Structure-Activity Relationships of [des-Arg7]Dynorphin A Analogues at the κ Opioid Receptor.

Dynorphin A (Dyn A) is an endogenous ligand for the opioid receptors with preference for the κ opioid receptor (KOR), and its structure-activity relationship (SAR) has been extensively studied at the KOR to develop selective potent agonists and antagonists. Numerous SAR studies have revealed that the Arg7 residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg7]Dyn A analogues found that Arg7 is not a key residue and even deletion of the residue does not affect biological activities at the KOR. In addition, it was also found that [des-Arg7]Dyn A(1-9)-NH2 is a minimum pharmacophore and its modification at the N-terminus leads to selective KOR antagonists. A lead ligand, 14, with high affinity and antagonist activity showed improved metabolic stability and could block antinociceptive effects of a KOR selective agonist, FE200665, in vivo, indicating high potential to treat KOR mediated disorders such as stress-induced relapse.

Enkephalin analogues with N-phenyl-N-(piperidin-2-ylmethyl)propionamide derivatives: Synthesis and biological evaluations.

N-Phenyl-N-(piperidin-2-ylmethyl)propionamide based bivalent ligands are unexplored for the design of opioid based ligands. Two series of hybrid molecules bearing N-phenyl-N-(piperidin-2-ylmethyl)propionamide derived small molecules conjugated with an enkephalin analogues with and without a linker (ß-alanine) were designed and synthesized. Both bivalent ligand series exhibited remarkable binding affinities from nanomolar to subnanomolar range at both µ and δ opioid receptors and displayed potent agonist activities as well. The replacement of Tyr with Dmt and introduction of a linker between the small molecule and enkephalin analogue resulted in highly potent ligands. Both series of ligands showed excellent binding affinities at both µ (0.6-0.9nM) and δ (0.2-1.2nM) opioid receptors respectively. Similarly, these bivalent ligands exhibited potent agonist activities in both MVD and GPI assays. Ligand 17 was evaluated for in vivo antinociceptive activity in non-injured rats following spinal administration. Ligand 17 was not significantly effective in alleviating acute pain. The most likely explanations for this low intrinsic efficacy in vivo despite high in vitro binding affinity, moderate in vitro activity are (i) low potency suggesting that higher doses are needed; (ii) differences in experimental design (i.e. non-neuronal, high receptor density for in vitro preparations versus CNS site of action in vitro); (iii) pharmacodynamics (i.e. engaging signalling pathways); (iv) pharmacokinetics (i.e. metabolic stability). In summary, our data suggest that further optimisation of this compound 17 is required to enhance intrinsic antinociceptive efficacy.

Design synthesis and structure-activity relationship of 5-substituted (tetrahydronaphthalen-2yl)methyl with N-phenyl-N-(piperidin-2-yl)propionamide derivatives as opioid ligands.

Here, we report the design, synthesis and structure activity relationship of novel small molecule opioid ligands based on 5-amino substituted (tetrahydronaphthalen-2-yl)methyl moiety with N-phenyl-N-(piperidin-2-yl)propionamide derivatives. We synthesized various molecules including amino, amide and hydroxy substitution on the 5th position of the (tetrahydronaphthalen-2-yl)methyl moiety. In our further designs we replaced the (tetrahydronaphthalen-2-yl)methyl moiety with benzyl and phenethyl moiety. These N-phenyl-N-(piperidin-2-yl)propionamide analogues showed moderate to good binding affinities (850-4 nM) and were selective towards the µ opioid receptor over the δ opioid receptors. From the structure activity relationship studies, we found that a hydroxyl substitution at the 5th position of (tetrahydronapthalen-2yl)methyl group, ligands 19 and 20, showed excellent binding affinities 4 and 5 nM, respectively, and 1000 fold selectivity towards the µ opioid relative to the delta opioid receptor. The ligand 19 showed potent agonist activities 75±21 nM, and 190±42 nM in the GPI and MVD assays. Surprisingly the fluoro analogue 20 showed good agonist activities in MVD assays 170±42 nM, in contrast to its binding affinity results.

Discovery of Novel Multifunctional Ligands with µ/δ Opioid Agonist/Neurokinin-1 (NK1) Antagonist Activities for the Treatment of Pain.

Multifunctional ligands with agonist bioactivities at µ/δ opioid receptors (MOR/DOR) and antagonist bioactivity at the neurokinin-1 receptor (NK1R) have been designed and synthesized. These peptide-based ligands are anticipated to produce better biological profiles (e.g., higher analgesic effect with significantly less adverse side effects) compared to those of existing drugs and to deliver better synergistic effects than coadministration of a mixture of multiple drugs. A systematic structure-activity relationship (SAR) study has been conducted to find multifunctional ligands with desired activities at three receptors. It has been found that introduction of Dmt (2,6-dimethyl-tyrosine) at the first position and NMePhe at the fourth position (ligand 3: H-Dmt-d-Ala-Gly-NMePhe-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) displays binding as well as functional selectivity for MOR over DOR while maintaining efficacy, potency, and antagonist activity at the NK1R. Dmt at the first position with Phe(4-F) at the fourth position (ligand 5: H-Dmt-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) exhibits balanced binding affinities at MOR and DOR though it has higher agonist activity at DOR over MOR. This study has led to the discovery of several novel ligands including 3 and 5 with excellent in vitro biological activity profiles. Metabolic stability studies in rat plasma with ligands 3, 5, and 7 (H-Tyr-d-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) showed that their stability depends on modifications at the first and fourth positions (3: T1/2 > 24 h; 5: T1/2 ≈ 6 h; 7: T1/2 > 2 h). Preliminary in vivo studies with these two ligands have shown promising antinociceptive activity.

