In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3’,5’-Bzl(CF3)2) which was a previously reported bifunctional compound with delta/mu opioid agonist and neurokinin-1 receptor antagonist activities, and with a half life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3’,5’-Bzl(CF3)2) was found to have effective bifunctional activities with a well-defined conformation with two β-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 ± 9 % of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, which have anti-opioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[D-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3′,5′-(CF 3 ) 2 -Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

IntroductionInadequate treatment of pain is an important and urgent problem, which needs to be addressed. Opiates are still the drugs of choice for the treatment of moderate to severe acute pain; however their use are generally accompanied by undesired side effects like analgesic tolerance. Several pharmacological studies have suggested the role of substance P, in pain transmission [1]. It has been observed that co-administration of neurokinin-1 receptor antagonists and opioid agonists augmented the acute effects of opioids and also prevented opioid induced tolerance [2]. In view of these and other observations we have designed novel bifunctional peptides with mixed opioid μ/δ agonist activity and neurokinin-1 antagonist activity [3]. Earlier reports of molecules with opioid μ/δ agonist activity and neurokinin-1 antagonist activity did not have balanced activity at both the receptor's systems [4]. In our studies the opioid pharmacophore chosen has the sequence H-Tyr-DAla-Gly-Phe which is a substructure of biphalin, a highly potent agonist at both δ and μ opioid receptors [5]. A 3,5-(bistrifluoromethyl) benzyl ester of N-acylated tryptophans was chosen as the NK1 pharmacophore [6]. These two pharmacophores were combined with amino acids acting as possible address moieties they could be part of the two pharmacophores.Sequences of some of the bifunctional peptides synthesized are represented below (Fig. 1) Results and DiscussionThe bifunctional peptide ligands were synthesized using solution phase chemistry. The synthetic strategy was started with coupling of tryptophan 3,5-(bistrifluoromethyl)benzyl ester with the respective Boc protected amino acid followed by subsequent chain elongation using BOP/HOBT/NMM method. The N α -Boc groups were deprotected by 100% TFA. The final crude peptides were purified by C 18 RP-HPLC (Vydac 10 mm x 250 mm, 10 μM) with a gradient of 30-70% CH 3 CN in aq. 0.1% TFA. NP30, with Gly as the linker showed excellent binding affinity at both δ and μ opioid receptors and rat NK1 receptors (K i = 4.7 nM, δ opioid; 0.29 nM, μ opioid; 4.2 nM, rNK1). It also showed potent δ and μ opioid agonist efficacies in the MVD and GPI assays binding assays with the IC 50 values of 21 and 26 nM, respectively. GPI assay clearly showed that NP30 was an antagonist against substance P stimulation with K e = 59 nM. NP34 with γAbu (Aminobutyric) at position 5 turned out to be the most potent at the GPI assay (K e = 0.96nM) among all the compounds synthesized. On the other hand substitution with DAla (NP40) at the same position resulted in drastic lowering of antagonistic activity in the GPI assays (K e = 610nM). However both NP34 and NP40 were highly potent in the binding affinity studies at the opioid and rat NK1 receptors. HHS Public AccessAuthor manuscript Adv Exp Med Biol. Author manuscript; available in PMC 2017 October 09. ConclusionSeveral bifunctional peptides with δ/μ opioid agonist pharmacophore and NK1 antagonist pharmacophore were designed and synthesized. Binding assays performed showed potent act...

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr1-DAla2-Gly3-Phe4-Gly5-Trp6-O-[3′,5′-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.

We describe the design and synthesis of novel bivalent ligands based on the conjugation of 4-anilidopiperidine derivatives with enkephalin analogues. The design of non-peptide analogues is explored with 5-amino substituted tetrahydronaphthalen-2yl)methyl containing 4-anilidopiperidine derivatives, while non-peptide-peptide ligands are explored by conjugating the C-terminus of enkephalin analogues (H-Xxx-DAla-Gly-Phe-OH) to the amino group of 4-anilidopiperidine small molecule derivatives with and without a linker. These novel bivalent ligands are evaluated for biological activities at μ and δ opioid receptors. They exhibit very good affinities at μ and δ opioid receptors, and potent agonist activities in MVD and GPI assays. Among these the lead bivalent ligand 17 showed excellent binding affinities (0.1 nM and 0.5 nM) at μ and δ opioid receptors respectively, and was found to have very potent agonist activities in MVD (56 +/- 5.9 nM) and GPI (4.6 +/− 1.9 nM) assays. In vivo the lead bivalent ligand 17 exhibited a short duration of action (< 15 min) comparable to 4-anilidopiperidine derivatives, and moderate analgesic activity. The ligand 17 has limited application against acute pain but may have utility in settings where a highly reversible analgesic is required.