Fentanyl-Type Antagonist of the µ-Opioid Receptor: Important Role of Axial Chirality in the Active Conformation.

In recent years, synthetic opioids have emerged as a predominant cause of drug-overdose-related fatalities, causing the "opioid crisis." To design safer therapeutic agents, we accidentally discovered µ-opioid receptor (MOR) antagonists based on fentanyl with a relatively uncomplicated chemical composition that potentiates structural modifications. Here, we showed the development of novel atropisomeric fentanyl analogues that exhibit more potent antagonistic activity against MOR than naloxone, a morphinan MOR antagonist. Derivatives displaying stable axial chirality were synthesized based on the amide structure of fentanyl. The aS- and aR-enantiomers exerted antagonistic and agonistic effects on the MOR, respectively, and each atropisomer interacted with the MOR by assuming a distinct binding mode through molecular docking. These findings suggest that introducing atropisomerism into fentanyl may serve as a key feature in the molecular design of future MOR antagonists to help mitigate the opioid crisis.

Searching for Synthetic Opioid Rescue Agents: Identification of a Potent Opioid Agonist with Reduced Respiratory Depression.

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 Tmax of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.

A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Synthesis and Evaluation of Antioxidant, Anti-Edematogenic and Antinociceptive Properties of Selenium-Sulfa Compounds.

Herein we describe results for the synthesis and synthetic application of 4-amino-3-(arylselenyl)benzenesulfonamides, and preliminary evaluation of antioxidant, anti-edematogenic and antinociceptive properties. This class of compounds was synthesized in good yields by a reaction of commercially available sulfanilamide and diorganyl diselenides in the presence of 10 mol% of I2 . Furthermore, the synthesized compound 4-amino-3-(phenylselenyl)benzenesulfonamide (3 a) was evaluated on complete Freund's adjuvant (CFA)-induced acute inflammatory pain. Dose- and time-response curves of antinociceptive effect of compound 3 a were performed using this experimental model. Also, the effect of compound 3 a was monitored in a hot-plate test to evaluate the acute non-inflammatory antinociception. The open-field test was performed to evaluate the locomotor and exploratory behaviors of mice. Oxidative stress markers, such as glutathione peroxidase activity; reactive species, non-protein thiols, and lipid peroxidation levels were performed to investigate the antioxidant action of compound 3 a. Our findings suggest that the antioxidant effect of compound 3 a may contribute to reducing the nociception and suppress the signaling pathways of inflammation on the local injury induced by CFA. Thus, compound 3 a reduced the paw edema as well as the hyperalgesic behavior in mice, being a promising therapeutic agent for the treatment of painful conditions.

Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists.

The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, were developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45128 as an inverse agonist. These compounds were then evaluated in vitro for their binding affinity by radioligand binding, their functional activity by 35S-GTPγS coupling, and their cAMP accumulation in cells expressing the human DOR. Both compounds demonstrated high binding affinity and selectivity at the DOR, and both displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45128). Together, these results demonstrate that we have successfully designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.

Development of Multifunctional and Orally Active Cyclic Peptide Agonists of Opioid/Neuropeptide FF Receptors that Produce Potent, Long-Lasting, and Peripherally Restricted Antinociception with Diminished Side Effects.

We previously reported that a multifunctional opioid/neuropeptide FF receptor agonist, DN-9, achieved peripherally restricted analgesia with reduced side effects. To develop stable and orally bioavailable analogues of DN-9, eight lactam-bridged cyclic analogues of DN-9 between positions 2 and 5 were designed, synthesized, and biologically evaluated. In vitro cAMP assays revealed that these analogues, except 7, were multifunctional ligands that activated opioid and neuropeptide FF receptors. Analogue 1 exhibited improved potency for κ-opioid and NPFF2 receptors. All analogues exhibited potent, long-lasting, and peripherally restricted antinociception in the tail-flick test without tolerance development after subcutaneous administration and produced oral analgesia. Oral administration of the optimized compound analogue 1 exhibited powerful, peripherally restricted antinociceptive effects in mouse models of acute, inflammatory, and neuropathic pain. Remarkably, orally administered analogue 1 had no significant side effects, such as tolerance, dependence, constipation, or respiratory depression, at effective analgesic doses.

A Novel Mitragynine Analog with Low-Efficacy Mu Opioid Receptor Agonism Displays Antinociception with Attenuated Adverse Effects.

