Discovery and Pharmacological Evaluation of a Diphenethylamine Derivative (HS665), a Highly Potent and Selective κ Opioid Receptor Agonist

Here we report on the design, synthesis, and biological characterization of novel κ opioid (KOP) receptor ligands of diphenethylamines. In opioid receptor binding and functional assays, the <i>N</i>-cyclobutylmethyl substituted derivative <b>4</b> (HS665) showed the highest affinity and selectivity for the KOP receptor and KOP agonist potency. Compound <b>4</b> inhibited acetic acid induced writhing after subcutaneous administration in mice via KOP receptor-mediated mechanisms, being equipotent as an analgesic to the KOP agonist U50,488

<i>In vitro</i> agonist potency and efficacy.

a<p>Membranes from CHO cells stably transfected with human MOP, DOP or KOP receptors were used.</p>b<p>CHO cells co-expressing chimeric G proteins and recombinant human MOP, DOP or KOP receptors.</p>c<p>E_max is expressed in percentage relative to maximal stimulation produced by DAMGO (MOP), DPDPE (DOP) or U69,593 (KOP).</p>d<p>Inactive up to 10 µM.</p>e<p>crc, concentration response curve.</p>f<p>ND, not determined due to very low binding affinity at the KOP receptor.</p><p>Values represent the mean ± SEM of at least three experiments each performed in duplicate or triplicate.</p

Structures of morphine, oxymorphone and N-substituted morphinans 1–6.

<p>Ph, phenyl.</p

Highly Potent and Selective New Diphenethylamines Interacting with the κ‑Opioid Receptor: Synthesis, Pharmacology, and Structure–Activity Relationships

We previously reported on a series of small molecules targeting the κ-opioid (KOP) receptor featuring a diphenethylamine scaffold and showed the promise of these ligands as effective analgesics with reduced liability for adverse effects. This study expands the structure–activity relationships on our original series by presenting several modifications in the lead compounds <b>1</b> (HS665) and <b>2</b> (HS666). A library of new diphenethylamines was designed, synthesized, and pharmacologically evaluated. In comparison with <b>1</b> and <b>2</b>, the KOP receptor affinity, selectivity, and agonist activity were modulated by introducing bulkier N-substituents, a 2-fluoro substitution, and additional hydroxyl groups at positions 3′ and 4′. Several analogues showed subnanomolar affinity and excellent KOP receptor selectivity acting as full or partial agonists, and one as an antagonist. The new diphenethylamines displayed antinociceptive efficacies with increased potencies than U50,488, <b>1</b> and <b>2</b> in the writhing assay and without inducing motor dysfunction after sc administration in mice

Dose-dependent antinociceptive effects produced by morphine, oxymorphone and <i>N</i>-methylmorphinans 1, 4 and 6.

<p>(A) Hot-plate test. (B) Tail-flick test. Hot-plate and tail-flick latencies (as %MPE) are shown at 30 min (peak of action) after s.c. drug administration to mice. Data are shown as the mean ± SEM (<i>n</i> = 5–6 mice per group).</p

Antinociceptive activities.

a<p>Antinociceptive potencies determined 30 min after s.c. drug administration in mice shown as ED₅₀ values with 95% confidence limits (95% CL) (<i>n</i> = 5–6 mice per group).</p

Opioid receptor binding affinities and selectivities at MOP, DOP and KOP receptors.

<p>Binding assays were performed with membranes from rat brain (MOP and DOP receptors) and guinea pig brain (KOP receptors).</p><p>Values represent the mean ± SEM of at least three experiments each performed in duplicate.</p

Time-course of antinociceptive effects produced by morphine, oxymorphone and <i>N</i>-methylmorphinans 1, 4 and 6.

<p>The effect of morphine (1.25–5 mg/kg), oxymorphone (0.2–1 mg/kg), and compounds <b>1</b> (0.05–0.5 mg/kg), <b>4</b> (0.5–5 mg/kg), and <b>6</b> (0.1–0.5 mg/kg) in the hot-plate test (A, left panel) and in the tail-flick test (B, right panel). Hot-plate and tail-flick latencies (in seconds) were determined in mice before (0 min) and after s.c. drug administration (30, 60 and 120 min). Data are shown as the mean ± SEM (<i>n</i> = 5–6 mice per group).</p

<i>In vitro</i> agonist activities at the MOP receptor of morphine, oxymorphone and <i>N</i>-methylmorphinans 1, 4 and 6.

<p>Concentration-response curves in (A) [³⁵S]GTPγS functional assay with membranes from CHO expressing human MOP receptor and (B) calcium mobilization experiments performed with CHO cells co-expressing the human MOP receptor and the Gα_qi5 protein. Activity is calculated as percentage of maximal stimulation produced by DAMGO. Data are shown as the mean ± SEM (<i>n</i>≥3).</p

Synthesis, Pharmacology, and Molecular Docking Studies on 6‑Desoxo‑<i>N</i>‑methylmorphinans as Potent μ‑Opioid Receptor Agonists

Position 6 of the morphinan skeleton plays a key role in the μ-opioid receptor (MOR) activity in vitro and in vivo. We describe the consequence of the 6-carbonyl group deletion in <i>N</i>-methylmorphinan-6-ones <b>1</b>–<b>4</b> on ligand–MOR interaction, signaling, and antinociception. While 6-desoxo compounds <b>1a</b>, <b>2a</b>, and <b>4a</b> show similar profiles to their 6-keto counterparts, the 6-desoxo-14-benzyloxy substituted <b>3a</b> displays significantly increased MOR binding and agonist potency and a distinct binding mode compared with its analogue <b>3</b>