Nitro-benzylideneoxymorphone, a bifunctional mu and delta opioid receptor ligand with high mu opioid receptor efficacy

Abstract

Introduction: There is a major societal need for analgesics with less tolerance, dependence, and abuse liability. Preclinical rodent studies suggest that bifunctional ligands with both mu (MOPr) and delta (DOPr) opioid peptide receptor activity may produce analgesia with reduced tolerance and other side effects. This study explores the structure-activity relationships (SAR) of our previously reported MOPr/DOPr lead, benzylideneoxymorphone (BOM) with C7-methylene-substituted analogs. Methods: Analogs were synthesized and tested in vitro for opioid receptor binding and efficacy. One compound, nitro-BOM (NBOM, 12) was evaluated for antinociceptive effects in the warm water tail withdrawal assay in C57BL/6 mice. Acute and chronic antinociception was determined, as was toxicologic effects on chronic administration. Molecular modeling experiments were performed using the Site Identification by Ligand Competitive Saturation (SILCS) method. Results: NBOM was found to be a potent MOPr agonist/DOPr partial agonist that produces high-efficacy antinociception. Antinociceptive tolerance was observed, as was weight loss; this toxicity was only observed with NBOM and not with BOM. Modeling supports the hypothesis that the increased MOPr efficacy of NBOM is due to the substituted benzylidene ring occupying a nonpolar region within the MOPr agonist state. Discussion: Though antinociceptive tolerance and non-specific toxicity was observed on repeated administration, NBOM provides an important new tool for understanding MOPr/DOPr pharmacology.

Keywords: SILCS; bifunctional analgesics; delta opioid receptor; dependence; mu opioid receptor; tolerance.

FIGURE 1

Opioid Analgesics and Bifunctional Lead Scaffolds. Currently available MOPr agonists (morphine, 1; oxymorphone, 2; oxycodone, 3; fentanyl, 4) and MOPr/DOPr-targeting bifunctional leads (7-E-benzylideneoxymorphone, BOM, 5; 7-E-benzylidenenaltrexone, BNTX, 6.

SCHEME 1

Reagents and conditions: (a) RCHO (6 equiv.), piperidine (2 equiv.), MeOH or EtOH, 120°C, 24 h (sealed tube). (b) RCHO (6 equiv.), piperidine (2 equiv.), EtOH, 160°C, 1 h (MW).

FIGURE 2

12 produces higher antinociceptive activity than the parent 5 in the WWTW assay in C57BL/6 mice. (A) Procedure for warm water tail withdrawal mouse tail-flick antinociceptive assay. Vehicle or drug was injected i. p. into naïve C57BL/6 mice followed by testing for antinociception—via measuring the tail withdrawal latency time—using 50°C warm water. (B) Dose-response curves for antinociception of 12, the parent compound 5, the standard MOPr agonist morphine, and vehicle. 12 is approximately 3-fold less potent than morphine in the WWTW antinociception assay. (C) Time course of antinociception following i. p. Administration of 10 mg/kg morphine—a standard MOPr agonist—and 32 mg/kg 12 both producing near-maximal latency. A one-way ANOVA to compare each dose of 5, 12, or morphine treatment to vehicle showed a significant effect of the drug dose on tail withdrawal latency [F (13,60) = 18.9; p < 0.001]. Post hoc comparisons using the Dunnett’s multiple comparisons test indicated that 10 mg/kg morphine, and 3.2 mg/kg, 10 mg/kg, and 32 mg/kg 12 significantly increased %MPE tail withdrawal latency. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

FIGURE 3

12 produces antinociception and antinociceptive tolerance mediated by MOPr in the WWTW assay. (A) Chronic administration paradigm for the antinociceptive tolerance experiment using the 50°C WWTW assay. Vehicle or drug was injected i. p. into naïve C57BL/L6 mice 3 times daily for up to 5 days followed by once-daily antinociception testing. (B) 12 in the presence of saline produces similar antinociceptive tolerance to morphine after chronic administration with both producing minimal antinociception by Day 3. (C) Mice treated with 12 lost significantly more weight than mice treated with morphine. (D and E) Pretreatment of C57BL/6 WT mice with 10 mg/kg naloxone before administration with 32 mg/kg 12 blocked antinociception (D) but not weight loss (E). (F and G) Treatment of C57BL/6 MOPr KO mice with 32 mg/kg 12 blocked antinociception (F), but did not alter weight loss induced by 12 (G). Insets: Arrow indicates activation; the bar indicates inhibition. A two-way ANOVA showed significant differences between days [F (5, 115) = 21.05, p < 0.0001] and drug treatment [F (4, 23) = 98.94, p < 0.0001]. Statistically significant differences in the follow-up post hoc Tukey analysis are indicated between days with *s and brackets in the drug color. Antinociception within drug groups is compared to baseline (BL) for drugs with * in the drug color. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

FIGURE 4

SILCS FragMaps overlaid on the (A and B) active and (C and D) inactive crystal structures of MOPr. The protein backbone is shown in a transparent gray cartoon with selected sidechains shown in atom colored CPK format. FragMap Color Code: Benzene (purple), Apolar (green), positive (cyan) at GFE energy contours of −1.2 kcal/mol.

FIGURE 5

(A) SILCS-MC poses of compounds 12 and 14 in the active receptor pocket of MOPr. Amino acid side chains are represented in atom colored CPK and ligands in licorice representation. (B) SILCS-MC poses of 14 and 10 in the active receptor pocket. Curved blue arrow represents a displaced orientation of the 4,5-epoxymorphinan core (BOM core) of 10 from the apolar site (A,C,D and E) Compounds 14, 12 and 10 represented in licorice representation with atom based GFE (blue letters—favorable GFE, red letters—GFE based penalties). FragMap Color Code: Benzene (purple), propane (green), methylammonium N positive (cyan), generic hydrogen-bond acceptor (red), generic hydrogen-bond donor (blue), methanol oxygen (olive green), and acetate O negative (orange) at GFE energy contours of −1.2 kcal/mol.