Design, synthesis, and preliminary evaluation of a potential synthetic opioid rescue agent.

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 (IC50 = 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.

Propionamide Derivatives as Dual µ-Opioid Receptor Agonists and σ1 Receptor Antagonists for the Treatment of Pain.

A new series of propionamide derivatives was developed as dual µ-opioid receptor agonists and σ1 receptor antagonists. Modification of a high-throughput screening hit originated a series of piperazinylcycloalkylmethyl propionamides, which were explored to overcome the challenge of achieving balanced dual activity and convenient drug-like properties. The lead compound identified, 18g, showed good analgesic effects in several animal models of both acute (paw pressure) and chronic (partial sciatic nerve ligation) pain, with reduced gastrointestinal effects in comparison with oxycodone.

Synthesis and Assessment of Fused ß-Carboline Derivatives as Kappa Opioid Receptor Agonists.

The synthesis of 5-formyl-6-aryl-6H-indolo[3,2,1-de][1,5] naphthyridine-2-carboxylates by reaction between 1-formyl-9H-ß-carbolines and cinnamaldehydes in the presence of pyrrolidine in water with microwave irradiation is described. Pharmacophoric modification of the formyl group offered several new fused ß-carboline derivatives, which were investigated for their κ-opioid receptor (KOR) agonistic activity. Two compounds 4 a and 4 c produced appreciable agonist activity on KOR with EC50 values of 46±19 and 134±9 nM, respectively. Moreover, compound-induced KOR signaling studies suggested both compounds to be extremely G-protein-biased agonists. The analgesic effect of 4 a was validated by the increase in tail flick latency in mice in a time-dependent manner, which was completely blocked by the KOR-selective antagonist norBNI. Moreover, unlike U50488, an unbiased full KOR agonist, 4 a did not induce sedation. The docking of 4 a with the human KOR was studied to rationalize the result.

Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a µ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects.

We previously identified a pyridomorphinan (6, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed µ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound 20 (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.

Design, synthesis and biological evaluation of N-hydroxy-aminobenzyloxyarylamide analogues as novel selective κ opioid receptor antagonists.

Aminobenzyloxyarylamide derivatives 1a-i and 2a-t were designed and synthesized as novel selective κ opioid receptor (KOR) antagonists. The benzoyl amide moiety of LY2456302 was changed into N-hydroxybenzamide and benzisoxazole-3(2H)-one to investigate whether it could increase the binding affinity or selectivity for KOR. All target compounds were evaluated in radioligand binding assays for opioid receptor binding affinity. These efforts led to the identification of compound 1c (κ Ki = 179.9 nM), which exhibited high affinity for KOR. Moreover, the selectivity of KOR over MOR and DOR increased nearly 2-fold and 7-fold, respectively, compared with (±)LY2456302.

Design and Synthesis of a Novel and Selective Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140).

κ opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of migraine and stress-related mood disorders including depression, anxiety, and drug abuse, thus the development of novel KOR antagonists with an improved potency/selectivity profile and medication-like duration of action has attracted the interest of the medicinal chemistry community. In this paper, we describe the discovery of 1-(6-ethyl-8-fluoro-4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)quinolin-2-yl)- N-(tetrahydro-2 H-pyran-4-yl)piperidin-4 amine (CYM-53093, BTRX-335140) as a potent and selective KOR antagonist, endowed with favorable in vitro ADMET and in vivo pharmacokinetic profiles and medication-like duration of action in rat pharmacodynamic experiments. Orally administered CYM-53093 showed robust efficacy in antagonizing KOR agonist-induced prolactin secretion and in tail-flick analgesia in mice. CYM-53093 exhibited a broad selectivity over a panel of off-target proteins. This compound is in phase 1 clinical trials for the treatment of neuropsychiatric disorders wherein dynorphin is thought to contribute to the underlying pathophysiology.

Synthesis and Biological Evaluation of Fentanyl Analogues Modified at Phenyl Groups with Alkyls.

A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce ß-arrestin2 recruitment. Compound 12 is a potent µ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak ß-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak ß-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

Pharmacological Research as a Key Component in Mitigating the Opioid Overdose Crisis.

The United States is experiencing an epidemic of opioid overdose deaths. Many of the recent fatalities are associated with illicitly manufactured fentanyl, which is being added to heroin and counterfeit pain pills. The crisis is further exacerbated by the emergence of an increasing number of novel synthetic opioids (NSOs), including various fentanyl analogs and non-fentanyl compounds that display potent agonist actions at the µ-opioid receptor. Importantly, most users are unaware of their exposure to fentanyl and NSOs. Stemming the tide of opioid-related fatalities will require a coordinated multidisciplinary response from policy makers, law enforcement personnel, first responders, treatment providers, family members, and scientists. To this end, basic research in pharmacology can contribute significantly to mitigating the crisis through efforts to characterize the biological effects of NSOs, discover more effective antidotes for overdose rescue, and develop safer medications for treating addiction and alleviating pain.

