BACKGROUND AND PURPOSENeuropeptide FF (NPFF) behaves as an endogenous opioid-modulating peptide. In the present study, the opioid and NPFF pharmacophore-containing chimeric peptide BN-9 was synthesized and pharmacologically characterized. EXPERIMENTAL APPROACHAgonist activities of BN-9 at opioid and NPFF receptors were characterized in in vitro cAMP assays. Antinociceptive activities of BN-9 were evaluated in the mouse tail-flick and formalin tests. Furthermore, its side effects were investigated in rotarod, antinociceptive tolerance, reward and gastrointestinal transit tests. KEY RESULTSBN-9 acted as a novel multifunctional agonist at μ, δ, κ, NPFF1 and NPFF2 receptors in cAMP assays. In the tail-flick test, BN-9 produced dose-related antinociception and was approximately equipotent to morphine; this antinociception was blocked by μ and κ receptor antagonists, but not by the δ receptor antagonist. In the formalin test, supraspinal administration of BN-9 produced significant analgesia. Notably, repeated administration of BN-9 produced analgesia without loss of potency over 8 days. In contrast, repeated i.c.v. co-administration of BN-9 with the NPFF receptor antagonist RF9 produced significant antinociceptive tolerance. Furthermore, i.c.v. BN-9 induced conditioned place preference. When given by the same routes, BN-9 had a more than eightfold higher ED 50 value for gastrointestinal transit inhibition compared with the ED 50 values for antinociception. CONCLUSIONS AND IMPLICATIONSBN-9 produced a robust, nontolerance-forming analgesia with limited inhibition of gastrointestinal transit. As BN-9 is able to activate both opioid and NPFF systems, this provides an interesting approach for the development of novel analgesics with minimal side effects.

A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural α-methylene-β-amino acids (Map) at position 3 or/and position 4. Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the μ-opioid receptor and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation and ERK1/2 phosphorylation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues, H-Tyr-Pro-Trp-(2-furyl)Map-NH(2) (analogue 12) exhibited the highest binding potency (K(i)(μ) = 0.221 nM) and efficacy (EC(50) = 0.0334 nM, E(max) = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that, compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the μ-opioid receptor.

We present herein for the first time the synthesis and preliminary biological evaluation of various modified chromanes via a rosin-derived tertiary amine-thiourea-catalyzed highly enantioselective Friedel-Crafts alkylation reaction.

Recently we reported the synthesis and structure-activity study of endomorphin-1 (EM-1) analogues containing novel, unnatural α-methylene-β-aminopropanoic acids (Map). In the present study, we describe new EM-1 analogues containing Dmt(1), (R/S)-βPro(2), and (ph)Map(4)/(2-furyl)Map(4). All of the analogues showed a high affinity for the μ-opioid receptor (MOR) and increased stability in mouse brain homogenates. Of the new compounds, Dmt(1)-(R)-βPro(2)-Trp(3)-(2-furyl)Map(4) (analogue 12) displayed the highest affinity toward MOR, in the picomolar range (Ki(μ) = 3.72 pM). Forskolin-induced cAMP accumulation assays indicated that this analogue displayed an extremely high agonistic potency, in the subpicomolar range (EC50 = 0.0421 pM, Emax = 99.5%). This compound also displayed stronger in vivo antinociceptive activity after iv administration when compared to morphine in the tail-flick test, which indicates that this analogue was able to cross the blood-brain barrier.

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