Design, syntheses, and pharmacological characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan analogues as opioid receptor ligands

Abstract

A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1'- and/or 4'-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6'- and/or 7'-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca(2+) increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6'-nitro group varied significantly in the different 'address' domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands.

Keywords: Antagonist; MOR; SAR; Selectivity.

Figure 1

Representative MOR antagonists used as therapeutic agents or under clinical/preclinical investigation.

Figure 2

Warm-water tail immersion assay in mice (n ≥ 6) at 56 ± 0.1 °C. All tested compounds were administered subcutaneously (s.c). (A) Antinociceptive effects of NAQ analogues 1–16. Compounds (10 mg/kg) were injected at Time 0. Twenty minutes after injection, tail flick was assessed with hot water. (B) Blockage of the antinociceptive effect of morphine by NAQ analogues 1–3, 5–16, and naltrexone (NTX). Tested compounds (1 mg/kg) were injected at Time 0. Five minutes later, morphine (10 mg/kg) were administered. Twenty minutes after morphine injection, tail flick was tested using hot water.

Figure 3

Ca²⁺ flux assays in G_qi5 transfected hMOR-CHO cells. (A) DAMGO dose-dependently increased intracellular Ca²⁺ levels. (B) Compounds 4 and 11 antagonized the intracellular Ca²⁺ increase triggered by activation of the MOR with DAMGO. The results shown are representative of at least three independent experiments.

Figure 4

Compound 11 (s.c.) in opioid-withdrawal assays in chronic morphine-exposed mice (n ≥ 6): (A) Escape jumps; (B) Wet-dog shakes. The first column in each figure represents placebo-pelleted mice while the second to the fifth represent morphine-pelleted mice. * indicates P < 0.05, compared to naltrexone (NTX, s.c).

Figure 5

Lowest interaction energy-associated NNQ poses after a 15-ns molecular dynamic simulation with three opioid receptors: (A) MOR; (B) KOR; (C) DOR. NNQ is represented by balls and sticks (green carbon atoms) and the interacting opioid receptor residues are shown as capped sticks (MOR = yellow; KOR = white; DOR = cyan). Ionic/dipole-dipole interactions and hydrogen bonds are shown with black dashed lines. Opioid receptor amino acid residues are labeled with their sequence number and Ballesteros-Weinstein index.

Scheme 1

Synthetic routes of isoquinoline-3-carboxylic acids 20, 23, and 29 with different spacer lengths.

Scheme 2

Synthetic routes of isoquinoline-3-carboxylic acids 31, 33, 34, and 36 with 4 ’ or 1’,4’-substitutions.

Scheme 3

Synthetic routes of isoquinoline-3-carboxylic acids 39, 44, and 46 with 1’-substitution.

Scheme 4

Synthetic routes of isoquinoline-3-carboxylic acids 48, 53, 55, 58, and 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 62 with 6 ’ and/or 7’-substitutions.

Scheme 5

Synthetic route of (S)-2-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride 61.

Scheme 6

Synthetic route used to generate the first generation of NAQ analogues 1–16.