR. The pharmacology of the combination of SAM and BUP was characterized at ratios like the molar ratios of both compounds at steady state in humans. In all assessments, SAM reduced the efficacy of BUP at the MOR without altering its potency. At the KOR, SAM had no significant effect on the activity of BUP. In bioluminescent resonance energy transfer assays, SAM, naltrexone, and naloxone were partial agonists when the MOR was coupled to the G α oB and G α z , and were antagonists when coupled to G α i At the KOR, SAM was a partial agonist activating G α oA and G α oB and a full agonist in stimulating G α z SAM inhibited BUP's recruitment of β -arrestin to the MOR, suggesting an attenuation of BUP's efficacy in activating G proteins correlated with an inhibition of β -arrestin recruitment. The collective data suggest that SAM attenuates the efficacy of BUP under all conditions tested at the MOR and DOR but had little effect on BUP activity at the KOR., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Patients receiving the cytokine immunotherapy, interferon-alpha (IFN-α) frequently present with neuropsychiatric consequences and cognitive impairments. Patients (25-80%) report symptoms of depression, including, anhedonia, irritability, fatigue and impaired motivation. Our lab has previously demonstrated treatment (170,000IU/kg sc, 3 times per week for 4weeks) of the pro-inflammatory cytokine, IFN-α, induced a depressive phenotype in rats in the forced swim test (FST). Here, we examine the biological mechanisms underlying behavioral changes induced by IFN-α, which may be reflective of mechanisms underlying inflammation associated depression. We also investigate the potential of 3-carboxamido seco-nalmefene (3CS-nalmefene), a novel opioid modulator (antagonist at mu and partial agonist at kappa and delta opioid receptors in vitro), to reverse IFN-α induced changes. In vitro radioligand receptor binding assays and the [ 35 S] GTPγS were performed to determine the affinity of 3CS-nalmefene for the mu, kappa and delta opioid receptors. IFN-α treatment increased circulating and central markers of inflammation and hypothalamic-pituitaryadrenal (HPA) axis activity (IL-6, IL-1β and corticosterone) while increasing immobility in the FST, impairing of object displacement learning in the object exploration task (OET), and decreasing neuronal proliferation and brain-derived neurotrophic factor (BDNF) in the hippocampus. Treatment with 3CS-nalmefene (0.3mg/kg/sc twice per day, 3 times per week for 4weeks) prevented IFN-α-induced immobility in the FST and impaired object displacement learning. In addition, 3CS-nalmefene prevented IFN-α-induced increases in inflammation and hyperactivity of the HPA-axis, the IFN-α-induced reduction in both neuronal proliferation and BDNF expression in the hippocampus. Overall, these preclinical data would support the hypothesis that opioid receptor modulation is a relevant target for treatment of depression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glycopeptides related to β-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H2O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (11s → 11sG → 11) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H2O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects.

Glycosylated β-endorphin analogues of various amphipathicity were studied in vitro and in vivo in mice. Opioid binding affinities of the O-linked glycopeptides (mono- or disaccharides) and unglycosylated peptide controls were measured in human receptors expressed in CHO cells. All were pan-agonists, binding to μ-, δ-, or κ-opioid receptors in the low nanomolar range (2.2-35 nM K(i)'s). The glycoside moiety was required for intravenous (i.v.) but not for intracerebroventricular (i.c.v.) activity. Circular dichroism and NMR indicated the degree of helicity in H2O, aqueous trifluoroethanol, or micelles. Glycosylation was essential for activity after i.v. administration. It was possible to manipulate the degree of helicity by the alteration of only two amino acid residues in the helical address region of the β-endorphin analogues without destroying μ-, δ-, or κ-agonism, but the antinociceptive activity after i.v. administration could not be directly correlated to the degree of helicity in micelles.