Mitragynine and 7-hydroxymitragynine (7OH) are the major alkaloids mediating the biological actions of the psychoactive plant kratom. To investigate the structure-activity relationships of mitragynine/7OH templates, we diversified the aromatic ring of the indole at the C9, C10, and C12 positions and investigated their G-protein and arrestin signaling mediated by mu opioid receptors (MOR). Three synthesized lead C9 analogs replacing the 9-OCH3 group with phenyl (4), methyl (5), or 3'-furanyl [6 (SC13)] substituents demonstrated partial agonism with a lower efficacy than DAMGO or morphine in heterologous G-protein assays and synaptic physiology. In assays limiting MOR reserve, the G-protein efficacy of all three was comparable to buprenorphine. 6 (SC13) showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning in mice. These results suggest the possibility of activating MOR minimally (G-protein Emax ≈ 10%) in cell lines while yet attaining maximal antinociception in vivo with reduced opioid liabilities.

Discovery of an M-Substituted N-Cyclopropylmethyl-7α-phenyl-6,14-endoethanotetrahydronorthebaine as a Selective, Potent, and Orally Active κ-Opioid Receptor Agonist with an Improved Central Nervous System Safety Profile.

The search for selective kappa opioid receptor (κOR) agonists with an improved safety profile is an area of interest in opioid research. In this work, a series of m-substituted analogs were designed, synthesized, and assayed, resulting in the identification of compound 6c (SLL-1206) as a κOR agonist with single-digit nanomolar activities. The subtype selectivity of compound 6c appeared to be a consequence of an enormous decrease in the affinity for µOR and δOR, rather than a significant increase in the affinity for κOR, which was not the case for SLL-039, another selective and potent κOR agonist identified in our previous work. Besides reduced central nervous system effects, SLL-1206 exhibited substantially improved physicochemical and pharmacokinetic properties compared with SLL-039, with increases of over 20-fold in aqueous solubility and approximately 40-fold in oral bioavailability in rats.

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.

Investigating the chemical impurity profiles of fentanyl preparations and precursors to identify chemical attribution signatures for synthetic method attribution.

From an analytical chemistry standpoint, determining the chemical attribution signatures (CAS) of synthetic reaction mixtures is an impurity profiling exercise. Identifying and understanding the impurity profile and CAS of these chemical agents would allow them to be exploited for chemical forensic information, such as how a particular chemical agent was synthesised. Being able to determine the synthetic route used to make a chemical agent allows for the possibility of batches of the agent, and individual incidents using that agent, to be forensically linked. This information is of particular benefit to agencies investigating the nefarious and illicit use of chemical agents. One such chemical agent of interest to law enforcement and national security agencies is fentanyl. In this study two acylation methods for the final step of fentanyl production, herein termed the Janssen and Siegfried methods, were investigated by liquid chromatography- high resolution mass spectrometry (LC-HRMS) and multivariate statistical analysis (MVA). From these data, fifty-five chemical impurities were identified. Of these, ten were specific CAS for the Janssen method, and five for the Siegfried method. Additionally, analytical data from four different literature methods for production of the fentanyl precursor 4-anilino-N-phenethylpiperidine (ANPP), were compared to the results obtained from the method of production (Valdez) used in this study. Comparison of the LC-HRMS data for these five methods allowed for four Valdez specific impurities to be identified. These may be useful CAS for the Valdez method of ANPP production.

Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain.

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.

Finding the proverbial needle: Non-targeted screening of synthetic opioids in equine plasma.

Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked samples inserted amongst authentic plasma samples, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a sample, as well as the confidence in detections that are made.

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing ß3-Lysine.

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a ß3-amino acid, (R)- and (S)-ß3-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-ß3-Lys-Phe-Phe-Asp]NH2 (RP-171) and Tyr-c[(S)-ß3-Lys-Phe-Phe-Asp]NH2, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp147, could explain its very low MOR affinity.

Towards compound identification of synthetic opioids in nontargeted screening using machine learning techniques.

The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian process regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the samples predicted within ±0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials.

Benzimidazole Derivative Ameliorates Opioid-Mediated Tolerance during Anticancer- Induced Neuropathic Pain in Mice.

BACKGROUND: Cancer is known to be the second significant cause of death worldwide. Chemotherapeutic agents such as platinum-based compounds are frequently used single-handedly or accompanied by additional chemotherapies to treat cancer patients. Chemotherapy-induced peripheral painful neuropathy is seen in around 40% of patients who are treated with platinum-based compounds, including cisplatin. This not only decreases the quality of life of patients but also patients' compliance with cisplatin. OBJECTIVES: Nalbuphine, an opioid, is frequently used to treat acute and chronic pain, coupled with cisplatin in cancer patients. However, long term use of nalbuphine induces tolerance to its analgesic effects. We employed the same strategy to induce tolerance in mice. METHODS: Here, we investigated analgesic effects of 2-[(pyrrolidin-1-yl) methyl]-1H-benzimidazole (BNZ), a benzimidazole derivative, on nalbuphine-induced tolerance during cisplatin-induced neuropathic pain using hot plate test, tail-flick tests and von Frey filament in mouse models. Furthermore, we investigated the effects of BNZ on the expression of Tumor Necrosis Factor-alpha (TNF-α) in the spinal cord. RESULTS: The results showed that BNZ reduced tolerance to analgesic effects of nalbuphine and TNF-α expression in mice. CONCLUSION: BNZ could be a potential drug candidate for the management of nalbuphine-induced tolerance in cisplatin-induced neuropathic pain.

