Structure-Activity Relationships of the Fentanyl Scaffold: Identification of Antagonists as Potential Opioid Overdose Reversal Agents.

Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist and its analogues (fentalogues) are increasingly found in illicit drug samples, both where the primary drug of abuse is an opioid and where it is not. The prevalence of fentalogues in the illicit drug market is thought to be the primary driver of the increased number of opioid-related overdose deaths since 2016. In fact, fentanyl and its analogues are involved in more than 70% of opioid-related overdoses. The standard opioid overdose rescue therapy naloxone is often insufficient to reverse opioid overdoses caused by fentalogue agonists under current treatment paradigms. However, the pharmacology of many fentalogues is unknown. Moreover, within the fentalogue series of compounds, it is possible that antagonists could be identified that might be superior to naloxone as opioid overdose reversal agents. In this report, we explore the pharmacology of 70 fentalogues and identify compounds that behave as MOR antagonists in vitro and demonstrate with one of these reversals of fentanyl-induced respiratory depression in the mouse. Such compounds could provide leads for the development of effective agents for the reversal of opioid overdose.

Opioid Antagonists from the Orvinol Series as Potential Reversal Agents for Opioid Overdose.

The opioid crisis continues to claim many lives, with a particular issue being the ready availability and use (whether intentional or accidental) of fentanyl and fentanyl analogues. Fentanyl is both potent and longer-acting than naloxone, the standard of care for overdose reversal, making it especially deadly. Consequently, there is interest in opioid reversal agents that are better able to counter its effects. The orvinol series of ligands are known for their high-affinity binding to opioid receptors and often extended duration of action; generally, compounds on this scaffold show agonist activity at the kappa and the mu-opioid receptor. Diprenorphine is an unusual member of this series being an antagonist at mu and only a partial agonist at kappa-opioid receptors. In this study, an orvinol antagonist, 14, was designed and synthesized that shows no agonist activity in vitro and is at least as good as naloxone at reversing the effects of mu-opioid receptor agonists in vivo.

SAR Matrices Enable Discovery of Mixed Efficacy µ-Opioid Receptor Agonist Peptidomimetics with Simplified Structures through an Aromatic-Amine Pharmacophore.

We previously described the development of potent µ-opioid receptor (MOR)-agonist/δ-opioid receptor (DOR)-antagonist peptidomimetic ligands as an approach toward effective analgesics with reduced side effects. In this series, a tetrahydroquinoline (THQ) or substituted phenyl is employed to link two key pharmacophore elements, a dimethyltyrosine amino acid and typically an aromatic pendant. Using new and previously reported analogues, we constructed a structure-activity relationship (SAR) matrix that probes the utility of previously reported amine pendants. This matrix reveals that the MOR-agonist/DOR-antagonist properties of these ligands do not change when a tetrahydroisoquinoline (THIQ) pendant is used, despite removal of substituents on the core phenyl ring. Based on this observation, we retained the THIQ pendant and replaced the phenyl core with simpler aliphatic chain structures. These simpler analogues proved to be potent MOR-agonists with high variability in their effects at the DOR and the κ-opioid receptor (KOR). These data show that the amine of the THIQ pendant may be a novel pharmacophore element that favors high MOR-efficacy, whereas the aromatic ring of the THIQ pendant may produce high MOR-potency. Combined, the two pharmacophores within the THIQ pendant may be a structurally efficient means of converting opioid peptides and peptidomimetics into potent and efficacious MOR-agonists.

In vitro pharmacology of fentanyl analogs at the human mu opioid receptor and their spectroscopic analysis.

Opioids are widely misused and account for almost half of overdose deaths in the United States. The cost in terms of lives, health care, and lost productivity is significant and has been declared a national crisis. Fentanyl is a highly potent mu opioid receptor (MOR) agonist and plays a significant role in the current opioid epidemic; fentanyl and its analogs (fentalogs) are increasingly becoming one of the biggest dangers in the opioid crisis. The availability of fentalogs in the illicit market is thought to play a significant role in the recent increase in opioid-related deaths. Although there is both rodent homolog in vivo and in vitro data for some fentalogs, prior to this publication very little was known about the pharmacology of many of these illicit compounds at the human MOR (hMOR). Using gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and in vitro assays, this study describes the spectral and pharmacological properties of 34 fentalogs. The reported spectra and chemical data will allow for easy identification of novel fentalogs in unknown or mixed samples. Taken together these data are useful for law enforcement and clinical workers as they will aid in the identification of fentalogs in unknown samples and can potentially be used to predict physiological effects after exposure.

Aromatic-Amine Pendants Produce Highly Potent and Efficacious Mixed Efficacy µ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Peptidomimetics with Enhanced Metabolic Stability.

