Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor

Abstract

Akuammine (1) and pseudoakuammigine (2) are indole alkaloids found in the seeds of the akuamma tree (Picralima nitida). Both alkaloids are weak agonists of the mu opioid receptor (μOR); however, they produce minimal effects in animal models of antinociception. To probe the interactions of 1 and 2 at the opioid receptors, we have prepared a collection of 22 semisynthetic derivatives. Evaluation of this collection at the μOR and kappa opioid receptor (κOR) revealed structural-activity relationship trends and derivatives with improved potency at the μOR. Most notably, the introduction of a phenethyl moiety to the N1 of 2 produces a 70-fold increase in potency and a 7-fold increase in selectivity for the μOR. The in vitro potency of this compound resulted in increased efficacy in the tail-flick and hot-plate assays of antinociception. The improved potency of these derivatives highlights the promise of exploring natural product scaffolds to probe the opioid receptors.

Figure 1.

The six major alkaloids (1-6) isolated from akuamma seeds.

Figure 2.

Pharmacological characterization of the akuammalogs at the μOR and κOR. Compounds 19-20 and 31-33 were screened in a full concentration-response radioligand binding assay at the μOR using [³H]DAMGO and akuammine (1) and pseudoakuammigine (2) as controls, respectively (A, C). Inhibition of forskolin-induced cAMP in a GloSensor assay in transfected HEK-293 cells at the μOR (B, D). All curves are representative of the averaged values from a minimum of three independent assays.

Figure 3.

Opioid receptor signaling properties of compound 33. The ability of 33 to inhibit cAMP at δOR (A) and κOR (B) with DPDPE and U50,488 as controls, respectively; Recruitment of β-arrestin-2 in a PathHunter assay at μOR of 33 with DAMGO as a control (C); Recruitment G_α-subtype screening of 2, 33, and DAMGO in hμOR. TRUPATH heatmaps demonstrate 33 and μOR agonists activate the G_i/o-class of transducers with varying levels of potency (D) and efficacy (E). Heatmap colors refer to mean log(EC₅₀) and normalized efficacy values. All curves are representative of the averaged values from a minimum of three independent assays.

Figure 4.

Antinociceptive effects of Compound 33. (A) Tail-flick assay. (B) Hot-plate assay, and antinociceptive effect of Compound 33 after the pretreatment with naloxone in (C) the Tail-flick assay and the (D) Hot-plate assay. (A-B). Separate groups of 3 mice received different doses of compound 33 (10, 80, 100mg/kg, subcutaneously), morphine (10mg/kg, subcutaneously), or vehicle. Time course of the antinociception response after dosing was measured. Data are expressed in mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs the control group. (C-D). Separate groups of 3 mice received naloxone (10 mg/kg, i.p.) or saline, followed by an injection of compound 33 (100 mg/kg, s.c.) or saline 15 min later. *p<0.05, **p<0.01, ***p<0.001 vs naloxone group.

Figure 5.

Effect of compound 33 on locomotor activity. Separate groups of 3 mice received different doses of compound 33 (50, 65, 80mg/kg subcutaneously). The mice were placed on an accelerating rotarod (4-40rpm over 300s) and the latency to fall was recorded. Both 65mg/kg and 80mg/kg of compound 33 significantly impaired motor activity. Data are expressed in mean ± SEM, ***p<0.001 vs the control group.

Figure 6.

Binding modes and interactions of 19 (cyan) and 33 (green) as compared to BU72 (orange) (A – B, respectively). Crystal structure of μOR co-crystallized with agonist BU-72 (PDB: 5C1M) was used as a starting point for all molecular docking simulation studies. Ligand receptor interactions are depicted as dashed lines: hydrogen bonds (magenta), salt-bridge (yellow), pi-pi stacking (blue). For clarity, ligand-receptor interactions are omitted for BU-72.

Scheme 1.

Semi-synthesis of (A) C10 analogues of 2 and (B) C11 analogues of 1. Reagents and conditions: (a) (CH₃)₂SiCHN₂, THF, MeOH, rt, 48 h (b) Ac₂O, DMAP, TEA, DCM, rt, 4 h (c) Tf₂NPh, DMAP, DCM, rt, 1 h (d) Zn(CN)₂, Pd(dppf)Cl₂, TEA, DMF, 120 °C, 24 h (e) boronic acid, K₂CO₃, Pd(PPh₃)₄, PhMe, MeOH, 80 °C, 4 – 30 h. (f) NBS, TFA, DCM, 0°C to rt, 5 h (g) NIS, TFA, DCM, 0°C to rt, 5 h. Numbers in parentheses represent isolated percent yields. All Suzuki coupling analogues (11 – 15 and 21 – 26) consist of para-substituted aryl rings except for 3-furanyl analogues 16 and 26.

Scheme 2.

Semi-synthesis of C16 akuamma alkaloid analogues. Reagents and conditions: (a) LAH, TFA, 0 °C to rt, 4 h (b) KOH, MeOH, rt, 1 h. Number in parentheses represent isolated yields.

Scheme 3.

Semi-synthesis of N1 pseudoakuammigine analogues. Reagents and conditions: (a) dimethyl acetal, TFA, TES, DCM, rt, 18 h. Number in parentheses represent isolated yields.