The impact and mechanism of ampakine CX1739 on protection against respiratory depression in rats.

Background: Abuse of analgesic and sedative drugs often leads to severe respiratory depression and sometimes death. Approximately 69,000 people worldwide die annually from opioid overdoses. Purpose: This work aimed to investigate whether CX1739 can be used for emergency treatment of acute respiratory depression due to drug abuse. Results: First, the results clarify that CX1739 is a low-impact ampakine that can safely activate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors without causing excito-neurotoxicity. Second, CX1739 rapidly crossed the blood-brain barrier (Tmax = 2 min), which meets the requirement of rapid onset of action in vivo. Our work provides preliminarily confirmation that high-dose intravenous administration of CX1739 can immediately reverse respiratory depression in animal models of respiratory depression caused by opioid agonist 030418, pentobarbital sodium and ethanol.

Phenylalanine Stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 Exhibit Distinctly Different Opioid Activity Profiles.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues' stereochemistry on the peptides' opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Editorial: 50 Years of Opioid Research and the International Narcotics Research Conference.

In the past 50 years, scientists have made considerable strides toward understanding how opioids act. This special issue of Molecular Pharmacology celebrates these 50 years of opioid research and the role that the International Narcotics Research Conference has played in driving this research, by bringing together review and original research articles that present historical highlights, the current state of the art, and perspectives on the future of opioid research. SIGNIFICANCE STATEMENT: Opioids have been used for thousands of years to manage pain and cause euphoria, but their use has been highly limited due to serious side effects. Deciphering the mechanisms of how opioids mediate beneficial and adverse physiological outcomes is essential for developing better treatments for pain and for opioid addiction.

Determination of 37 fentanyl analogues and novel synthetic opioids in hair by UHPLC-MS/MS and its application to authentic cases.

The recent emergence of new fentanyl analogues and synthetic opioids on the drug market poses a global public health threat. However, these compounds cannot typically be identified using existing analytical methods. In this study, we aimed to develop and validate a rapid and sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 37 fentanyl analogues and novel synthetic opioids in hair samples. Hair samples (20 mg) were extracted by cryogenic grinding in an extraction medium of methanol, acetonitrile, and 2 mmol/L ammonium acetate (pH 5.3). Following centrifugation of the samples, the analytes were separated using a WATERS Acquity UPLC HSS T3 column. The limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.5 to 2.5 pg/mg and from 2 to 5 pg/mg, respectively. The intraday and interday precisions were within 13.32% at LOQ, low, medium, and high levels. The accuracies were within the range of 85.63-116.1%. The extraction recoveries were in the range of 89.42-119.68%, and the matrix effects were within the range of 44.81-119.77%. Furthermore, the method was successfully applied to the detection and quantification of fentanyl and sufentanil in hair samples from two authentic cases. Thus, this method has great potential for detecting fentanyl analogues and novel synthetic opioids in forensic work.

Synthesis and pharmacological evaluation of novel cis and trans 3-substituted anilidopiperidines.

BACKGROUND: 4-Anilidopiperidine class of synthetic opioid analgesics, with it's representative fentanyl, are by far the most potent and clinically significant for the treatment of the severe chronic and surgical pain. However, side effects of µ-opioids are often quite serious. In order to improve the pharmacological profile of this class of opioid analgesics, a novel fentanyl analogs were designed, synthesized and evaluated in vivo for their antinociceptive activity. METHODS: The title compounds were prepared using known synthetic transformations, including N-bromoacetamide mediated Hofmann rearrangement, highly selective carbamate cleavage with trimethylsilyl iodide and dehydration of carboxamide group to nitrile in the presence of SOCl2. The antinociceptive activity of the synthesized compounds was determined by tail-immersion and formalin test. RESULTS: The scalable synthetic route towards novel fentanyl analogs bearing nitrogen groups in position C3 of piperidine ring is designed. In addition, Hofmann rearrangement was substantially improved for the more efficient synthesis of previously published 3-substituted fentanyl analogs. The series of ten fentanyl analogs was tested in vivo for their antinociceptive activity. The most potent compound of the series was found to be cis-4, based on the determined ED50 values in tail-immersion test. CONCLUSION: Of ten compounds tested for their antinociceptive activity, compound cis-4 is characterized by high potency, rapid beginning and short duration of action and due to this might be incorporated in different pharmaceutical forms.