Design and synthesis of novel bivalent ligands (MOR and DOR) by conjugation of enkephalin analogues with 4-anilidopiperidine derivatives.

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.

Discovery of 5-substituted tetrahydronaphthalen-2yl-methyl with N-phenyl-N-(piperidin-4-yl)propionamide derivatives as potent opioid receptor ligands.

A new series of novel opioid ligands have been designed and synthesized based on the 4-anilidopiperidine scaffold containing a 5-substituted tetrahydronaphthalen-2yl)methyl group with different N-phenyl-N-(piperidin-4-yl)propionamide derivatives to study the biological effects of these substituents on µ and δ opioid receptor interactions. Recently our group reported novel 4-anilidopiperidine analogues, in which several aromatic ring-contained amino acids were conjugated with N-phenyl-N-(piperidin-4-yl)propionamide and examined their biological activities at the µ and δ opioid receptors. In continuation of our efforts in these novel 4-anilidopiperidine analogues, we took a peptidomimetic approach in the present design, in which we substituted aromatic amino acids with tetrahydronaphthalen-2yl methyl moiety with amino, amide and hydroxyl substitutions at the 5th position. In in vitro assays these ligands, showed very good binding affinity and highly selective toward the µ opioid receptor. Among these, the lead ligand 20 showed excellent binding affinity (2 nM) and 5000 fold selectivity toward the µ opioid receptor, as well as functional selectivity in GPI assays (55.20 ± 4.30 nM) and weak or no agonist activities in MVD assays. Based on the in vitro bioassay results the lead compound 20 was chosen for in vivo assessment for efficacy in naïve rats after intrathecal administration. Compound 20 was not significantly effective in alleviating acute pain. This discrepancy between high in vitro binding affinity, moderate in vitro activity, and low in vivo activity may reflect differences in pharmacodynamics (i.e., engaging signaling pathways) or pharmacokinetics (i.e., metabolic stability). In sum, our data suggest that further optimization of this compound 20 is required to enhance in vivo activity.

Design, synthesis and biological evaluation of multifunctional ligands targeting opioid and bradykinin 2 receptors.

We report here the design and synthesis of novel multifunctional ligands that act as (µ/δ) opioid agonists and bradykinin 2 receptor antagonists. These multifunctional ligands were designed to interact with the multiple receptors to show an enhanced analgesic effect, with no opioid-induced tolerance. We designed our multifunctional ligands based on the well-known second generation bradykinin 2 receptor antagonist Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) and the opioid enkephalin analogues Tyr-DAla-Phe, Tyr-DAla-Gly-Phe and Tyr-Pro-Phe. We explored the conjugation of opioid pharmacophore to the Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) in various positions with and without a linker. These bifunctional ligands showed very good binding affinity towards the both µ and δ opioid receptors. Among these bifunctional ligands 8, 11 and 12 showed excellent and balanced binding affinity at both µ and δ opioid receptors (0.5 nM, 2.0 nM; 0.3 nM, 2 nM; 2 nM and 3 nM), respectively. On the other hand these bifunctional ligands showed very weak and no binding affinity for rat brain bradykinin 2 receptors. Similarly, the Hoe 140 showed very low affinity (>10,000 nM and 9,000 nM) against [(3)H] BK binding in rat brain membranes and in HEK293 cells, respectively. In contrast, the Hoe 140 showed very good binding affinity in guinea pig ileum (0.43 nM) similar to that of previously reported. The bradykinin 2 receptors are known to be present in rat brain membrane, guinea pig ileum (GPI) and rabbit jugular vein. Previously the binding affinity of Hoe 140 for bradykinin 2 receptor was reported using guinea pig ileum. The above results suggest that the bradykinin 2 receptors present in rat brain membrane are a different sub type than the bradykinin 2 receptor present in guinea pig ileum (GPI).

Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities.

Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.

Novel cyclic biphalin analogue with improved antinociceptive properties.

Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent µ/δ mixed receptor affinities (K i (δ) = 5.2 nM; K i (µ) = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities.

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-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.

Effect of anchoring 4-anilidopiperidines to opioid peptides.

We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.

Biological active analogues of the opioid peptide biphalin: mixed α/ß(3)-peptides.

Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-ß(3) amino acids. The derivative 1 containing hß(3) Phe in place of Phe showed good µ- and δ-receptor affinities (Ki(δ) = 0.72 nM; Ki(µ) = 1.1 nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.

