Caged Naloxone: Synthesis, Characterization, and Stability of 3‑<i>O</i>‑(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX)

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3-<i>O</i>-(4,5-dimethoxy-2-nitrophenyl)­carboxymethyl naloxone (also referred to as “caged naloxone”, 3-<i>O</i>-(α-carboxy-6-nitroveratryl)­naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of α<i>R</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> and α<i>S</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> diastereomers. Using long-range and heteronuclear NMR correlations, the ¹H NMR and ¹³C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products

The Importance of Hydrogen Bonding and Aromatic Stacking to the Affinity and Efficacy of Cannabinoid Receptor CB₂ Antagonist, 5‑​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)methyl]‑​<i>N</i>‑​[(1<i>S</i>,​2<i>S</i>,​4<i>R</i>)‑​1,​3,​3-​trimethylbicyclo[2.2.1]hept-​2-​yl]-​1H-​pyrazole-​3-​carboxamide (SR144528)

Despite the therapeutic promise of the subnanomolar affinity cannabinoid CB₂ antagonist, 5-​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)­methyl]-<i>​N</i>-​[(1<i>S</i>,​2<i>S</i>,​4<i>R</i>)-​1,​3,​3-​trimethylbicyclo­[2.2.1]­hept-​2-​yl]-​1<i>H</i>-​pyrazole-​3-​carboxamide (SR144528, <b>1</b>), little is known about its binding site interactions and no primary interaction site for <b>1</b> at CB2 has been identified. We report here the results of Glide docking studies in our cannabinoid CB₂ inactive state model that were then tested via compound synthesis, binding, and functional assays. Our results show that the amide functional group of <b>1</b> is critical to its CB2 affinity and efficacy and that aromatic stacking interactions in the TMH5/6 aromatic cluster of CB2 are also important. Molecular modifications that increased the positive electrostatic potential in the region between the fenchyl and aromatic rings led to more efficacious compounds. This result is consistent with the EC-3 loop negatively charged amino acid, D275 (identified via Glide docking studies) acting as the primary interaction site for <b>1</b> and its analogues

Peripherally Selective Cannabinoid 1 Receptor (CB1R) Agonists for the Treatment of Neuropathic Pain

Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with <1% blood–brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (<b>1</b>) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ∼0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene’s antiallodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain

Novel Synthesis and Pharmacological Characterization of NOP Receptor Agonist 8-[(1<i>S</i>,3a<i>S</i>)-2,3,3a,4,5,6-Hexahydro-1<i>H</i>-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198)

The nociceptin/orphanin FQ opioid peptide (NOP) receptor is a widely expressed GPCR involved in the modulation of pain, anxiety, and motor behaviors. Dissecting the functional properties of this receptor is limited by the lack of systemically active ligands that are brain permeant. The small molecule NOP receptor-selective, full agonist 8-[(1<i>S</i>,3a<i>S</i>)-2,3,3a,4,5,6-hexahydro-1<i>H</i>-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198) hydrochloride is an active, brain penetrant ligand, but its difficult and cost-prohibitive synthesis limits its widespread use and availability for animal studies. Here, we detail a more efficient and convenient method of synthesis, and use both in vitro and in vivo pharmacological assays to fully characterize this ligand. Specifically, we characterize the pharmacodynamics of Ro 64-6198 in cAMP and G-protein coupling in vitro and examine, for the first time, the effects of nociceptin/orphanin FQ and Ro 64-6198 in arrestin recruitment assays. Further, we examine the effects of Ro 64-6198 on analgesia, anxiety, and locomotor responses in vivo. This new synthesis and pharmacological characterization provide additional insights into the useful, systemically active, NOP receptor agonist Ro 64-6198

Identification of the GPR55 Antagonist Binding Site Using a Novel Set of High-Potency GPR55 Selective Ligands

GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ∼300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC₅₀ values in the range of 0.16–2.72 μM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists