Exploring substitution effects on the potential dominant conformations of NBF derivatives leading to functional conversion at the mu opioid receptor

Abstract

We previously identified NBF (β-configuration at C6) and its 6α-counterpart as mu opioid receptor (MOR) antagonists. To explore the effect of C6 conformation of the epoxymorphinan ring on their MOR function, five pairs of NBF derivatives bearing both 6α and 6β configurations with substitutions on the 3'-position of the benzofuran ring were synthesized. In vitro and in vivo studies demonstrated that compounds carrying phenyl and 4-pyridine substituents retained their antagonistic properties independent of the C6 configuration. Halogen and methyl substituents with the 6α-configuration remained as MOR antagonists, while their 6β-counterparts switched to MOR agonists. Molecular modeling studies indicated that the C6 configuration and structural modification may collectively decide the orientation of the benzofuran ring, leading to conformation retention or a switch within the MOR binding pocket. These results together aid the understanding of the NBF structure-activity relationship (SAR) and provide insights for functional conversion at the MOR, supporting future endeavors to develop novel MOR ligands.

Fig. 1. Chemical structures of MOR-targeted agonist INTA, antagonist NAN, partial agonist NBF and its 6α-counterpart (compound 1).

Scheme 1. Synthetic route for target compounds.

Fig. 2. Warm-water tail immersion assay results of NBF-derivatives as agonists at a single dose of 10 mg kg⁻¹ (s.c.). Vehicle and morphine were used as the negative and positive controls, respectively. Data are presented as mean values ± SD. ****p < 0.0001, compared to vehicle (s.c.).

Fig. 3. Warm-water tail immersion assay results of NBF-derivatives as antagonists at a single dose of 10 mg kg⁻¹ (s.c.) in the presence of morphine (10 mg kg⁻¹, s.c.). Vehicle and morphine were used as the negative and positive controls, respectively. Data are presented as mean values ± SD. *p < 0.01, ****p < 0.0001, compared to vehicle + morphine (s.c.).

Fig. 4. Binding mode of (A) compounds 2, 3, and 4 with the inactive MOR receptor; (B) compounds 5 and 6 with the inactive MOR receptor; (C) compounds 8, 9, and 10 with the active MOR receptor; and (D) compounds 11 and 12 with the inactive MOR receptor (PDB; 4DKL). The MOR is shown as grey cartoons. NBF derivatives and key amino acid residues are shown on the sticks. Carbon atoms: 2, 8, (magenta); 3, 9, (cyan); 4, 10 (blue); 5, 11 (green); 6, 12 (orange); key amino acid residues (yellow); oxygen atoms (red); and nitrogen atoms (blue).

Fig. 5. Binding mode of (A) compound 3 and the inactive MOR; (B) compound 9 and the active MOR with key residues in the binding pocket after MD simulation. The MOR is shown as grey and cyan cartoons. Compound 3, compound 9 and key amino acid residues are shown on the sticks. Carbon atoms: compound 3 (magenta), 9 (yellow); key amino acid residues (green and salmon); oxygen atoms (red); and nitrogen atoms (blue).