We previously reported a novel SAR campaign that converted a metabolically unstable series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist bicyclic core peptidomimetics with promising analgesic activity and reduced abuse liabilities into a more stable series of benzylic core analogues. Herein, we expanded the SAR of that campaign and determined that the incorporation of amines into the benzylic pendant produces enhanced MOR-efficacy in this series, whereas the reincorporation of an aromatic ring into the pendant enhanced MOR-potency. Two compounds, which contain a piperidine (14) or an isoindoline (17) pendant, retained the desired opioid profile in vitro, possessed metabolic half-lives of greater than 1 h in mouse liver microsomes (MLMs), and were active antinociceptive agents in the acetic acid stretch assay (AASA) at subcutaneous doses of 1 mg/kg.

Structure-Activity Relationships of 7-Substituted Dimethyltyrosine-Tetrahydroisoquinoline Opioid Peptidomimetics.

The opioid receptors modulate a variety of biological functions, including pain, mood, and reward. As a result, opioid ligands are being explored as potential therapeutics for a variety of indications. Multifunctional opioid ligands, which act simultaneously at more than one type of opioid receptor, show promise for use in the treatment of addiction, pain, and other conditions. Previously, we reported the creation of bifunctional kappa opioid receptor (KOR) agonist/mu opioid receptor (MOR) partial agonist ligands from the classically delta opioid receptor (DOR) antagonist selective dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold through the addition of a 7-benzyl pendant on the tetrahydroisoquinoline ring. This study further explores the structure-activity relationships surrounding 7-position pendants on the Dmt-Tiq scaffold. Some analogues maintain a KOR agonist/MOR partial agonist profile, which is being explored in the development of a treatment for cocaine addiction. Others display a MOR agonist/DOR antagonist profile, which has potential to be used in the creation of a less addictive pain medication. Ultimately, we report the synthesis and in vitro evaluation of novel opioid ligands with a variety of multifunctional profiles.

Novel Dimethyltyrosine-Tetrahydroisoquinoline Peptidomimetics with Aromatic Tetrahydroisoquinoline Substitutions Show in Vitro Kappa and Mu Opioid Receptor Agonism.

The dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold was originally developed in the production of selective delta opioid receptor (DOR) antagonists. Installation of a 7-benzyl pendant on the tetrahydroisoquinoline core of this classic opioid scaffold introduced kappa opioid receptor (KOR) agonism. Further modification of this pendant resulted in retention of KOR agonism and the addition of mu opioid receptor (MOR) partial agonism, a bifunctional profile with potential to be used in the treatment of cocaine addiction.

Structural Simplification of a Tetrahydroquinoline-Core Peptidomimetic µ-Opioid Receptor (MOR) Agonist/δ-Opioid Receptor (DOR) Antagonist Produces Improved Metabolic Stability.

We have previously reported a series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands to serve as potential nonaddictive opioid analgesics. These ligands have been shown to be active in vivo, do not manifest withdrawal syndromes or reward behavior in conditioned-place preference assays in mice, and do not produce dependence. Although these attributes are promising, these analogues exhibit poor metabolic stability in mouse liver microsomes, likely due to the central tetrahydroquinoline scaffold in this series. As such, a structure-activity relationship (SAR) campaign was pursued to improve their metabolic stability. This resulted in a shift from our original bicyclic tetrahydroquinoline core to a monocyclic benzylic-core system. By eliminating one of the rings in this scaffold and exploring the SAR of this new core, two promising analogues were discovered. These analogues (5l and 5m) had potency and efficacy values at MOR better or comparable to morphine, retained their DOR-antagonist properties, and showed a 10-fold improvement in metabolic stability.

Dual Pharmacophores Explored via Structure-Activity Relationship (SAR) Matrix: Insights into Potent, Bifunctional Opioid Ligand Design.

Short-acting µ-opioid receptor (MOR) agonists have long been used for the treatment of severe, breakthrough pain. However, selective MOR agonists including fentanyl and morphine derivatives are limited clinically due to high risks of dependence, tolerance, and respiratory depression. We recently reported the development of a long-acting, bifunctional MOR agonist/δ-opioid receptor (DOR) antagonist analgesic devoid of tolerance or dependence in mice (AAH8, henceforth referred to as 2B). To address the need for short-acting treatments for breakthrough pain, we present a series of novel, short-acting, high-potency MOR agonist/DOR antagonist ligands with antinociceptive activity in vivo. In this study, we utilized a two-dimensional structure-activity relationship matrix to identify pharmacological trends attributable to combinations of two key pharmacophore elements within the chemotype. This work enhances our ability to modulate efficacy at MOR and DOR, accessing a variety of bifunctional profiles while maintaining high affinity and potency at both receptors.

Synthesis and Pharmacological Evaluation of Novel C-8 Substituted Tetrahydroquinolines as Balanced-Affinity Mu/Delta Opioid Ligands for the Treatment of Pain.