A series of levo- and dextromorphinan pairs have been synthesized and evaluated for their affinities to the mu, kappa, and delta opioid receptors, the N-methyl-D-aspartate (NMDA) channel, and sigma 1 and 2 receptors. It was found that levo isomers tended to have higher affinities at the opioid receptors and moderate to high affinities to the NMDA and sigma receptors, while dextro isomers tended to have lower affinities to the opioid receptors but comparatively higher affinities to the NMDA and sigma receptors. This series of compounds have interesting and complex pharmacological profiles, and merit further investigation as potential therapies for drug abuse treatment.

Previous studies with aminothiazolomorphinans suggested that this class of opioid ligands may be useful as a potential pharmacotherapeutic to decrease drug abuse. Novel aminothiazole derivatives of cyclorphan were prepared to evaluate a series of aminothiazolomorphinans with varying pharmacological properties at the κ opioid receptor (KOR) and μ opioid receptor (MOR). This study was focused on exploring the regioisomeric analogs with the aminothiazole on the C-ring of the morphinan skeleton. Receptor binding and [(35)S]GTPγS binding assays were used to characterize the affinity and pharmacological properties of the aminothiazolomorphinans. Intracranial self-stimulation (ICSS) was used to compare the effects of a representative aminothiazolomorphinan with the morphinan mixed-KOR/MOR agonist butorphan (MCL-101) on brain-stimulation reward.

Derivatives of the lead compound N-BPE-8-CAC (1) where each CH of the biphenyl group was individually replaced by N were prepared in hopes of identifying high affinity ligands with improved aqueous solubility. Compared to 1, binding affinities of the five possible pyridinyl derivatives for the μ opioid receptor were between threefold lower to fivefold higher with the Ki of the most potent compound being 0.064 nM. Docking of 8-CAC (2) into the unliganded binding site of the mouse μ opioid receptor (pdb: 4DKL) revealed that 8-CAC and β-FNA (from 4DKL) make nearly identical interactions with the receptor. However, for 1 and the new pyridinyl derivatives 4-8, binding is not tolerated in the 8-CAC binding mode due to the steric constraints of the large N-substituents. Either an alternative binding mode or rearrangement of the protein to accommodate these modifications may account for their high binding affinity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of 3-benzylamino-3-desoxymorphinan (I) and 3-benzylamino-3-desoxymorphine (II) derivatives were synthesized and evaluated for their binding affinities, and functional activity data are presented at MOR, KOR, and DOR. Some of these ligands were found to have high binding affinity at MOR and KOR and displayed increased selectivity at MOR over KOR and DOR compared to butorphan or cyclorphan. The most selective compound, 3-(3'-hydroxybenzyl)amino-17-methylmorphinan (4g) (24-fold MOR to KOR and 1700-fold MOR to DOR) also showed high binding affinity (0.42 nM to MOR) and was a full agonist in the [(35)S]GTPγS binding assay. 2-(3'-Hydroxybenzyl)amino-17-cyclopropylmethylmorphinan (17) was found to be a KOR-selective ligand (150-fold over MOR and >10000-fold over the DORs). Most 3-benzylaminomorphinan derivatives were partial agonists at MOR and full agonists at KOR in the [(35)S]GTPγS binding assay.

Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ-agonist, and one related to DAMGO, a highly selective μ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its β-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human μ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55 °C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations., (© 2011 John Wiley & Sons A/S.)

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling., (Copyright © 2011 International Association for the Study of Pain. All rights reserved.)

A series of N-substituted and N'-substituted aminothiazole-derived morphinans (5) were synthesized for expanding the structure-activity relationships of aminothiazolo-morphinans. Although their affinities were somewhat lower than their prototype aminothiazolo-N-cyclopropylmorphinan (3), 3-aminothiazole derivatives of cyclorphan (1) containing a primary amino group displayed high affinity and selectivity at the κ and μ opioid receptors. [(35)S]GTPγS binding assays showed that the aminothiazolomorphinans were κ agonists with mixed agonist and antagonist activity at the μ opioid receptor. These novel N'-monosubstituted aminothiazole-derived morphinans may be valuable for the development of drug abuse medications.

