Design, Synthesis, and In Vitro Characterization of Proteolytically-Stable Opioid-Neurotensin Hybrid Peptidomimetics

Abstract

Linking an opioid to a nonopioid pharmacophore represents a promising approach for reducing opioid-induced side effects during pain management. Herein, we describe the optimization of the previously reported opioid-neurotensin hybrids (OPNT-hybrids), SBL-OPNT-05 & -10, containing the μ-/δ-opioid agonist H-Dmt-d-Arg-Aba-β-Ala-NH₂ and NT(8-13) analogs optimized for NTS2 affinity. In the present work, the constrained dipeptide Aba-β-Ala was modified to investigate the optimal linker length between the two pharmacophores, as well as the effect of expanding the aromatic moiety within constrained dipeptide analogs, via the inclusion of a naphthyl moiety. Additionally, the N-terminal Arg residue of the NT(8-13) pharmacophore was substituted with β³ hArg. For all analogs, affinity was determined at the MOP, DOP, NTS1, and NTS2 receptors. Several of the hybrid ligands showed a subnanomolar affinity for MOP, improved binding for DOP compared to SBL-OPNT-05 & -10, as well as an excellent NTS2-affinity with high selectivity over NTS1. Subsequently, the G_αi1 and β-arrestin-2 pathways were evaluated for all hybrids, along with their stability in rat plasma. Upon MOP activation, SBL-OPNT-13 and -18 were the least effective at recruiting β-arrestin-2 (E _max = 17 and 12%, respectively), while both compounds were also found to be partial agonists at the G_αi1 pathway, despite improved potency compared to DAMGO. Importantly, these analogs also showed a half-life in rat plasma in excess of 48 h, making them valuable tools for future in vivo investigations.

Figure 1

Structures of the two lead hybrids SBL-OPNT-05 and SBL-OPNT-10 (including in vitro pharmacological data) with the opioid pharmacophore framed in orange and the NT pharmacophore framed in blue.

Scheme 1. Synthesis of Boc-Dmt-OH

(a) SOCl₂ (1.1 equiv), MeOH (anhydrous, 1.0 M), reflux, o.n., quant.; (b) TBDMSCl (1.2 equiv), imidazole (1.2 equiv), CH₂Cl₂ (anhydrous, 0.2 M), r.t., o.n., 97%; (c) picolinic acid (1.2 equiv), TBTU (1.2 equiv), DIPEA (2.5 equiv), CH₂Cl₂ (anhydrous, 0.2 M), r.t., o.n., 73%; (d) MeI (5.0 equiv), K₂CO₃ (3.0 equiv), Pd(OAc)₂ (5 mol %), toluene (0.25 M), 120 °C, 48 h; 69%; (e) HCl_conc (17.0 equiv), H₂O (0.7 M), 0 °C, 90 °C, 24 h; (f) Boc₂O (1.1 equiv), Et₃N (1.1 equiv), THF, r.t., o.n., 54% (over two steps).

Scheme 2. Synthesis of Fmoc-Aba-Gly-OH and Fmoc-1Ana-Gly-OH

(a) Methyl 2-(succinimidooxy)carbonyl-benzoate (1.1 equiv), Na₂CO₃ (3.0 equiv), MeCN/H₂O (3:2 v/v, 0.05 M), r.t., o.n., 90 and 96%; (b) HCl H-Gly-OEt (1.1 equiv), TBTU (1.1 equiv), Et₃N (3.0 equiv), CH₂Cl₂ (anhydrous, 0.2 M), r.t., 1 h 30 min, 94 & 65%; (c) 1 N HCl/acetone (1:1 v/v, 0.05 M), 90 °C, 4h30, quant. and 92%; (d) paraformaldehyde (15.0 equiv), pTosOH (0.1 equiv), toluene (0.05 M), reflux, 1 h 30 min, 93 and 73%; (e) triflic acid (5.0 equiv), CH₂Cl₂ (anhydrous, 0.1 M), r.t., 1 h, argon, quant. and quant.; (f) hydrazine (6.6 equiv), EtOH (0.1 M), reflux, 30 min; (g) FmocOSu (1.1 equiv), Na₂CO₃ (1.2 equiv), H₂O/acetone (1:1 v/v, 0.1 M), r.t., o.n., 64 and 48% (over two steps).

Scheme 3. Synthesis of Fmoc-Aba-β-Ala-OH (n = 1), Fmoc-1Ana-β-Ala-OH (n = 1), Fmoc-Aba-GABA–OH (n = 2) & Fmoc-1Ana-GABA–OH (n = 2)

(a) Methyl 2-(succinimidooxy)carbonyl-benzoate (1.1 equiv), Na₂CO₃ (3.0 equiv), MeCN/H₂O (3:2 v/v, 0.05 M), r.t., o.n., 90 and 96%; (b) HCl H-β-Ala-OEt or HCl H-GABA-OEt (1.1 equiv), TBTU (1.1 equiv), Et₃N (3.0 equiv), CH₂Cl₂ (anhydrous, 0.2 M), r.t., 1 h 30 min, quant. & 74% (for Phe), 65 and 94% (for 1-Nal); (c) 1,3,5-trioxane (6.6 equiv), P₂O₅ (22.2 equiv), H₃PO₄ (85% aq., 10.7 equiv), benzene/AcOH (3:2 v/v, 0.025 M), 115 °C, 1 h 30 min, 63 and 79% (for Phe), 82 and 65% (for 1-Nal); (d) 1 N HCl/acetone (1:1 v/v, 0.05 M), 90 °C, o.n., 87 & 91% (for Phe), quant. and 75% (for 1-Nal); (e) hydrazine (6.6 equiv), EtOH (0.1 M), reflux, 30 min; (f) FmocOSu (1.1 equiv), Na₂CO₃ (1.2 equiv), H₂O/acetone (1:1 v/v, 0.1 M), r.t., o.n., 37 and 59% (for Phe), 54 and 76% (for 1-Nal) (over two steps).

Figure 2

Concentration–response curves for hybrid ligands on the G_αi1, and β-arrestin-2 pathways following MOP activation. Schematic representation of the BRET-based assay used to assess G-protein activation showing loss of BRET² signal upon activation (A) or recruitment of β-arrestin-2 illustrating a gain in BRET² signal after receptor stimulation (B). Ligand-triggered G_αi1 engagement on MOP (C). Ligand-induced β-arrestin-2 recruitment to MOP (D). Each set represents the mean of at least three independent experiments and is expressed as mean ± SEM.

Figure 3

Concentration–response curves for hybrid ligands on the G_αi1, and β-arrestin-2 pathways following DOP activation. DOP (A) is monitored using the BRET-based G protein dissociation assay or DOP (B) using the BRET-based β-arrestin-2 recruitment assay. Each set represents the mean of at least three independent experiments and is expressed as mean ± SEM.

Figure 4

Concentration–response curves for hybrid ligands on the G_αi1 pathways following KOP activation. Ligand-triggered G_αi1 engagement at KOP was evaluated using BRET-based biosensors. Each set represents the mean of at least three independent experiments and is expressed as mean ± SEM.

Figure 5

Plasma stability of hybrid ligands and their corresponding NT moiety compared to NT(8–13). Degradation curves were generated following incubation of the OPNT hybrids in rat plasma at 37 °C for up to 15 min (A) or 48 h (B). Each series represents the mean of three independent experiments and is expressed as mean ± SEM.