Synthesis, Pharmacology, and Molecular Docking Studies on 6-Desoxo-N-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 N-methylmorphinan-6-ones 1–4 on ligand–MOR interaction, signaling, and antinociception. While 6-desoxo compounds 1a, 2a, and 4a show similar profiles to their 6-keto counterparts, the 6-desoxo-14-benzyloxy substituted 3a displays significantly increased MOR binding and agonist potency and a distinct binding mode compared with its analogue 3.


Hot-plate assay
The hot-plate assay was performed as described. 5 Each mouse was placed on a UB 35100 hot/cold plate (Ugo Basile s.r.l., Varese, Italy) kept at 55C, and the occurrence of a nociceptive response (licking or shaking a paw, jumping) was observed. To confine the mice to a certain observation area, a colourless plastic cylinder of 20 cm diameter was placed on the hot plate. In order to avoid possible tissue injury, a cut-off time of 12 s was used. Hot-plate latencies were measured before (basal latency, BL) and 30, 60 and 120 min after drug or vehicle sc administration (test latency, TL). For establishing the dose-response effect, the antinociceptive response was expressed as percent of x 100 for each dose tested. The dose necessary to produce a 50% MPE (ED50) and 95% confidence limits (95% CL) were calculated using the method of Litchfield and Wilcoxon. 6

Data Analysis
Experimental data were analyzed and graphically processed using the GraphPad Prism 5.0 Software (GraphPad Prism Software Inc., San Diego, CA), and are presented as means ± SEM. Data were statistically evaluated using one-way ANOVA with Tukey's post hoc test or two-way ANOVA for multiple comparisons, with significance set at P < 0.05.

Hardware and software specifications
Molecular modeling study was performed utilizing a Fujitsu CELSIUS R940 workstation, equipped with an Intel Xeon E5-2620 v3 CPU and 16 GB of RAM, and running Microsoft Windows 8.1 operating system. For the docking study, preparation of ligands was conducted using LigandScout 7 (version 3.1) from Inte:Ligand (http://www.inteligand.com), and OpenEye's conformer ensemble generator OMEGA. 8 For the assignment of partial atomic charges the toolkit QUACPAC 9 (version 1.7.0.2) from OpenEye (http://www.eyesopen.com) was employed. Molecules were docked employing GOLD 10,11 (version 5.2) from the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/solutions/csd-discovery/Components/Gold/). Evaluation of docking solutions was performed within LigandScout, which was also used for visualization purposes.

Ligand preparation
First, the ligand preparation was performed with the import of the smiles codes of compounds into LigandScout, followed by checking the protonation states, along with the strain energy, as the molecules were submitted to further processing, ensuing that the three-dimensional (3D) geometry was relaxed. Next, conformational models were calculated within LigandScout (RMS distance: 0.1; maximal number of conformers: 5). Furthermore, partial atomic charges were assigned from the MMFF94, 12 by running OpenEye's toolkit QUACPAC in default mode.

Molecular docking
Molecular docking of compounds to the active structure of the MOR was conducted as recently reported, 13 with some modifications as described. The X-ray crystal structure of the active MOR conformation was utilized (PDB accession code: 5C1M), 14 accessible via the web portal of the Protein Data Bank 15 (http://www.rcsb.org/pdb/). The amino acid residue D147 was assigned as constraint, as its relevance for the recognition of small drug-like molecules by the receptor was stressed. 16,17 Three water molecules were included during the docking runs, i.e. HOH505, HOH526 and HOH538, accounting the results reported by Huang et al, 14 which suggest a hydrogen-bonding network. Specifically, these water molecules are involved by mediating a polar contact from the receptor to the co-crystallized ligand, the morphinan BU72. 14 Prior to docking, the preparation of the valuable and substantial 3D structure of MOR was performed within GOLD. Furthermore, the implemented consensus scoring protocol "Chemscore-GS" was employed in this study, following that the settings of the genetic algorithm (GA), a cornerstone of the docking program GOLD, were assigned to a considerably exhaustive variant (GA runs: 20; GA efficiency: 200%), as outlined elsewhere. 18 In addition, the docking runs were performed with up to five conformers per ligand, and S7 by employing adjusted settings for the ligand flexibility, as enhanced flexibility was enabled ("flip pyramidal N", along with "flip ring corners"). In total, five independent runs were conducted, and the three top-ranked docking solutions per molecule and conformer from each of the five runs were collected.

Evaluation
Critical non-covalent interactions between the compounds and the MOR were surmised by inferring 3D pharmacophores or pharmacophore models within LigandScout, as interesting variant to derive key findings from the retrieved poses. 7