Bifunctional ligands containing an ester linkage between morphine and the δ-selective pharmacophore Dmt-Tic were synthesized, and their binding affinity and functional bioactivity at the μ, δ and κ opioid receptors determined. Bifunctional ligands containing or not a spacer of β-alanine between the two pharmacophores lose the μ agonism deriving from morphine becoming partial μ agonists 4 or μ antagonists 5. Partial κ agonism is evidenced only for compound 4. Finally, both compounds showed potent δ antagonism., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, we generated a mutant mouse with brain neuron-specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.

Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist / δ antagonist, H-Dmt-Tic-Gly-NH-Bzl. Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (-Cl, -CH(3), partially -NO(2), inactive -NH(2)) was found to give a more potent μ agonist / antagonist effect associated with a relatively unmodified δ antagonist activity (pA(2) = 8.28-9.02). Increasing the steric hindrance of the benzyl group (using diphenylmethyl and tetrahydroisoquinoline functionalities) substantially maintained the μ agonist and δ antagonist activities of the lead compound. Finally and quite unexpectedly D-Tic2, considered as a wrong opioid message now; inserted into the reference compound in lieu of L-Tic, provided a μ agonist / δ agonist better than our reference ligand (H-Dmt-Tic-Gly-NH-Ph) and was endowed with the same pharmacological profile.

The phenolic group of the potent mu and kappa opioid morphinan agonist/antagonists cyclorphan and butorphan was replaced by phenylamino and benzylamino groups including compounds with para-substituents in the benzene ring. These compounds are highly potent mu and kappa ligands, e.g., p-methoxyphenylaminocyclorphan showing a K(i) of 0.026 nM at the mu receptor and a K(i) of 0.03 nM at the kappa receptor. Phenyl carbamates and phenylureas were synthesized and investigated. Selective o-formylation of butorphan and levorphanol was achieved. This reaction opened the way to a large set of 2-substituted 3-hydroxymorphinans, including 2-hydroxymethyl-, 2-aminomethyl-, and N-substituted 2-aminomethyl-3-hydroxymorphinans. Bivalent ligands bridged in the 2-position were also synthesized and connected with secondary and tertiary aminomethyl groups, amide bonds, and hydroxymethylene groups, respectively. Although most of the 2-substituted morphinans showed considerably lower affinities compared to their parent compounds, the bivalent ligand approach led to significantly higher affinities compared to the univalent 2-substituted morphinans.

Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the mu, delta, and kappa opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer length and structure show potent binding profiles with the most potent compound (4b), having K(i) values of 0.47 nM for both the mu and kappa opioid receptors, and 4a, having K(i) values of 0.95 and 0.62 nM for the mu and kappa receptors, respectively. Both 4a and 4b were partial agonists at the kappa and micro receptors in the [(35)S]GTPgammaS binding assay.

We synthesized several hydrophobic esters and ethers of butorphanol and assessed their affinities at opioid receptors in CHO cell membranes. Tested compounds displayed moderate to high affinities to the mu and kappa receptors. The findings accord with previous evidence of a lipophilic binding pocket in the opioid receptors that can be accessed to afford good binding affinity without the need for a phenolic hydrogen-bond donor group. The most potent (K(i)=61 pM at mu and 48 pM at kappa) novel agent was (-)-N-cyclobutylmethylmorphinan-3-yl-14-ol phenoxyacetate (4d).

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.

To further extend the structure-activity relationships of levorphanol, two series of novel morphinans were prepared by incorporation of an indole or aminothiazole fragment to the hexyl ring (ring C) in levorphanol. Such morphinans differed from previously reported ligands in that such indole- or aminothiazole-containing morphinans displayed enhanced binding affinity to the delta opioid receptor, while the affinity to kappa and micro receptors was slightly reduced.

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.

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.