Chiral Effect of a Phe Residue in Position 3 of the Dmt1-L(or D)-Tic2 Analogues on Opioid Functional Activities.

In this letter, we describe a structure-activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-L(or D)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the µ opioid receptor.

cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues: part 2.

Recently, we reported synthesis and activity of a constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) and related linear models containing the cis-4-amino-L-proline (cAmp) in place of native Pro(2). In the present article, the adopted rationale is the possible modulation of the receptor affinity of the cAmp containing EM-2 analogues by assigning a different stereochemistry to the Phe(3) and Phe(4) residues present in the ring. Thus, eight more analogues with different absolute configuration at the chiral center of the aromatic residues in positions 3 and 4 have been synthesized and their opioid activity examined. The stereochemical change at the α-carbon atoms leads to a meaningful enhancement of the affinity and activity toward µ opioid receptors with respect to the prototype compound 9: e.g., 9a, K(i)(µ) = 63 nM, GPI (IC(50)) = 480 nM; 9b, K(i)(µ) = 38 nM, GPI (IC(50)) = 330 nM.

The cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues.

Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the µ opioid receptor, the most responsible for analgesic effects in the central nervous system. The presence of the Pro(2) represents a crucial factor for the ligand structural and conformational properties. Proline is in fact an efficient stereochemical spacer, capable of inducing favorable spatial orientation of aromatic rings, a key factor for ligand recognition and interaction with receptors. Here the Pro(2) has been replaced by 4(S)-NH(2)-2(S)-proline (cAmp), a proline/GABA cis-chimera residue. This bivalent amino acid maintains the capacity to influenc the tetrapeptide conformation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and κ opioid receptors and bind only poorly to the µ receptors (for cyclopeptide 9: K(i)(µ) = 660 nM; GPI (IC(50)) = 1.4% at 1 µM; for linear tetrapeptide acid 13: K(i)(µ) = 2000 nM; GPI (IC(50)) = 0% at 1 µM; for linear tetrapeptide amide 15: K(i)(µ) = 310 nM; GPI (IC(50)) = 894 nM).

Afferent drive elicits ongoing pain in a model of advanced osteoarthritis.

Osteoarthritis (OA) is a chronic condition characterized by pain during joint movement. Additionally, patients with advanced disease experience pain at rest (ie, ongoing pain) that is generally resistant to nonsteroidal antiinflammatory drugs. Injection of monosodium iodoacetate (MIA) into the intraarticular space of the rodent knee is a well-established model of OA that elicits weight-bearing asymmetry and referred tactile and thermal hypersensitivity. Whether ongoing pain is present in this model is unknown. Additionally, the possible relationship of ongoing pain to MIA dose is not known. MIA produced weight asymmetry, joint osteolysis, and cartilage erosion across a range of doses (1, 3, and 4.8 mg). However, only rats treated with the highest dose of MIA showed conditioned place preference to a context paired with intraarticular lidocaine, indicating relief from ongoing pain. Diclofenac blocked the MIA-induced weight asymmetry but failed to block MIA-induced ongoing pain. Systemic AMG9810, a transient receptor potential V1 channel (TRPV1) antagonist, effectively blocked thermal hypersensitivity, but failed to block high-dose MIA-induced weight asymmetry or ongoing pain. Additionally, systemic or intraarticular HC030031, a TRPA1 antagonist, failed to block high-dose MIA-induced weight asymmetry or ongoing pain. Our studies suggest that a high dose of intraarticular MIA induces ongoing pain originating from the site of injury that is dependent on afferent fiber activity but apparently independent of TRPV1 or TRPA1 activation. Identification of mechanisms driving ongoing pain may enable development of improved treatments for patients with severe OA pain and diminish the need for joint replacement surgery.

Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands.

Newly designed bivalent ligands-opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials-carboxy-derivatives of Fentanyl (1a-1c) was developed. These products have been transformed to 'isoimidium perchlorates' (2a-c). The new isoimidium perchlorates have been successfully implemented in nucleophilic addition reactions, with l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to give the target compounds-amides (3a-c). Perchlorates (2a-c) successfully undergo reactions with other nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited µ-opioid agonist activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a new series of opioid agonist/NK1-antagonist compounds.

Discovery of a potent and efficacious peptide derivative for δ/µ opioid agonist/neurokinin 1 antagonist activity with a 2',6'-dimethyl-L-tyrosine: in vitro, in vivo, and NMR-based structural studies.

Multivalent ligands with δ/µ opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and had significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt(1) incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus) and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug.

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers.

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4' positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-L-phenylalanine residues at 4 and 4' positions. Binding values are: Kµ(i)=0.51 nM and Kδ(i)=12.8 nM for compound 9, Kµ(i)=0.09 nM and Kδ(i)=0.11 nM for analogue 10.

Development of potent µ and δ opioid agonists with high lipophilicity.

An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-ylpropionamide moiety at the C-terminal was performed and has resulted in highly potent ligands at µ and δ opioid receptors. In general, ligands with the substitution of D-Nle(2) and halogenation of the aromatic ring of Phe(4) showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.