The use of opioids for the treatment of pain, while largely effective, is limited by detrimental side effects including analgesic tolerance, physical dependence, and euphoria, which may lead to opioid abuse. Studies have shown that compounds with a µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist profile reduce or eliminate some of these side effects including the development of tolerance and dependence. Herein we report the synthesis and pharmacological evaluation of a series of tetrahydroquinoline-based peptidomimetics with substitutions at the C-8 position. Relative to our lead peptidomimetic with no C-8 substitution, this series affords an increase in DOR affinity and provides greater balance in MOR and DOR binding affinities. Moreover, compounds with carbonyl moieties at C-8 display the desired MOR agonist/DOR antagonist profile whereas alkyl substitutions elicit modest DOR agonism. Several compounds in this series produce a robust antinociceptive effect in vivo and show antinociceptive activity for greater than 2 h after intraperitoneal administration in mice.

Multifunctional Opioid Ligands.

The opioid receptor system plays a major role in the regulation of mood, reward, and pain. The opioid receptors therefore make attractive targets for the treatment of many different conditions, including pain, depression, and addiction. However, stimulation or blockade of any one opioid receptor type often leads to on-target adverse effects that limit the clinical utility of a selective opioid agonist or antagonist. Literature precedent suggests that the opioid receptors do not act in isolation and that interactions among the opioid receptors and between the opioid receptors and other proteins may produce clinically useful targets. Multifunctional ligands have the potential to elicit desired outcomes with reduced adverse effects by allowing for the activation of specific receptor conformations and/or signaling pathways promoted as a result of receptor oligomerization or crosstalk. In this chapter, we describe several classes of multifunctional ligands that interact with at least one opioid receptor. These ligands have been designed for biochemical exploration and the treatment of a wide variety of conditions, including multiple kinds of pain, depression, anxiety, addiction, and gastrointestinal disorders. The structures, pharmacological utility, and therapeutic drawbacks of these classes of ligands are discussed.

In vivo effects of µ-opioid receptor agonist/δ-opioid receptor antagonist peptidomimetics following acute and repeated administration.

BACKGROUND AND PURPOSE: Agonists at µ-opioid receptors (µ-receptors) are used for pain management but produce adverse effects including tolerance, dependence and euphoria. The co-administration of a µ-receptor agonist with a δ-opioid receptor (δ-receptor) antagonist has been shown to produce antinociception with reduced development of some side effects. We characterized the effects of three µ-receptor agonist/δ-receptor antagonist peptidomimetics in vivo after acute and repeated administration to determine if this profile provides a viable alternative to traditional opioid analgesics. EXPERIMENTAL APPROACH: Three µ-receptor agonist / δ-receptor antagonist peptidomimetics, AAH8, AMB46 and AMB47, and morphine were evaluated for the development of tolerance and dependence after 5 days of twice daily treatment with escalating doses of drug (10-50 mg·kg-1 ). Antinociceptive effects were measured in the warm water tail withdrawal assay before and after repeated drug treatment. Physical dependence was evaluated by naltrexone-precipitated withdrawal jumping. The rewarding effects of AAH8 were evaluated using a conditioned place preference (CPP) assay with twice daily conditioning sessions performed for 5 days. KEY RESULTS: Morphine, AAH8, AMB47 and AMB46 all demonstrated acute antinociceptive effects, but repeated administration only produced tolerance in animals treated with morphine and AMB46. Injection of naltrexone precipitated fewer jumps in mice treated repeatedly with AAH8 as compared with morphine, AMB47 or AMB46. Conditioning with morphine, but not AAH8, produced significant CPP. CONCLUSIONS AND IMPLICATIONS: AAH8 may be a better alternative than traditional opioid analgesics, producing antinociception with less development of tolerance and dependence and may be less rewarding than morphine.

Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy µ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands.

N-Acetylation of the tetrahydroquinoline (THQ) core of a series of µ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands increases DOR affinity, resulting in ligands with balanced MOR and DOR affinities. We report a series of N-substituted THQ analogues that incorporate various carbonyl-containing moieties to maintain DOR affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors. 4h produced in vivo antinociception (ip) for 1 h at 10 mg/kg.

The behavioral effects of a mixed efficacy antinociceptive peptide, VRP26, following chronic administration in mice.