A series of mu-agonist DAMGO analogs were synthesized and pharmacologically characterized to test the 'biousian' hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus via carboxamide and simple glycoside additions. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO (1) to l-serine amide (2), l-serine amide beta-d-xyloside (3), l-serine amide beta-d-glucoside (4), and finally to l-serine amide beta-lactoside (5). Opioid binding and mouse tail-flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A(50) approximately 0.2 micromol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A(50) approximately 1.0 micromol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A(50) values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A(50) versus e(-Awater/Alipid)), and provided either a V-shaped or a U-shaped curve, as predicted by the 'biousian' hypothesis. The mu-selective receptor profile was maintained (K(i)'s = 0.66-1.3 nm) upon modifications at the C-terminus. The optimal 'degree of glycosylation' for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface-derived amphipathicity values.

Here, we report the synthesis of a designed multi-pharmacophore ligand derived from the linkage of a delta selective peptide antagonist (Dmt-Tic) and a mu/kappa morphinan agonist butorphan (MCL 101) through a two methylene spacer. The new compound MCL 450 maintains the same characteristics as those the two reference compounds. MCL 450 represents a useful starting point for the synthesis of other multiple opioid ligands endowed with analgesic properties with low tolerance and dependence.

A series of homo- and heterodimeric ligands containing kappa agonist and mu agonist/antagonist pharmacophores joined by a linker chain of varying lengths was synthesized and evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors. The functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. The data suggest that the stereochemistry of the pharmacophores, the N-substituents of the pharmacophore, ester linkages, and the spacer length were crucial factors for optimum interactions of such ligands at opioid receptor binding sites. These novel ligands as well as their pharmacological properties will serve as the basis for our continuing investigation of such bivalent ligands as probes of the opioid receptor oligomerization phenomena and for in vivo studies as analgesics.

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.

A series of 10-ketomorphinan analogues were synthesized, and their binding affinity at all three opioid receptors was investigated. In most cases, high affinity at micro and kappa receptors, and lower affinity at delta receptor was observed, resulting in good selectivity for micro and kappa receptors. A wide range of substituents can be accommodated on the nitrogen position. The N-(S)-tetrahydrofurfuryl analogue 11 displayed the highest affinity at all three receptors. The N-cyclobutylmethyl analogue 13 gave both high affinity and selectivity at kappa receptor, and N-2-phenylethyl analogue 18 exhibited good affinity and selectivity at micro receptor. Further modifications of the 3-substituent indicated that one H-bond donor was an essential requirement for good affinity at micro and kappa receptors. Similar modifications were investigated at the 3-OH group of morphinans: levorphanol (2a), cyclorphan (2b), and MCL-101 (2c) lacking the 10-keto group. The 3-amino bioisosteric analogues (40 and 41) displayed reasonably good affinity at micro and kappa receptors. The 3-carboxamido replacement (compounds 46-48) in the morphinan subseries resulted in similar affinities comparable to their corresponding 3-OH congeners. The high affinity of these carboxamido analogues, along with their greater lipophilicity and metabolic stability, make them promising candidates for further pharmacological investigation.

A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for micro, delta, and kappa opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical morphinan pharmacophores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that the affinity of these ligands was sensitive to the character and length of the spacer. Compound 13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer were the most potent ligands in this series, displaying excellent affinities at micro and kappa receptors (K(i) = 0.09-0.2 nM at micro and K(i) = 0.078-0.049 nM at kappa), which were comparable to the parent compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a long-chain ester group, had affinity at both micro and kappa receptors almost identical to that of the parent ligand 2. In the [(35)S]GTPgammaS binding assay, ligands 13, 17, and 19 and their parent morphinans 1 and 2 stimulated [(35)S]GTPgammaS binding mediated by the micro and kappa receptors. Compounds 13 and 17 were full kappa agonists and partial micro agonists, while compound 19 was a partial agonist at both micro and kappa receptors. These novel ligands, as well as their interesting pharmacological properties, will serve as the basis for our continuing investigation of the dimeric ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new avenues for the characterization of opioid receptors.