Cocaine reward is reduced by decreased expression of receptor-type protein tyrosine phosphatase D (PTPRD) and by a novel PTPRD antagonist.

Receptor-type protein tyrosine phosphatase D (PTPRD) is a neuronal cell-adhesion molecule/synaptic specifier that has been implicated in addiction vulnerability and stimulant reward by human genomewide association and mouse cocaine-conditioned place-preference data. However, there have been no reports of effects of reduced expression on cocaine self-administration. There have been no reports of PTPRD targeting by any small molecule. There are no data about behavioral effects of any PTPRD ligand. We now report (i) robust effects of heterozygous PTPRD KO on cocaine self-administration (These data substantially extend prior conditioned place-preference data and add to the rationale for PTPRD as a target for addiction therapeutics.); (ii) identification of 7-butoxy illudalic acid analog (7-BIA) as a small molecule that targets PTPRD and inhibits its phosphatase with some specificity; (iii) lack of toxicity when 7-BIA is administered to mice acutely or with repeated dosing; (iv) reduced cocaine-conditioned place preference when 7-BIA is administered before conditioning sessions; and (v) reductions in well-established cocaine self-administration when 7-BIA is administered before a session (in WT, not PTPRD heterozygous KOs). These results add to support for PTPRD as a target for medications to combat cocaine use disorders. 7-BIA provides a lead compound for addiction therapeutics.

Synthesis of 7ß-hydroxy-8-ketone opioid derivatives with antagonist activity at mu- and delta-opioid receptors.

Despite extensive years of research, the direct oxidation of the 7,8-double bond of opioids has so far received little attention and knowledge about the effects of this modification on activity at the different opioid receptors is scarce. We herein report that potassium permanganate supported on iron(II) sulfate heptahydrate can be used as a convenient oxidant in the one-step, heterogeneous conversion of Δ7,8-opioids to the corresponding 7ß-hydroxy-8-ketones. Details of the reaction mechanism are given and the effects of the substituent at position 6 of several opioids on the reaction outcome is discussed. The opioid hydroxy ketones prepared are antagonists at the mu- and delta-opioid receptors. Docking simulations and detailed structure-activity analysis revealed that the presence of the 7ß-hydroxy-8-ketone functionality in the prepared compounds can be used to gain activity towards the delta opioid receptor. The 7ß-hydroxy-8-ketones prepared with this method can also be regarded as versatile intermediates for the synthesis of other opioids of interest.

Opioid Receptor Modulators with a Cinnamyl Group.

To obtain selective and potent opioid receptor ligands, we synthesized dehydro derivatives of alvimopan and found compound (28f), a selective but modest affinity MOR antagonist weaker than alvimopan (1). We replaced the arylpiperidine unit by an arylpiperazine to obtain the 1-(α-carboxycinnamyl)-4-arylpiperazines like 13h, which to our surprise had no MOR or DOR activity but was a KOR agonist with moderate affinity. In contrast, literature examples of arylpiperazines 4 and 5 were reported to be pan opioid receptor antagonists, while 6 was a MOR agonist. Two compounds (13l and 11b) showed analgesic response in tail flick test which was blocked by pretreatment with norbinaltorphimine (norBNI). Among 10 1-(α-carboxycinnamyl)-4-arylpiperidines, compound 28g and five others were specific MOR antagonists. Interestingly, compound 26b of this series was found to be more potent than naloxone but weaker than 1. Docking studies have explained differential activities of the above piperazines and piperidines.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

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 [35S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile5,6deltorphin 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.

Design, synthesis and biological evaluation of aminobenzyloxyarylamide derivatives as selective κ opioid receptor antagonists.

Opioid receptors play an important role in both behavioral and mood functions. Based on the structural modification of LY2456302, a series of aminobenzyloxyarylamide derivatives were designed and synthesized as κ opioid receptor antagonists. The κ opioid receptor binding ability of these compounds were evaluated with opioid receptors binding assays. Compounds 1a-d showed high affinity for κ opioid receptor. Especially for compound 1c, exhibited a significant Ki value of 15.7 nM for κ opioid receptor binding and a higher selectivity over µ and δ opioid receptors compared to (±)LY2456302. In addition, compound 1c also showed potent κ antagonist activity with κ IC50 = 9.32 nM in [35S]GTP-γ-S functional assay. The potential use of the representative compounds as antidepressants was also investigated. The most potent compound 1c not only exhibited potent antidepressant activity in the mice forced swimming test, but also displayed the effect of anti-anxiety in the elevated plus-maze test.

Fluorine-18-Labeled Antagonist for PET Imaging of Kappa Opioid Receptors.

Kappa opioid receptor (KOR) antagonists are potential drug candidates for diseases such as treatment-refractory depression, anxiety, and addictive disorders. PET imaging radiotracers for KOR can be used in occupancy study to facilitate drug development, and to investigate the roles of KOR in health and diseases. We have previously developed two 11C-labeled antagonist radiotracers with high affinity and selectivity toward KOR. What is limiting their wide applications is the short half-life of 11C. Herein, we report the synthesis of a first 18F-labeled KOR antagonist radiotracer and the initial PET imaging study in a nonhuman primate.

(+)-and (-)-Phenazocine enantiomers: Evaluation of their dual opioid agonist/σ1 antagonist properties and antinociceptive effects.

cis-N-Substituted N-normetazocine enantiomers possess peculiar pharmacological profiles. Indeed, dextro enantiomers bind with high affinity σ1 receptor while opposite enantiomers bind opioid receptors. In spite of their stereochemistry, cis-N-2-phenylethyl N-normetazocine (phenazocine) enantiomers showed mixed opioid/σ1 receptor profiles and a significant in vivo analgesia. To the best of our knowledge, there is no information available regarding the evaluation of σ1 pharmacological profile in the antinociceptive effects of (+)- and (-)-phenazocine. Therefore, the present study was designed to ascertain this component by in vitro and in vivo studies. In particular, we tested the σ1 affinity of both enantiomers by a predictive binding assay in absence or presence of phenytoin (DPH). Our results showed that DPH (1 mM) did not increase the σ1 receptor affinity of (+)-and (-)-phenazocine (Ki = 3.8 ± 0.4 nM, Ki = 85 ± 2.0 nM, respectively) suggesting a σ1 antagonist profile of both enantiomers. This σ1 antagonistic component of two phenazocine enantiomers was corroborated by in vivo studies in which the selective σ1 receptor agonist PRE-084, was able to unmask their σ1 antagonistic component associated with the opioid activity. The σ1 antagonistic component of (+)- and (-)-phenazocine may justify their analgesic activity and it suggests that they may constitute useful lead compounds to develop new ligands with this dual activity.

Structure-Activity Relationships of [des-Arg7]Dynorphin A Analogues at the κ Opioid Receptor.

Dynorphin A (Dyn A) is an endogenous ligand for the opioid receptors with preference for the κ opioid receptor (KOR), and its structure-activity relationship (SAR) has been extensively studied at the KOR to develop selective potent agonists and antagonists. Numerous SAR studies have revealed that the Arg7 residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg7]Dyn A analogues found that Arg7 is not a key residue and even deletion of the residue does not affect biological activities at the KOR. In addition, it was also found that [des-Arg7]Dyn A(1-9)-NH2 is a minimum pharmacophore and its modification at the N-terminus leads to selective KOR antagonists. A lead ligand, 14, with high affinity and antagonist activity showed improved metabolic stability and could block antinociceptive effects of a KOR selective agonist, FE200665, in vivo, indicating high potential to treat KOR mediated disorders such as stress-induced relapse.

Design, Synthesis, and Biological Evaluation of Structurally Rigid Analogues of 4-(3-Hydroxyphenyl)piperidine Opioid Receptor Antagonists.

In order to gain additional information concerning the active conformation of the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1) class of opioid receptor antagonists, procedures were developed for the synthesis of structurally rigid N-substituted-6-(3-hydroxyphenyl)3-azabicyclo[3.1.0]hexane and 3-methyl-4-(3-hydroxyphenyl)-4-azabicyclo[4.1.0]heptanes. Evaluation of the conformationally constrained series in a [35S]GTPγS assay showed that structural rigid compounds having the 3-hydroxyphenyl group locked in the piperidine equatorial orientation had potencies equal to or better than similar compounds having more flexible structures similar to 1. The studies of the rigid compounds also suggested that the 3-methyl group present in compound 1 type antagonists may not be necessary for their pure opioid antagonist properties.

Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators.

Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit ß-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids.

Synthesis and evaluation of novel opioid ligands with a C-homomorphinan skeleton.

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.

A selective delta opioid receptor antagonist based on a stilbene core.

Studies of directed ortho metalation reactions on an aromatic substrate with multiple potential directing groups have identified conditions that favor either of two regioisomers. One of these regioisomers has been converted to an analogue of the stilbene pawhuskin A, and been shown to have high selectivity as an antagonist of the delta opioid receptor. Docking studies have suggested that this compound can adopt a conformation similar to naltrindole, a known delta antagonist.