RATIONALE: VRP26 displays mu opioid receptor agonist and delta opioid receptor antagonist activity in vitro, a pharmacological profile purported to produce reduced tolerance, dependence, and rewarding effects. We hypothesized that VRP26 would display reduced adverse effects after chronic administration as compared with the traditional opioid analgesic fentanyl. OBJECTIVE: The aim of this study is to explore the development of tolerance, dependence, and conditioned place preference of VRP26 as compared with the traditional opioid analgesic fentanyl. METHODS: The antinociceptive effects of VRP26 and fentanyl were assessed using the mouse warm water tail withdrawal (WWTW) assay. Measurement of antinociceptive tolerance and physical dependence occurred after 7 days of continuous administration of either fentanyl (0.3 mg/kg/day) or VRP26 (10 mg/kg/day); tolerance was measured by a shift in the antinociceptive dose response curve in the WWTW assay. Physical dependence was determined by observation of withdrawal symptoms after precipitated withdrawal. Rewarding effects were measured by the ability of VRP26 or fentanyl to produce conditioned place preference. RESULTS: Fentanyl produced significant tolerance and dependence, as well as significant conditioned place preference. VRP26 produced neither tolerance nor physical dependence, nor did it produce significant conditioned place preference. CONCLUSIONS: These results suggest that chronic treatment with VRP26 may produce less tolerance or physical dependence than chronic treatment with clinically available mu opioid analgesics such as fentanyl. Additionally, VRP26 produces less rewarding effects than fentanyl. This desirable in vivo profile may be due to the mixed efficacy nature of VRP26 and could provide the framework for safer opioid analgesics.

Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy µ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

In a previously described peptidomimetic series, we reported the development of bifunctional µ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.

Asymmetric synthesis and in vitro and in vivo activity of tetrahydroquinolines featuring a diverse set of polar substitutions at the 6 position as mixed-efficacy µ opioid receptor/δ opioid receptor ligands.

We previously reported a small series of mixed-efficacy µ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist peptidomimetics featuring a tetrahydroquinoline scaffold and showed the promise of this series as effective analgesics after intraperitoneal administration in mice. We report here an expanded structure-activity relationship study of the pendant region of these compounds and focus in particular on the incorporation of heteroatoms into this side chain. These analogues provide new insight into the binding requirements for this scaffold at MOR, DOR, and the κ opioid receptor (KOR), and several of them (10j, 10k, 10m, and 10n) significantly improve upon the overall MOR agonist/DOR antagonist profile of our previous compounds. In vivo data for 10j, 10k, 10m, and 10n are also reported and show the antinociceptive potency and duration of action of compounds 10j and 10m to be comparable to those of morphine.

Development of a bioavailable µ opioid receptor (MOPr) agonist, δ opioid receptor (DOPr) antagonist peptide that evokes antinociception without development of acute tolerance.

We have previously described a cyclic tetrapeptide, 1, that displays µ opioid receptor (MOPr) agonist and δ opioid receptor (DOPr) antagonist activity, a profile associated with a reduced incidence of opioid tolerance and dependence. Like many peptides, 1 has poor bioavailability. We describe here an analogue of 1 with an added C-terminal ß-glucosylserine residue, Ser(ß-Glc)NH2, a modification that has previously been shown to improve bioavailability of opioid peptides. The resulting peptide, 4, exhibits full antinociceptive efficacy in the mouse warm water tail withdrawal assay after intraperitoneal administration with potency similar to that of morphine. Further, 4 does not give rise to acute tolerance and thus represents a promising lead for the development of opioid analgesics with reduced side effects.

Translation of structure-activity relationships from cyclic mixed efficacy opioid peptides to linear analogues.

Most opioid analgesics used in the treatment of pain are mu opioid receptor (MOR) agonists. While effective, there are significant drawbacks to opioid use, including the development of tolerance and dependence. However, the coadministration of a MOR agonist with a delta opioid receptor (DOR) antagonist slows the development of MOR-related side effects, while maintaining analgesia. We have previously reported a series of cyclic mixed efficacy MOR agonist/DOR antagonist ligands. Here we describe the transfer of key features from these cyclic analogs to linear sequences. Using the linear MOR/DOR agonist, Tyr-DThr-Gly-Phe-Leu-Ser-NH2 (DTLES), as a lead scaffold, we replaced Phe(4) with bulkier and/or constrained aromatic residues shown to confer DOR antagonism in our cyclic ligands. These replacements failed to confer DOR antagonism in the DTLES analogs, presumably because the more flexible linear ligands can adopt binding poses that will fit in the narrow binding pocket of the active conformations of both MOR and DOR. Nonetheless, the pharmacological profile observed in this series, high affinity and efficacy for MOR and DOR with selectivity relative to KOR, has also been shown to reduce the development of unwanted side effects. We further modified our lead MOR/DOR agonist with a C-terminal glucoserine to improve bioavailability. The resulting ligand displayed high efficacy and potency at both MOR and DOR and no efficacy at KOR.

Opioid peptidomimetics: leads for the design of bioavailable mixed efficacy µ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands.

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a µ opioid receptor (MOR) agonist, δ opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood-brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.

Modulation of opioid receptor ligand affinity and efficacy using active and inactive state receptor models.

Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however, chronic use results in the development of tolerance and dependence. It has been demonstrated that coadministration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side-effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH(2), and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys(5) for L-Cys(5), generated analog 13, which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure.